Best Practice & Research Clinical Haematology Volume 24, Issue 1 March 2011

CLINICAL HAEMATOLOGY The Best Practice & Research series aims to provide a topical serial publication describing and i...

Author: W. Fibbe

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CLINICAL HAEMATOLOGY The Best Practice & Research series aims to provide a topical serial publication describing and integrating the results from the latest original research articles into practical, evidence-based review articles. These articles seek to address the key clinical issues of diagnosis, treatment and patient management. Each issue follows a problem-orientated approach which focuses on the key questions to be addressed, clearly defining what is known and not known. Management is described in practical terms so that it can be applied to the individual patient. The serial is aimed at the physician in both practice and training. Best Practice & Research Clinical Haematology is abstracted and indexed in the following sources: Index Medicus, Medline, PubMed, Current Contents/Clinical Medicine, Current Contents/Life Sciences, Science Citation Index, SciSearch, Research Alert and EMBASE/Excerpta Medica.

Editor-in-Chief Jacob M. Rowe MD Dresner Professor of Hemato-oncology; Technion Israel Institute of Technology Chief, Department of Hematology and Bone Marrow Transplant Rambam Medical Center Haifa 31096, Israel EDITORIAL BOARD T. Barbui, Italy I. M. Franklin, UK D. G. Gilliland, USA A. Gratwohl, Switzerland C. Hershko, Israel

V. Hoffbrand, UK V. J. Marder, USA M. S. Tallman, USA J. C. Wade, USA

Best Practice & Research Clinical Haematology 24 (2011) 1–2

Contents lists available at ScienceDirect

Best Practice & Research Clinical Haematology journal homepage: www.elsevier.com/locate/beha

Preface

Introduction

Mesenchymal stromal cells (MSCs) were recognized more than 40 years ago by Alexander Friedenstein, who described a population of adherent cells from the bone marrow which were non phagocytic, had a fibroblastic appearance and that exhibited in-vitro multi-lineage differentiation into bone, cartilage, adipose tissue, tendon and muscle. Following transplantation under the kidney capsule, these cells gave rise to ossicles that contained multiple mesenchymal lineages. In recent years, there has been a renewed interest in MSCs that is mainly driven by their potential for clinical application in a variety of disorders. A vast amount of data suggest that MSCs can be applied for tissue regeneration, for instance to repair bone, cartilage or muscle defects. Perhaps more importantly, MSCs have been demonstrated to regulate immune responses in the context of tissue injury, autoimmunity and alloimmunity in particular transplantation. MSCs have been used therapeutically in clinical trials in stem cell transplantation, with the aim to promote engraftment or to prevent or treat acute graft-versus-host disease. Following encouraging experiments in experimental animals suggesting that MSC treatment may alleviate the symptoms in several models of autoimmune conditions, such as Crohn’s disease and Systemic Lupus Erythematosus, clinical trials have now began to explore their potential efficacy in the setting of human autoimmune disorders. The possibility to expand the cells ex-vivo under appropriate culture conditions has greatly facilitated their clinical use. At present, most MSC products are expanded in media containing fetal calf serum or platelet lysate. Under these conditions, cells can be culture expanded to numbers required for clinical use within a period of several weeks. However, the effects of culture conditions or donor selection on the functional and therapeutic properties of clinical grade MSCs is still unclear. Very few markers are available for the prospective isolation of MSCs and in most instances, isolation still relies on their ability to adhere to plastic surfaces. An enrichment of CFU-F activity has been reported for markers including STRO1 and the low affinity Nerve Growth Factor Receptor CD271. However, these markers are rarely used for prospective isolation. The clinical potential of MSCs is also determined by host factors and it has been suggested that MSCs need to become activated in the host environment in order to mediate their immune modulatory effects. Heterogeneity in host factors, in particular the availability of an appropriate pro-inflammatory environment, may explain the variability that is observed in clinical trials. It is therefore attempted to mimic the pro-inflammatory environment by the addition of cytokines, including interferon gamma. I am happy that leading experts in the field of MSC biology, translation and clinical application have accepted the invitation to contribute to this special issue. It covers not only the biology of murine and human MSCs, their ex-vivo expansion and their role in the hematopoietic microenvironment, but also their potential for clinical application in autoimmune disorders, neuroregenerative disorders and allogeneic stem cell and cord blood transplantation.

1521-6926/$ – see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.beha.2011.02.001

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Preface / Best Practice & Research Clinical Haematology 24 (2011) 1–2

Willem E. Fibbe, Prof.* Leiden University Medical Centre, Department of Immunohematology and Blood Transfusion, Albinusdreef 2, Building 1, E3-Q P.O. Box 9600, 2300 RC Leiden, Netherlands Tel.: þ31 71 5263827; Fax: þ31 71 5265267. E-mail address: [email protected]




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Multipotent adult progenitor cells Abhishek Sohni, MSc, Doctoral fellow *, Catherine M. Verfaillie, MD, Director, Stem cell institute Leuven * Interdepartementaal Stamcelinstituut, Katholieke Universiteit Leuven, O&N1, bus 804, Herestraat 49, 3000 Leuven, Belgium

Keywords: MAPC pluripotency reprogramming

We here discuss the potency and characteristics of various adult derived adherent stem cells with special focus on multipotent adult progenitor cells (MAPC) isolated first in 2002 in our lab. We describe the potency of MAPC, our current understanding in relationship with novel insights gained in epigenetic modifications that increase cellular potency, and their possible clinical applications. Ó 2011 Published by Elsevier Ltd.

Introduction A stem cell is characterized by its unique ability to self-renew, some without senescence. When selfrenewal divisions are symmetrical, the two daughter cells become stem cells, leading to expansion of the stem cell pool. In postnatal life, most stem cell divisions are asymmetrical, yielding one stem cell and a more differentiated cell, or progenitor cell, which has limited self-renewal ability. The potency of a stem cell is defined by its ability to divide and produce one to many different cell types. The zygote is totipotent as it can contribute to the cells of embryonic as well as extraembryonic tissue. An embryonic stem cell (ESC), derived from the inner cell mass (ICM) of the embryo, is pluripotent as it differentiates to all the cell types of embryo proper, but no longer the trophoblast. Stem cells derived from different tissues are multipotent as they can give rise to all cells of the tissue they are derived from, but not cells of other tissues. The third part of the definition of stem cells is that they can functionally repopulate a tissue (even undamaged) in vivo. The most extensively studied adult stem cell is the hematopoietic stem cell (HSC) – isolated from bone marrow (BM) [1], from peripheral blood following mobilization from the BM or from umbilical cord blood (UCB) – which can replenish the entire blood lineage of lethally irradiated organisms upon transplantation. Since the characterization of HSC, many other adult stem cells have been defined,

* Corresponding authors. Tel.: þ32 16 33 02 95; Fax: þ32 16 33 02 94. E-mail addresses: [email protected] (A. Sohni), [email protected] (C.M. Verfaillie). 1521-6926/$ – see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.beha.2011.01.006

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A. Sohni, C.M. Verfaillie / Best Practice & Research Clinical Haematology 24 (2011) 3–11

including neural stem cells (NSC) in the hippocampus and subventricular zone of adult brain [2–5], gastrointestinal stem cells [6], epidermal stem cells [7,8], among others. Friedenstein and colleagues demonstrated in 1970 that a population of fibroblast like cells can be isolated from BM, that can form bone and cartilage and reconstitute the bone marrow microenvironment [9], which later were called mesenchymal stem cells or marrow stromal cells (MSC) [10,11]. In a recent study by the Bianco group, it was shown that MSC isolated freshly from BM can recreate bone and the bone marrow microenvironment, from which MSC can again be isolated, demonstrating for the first time that MSC indeed have stem cell properties [12]. Although it is generally accepted that the adult stem cells named above have a restricted differentiation ability; i.e. they generate cells from the tissue from which they were derived, since the late 1990s, many studies have suggested that under some conditions, adult tissue derived stem cells may have broader differentiation potency, also termed stem cell plasticity. Jiang et al. in 2002 cultureisolated such an adult cell from bone marrow, called Multipotent Adult Progenitor Cells [13]. An update on our understanding of the characteristics and origin of MAPC will be given, specifically describing rodent MAPC, and MAPC will be placed in relationship to current knowledge on different stem cells and ability of cells to acquire novel fates. Embryo and non-embryo-derived stem cells Embryonic tissue derived stem cell The most extensively studied and characterized embryo derived stem cells are the ESC. ESC are classically derived from the ICM of the blastocyst at E 3.5 and E 4–5 in mouse and man, respectively. Murine (m) and human (h) ESC are pluripotent [14,15]. This can be demonstrated by tetraploid complementation studies in mouse, where mESC give rise to all cells of the somatic and germline lineages [16,17]. Another method to demonstrate pluripotency is by formation of teratomas from either hESC or mESC, containing cells of all three somatic germ layers. Stem cell populations have also been isolated from other stages of early murine conspectuses, including from the 4–8 morula [18] stage and from the pre-gastrula stage from mouse embryos [19].The former are like mESC – pluripotent, as they contribute to all the somatic and germline cell types, and are maintained in vitro like ICM derived mESC. Cells isolated from pre-gastrula embryos, also termed epiblast-stem cells (EpiSC) express like mESC, Oct4, Nanog and Ssea-1, and form teratomas. However unlike mESC, EpiSC do not require feeders or leukemia inhibitory factor (Lif) for culture but can be expanded in the presence of Activin/nodal signaling [19]. In contrast to preblastocyst derived stem cells, EpiSC were unable to contribute to somatic cells and the germline following injection into blastocyst or following morula aggregation [18,19]. It is believed that hESC may be more akin to mEpiSC, based on gene expression profiling data and similar culture conditions required to maintain hESC in vitro [19]. Jannet Rossant’s group isolated stem cells from extraembryonic endoderm called XEN [20]. These cells express Sox7, Hnf4, Gata4 and Foxa2 but lack expression of Oct4 and Nanog [20]. In a chimera assay, XEN cells contributed to the parietal endoderm and to the parietal yolk sac at later stages during embryo development [20]. More recently, Debeb et al. isolated an other population of Oct4 expressing cells from rat blastocysts, termed extraembryonic endodermal precursor cells or XEN-P cells. Rat XENP cells, like rat or mouse ESC, express Oct4 and Ssea-1 and require leukemia inhibitory factor (Lif) for ex vivo maintenance. However, XEN-P cells do not express Nanog or Sox2, but express Sox7, Sox17, Gata4 and Gata6, similar to the hypoblast fraction of cells within the ICM that also do not express Nanog, but express Gata6 [21]. Upon morula aggregation or injection in the blastocyst, XEN-P cells contribute to primitive/visceral and parietal extraembryonic endodermal lineages but not the embryo proper [21], and even though they form tumors when injected postnataly, the tumors are akin to yolk sac tumors, not teratoma [22]. Yet another cell type of the embryonic lineage is the trophoblast stem cell. As the name suggests, they are derivative of the trophectoderm of the blastocyst, and require fibroblast growth factor-4 (FGF4) for ex vivo maintenance. These cells contribute to the embryo part of the placenta, an important cell lineage for the embryo survival in utero [23]. Matsui et al. isolated embryonic germinal stem cells (EGC) with potency similar to ESC [24]. These cells were derived from primodial germ cells of 8.5 days post


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coitum embryos and exhibit properties similar to ESC including germ line transmission, teratoma formation and chimeric contribution to embryo proper [24]. EGC have also been isolated from human abortuses, and like their mouse counterparts exhibit many characteristics of ESC [25]. Stem cells derived from non-embryonic tissues As many of the stem cells discussed below will be discussed at length in other sections of this special issue, the description of stem cells from non-embryonic tissue, except for MSC, will be very brief. HSC, isolated from BM, blood or UCB, have been the best characterized. HSC can be purified to homogeneity in mouse and highly enriched in human, based on a complement of cell surface antigens, and many signals that govern their quiescence and differentiation have been identified. Likewise, stem cells that generate skin and its appendages and gastrointestinal stem cells can be purified to near homogeneity based on cell surface antigens [6–8]. Based on these cell surface determinants as well as based on the fact that they proliferate rarely, and hence retain bromodeoxyuridine (BrdU) long term has allowed investigators to identify their position in the different tissues [26–28]. This has lead to characterization of the niche cells surrounding the stem cells. These studies have indicated that signals that govern selfrenewal and differentiation of HSC (Wnt’s, TGFb-family members, Notch-signaling) are highly similar for epidermal and gastrointestinal stem cells. Similarly, NSC have been isolated from postnatal brain where they are located in the subventricular zone (SVZ) and the hippocampus. Even though prospective isolation as for HSC, and epidermal and gastrointestinal stem cells is less completely defined, the location of NSC in the SVZ has been defined. Morphogens such as Wnt’s, TGFb-family members and signals via the Notch system also play a role in NSC homeostasis and differentiation. In the 1970s Friedenstein et al. demonstrated that aside from HSC another population of cells could be cultivated from bone marrow, that had the appearance of fibroblasts, but can generate aside from fibroblasts, also adipocytes, chondrocytes and osteocytes [9,29]. These cells were initially termed fibroblast colony forming cells (CFU-F), and were later renamed by Caplan and colleagues and Prockop and colleagues as mesenchymal stem cells or MSC [10,11]. MSC are commonly isolated from BM, and since the initial description by Friedenstein, also from many other tissues, such as adipose tissue, fetal lung, placenta, Wharton’s jelly and UCB, among others [30–33], by culturing cells from these tissues on plastic dishes, to which they attach. MSC give rise to fibroblasts, adipocytes, chondrocytes, osteocytes, smooth muscle cells, and hematopoietic supportive “stromal” cells [9–11,34]. Some have also suggested that MSC may give rise to endothelial cells and cardiac muscle cells, two other mesodermal cell types, although this notion remains contested [35–38]. During the past 3–4 years, more insights have been gained related to the origin of MSC, as well as their stem cell capacities. Studies from the Bianco and the Peault groups have demonstrated that MSC in different tissues are located surrounding blood vessels and are a subpopulation of pericytes [12,39]. They can be prospectively isolated based on expression of the cell surface antigen CD146 [12], and as alluded to earlier, can be grafted in vivo where they reconstitute bone tissue from which they can be re-isolated, demonstrating that they represent true stem cells. Although the location of MSC in vivo has been defined, what the signals are that regulate the homeostasis of MSC in vivo has yet to be defined. MSC, isolated by culturing in the presence of serum on plastic surfaces, are thought to contain only a small fraction of the cells that can be prospectively isolated, as transcriptome studies have shown that the expressed gene profile changes very fast upon culturing MSC in vitro (PJ Simons, personal communication). Whether these cultureinduced changes may also be responsible for changing/broadening the fate of MSC, will be discussed below. Stem cells derived from non-embryonic tissues with broader potential During the last 10 years, a number of groups have isolated cells with broader differentiation ability than MSC from BM and other tissues, such amniotic fluid and UCB, cells by plastic adherence, as is done for MSC. These include MAPC (to be discussed further below), very small embryonic stem cell like (VSEL) stem cells, marrow isolated adult multilineage inducible (MIAMI), multipotent adult stem cells (MASC), amnitotic fluid stem cells (AFS), and unrestricted somatic stem cells (USSC), among others [40–43]. Cell surface phenotype and differentiation potential has been summarized in Table 1. VSEL, are

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Table 1 Adherent adult stem cells with cell surface phenotype and differentiation capacity. Cell type

Derived from

Potency

Surface phenotype

Reference

Human mesenchymal stem cell (MSC)a

Bone marrow, cord blood, tissue resident

Mesenchymal lineage

[10–12]

Human marrow isolated adult multilineage inducible cells (MIAMI) Human Unrestricted somatic stem cells (USSC) Human very small embryonic like (VSEL)

Bone marrow

Mesoderm, endoderm and ectoderm

CD105þ, CD90þ, CD73þ, CD79a, CD45, CD34, CD19, CD14, CD11b, HLA-DRCD164þ, CD122þ, CD81þ, SSEA4þ, CD45, CD34, c-Kit-,

Cord blood

Mesoderm, endoderm and ectoderm Neuroectoder, pancreatic cells, cardiomyocytes

CD10 low, Flk1 low, CD45, CD34, c-kitCXCR4þ, AC133þ, Sca-1þ, CD45-, SSEA1þ(mouse), SSEA4þ(Human), Lin-, MHC-I-

Pancreatic cells, neuronal, mesenchymal

CD49bþ/CD90þ, CD13þ, CD105 low, CD73 low, CD44 low, HLA-ABC low, CD133, CD45, CD34, CD14, HLA-DRCD105þ, CD90þ, CD73þ, CD44þ, CD29þ, MHC-Iþ, c-Kitþ, MHC-II low, CD133, CD45, CD34 c-Kitþ, CD9þ, CD13þ, CD31þ, CD44, MHC-I-, CD45, Thy1-

Human Multipotent adult stem cells (MASC)

Umbilical cord blood, bone marrow mobilized Bone marrow, heart, liver

Human amniotic fluid stem cell (AFS)

Amniotic fluid

Mesenchymal, neuronal, endothelial, hepatic

Rodent multipotent adult progenitor cells (MAPC)

Bone marrow, muscle, brain

Mesoderm, endoderm and ectoderm

a

[41]

[46] [42,43]

[40]

[45]

[47,49, 61,79]

Phenotype based on the International society for Cellular therapy criteria.

cells derived from bone marrow (mouse) and cord blood (Human) [42,43]. While human cord blood derived VSEL cells cannot be expanded ex vivo [42] their mouse counter parts can be grown as spheres in co-culture on C2C12 cells. These cells have been shown to differentiate to cardiomyocytes, neuroectoderm and pancreas [43,44]. They reportedly express Oct4 and Nanog, two transcription factors that govern pluripotency of ESC. MASC are isolated from BM, heart and liver [40]. MASC can divide in vitro for more than 40 population doublings without shortening of telomere length, and can differentiate into neuroectodermal, hepatic and mesodermal lineages in vitro. MASC cells were also reported to express Sox2, Oct4 and Nanog. AFS are isolated from amniotic fluid, express Oct4 and Nanog, proliferate well beyond 100 population doublings and generate mesenchymal, endothelial, hepatic and neuronal lineages [45]. The other cell populations, MIAMI and USSC, by contrast do not express markers associated with pluripotency such as Oct4, Nanog and Sox2. MIAMI cells, isolated from BM, can proliferate extensively, and give rise to pancreatic and neuronal lineages apart from mesenchymal cells [41]. USSC in vitro have the capacity to form fat, bone and cartilage tissue and can generate hepatocyte and neuroectodermal cell types [46]. When implanted in pre immune sheep USSC can contribute to chondrocytes, cardiomyocytes, hematopoietic cells and certain neuron-like cells [46]. Culture conditions for all these cell populations are relatively similar, even though differences exist in cytokines and serum used and ambient oxygen levels. Whether these differences in culture conditions are responsible for the differences between MASC, MIAMI, AFS, USSC and VSEL populations, and between these cell populations and “classical” MSC is unknown. Isolation and characteristics of rodent MAPC We will here discuss the isolation of MAPC from the bone marrow of rat and mouse. An extensive protocol for the isolation of rodent MAPC has recently been published Subramanian et al. [47]; hence we will only briefly highlight the isolation procedure here. Rodent bone marrow cells are flushed from the long bones of young rodents, and plated with EGF, PDGF-BB, LIF in 2% serum containing medium on plastic plates for 4 weeks. Subsequently the cells are depleted of CD45 cells and subcloned at very low


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densities (5 cells/well of 96 well plate) [13,47,48] After 6–8 weeks, clones of cells with MAPC morphology and typical MAPC gene expression (Oct4) and cell surface characteristics (SSEA1 and CD31 for rat MAPC, for example) can be detected. To generate clonal populations, such cell populations can be re-cloned at 0.8 cells/well. Compared with the isolations described by Jiang et al. in 2002, the current protocol differs in that isolations and maintenance of rodent MAPC are done using fetal calf serum under hypoxic conditions. Murine and rat MAPC do not express CD45, a hematopoietic marker nor MHC class II and low to negligible levels of MHC class I. Mouse MAPC express c-Kit, EpCam and CD9 and lack CD34 and CD44, whereas rat MAPC express CD31, SSEA1, and lack CD44. Clones of rat MAPC express Oct4 at levels nearly equivalent to that of mouse embryonic stem cells, and mouse MAPC at levels 10–20% of that in ESC [49]. Of note rodent MAPC do not express Nanog or Sox2. Rodent MAPC differentiate into cells of all the three germ layers [13,49]. Some of these aspects will be highlighted here. Cardiovascular system The cardiovascular system is made up of tissue of the heart and blood vessels. MAPC differentiate robustly to endothelium and smooth muscle making up the components of blood vessel. Aranguren et al. demonstrated that MAPC can be differentiated to both venous and arterial endothelial cells [50] which was confirmed further by Liu et al. and Xu et al. [51, 52]. In a functional analysis study, Aranguren et al. also demonstrated that undifferentiated MAPC could restore muscle function as well as blood flow in a limb ischemia model in part by direct contribution to smooth muscle and endothelium, as well as skeletal muscle, and in part by elaborating trophic factors that enhance endogenous angiogenesis and myogenesis [53]. Ross et al. demonstrated that MAPC when treated with TGF-b and PDGFbb under serum free conditions can form smooth muscle cells (SMC) [54]. These MAPC-SMC express genes such as Transgelin (Tagln or Sm22), Calponin h-1 (CNN1) and more mature markers of smooth muscle such as alpha smooth muscle actin (aSMA) and smooth muscle myosin heavy chain (SM-MHC). MAPC-SMC function like primary mature smooth muscle cells under mechanical stress and chemical stimulation, and express calcium channels similar to those found on mature smooth muscle cells [54]. Tolar et al. and Pelacho et al. demonstrated that when mouse MAPC were grafted in an acute myocardial infract in mice, functional improvement occurred, even though no direct contribution of the MAPC to cardiac muscle was observed, but because of trophic factors generated by MAPC that decreased the infarct size and enhanced angiogenesis [55,56]. Hematopoietic system Serafini et al. demonstrated that when mouse MAPC were injected into lethally irradiated mice, reconstitution of the hematopoietic system could be seen, with generation of HSC that could repopulate secondary recipients. However, 1000 fold more MAPC were required compared to control (KTLS) HSC for reconstitution [57]. Osteoblast differentiation Maes et al. demonstrated that like MSC, MAPC can generate osteoblasts, which is enhanced by placenta-like growth factor [58]. Unpublished studies have also shown that rodent MAPC can differentiate into adipocytes, again akin to MSC. Hepatic differentiation MAPC have been instrumental in development of robust protocol [59] for differentiation to functional hepatocyte-like cells. This protocol is not only applicable for differentiation of MAPC to liver cells, but also can be adapted for mouse and human ESC [60]. Roelandt et al. have demonstrated that by mimicking early embryonic developmental cues that MAPC and ESC can be differentiated to functional hepatocyte-like cells. These hepatocyte-like cells express early hepatic markers such as Afp, Ttr, Alb and mature genes such as G6P, Cyp1a2, and the coagulation Factor-V. Roelandt et al. also demonstrated that

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these MAPC derived hepatocyte-like cells can secrete albumin, produce urea, store glycogen, and express functionally active cytchrome p450 enzymes [60]. Neural differentiation Jiang et al. in 2002 demonstrated that rat and mouse MAPC can differentiate to the neuroectodermal lineage, generating neurons with electrophysiological properties similar to CNS neurons [61]. More recently, differentiation toward neural stem cell-like cells has been achieved using a modified Conti et al. protocol [62], although maturation to neurons and astrocytes has not yet been achieved [49]. Immunomodulatory role of rodent MAPC Immunomodulation has been associated with certain adult stem cells and has been extensively studied in MSC. MSC derived from BM as well as UCB lack MHC-II and co-stimulatory molecules CD40, CD40L (ligand), CD80 & CD86 on their cell surface [63–65]. MSC inhibit the function of T-cells, B-cells and dendritic cells, and are therefore being tested clinically in immune disorders such as graft versus host disease, and Chrohn’s disease, among others [63,66]. Clones of murine and rat MAPC also demonstrate such immunomodulatory effect. Kovacsovics-Bankowski et al. have demonstrated that clinical scale expanded rat MAPC inhibited GVHD (graft versus host disease) response [67]. These rat MAPC were not sensitive to NK-cell lysis and could in a dose dependent manner suppress T-cell proliferation in mixed lymphocyte response (MLR) [67]. Similarly, mouse MAPC can inhibit in vivo Tcell alloresponse and GVHD, however, the GVHD suppression was only found upon intrasplenic delivery of the cells [68], or a popliteal lymph node assay [69] and not systemic delivery [68]. What is the developmental analog of rodent MAPC? Although rodent MAPC share certain characteristics with MSC, a transcriptome study comparing MAPC to MSC and ESC[49], demonstrated that the expressed gene profile of MSC and rat and mouse MAPC differs significantly. Importantly, mouse and rat MAPC express pluripotency markers such as Oct4, c-Myc, Klf4, Sall4, and a number of ESC-associated transcripts (Ecats) [49] similar to those found in ESC, but not expressed in mouse MSC. However, unlike ESC, MAPC do not express Nanog and some Ecats. Interestingly, MAPC express a number of endodermal genes such as Sox17, Foxa2, Hnf1b, Gata4 and Gata6, as well as Sox7 [49]. We concluded from this study that MAPC were most equivalent to extraembryonic endoderm cells (XEN) cells, described by the Rossant group. In 2009 Debeb et al. described the direct isolation from rat blastocysts of extraembryonic progenitor cells, termed XEN-P cells, to which MAPC likely most closely compare. Like XEN-P cells, but unlike XEN cells, MAPC express Oct4, Hnf1b and not Hnf4a, and Ssea-1, and require LIF for their in vitro maintenance [21,49]. Like XEN-P cells and XEN cells, MAPC express Gata4, Gata6, Sox7 and Sox17, but not Nanog. Rodent MAPC express high levels of the pluripotency gene Oct4 whose expression is limited to ESC and cells of the gonads in adult [70]. Oct4 is not required in postnatal life and is not expressed outside of the gonads in adult tissues [70]. We have evidence that Oct4 is not expressed in fresh rat or mouse BM, but that the expression is acquired during in vitro culture. Such a “reprogramming event” is in line with a number of recent observations. Takahashi et al., demonstrated that it is possible to reprogram terminally differentiated adult somatic cell such as fibroblasts to ESC-like called induced pluripotent (iPS) cells by introduction of four transcription factors [71]. Since then, reprogramming has also been achieved by transfection of the factors as proteins, and by using fewer transcription factors in combination with small molecules that affect the epigenetic state of cells. In addition, several groups have demonstrated that culture of spermatogonial stem cells, that express Oct4 but not Nanog and Sox2, under ESC culture conditions can reprogram these single lineage stem cells to cell with most if not all features of ESC [24,72–75], We therefore hypothesize that the rodent MAPC is induced by the longterm culture leading to de-differentiation of rare cells to cells that express Oct4 as well as primitive endoderm genes. How closely related are MAPC and XEN-P cells, i.e. whether MAPC are a cultureinduced reprogramming of BM cells to hypoblast-like cells, is currently being evaluated.


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Isolation, characteristics of human MAPC We and others have also isolated MAPC from human BM. A full description of the isolation and in vitro differentiation ability of human MAPC, in comparison with MSC and Mesoangioblasts, including a transcriptome comparison is forthcoming. Human MAPC can be expanded long-term (more than 70 population doublings), and unlike MSC can robustly generate endothelial cells [50,76]. Compared with rodent MAPC human MAPC are cultured without LIF, but like rodent MAPC with FCS, EGF and PDGF-BB. Like MSC they have extensive immunomodulatory properties in vitro and in vivo [67,77] [and manuscript in preparation]. When grafted in vivo in ischemia models, human MAPC like rodent MAPC, significantly increase angiogenesis and endogenous stem cell proliferation, leading to improved CNS [78], cardiac [77] and skeletal muscle function [53]. This has lead to the creation of cGMP-grade human MAPC, termed MultiStemÒ, which is currently being evaluated in phase I clinical trials. Future prospective The premise of studying stem cells is their potential for future cell based therapies for various pathologies. Rodent and murine models and stem cells derived from them have been used widely as an excellent tool to understand the development and pathogenesis of a disease. Their role in human therapies is limited to initial screens. Nevertheless, the studies relating to tissue specific differentiation of rodent and murine stem cells has been instrumental in understanding and devising better protocols for human stem cell differentiation. Rodent and murine MAPC are being used to study the molecular mechanisms underlying tissue development. The clinical use of human embryonic stem cells is limited not only due to their tumorigenicity in their undifferentiated state but also have ethical limiations. While on the other hand, adult stem cells have more limited differentiation capacity and therefore also less tumorigenic potential, are being considered for clinical use. MAPC of human origin (MultiStemÒ) are currently being evaluated in a number of preclinical vascular disease models, as well as in a phase I clinical trial in patients with acute myocardial infarct, and stroke. It has been demonstrated that MultiStemÒ are tolerated well and positively affect cardiac function [77], even though the functional effects will need to be further established by phase II and III clinical trials. The immunomodulatory properties of MultiStemÒ are currently being tested clinically in a phase I trial to prevent GVHD and in a phase I trial for Crohn’s disease, as well as in preclinical models of solid organ transplantation, among others. The very extensive expansion potential of MultiStemÒ, and their immunomodulatory properties, should allow for clinical trials to be done in an allogeneic setting and using a single donor derived bank of cells, limiting the variability of the graft when multiple donors are required or when autologous cells are used. Their outspoken proangiogenic phenotype also suggests that MultiStemÒ are a good cell source for therapy of ischemic disorders. Conflict of interest statement Catherine M. Verfaillie is a consultant to Athersys Inc. References [1] Spangrude GJ, Scollay R. A simplified method for enrichment of mouse hematopoietic stem cells. Exp Hematol 1990;18: 920–6. [2] Burns AJ, Pasricha PJ, Young HM. Enteric neural crest-derived cells and neural stem cells: biology and therapeutic potential. Neurogastroenterol Motil 2004;16(Suppl. 1):3–7. [3] Joseph NM, Mukouyama YS, Mosher JT, et al. Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to schwann cells. Development 2004;131:5599–612. [4] Zhang J, Duan X, Zhang H, et al. Isolation of neural crest-derived stem cells from rat embryonic mandibular processes. Biol Cell 2006;98:567–75. [5] Brandl C, Florian C, Driemel O, et al. Identification of neural crest-derived stem cell-like cells from the corneal limbus of juvenile mice. Exp Eye Res 2009;89:209–17. [6] Barker N, Huch M, Kujala P, et al. Lgr5(þve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 2010;6:25–36.

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[7] Hunt DP, Jahoda C, Chandran S. Multipotent skin-derived precursors: from biology to clinical translation. Curr Opin Biotechnol 2009;20:522–30. [8] Toma JG, McKenzie IA, Bagli D, Miller FD. Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells 2005;23:727–37. [9] Friedenstein AJ, Chailakhyan RK, Latsinik NV, et al. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 1974;17:331–40. [10] Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–50. *[11] Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–4. [12] Sacchetti B, Funari A, Michienzi S, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007;131:324–36. *[13] Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–9. *[14] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981;292:154–6. [15] Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981;78:7634–8. [16] Nagy A, Rossant J, Nagy R, et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A 1993;90:8424–8. [17] Nagy A, Gocza E, Diaz EM, et al. Embryonic stem cells alone are able to support fetal development in the mouse. Development 1990;110:815–21. [18] Tesar PJ. Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos. Proc Natl Acad Sci U S A 2005;102:8239–44. [19] Tesar PJ, Chenoweth JG, Brook FA, et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 2007;448:196–9. [20] Kunath T, Arnaud D, Uy GD, et al. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 2005;132:1649–61. *[21] Debeb BG, Galat V, Epple-Farmer J, et al. Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines. PLoS One 2009;4:e7216. [22] Galat V, Binas B, Iannaccone S, et al. Developmental potential of rat extraembryonic stem cells. Stem Cells Dev 2009;18: 1309–18. [23] Tanaka S, Kunath T, Hadjantonakis AK, et al. Promotion of trophoblast stem cell proliferation by FGF4. Science 1998;282:2072–5. *[24] Matsui Y, Zsebo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992;70:841–7. [25] Shamblott MJ, Axelman J, Wang S, et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 1998;95:13726–31. [26] Gage FH. Mammalian neural stem cells. Science 2000;287:1433–8. [27] Altman J, Das GD. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 1965;124:319–35. [28] Altman J. Are new neurons formed in the brains of adult mammals? Science 1962;135:1127–8. *[29] Friedenstein AJ, Deriglasova UF, Kulagina NN, et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 1974;2:83–92. [30] Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 2000;109:235–42. [31] De Ugarte DA, Morizono K, Elbarbary A, et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs 2003;174:101–9. [32] Anzalone R, Iacono ML, Corrao S, et al. New emerging potentials for human Wharton’s jelly mesenchymal stem cells: immunological features and hepatocyte-like differentiative capacity. Stem Cells Dev 2010;19:423–38. [33] Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:1294–301. [34] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143–7. [35] Au P, Tam J, Fukumura D, et al. Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood 2008;111:4551–8. [36] Dufourcq P, Descamps B, Tojais NF, et al. Secreted frizzled-related protein-1 enhances mesenchymal stem cell function in angiogenesis and contributes to neovessel maturation. Stem Cells 2008;26:2991–3001. [37] Oswald J, Boxberger S, Jorgensen B, et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 2004;22:377–84. [38] Makino S, Fukuda K, Miyoshi S, et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103:697–705. [39] Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3:301–13. [40] Beltrami AP, Cesselli D, Bergamin N, et al. Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow). Blood 2007;110:3438–46. [41] D’Ippolito G, Diabira S, Howard GA, et al. Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci 2004;117:2971–81. [42] Kucia M, Halasa M, Wysoczynski M, et al. Morphological and molecular characterization of novel population of CXCR4þ SSEA-4þ Oct-4þ very small embryonic-like cells purified from human cord blood: preliminary report. Leukemia 2007; 21:297–303. [43] Kucia M, Reca R, Campbell FR, et al. A population of very small embryonic-like (VSEL) CXCR4(þ)SSEA-1(þ)Oct-4þ stem cells identified in adult bone marrow. Leukemia 2006;20:857–69. [44] Wojakowski W, Tendera M, Kucia M, et al. Cardiomyocyte differentiation of bone marrow-derived Oct-4þCXCR4þSSEA1þ very small embryonic-like stem cells. Int J Oncol 2010;37:237–47.


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[45] De Coppi P, Bartsch Jr G, Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100–6. [46] Kogler G, Sensken S, Airey JA, et al. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 2004;200:123–35. [47] Subramanian K, Geraerts M, Pauwelyn KA, et al. Isolation procedure and characterization of multipotent adult progenitor cells from rat bone marrow. Methods Mol Biol 2010;636:55–78. [48] Breyer A, Estharabadi N, Oki M, et al. Multipotent adult progenitor cell isolation and culture procedures. Exp Hematol 2006;34:1596–601. *[49] Ulloa-Montoya F, Kidder BL, Pauwelyn KA, et al. Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity. Genome Biol 2007;8:R163. [50] Aranguren XL, Luttun A, Clavel C, et al. In vitro and in vivo arterial differentiation of human multipotent adult progenitor cells. Blood 2007;109:2634–42. [51] Xu J, Liu X, Jiang Y, et al. MAPK/ERK signalling mediates VEGF-induced bone marrow stem cell differentiation into endothelial cell. J Cell Mol Med 2008;12:2395–406. [52] Liu Z, Jiang Y, Hao H, et al. Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells. Am J Physiol Heart Circ Physiol 2007;293:H1760–5. [53] Aranguren XL, McCue JD, Hendrickx B, et al. Multipotent adult progenitor cells sustain function of ischemic limbs in mice. J Clin Invest 2008;118:505–14. [54] Ross JJ, Hong Z, Willenbring B, et al. Cytokine-induced differentiation of multipotent adult progenitor cells into functional smooth muscle cells. J Clin Invest 2006;116:3139–49. [55] Pelacho B, Nakamura Y, Zhang J, et al. Multipotent adult progenitor cell transplantation increases vascularity and improves left ventricular function after myocardial infarction. J Tissue Eng Regen Med 2007;1:51–9. [56] Tolar J, Wang X, Braunlin E, et al. The host immune response is essential for the beneficial effect of adult stem cells after myocardial ischemia. Exp Hematol 2007;35:682–90. [57] Serafini M, Dylla SJ, Oki M, et al. Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to longterm hematopoietic stem cells. J Exp Med 2007;204:129–39. [58] Maes C, Coenegrachts L, Stockmans I, et al. Placental growth factor mediates mesenchymal cell development, cartilage turnover, and bone remodeling during fracture repair. J Clin Invest 2006;116:1230–42. [59] Roelandt P, Sancho-Bru P, Pauwelyn K, et al. Differentiation of rat multipotent adult progenitor cells to functional hepatocyte-like cells by mimicking embryonic liver development. Nat Protoc 2010;5:1324–36. [60] Roelandt P, Pauwelyn KA, Sancho-Bru P, et al. Human embryonic and rat adult stem cells with primitive endoderm-like phenotype can be fated to definitive endoderm, and finally hepatocyte-like cells. PLoS One 2010;5:e12101. [61] Jiang Y, Vaessen B, Lenvik T, et al. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 2002;30:896–904. [62] Conti L, Pollard SM, Gorba T, et al. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol 2005;3:e283. [63] Wang M, Yang Y, Yang D, et al. The immunomodulatory activity of human umbilical cord blood-derived mesenchymal stem cells in vitro. Immunology 2009;126:220–32. [64] Lee RH, Kim B, Choi I, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004;14:311–24. [65] Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 2003;75:389–97. [66] Jiang XX, Zhang Y, Liu B, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 2005;105:4120–6. [67] Kovacsovics-Bankowski M, Streeter PR, Mauch KA, et al. Clinical scale expanded adult pluripotent stem cells prevent graft-versus-host disease. Cell Immunol 2009;255:55–60. [68] Highfill SL, Kelly RM, O’Shaughnessy MJ, et al. Multipotent adult progenitor cells can suppress graft-versus-host disease via prostaglandin E2 synthesis and only if localized to sites of allopriming. Blood 2009;114:693–701. [69] Luyckx A, De Somer L, Rutgeerts O, et al. Mouse MAPC-mediated immunomodulation: cell-line dependent variation. Exp Hematol 2010;38:1–2. [70] Lengner CJ, Camargo FD, Hochedlinger K, et al. Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell 2007;1:403–15. *[71] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–76. [72] Kanatsu-Shinohara M, Inoue K, Lee J, et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell 2004; 119:1001–12. [73] Guan K, Nayernia K, Maier LS, et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006;440:1199–203. [74] Seandel M, James D, Shmelkov SV, et al. Generation of functional multipotent adult stem cells from GPR125þ germline progenitors. Nature 2007;449:346–50. [75] Labosky PA, Barlow DP, Hogan BL. Mouse embryonic germ (EG) cell lines: transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines. Development 1994;120:3197–204. *[76] Reyes M, Dudek A, Jahagirdar B, et al. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 2002;109:337–46. [77] Van’t Hof W, Mal N, Huang Y, et al. Direct delivery of syngeneic and allogeneic large-scale expanded multipotent adult progenitor cells improves cardiac function after myocardial infarct. Cytotherapy 2007;9:477–87. [78] Walker PA, Shah SK, Jimenez F, et al. Intravenous multipotent adult progenitor cell therapy for traumatic brain injury: preserving the blood brain barrier via an interaction with splenocytes. Exp Neurol; 2010. *[79] Schwartz RE, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002;109:1291–302.




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Prospective identification and isolation of murine bone marrow derived multipotent mesenchymal progenitor cells Fernando Anjos-Afonso, Ph.D., Senior Research Fellow *, Dominique Bonnet, Ph.D., Senior Group Leader * Haematopoietic Stem Cell Lab, Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom

Keywords: mesenchymal stroma cell (MSC) mesenchymal progenitor cell (MPC) sca-1 multipotent differentiation pericyte adventitial reticular cell vascular smooth muscle cell (VSMC)

Enormous confusion still exists in the scientific community regarding the in vivo identity of putative bone marrow (BM) multipotent mesenchymal progenitor cells (MPCs). There is still lack of consensus between laboratories on this issue but recent advancements in this field have shed light on the identity of these cells in humans. However, in mice there are limited and reproducible data available that convincingly define prospectively these cells in vivo. In this review we will critically address: 1) important considerations on how to interpret MPC nomenclature, heterogeneity and differentiation abilities; 2) potential surface antigens that could aid in the isolation of MPC from mouse BM; 3) and their topography and prospective cellular relationship with pericytes, adventitial reticulocytes (ARCs) and vascular smooth muscle cells (VSMCs). Ó 2010 Elsevier Ltd. All rights reserved.

A brief historical and nomenclatural background Detailed introduction of the history of MSCs and their definition, differentiation, immuno-modulatory properties and potential use of these cells in different disease models are covered by other contributors of these series of reviews. We will briefly introduce these cells and the nomenclature used here. Mesenchymal cells are fundamental cells that form the connective tissue throughout the body. In the bone marrow (BM) they are thought to be mainly mesodermally but also neuro-epithelially derived [1–3]. Many scientists believe that putative mesenchymal stem or progenitor cells exist in adult organisms and they are the founders of fibroblasts, osteoblasts, chondrocytes, adipocytes and smooth

* Corresponding authors. Tel.: þ44 (0) 20 7269 3281; Fax: þ44 (0) 20 7269 3581. E-mail addresses: [email protected] (F. Anjos-Afonso), [email protected] (D. Bonnet). 1521-6926/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2010.11.003

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F. Anjos-Afonso, D. Bonnet / Best Practice & Research Clinical Haematology 24 (2011) 13–24

muscle cells in vivo. The definitive evidence that bone marrow includes cells that can generate connective tissue-forming cells was originally provided by the pivotal work of Friedenstein and his coworkers [4,5]. First, they demonstrated by heterotopic transplantation the existence of a minor population of cells in human BM that are precursors of osteoblasts [4,5]. These cells were distinguishable from the majority of haematopoietic cells by their rapid adherence to plastic and by the elongated fibroblast-like appearance in culture [4,5]. Then, they were able to show that seeding BM cells at clonal level resulted in the formation of colonies initiated by single cells, named the colony-forming unit fibroblastic, CFU-Fs. CFU-Fs have since been used as the hallmark for the quality and growth potential of human MSC isolates in vitro. However, in the murine system CFU-Fs are highly contaminated with haematopoietic cells, at least in early cultures initiated with unfractionated BM [6,7] making this assay inappropriate as a predictive factor for the quality and growth potential of murine stromal cells. Since then, others have extended these observations supporting the finding that the cells identified by Friedenstein were multipotent. In particular work done by Pittenger et al., showed that tri-lineage potential (osteoblast, chondrocyte and adipocyte lineages) clones were present in human BM and provided a substantial description of the cell surface phenotype of these cells [8]. However, the nomenclature used to describe multipotent mesenchymal stromal cells has varied throughout the years and until today there is still no universal consensus to name them. These cells originally termed as “osteogenic stem cells” by Friedenstein [4], were then being introduced as “Mesenchymal Stem Cells” by Arnold Caplan [9]. The latter, although not the most appropriate designation (discussed below), became more widely used by many scientists. In this review we followed the recommendations of the International Society for Cellular Therapy, which proposed the use of “Multipotent Mesenchymal Stromal Cell” (MSC) [10]. This is due to the fact that the current known markers used to isolate these cells are shared by a variety of different mesenchymal cell-types. Consequently, the resulting cultures are stromal cultures that might contain a proportion of cells with immature features, which could be the “Mesenchymal Progenitor Cells (MPCs)” as initially suggested by Dennis et al. [11]. Some important considerations Before starting a detailed examination on the main headlines of this review, we would like to bring up some important issues that could help new investigators in the field to better understand the biology of MSCs and how to interpret the available data with a cautious and critical view. From man to mice? MSC biology is one of those unorthodox fields where we have a better understanding of the human than the murine system. Mouse strain variations and more difficult methods to culture murine MSCs (as they depend on the haematopoietic contaminants to thrive in early cultures) [6,7,12,13]; have hindered the understanding of these cells in mice. Consequently, some examples given in this review will be based on human studies. However, we would like to stress that findings from the human studies are not necessarily the same in mice. There are numerous examples of this, for instance: Stro-1, one of the best-known human MSC markers has no mouse counterpart; the cell surface epitopes and in vitro differentiation capacities vary between mouse strains, especially the (reduced) capacity of murine MSCs (mMSCs) to form cartilage-like tissue [12,13]; and mMSCs are karyotypically very unstable in culture right from early cultures whereas human MSC are not [14–16]. Therefore, when analysing data from human studies, a cautious inspection is required to apply the biology to mMSCs and vice-versa. Can we call MSCs “Stem Cells”? If we use the very stringent definition of a “Stem Cell”, which is defined as “at single cell level, a cell capable not only to give rise to different type of progenies but also of self-renewal capacity in vivo assayed by serial transplantation” then presently, MSCs are not fulfilling these rigorous criteria (routinely used to evaluate Haematopoietic Stem Cells (HSCs) function). Indeed, the self-renewal capacity of MSCs has been merely associated with their continuous growth in culture with the preservation of in vitro differentiation after multiple cell passages. However, these in vitro features correlate


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poorly with results of in vivo differentiation assays as exemplified by a recent work by Bianco and coworkers showing that the generation of bone tissue in vitro does not appear to reflect the capacity of forming bone (also of forming stroma and adipocytes) in vivo, and only 10% of the clones were able to do so [17]. Unlike HSCs, which can be systemically transplanted in vivo with high engraftment efficiency, MSCs have to be expanded in sufficient numbers and must be transplanted locally to exert their in vivo functions. One could argue that the feeble knowledge on their identity has hampered their use at single cell level. We still don’t know if all cultured cells have similar regenerative capacity for different lineages in vivo even when prospectively isolated using the current known surface markers. Furthermore, we have to consider that the turnover of cells in connective tissue in an adult organism is relatively low and consequently the rate of self-renewal divisions for the putative MPC in vivo under homeostasis (assuming that really takes place) should also be very low. Moreover, there is no data supporting that in vivo serial transplantation is feasible and therefore assessing self-renewal and multipotency in vivo of MPCs or other stromal cells might require different assays from those applied for HSCs. That being said, evidence for self-renewal of MSCs had recently emerged. Paolo Bianco’s team has shown that clonogenic human CD146þ BM derived cells were able to self-renew. When clonally derived cells were implanted subcutaneously into immunodeficient mice they were able to retrieve a minor population of cells weeks later with a similar phenotype as the initial inoculum and re-grow these cells at a clonal level [18]. Overall, there is a lack of definite data supporting the idea that MSCs isolated from tissues other than BM contain “Stem Cells”. We would also like to call the attention of the readers to the claims of MSC multipotency in numerous publications based solely on in vitro assays. As mentioned above, the outcome of these assays does not necessarily correlate with in vivo differentiation potential of MSCs. Furthermore, most of the data published relied heavily on representative figures of specific lineage staining without any accompanied quantification. As an example, it is often found figures showing just very few Oil Red O positive cells (that depicts adipocytes) claiming that the MSCs in question have adipogenic differentiation. Such data can be misleading and create a barrier for the readers to compare data from different laboratories while also being biologically meaningless. Assessment of the degree of differentiation from MSCs to each specific lineage by quantificative methods should be implemented to be able to evaluate the potential of the cells isolated using different methods and from different tissues.

Expansion/Differentiation and heterogeneity Currently, most of the in vitro culture and expansion protocols are based on a basic medium supplemented with 10–20% of selected batches of Foetal Bovine Serum, which certainly does not constitute the most suitable way to maintain the undifferentiated state of MSCs. This is crucial, as the interpretation of the immense data available on their heterogeneity and differentiation potential depend on how MSCs are grown. BM MSCs are widely perceived as a heterogeneous population of cells in vitro, despite the homogeneous expression of most surface antigens used to describe them. This view has been supported by many reports showing that at clonal level not all the cells have the same in vitro differentiation potential [11,19–21]. One of the earliest works reporting this observation was from Muraglia et al., which demonstrated that approximately 30% of all human MSC clones exhibit a tri-lineage differentiation potential [19]. A more recent study using mouse cells from O’Connor’s group reported that tri-lineage MSCs accounted for nearly 50% of the CFU-Fs [20]. The most prominent study on the heterogeneity of cultured MSCs comes from Prockop’s group. They showed that at steady state, MSC cultures contain a minor population of small, agranular and quiescent cells (named RS-1 cells). These RS-1 cells express an antigenic profile that is different from the most abundant fast growing and committed precursors (mature-MSCs) and for example, express the high-affinity nerve growth factor receptor, Trk [22]. When studying a precursor-progeny relationship between RS-1 and other cell-types, it was concluded that the high proliferative capacity of mature-MSCs depends on the presence of RS-1 cells [22]. Moreover, RS-1 cells display a more robust differentiation capacity than mature-MSCs. It seems that RS-1 cells may

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represent a subset of uncommitted cells [22] and therefore RS-1 sub-population could be the putative MPC enriched fraction. The assumption that cultured MSCs are heterogeneous is well supported but whether it is the same for cells in vivo is debatable. Most current methodologies applied for in vitro differentiation assays require a certain cell number and density to drive the cells to a particular lineage [8,12,13,21–23]. It is thus necessary to expand these cells beforehand in order to accomplish in vitro assays. One must consider that in vitro expansion with commonly used medium might cause a substantial number of MSCs to lose their differentiation capacity, even at early passage. This has been supported by many studies which show not only that the differentiation capacity of cultured MSCs is gradually reduced upon cell passaging [24–26], but also that, as MSC culture are passaged, they become heterogeneous as they expand and contain at least 2 subpopulations of cells: small, rapidly self-renewing MSCs (RS-1 MSCs) and larger, slowly renewing MSCs (mature-MSC) [22]. Without a more in-depth knowledge on how to maintain the primitive features of MSCs we have to consider the possibility that MSCs might not be as heterogeneous in vivo. Indeed, some authors have questioned the concept of the heterogeneity of MSC. Jones et al., showed that by using a combinatorial analysis of surface antigens by flow-cytometry that most cells that have CFU-F capacity express many of the commonly known MSC markers uniformly [27,28]. They showed this by first positively selecting bone marrow cells using an antibody that recognises fibroblasts (anti-D7-FIB antibody) and then eliminating haematopoietic cells from this analysis. As a result, the remaining cell fraction expresses CD73 (ecto-50 -nucleotidase), CD105 (endoglin), CD90 (Thy-1) and very interestingly, another nerve growth factor receptor, CD271 almost homogeneously [27,28]. Similarly, another paper reported that most of the human BM CFU-F activity comes from the non-haematopoietic CD271þ fraction [29]. Nevertheless, it is likely that some of these markers are shared in vivo with other lineage specific mesenchymal precursors (or even their subsequent progenies), like adipocyte-precursors whose phenotypes are not well defined for human cells and fibroblasts that have limited differentiation capacities [18,30]. It is possible that these lineage specific precursors and other cell-types can still form CFU-Fs. Until we can distinguish all the differentiation stages phenotypically for the different mesenchymal cell lineages using surface markers or other means, then apply these measures to further dissect the non-haematopoietic/endothelial CD271þ or CD73þ or other similar BM populations, we can not be sure that these populations are homogeneous. What is clear at this stage is that the heterogeneity observed from MSC cultures is likely a consequence of inefficient in vitro culture systems that are unable to maintain the original features of these cells. Potential surface antigens that could aid the isolation of mouse BM derived MSCs In this section we will summarise what is currently known about the phenotype of mMSCs mostly from data generated by analysis of cultured cells. Before going into more details, a few important issues should be highlighted. First, most of current surface antigens known to be expressed on MSCs are not specific to MSCs; there is a myriad of positive markers that are also shared by different cell types. Secondly, the expression of some of the positive markers changes during in vitro culture. In addition to the examples mentioned above, the expression of Stro-1 and CD271 are down regulated in culture [27,28,31,32]. In the murine system, the reverse also happens: some antigens that are highly expressed on cultured cells are difficult to detect directly from BM cells (see below). Thirdly, some of the antigens are not universal for all the mouse strains. Altogether these impede the establishment of a panel of surface antigens that can be used consistently to distinguish the most primitive sub-fraction from the rest of the cultured cells and the prospective isolation of these cells from BM of different mouse strains. MPC: how to find the needle in a haystack? There is a general consensus that, like in human cells, mMSCs do not express most common surface antigens that are found on haematopoietic and endothelial cells. These include: CD11b (Mac-1), Ter119 (an erythroid lineage marker), CD45 (protein tyrosine phosphatase, receptor type C; a pan haematopoietic marker) and CD31 (PECAM-1; endothelial and haematopoietic marker) [6,7,12,13]. Therefore, the common view is that mMSCs are confined in the CD45CD11bTer119CD31 fraction. Other


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surface antigens that are commonly found in different stem/progenitor populations, such as CD117 (c-Kit; receptor for Stem Cell Factor, SCF), are not found on cultured MSCs [6,7,12,13,33]. Although CD90 (Thy-1) is expressed in human MSCs, the expression of this antigen has been repeatedly reported by many groups, including ours, to be absent on mMSCs derived from most commonly used mouse strains including C57BL/6 mice [6,7,12,13,33]. However, two recent reports have reported CD90 expression in mMSCs from C57BL/6 mice [34,35]. The reason for this discrepancy is unclear and it deserves further analysis. The CD34 (a protein in the sialomucin family) antigen is known to be expressed on cultured MSCs derived from some mouse strains like C57BL/6, FVB/N and NOD/SCID, but not in DAB1 and BALB/c mice [7,12]. Thus, CD34 cannot be used as a universal cross-strain positive surface antigen for identifying mMSCs in BM. The other two highly expressed antigens that are commonly found on both cultured and freshly isolated human MSCs, CD105 and CD73, can also be found in cultured mMSCs derived from different strains [7,12,33] albeit at different levels of expression (Anjos-Afonso, unpublished data). Unfortunately, none of these antigens are very useful in prospectively isolating an enriched population of mMPCs. As an example, the CD73 antigen is difficult to detect in fresh cells from NOD/SCID and ROSA26 mice (C57BL/6 background) (Anjos-Afonso, unpublished data). Interestingly, CD73 is highly expressed on cultured cells derived from the same mouse strains, highlighting the upregulation of CD73 upon culture as mentioned previously (Anjos-Afonso, unpublished data). Our group has found that CD105 is expressed at low levels in freshly isolated BM CD45CD11bTer119CD31 fraction from the aforementioned mouse strains. However, CD105 has been reported to be undetectable in mouse calvaria in vivo or calvaria-derived cells in vitro [36]. Sca-1, the best marker in the mouse system? Sca-1 (stem cell antigen) is a well-known marker used to enrich adult murine HSCs and can be used to isolate a nearly pure HSC population when used in conjunction with additional markers. Sca-1 is an 18-kDa mouse GPI-AP (glycosyl phosphatidylinositol-anchored cell surface protein) of the Ly-6 gene family [37]. Despite our lack of knowledge regarding its physiological role, Sca-1 is used regularly in conjunction with negative selection against mature markers to enrich stem and progenitor haematopoietic cells. There is some solid evidence supporting the idea that Sca-1 could be also a potential positive marker for the identification of mMPCs. First, Sca-1/ mice exhibit age-related osteoporosis characterised by weakening in bone material, microarchitectural, and mechanical properties [38]. Decreased osteoprogenitors, osteoblasts and bone formation are apparent in these mutant mice and are the result of reduced numbers of MSCs. Also, Sca-1/ mice display reduced adipogenesis in vitro [38]. In addition to these findings, some reports have shown that the tri-lineage potential of cells isolated based on Sca1 expression alone [34–36,39] or in combination with other antigens is confined to the Sca-1þ fraction whereas the Sca-1 fraction displays mainly osteo- and chrondrogenic potential [34–36,39]. Unfortunately, Sca-1 expression varies between mouse strains. Strains with the Ly6.2 variants, such as C57BL/6, FVB/N and 129 have a higher percentage of Sca-1 expressing cells in fresh BM cells [40,41], whereas in Ly6.1 strains such as BALB/c and DAB1 display lower percentage of Sca-1þ cells in vivo and an absent-low Sca-1 expression in cultured MSCs [12,41]. The NOD/SCID mouse strain and its variants are widely used mouse models in the study of human haematopoiesis in vivo and there is an increased interest in dissecting how human HSCs interact with cells from the BM niche. Although Sca-1 is highly expressed on cultured MSCs isolated from these immunodeficient mice [7,13], the percentage of Sca-1þ cells in the BM CD45CD11bTer119CD31 is much lower than in C57BL/6 mice (Anjos-Afonso, unpublished data). Even though Sca-1 is one of the best surface antigens known to date, this limits its use as a cross-strain marker to enriched mMPCs. Putting aside this limitation, Sca-1 has been used in combination with other markers such as CD166 (Alcam), CD51 (integrin alpha V) and PDGFRa (plateletderived growth factor receptor alpha) to further enrich mMPCs in vivo [34,35,39,42]. What other markers could be used? An early study from Suda’s group showed that most of the multilineage differentiation capacity of foetal limb perichondrium MSCs resides mainly in the CD166þ fraction [42]. However, our group

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has been unable to identify co-expression of CD166 with Sca-1 in the adult C57BL/6 BM CD45CD11bTer119CD31 fraction. This lack of co-expression has recently been confirmed by the aforementioned authors, demonstrating that adult BM CD45Ter119CD166þ cells are an osteoblastenriched population instead [35]. CD51 is also known be expressed in osteoblasts and has been used to enrich osteoblasts from the BM [39,43], and when combined with Sca-1, a double positive subpopulation can be detected [43]. However, there is no functional data available demonstrating that this double positive subpopulation is enriched for mMPCs. Both PDGFRa and PDGFRb are expressed by different mesenchymal cells. Not only is the PDGF signalling pathway important in promoting survival and proliferation in mesenchymal cells [44,45], but these receptors have also been used in combination with VEGFR2 (vascular endothelial growth factor receptor 2) to identify the point of lineage divergence between lateral and paraxial mesoderm [1]. This makes them ideal as prospective positive markers. When combined with Sca-1 staining, Matsuzaki’s group have recently demonstrated that the CD45Ter119þSca-1þPDGFRaþ BM subpopulation, derived from C57BL/6, is enriched with MPCs with tri-lineage capacity whereas other cellular fractions have a more restricted differentiation potential, mainly lacking fat-forming capacity [34]. Moreover, this population can be found in different mouse strains like DBA-1 and BALB/c mice. In this study the level of Sca-1 expression was similar between all strains tested [34]. The reason for the observed differences from other published studies is currently not clear. Then, the authors went on showing that this population isolated from green fluorescent protein (GFP) mice when injected intravenously into non-GFP recipients, the inoculated cells were able to give rise to some GFPþ osteoblasts and adipocytes in vivo. Although this study provides the first plausible enrichment of mMPC using Sca-1 and PDGFRa expression, there are some caveats. First, the subpopulation in question, Sca1þPDGFRaþ, is almost undetectable in the BM and most of the study was performed using cells isolated from bone fragments after collagenase digestion. As such, the isolated cells are mainly bone-derived and not BM derived. It was assumed that the BM has a similar subpopulation based on a histological staining locating two Sca-1þPDGFRaþ cells near a BM vasculature [34]. This assumption is only valid if similar in vitro and in vivo functions are demonstrated and compared between the two populations. Secondly, the seeding efficiency of these cells was very low, reaching w25 CFU-Fs/1000 purified cells. While this frequency is comparable from human studies using non-haematopoietic/endothelial CD164þ or CD73þ BM cells [18,46], one must bear in mind that the Sca-1þPDGFRaþ population was extracted from bone fragments and as the frequency of this population is w40x lower in the BM, this suggests that the seeding efficiency should be lower than 1 CFU-Fs/1000 purified cells. Most importantly, it is known that murine MSCs depend on the haematopoietic contaminants to thrive in early cultures. Consequently, we cannot exclude that non-Sca-1þPDGFRaþ fractions could also contain some mesenchymal progenitors unable to survive in culture and therefore excluded from the analysis. Thirdly, most of the clonally derived populations showed very weak adipogenic differentiation capacity in vitro [34], implying that the Sca-1þPDGFRaþ population it is still heterogeneous. A recent paper showed that the bone-fragment derived non-hematopoietic/endothelial Sca-1þPDGFRaþ cells have similar gene expression profile to Sca-1þCD166 population despite the lack of chondrogenic differentiation potential by the latter [35]. Altogether it highlights the possibility for the Sca1þPDGFRaþ subpopulation to be the fraction where most mMPCs might reside. That said, the literature available is too limited at this stage to make any absolute claims. Moreover there is a necessity to find more stable, universal (cross-strain) and highly expressed surface antigen (specially in fresh BM cells) that could substitute or complement the currently known murine markers. MSC topography and the prospective cellular relationship with adventitial reticulocytes, pericytes and vascular smooth muscle cells All the same or different members of the same family with the same outfit? Recently, many investigators have used immunohistology to revisit the topography of MSCs in vivo [18,28] and their findings suggest that MSCs may be identical to BM adventitial reticulocytes (ARCs) [47–49]. The morphological and phenotypical similarities between MSCs and ARCs are well supported (e.g. both express CD10, CD13, CD271, CD146, tissue-non specific Alkaline Phosphatase (AP))


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[8,18,27,28,47,48]. Findings from these recent reports are similar to that published in the early 90’s when Colombo’s group first used CD271 to locate potential BM stromal cells in human trephine sections [48]. They found that CD271þ cells have an oval nucleus and a scant cytoplasm with long dendrites that intermingle with the haematopoietic cells. They also found that a substantial number of CD271þ cells are lining in the abluminal side of sinus endothelial cells and provide the scaffold for the haematopoietic marrow [48]. Moreover, they showed that the number of CD271 expressing cells is correlated with the traditional reticulin, vimentin, CD13 and AP expression [48], thus providing the first compelling evidence that CD271 expressing cells represent ARCs. Some investigators have also suggested that MSCs might be vascular pericytes [50–52]. This suggestion has a basis in that: historically, ARCs have also been also named pericytes of the venous sinusoids [47,48] as they are found, though not exclusively, in the perivascular areas; human Stro1þCD106þ or Stro-1þCD146þ express aSMA (alpha smooth muscle actin; a marker that is expressed by most, but not all, pericytes and in VSMCs) or were positive for the 3G5 antigen, which is thought to be specific for pericytes [31,53]; pericytes have been shown to be able to differentiate into adipocytes, chondrocytes and osteoblasts [54,55]; and several antigens that are expressed in MSCs are also found to be expressed in pericytes including: CD13, CD73, CD146, Stro-1, CD90, aSMA, etc [50,53,56–59]. With respect to these findings, some authors suggest that the reason why MSC populations could be isolated from different tissues other than BM is because of the presence of tissue-resident vascular pericytes as they form a subendothelial network that spans the microvasculature in most tissues [50–52]. However, VSMCs also share some of the markers of pericytes, such as CD146, PDGFRs, CD13, NG2 and CD105 and therefore it could be argued that they are also similar to MSCs [1,60–65]. Furthermore, VSMCs have also been shown to have tri-lineage differentiation capacities in vitro [66–68]. Apart from pericytes and VSMCs, progenitors with multilineage capacities have also been isolated from the mesenchyme of tunica adventitia (discussed below) [69,70]. This makes it difficult to interpret and distinguish some of the results published when different mesenchymal cell-types that have overlapping features are present in the same tissue and can be co-isolated using the current protocols. As such, stromal cells obtained based on selection with one positive marker or simply by adherence to plastic, are likely to contain different mesenchymal cell-types such as pericytes, VSMCs and adventitia progenitors (if we assume that these cells are distinct from each other). One must acknowledge that there is indeed a high degree of similarity between MSCs and these other cell-types. Moreover, it seems that most MSCs appear to have perivascular topography [18,47,53], although MSC cultures could also be obtained from articular cartilages, which are avascular [71]. However, it is worth noting that some of the data available might have led to presumptive conclusions and one should thus consider the following: 1. Two simple facts that are often forgotten are that all cell-types mentioned are mesenchyme by nature and therefore, it is not surprising that they share numerous cell surface antigens. Second, most tissues, including BM, are very vascular and it is not surprising that stromal cells may have perivascular localization as their main function is to form the supporting network of other structures, including the vascular system. 2. As described previously, some of the markers used to identify MSCs can be up regulated upon cultivation. Hence, all these mesenchymal cell-types that seem to share a list of common antigens could be a consequence of culture. Currently, there is limited data available where multiparameter analyses have been conducted in situ or with freshly isolated cells and then applied these mutiantigen staining approaches for the subsequent isolation of the cells in question. This raises the possibility that some “shared” surface antigens may be actually be useful to distinguish the aforementioned cell-types prior to cultivation. 3. The in vitro differentiation assays that are used to demonstrate the “plastic” potential of the cells might not be the most accurate assays to determine MSC potential, and could be an artifact from the in vitro culture employed. It has been reported that more mature mesenchymal cell-types, such as adipocytes, chondrocytes and osteoblasts seem to be able to de-differentiate and then convert into another lineage in response to inductive extracellular cues [71–74]. In this context, VSMCs have also been shown to be prone to de-differentiation during in vitro cultivation, and consequently being able to re-direct them to adipo- or osteo- or chondrocytic lineages in vitro [66–68]. One could argue that this

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could also happen with pericytes although there is no formal data available to support this view yet. Interestingly, it has been shown that rat aortic smooth muscle cells can become pericytes during angiogenesis in vitro [75]. 4. Variation in expression of markers used to characterise these cell-types exists in other tissues. Interpreting data from experiments in tissues other than BM might not necessarily relate to the same cells derived from BM. In disguise, can we hunt them down? The question remains whether we can delineate the differences between all these cell-types. At very least we can attempt to exemplify their differences. For this, one must first define pericytes: the cells that are embedded within the vascular basement membrane of blood microvessels where they make specific focal contacts with the endothelium [76,77]. Generally, the arteries and veins are surrounded by single or multiple layers of VSMCs (tunica media), whereas the smallest capillaries are partially covered by single pericytes. The latter are found around capillaries, pre-capillary arterioles, post-capillary venules, and collecting venules. The distinction between pericyte and VSMC morphology and location is not absolute but exists as a continuum of properties ranging from the usual VSMC to the typical pericyte, distributed along intermediate size to small vessels [76,77]. The morphological features are important because in a few studies where multipotent mesenchymal cells were isolated based on CD146 expression, the demonstration of their in situ localisation seems to have depicted VSMCs (with multinuclei layer of cells surrounding endothelial cells) instead of pericytes claimed by the authors of these reports [50,69]. Typically pericytes are positive for CD13, NG2, desmin, aSMA, PDGFRb, CD146 (human) and for the species-specific 3G5 antibody staining (human and bovine) [50,53,56,77–79]. However, none of these markers are pan-pericyte markers, as already mentioned, with CD13, NG2, desmin, aSMA, PDGFRb and CD146 also being expressed in VSMCs [60–65]. Moreover, the expression of some of these markers is dynamic and varies between tissues. The classic example is the expression of aSMA, which is not found in both bovine retina and rat mesenteric mid-capillary pericytes [80]. Skin and CNS pericytes have almost no expression of aSMA [77,81]. Similarly, NG2 expression is restricted to arteriolar and capillary perivascular cells and is absent on venous pericytes in rats [82]. Endoglin, a prominent MSC marker is also expressed at low levels in human cultured and arterial tissue VSMCs [60,61], and therefore makes its use inappropriate to differentiate between pericytes and VSMCs. The usefulness of other conventional markers like CD90 and CD73 is still unclear. In most studies CD90 has been shown to be expressed on pericytes [50,57,69] but convincing data are lacking on its in situ expression in VSMCs. However, commercially available human VSMC lines do not express CD90. Overall, the available data seem to indicate that CD90 might be useful for this lineage delineation but further studies are required to confirm this idea. There is also ambiguous data concerning whether CD73 is present in pericytes. Human dermal- [79] and adipose- [69] derived pericytes appear to express low levels of CD73, while high expression has been found on cells derived from human skeletal muscle [50] but absent in mouse retinal pericytes [58]. Again, expression data of CD73 in VSMCs is lacking. What can then be used to identify putative MPCs or immature mesenchymal cells from typical pericytes? An evaluation of the literature and our experimental data indicates that desmin expression might be an appropriate candidate. Freshly isolated and cultured cells while sub-confluent (desmin expression can be artificially induced if the cells are being cultured in constant confluency) do not express desmin in both human and murine MSCs at the protein level [7,83,84,85]. Desmin is expressed in pericytes, albeit at different levels depending on their maturity status [76,81]. As such, reporter mice for desmin might help to address this question. If we consider that Sca-1 is the primary surface antigen for mMSCs and NG2 is expressed in all typical BM pericytes then, according to our findings, putative mMPCs are unlikely to be pericytes as we found little or no co-expression of these two antigens in freshly isolated BM cells (Anjos-Afonso, unpublished data). Validation on whether NG2 can depict all typical pericytes is necessary to confirm this observation. Nevertheless, recent findings examining progenitors from muscle and adipose tissues might support our observations. Mesenchymal progenitors derived from adult skeletal muscle (defined as non-haematopoietic/endothelial/muscle Sca-1þCD90þPDGFRbþ cells that reside in the interstitial


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space of muscle tissue) have been shown to have tri-lineage differentiation potential [86]. These cells of mesenchymal origin (vimentinþ) do not express any other pericytic marker including NG2 and aSMA [86]. These cells phenotypically resemble progenitors identified in mouse aortica adventitia (Sca1þPDGFRbþ cells), which do not express smooth muscle cell markers but can easy differentiate into VSMCs upon PDGF stimulation [86]. Unfortunately, the tri-lineage differentiation capacity was not demonstrated in this study. Interestingly, high adipogenic differentiation capacity can also be achieved from adventitial cells (CD34þaSMACD90þCD146) isolated from human adipose tissue, which is distinctive from what has been suggested as “pericytes” by the lack of CD146 expression [69]. Furthermore, retinal and brain pericytes have different morphology than conventional MSCs in vitro [87,88]. These pericytes have smooth muscle cell-like morphology and upon reaching confluency they form multilayered areas, which retract and form nodules, the extracellular matrix of which becomes mineralised [55,87,88]. The mineralised matrix by the microvascular pericytes is associated with the expression of markers of the osteoblast lineage (AP, bone sialoprotein, osteocalcin, osteonectin and osteopontin) [55,87,88]. These features are not found in conventional MSC cultures. In addition, despite reports claiming that pericytes have adipogenic potential in vitro, stimulated pericytes are hardly stained by Oil Red O dye (which stains for neutral triglycerides and lipids) upon closer inspection [50,54,55,87,88]. Altogether, it suggests not only that typical pericytes may be distinct from putative MPCs but they also don’t seem to have the same degree of tri-lineage potential that is found in conventional BM derived MSC cultures. Conclusions In summary, we currently believe that the murine BM stromal compartment is largely unexplored compared to their human equivalent. The phenotype of the potential BM MPC is still vague but most probably confine to the Sca-1þPDGFRbþ fraction. Moreover, the data available points to the idea that MSCs are related to both progenitors from adventitia and pericytes, (perhaps slightly closer to the former) but further apart from classical VSMCs. We might hypothesise that in adult BM the primitive mesenchymal compartment is composed of a myriad of cell-types with similar phenotypes and differentiation potentials and even perhaps with the capacity of lineage inter-convertibility in vivo rather than one identifiable mesenchymal progenitor population, thus explaining the challenges/ hurdles that this field had faced over the years trying to identify/isolate these putative cells in vivo. Conflict of interest statement No conflicts of interest to declare. Acknowledgments We would like to thank Erin Currie and Katie Foster for their valuable comments on the manuscript. Cancer Research UK supported this work. References [1] Sakurai H, Era T, Jakt LM, et al. In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility. Stem Cells 2006;24:575–86. [2] Takashima Y, Era T, Nakao K, et al. Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 2007; 129:1377–88. [3] Morikawa S, Mabuchi Y, Niibe K, et al. Development of mesenchymal stem cells partially originate from the neural crest. Biochem Biophys Res Commun 2009;379:1114–9. [4] Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 1987;20:263–72. [5] Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 1970;3:393–403. [6] Phinney DG, Kopen G, Isaacson RL, et al. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem 1999;72:570–85. [7] Anjos-Afonso F, Siapati EK, Bonnet D. In vivo contribution of murine mesenchymal stem cells into multiple cell-types under minimal damage conditions. J Cell Sci 2004;117:5655–64.

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[8] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143–7. [9] Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–50. [10] Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 2006;8:315–7. [11] Dennis JE, Merriam A, Awadallah A, et al. A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse. J Bone Miner Res 1999;14:700–9. *[12] Peister A, Mellad JA, Larson BL, et al. Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 2004;103:1662–8. [13] Anjos-Afonso F, Bonnet D. Isolation, culture, and differentiation potential of mouse marrow stromal cells. Curr Protoc Stem Cell Biol; 2008 [Chapter 2]:Unit 2B 3. [14] Bernardo ME, Zaffaroni N, Novara F, et al. Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms. Cancer Res 2007; 67:9142–9. [15] Miura M, Miura Y, Padilla-Nash HM, et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 2006;24:1095–103. [16] Aguilar S, Nye E, Chan J, et al. Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung. Stem Cells 2007;25:1586–94. [17] Bianco P, Kuznetsov SA, Riminucci M, et al. Postnatal skeletal stem cells. Methods Enzymol 2006;419:117–48. *[18] Sacchetti B, Funari A, Michienzi S, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007;131:324–36. *[19] Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 2000;113:1161–6. [20] Russell KC, Phinney DG, Lacey MR, et al. In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment. Stem Cells 2010;28:788–98. *[21] Smith JR, Pochampally R, Perry A, et al. Isolation of a highly clonogenic and multipotential subfraction of adult stem cells from bone marrow stroma. Stem Cells 2004;22:823–31. *[22] Colter DC, Sekiya I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A 2001;98:7841–5. [23] McBeath R, Pirone DM, Nelson CM, et al. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 2004;6:483–95. [24] Banfi A, Muraglia A, Dozin B, et al. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 2000;28:707–15. [25] Bonab MM, Alimoghaddam K, Talebian F, et al. Aging of mesenchymal stem cell in vitro. BMC Cell Biol 2006;7:14. [26] Kretlow JD, Jin YQ, Liu W, et al. Donor age and cell passage affects differentiation potential of murine bone marrowderived stem cells. BMC Cell Biol 2008;9:60. [27] Jones EA, English A, Kinsey SE, et al. Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow. Cytometry B Clin Cytom 2006;70:391–9. [28] Jones E, McGonagle D. Human bone marrow mesenchymal stem cells in vivo. Rheumatology 2008;47:126–31. [29] Quirici N, Soligo D, Bossolasco P, et al. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 2002;30:783–91. [30] Jones S, Horwood N, Cope A, et al. The antiproliferative effect of mesenchymal stem cells is a fundamental property shared by all stromal cells. J Immunol 2007;179:2824–31. [31] Gronthos S, Zannettino AC, Hay SJ, et al. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci 2003;116:1827–35. [32] Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991;78:55–62. [33] da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 2006;119:2204–13. *[34] Morikawa S, Mabuchi Y, Kubota Y, et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J Exp Med 2009;206:2483–96. [35] Nakamura Y, Arai F, Iwasaki H, et al. Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells. Blood 2010; doi:10.1182/blood-2009-08-239194. [36] Steenhuis P, Pettway GJ, Ignelzi Jr MA. Cell surface expression of stem cell antigen-1 (Sca-1) distinguishes osteo-, chondro-, and adipoprogenitors in fetal mouse calvaria. Calcif Tissue Int 2008;82:44–56. [37] LeClair KP, Palfree RG, Flood PM, et al. Isolation of a murine Ly-6 cDNA reveals a new multigene family. EMBO J 1986; 5:3227–34. *[38] Bonyadi M, Waldman SD, Liu D, et al. Mesenchymal progenitor self-renewal deficiency leads to age-dependent osteoporosis in Sca-1/Ly-6A null mice. Proc Natl Acad Sci U S A 2003;100:5840–5. [39] Lundberg P, Allison SJ, Lee NJ, et al. Greater bone formation of Y2 knockout mice is associated with increased osteoprogenitor numbers and altered Y1 receptor expression. J Biol Chem 2007;282:19082–91. [40] Uchida N, Weissman IL. Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1þ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J Exp Med 1992;175:175–84. [41] Spangrude GJ, Brooks DM. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells. Blood 1993;82:3327–32. [42] Arai F, Ohneda O, Miyamoto T, et al. Mesenchymal stem cells in perichondrium express activated leukocyte cell adhesion molecule and participate in bone marrow formation. J Exp Med 2002;195:1549–63. [43] Winkler IG, Barbier V, Wadley R, et al. Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: serially reconstituting hematopoietic stem cells reside in distinct non-perfused niches. Blood 2010; doi:10.1182/ blood-2009-07-233437.


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[44] Andrae J, Gallini R, Betsholtz C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev 2008;22: 1276–312. [45] Betsholtz C. Biology of platelet-derived growth factors in development. Birth Defects Res C Embryo Today 2003;69:272–85. [46] Boiret N, Rapatel C, Veyrat-Masson R, et al. Characterization of nonexpanded mesenchymal progenitor cells from normal adult human bone marrow. Exp Hematol 2005;33:219–25. [47] Bianco P, Riminucci M, Gronthos S, et al. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19:180–92. [48] Cattoretti G, Schiro R, Orazi A, et al. Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro. Blood 1993;81:1726–38. [49] Caneva L, Soligo D, Cattoretti G, et al. Immuno-electron microscopy characterization of human bone marrow stromal cells with anti-NGFR antibodies. Blood Cells Mol Dis 1995;21:73–85. *[50] Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3:301–13. [51] Chen CW, Montelatici E, Crisan M, et al. Perivascular multi-lineage progenitor cells in human organs: regenerative units, cytokine sources or both? Cytokine Growth Factor Rev 2009;20:429–34. [52] da Silva Meirelles L, Caplan AI, Nardi NB. In search of the in vivo identity of mesenchymal stem cells. Stem Cells 2008; 26:2287–99. [53] Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 2003;18:696–704. [54] Farrington-Rock C, Crofts NJ, Doherty MJ, et al. Chondrogenic and adipogenic potential of microvascular pericytes. Circulation 2004;12(110):2226–32. [55] Doherty MJ, Ashton BA, Walsh S, et al. Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone Miner Res 1998;13:828–38. [56] Bagley RG, Weber W, Rouleau C, et al. Pericytes and endothelial precursor cells: cellular interactions and contributions to malignancy. Cancer Res 2005;65:9741–50. [57] Oishi K, Kamiyashiki T, Ito Y. Isometric contraction of microvascular pericytes from mouse brain parenchyma. Microvasc Res 2007;73:20–8. [58] Scheef EA, Sorenson CM, Sheibani N. Attenuation of proliferation and migration of retinal pericytes in the absence of thrombospondin-1. Am J Physiol Cell Physiol 2009;296:724–34. [59] Ozerdem U, Stallcup WB. Early contribution of pericytes to angiogenic sprouting and tube formation. Angiogenesis 2003; 6:241–9. [60] Adam PJ, Clesham GJ, Weissberg PL. Expression of endoglin mRNA and protein in human vascular smooth muscle cells. Biochem Biophys Res Commun 1998;247:33–7. [61] Ma X, Labinaz M, Goldstein J, et al. Endoglin is overexpressed after arterial injury and is required for transforming growth factor-beta-induced inhibition of smooth muscle cell migration. Arterioscler Thromb Vasc Biol 2000;20:2546–52. [62] Seftalioglu A, Karakoc L. Expression of CD146 adhesion molecules (MUC18 or MCAM) in the thymic microenvironment. Acta Histochem 2000;102:69–83. [63] Ozerdem U, Grako KA, Dahlin-Huppe K, et al. NG2 proteoglycan is expressed exclusively by mural cells during vascular morphogenesis. Dev Dyn 2001;222:218–27. [64] Grako KA, Stallcup WB. Participation of the NG2 proteoglycan in rat aortic smooth muscle cell responses to plateletderived growth factor. Exp Cell Res 1995;221:231–40. [65] Kappert K, Paulsson J, Sparwel J, et al. Dynamic changes in the expression of DEP-1 and other PDGF receptor-antagonizing PTPs during onset and termination of neointima formation. FASEB J 2007;21:523–34. [66] Iyemere VP, Proudfoot D, Weissberg PL, et al. Vascular smooth muscle cell phenotypic plasticity and the regulation of vascular calcification. J Intern Med 2006;260:192–210. [67] Davies JD, Carpenter KL, Challis IR, et al. Adipocytic differentiation and liver x receptor pathways regulate the accumulation of triacylglycerols in human vascular smooth muscle cells. J Biol Chem 2005;280:3911–9. [68] Tintut Y, Alfonso Z, Saini T, et al. Multilineage potential of cells from the artery wall. Circulation 2003;108:2505–10. [69] Zimmerlin L, Donnenberg VS, Pfeifer ME, et al. Stromal vascular progenitors in adult human adipose tissue. Cytometry A 2010;77:22–30. *[70] Hu Y, Zhang Z, Torsney E, et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest 2004;113:1258–65. [71] Barbero A, Ploegert S, Heberer M, et al. Plasticity of clonal populations of dedifferentiated adult human articular chondrocytes. Arthritis Rheum 2003;48:1315–25. [72] Bennett JH, Joyner CJ, Triffitt JT, et al. Adipocytic cells cultured from marrow have osteogenic potential. J Cell Sci 1991; 99:131–9. [73] Song L, Tuan RS. Transdifferentiation potential of human mesenchymal stem cells derived from bone marrow. FASEB J 2004;18:980–2. [74] Park SR, Oreffo RO, Triffitt JT. Interconversion potential of cloned human marrow adipocytes in vitro. Bone 1999;24:549–54. [75] Nicosia RF, Villaschi S. Rat aortic smooth muscle cells become pericytes during angiogenesis in vitro. Lab Invest 1995; 73:658–66. [76] Hall AP. Review of the pericyte during angiogenesis and its role in cancer and diabetic retinopathy. Toxicol Pathol 2006; 34:763–75. [77] Armulik A, Abramsson A, Betsholtz C. Endothelial/pericyte interactions. Circ Res 2005;97:512–23. [78] Li Q, Yu Y, Bischoff J, et al. Differential expression of CD146 in tissues and endothelial cells derived from infantile haemangioma and normal human skin. J Pathol 2003;201:296–302. [79] Paquet-Fifield S, Schluter H, Li A, et al. A role for pericytes as microenvironmental regulators of human skin tissue regeneration. J Clin Invest 2009;119:2795–806. [80] Nehls V, Drenckhahn D. Heterogeneity of microvascular pericytes for smooth muscle type alpha-actin. J Cell Biol 1991; 113:147–54.

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[81] Hellstrom M, Kalen M, Lindahl P, et al. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 1999;126:3047–55. [82] Murfee WL, Skalak TC, Peirce SM. Differential arterial/venous expression of NG2 proteoglycan in perivascular cells along microvessels: identifying a venule-specific phenotype. Microcirculation 2005;12:151–60. [83] Chan J, O’Donoghue K, Gavina M, et al. Galectin-1 induces skeletal muscle differentiation in human fetal mesenchymal stem cells and increases muscle regeneration. Stem Cells 2006;24:1879–91. [84] Burlacu A, Rosca AM, Maniu H, et al. Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells. Eur J Cell Biol 2008;87:173–84. [85] Delorme B, Ringe J, Pontikoglou C, et al. Specific lineage-priming of bone marrow mesenchymal stem cells provides the molecular framework for their plasticity. Stem Cells 2009;27:1142–51. *[86] Uezumi A, Fukada S, Yamamoto N, et al. Mesenchymal progenitors distinct from satellite cells contribute to ectopic fat cell formation in skeletal muscle. Nat Cell Biol 2010;12:143–52. [87] Collett G, Wood A, Alexander MY, et al. Receptor tyrosine kinase Axl modulates the osteogenic differentiation of pericytes. Circ Res 2003;30(92):1123–9. [88] Howson KM, Aplin AC, Gelati M, et al. The postnatal rat aorta contains pericyte progenitor cells that form spheroidal colonies in suspension culture. Am J Physiol Cell Physiol 2005;289:1396–407.




3

Prospective isolation of human MSC Abhishek Harichandan, MSc, Hans-Jörg Bühring, PhD, Head of Laboratory for Stem Cell Research * University Clinic of Tübingen, Department of Internal Medicine II, Division of Haematology, Immunology, Oncology, Rheumatology, and Pulmonology, Laboratory for Stem Cell Research, Otfried-Müller-Str. 10, 72076 Tübingen, Germany

Keywords: mesenchymal/stromal stem cells MSC prospective isolation surface antigens MSC subsets

Conventionally, mesenchymal/stromal stem cells (MSC) are functionally isolated from primary tissue based on their capacity to adhere to the plastic surface. This isolation procedure is hampered by the unpredictable influence of secreted molecules or interactions with co-cultured hematopoietic and other unrelated cells as well as by the arbitrarily selected removal time of non-adherent cells prior to expansion of MSC. Early removal of non-adherent cells may result in the elimination of a late adhering MSC subsets and late removal increases the influence of undesired cells on the growth and differentiation of MSC. Finally, in conventional protocols MSC are co-expanded together with macrophages, endothelial cells and other adherent cells. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSC based on the selective expression or absence of surface markers. Here we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow-derived MSC. Ó 2011 Elsevier Ltd. All rights reserved.

Characteristics of bone marrow-derived MSC Mesenchymal stem/stromal cells (MSC) are multipotent cells, which are able to form fibroblast-like colonies (CFU-F) [1,2]. After expansion in culture, bone marrow-derived MSC express the surface markers CD29, CD73, CD90, CD105, CD106, CD140b, and CD166 but lack CD31, CD45, CD34, CD133, and MHC class II expression [3–5]. They are not only able to differentiate into osteoblasts, adipocytes, and chondrocytes, but also into cells of non-mesodermal lineages including hepatocytes, neuron-like and pancreatic-like cells [6–11]. Because of their microenvironment forming ability and multi-lineage * Corresponding author. Tel.: þ49 7071 2982730; Fax: þ49 7071 292730. E-mail address: [email protected] (H.-J. Bühring). 1521-6926/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2011.01.001

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differentiation capacity, they present an attractive cell source for co-transplantation with hematopoietic stem cells as well as for replacement therapy of damaged tissues in patients with osteoarthritis, spinal cord injury, cardiovascular, neurological, and immunological diseases [12–15]. MSC from other tissues Originally, MSC were derived from cultured plastic-adherent bone marrow cells but meanwhile, a number of other tissues have been identified that contain MSC at varying frequencies and with varying differentiation capacities. Additional sources with MSC potential include placenta, adipose tissue, peripheral blood, umbilical blood, amniotic fluid, fetal hepatic and pulmonary tissue, skin, and prostate [3,16–25]. Although cultured MSC from all sources appear to be negative for CD31, CD45, CD80, and uniformly express CD9, CD10, CD13, CD29, CD73, CD90, CD105, and CD106, a more tissue-specific expression of other surface antigens has been reported. For example, only adipose tissue-derived MSC express high levels of CD34, and only placenta-derived MSC but not bone marrow-derived MSC are positive for SSEA-4, and TRA-1-81 [17,26]. In contrast, bone marrow-derived MSC but not placentaderived MSC express high levels of CD271 and tissue non-specific alkaline phosphatase (TNAP) [6,27–29]. MSC from different sources do not only display differential expression patterns of surface antigens but also vary in their differentiation capacity. In a recent publication it was demonstrated that bone marrow-derived MSC display a better chondrogenic differentiation potential compared with MSC of other sources [30]. As MSC represent an attractive tool for cartilage tissue repair strategies, bone marrow is considered as the preferred MSC source for these therapeutic approaches [30]. Isolation procedures of MSC Functional isolation of MSC Conventional procedures to prepare MSC for research and clinical purposes rely on the expansion of unselected bone marrow cells based on their capacity to adhere to the plastic surface in culture dishes. These functionally isolated MSC are expanded in defined media in the presence of platelet lysate or other growth factor compositions [4,31–33]. This isolation procedure is accompanied by several limitations including i) undesired interactions of MSC with hematopoietic cells and their released growth factors in the first culture period, ii) the challenging decision to define the optimal time point of removal of nonadherent cells and replacement with fresh media, and iii) the co-expansion of other adherent cells – mainly macrophages and endothelial cells – during the expansion period. In addition, functionally isolated MSC do not provide any information about the antigenic composition of the starting population. As a consequence, most of publications describe retrospective antigen expression profiles of MSC progeny but not of the initiating cells. Not surprisingly, a variety of surface markers such as CD109, CD166 (however, this molecule is expressed on CD56þ MSC in primary BM), and CD318, are exclusively found on cultured MSC but not on their primary counterparts [6,34]. Vice versa, other markers like CD271 or CD56, which are known to be highly expressed on primary MSC or MSC subsets, are rapidly downregulated in culture [6]. Despite the limitations of functional isolation protocols, these procedures are still prevalent for large-scale MSC preparations in clinical settings because expensive GMP-manufactured antibodies for immunoselection are not required. Prospective isolation of MSC In contrast to functional isolation procedures, the prospective isolation of MSC allows a precise definition of the starting population. This isolation procedure also precludes the potential adverse effect of co-cultured hematopoietic cells and avoids the potential removal of important MSC subsets together with other non-adherent cells. In addition, no other adherent cells are co-cultured that may interfere with the expansion of MSC. To isolate MSC from primary bone marrow or other tissue, several markers were identified, which are suitable to enrich for these cells. These markers include antibodies against a variety of surface molecules including CD49a, CD63, CD73 (SH3/SH4), CD105 (SH2), CD106, CD140b, CD271, TNAP, Hsp90-beta, as well as orphan antigens defined by antibodies STRO-1, W3D5,


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Table 1 List of antigens/antibodies used for the prospective isolation of MSC. Markers used A) Known antigens for positive selection CD9 (MRP-1; MIC3) CD10 (Neprilysin; CALLA) CD26 (DPP4) CD34 (MY10; gp105-120)) CD44 (PGP-1; ECMR-3) CD49a (Integrin a1) CD49e (Integrin a5) CD56 (NCAM) CD63 (MLA1; TSPAN30) CD73 (NT5E) CD90 (Thy-1)

CD105 (Endoglin)

CD106 (VCAM-1) CD117 (C-Kit) CD130 (gp130) CD140b (PDGFRB) CD146 (MCAM)

CD166 (ALCAM)

CD200 (MRC, OX2) CD271 (LNGFR)

CD309 (Flk-1; VEGFR-2) CD349 (Frizzled-9) ALDH GD2 (Neural Ganglioside) HSP90beta Integrin alphaV/beta5 SSEA-4 TNAP B) Unknown (antibody-defined) antigens for positive selection. 3G5 D7-FIB STRO-1 W5C5a C) Known antigens for negative selection CD3 (T-cell surface glycoprotein) CD14 (LPS receptor) CD31 (PECAM-1)

Tissue

References

Synovial membrane Placenta Placenta Adipose tissue Bone marrow Bone marrow Bone marrow Bone marrow Bone marrow Bone marrow Adipose tissue Bone marrow Synovial membrane Endometrium Synovial membrane Bone marrow Cartilage Endometrium Wharton’s jelly Bone marrow Umbilical cord Amniotic fluid Bone marrow Endometrium Bone marrow Adipose tissue Endometrium Synovial membrane Cartilage Bone marrow Fetal membranes Bone marrow Amnion Bone marrow Chorion Adipose tissue Bone marrow Placenta Bone marrow Bone Marrow Umbilical chord Bone marrow Bone marrow Bone marrow Bone marrow

[44] [17] [17] [22–24,45–47] [48] [49–53] [54] [6,27,28] [52] [55–57] [23,24] [58] [44] [59] [60] [48,56,57,61–63] [64] [65] [66] [67,68] [21] [36] [55] [69] [55,70] [16,71] [59,65,69] [44] [64] [52] [3] [55] [3] [6,27,28,33,37,63,72–75] [3] [45] [76] [17] [35] [77] [20] [78] [55] [79] [6,27–29]

Adipose tissue Bone marrow Bone marrow Adipose tissue Bone marrow

[16] [74,80] [29,49,52,67,68,81–87] [16] [37]

Peripheral blood Peripheral blood Bone marrow Adipose tissue

[19] [19] [76] [22–24] (continued on next page)

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Table 1 (continued ) Markers used

Tissue

References

CD34 (Hematopoietic progenitor cell antigen)

Bone marrow Peripheral blood Bone marrow Lung Adipose tissue Adipose tissue Adipose tissue Adipose tissue Bone marrow Bone marrow

[29,76,87–89] [19] [49,54,62,74,81,87,88,90] [18] [23] [23,24] [22] [23,24] [49,81,83,87,90] [33,63,90]

CD45 (Leukocyte common antigen)

CD105 (Endoglin) CD144 (Cadherin-5) CD146 (MCAM) CD235a (GlycophorinA) Lin (various antigens) a

Manuscript in preparation.

W5C5 (Table 1A and B). In some cases, MSC were not or not only selected by their immunophenotype but rather by functional features like the enzymatic activity of aldehyde dehydrogenase, which is known to be increased in stem cells of many tissues [35]. Employing Aldefluor as a specific dye to monitor this enzyme activity, a 9.5 fold enrichment of bone marrow-derived MSC in the Aldefluorbright population was achieved (Table 1A) [35]. In other approaches, bone marrow-derived MSC were enriched by negative selection, employing markers such as CD14, CD34, CD45, and/or CD235 (glycophorin A) and other “lineage-negative” markers (Table 1C). Distinct markers are required for the selection of MSC from other sources than bone marrow because of some unique phenotypic peculiarities. For example, placenta-derived MSC are preferentially isolated using antibodies against CD349 (frizzled-9), SSEA-4, and TRA-1-81, which is in contrast to their bone marrow-derived counterparts that are preferably isolated by CD271 or TNAP selection [17]. Other markers such as CD34 and CD117 are more suitable to select adipose- and amniotic fluid-derived MSC, respectively (Table 1A) [36]. The CFU-F assay is the most frequently used test to analyze the clonogenic potential of prospectively isolated MSC [1,2]. Candidate antibodies selective for MSC can be evaluated by screening their reactivity with cell populations that express established key MSC markers such as CD271 or STRO-1. An example is shown in Fig. 1, showing bone marrow cells double stained of with antibodies against CD271 and CD140b (PDGF receptor-beta). The FACS plot demonstrates that only CD271bright but not CD271dim cells give rise to clonogenic MSC and that these populations differ considerably in their morphological appearance. Giemsa staining shows that CD271bright cells are characterized by a relatively bright nuclear staining and a high cytoplasmic content, compared to the immature lymphoblastoid appearance with darker nuclear staining of CD271dim cells. The plot additionally shows that CD140b is a more selective marker for MSC isolation than CD271, because this molecule is expressed only on CD271bright but not CD271dim cells. As expected, clonogenic cells (CFU-F) were exclusively found in the CD140bþ population. Using this screening approach, additional antibodies with specificity for MSC have been identified [6,17,27,28,37] and may be discovered in the future. Prospective isolation of MSC subsets Several groups have reported that MSC are heterogeneous with respect to their growth and differentiation potential [34,38,39]. However, little information exists about markers that discriminate between developmentally, functionally, and morphologically distinct MSC subsets. Recently, we introduced a monoclonal antibody against CD56 that recognizes a distinct MSC subset with high selectivity [6]. This antibody (termed 39D5) detects a CD56 epitope, which is not expressed on NK cells (Fig. 2A) but is highly expressed on about 0.5–15% of CD271bright cells (Fig. 2B). Giemsa staining revealed that CD56 cells contain a large bright cytoplasm with vacuoles, whereas cells of the CD56þ subset contain a smaller cytoplasm with basophilic granules (Fig. 2C). Interestingly, cells of the CD56þ population were about two times more clonogenic than cells of the CD56 subset (Fig. 2D). Further analysis has shown that this increased frequency of clonogenic cells is correlated with an increased proliferation rate [6]. Surface marker expression analysis of sorted CD56þ and CD56 cells revealed that


Fig. 1. Morphological features and clonogenic capacity of sorted CD271brightCD140bþ and CD271dimCD140b bone marrow cells. Cells were stained with anti-CD271 and anti-CD140b, gated on the indicated populations, and sorted by flow cytometry. Fourteen days after culture in serum-free, b-FGF containing medium, the resulting colonies were enumerated and CFU-F numbers normalized to 5.000 plated cells. Note that CFU-F were exclusively found in the CD140bþ subset and only cells of this subset gave rise to fibroblast-like cells. The morphology of CD271dim and CD271bright cells was evaluated by staining of sorted cells with Giemsa.

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Fig. 2. CD56 defines a subset of MSC. A) CD56 epitope NCAM16.2 but not 39D5 is expressed on natural killer cells from peripheral blood. B) CD56 is expressed on cells of a CD271bright MSC subset. C) CD271brightCD56þ and CD271brightCD56 BM cells are clonogenic. CFU-F derived from 500 FACS-sorted cells were stained and scored as described. Data represent the mean CFU-F numbers of three different experiments (* ¼ p < 0.01). D) Morphology of CD271brightCD56 and CD271brightCD56þ cells. Subsets were sorted, cytocentrifuged, stained with Giemsa solution, and scored on a Zeiss Axiovert 200 microscope. Note the presence of basophilic-like granules in cells of the CD271brightCD56þ population.

only CD56þ cells but not CD56 cells coexpress CD166, and only a subset of CD56 cells but not CD56þ cells express CD349 (frizzled-9) [6]. However, when these cells were put into culture, both cell types expressed high levels of CD166 and moderate levels of CD349 (Fig. 3). The tumor antigen CDCP1 (CD318), which is not expressed on primary MSC, was upregulated after culture of cells from both MSC populations, whereas CD271 and CD56 were rapidly downregulated. When cultured cells of both populations were induced to differentiate into defined cell lineages, only MSC derived from the CD56 population were able to differentiate into adipocytes [6]. This population was also the major source for


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Fig. 3. Expression of selected markers on cultured MSC derived from sorted CD271brightCD56 and CD271brightCD56þ cells. Sorted MSC were cultured on gelatine-coated flasks and cultured in b-FGF containing serum free medium. After reaching confluence, cells were stained with antibodies against the indicated antigens. Note the rapid downregulation of CD56 and CD271 after culture and the upregulation of CD318.

MSC with osteogenic differentiation potential. In contrast, only MSC from the CD56þ subset effectively gave rise to chondrocytes, suggesting that this subpopulation is the preferred source for therapeutic approaches in the field of cartilage tissue repair [6]. We have previously shown that CD56þ MSC express low levels of TNAP, a molecule that is upregulated during osteogenic differentiation [6]. In a model proposed by Gronthos et al. cell surface expression of TNAP is absent on early STRO-1þ stem cells but upregulated during osteogenic differentiation [40]. This STRO-1þTNAP population may correspond to the recently described CD56þTNAP/ dim subset, which was identified by our group. In agreement with this hypothesis, cells of the CD56þ subset mature at a later time point into osteoblasts compared to CD56 cells. We therefore propose an extended model, in which STRO-1þCD56þTNAP/dim MSC represent an immature precursor with multi-lineage differentiation capacity. Cells committed to the chondrocyte lineage diverge at very early (CD56þ) stages of MSC differentiation. This chondrogenic potential, which is rapidly lost upon differentiation into TNAPþCD56 cells, is accompanied by the induction of the adipogenic differentiation potential and by the enhancement of the osteogenic differentiation capacity. Several reports underline the important role of CD56 expression on fibroblasts to support the growth of hematopoietic stem cells [41–43]. The contribution of CD56 was initially described in mouse and monkey [41,42], but a more recent report showed that CD56 expressed on a mouse stromal line plays a crucial role to support human hematopoiesis in vitro and in vivo [43]. The authors showed that co-culture of CD34þCD38 cord blood cells with a CD56þ stromal cell line resulted in a significantly greater expansion rate of CD34þ hematopoietic cells compared to stromal cells, which did not express CD56. This enhancing effect could be blocked by the addition of an inhibitory anti-CD56 antibody, suggesting that direct interactions between CD56 molecules from different cells are essential. It remains open, whether CD56 on human MSC plays a similar hematopoiesis supporting activity. As human hematopoietic stem cells do not express CD56, it is unlikely that a potential supporting effect is caused by homotypic interactions between CD56 molecules on stromal and hematopoietic cells. Rather, CD56þ stromal cells may interact with extracellular matrix components such as heparin sulfate or chondroitin sulfate proteoglycans. We have recently shown that SSEA-3, but not SSEA-4, TRA-1-60 and TRA-1-81, is a candidate marker for MSC derived from primary femur derived bone marrow [27]. To verify this assumption, bone marrow cells were stained with CD271, SSEA-3, and CD56 and gated on CD271brightSSEA3CD56, CD271brightSSEA-3þCD56, and CD271brightSSEA-3CD56þ cells (Fig. 4A). The clonogenic potential and the differentiation capacity of the sorted populations were determined by CFU-F assays and appropriate differentiation protocols. As shown in Fig. 4, clonogenic cells were about 44-fold enriched for CFU-F in the CD271brightSSEA-3þCD56 population, 83-fold in the CD271brightSSEA3CD56þ population, but only about 2-fold in the CD271brightSSEA-3CD56 fraction. Not surprisingly,

32 A. Harichandan, H.-J. Bühring / Best Practice & Research Clinical Haematology 24 (2011) 25–36

Fig. 4. SSEA-3 defines a subset of MSC complementary to CD56. A) Display of CD56 versus SSEA-3 of triple stained bone marrow cells gated on the CD271bright cells. Note that CD56þ MSC lack coexpression of SSEA-3. B) Culture of sorted bone marrow cells gated on the indicated populations (Passage 0). Cells were sorted on a FACS Aria cell sorter (Becton Dickinson) and transferred into T25 flask and cultured for 12 days. C) Osteogenic differentiation of MSC. After 10 days of culture in differentiation medium, osteoblasts were stained for alkaline phosphatase activity. D) Adipogenic differentiation of MSC. After 21 days of culture in differentiation medium, adipocytes were stained for neutral lipids with Oil Red O dye and visualized on a Zeiss Axiovert 200 microscope. Note the exclusive presence of adipocytes in the CD56- subsets. E) Clonogenic capacity of sorted BM cells. Defined cell numbers of the sorted BM cells were plated on gelatine-coated flasks and cultured serum free in b-FGF containing medium. 12 days after culture, the colonies were enumerated. CFU-F numbers were normalized to 2000 plated cells.


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CD271brightSSEA-3þCD56 MSC gave rise to osteoblasts and adipocytes (Fig. 4C and D), but not to chondrocytes (not shown). In contrast, CD271brightSSEA-3CD56þ cells were able to differentiate into chondrocytes (not shown) but not into adipocytes. Collectively, SSEA-3 is a suitable and selective marker for the isolation of osteoblast and adipocyte precursors, whereas CD56 is the more appropriate target for the isolation of chondrocyte precursors. Isolation of single MSC We have recently described the prospective isolation of MSC subsets from primary tissue using antibodies against molecules, which are selectively expressed on the surface of these subsets [6,28]. Although phenotypically distinct MSC subsets exhibit properties, which are unique with regard to their proliferation and differentiation capacity, there is still a broad heterogeneity at the clonal level [6]. Heterogeneity of individual MSC clones has been reported by several groups, who demonstrated that the developmental and proliferative potential was highest in cells giving rise to large colonies, whereas small-sized colonies were derived from cells with limited differentiation and proliferation capacity [6]. Sorting of single cells into culture plates does not only provide information about the growth characteristics of individual MSC clones but also about the frequency of MSC clones with defined differentiation potential. Pittenger et al. described that almost 100% of colonies derived from single bone marrow cells underwent osteogenic differentiation, about 80% of the colonies revealed adipogenic differentiation potential, but only 30% of the colonies showed chondrogenic differentiation potential. Our group was successful to isolate clones with capacity for osteoblast but not adipocyte differentiation as well as for adipocyte but not osteoblast differentiation [6]. Further experiments are required to determine the frequency of MSC with multipotent differentiation capacity as well as of MSC with restricted differentiation potential. These analyses may contribute to customize complex models of MSC maturation and differentiation, similar to those proposed for cells of the hematopoietic system. Concluding remarks Conventionally, MSC are functionally isolated by their capacity to adhere to the surface of culture plates. The resulting cells are poorly defined and give rise to a heterogeneous mixture of cells including MSC, reticular cells, macrophages, and endothelial cells. To gain information about the starting population, several markers have been introduced to prospectively isolate MSC and their subsets. A similar degree of hierarchy and progenitor cell heterogeneity may exist among MSC as described for the hematopoietic system. The identification of MSC subsets and clonal analysis of individual MSC may contribute to gain more insight in the heterogeneity of MSC from different tissues.

Practice points Bone marrow-derived CD56þ MSC may be considered as a promising starting population for therapeutic approaches in the field of cartilage tissue repair. Bone marrow-derived SSEA-3þ MSC may be considered as a promising starting population for therapeutic approaches in the field of bone repair.

Research agenda Novel MSC-reactive antibodies combined with clonal analysis of individual MSC may contribute to establish a developmental scheme similar to the hematopoietic system.

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Conflict of interest None to declare. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich SFB-685 (Immunotherapy: Molecular Basis and Clinical Applications) project C10: Development of therapeutic antibodies for the elimination of tumor stem cells; by the DFG project BU 516/2-1: Identifizierung und funktionelle Untersuchung von MSC-spezifischen Molekülen; and by the DFG project SK49/10-1: Identification and functional analysis of human adult spermatogonial and germ line stem cells. The authors would like to thank Kavitha Sivasubramaniyan for her critical revision of the manuscript and acknowledge Drs. Uwe Ochs and Peter de Zwart for supply of bone marrow samples. References [1] Friedenstein AJ, Deriglasova UF, Kulagina NN, et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 1974;2:83–92. [2] Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 1976;4:267–74. [3] Soncini M, Vertua E, Gibelli L, et al. Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue Eng Regener Med 2007;1:296–305. [4] Schallmoser K, Bartmann C, Rohde E, et al. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 2007;47:1436–46. *[5] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143–7. *[6] Battula VL, Treml S, Bareiss PM, et al. Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1. Haematologica 2009;94:173–84. [7] Aurich H, Sgodda M, Kaltwasser P, et al. Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo. Gut 2009;58:570–81. [8] Hou L, Cao H, Wang D, et al. Induction of umbilical cord blood mesenchymal stem cells into neuron-like cells in vitro. Int J Hematol 2003;78:256–61. [9] Yang XS, Wu HX, Xiao B. Human mesenchymal stem cells differentiate into neuron-like cells and show SMN protein expression. Zhonghua Yi Xue Za Zhi 2005;85:1125–8. [10] Sun Y, Chen L, Hou XG, et al. Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitro. Chin Med J (Engl.) 2007;120:771–6. [11] Xie QP, Huang H, Xu B, et al. Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro. Differentiation 2009;77:483–91. [12] Amado LC, Saliaris AP, Schuleri KH, et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A 2005;102:11474–9. [13] Rojas M, Xu J, Woods CR, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol 2005;33:145–52. [14] Maitra B, Szekely E, Gjini K, et al. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant 2004;33:597–604. [15] Parr AM, Tator CH, Keating A. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transplant 2007;40:609–19. [16] Zannettino AC, Paton S, Arthur A, et al. Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo. J Cell Physiol 2008;214:413–21. [17] Battula VL, Treml S, Abele H, Bühring HJ. Prospective isolation and characterization of mesenchymal stem cells from human placenta using a frizzled-9-specific monoclonal antibody. Differentiation 2008;76:326–36. [18] Martin J, Helm K, Ruegg P, et al. Adult lung side population cells have mesenchymal stem cell potential. Cytotherapy 2008;10:140–51. [19] Zvaifler NJ, Marinova-Mutafchieva L, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res 2000;2:477–88. [20] Jin HJ, Nam HY, Bae YK, et al. GD2 expression is closely associated with neuronal differentiation of human umbilical cord blood-derived mesenchymal stem cells. Cell Mol Life Sci 2010;67:1845–58. [21] Lu LL, Liu YJ, Yang SG, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica 2006;91:1017–26. [22] Traktuev DO, Merfeld-Clauss S, Li J, et al. A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res 2008;102:77–85. [23] Yoshimura K, Shigeura T, Matsumoto D, et al. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol 2006;208:64–76. [24] Lin K, Matsubara Y, Masuda Y, et al. Characterization of adipose tissue-derived cells isolated with the Celution system. Cytotherapy 2008;10:417–26.


35

[25] Hu Y, Liao L, Wang Q, et al. Isolation and identification of mesenchymal stem cells from human fetal pancreas. J Lab Clin Med 2003;141:342–9. [26] Yen BL, Huang HI, Chien CC, et al. Isolation of multipotent cells from human term placenta. Stem Cells 2005;23:3–9. [27] Sobiesiak M, Sivasubramaniyan K, Hermann C, et al. The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase. Stem Cells Dev 2010;19:669–77. [28] Bühring HJ, Treml S, Cerabona F, et al. Phenotypic characterization of distinct human bone marrow-derived MSC subsets. Ann N Y Acad Sci 2009;1176:124–34. [29] Gronthos S, Fitter S, Diamond P, et al. A novel monoclonal antibody (STRO-3) identifies an isoform of tissue nonspecific alkaline phosphatase expressed by multipotent bone marrow stromal stem cells. Stem Cells Dev 2007;16:953–63. *[30] Bernardo ME, Emons JA, Karperien M, et al. Human mesenchymal stem cells derived from bone marrow display a better chondrogenic differentiation compared with other sources. Connect Tissue Res 2007;48:132–40. *[31] Battula VL, Bareiss PM, Treml S, et al. Human placenta and bone marrow derived MSC cultured in serum-free, b-FGFcontaining medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation. Differentiation 2007;75:279–91. [32] Muller I, Kordowich S, Holzwarth C, et al. Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM. Cytotherapy 2006;8:437–44. [33] Bieback K, Hecker A, Kocaomer A, et al. Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 2009;27:2331–41. [34] Vogel W, Grunebach F, Messam CA, et al. Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells. Haematologica 2003;88:126–33. [35] Gentry T, Foster S, Winstead L, et al. Simultaneous isolation of human BM hematopoietic, endothelial and mesenchymal progenitor cells by flow sorting based on aldehyde dehydrogenase activity: implications for cell therapy. Cytotherapy 2007;9:259–74. [36] De Coppi P, Bartsch Jr G, Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:100–6. [37] Bühring HJ, Battula VL, Treml S, et al. Novel markers for the prospective isolation of human MSC. Ann N Y Acad Sci 2007; 1106:262–71. [38] Krinner A, Hoffmann M, Loeffler M, et al. Individual fates of mesenchymal stem cells in vitro. BMC Syst Biol 2010;4:73. [39] Lin CS, Xin ZC, Deng CH, et al. Defining adipose tissue-derived stem cells in tissue and in culture. Histol Histopathol 2010; 25:807–15. *[40] Gronthos S, Zannettino AC, Graves SE, et al. Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells. J Bone Miner Res 1999;14:47–56. *[41] Wang X, Hisha H, Taketani S, et al. Neural cell adhesion molecule contributes to hemopoiesis-supporting capacity of stromal cell lines. Stem Cells 2005;23:1389–99. [42] Kato J, Hisha H, Wang XL, et al. Contribution of neural cell adhesion molecule (NCAM) to hemopoietic system in monkeys. Ann Hematol 2008;87:797–807. [43] Wang X, Hisha H, Mizokami T, et al. Mouse mesenchymal stem cells can support human hematopoiesis both in vitro and in vivo: the crucial role of neural cell adhesion molecule. Haematologica 2010;95:884–91. [44] Fickert S, Fiedler J, Brenner RE. Identification, quantification and isolation of mesenchymal progenitor cells from osteoarthritic synovium by fluorescence automated cell sorting. Osteoarthritis Cartilage 2003;11:790–800. [45] Quirici N, Scavullo C, de Girolamo L, et al. Anti-L-NGFR and -CD34 monoclonal antibodies identify multipotent mesenchymal stem cells in human adipose tissue. Stem Cells Dev 2010;19:915–25. [46] Mitchell JB, McIntosh K, Zvonic S, et al. Immunophenotype of human adipose-derived cells: temporal changes in stromalassociated and stem cell-associated markers. Stem Cells 2006;24:376–85. [47] Varma MJ, Breuls RG, Schouten TE, et al. Phenotypical and functional characterization of freshly isolated adipose tissuederived stem cells. Stem Cells Dev 2007;16:91–104. [48] Martins AA, Paiva A, Morgado JM, et al. Quantification and immunophenotypic characterization of bone marrow and umbilical cord blood mesenchymal stem cells by multicolor flow cytometry. Transplant Proc 2009;41:943–6. [49] Letchford J, Cardwell AM, Stewart K, et al. Isolation of C15: a novel antibody generated by phage display against mesenchymal stem cell-enriched fractions of adult human marrow. J Immunol Methods 2006;308:124–37. [50] Gindraux F, Selmani Z, Obert L, et al. Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell markers can be directly enriched by using the CD49a molecule. Cell Tissue Res 2007;327:471–83. [51] Rider DA, Nalathamby T, Nurcombe V, Cool SM. Selection using the alpha-1 integrin (CD49a) enhances the multipotentiality of the mesenchymal stem cell population from heterogeneous bone marrow stromal cells. J Mol Histol 2007; 38:449–58. [52] Stewart K, Monk P, Walsh S, et al. STRO-1, HOP-26 (CD63), CD49a and SB-10 (CD166) as markers of primitive human marrow stromal cells and their more differentiated progeny: a comparative investigation in vitro. Cell Tissue Res 2003;313:281–90. [53] Deschaseaux F, Gindraux F, Saadi R, et al. Direct selection of human bone marrow mesenchymal stem cells using an antiCD49a antibody reveals their CD45med, low phenotype. Br J Haematol 2003;122:506–17. *[54] Baksh D, Zandstra PW, Davies JE. A non-contact suspension culture approach to the culture of osteogenic cells derived from a CD49elow subpopulation of human bone marrow-derived cells. Biotechnol Bioeng 2007;98:1195–208. [55] Delorme B, Ringe J, Gallay N, et al. Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells. Blood 2008;111:2631–5. [56] Odabas S, Sayar F, Guven G, et al. Separation of mesenchymal stem cells with magnetic nanosorbents carrying CD105 and CD73 antibodies in flow-through and batch systems. J Chromatogr B Anal Technol Biomed Life Sci 2008;861:74–80. [57] Liu PG, Zhou DB, Shen T. Identification of human bone marrow mesenchymal stem cells: preparation and utilization of two monoclonal antibodies against SH2, SH3. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2005;13:656–9. [58] Campioni D, Lanza F, Moretti S, et al. Loss of Thy-1 (CD90) antigen expression on mesenchymal stromal cells from hematologic malignancies is induced by in vitro angiogenic stimuli and is associated with peculiar functional and phenotypic characteristics. Cytotherapy 2008;10:69–82.

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*[59] Schwab KE, Hutchinson P, Gargett CE. Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells. Hum Reprod 2008;23:934–43. [60] Arufe MC, De la FA, Fuentes-Boquete I, et al. Differentiation of synovial CD-105(þ) human mesenchymal stem cells into chondrocyte-like cells through spheroid formation. J Cell Biochem 2009;108:145–55. [61] Aslan H, Zilberman Y, Kandel L, et al. Osteogenic differentiation of noncultured immunoisolated bone marrow-derived CD105þ cells. Stem Cells 2006;24:1728–37. [62] Kastrinaki MC, Andreakou I, Charbord P, Papadaki HA. Isolation of human bone marrow mesenchymal stem cells using different membrane markers: comparison of colony/cloning efficiency, differentiation potential, and molecular profile. Tissue Eng Part C Methods 2008;14:333–9. [63] Jarocha D, Lukasiewicz E, Majka M. Advantage of mesenchymal stem cells (MSC) expansion directly from purified bone marrow CD105þ and CD271þ cells. Folia Histochem Cytobiol 2008;46:307–14. [64] Alsalameh S, Amin R, Gemba T, Lotz M. Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage. Arthritis Rheum 2004;50:1522–32. [65] Tsuji S, Yoshimoto M, Takahashi K, et al. Side population cells contribute to the genesis of human endometrium. Fertil Steril 2008;90:1528–37. [66] Conconi MT, Burra P, Di Liddo R, et al. CD105(þ) cells from Wharton’s jelly show in vitro and in vivo myogenic differentiative potential. Int J Mol Med 2006;18:1089–96. [67] Gronthos S, Zannettino AC. A method to isolate and purify human bone marrow stromal stem cells. Methods Mol Biol 2008;449:45–57. *[68] Gronthos S, Zannettino AC, Hay SJ, et al. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci 2003;116:1827–35. [69] Schwab KE, Gargett CE. Co-expression of two perivascular cell markers isolates mesenchymal stem-like cells from human endometrium. Hum Reprod 2007;22:2903–11. [70] Sorrentino A, Ferracin M, Castelli G, et al. Isolation and characterization of CD146þ multipotent mesenchymal stromal cells. Exp Hematol 2008;36:1035–46. [71] Astori G, Vignati F, Bardelli S, et al. In vitro" and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells. J Transl Med 2007;5: 55. [72] Poloni A, Maurizi G, Rosini V, et al. Selection of CD271(þ) cells and human AB serum allows a large expansion of mesenchymal stromal cells from human bone marrow. Cytotherapy 2009;11:153–62. [73] Horn P, Bork S, Diehlmann A, et al. Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis. Cytotherapy 2008;10:676–85. [74] Jones EA, English A, Kinsey SE, et al. Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow. Cytometry B Clin Cytom 2006;70: 391–9. [75] Quirici N, Soligo D, Bossolasco P, et al. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 2002;30:783–91. [76] Liu L, Sun Z, Chen B, et al. Ex vivo expansion and in vivo infusion of bone marrow-derived Flk-1þCD31-. Stem Cells Dev 2006;15:349–57. [77] Martinez C, Hofmann TJ, Marino R, et al. Human bone marrow mesenchymal stromal cells express the neural ganglioside GD2: a novel surface marker for the identification of MSCs. Blood 2007;109:4245–8. [78] Gronthos S, McCarty R, Mrozik K, et al. Heat shock protein-90 beta is expressed at the surface of multipotential mesenchymal precursor cells: generation of a novel monoclonal antibody, STRO-4, with specificity for mesenchymal precursor cells from human and ovine tissues. Stem Cells Dev 2009;18:1253–62. [79] Gang EJ, Bosnakovski D, Figueiredo CA, et al. SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood 2007; 109:1743–51. [80] Jones EA, Kinsey SE, English A, et al. Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells. Arthritis Rheum 2002;46:3349–60. [81] Peiffer I, Eid P, Barbet R, et al. A sub-population of high proliferative potential-quiescent human mesenchymal stem cells is under the reversible control of interferon alpha/beta. Leukemia 2007;21:714–24. [82] Psaltis PJ, Paton S, See F, et al. Enrichment for STRO-1 expression enhances the cardiovascular paracrine activity of human bone marrow-derived mesenchymal cell populations. J Cell Physiol 2010;223:530–40. *[83] Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991;78:55–62. [84] Gronthos S, Graves SE, Ohta S, Simmons PJ. The STRO-1þ fraction of adult human bone marrow contains the osteogenic precursors. Blood 1994;84:4164–73. [85] Encina NR, Billotte WG, Hofmann MC. Immunomagnetic isolation of osteoprogenitors from human bone marrow stroma. Lab Invest 1999;79:449–57. [86] Dennis JE, Carbillet JP, Caplan AI, Charbord P. The STRO-1þ marrow cell population is multipotential. Cells Tissues Organs 2002;170:73–82. [87] Zannettino AC, Paton S, Kortesidis A, et al. Human mulipotential mesenchymal/stromal stem cells are derived from a discrete subpopulation of STRO-1bright/CD34/CD45(-)/glycophorin-A-bone marrow cells. Haematologica 2007;92: 1707–8. [88] Kaiser S, Hackanson B, Follo M, et al. BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype. Cytotherapy 2007;9:439–50. [89] Huss R. Perspectives on the morphology and biology of CD34-negative stem cells. J Hematother Stem Cell Res 2000;9:783– 93. [90] Tondreau T, Lagneaux L, Dejeneffe M, et al. Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential. Cytotherapy 2004;6:372–9.




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Osteoprogenitors and the hematopoietic microenvironment Paolo Bianco, Professor of Pathology a, b, *, Benedetto Sacchetti, Ph.D Postdoctoral Fellow a, b, Mara Riminucci, Associate Professor of Pathology a, b a b

Department of Molecular Medicine, La Sapienza University, 00161 Rome, Italy Biomedical Science Park San Raffaele, 00128 Rome, Italy

Keywords: Hematopoietic stem cell Hematopoietic niche Mesenchymal stem cell Bone marrow stromal cell

The identification of skeletal progenitor cells in the human bone marrow (so-called mesenchymal stem cells) by anatomy and phenotype (CD146-expressing, adventitial reticular cells) has coincided with the recognition that the ability to transfer the hematopoietic microenvironment is an inherent property of skeletal progenitor cells. Inasmuch as these cells generate osteoblasts, associate with sinusoids (the assembly of which they dynamically direct), and coincide with, and self-renew into, stromal reticular cells, these cells are pivotal organizers of the hematopoietic microenvironment. Their nature as osteogenic cells and sinusoidal location reconcile the dual view of endosteal surfaces and sinusoidal walls as the hematopoietic stem cell “niches”, and highlight the dynamic nature of a niche/microenvironment essentially maintained by cells with properties of progenitors/stem cells for skeletal tissues. This view brings the long recognized, and somewhat mysterious, interaction between bone and bone marrow into a new perspective, where two stem cells interact with each other at the same niche. Ó 2011 Published by Elsevier Ltd.

A seminal experiment marking the birth of two key concepts A single seminal experiment [1] stands at the date and place of birth of two lines of investigation that were to become, and currently are, the focus of extensive attention. The first line of investigation lead to the large wealth of ex vivo and in vivo studies aiming at elucidating the role of microenvironmental cues in regulating not only hematopoiesis, but also hematopoietic stem cell function – what

* Corresponding author. Department of Molecular Medicine, La Sapienza University of Rome, Division of Pathology, Viale Regina Elena 324, 00161 Rome, Italy. Tel.: þ3906 444 1049; Fax: þ3906 4940896. E-mail address: [email protected] (P. Bianco). 1521-6926/$ – see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.beha.2011.01.005

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is presently often referred to as the hematopoietic “niche”. The second line was to develop into the concept that bone marrow includes a second type of stem cell, in addition to hematopoietic stem cells (HSCs), referred to as “osteogenic” [2], “stromal” [3], or “skeletal” [4,5] stem cells, prior to their rediscovery as “mesenchymal stem cells” [6,7]. The experiment [1] consisted in transplanting bone-less fragments of mammalian bone marrow at heterotopic sites. The general biological question being pursued by Tavassoli and Crosby was to determine why hematopoiesis is restricted to bone as the dominant site in post-natal mammals. Within this general context, the specific question tackled by a specific experimental approach (heterotopic transplantation) was whether hematopoiesis can indeed be transplanted to a non-hematopoietic, nonskeletal site. The answer that nature provided was, at a glance, cryptic and obscure, seemingly irrelevant to the question being asked – can hematopoiesis be established heterotopically? That answer was – “there is an osteogenic potential in the bone marrow”. Since the time of these findings, investigators have failed to grasp the relevance of nature’s answer to the investigators’ quest. Hematopoiesis can indeed be transferred to heterotopic sites, if the osteogenic potential of bone marrow is also transferred. Many years later, we can now understand the meaning. It is cells endowed with that potential (hence, a population of skeletal progenitors), which establishes the hematopoietic microenvironment. Indeed, a most conspicuous result of the Tavassoli and Crosby experiment was that heterotopic transplantation of marrow leads to formation of bone. Actually, of an architecturally sound “ossicle,” with a true shell of cortical bone, a cavity, and hematopoietic tissue contained within it. This observation provided the foundation for subsequent work by Friedenstein and others [8–10], who assigned the osteogenic potential revealed by the Tavassoli and Crosby experiment to non-hematopoietic, stromal cells contained within the bulk of hematopoietic tissue. It was the work of Friedenstein and coworkers that established the concept that bone marrow stromal cells would include a population of non-hematopoietic, skeletogenic, multipotent progenitors able to generate, in vivo, histology-proven skeletal tissues. This concept was much later popularized, and to a large extent corrupted, into the quite different concept of “mesenchymal stem cells”. Read through the most common interpretation of the concept, “mesenchymal stem cells” would be ubiquitous and equipotent cells found throughout the body and capable of generating virtually every mesoderm derivative (beyond skeletal tissues, including also muscle, endothelial cells, cardiomyocytes, etc.) and even non-mesoderm derivatives such as liver cells (endoderm derivatives) and neurons (neuroectoderm derivatives) (reviewed in [11]). While revealing the osteogenic potential of bone-free marrow, Tavassoli and Crosby’s work established at the same time the notion that indeed, hematopoiesis can be established at heterotopic sites, but that it is dependent on the establishment of a bone structure first. Again, this observation emphasized that close link between bone and marrow that had attracted the investigators’ attention, and before that, the attention of philosophers and poets alike (from Aristotle to Cervantes and Shakespeare), long before the simple observations of a young unsettled Italian fellow (Giulio Bizzozero) and an established German professor (Ernst Neumann) would ascribe to bone marrow the function of making blood cells [12]. This sets the stage for the concept of a hematopoietic “niche” in bone, first proposed by Schofield in 1978 [13], and inherited by present-day experimental work, however, leaving unsolved the problem of which cell(s) would in fact provide the “niche”. The multiple meanings of a niche In spite of its universal usage, the term niche does not actually convey a univocal meaning. In Schofield’s view, it meant the components that would ensure constant regeneration of hematopoietic tissue (and hematopoietic progenitors) in bone through renewal of stem cells. Inherited by what was to become a stem cell field encompassing a much broader array of stem cell types and dependent systems, a “niche” is the set of spatially defined environmental cues dictating self-renewal (hence, asymmetric division) of stem cells, in any tissue where stem cells exist and operate [14]. In a modified view (e.g., [15]), which has gained wide acceptance in hematology, a “niche” is the site where hematopoietic stem cells are kept quiescent (rather than self-renew; that is, the site where they do not divide rather than the site where they divide asymmetrically). Finally, in most studies attempting to grasp the microanatomy of a microanatomically defined entity, it is the place where one can localize bona fide HSCs (whether functionally or phenotypically defined, and regardless, of course, of any consideration


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of their kinetics) [16–19]. As related to the specific biology of hematopoiesis, therefore, the “niche” remains a quite effective and readily communicated word, but also a quite elusive concept of an elusive entity. In contrast, one faces much less uncertainty when trying to define the “hematopoietic microenvironment.” By this, one means the set of local cues provided by cells of non-hematopoietic origin and dictating the development of long-term hematopoiesis – either in vitro, as per the seminal experiments of Dexter and Allen [20,21], or in vivo, as per a line of investigation that goes from Tavassoli and Crosby (and their largely unknown XIX century antecedent, Goujon [22]), through the work of Friedenstein and coworkers, to our very day. Heterotopic transfer of the HME Transplantation has long been the mainstay of in vivo experimental dissection of crucial components of the hematopoietic microenvironment, through a series of experiments sharing the histology-proven establishment of hematopoietic tissue at heterotopic sites as the critical readout. Progressive refinement of the same fundamental approach over time led from transplantation of entire bone marrow fragments [1] to transplantation of non-hematopoietic, adherent stromal cells, to transplantation of single clones of stromal cells [8]. These studies clarified that the osteogenic potential associated with bone marrow demonstrated by Tavassoli and Crosby is in fact associated with non-hematopoietic stromal cells that can be grown in vitro, and indeed with single clonogenic stromal cells (Colony Forming Unit-Fibroblast, CFU-F) capable of giving rise to multiple non-hematopoietic cell types (the osteogenic stem cells of Friedenstein and Owen). At the same time, these experiments progressively narrowed the focus on cells that actually transfer the hematopoietic microenvironment. In all of these experiments, bone of donor origin is established along with hematopoietic tissue of host origin, giving rise to an artificial bone/bone marrow organ that exhibits reverse chimerism (“reverse” bone marrow transplantation) as compared to the chimeric bone marrow generated by transplantation of hematopoietic progenitors. Distinguishing individual stromal cells hidden amongst hematopoietic cells within the heterotopic ossicles, in order to determine their donor or host origin, remained essentially unfeasible, or perhaps unaddressed, until recently when their donor origin was unequivocally demonstrated [23]. In all of these experiments, bone is formed before a network of sinusoids is established, and hematopoiesis does not appear until such network is developed. One of the appealing aspects of heterotopic transplantation systems rests indeed with their ability to recapitulate a temporal sequence of events in which significant hints can be encrypted. In such systems, as well as in individual bone rudiments developing orthotopically in vivo, hematopoietic colonization is the result of seeding of blood-borne hematopoietic progenitor/stem cells (Fig. 1). Hence, the need for sinusoids is not surprising, and yet, the specific anatomy and flow characteristics of sinusoids, as opposed to capillaries, are often not appreciated. Slow flow and marginal, laminar flow of blood-borne cells are maximized in a sinusoid-type vessel, facilitating vascular egress of blood-borne cells. The apparent requirement for sinusoids in order for hematopoiesis to develop heterotopically finds here a simple explanation in simple laws of fluid dynamics. What remains less readily understood is what turns newly forming blood vessel into sinusoids, rather than capillaries. In heterotopic systems, the endothelial lining is provided by neighboring host vessels, and indeed, sinusoids that are established in the developing chimeric bone marrow are made of the same kind of endothelial cell that lines host capillaries just outside the boundaries of the developing ossicle. Hence, a local cue(s) must be operating, and must be provided at least in part by the transplanted, donor cells. In spite of the temporal link between bone formation and establishment of the hematopoietic microenvironment within heterotopic ossicles, the latter is not at all an obligate consequence of the former. Of note, bone marrow stromal cells from caPPR mice (constitutively active PTH/PTHrP receptor driven by the bone-specific type I collagen promoter [24], one of the two original disclosers of the osteoblastic niche [25]) are inefficient in transferring the hematopoietic microenvironment. In humans, several kinds of cell strains that can generate genuine osteoblasts and bone upon in vivo transplantation fail to establish a hematopoietic microenvironment. This is the case of cells derived in culture from bone surfaces rather than from bone marrow proper, such as trabecular bone cells, or periosteal cells [23], and ms in preparation. Lack of support of the hematopoietic microenvironment also occurs when cells are pushed to extensive in vitro proliferation through mitogens such as FGF-2

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Fig. 1. (A) Heterotopic transplant of human CD146-purified osteoprogenitors. 4 weeks post-transplant, demonstrating formation of bone and mesenchymal tissue, within which a nascent hematopoietic spot is seen, around a developing, sinusoidal type vessel (sin) coated with human CD146-expressing adventitial reticular cells. ost, osteoid tissue (unmineralized early bone). Top, hematoxylin/ eosin staining. Bottom and right, hCD146 immunostaining. (B) Heterotopic transplant at 7 weeks, showing clusters of granulopoiesis (h) and megakaryocytes (mk) around a sinusoid (sin), coated with CD146-expressing, human adventitial reticular cells (arrows). hac, hydroxyapatite/tricalcium phosphate carrier.


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[23]. Interestingly, human cell strains that form bone, but do not transfer the hematopoietic microenvironment, regularly include a low proportion of CD146-expresing cells, and yet express another marker of “MSCs”, CD63, at high levels. CD146 and adventitial reticular cells For a long time, the identity of the single cells comprised within the bone marrow stroma that are endowed with properties of multipotent skeletal progenitors, and are able at the same time to transfer the hematopoietic microenvironment, remained elusive. This was largely due to the lack of markers suited not only for isolation and enrichment of CFU-Fs (which can be accomplished using multiple markers, first and foremost STRO-1 [26], but also many others including, for example, endoglin (CD105) or alpha1 integrin [27,28]), but rather, and more importantly, for identifying the anatomy of those cells that are explanted as CFU-Fs from intact tissues through correlative studies. Likewise, a marker suited at the same time for CFU-F isolation and histology and for following their fate over culture and after in vivo transplantation is essential. In both cases, the marker should be suited for direct histological analysis of tissues comprising hard phases such as bone, or even harder phases such as the inorganic mineralized scaffolds used to load cells to be transplanted. This, of course, applies with specific relevance to studies on human cells and tissues, which necessarily elude more stringent and sophisticated, genetic approaches of lineage tracing. A breakthrough came when CD146/MCAM (Melanoma Cell Adhesion Molecule [29]), a cell adhesion molecule of the immunoglobulin super family, turned out to represent one such marker. CD146 is expressed at high levels in all human bone marrow stromal cells that can be assayed as CFU-Fs, and therefore in all human skeletal progenitors, prior to seeding in culture and throughout culture of the progeny of seeded cells. In situ, osteoblasts and bone-lining cells do not express CD146. CD146/MCAM expression is restricted to adventitial cells in the bone marrow microvasculature, particularly in cells known from classical histology as adventitial reticular cells. First identified by SEM studies in rodents and lagomorphs [30], adventitial reticular cells were later noted for their characteristic expression of alkaline phosphatase in rodents [31,32] and humans [33,34]. Long suspected of being implicated with hematopoietic support based on morphological evidence alone, adventitial reticular cells produce large amounts of stromal cell-derived factor-1/CXCL12 (the ligand for CXCR4) both in humans [23] and in the mouse (CAR cells [35]), and on this updated basis, they have been suggested to contribute to an HSC niche. The term adventitial alludes to their position in the sinusoid wall, where they reside abluminally to endothelial cells. The term reticular, in turn, signifies the branched morphology, which results from a wealth of thin processes projected from the cell body away from the sinus wall into the adjacent hematopoietic tissue (Fig. 2). Once isolated based on immuno selection for CD146, adventitial reticular cells behave as clonogenic progenitors (CFU-Fs), and generate a clonal progeny of cells, which in turn express CD146, and can be induced to osteogenic differentiation. Of note, upregulation of osteoblastic markers is coupled with down regulation of CD146 in vitro. Upon transplantation in vivo, CD146 positive cells generate bone and CD146 negative osteoblasts, guide the formation of sinusoids, and also selfrenew into CD146 positive human adventitial reticular cells that line the sinusoids, around which hematopoiesis is established. Hence, CD146-expressing adventitial reticular cells are skeletal progenitor/stem cells. Not only do they transfer, but they generate and organize the hematopoietic microenvironment (sought by blood-borne hematopoietic progenitors). They generate osteoblasts (and bone), direct the formation of sinusoids, and establish themselves along their walls. All three cell types that have been implicated with the “niche” affair, in fact, depend on one, which generates two of the three (osteoblasts and reticular cells), and organizes the third (sinusoidal endothelium). Three players for a dual niche Until very recently, the dominant view of the HSC niche entertained in the relevant literature has been polarized to a dual identity of HSC niches; i.e., an osteoblastic niche, and a sinusoidal or endothelial niche (reviewed in [36]). It was the development of two murine models in the last decade that potently revived interest in the hematopoietic “niche”, while at the same time projecting osteoblasts at center stage of its conceptual view [25,37]. In both models, a measurable excess of functional HSCs was coupled to a measurable excess in bone tissue and in osteoblast numbers. Of note, beyond this

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Fig. 2. Cartoon depicting the position of osteoprogenitors (adventitial reticular cells) at the wall of sinusoids, and their physical contact with both immigrant, and maturing, hematopoietic cells. obs, osteoblasts.

correlation, one of the two models (caPPR mice) features additional phenotypic changes. The latter point adds some complexities to the overall interpretation of how the interplay between bone and hematopoiesis is portrayed. For example, the development of marrow cavities, and therefore of hematopoietic tissue proper, is delayed in caPPR mice due to exuberant bone formation within presumptive marrow space [38]. For this reason, the biological age of the bone marrow in each bone of caPPR mice is younger than in wild-type mice. This circumstance, and the physical constraint placed by occupancy of space in the bone marrow cavity might contribute to explain an increased number of assayable HSCs in these mice, as complementary to an inhibited development of hematopoietic tissue, and to a reduced total hematopoietic cell mass. In addition, upon heterotopic transplantation, the bone marrow stroma from caPPR mice results in establishment of heterotopic bone and osteoblasts with the same efficiency as wild-type stroma, but quite paradoxically, it fails to establish a heterotopic microenvironment able to support hematopoiesis proper, at variance with wild-type stromal cells [38]. This is associated with a reduced content in stromal progenitors (CFU-Fs) in caPPR vs. wild-type mice, and an enhanced expansion of their progeny through culture, suggesting that the “mesenchymal” stem cell compartment, besides the HSC compartment, also undergoes major changes in size and kinetics in caPPR mice, suggesting a potential interplay of the two stem cell compartments. To account for the existence of two putative niches, one then has to envision different functional models, in which different niches are either equivalent, or functionally distinct. In the first scenario, HSCs would reside both at endosteal surfaces, where they would rely on cues provided by osteoblasts, and at sinusoidal surfaces, where they would be exposed to endothelial cues. In this case, there would be no defining cell type of the HSC niche, and it would be difficult to explain why hematopoiesis occurs specifically in bone, given the ubiquitous existence of endothelial cells. In the second scenario, in contrast, one would have to invoke dynamic exchanges between the endosteal and sinusoidal niche, and migration of stem cells in both directions as events underpinning the alternative behaviors of HSCs at either microanatomical site. Finally, one could postulate that each endosteal niche is also, at the same time, a sinusoidal niche, a view that careful consideration of the true anatomy of the microcirculation in bone would thoroughly support. However, the reverse would not be true, as sinusoids may in fact exist at a significant distance from any endosteal surface. Osteoblasts vs. osteogenic lineage cells Plausible, and even possible, as at least some of these models can be, they all rely on a rather loose definition of the term, “osteoblast”, and an even more casual use of the term. An “osteoblast” is defined


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as primarily a non-mitotic cell of a characteristic morphology lying on a bone-forming surface, in the process of producing copious amount of matrix that becomes physiologically mineralized [39]. In the relevant literature, endosteal surfaces and osteoblastic surfaces are used interchangeably, as if every square micron of the endosteum was covered by osteoblasts. In fact, proportions of bone surfaces covered by osteoblasts vary remarkably at different sites, ages, and across species. Whereas w90% of the “endosteal” surface of the bone trabeculae in a mouse primary spongiosa is covered by osteoblasts, only 0.1–7.3% of the trabecular surface of the adult human cancellous bone that host’s hematopoiesis is lined by osteoblasts [40]. In addition, osteoblasts only exist, at any bone surface in adult human bone, for about 3 weeks, the time window of a single bone-remodeling event. This would imply that taking the notion of an “osteoblastic” niche literally, not the HSCs, but the niche itself, would migrate from one site to another constantly. While again conceivable, this view would remain at odds with the very fundamental idea of a “niche” as a steady state, anatomical environment. In fact, not all cells that line the endosteal surface are osteoblasts. The vast majority of them, referred to as “bone-lining cells”, are attenuated cells that belong to the same lineage as osteoblasts, share the same type of anatomical location at bone surfaces as osteoblasts, and yet are not engaged in the defining function of an osteoblast – deposition and mineralization of bone matrix [39]. Bone-lining cells are established at bone surfaces at the end of a bone formation cycle, and can be seen as a further differentiation stage of cells that once was osteoblasts. In this respect, they are equivalent to cells that at the end of a bone formation cycle remain entombed within the bone matrix – osteocytes. Bone-lining cells can be distinguished from osteoblasts based on their morphology (flat and elongated, rather than plump), lack of expression of alkaline phosphatase, and reduced biosynthetic activity. This can be assessed, histologically, by the low abundance of mRNAs encoding major collagenous (COL1A1, COL1A2) and noncollagenous proteins (small leucine-rich proteoglycans, osteonectin, osteopontin, bone sialoprotein, osteocalcin) in bone matrix, all expressed at high levels, albeit with local variation reflecting specific functional stages in mature, active osteoblasts. Nonetheless, osteoblasts remain defined essentially by their morphology and position, which are indeed necessary and sufficient hallmarks of osteoblasts, and provide the key criteria whereby osteoblastic activity is measured by histomorphometry as relevant to the clinical assessment of bone metabolism [40]. All osteoblasts are cells of osteogenic lineage, but the reverse does not apply. Besides osteocytes and bone-lining cells, cells of osteogenic lineage include osteoprogenitor cells, found within the bone marrow stroma. At variance with the hematopoetic lineages, the osteogenic lineage does not feature rigorously discrete differentiation stages, and arrays of markers otherwise used to denote such stages are not always precisely defined. Alkaline phosphatase, for example, the best known marker of “osteoblasts”, is not unique to osteoblasts either in the organism (the very same isoenzyme is expressed in liver and kidney) or within the bone/bone marrow organ (where perivascular cells also express it, as do subsets of cells transitioning from the osteoblastic state to an osteocyte or a bonelining cell). Whereas there is usually no ambiguity in defining an osteoblast in histological sections, markers are necessary to distinguish osteoblasts from other cells in the same lineage ex vivo. Definition of markers apt to distinguish, in such settings, osteoblasts from osteoprogenitors are particularly meaningful when one aims to dissect the relative merit of osteoblasts or their progenitors with respect to the hematopoietic function. Stromal cells are osteoprogenitors, and are vascular cells Of note, all the views of the dual niche, their conceptual interpretations, and even their diagrammatic representations in the literature, invariably do include non-osteoblastic, non-endothelial, stromal cells, but fail to assign them a role or even a defined anatomical position. Cartoons aiming at conveying the concept of a dual niche have repeatedly depicted HSCs as localized to either endosteal surfaces covered by osteoblasts, or to sinusoidal surfaces. However, stromal cells are envisioned and commonly represented as physically residing away from any defined tissue landmark such as the endosteum or sinusoids, and they are even drawn, perhaps inadvertently but probably not by chance, in the way that best conveys in a drawing our subliminal idea of undefinedness – neither black nor white, nor truly colorful – just gray [36]. In fact, stromal cells are as little in focus in current views of the dual niche, as they are in the concepts that are the object of pictorial representation. Difficult to

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visualize due to both their inherent, unique morphology and the limited awareness of markers suited for their visualization, stromal cells in the bone marrow have remained the most elusive anatomical component of the bone/bone marrow organ. This is certainly uncanny considering how popular their use has been in bone biology and experimental hematology for at least 3 decades. The single most important fact that has remained elusive about the identity of “stromal cells” is, specifically, their perivascular location. Indeed, it is the whole structure of the bone marrow, and its unique vascular pattern, that is commonly overlooked whenever “stroma”, “niches” and “microenvironments” are considered – anatomically, the whole bone marrow is but a hugely extended, branched perivascular space adjacent to a hugely extended, branched sinusoidal network. Hints, nonetheless, are abundant in the earlier literature. The very name “adventitial” recalls the association of bone marrow “reticular” cells with sinusoidal walls. Once the nature of adventitial reticular cells as osteoprogenitors (i.e., “mesenchymal stem cells”, or “skeletal stem cells”) is finally recognized, it then follows that the sinus wall is the site (the niche, for some) where mesenchymal/skeletal stem cells reside. Given that HSCs also reside at the sinus wall [41], it also follows that the two kinds of stem cells – hematopoietic and stromal – known to reside and function within the bone marrow, share one and the same “niche”. It also follows, that the conceptual puzzle arising from the “duality” of HSC niches – endosteal vs. sinusoidal, osteoblastic vs. endothelial – can perhaps be solved. The sinus wall does not include endothelial cells only, but also osteogenic cells. Osteogenic cells (not osteoblasts, but progenitors of osteoblasts) are not found at bone surfaces only, but also at sinus walls, next to endothelial cells. Stated in a different way, it is not an osteoblast, but osteoblast progenitor, and not endothelial, but subendothelial cells that actually establish and transfer the hematopoietic microenvironment. Of bone and fat and blood Assuming that hematopoietic tissue is the product of HSCs, bone marrow adipocytes and bone itself is the product of skeletal (mesenchymal, stromal) stem cells. The two systems coexist physically in the bone/bone marrow organ, and adapt to each other. Noted examples of this adaptation are Neumann’s law (the replacement of inactive bone marrow territories with fat as an age-dependent phenomenon), the porotic hyperostosis seen in congenital hemolytic anemias (where bone is displaced by hematopoiesis) or even osteoporosis (where bone is displaced by marrow fat) [42]. Of note, adventitial reticular cells are the direct progenitors of bone marrow adipocytes, both in rodents and lagomorphs and in humans, so that adipocytes themselves are also perivascular (perisinusoidal) cells in the bone marrow [30]. Accumulation of lipid in adventitial reticular cells follows any reduction of hematopoietic cell mass, either simply age-related, or, for example, induced by myeloablation [43]. Not only is fatty marrow hematopoietically inactive, but also adipocytes themselves may be negative regulators of hematopoietic stem cell function [44]. Conversion of adventitial reticular cells into adipocytes replaces a cell with a thin, imperceptible body with a conspicuous, space-occupying fat cell. This event collapses the sinusoid and therefore alters blood flow in the bone marrow, mediating the correlation between blood flow and hematopoietic activity. In this way, adventitial reticular cells (the specialized mural cells of the specialized vessels we refer to as bone marrow sinusoids) mediate a mechanism for remodeling the microvascular network that is likely unique to the bone marrow. In all other tissues, loss of pericytes leads to vessel pruning, an irreversible simplification of the vascular network active in the maturation stage following any angiogenic event, whether developmental or post-natal. In post-natal bone marrow, adipose conversion of reticular cells (sinus pericytes) permits a reversible pruning of the sinusoidal network: sinusoids collapse due to the formation of adipocytes at their wall, but subsequent loss of lipid at the single cell level will restore the phenotype of adventitial reticular cells; the sinusoid will then become patent again, an hematopoiesis at one particular perisinusoidal location will resume. Reversibility of the adipose-reticular phenotype shift permits reversibility of sinusoid patency, blood flow and dependent hematopoietic activity. The microvascular connection As hematopoiesis is ultimately directed by blood-borne cells that immigrate to the bone marrow, and culminates with blood-borne cells that egress the bone marrow, it seems even trivial to consider


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that the bone marrow microvasculature would be at center stage of hematopoiesis, and therefore of any niche or microenvironment related to it. The connection between vascularity and hematopoiesis, if one looks at development and disease, is even more complex and more clear: intravascular erythropoiesis in birds and in human idiopathic myelofibrosis, Wolff’s islands and the hemogenic endothelium of the Aorta-Gonad-Meseonephron [45–47], the sinusoidal hematopoiesis in fetal liver and the colonization of liver sinusoids in leukemia and other hematologic malignancies, all reveal, indirectly, a general, multi-faceted, microvascular connection. Because of their position, adventitial reticular cells are the first cells in the extravascular space of the bone marrow seen by any immigrant cell. They are contractile cells, ideally positioned to regulate traffic across the sinusoidal wall in both directions. Their contractile nature is very well in keeping with their nature as a local adaptation of microvascular mural cells/pericytes, otherwise found in all tissues, and long surmised to represent local tissue progenitors [48–51]. Whereas the lineage, nature, and potential diversity of pericytes across tissues remains to be defined (any reference to “pericytes” as a uniform category, or even a uniform cell type [52,53] is simply unwarranted), each and every feature so far indicated as characteristic of a “pericyte” is indeed found in bone marrow osteoprogenitors. Expression of a host of smooth muscle gene products, of extracellular matrix proteins characteristic of basement membrane, and of growth factor receptors characteristic of mural cells is uniquely blended, in the transcriptome of bone marrow osteoprogenitors, with the expression of markers signifying a commitment to the osteogenic lineage, but not a mature osteoblastic phenotype [23]. Factors that act as mitogens for pericytes, such as FGF-2 or sphingosine-1-phosphate signaling through EDG receptors 1–3 (reviewed in [54,55]), are potent stimulators of proliferation of bone marrow skeletal progenitors, and inhibitors of pericyte proliferation such as TGF-beta1 [56], act likewise in cultures of skeletal progenitor cells. This suggests that the role that these and other factors play in the recruitment of mural cells to the nascent microvascular wall in many settings in development and post-natal growth, may be directly similar to the role that the same factors play in the recruitment of skeletal progenitors to the sinusoidal wall in the bone marrow. Mesenchymal cells recruited to a pericyte fate, enter a state of quiescence – they stop proliferating, do not become apoptotic, and do not differentiate into mature cell types of mesoderm lineages. Establishment of adventitial reticular cells at the sinusoidal wall thus represents a special case of a general paradigm. The best-characterized function of mural cells/pericytes is the stabilization and remodeling of vascular plexuses. Recruitment of pericytes prevents pruning of primitive vessels, stabilizes endothelial cells, and stabilizes pericytes themselves [54,55,57,58]. One of the key effectors of this function is Angiopoietin-1, the ligand of the receptor tyrosine kinase, Tie-2, expressed in endothelial cells. Angiopoietin-1 is a product of mesenchymal cells en route to becoming mural cells, and its ablation in mice results in a lethal vascular phenotype noted for the loss of remodeling of primary vascular lattices [59]. Of note, Tie-2 is also expressed in HSCs, and Ang-1 has been reported to induce HSC quiescence [15]; i.e., to retain HSCs within a functional, if not anatomical, “niche”. Therefore, the observation that skeletal progenitors are the prime producers of Ang-1 in human bone marrow [23] links directly their mural cell nature and function with a role in HSC regulation. More importantly, expression of Ang-1 in skeletal progenitors is itself tightly regulated in relation to cell differentiation. Once induced to differentiate to mature osteoblasts in vitro, or to proliferate, skeletal progenitors down regulate production of Ang-1 [23]. This posits a simple, and yet overlooked, scenario. Any property that functionally defines the HSC “niche”, like for example Ang-1, is of course itself the subject of regulation. As applied to gene products that are expressed in skeletal progenitors, regulation of the HSC niche or microenvironment, and regulation of proliferation and differentiation of skeletal progenitors are linked to one another. The long-recognized interaction of bone and its strange bedfellow, the bone marrow, thus turns into an interaction of two stem cells: both residing at sinusoidal walls, both subject to regulation, and both directing the growth and differentiation and physiology of bone and hematopoietic marrow. A tale of two tissues, and a tale of two stem cells. Conflict of interest None to declare.

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Acknowledgments This work was supported in part by grants from Associazione Italiana Ricerca sul cancro AIRC, Fondazione Roma, European Union (GENOSTEM), and Ministry of University and Research of Italy to PB.

References *[1] Tavassoli M, Crosby WH. Transplantation of marrow to extramedullary sites. Science 1968;161(836):54–6. *[2] Friedenstein AJ. Osteogenic stem cells in bone marrow. In: Heersche JNM, Kanis JA, editors. Bone and mineral research. Elsevier; 1990. [3] Owen M, Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp 1988;136:42–60. [4] Bianco P, Robey PG. Skeletal stem cells. In: Lanza RP, editor. Handbook of adult and fetal stem cells. San Diego: Academic Press; 2004. p. 415–24. [5] Bianco P, Kuznetsov SA, Riminucci M, et al. Postnatal skeletal stem cells. Methods Enzymol 2006;419:117–48. [6] Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–50. [7] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143–7. [8] Friedenstein AJ, Chailakhyan RK, Latsinik NV, et al. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 1974;17(4):331–40. [9] Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 1987;20:263–72. [10] Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 1970;3:393–403. *[11] Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2008;2 (4):313–9. [12] Tavassoli M, Yoffey JM. Bone marrow: structure and function. New York, NY: Alan R Liss; 1983. *[13] Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4 (1–2):7–25. [14] Watt FM, Hogan BLM. Out of Eden: stem cells and their niches. Science 2000;287:1427–30. *[15] Arai F, Hirao A, Ohmura M, et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118(2):149–61. [16] Lord BI, Testa NG, Hendry JH. The relative spatial distributions of CFUs and CFUc in the normal mouse femur. Blood 1975; 46:65–72. [17] Gong JK. Endosteal marrow: a rich source of hematopoieticstem cells. Science 1978;199:1443–5. [18] Lo Celso C, Fleming HE, Wu JW, et al. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 2009;457(7225):92–6. [19] Xie Y, Yin T, Wiegraebe W, et al. Detection of functional haematopoietic stem cell nicheusing real-time imaging. Nature 2009;457:97–101. [20] Dexter TM, Testa NG. Differentiation and proliferation of hemopoietic cells in culture. In: Prescott DM, editor. Methods in cell biology. New York, NY: Academic Press; 1976. p. 387. [21] Dexter TM, Allen TD, Lajtha LG. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 1977;91:335–44. [22] Goujon E. Recherches experimentales sur les proprietes physiologiques de la moelle des os. J de L’Anat et de La Physiol 1869;6:399–412. *[23] Sacchetti B, Funari A, Michienzi S, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007;131:324–36. [24] Calvi LM, et al. Activated parathyroid hormone/parathyroid hormone – related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone. J Clin Invest 2001;107:277–86. *[25] Calvi LM, Adams GB, Weibrecht KW, et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003; 425(6960):841–6. [26] Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991;78(1):55–62. [27] Barry FP. Biology and clinical applications of mesenchymal stem cells. Birth Defects Res C Embryo Today 2003;69(3): 250–6. [28] Deschaseaux F, Remy-Martin JP, Charbord P. Adhesion of CD34 þ marrow precursors to human stroma is related to alphaSM actin expression by human marrow myofibroblasts. J Hematother Stem Cell Res 2001;10(2):291–302. [29] Shih IM. The role of CD146 (Mel-CAM) in biology and pathology. J Pathol 1999;189(1):4–11. [30] Weiss L. The hematopoietic microenvironment of the bone marrow: an ultrastructural study of the stroma in rats. Anat Rec 1976;186(2):161–84. [31] Westen H, Bainton DF. Association of alkaline-phosphatase-positive reticulum cells in bone marrow with granulocytic precursors. J Exp Med 1979;150:919–37. [32] Bianco P, Riminucci M, Bonucci E, et al. Bone sialoprotein (BSP) secretion and osteoblast differentiation: relationship to bromodeoxyuridine incorporation, alkaline phosphatase, and matrix deposition. J Histochem Cytochem 1993;41(2):183– 91. [33] Bianco P, Boyde A. Confocal images of marrow stromal (Westen-Bainton) cells. Histochemistry 1993;100(2):93–9. [34] Bianco P, Bradbeer JN, Riminucci M, et al. Marrow stromal (Western-Bainton) cells: identification, morphometry, confocal imaging and changes in disease. Bone 1993;14(3):315–20.


47

*[35] Sugiyama T, Kohara H, Noda M, et al. Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity 2006;25(6):977–88. [36] Moore KA, Lemischka IR. Stem cells and their niches. Science 2006;311(5769):1880–5. *[37] Zhang J, et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425(6960): 836–41. [38] Kuznetsov SA, Riminucci M, Ziran N, et al. The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow. J Cell Biol 2004;167(6):1113–22. [39] Gehron Robey P, Bianco P. The cellular biology and molecular biochemistry of bone. In: Favus MA, Coe F, editors. Disorders of bone and mineral metabolism. Philadelphia, PA: Lippincott Williams and Wilkins; 2002. p. 199–226. [40] Weinstein RS. Clinical use of bone biopsy. In: Favus MJ, Coes FL, editors. Disorders of bone and mineral metabolism. Philadelphia, PA: Lippincott Williams & Wilkins; 2002. p. 469–85. *[41] Kiel MJ, Yilmaz OH, Iwashita T, et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121(7):1109–21. [42] Bianco P, Riminucci M, Kuznetsov S, et al. Multipotential cells in the bone marrow stroma: regulation in the context of organ physiology. Crit Rev Eukaryot Gene Expr 1999;9(2):159–73. [43] Bianco P, Costantini M, Dearden LC, et al. Alkaline phosphatase positive precursors of adipocytes in the human bone marrow. Br J Haematol 1988;68(4):401–3. [44] Naveiras O, Nardi V, Wenzel PL, et al. Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment. Nature 2009;460:259–63. [45] Bertrand JY, Giroux S, Golub R, et al. Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin. Proc Natl Acad Sci U S A 2005;102(1):134–9. [46] Godin I, Cumano A. The hare and the tortoise: an embryonic haematopoietic race. Nat Rev Immunol 2002;2:593–604. [47] Cumano A, Godin I. Ontogeny of the hematopoietic system. Annu Rev Immunol 2007;25:745–85. [48] Diaz-Flores L, Gutierrez R, Gonzalez P, et al. Inducible perivascular cells contribute to the neochondrogenesis in grafted perichondrium. Anat Rec 1991;229(1):1–8. [49] Diaz-Flores L, Gutierrez R, Lopez-Alonso A, et al. Pericytes as a supplementary source of osteoblasts in periosteal osteogenesis. Clin Orthop Relat Res 1992;275:280–6. [50] Díaz-Flores L, Gutiérrez R, Varela H, et al. Microvascular pericytes: a review of their morphological and functional characteristics. Histol Histopathol 1991;6(2):269–86. [51] Díaz-Flores L, Martín Herrera AI, García Montelongo R, et al. Role of pericytes and endothelial cells in tissue repair and related pathological processes. J Cutan Pathol 1990;17(3):191–2. [52] Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3(3):301–13. [53] Caplan AI. All MSCs are pericytes? Cell Stem Cell 2008;3(3):229–30. [54] Jain RK, Booth MF. What brings pericytes to tumor vessels? J Clin Invest 2003;112(8):1134–6. [55] Jain RK. Molecular regulation of vessel maturation. Nat Med 2003;9(6):685–93. [56] Antonelli-Orlidge A, Saunders KB, Smith SR, et al. An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci U S A 1989;86(12):4544–8. [57] Hirschi KK, D’Amore PA. Control of angiogenesis by the pericyte: molecular mechanisms and significance. EXS 1997;79: 419–28. [58] Hirschi KK, D’Amore PA. Pericytes in the microvasculature. Cardiovasc Res 1996;32(4):687–98. [59] Suri C, Jones PF, Patan S, et al. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 1996;87(7):1171–80.




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Mesenchymal stem cells and autoimmune diseases Francesco Dazzi, MD, Professor of Stem Cell Biology, Consultant Haematologist a, *, Mauro Krampera, MD, PhD, Assistant Professor, Consultant Haematologist b a b

Stem Cell Biology, Haematology Centre, Department of Medicine, Imperial College, London, UK Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Italy

Keywords: mesenchymal stem cells autoimmune diseases inflammation cell therapy

Mesenchymal stem cell (MSC) immunosuppressive properties offer a potentially attractive therapeutic modality for autoimmune diseases. MSC inhibit virtually all types of immune responses in vitro and prevent the induction of disease in several experimental models of autoimmunity. However, the processes involved in the pathogenesis of human diseases are more complicated and treatment cannot be administered before disease induction. In autoimmune diseases persistent antigenic stimulation recruits endogenous MSC to the site of lesion that contribute to the fibrotic evolution. Therefore, administering MSC to a chronic inflammatory disorder may not be desirable. In fact, MSC are not constitutively immunosuppressive but require a ‘licensing’ step provided by molecules of acute phase inflammation, like IFNg and TNF-a, or toll-like receptor (TLR) ligands. Conversely, different cytokines and/or the stimulation of selective TLR make MSC to become immunostimulatory. Therefore, dissecting the inflammatory environment in autoimmune diseases will identify the best conditions amenable to successful MSC therapy. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

Chronic inflammatory autoimmune diseases impart a massive burden on health services worldwide. Historically, therapeutic strategies have centred on non-selective systemic immune suppression. The clinical trials investigating the effects of depleting T cells turned out to be disappointing [1,2], whilst the depletion of CD20 expressing B cells has been more promising [3,4] with durability of clinical responses relating to the efficiency of the depletion phase and the timing of the re-emergence of pathogenic clonotypes. A number of targeted therapies interfering with key pathogenetic events like cytokines or homing receptors have been developed with considerable success in some diseases. * Corresponding author. Haematology Centre, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. E-mail address: [email protected] (F. Dazzi). 1521-6926/$ – see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2011.01.002

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F. Dazzi, M. Krampera / Best Practice & Research Clinical Haematology 24 (2011) 49–57

However, these approaches are expensive and none of them consistently lead to prolonged periods of drug-free remission [5,6]. Thus, curative therapy remains a major unmet need in the management of chronic inflammatory disorders. Cell therapies: options for autoimmune diseases In alternative to conventional immunosuppression, cell therapy-based approaches have more recently been tested with a view of restoring the integrity of immuno-regulatory networks and, at the same time, preserving a pool of memory cells capable of responding to environmental pathogens. Haematopoietic stem cell transplantation (HSCT) is a treatment aimed at resetting the deregulated immune system of patients with severe autoimmune diseases. HSCT induces alterations of the immune system which are beyond the effects of a dose-escalating immunosuppressive approach by creating space for a new immunological repertoire, generated as a result of the reinfused and/or residual haematopoietic stem cells [7]. Although the conditions to reset patient immune repertoire may also restore immune regulation, alternative tactics have been proposed to specifically transfuse cells with immunomodulatory activity. Regulatory T (Treg) cells play a crucial role in the maintenance of peripheral tolerance and modulate susceptibility to autoimmune disease [8,9]. The infusion of Treg cells can ameliorate immune mediated diseases in pre-clinical model but the hurdles of their ex vivo expansion has prevented their application in humans. Natural killer T (NKT) cells use an invariant T-cell receptor (invariant NKT, iNKT) which interact with synthetic glycolipids such as a-galactosylceramide in the context of the monomorphic CD1d antigenpresenting molecule. iNKT cells have the unique capacity to rapidly produce large amounts of both T helper (Th) 1 and Th2 cytokines through which they regulate autoimmune, allergic, antimicrobial, and antitumour immune responses [10]. The activation of iNKT cells with a-galactosylceramide has been tested with some success in animal models. Also monocytes/macrophages can negatively regulate immune functions when exposed to particular environments [11,12]. Similarly, myeloid cells with immunosuppressive properties have been originally identified in the tumour setting – characterised by the expression of CD11b and Gr-1 – which involves arginase and nitric oxide synthase as the main effector pathways [13]. Much attention has been paid to the potential exploitation of the immunosuppressive properties of mesenchymal stromal cells (MSC). As extensively reviewed in this issue, MSC have been described to inhibit a variety of cell types mediating both adaptive and innate immunity. The effects on T lymphocytes are probably the best characterised. MSC non-selectively and non-specifically suppress CD4þ and CD8þ T lymphocytes independently of whether they are naïve or antigen-experienced [14], their functional state [15] or the type of T-cell receptor expressed [16]. Similarly, MSC appear to inhibit the differentiation and antibody production of B cells [17–19] as well as the activation and expansion of natural killer (NK) cells [20]. Although MSC have no effect on neutrophil phagocytosis and chemotaxis, they protect them from apoptosis and inhibit their production of reactive oxygen species [21]. A further inhibitory activity is directed at pro-inflammatory monocytes/macrophages and the differentiation of monocytes into mature dendritic cells [22]. The anti-inflammatory properties of MSC can be partly ascribed to a potent anti-proliferative activity that is associated with the inhibition of cyclin D2. The capacity of T cell to proliferate appears to be rescued by the addition of exogenous interleukin-2 (IL-2) [23], thus indicating that the inhibition is reversible. The evidence that T cells are only temporarily inhibited in their proliferative and functional capacity suggests that MSC do not cause cell death [24,25]. There is evidence demonstrating that MSC protect from apoptosis not only cells of the immune system but a number of cell types of different tissue origin, including malignant cells. Such an activity is well in accord with the notion that MSC exhibit a cytoprotective effect which explains their ability to promote tissue repair. MSC for the treatment of autoimmune diseases The first evidence that MSC could exert an immunosuppressive activity in vivo was obtained in the setting of organ transplantation in non-human primates, whereby the administration of MSC at the


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time of skin allograft proved able to prolong its survival [26]. This and other similar pre-clinical experiences paved the way to their exploitation in the clinical setting in acute graft-versus-host disease (GvHD) after allogeneic HSCT. The spectacular results obtained in an otherwise untreatable and fatal disease [27] have fuelled an enormous interest to better understand the principles of MSC therapies and test their properties in other diseases. Because of the immune mediated pathogenesis and the remarkable social burden, autoimmune diseases are considered a priority target. However, the circumstances for the use of MSC in these conditions remains to be established, not only because the prognosis of these disorders is not so poor to ethically justify a potentially harmful experimental treatment, but especially because there are a number of issues, which the animal models have highlighted, that need to be clarified. One of the first issues is the source of MSC. MSC for the treatment of steroid-refractory acute GvHD has unavoidably to come from a third-party donor because the severity of the condition would not consent sufficient time to produce MSC from the original donor unless this is done before the transplant. Furthermore, the profound immunocompromised status of these patients makes allogeneic MSC unlikely to be rejected [28]. In contrast, the choice of treating a chronic condition is not generally under any similar clinical pressure and the ability of autoimmune disease patients to respond at least to alloantigens is certainly fully preserved. Therefore, the choice of using the patient both as a donor and recipient of MSC is fully justified. However, there is not sufficient information to definitely choose the autologous strategy. First, there is no proof that MSC need to engraft or at least stay in the host for long time to produce the therapeutic efficacy. After injection, MSC home to the site of inflammation where they act via paracrine mechanisms and cannot be detected any longer only a day or two after their infusion [29]. In accord, a few pre-clinical models have employed MSC originating from different species of the recipient and still observed a remarkable therapeutic efficacy [30]. In the perspective of using autologous MSC, an important question that must be addressed is whether MSC from patients with autoimmune diseases maintain their ability to exert the beneficial activity. The behaviour of MSC obtained from patients with a range of autoimmune diseases of rheumatological origin does not seem to differ from those of normal healthy controls [31]. Further studies conducted on MSC from patients with Crohn’s disease [32] and multiple sclerosis [33] confirm this notion. In contrast, MSC from patients with aplastic anaemia have been reported to lack in immunosuppressive abilities [34]. Unfortunately, the assays employed to assess MSC immunosuppression have not been proven to essentially reflect their in vivo therapeutic activity thus making it difficult to draw any conclusion. In pre-clinical models, MSC have been observed to delay the onset of diabetes in non-obese diabetic mice (NOD) if MSC are obtained from healthy but not NOD mice [35]. Despite the several aspects that still require attention, the experience of using MSC in pre-clinical models of autoimmune diseases has been encouraging since the very first demonstration that MSC can prevent the generation of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis [36]. In this model, MSC determine a substantial decrease in the number of inflammatory infiltrates and reduce demyelination and axonal loss. In the first published study the therapeutic efficacy was maximal when MSC were administered at disease onset but no effect was documented after disease stabilisation. However, adipose tissue-derived MSC appear to be able to prevent the onset of the disease as well as to decrease the severity of the neurological defect after disease stabilisation [37]. This effect may relate to the peculiar capability of adipose tissue-derived MSC of migrating to the inflammatory sites of the central nervous system, thus controlling local inflammatory and sustaining glial cell regeneration. The results in the clinical setting, according to a very recent publication, are very promising. Autologous bone marrow-derived MSC, given intrathecally in 10 patients with advanced multiple sclerosis, produced clinical improvement in nearly 50% of the patients although no benefit could be documented at radiologic investigations [38]. However, the experience of a previous pilot study was less enthusiastic [39]. Chemically induced (trinitrobenzene sulfonic acid) autoimmune colitis may be significantly improved by the systemic infusion of adipose tissue-derived MSC, as a consequence of the inhibition of T helper 1-type immune response and the expansion of T regulatory cells [40]. In a model of multiorgan autoimmunity caused by a deficiency in regulatory T cells, MSC produced a remarkable improvement in the histopathology of the distal ileum [41]. Also in this case, the animal studies have been followed by

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encouraging clinical results in which third-party adipose-derived MSC were administered locally for the treatment of complex perianal fistula in patients with Crohn’s disease [42]. More recently, autologous bone marrow-derived MSC were tested in a phase I study for the treatment refractory luminal Crohn’s disease [43]. It is too early to draw any conclusions but inflammatory bowel diseases appear to be a responsive target and consequently a number of groups worldwide are concentrating their efforts in this direction. The initial clinical experience in systemic lupus erythematosus (SLE) might support the exploitation of MSC in autoimmune diseases but also highlight the issue of the necessity of choosing the appropriate type of MSC. An initial study investigated the effect of autologous MSC in two patients with SLE. Despite an increased in regulatory T cells no beneficial effect on disease activity was observed [44]. A few month later two studies were published by another group utilising allogeneic bone marrow MSC [45] or MSC obtained from umbilical cord [46]. In both cases MSC produced an improvement in clinical and laboratory parameters, thus suggesting that, at least in some forms of SLE, MSC function could be impaired by the underlying disease. Interesting information has been learnt from the studies in collagen induced arthritis (CIA). The results of the initial studies were in complete disagreement, whereby whilst in one case MSC effectively protected from the disease [47], in the other study the administration of MSC was in fact worsening the clinical parameters [48]. The differences could be partly reconciled with the fact that MSC were administered at different times after disease induction when changes in the inflammatory environment may influence MSC activities. The same concept could also explain why, in the majority of cases, MSC can prevent but not necessarily affect an established disease. The studies on MSC in GvHD have produced important results to clarify the modalities of MSC therapeutics and interpret some of the discrepancies. One of the first study observed that the infusion of MSC is not sufficient to prevent the development of GvHD [49]. Another study, using MSC of human umbilical cord blood origin, observed that the GvHD produced by human T cells in NOD/SCID mice could not be prevented when MSC were given right at the beginning of the lymphocyte infusion. In contrast, GvHD pathology and symptoms were fully prevented if MSC were given in multiple doses at weekly intervals although no effect was detectable if MSC were administered too late in the course of the disease [50]. These apparently conflicting data were resolved when MSC were tested for their ability to prevent GvHD when given at different times in one single dose. It was observed that only when MSC were infused at day þ2 or þ20, they significantly increased the survival of recipient mice [51]. At these times, the levels of IFNg were particularly high in this model and therefore, as we will extensively discuss in the next section, likely to promote MSC immunosuppressive activity. The clinical studies are fully in accord with these data. Whilst the administration of MSC at the time of HSC transplant did not change the frequency of acute or chronic GvHD [52], their administration at the time of full-blown disease resulted in the control of the disease in a large proportion of cases [27]. Inflammatory environment and plasticity of MSC immunomodulatory activity At this stage, to better understand the results described in the previous section, it becomes fundamental to discuss more extensively whether and how the surrounding environment affects the MSC immunosuppressive activity. The immunosuppressive activity of MSC was initially observed when MSC were added to cultures of activated immune cells. However, MSC are not constitutively inhibitory, but they acquire their immunosuppressive functions after being exposed to the inflammatory environment. This important principle stemmed from the seminal observation that anti–IFN-g receptor antibodies can revert the suppressive effect of MSC. The various techniques to activate an immune response in vitro invariably elicit both T cells and monocytes thus generating the release of high concentrations of IFNg [18] which in turns activate the immunosuppressive activity of MSC (Fig. 1). This ‘licensing’ step is a fundamental requirement to induce MSC mediated immunosuppression [53]. However, the role of IFNg is more complex than just being an activating agent tout-court. Its level and the contemporary presence of other inflammatory cytokines can change the functional profile of MSC. In fact, IFNg can enable MSC to act as antigen-presenting cells [54,55]. Mice immunised with ovalbumin-pulsed IFNg-treated MSC developed antigen-specific cytotoxic CD8þ T cells. Similarly, IFNg-treated human MSC induce significant IL-2 production when co-cultured with HLA-restricted


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Fig. 1. MSC immunosuppressive activity is modulated by cytokines and Toll-like receptor (TLR) ligands. Upper panel: The inhibitory activity of MSC on T cell proliferation requires a ‘licensing’ step that can be provided by IFNg. In fact, the presence of anti-IFNreceptor antibodies in culture impairs the immunosuppressive activity (from Ref. [18]). Lower panel: MSC activity on T cell proliferation can be polarised by stimulating different TLR. In condition of T cell activation (A) MSC acquire the ability to suppress. Similarly, the stimulation of TLR3 stimulates such activity (C), whilst the use of TLR4 agonists prevents the acquisition of MSC mediated inhibitory properties (from Ref. [66]).

influenza-specific T-cell hybridomas. More interestingly, other data indicate that the antigen-presenting property of MSC develop only within a narrow window, at low levels of interferon-gamma. As IFNg levels increase, decrease in MHC class II molecule expression on MSC decreases with the loss of alloreactive inducing activity [54]. More recent studies demonstrated that MSC, like dendritic cells, can even cross-present exogenous antigen and induce an effective CD8þ T-cell immune response [56]. However, the physiological relevance of MSC as antigen-presenting-cells has to be elucidated and whether this is a property that could be exploited for cellular vaccination remains equally unclear. There are other inflammatory cytokines, such as TNF-a or IL-1b, that can license MSC immunosuppression [57] and determine substantial changes in their immunophenotypic profile. IFNg, TNF-a or IL-1b induce the upregulation of HLA-class I, ICAM-1 and VCAM-1 and the de novo expression of both HLA-class II and the inhibitor ligand B7-H1. Although these changes may promote APC-like function, they are also critical for MSC immunosuppressive properties [57]. Some of these discrepancies can be partly explain by the observations that different cytokine combinations can produce different effects. Whilst IFNg alone is sufficient to induce IDO and B7-H1 upregulation, when in combination with TNFa it induces HGF production and the two cytokines act synergistically in the induction of COX2 [58]. In addition, MSC can secrete SOD3, an anti-inflammatory enzyme involved in the catabolism of superoxide anion, which is regulated following exposure to IFNg and TNF-a [59]. Although in a typical inflammatory environment MSC acquire an immunosuppressive function, not all inflammatory milieux produce the same outcome. One of the very first observations in MSC immunobiology was that MSC can indeed acquire immunosuppressive or immunostimulating properties [60]. More recent studies have indicated an important role for toll-like receptors (TLR). TLR are membrane-spanning, non-catalytic receptors that recognise structurally conserved molecules derived from microbes and mediate the activation of immune responses of both innate and adaptive type. MSC express a large number of TLR the stimulation of which has been shown to profoundly affect MSC immunomodulatory properties as well as their migratory phenotype. This aspect is of particular relevance in the therapeutic setting because TLR activation has been implicated in the pathogenesis of

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a number of inflammatory diseases whereby they can initiate or sustain the chronic inflammatory process. Therefore, depending on the pattern of TLR stimulation, MSC can acquire different functional properties (reviewed in Ref. [61]). It is well established that MSC express a number of TLR both at RNA and protein level. High mRNA expression of TLR 1, 2, 3, 4, 5, and 6 has been consistently detected, whilst TLR2, TLR3, TLR4, TLR7, and TLR9 has been reported by flow cytometry. The expression of some TLR is affected also by the oxygen level. The expression of TLR on MSC is certainly functional. It has been shown that TLR3 and TLR4 binding antagonises MSC immunosuppressive activity by interfering with the Jagged1-Notch1 loop [62]. However, if TLR 3 and TLR4 activation is effected on isolated MSC before they are added to the T-cell response cultures, an opposite effect has been observed whereby TLR stimulation boosts MSC immunosuppressive activity [63]. In addition, exposure of MSC to poly(I:C) – a TL3 ligand – or LPS – a TLR4 ligand – induces the production of pro-inflammatory mediators, such as IL-1, IL-6, IL-8, and CCL5 together with the expression of inducible NO synthase (iNOS), and TNF-related apoptosis-inducing ligand (TRAIL). TLR activation of MSC might generate an inflammatory site to attract innate immunity cells, such as neutrophils, thus promoting defences against pathogens [64]. More recently, it has been proposed that the complexity of TLR-mediated stimulation of MSC could be revisited by an analogy with the functional status of monocytes/macrophages. Although in a simplified approach, it has been established that stimulation of monocytes with specific cytokines or TLR agonists polarises them into a classical M1 pro-inflammatory phenotype, while others promote their alternate M2 phenotype associated with anti-inflammatory and tissue repair activity [65]. Utilising low-level, short-term TLR-priming protocol, Waterman and colleagues noted nearly opposite effects on human MSC following stimulation of TLR3 or TLR4. TLR4-primed MSC exhibit a mostly proinflammatory profile with increased levels of molecules like IL-6, IL-8, or TGFb (MSC1), whilst TLR3primed MSC develop the characteristics of immunosuppressive cells producing IL-10, IDO and PGE2 (MSC2) [66]. As a consequence of MSC polarisation, T cell inhibition may be achieved only following MSC2 co-culture, while T lymphocyte activation may occur only in co-culture with MSC1. It should be noted however that longer exposure to TLR ligands and/or the concomitant presence of other cytokine is likely to add layers of complexity to this simplified classification (Fig. 1). The network of stimuli affecting MSC activity is largely unknown but the notion that MSC are functionally plastic has fundamental implications to understand their physiology, its role in pathological processes and the factors that influence their therapeutic exploitation. In fact, some of the molecules that modulate MSC immunological properties also affect the ability to differentiate along the mesenchymal lineage, thus regulating stem cell renewal and tissue repair. Should MSC be used for autoimmune diseases? The rationale for using MSC in autoimmune disease would be to deliver a potent but local immunosuppressive and anti-inflammatory activity. The lesson we have learnt from the concept of MSC polarisation towards an immunosuppressive or immunostimulating phenotype has profound implication on the strategies we should adopt to identify the patients who are more likely to respond to MSC therapies. The inflammatory environment to which MSC are exposed is in fact capable of shaping their properties in completely opposite directions. It is important to remember that the same molecules (cytokines and TLR ligands) that modulate MSC activity are prominently involved in the pathogenesis of autoimmune diseases. We believe that a careful characterisation of the patients’ microenvironment is fundamental to identify the ‘signature’ of those more likely to benefit of this cell therapy. Another factor to be considered is that in chronic inflammatory autoimmunity, the disease is often associated with fibrotic changes that result from the recruitment of mesenchymal stromal cells to the site of the lesion. Interestingly, these cells often share the same properties of bone marrow MSC because they exhibit multipotential differentiation ability and once expanded they can also immunosuppress [67]. However, the fact that despite their accumulation the disease persists, suggests that the microenvironment impairs their functions. Studies in mice have shown that mesenchymal cell targeting by TNF is a key pathogenic mechanism in chronic inflammatory joint and intestinal diseases. Therefore, the adoptive transfer of MSC to patients with disorders characterised by ‘mesenchymal cell pathologies’ could eventually add fuel to the pathological process and exacerbate the disease.


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Fig. 2. The inflammatory microenvironment in autoimmunity conditions MSC plasticity. The inflammatory environment associated with autoimmune diseases is supposed to modify the way by which MSC intervenes on the inflammatory process. If MSC are infused during acute inflammation, the microenvironment (M1-type monocytes) actively ‘licenses’ MSC to inhibit the effector T cells responsible for tissue damage. In case of antigen persistence the players involved in inflammation change and produce fibrosis. We propose that, if MSC are infused to patients during this phase the microenvironment does not provide the ‘licensing’ and/or favours the recruitment of MSC to the fibrotic process.

Fortunately, the limited clinical experience reported so far seem to exclude overt aggravation of the underlying condition. Several investigations are currently in progress to identify molecules with the ability to revert the process of fibrosis which could eventually restore the original tissue with or without the intervention of exogenous MSC. In conclusion, as summarised in Fig. 2, the inflammatory environment to which MSC are exposed is a fundamental factor influencing MSC functions. However, it should also be noted that under some circumstances the lack of therapeutic effects could be ascribed to a weak activity of MSC on the underlying disease and/or to the fact that the disease, when established, is completely insensitive to the immunosuppressive activity. Conflict of interest statement None to declare.

References [1] Moreland LW, Bucy RP, Tilden A, et al. Use of a chimeric monoclonal anti-CD4 antibody in patients with refractory rheumatoid arthritis. Arthritis Rheum 1993 Mar;36(3):307–18. [2] van der Lubbe PA, Dijkmans BA, Markusse HM, et al. A randomized, double-blind, placebo-controlled study of CD4 monoclonal antibody therapy in early rheumatoid arthritis. Arthritis Rheum 1995 Aug;38(8):1097–106. [3] Leandro MJ, Edwards JC, Cambridge G, et al. An open study of B lymphocyte depletion in systemic lupus erythematosus. Arthritis Rheum 2002 Oct;46(10):2673–7. [4] Edwards JC, Szczepanski L, Szechinski J, et al. Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med 2004 Jun 17;350(25):2572–81.

56


[5] Hyrich KL, Symmons DP, Watson KD, et al. Comparison of the response to infliximab or etanercept monotherapy with the response to cotherapy with methotrexate or another disease-modifying antirheumatic drug in patients with rheumatoid arthritis: results from the British Society for Rheumatology Biologics Register. Arthritis Rheum 2006 Jun;54(6):1786–94. [6] Listing J, Strangfeld A, Rau R, et al. Clinical and functional remission: even though biologics are superior to conventional DMARDs overall success rates remain low-results from RABBIT, the German biologics register. Arthritis Res Ther 2006;8 (3):R66. *[7] Sykes M, Nikolic B. Treatment of severe autoimmune disease by stem-cell transplantation. Nature 2005 Jun 2;435(7042): 620–7. [8] Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 1995 Aug 1;155(3):1151–64. [9] Ehrenstein MR, Evans JG, Singh A, et al. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNF alpha therapy. J Exp Med 2004 Aug 2;200(3):277–85. [10] Godfrey DI, Kronenberg M. Going both ways: immune regulation via CD1d-dependent NKT cells. J Clin Invest 2004 Nov; 114(10):1379–88. [11] Rutella S, Danese S, Leone G. Tolerogenic dendritic cells: cytokine modulation comes of age. Blood 2006 Sep 1;108(5): 1435–40. [12] Mellor AL, Munn DH. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat Rev Immunol 2004 Oct;4(10):762–74. [13] Zea AH, Rodriguez PC, Atkins MB, et al. Arginase-producing myeloid suppressor cells in renal cell carcinoma patients: a mechanism of tumor evasion. Cancer Res 2005 Apr 15;65(8):3044–8. [14] Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide. Blood 2003 May 1;101(9):3722–9. [15] Ghannam S, Pene J, Torcy-Moquet G, et al. Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype. J Immunol 2010 Jul 1;185(1):302–12. [16] Prigione I, Benvenuto F, Bocca P, et al. Reciprocal interactions between human mesenchymal stem cells and gammadelta T cells or invariant natural killer T cells. Stem Cells 2009 Mar;27(3):693–702. [17] Glennie S, Soeiro I, Dyson PJ, et al. Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood 2005 Apr 1;105(7):2821–7. *[18] Krampera M, Cosmi L, Angeli R, et al. Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells 2006 Feb;24(2):386–98. [19] Corcione A, Benvenuto F, Ferretti E, et al. Human mesenchymal stem cells modulate B cell functions. Blood; 2005 Sep 1. [20] Spaggiari GM, Capobianco A, Becchetti S, et al. Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 2006 Feb 15;107(4):1484–90. [21] Morandi F, Raffaghello L, Bianchi G, et al. Immunogenicity of human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigens. Stem Cells 2008 May;26(5):1275–87. [22] Ramasamy R, Fazekasova H, Lam EW, et al. Mesenchymal stem cells inhibit dendritic cell differentiation and function by preventing entry into the cell cycle. Transplantation 2007 Jan 15;83(1):71–6. [23] Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002 May 15;99(10):3838–43. *[24] Jones S, Horwood N, Cope A, et al. The antiproliferative effect of mesenchymal stem cells is a fundamental property shared by all stromal cells. J Immunol 2007 Sep 1;179(5):2824–31. [25] Benvenuto F, Ferrari S, Gerdoni E, et al. Human mesenchymal stem cells promote survival of T cells in a quiescent state. Stem Cells 2007 Jul;25(7):1753–60. [26] Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002 Jan;30(1):42–8. *[27] Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versushost disease: a phase II study. Lancet 2008 May 10;371(9624):1579–86. [28] Nauta AJ, Westerhuis G, Kruisselbrink AB, et al. Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 2006 Sep 15;108(6):2114–20. [29] Togel F, Hu Z, Weiss K, et al. Amelioration of acute renal failure by stem cell therapy–paracrine secretion versus transdifferentiation into resident cells: administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation–independent mechanisms. J Am Soc Nephrol 2005 May;16(5):1153–63. Am J Physiol Renal Physiol [E-pub February 15, 2005]. [30] Gonzalez-Rey E, Anderson P, Gonzalez MA, et al. Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis. Gut 2009 Jul;58(7):929–39. [31] Bocelli-Tyndall C, Bracci L, Spagnoli G, et al. Bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donors and auto-immune disease patients reduce the proliferation of autologous- and allogeneic-stimulated lymphocytes in vitro. Rheumatology (Oxford); 2006 Aug 18. [32] Bernardo ME, Avanzini MA, Ciccocioppo R, et al. Phenotypical/functional characterization of in vitro-expanded mesenchymal stromal cells from patients with Crohn’s disease. Cytotherapy 2009;11(7):825–36. [33] Mallam E, Kemp K, Wilkins A, et al. Characterization of in vitro expanded bone marrow-derived mesenchymal stem cells from patients with multiple sclerosis. Mult Scler 2010 Aug;16(8):909–18. [34] Bacigalupo A, Valle M, Podesta M, et al. T-cell suppression mediated by mesenchymal stem cells is deficient in patients with severe aplastic anemia. Exp Hematol 2005 Jul;33(7):819–27. [35] Fiorina P, Jurewicz M, Augello A, et al. Immunomodulatory function of bone marrow-derived mesenchymal stem cells in experimental autoimmune type 1 diabetes. J Immunol 2009 Jul 15;183(2):993–1004. *[36] Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 2005 Sep 1;106(5):1755–61.


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[37] Constantin G, Marconi S, Rossi B, et al. Adipose-derived mesenchymal stem cells ameliorate chronic experimental autoimmune encephalomyelitis. Stem Cells 2009 Oct;27(10):2624–35. [38] Yamout B, Hourani R, Salti H, et al. Bone marrow mesenchymal stem cell transplantation in patients with multiple sclerosis: a pilot study. J Neuroimmunol 2010 Oct 8;227(1–2):185–9. [39] Mohyeddin Bonab M, Yazdanbakhsh S, Lotfi J, et al. Does mesenchymal stem cell therapy help multiple sclerosis patients? Report of a pilot study. Iran J Immunol 2007 Mar;4(1):50–7. [40] Gonzalez MA, Gonzalez-Rey E, Rico L, et al. Treatment of experimental arthritis by inducing immune tolerance with human adipose-derived mesenchymal stem cells. Arthritis Rheum 2009 Apr;60(4):1006–19. *[41] Parekkadan B, Tilles AW, Yarmush ML. Bone marrow-derived mesenchymal stem cells ameliorate autoimmune enteropathy independently of regulatory T cells. Stem Cells 2008 Jul;26(7):1913–9. [42] Garcia-Olmo D, Herreros D, Pascual I, et al. Expanded adipose-derived stem cells for the treatment of complex perianal fistula: a phase II clinical trial. Dis Colon Rectum 2009 Jan;52(1):79–86. *[43] Duijvestein M, Vos AC, Roelofs H, et al. Autologous bone marrow-derived mesenchymal stromal cell treatment for refractory luminal Crohn’s disease: results of a phase I study. Gut; 2010 Oct 4. *[44] Carrion F, Nova E, Ruiz C, et al. Autologous mesenchymal stem cell treatment increased T regulatory cells with no effect on disease activity in two systemic lupus erythematosus patients. Lupus 2010 Mar;19(3):317–22. [45] Liang J, Zhang H, Hua B, et al. Allogenic mesenchymal stem cells transplantation in refractory systemic lupus erythematosus: a pilot clinical study. Ann Rheum Dis 2010 Aug;69(8):1423–9. [46] Sun L, Wang D, Liang J, et al. Umbilical cord mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus. Arthritis Rheum 2010 Aug;62(8):2467–75. *[47] Augello A, Tasso R, Negrini SM, et al. Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis. Arthritis Rheum 2007 Apr;56(4):1175–86. [48] Djouad F, Fritz V, Apparailly F, et al. Reversal of the immunosuppressive properties of mesenchymal stem cells by tumor necrosis factor alpha in collagen-induced arthritis. Arthritis Rheum 2005 May;52(5):1595–603. [49] Sudres M, Norol F, Trenado A, et al. Bone marrow mesenchymal stem cells suppress lymphocyte proliferation in vitro but fail to prevent graft-versus-host disease in mice. J Immunol 2006 Jun 15;176(12):7761–7. [50] Tisato V, Naresh K, Girdlestone J, et al. Mesenchymal stem cells of cord blood origin are effective at preventing but not treating graft-versus-host disease. Leukemia 2007 Sep;21(9):1992–9. *[51] Polchert D, Sobinsky J, Douglas G, et al. IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease. Eur J Immunol 2008 Jun;38(6):1745–55. [52] Lazarus HM, Koc ON, Devine SM, et al. Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol Blood Marrow Transplant 2005 May;11(5): 389–98. [53] Dazzi F, Marelli-Berg FM. Mesenchymal stem cells for graft-versus-host disease: close encounters with T cells. Eur J Immunol 2008 Jun;38(6):1479–82. [54] Chan JL, Tang KC, Patel AP, et al. Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-{gamma}. Blood 2006 Jun 15;107(12):4817–24. [55] Stagg J, Pommey S, Eliopoulos N, et al. Interferon-gamma-stimulated marrow stromal cells: a new type of nonhematopoietic antigen-presenting cell. Blood 2006 Mar 15;107(6):2570–7. [56] Francois M, Romieu-Mourez R, Stock-Martineau S, et al. Mesenchymal stromal cells cross-present soluble exogenous antigens as part of their antigen-presenting cell properties. Blood 2009 Sep 24;114(13):2632–8. [57] Ren G, Zhang L, Zhao X, et al. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2008 Feb 7;2(2):141–50. [58] English K, Barry FP, Field-Corbett CP, et al. IFN-gamma and TNF-alpha differentially regulate immunomodulation by murine mesenchymal stem cells. Immunol Lett 2007 Jun 15;110(2):91–100. [59] Kemp K, Gray E, Mallam E, et al. Inflammatory cytokine induced regulation of superoxide dismutase 3 expression by human mesenchymal stem cells. Stem Cell Rev 2010 Dec;6(4):548–59. [60] Le Blanc K, Tammik L, Sundberg B, et al. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003 Jan;57(1):11–20. [61] DelaRosa O, Lombardo E. Modulation of adult mesenchymal stem cells activity by toll-like receptors: implications on therapeutic potential. Mediators Inflamm 2010;2010:865601. [62] Liotta F, Angeli R, Cosmi L, et al. Toll-like receptors 3 and 4 are expressed by human bone marrow-derived mesenchymal stem cells and can inhibit their T-cell modulatory activity by impairing Notch signaling. Stem Cells 2008 Jan;26 (1):279–89. [63] Opitz CA, Litzenburger UM, Lutz C, et al. Toll-like receptor engagement enhances the immunosuppressive properties of human bone marrow-derived mesenchymal stem cells by inducing indoleamine-2,3-dioxygenase-1 via interferon-beta and protein kinase R. Stem Cells 2009 Apr;27(4):909–19. [64] Romieu-Mourez R, Francois M, Boivin MN, et al. Cytokine modulation of TLR expression and activation in mesenchymal stromal cells leads to a proinflammatory phenotype. J Immunol 2009 Jun 15;182(12):7963–73. [65] Mantovani A, Sica A, Locati M. Macrophage polarization comes of age. Immunity 2005 Oct;23(4):344–6. [66] Waterman RS, Tomchuck SL, Henkle SL, et al. A new mesenchymal stem cell (MSC) paradigm: polarization into a proinflammatory MSC1 or an Immunosuppressive MSC2 phenotype. PLoS One 2010;5(4):e10088. [67] Haniffa MA, Collin MP, Buckley CD, et al. Mesenchymal stem cells: the fibroblasts’ new clothes? Haematologica 2009 Feb; 94(2):258–63.




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Neuroprotective features of mesenchymal stem cells Antonio Uccelli, MD a, b, c, *, Federica Benvenuto, PhD a, b, Alice Laroni, MD a, Debora Giunti, PhD a a

Department of Neurosciences, Ophthalmology and Genetics, University of Genoa, Via De Toni 5, 16132 Genoa, Italy Center of Excellence for Biomedical Research, University of Genoa, Italy c Advanced Biotechnology Center (ABC), Genoa, Italy b

Keywords: mesenchymal stem cells (MSC) experimental autoimmune encephalomyelitis (EAE) central nervous system (CNS): neurons microglia oligodendrocytes neuroprotection

Bone marrow (BM) derived mesenchymal stem cells (MSC) differentiate into cells of the mesodermal lineage but also, under certain experimental circumstances, into cells of the neuronal and glial lineage. Their therapeutic translation has been significantly boosted by the demonstration that MSC display significant also anti-proliferative, anti-inflammatory and anti-apoptotic features. These properties have been exploited in the effective treatment of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis where the inhibition of the autoimmune response resulted in a significant neuroprotection. A significant rescue of neural cells has been achieved also when MSC were administered in experimental brain ischemia and in animals undergoing brain or spinal cord injury. In these experimental conditions BM-MSC therapeutic effects are likely to depend on paracrine mechanisms mediated by the release of growth factors, anti-apoptotic molecules and anti-inflammatory cytokines creating a favorable environment for the regeneration of neurons, remyelination and improvement of cerebral flow. For potential clinical application BM-MSC offer significant practical advantages over other types of stem cells since they can be obtained from the adult BM and can be easily cultured and expanded in vitro under GMP conditions displaying a very low risk of malignant transformation. This review discusses the targets and mechanisms of BM-MSC mediated neuroprotection. Ó 2011 Elsevier Ltd. All rights reserved.

* Corresponding author. Department of Neurosciences Ophthalmology and Genetics, University of Genoa, Via De Toni 5, 16132 Genoa, Italy. Tel.: þ39 0 103537028; Fax: þ39 0 103538639. E-mail addresses: [email protected], [email protected] (A. Uccelli). 1521-6926/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2011.01.004

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Introduction Mesenchymal stem cells (MSC) were firstly identified by Friedenstein as stromal cells from the bone marrow (BM) and described as spindle-shaped cells in culture [1]. In this organ MSC represent a very rare population, less than 0,1% of nucleated cells representing progenitor cells of the stromal lineage at different stages of differentiation. They take part to the bone marrow microenviroment within the haematopoietic niche, supporting the mainteinance of hematopoietic stem cell (HSC) pool and the differentiation programs of blood born cells [2,3]. Beside their trophic role in the haematopoeitic niche, MSC are likely to play a similar role in other tissues where they display also an anti-proliferative activity resulting in tissue homeostasis [4]. In addition to adult bone marrow, MSC have been identified in several tissues and organs of either fetal or adult origin [5–7]. BM-derived MSC differentiate into fat, bone, and cartilage but can also transdifferentiate into embriological unrelated tissues [8]. They are easily cultured in vitro under appropriate conditions, grow as adherent cells until confluence resulting in yields sufficient for clinical exploitation for cell therapy or gene therapy strategies [9]. MSC for the tratment of experimental autoimmunity of the central nervous sytem Several studies showed that MSC possess immunomodulating properties exerted in vitro on cells populations of both adpative and innate immunity [10]. These features were together with the reported ability of MSC to transdifferentiate into neural cells [11] and migrate, although to a limited extent, to the central nervous sytem (CNS) [12] induced researchers to exploit them for the treatment of experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis (MS). Intravenous (i.v.) infusion of MSC improved the clinical course of EAE [13,14]. In this model injected MSC on one side induced peripheral T cell tolerance to myelin proteins thus reducing migration of pathogenic T cells to the CNS and, on the other side, homed themselves to the CNS where they preserved axons and reduced demyelination. In other studies it was observed that following administration in mice with EAE, MSC exert an effect on oligodendrocytes enhancing remyelination possibly through the release of neurtrophins such as Brain Derived Neurotrophic Factor [15–17]. Immunomodulating properties of MSC are not the only mechanisms that could explain their therapeutic plasticity. The heterogenity of MSC, expressing a large number of regulatory proteins, may explain their wide therapeutic features and their capacity to respond differently to injuries depending on the microenvironment and despite their low engraftment in vivo [18]. MSC produce cytokines and a variety of soluble factors regulating several biological activities as demonstrated by their transcriptome analysis [19], which suggests their ability to support survival of non-proliferating hematopoietic stem cells (HSC) within the niche, thus playing a major role in the maintenance of local homeostasis [3]. Neuroprotective properties of MSC MSC initially attracted interest for their presumed ability to home into injured tissues and differentiate into multiple cellular phenotypes in vivo. This prediction was challenged by recent observations indicating that only small numbers of the infused cells engraft into tissues, even if damaged and they quickly disappear quickly [20] and that just MSC supernatant suffices to block fulminant hepatic failure [21]. These observations together with in vitro studies showing that significant biological effects on target cells could be achieved in several experimental conditions without the need of cell contact have led to the hypothesis that MSCs therapeutic plasticity relies greatly on the paracrine release of molecules. Several groups have demonstrated that MSC can rescue neurons from apoptosis in vitro [22– 24], a mechanism that together with their ability to inhibit immune-mediated damage, can explain the reduced axonal loss observed in mice with EAE treated with MSC [14,25,26]. This anti-apoptotic effect, together with the reported capacity of releasing neurotrophic molecules may well explain the remarkable effect obtained with the administration of MSC, either i.v. or locally, in experimental models of stroke and spinal cord injury. For example, direct transplantation of bone


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marrow pluripotent cells has been reported to promote remyelination[27]. In another study the injection of MSC into the lumbar spine of rats led to the infiltration of the spinal cord parenchyma following complete transection and motor improvement [28]. An improvement of motorneurons survival was observed following transplantation into the lumbar spinal cord in transgenic SOD1 (G93A) mice, a model for human amyotrophic lateral sclerosis [29]. In this study, authors observed a delay of death and improved motor performances upon MSC transplantation. In a model of stroke the injection of MSC significantly improved functional outcome in rats reducing apoptosis and promoting the induction of local neurogenesis [30]. MSC secrete a number of growth factors and cytokines, which normally support hematopoietic progenitors to proliferate and differentiate [31]. Besides, MSC may be directly involved in promoting plasticity of the ischemic damaged neurons or in stimulating glial cells to secrete neurotrophins such as BDNF and NGF, in the reduction of apoptosis in the penumbral zone of the lesion and the proliferation of the endogenous cells in the subventricular zone [32]. An indirect effect involving the recruitment of local progenitors was suggested also by the implantation of human MSC in the mouse hippocampus, which stimulated proliferation, migration and differentiation of the endogenous neural stem cells that survived as more differentiated neural cells [33]. The effects produced by the MSC are presumably explained by their secretion of different trophic factors including NGF, VEGF, CNTF and FGF-2 and BMI-1 that probably account for the increase in neurogenesis acting directly on local precursors but also activating nearby astocytes, which independently may increase neurogenesis [33].

Fig. 1. The main neuroprotective effects are depicted. Dashed lines indicate the current lack of strong evidence for that phenomenon.

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In a mouse model of global ischemia the administration of human MSC resulted in the improvement of neurological function and prevented neuronal cell death in the hippocampus. Authors claimed an indirect effect mediated by local microglia, which, following MSC administration, displayed an enhanced expression of the neuroprotective factor Ym1 and other trophic molecules including IGF and Gal-3 [34]. The observation that MSC neuroprotective effect may occur through the interaction with local neural cells is consistent with experiments addressing the interaction between MSC and microglia. The latter, upon activation, produces a variety of inflammatory molecules, including nitric oxide (NO), tumor necrosis factor (TNF), interleukin 1-b (IL1- b), and reactive oxygen species (ROS) that have been closely associated with the pathogenesis of neural damage resulting from ischemia, inflammation and primary neurodegeneration. Zhou et al demonstrated that human MSC are able to inhibit LPS-stimulated microglia activation and the production of inflammatory factors through diffusible molecules and that human MSC sense inflammatory molecules, released by the activated microglia, thus increasing significantly the production of neurotrophic factors, which are likely to be involved in neuroprotection [35]. Consistent with these results, Kim and colleagues demonstrated that cell death of dopaminergic neurons induced by activated microglia is inhibited by the addition of human MSC [36,37]. Regardless of the role of inhibition of microglia activation in the neuroprotective effects observed following MSC transplantation in several experimental models, it has been clearly shown that i.v. administration of MSC suffices to significantly revert the upregulation of molecules involved in oxyradical detoxification occurring during the acute phase of EAE [38]. Interestingly, a similar turnaround of the increase of oxidative stress associated proteins was observed following coculture of neurons exposed to H2O2 in the presence of MSC [38] suggesting that proinflammatory and oxidative stress molecules released by macrophages and microglia and known to damage neurons during neuroinflammation can be inhibited by MSC. Microglia is not the only neural cell type targeted by MSC. In fact it has been shown in vitro that MSC can instruct neural precursor cells (NPC) to enter an oligodendrogenic program while inhibiting their ability to differentiate into astrocytes [36,39]. These results support the in vivo observation that in EAE MSC administration leads to some degree of remyelination [15,16,39]. Conclusions In conclusion, experimental evidence in preclinical model of neurological diseases suggests that MSC are a promising approach to achieve neural repair and protection. However, current data do not support the possibility that most of the reported effects occur through cell replacement. Many other paracrine mechanisms, including a potent anti-inflammatory capacity, the direct release of antiapoptotic and neurotrophic factors, the ability to induce other cells, such as microglia, to acquire a protective phenotype and to induce proliferation of local neural progenitor cells possibly leading to remyelination, are likely to sustain the protective effects observed in preclinical models (Fig. 1). Because the clinical translation into humans has been based so far mainly on their given immunomodulatory functions [40] or on their ability to support hematopoiesis [41], properly designed and carefully controlled clinical studies will have to confirm the therapeutic relevance of these neuroprotective features. Conflict of interest statement Authors do not have any conflict of interest concerning the topic of this review. Some of the results discussed here were obtained from research supported by grants from the Fondazione Italiana Sclerosi Multipla (FISM) (AU), the Italian Ministry of Health (Ricerca Finalizzata) (AU), the Italian Ministry of the University and Scientific Research (MIUR) (AU), the ‘Progetto LIMONTE’ (AU) and the Fondazione CARIGE (AU). Funding The funding sources were not involved in any decision about how to perform research described in the article or in the writing of the article.


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References [1] Friedenstein AJ, Chailakhyan RK, Latsinik NV, et al. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 1974;17(4):331–40. [2] Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol 2006;6(2):93–106. *[3] Mendez-Ferrer S, Michurina TV, Ferraro F, et al. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 2010;466(7308):829–34. [4] Jones S, Horwood N, Cope A, Dazzi F. The antiproliferative effect of mesenchymal stem cells is a fundamental property shared by all stromal cells. J Immunol 2007;179(5):2824–31. [5] Campagnoli C, Roberts IA, Kumar S, et al. Identification of mesenchymal stem/progenitor cells in human first- trimester fetal blood, liver, and bone marrow. Blood 2001;98(8):2396–402. [6] Sessarego N, Parodi A, Podesta M, et al. Multipotent mesenchymal stromal cells from amniotic fluid: solid perspectives for clinical application. Haematologica 2008;93(3):339–46. [7] Meirelles LdS, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 2006;119(11):2204–13. [8] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284(5411):143–7. [9] Sotiropoulou PA, Perez SA, Gritzapis AD, et al. Interactions Between Human Mesenchymal Stem Cells and Natural Killer Cells. Stem Cells 2006;24(1):74–85. *[10] Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008;8(9):726–36. [11] Kopen GC, Prockop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 1999;96(19):10711–6. [12] Devine SM, Cobbs C, Jennings M, et al. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into non-human primates. Blood 2003;101(8):2999–3001. *[13] Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T cell anergy. Blood 2005;106(5):1755–61. [14] Gerdoni E, Gallo B, Casazza S, et al. Mesenchymal stem cells effectively modulate pathogenic immune response in experimental autoimmune encephalomyelitis. Ann Neurol 2007;61(3):219–27. [15] Zhang J, Li Y, Chen J, et al. Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice. Exp Neurol 2005;195(1):16–26. [16] Constantin G, Marconi S, Rossi B, et al. Adipose-Derived Mesenchymal Stem Cells Ameliorate Chronic Experimental Autoimmune Encephalomyelitis. Stem Cells 2009;27:11. [17] Bai L, Lennon DP, Eaton V, et al. Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune response and promote endogenous repair in animal models of multiple sclerosis. Glia 2009;57(11):1192–203. [18] Phinney DG, Prockop DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair–current views. Stem Cells 2007;25(11):2896–902. [19] Phinney DG, Hill K, Michelson C, et al. Biological activities encoded by the murine mesenchymal stem cell transcriptome provide a basis for their developmental potential and broad therapeutic efficacy. Stem Cells 2006;24(1):186–98. *[20] Lee RH, Pulin AA, Seo MJ, et al. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 2009;5(1):54–63. *[21] Parekkadan B, van Poll D, Suganuma K, et al. Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure. PLoS ONE 2007;2(9):e941. [22] Scuteri A, Cassetti A, Tredici G. Adult mesenchymal stem cells rescue dorsal root ganglia neurons from dying. Brain Res 2006;1116(1):75–81. [23] Crigler L, Robey RC, Asawachaicharn A, et al. Human mesenchymal stem cell subpopulations express a variety of neuroregulatory molecules and promote neuronal cell survival and neuritogenesis. Exp Neurol 2006;198(1):54–64. [24] Wilkins A, Kemp K, Ginty M, et al. Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro. Stem cell res 2009;3(1):63–70. [25] Zhang J, Li Y, Lu M, et al. Bone marrow stromal cells reduce axonal loss in experimental autoimmune encephalomyelitis mice. J neurosci res 2006;84(3):587–95. [26] Kassis I, Grigoriadis N, Gowda-Kurkalli B, et al. Neuroprotection and immunomodulation with mesenchymal stem cells in chronic experimental autoimmune encephalomyelitis. Arch Neurol 2008;65(6):753–61. [27] Akiyama Y, Radtke C, Honmou O, Kocsis JD. Remyelination of the spinal cord following intravenous delivery of bone marrow cells. Glia 2002;39(3):229–36. [28] Satake K, Lou J, Lenke LG. Migration of mesenchymal stem cells through cerebrospinal fluid into injured spinal cord tissue. Spine 2004;29(18):1971–9. [29] Vercelli A, Mereuta OM, Garbossa D, et al. Human mesenchymal stem cell transplantation extends survival, improves motor performance and decreases neuroinflammation in mouse model of amyotrophic lateral sclerosis. Neurobiol disease 2008;31(3):395–405. [30] Chen J, Li Y, Katakowski M, et al. Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat. J neurosci res 2003;73(6):778–86. [31] Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J cellular biochem 2006;98(5):1076–84. [32] Li Y, Chen J, Chen XG, et al. Human marrow stromal cell therapy for stroke in rat: Neurotrophins and functional recovery. Neurology 2002;59:514–23. [33] Munoz JR, Stoutenger BR, Robinson AP, et al. Human stem/progenitor cells from bone marrow promote neurogenesis of endogenous neural stem cells in the hippocampus of mice. Proc Natl Acad Sci USA 2005;102(50):18171–6. *[34] Ohtaki H, Ylostalo JH, Foraker JE, et al. Stem/progenitor cells from bone marrow decrease neuronal death in global ischemia by modulation of inflammatory/immune responses. Proc Natl Acad Sci USA 2008;105(38):14638–43. [35] Zhou C, Zhang C, Chi S, et al. Effects of human marrow stromal cells on activation of microglial cells and production of inflammatory factors induced by lipopolysaccharide. Brain res 2009;1269:23–30.

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[36] Rivera FJ, Couillard-Despres S, Pedre X, et al. Mesenchymal stem cells instruct oligodendrogenic fate decision on adult neural stem cells. Stem Cells 2006;24(10):2209–19. [37] Kim YJ, Park HJ, Lee G, et al. Neuroprotective effects of human mesenchymal stem cells on dopaminergic neurons through anti-inflammatory action. Glia 2009;57(1):13–23. [38] Lanza C, Morando S, Voci A, et al. Neuroprotective mesenchymal stem cells are endowed with a potent antioxidant effect in vivo. J neurochem 2009;110(5):1674–84. [39] Bai L, Caplan A, Lennon D, Miller RH. Human mesenchymal stem cells signals regulate neural stem cell fate. Neurochem res 2007;32(2):353–62. *[40] Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versushost disease: a phase II study. The Lancet 2008;371(9624):1579–86. [41] Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000;18(2):307–16.




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Mesenchymal stem cells for treatment of acute and chronic graft-versus-host disease, tissue toxicity and hemorrhages O. Ringden, MD, PhD, Professor in Transplantation Immunology *, K. Le Blanc, MD, PhD, Professor in Clinical Stem Cell Research Center for Allogeneic Stem Cell Transplantation, Division of Clinical Immunology, and Hematology Center, Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, F79, SE-141 86 Stockholm, Sweden

Keywords: Mesenchymal stem cells Hematopoietic stem cell transplantation Graft-versus-host disease Hemorrhagic cystitis Tissue toxicity

Mesenchymal stem cells (MSCs) have immunomodulatory effects and low immunogenicity. MSCs inhibit T-cell alloreactivity in vitro. Immune inhibition is caused by soluble factors. MSCs affect almost all cells of the immune system. They are safe to infuse in humans with no acute toxicity and no ectopic tissue formation. We treated patients with life-threatening acute graft-versus-host disease (GVHD) not responding to conventional immunosuppressive therapy with MSCs. Approximately half of the patients responded. HLA-identical or third party MSCs were equally effective. Children tended to have a better response compared to adults. MSCs have also been used for chronic GVHD with positive effects. MSCs also reversed tissue toxicity such as hemorrhagic cystitis, pneumomediastinum and colon perforation with peritonitis. A patient with extensive hemorrhages was successfully treated with repeated doses of MSCs pooled from two donors. This may indicate that MSCs apart from wound healing may stimulate clotting and vasoconstriction. To conclude, MSCs is a novel treatment that may be used for GVHD, tissue toxicity and hemorrhages because of its immune inhibitory and anti-inflammatory effects. Ó 2011 Published by Elsevier Ltd.

Graft-versus-host disease and toxicity in hematopoietic stem cell transplantation Allogeneic hematopoietic stem cell transplantation (HSCT) is mainly used to treat hematopoietic malignancies such as leukemia, but can also be used for severe aplastic anemia and some rare inborn

* Corresponding author. Tel.: þ46 8 585 826 72; Fax: þ46 8 746 6699. E-mail address: [email protected] (O. Ringden). 1521-6926/$ – see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.beha.2011.01.003

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errors of metabolism [1–3]. Graft-versus-host disease (GVHD) in its acute or chronic form causes considerable morbidity and mortality after HSCT [4,5]. T-cells from the graft are responsible for triggering GVHD and proliferate after activation by recipient major histocompatibility class I or class II antigens, and minor antigenic peptides [6]. Antigen-presenting cells, including dendritic cells of host origin, present the antigens to CD4þ T-cells in association with human leukocyte antigen system (HLA) class II molecules. Cytokines including IL-1 stimulate CD4þ T-cells, which release IL-2. IL-2 stimulates CD8þ T-cells, which react with HLA class I positive targets. Natural killer (NK-cells) and macrophages also participate in the development of acute GVHD. Activated CD4þ cells produce interferon-g which enhances HLA class II expression on epithelial cells and macrophages, which further stimulates T- and NK-cell activation. The main target organs for acute GVHD are skin, gastrointestinal tract, liver and the lymphohematopoietic system. GVHD may be abolished if the graft is T-cell depleted [7,8]. Using non-manipulated grafts, GVHD needs to be prevented by immunosuppressive drugs. The most commonly used drugs are cyclosporine in combination with a short arm of methotrexate [9,10]. Mild or moderate acute GVHD is easily reversed using high doses of steroids. However, more severe acute GVHD grade III is more difficult to overcome and grade IV is life-threatening. If high-dose steroids fails, second line therapy including anti-IL-2 receptor antibodies, monoclonal anti-CD3 antibodies, antibodies against tumor necrosis factor-a, recombinant human IL-1 receptor antibodies, methotrexate, rapamycin and a large variety of treatments have been tried [11–15]. Responses to these therapies are generally poor with a high mortality of hemorrhages, multiorgan failure, and infectious complications [16]. Chronic GVHD may appear as a continuation of the acute form, but may also be quiescent [4,17]. If often appears later in the course, three months or more, after HSCT. Chronic GVHD resembles autoimmune disorders and skin disease, keratoconjunctivits sicca, oral mucositis, strictures, malabsorption, liver disease, obstructive bronchiolitis, may appear. Chronic GVHD is also associated with immunodeficiency with frequent bacterial and viral infections. Chronic GVHD may be treated with steroids, cyclosporine, tacrolimus, extracorporeal photopheresis, azathioprine, 1 Gy of total body irradiation, thalidomide, mycophenolate mofetil, sirolimus, and anti-B-cell antibodies [4,18–22]. Acute and especially chronic GVHD have a pronounced antileukemic and antitumor effect [23–25]. The allogeneic graft-versus-tumor effect is one of the reasons to perform HSCT in patients with malignancies. The other reason is that high doses of chemotherapy and irradiation can be given disregarding hematopoietic bone marrow aplasia, which is rescued by HSCT from a healthy donor. Conditioning by chemo radiotherapy prior to HSCT may also induce toxicity to other tissues than bone marrow, such as mucositis, gastroenteritis, liver toxicity, pneumonitis and hemorrhagic cystitis, among other complications. Rationale to use mesenchymal stem cells for graft-versus-host disease and tissue toxicity after hematopoietic stem cell transplantation Mesenchymal stem cells (MSCs) have raised interest in regenerative medicine because they can differentiate to several mesenchymal tissues and participate in wound healing, support hematopoiesis and participate in the marrow microenvironment [26]. MSCs have immunomodulatory effects and also low immunogenicity, which may make them useful for transplantation. MSCs inhibit T-cell alloreactivity in mixed lymphocyte cultures (MLCs) or by mitogens [27–29]. MSCs inhibited allogeneic specific cytotoxic T-cell lysis if they were added early in the MLC, but not when they were added in the cytotoxic phase [30]. Inhibition was caused by soluble factors including hepatocyte growth factor and transforming growth factor-a, prostaglandin E2, IL-10 and indoleamine2,3-dioxygenase [31–34]. MSCs affect almost all cells of the immune system, T-cells, B-cells, dendritic cells and NK-cells [26]. Mesenchymal stem cells for treatment of severe acute graft-versus-host disease It was demonstrated that MSCs were safe to infuse in humans with no acute toxicity or no ectopic tissue formation [35–37]. Furthermore, MSCs prolonged skin allograft survival in baboons [27]. Because of these findings and the suppression of alloantigens in vitro, we decided to treat patients with lifethreatening acute GVHD not responding to conventional immunosuppressive therapy with MSCs


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[38,39]. In the first patient with grade IV acute GVHD, we observed a dramatic response in the gastroenteral tract and liver. However, when cyclosporine was discontinued, acute GVHD reappeared. Reinstitution of cyclosporine and a second infusion of MSCs again reversed acute GVHD [40]. Based on the in vitro findings that MSCs were equally suppressive in MLC whether they were HLA-identical, haploidentical or completely HLA mismatched, we used third party MSCs for practical reasons. Third party MSCs can be stored frozen and be ready available for patients who develop steroid-resistant acute GVHD. After this initially successful case, we found that MSCs improve acute GVHD in some, although not all patients. In our first series of eight patients with grades III-IV steroid-refractory acute GVHD, responses were seen in six of the patients [38,39]. Two patients died soon after MSC treatment with no obvious response. In one of them, MSC donor DNA was detected in colon and a mesenteric lymph node. Survival in patients treated with MSCs was significantly better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSCs during the same time period (p ¼ 0.03). The results from this pilot study inspired a European phase II study in which 55 patients with therapy-resistant acute GVHD were treated with MSCs [41]. The patients received a median dose of bone marrow-derived MSCs of 1.4 106 (0.4–9 106) cells/kg body weight. Twentyseven patients received one dose, 22 received two doses, and six patients 3–5 doses. MSCs were obtained from HLA-identical sibling donors (n ¼ 5), haploidentical donors (n ¼ 18), and third party HLA-mismatched donors (n ¼ 69). Complete response was obtained in 30 patients and nine had a partial response. Response rate was not related to MSC donor and recipient HLA match. Complete responders had an overall survival two years after MSC infusion of 52%, as opposed to 16% for partial and non-responders (p ¼ 0.018). Children tended to have a better response, 21/25 (68%), compared to 18/30 (43%, p ¼ 0.07) in adults. Fang and co-workers used human adipose tissue-derived MSCs for steroid refractory acute GVHD [42]. They treated six patients with a dose of 1 106/kg of adipose-derived MSCs. No side-effects were noted. Two patients received HLA-haploidentical MSCs, and four patients received third party MSCs. Acute GVHD disappeared completely in 5/6 of the patients. One of the responders died from leukemic relapse. The patient who did not respond to MSC therapy died from multiorgan failure. Ho and co-workers reported three patients treated with MSC using a dose ranging from 0.92 to 1.34 106/kg. Two patients showed a response, but one patient died twelve days after MSC infusion [43]. Kebriaei and co-workers did a randomized study where grades II-IV acute GVHD patients were randomized to receive two treatments of MSCs (ProchymalÒ) in a dose of either 2 or 8 106 MSCs/kg [44]. Median age was 52 years in the 32 adult patients enrolled in the study. Twenty-one patients had a grade II, eight had a grade III, and three had grade IV acute GVHD. An initial response to MSC therapy was seen in 94% with a complete response in 77%. There were no differences in safety or efficacy between the low and the high MSC dose groups. As an alternative to MSCs expanded in fetal calf serum, platelet lysate medium has also been used. von Bonin and co-workers reported experience of 13 adult patients treated with MSCs expanded in platelet lysate medium for steroid-refractory acute GVHD. Complete response was initially seen in two patients (15%). The remaining 11 patients received additional immunosuppressive therapy and further MSC infusions, and partial response was seen in 5/11 of those additional patients [45]. In another study on platelet-lysate generated MSC, eleven children were treated for severe resistant acute or chronic GVHD [46]. Median dose was 1.2 106 MSCs/kg. Overall response was obtained in 71% of the children with a complete response in 24%. Four patients had GVHD recurrence between two and five months after MSC infusion. A recent study confirmed encouraging results using MSCs in pediatric patients [47]. They used Prochymal in twelve children 0.4 to 15 years of age, treated for therapy-resistant grade III and IV acute GVHD on compassionate use basis. The MSC dose was 8 106 cells/kg in two patients and 2 106 cells/kg in the remaining patients. The cells were given twice a week for four weeks. Overall, 7 (58%) patients had complete response, 2 (17%) partial response and 3 (25%) mixed response. The cumulative incidence of survival at 100 days from the initiation of Prochymal therapy was 58%. Complete resolution of gastrointestinal symptoms occurred in 9 (75%) of the children. All these studies with varying numbers of patients treated for acute GVHD suggest that complete and partial responses can be obtained in a majority of patients. HLA-compatibility between MSC donor and recipient does not seem to be of major importance. A cell dose between 1 and 2 106 cells/kg

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appears as effective as higher doses. Whether a particular source of stem cells is superior, bone marrow versus adipose-derived MSCs or MSCs cultured in medium containing fetal calf serum or platelet lysate medium, cannot be concluded from the available data. Furthermore, if the number of passages of MSCs is of importance for response or not remains to be answered. A higher response rate in children than adults may be explained by a better healing capacity and that children can tolerate severe acute GVHD better, compared to adults. From the data of Kebriaei and co-workers [44], it seems that there is a better response if patients are treated for milder acute GVHD. Otherwise, we do not know why some patients respond and others do not. To answer this question, a better understanding how MSC repair damaged tissue is needed. For instance, is the effect direct or indirect? Are accessory cells needed for this process? MSC donor DNA has been detected in target tissues of patients with GVHD at the site of injury [39]. This may suggest that MSCs exert their immunomodulatory and healing effect at the site of injury. However, in some patients MSC donor DNA cannot be detected after infusion. More data from MSC responders and non-responders is required as well as an understanding of how MSC are distributed after intravenous infusion, Furthermore, studies regarding optimal dosing, timing and how to match different donors with the different kinds of patients are required. Future research and the ongoing randomized trials will hopefully shed light on these issues. Mesenchymal stem cells for treatment of chronic graft-versus-host disease As for acute GVHD, there is a variety of treatments used for chronic GVHD because of the poor response in refractory disease. Due to the difficulty to evaluate clinical responses, there is a scarcity of prospective randomized studies of the various treatments [19,48]. Because of the difficulties in the evaluation of response and outcome regarding chronic GVHD, the National Institutes of Health (NIH) formed a consensus on how to measure response in different organs with chronic GVHD involvement [49]. Chronic GVHD resembles autoimmune disorders. Furthermore, MSCs showed positive effects in several experimental autoimmune models [50–53]. There is also an association between acute and chronic GVHD [54–56]. Some risk-factors, target organs and pathophysiology is similar and MSCs also showed effects in treatment of acute GVHD [38,39,41,42,44,46]. Therefore, it seems logical to try MSCs for chronic GVHD [57]. We first used MSCs for treatment in extensive chronic GVHD in a patient with lichenoid changes all over the skin and slight liver disease [39]. He was given 1 106 MSC/kg on day 153 after HSCT. The lichenoid skin changes did not improve, but the liver enzymes declined. He later died of Epstein-Barr virus post transplant lymphoproliferative disorder (PTLD). Subsequently, Zhou and co-workers reported on four patients with sclerodermatous chronic GVHD, who gradually improved after treatment with MSCs [58]. More recently, Weng et al presented their experiments using MSCs in the treatment of 19 patients with refractory chronic GVHD [59]. The median dose of MSCs was 0.6 106 cells/kg and 14/19 showed partial or complete responses (34%). Immunosuppression given for chronic GVHD could be discontinued in five patients within a median of 324 days after MSC infusion. The response rates were graded according to the NIH scoring system [49]. The cumulative response rate for skin was 78%. Among three patients with scleroderma, one had a partial response. Cumulative responses in oral mucosa, liver and gastrointestinal tract were between 90 and 100%. Two-year survival rate was 78%. B-cells are involved in chronic GVHD [60]. The effects of MSCs on B-cells may therefore be of importance. It was reported that MSCs inhibited B-cell proliferation in vitro in an MSC/lymphocyte ratio of 1:1 [61]. However, it may be questioned if this concentration of MSCs compared to lymphocytes is relevant for the in vivo situation. Anyhow, another study found that MSCs arrested B-cell proliferation in the G0/G1 phase of the cell cycle [62]. We studied an ultimate B-cell function, IgG secretion, and found that MSCs stimulated blood and splenic B-cell IgG production [63]. Thus, the value of in vitro data for prediction of in vivo responses in chronic GVHD is likely to be limited, since T-cells are inhibited and B-cells may be activated. In vivo studies in experimental animals and in patients treated with MSCs should address this point. Mesenchymal stem cells for treatment of tissue toxicity and hemorrhages Because MSCs localize to target organs and heal acute GVHD, we also treated patients with severe tissue toxicity with infusions of MSC [64]. Life-threatening grade 5 hemorrhagic cystitis was the


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indication for treatment with MSCs in the first two patients [65]. The two patients needed massive transfusions of erythrocytes, platelets and fresh frozen plasma. After infusion of MSCs, both patients had dramatically reduced transfusion requirements [64]. In spite of this, both patients died of multiorgan failure. One of them received MSCs consecutively from two donors. At autopsy, donor DNA from the two donors could be demonstrated in the bladder tissue. This patient had a bladder perforation, which seemed healed at autopsy. In total, we have now treated 12 patients for severe hemorrhagic cystitis after HSCT. Seven of these patients have been reported previously [64]. As mentioned above, two patients died of multiorgan failure. Two patients did not respond to MSC infusions. However, in the remaining eight patients, gross hematuria disappeared after median 3 (1-14) days. The two most recent responders received MSCs pooled from two donors. The reason to pool MSCs from several donors was that when we pooled MSCs from several donors in vitro, we could improve the response rate in mixed lymphocyte cultures [66]. Therefore, this may suggest that pooling of MSCs from several donors might improve the response rate. In two HSCT patients, pneumomediastinum resolved after treatment with MSCs [64]. Colon perforation and peritonitis was reversed in an elderly female with severe gastrointestinal GVHD. Peritonitis and abdominal muscle defense developed on day 70 after HSCT. X-ray showed intra-abdominal air. The patient refused surgery. She was treated with antibiotics and 1 106 MSCs/kg from a third party donor. Six days after MSC infusion peritonitis and abdominal muscle defence disappearted. X-ray showed that abdominal gas had markedly decreased and had completely disappeared 11 days after MSC infusion. As second colon perforation was also reversed by MSCs. Unfortunately, the patient died of invasive fungal infection. A 61-year old male with myelofibrosis developed extensive hemorrhages from the proximal jejunum during the neutropenic phase after HSCT using an unrelated donor [67]. Due to multi-specific anti-HLA antibodies, he was refractory to platelet transfusions. Surgery was impossible for technical reasons and platelet refractoriness. He was given 2 106 MSCs/kg pooled from two donors. Prior to MSC infusion, he was daily given several erythrocyte units, platelets from HLA-identical donors and several units of fresh frozen plasma. After MSC infusions, hemorrhages stopped and he only required occasional erythrocyte and platelet transfusions. He experienced two additional hemorrhages, which were stopped by MSC infusions. Due to hematopoietic graft failure, he was regrafted with a new donor. Subsequent to this, he again had hemorrhage during the pancytopenia that was treated by MSCs. Due to poor graft function, despite complete donor chimerism, he was given a T-cell depleted bone marrow boost from the same donor. He is now six months after retransplantation, alive and well with stable hematopoietic engraftment. From this patient and the experience using MSCs for the treatment of hemorrhagic cystitis, it is obvious that MSCs can heal damaged tissue and stop hemorrhages. One may speculate if MSCs, apart from wound healing, stimulate clotting and also induce vasoconstriction. Adverse effects All studies have uniformly shown that it is safe to infuse MSCs i.v. and no side-effects have been observed. It has been debated whether MSCs with their immunomodulatory effects increase the risk of leukemic relapse, following HSCT. One study indicated that patients co-infused with MSCs at the time of HSCT had an increased risk of leukemic relapse [68]. However, it is expected that any treatment that decreases acute and especially chronic GVHD also will decrease the graft-versus-leukemia effect [23–25]. Patients with acute and chronic GVHD are susceptible to opportunistic infections. MSCs also decrease the immune function of all cells of the immune system [26]. It has therefore been discussed if MSCs may increase the risk of infection in these already severely immunocompromised HSCT patients. However, MSCs did not decrease the effects of virus-specific T-cell responses to cytomegalovirus and Epstein-Barr virus, in contrast to the effects on alloantigen cytotoxic T-cells [69]. Conclusion The dramatic effects seen after i.v. infusion of MSCs in certain patients have opened up the field to treat patients with acute and chronic GVHD, tissue toxicity and hemorrhages after HSCT. Based on these findings, MSCs may also be used for autoimmune disorders such as inflammatory bowel disease

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and other indications. MSCs may also be tried in patients with gastrointestinal perforations and peritonitis, where surgery is not possible for various reasons. Pilot studies have shown that MSCs may be an effective tool. Now it is time for randomized studies to prove its efficacy. Randomized studies have been performed for the treatment of severe acute GVHD and others are ongoing. Randomized studies are also needed to prove its efficacy in patients with chronic GVHD and tissue toxicity. Studies are also needed how to optimize the use of MSC. Conflict of interest statement None to declare. Acknowledgments We thank Inger Hammarberg for excellent typing of this manuscript. The study was supported by grants from the Swedish Cancer Society, the Children’s Cancer Foundation, the Swedish Research Council, the Tobias Foundation, the Cancer Society in Stockholm, the Swedish Society of Medicine, the Stockholm County Council, the Sven and Ebba-Christina Hagbergs Foundation, and Karolinska Institutet. References [1] Ringden O, Remberger M, Svahn BM, et al. Allogeneic hematopoietic stem cell transplantation for inherited disorders: experience in a single center. Transplantation 2006;81:718–25. [2] Storb R, Thomas ED, Weiden PL, et al. Aplastic anemia treated by allogeneic bone marrow transplantation: a report on 49 new cases from Seattle. Blood 1976;48:817–41. [3] Thomas ED, Buckner CD, Banaji M, et al. One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 1977;49:511–33. [4] Ringden O, Deeg HJ. Clinical spectrum of graft-versus-host disease. In: Ferrara JLM, Deeg HJ, Burakoff S, editors. Graft vs host disease. ed. 2nd. New York: Marcel Dekker, Inc; 1996. p. 525–59. [5] Storb R, Thomas ED. Graft-versus-host disease in dog and man: the seattle experience. Immunol Rev. 1985;88:215–38. [6] Ferrara JL, Levy R, Chao NJ. Pathophysiologic mechanisms of acute graft-vs.-host disease. Biol Blood Marrow Transplant 1999;5:347–56. [7] Marmont AM, Horowitz MM, Gale RP, et al. T-cell depletion of HLA-identical transplants in leukemia. Blood 1991;78:2120–30. [8] Prentice HG, Blacklock HA, Janossy G, et al. Depletion of T lymphocytes in donor marrow prevents significant graftversus-host disease in matched allogeneic leukaemic marrow transplant recipients. Lancet 1984;1:472–6. [9] Ringden O, Horowitz MM, Sondel P, et al. Methotrexate, cyclosporine, or both to prevent graft-versus-host disease after HLA-identical sibling bone marrow transplants for early leukemia? Blood 1993;81:1094–101. [10] Storb R, Deeg HJ, Pepe M, et al. Methotrexate and cyclosporine versus cyclosporine alone for prophylaxis of graft-versushost disease in patients given HLA-identical marrow grafts for leukemia: long-term follow-up of a controlled trial. Blood 1989;73:1729–34. [11] Anasetti C, Hansen JA, Waldmann TA, et al. Treatment of acute graft-versus-host disease with humanized anti-tac: an antibody that binds to the interleukin-2 receptor. Blood 1994;84:1320–7. [12] Benito AI, Furlong T, Martin PJ, et al. Sirolimus (rapamycin) for the treatment of steroid-refractory acute graft-versus-host disease. Transplantation 2001;72:1924–9. [13] Deeg HJ, Blazar BR, Bolwell BJ, et al. Treatment of steroid-refractory acute graft-versus-host disease with anti-CD147 monoclonal antibody ABX-CBL. Blood 2001;98:2052–8. [14] Herve P, Wijdenes J, Bergerat JP, et al. Treatment of corticosteroid resistant acute graft-versus-host disease by in vivo administration of anti-interleukin-2 receptor monoclonal antibody (B-B10). Blood 1990;75:1017–23. [15] Kobbe G, Schneider P, Rohr U, et al. Treatment of severe steroid refractory acute graft-versus-host disease with infliximab, a chimeric human/mouse antiTNFalpha antibody. Bone Marrow Transplant 2001;28:47–9. [16] Remberger M, Aschan J, Barkholt L, et al. Treatment of severe acute graft-versus-host disease with anti-thymocyte globulin. Clin Transplant 2001;15:147–53. [17] Sullivan KM, Shulman HM, Storb R, et al. Chronic graft-versus-host disease in 52 patients: adverse natural course and successful treatment with combination immunosuppression. Blood 1981;57:267–76. [18] Canninga-van Dijk MR, van der Straaten HM, Fijnheer R, et al. Anti-CD20 monoclonal antibody treatment in 6 patients with therapy-refractory chronic graft-versus-host disease. Blood 2004;104:2603–6. [19] Flowers ME, Apperley JF, van Besien K, et al. A multicenter prospective phase 2 randomized study of extracorporeal photopheresis for treatment of chronic graft-versus-host disease. Blood 2008;112:2667–74. [20] Greinix HT, Volc-Platzer B, Kalhs P, et al. Extracorporeal photochemotherapy in the treatment of severe steroid-refractory acute graft-versus-host disease: a pilot study. Blood 2000;96:2426–31. [21] Koc S, Leisenring W, Flowers ME, et al. Therapy for chronic graft-versus-host disease: a randomized trial comparing cyclosporine plus prednisone versus prednisone alone. Blood 2002;100:48–51. [22] Socie G, Devergie A, Cosset JM, et al. Low-dose (one gray) total-lymphoid irradiation for extensive, drug-resistant chronic graft-versus-host disease. Transplantation 1990;49:657–8.


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[23] Horowitz MM, Gale RP, Sondel PM, et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75:555–62. [24] Ringden O, Karlsson H, Olsson R, et al. The allogeneic graft-versus-cancer effect. Br J Haematol 2009;147:614–33. [25] Weiden PL, Flournoy N, Thomas ED, et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 1979;300:1068–73. [26] Le Blanc K, Ringden O. Immunobiology of human mesenchymal stem cells and future use in hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005;11:321–34. *[27] Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:42–8. *[28] Le Blanc K, Tammik L, Sundberg B, et al. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003;57:11–20. [29] Tse WT, Pendleton JD, Beyer WM, et al. Suppression of allogeneic T-cell proliferation by human marrow stromal cells: implications in transplantation. Transplantation 2003;75:389–97. *[30] Rasmusson I, Ringden O, Sundberg B, et al. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation 2003;76:1208–13. *[31] Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005;105: 1815–22. [32] Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002;99:3838–43. *[33] Meisel R, Zibert A, Laryea M, et al. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood 2004;103:4619–21. [34] Rasmusson I, Ringden O, Sundberg B, et al. Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp Cell Res. 2005;305:33–41. [35] Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000;18:307–16. [36] Koc ON, Peters C, Aubourg P, et al. Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases. Exp Hematol 1999;27:1675–81. [37] Lazarus HM, Haynesworth SE, Gerson SL, et al. Ex vivo expansion and subsequent infusion of human bone marrowderived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant 1995;16:557–64. [38] Le Blanc K, Rasmusson I, Sundberg B, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004;363:1439–41. *[39] Ringden O, Uzunel M, Rasmusson I, et al. Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation 2006;81:1390–7. [40] Ringden O, Le Blanc K. Mesenchymal stem cells combined with cyclosporine inhibits cytotoxic T cells. Biol Blood Marrow Transplant 2006;12:693–4. *[41] Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versushost disease: a phase II study. Lancet 2008;371:1579–86. [42] Fang B, Song Y, Liao L, et al. Favorable response to human adipose tissue-derived mesenchymal stem cells in steroidrefractory acute graft-versus-host disease. Transplant Proc. 2007;39:3358–62. [43] Ho S-J, Dyson P, Rawling T, et al. Mesenchymal stem cells for treatment of steroid-resistant graft-versus-host disease. Biol Blood Marrow Transplant 2007;13:46–7. [44] Kebriaei P, Isola L, Bahceci E, et al. Adult human mesenchymal stem cells added to corticosteroid therapy for the treatment of acute graft-versus-host disease. Biol Blood Marrow Transplant 2009;15:804–11. [45] von Bonin M, Stolzel F, Goedecke A, et al. Treatment of refractory acute GVHD with third-party MSC expanded in platelet lysate-containing medium. Bone Marrow Transplant 2009;43:245–51. [46] Lucchini G, Introna M, Dander E, et al. Platelet-lysate-expanded mesenchymal stromal cells as a salvage therapy for severe resistant graft-versus-host disease in a pediatric population. Biol Blood Marrow Transplant 2010;16:1293–301. [47] Prasad VK, Lucas KG, Kleiner GI, et al. Efficacy and safety of ex-vivo cultured adult human mesenchymal stem cells (prochymal(TM)) in pediatric patients with severe refractory acute graft-versus-host disease in a compassionate use study. Biol Blood Marrow Transplant; 2010, May 7 [Epub ahead of print]. [48] Sullivan KM, Witherspoon RP, Storb R, et al. Prednisone and azathioprine compared with prednisone and placebo for treatment of chronic graft-v-host disease: prognostic influence of prolonged thrombocytopenia after allogeneic marrow transplantation. Blood 1988;72:546–54. [49] Pavletic SZ, Martin P, Lee SJ, et al. Measuring therapeutic response in chronic graft-versus-host disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: IV. Biol Blood Marrow Transplant 2006;12:252–66. Response Criteria Working Group report. [50] Gonzalez MA, Gonzalez-Rey E, Rico L, et al. Adipose-derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology 2009;136:978–89. [51] Parekkadan B, Tilles AW, Yarmush ML. Bone marrow-derived mesenchymal stem cells ameliorate autoimmune enteropathy independently of regulatory T cells. Stem Cells 2008;26:1913–9. *[52] Sun L, Akiyama K, Zhang H, et al. Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans. Stem Cells 2009;27:1421–32. [53] Zappia E, Casazza S, Pedemonte E, et al. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood 2005;106:1755–61. [54] Atkinson K, Farewell V, Storb R, et al. Analysis of late infections after human bone marrow transplantation: role of genotypic nonidentity between marrow donor and recipient and of nonspecific suppressor cells in patients with chronic graft-versus-host disease. Blood 1982;60:714–20.

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[55] Ringden O, Paulin T, Lonnqvist B, et al. An analysis of factors predisposing to chronic graft-versus-host disease. Exp Hematol 1985;13:1062–7. [56] Storb R, Prentice RL, Sullivan KM, et al. Predictive factors in chronic graft-versus-host disease in patients with aplastic anemia treated by marrow transplantation from HLA-identical siblings. Ann Intern Med 1983;98:461–6. [57] Ringden O, Keating A. Mesenchymal stem cells as treatment for chronic graft-versus-host disease. Bone Marrow Transplant. in press. [58] Zhou H, Guo M, Bian C, et al. Efficacy of bone marrow-derived mesenchymal stem cells in the treatment of sclerodermatous chronic graft-versus-host disease: clinical report. Biol Blood Marrow Transplant 2010;16:403–12. [59] Weng J, Du X, Geng S, et al. Mesenchymal stem cell as salvage treatment for refractory chronic graft-versus-host disease. Bone Marrow Transplant; 6 September 2010. E-pub ahead of print. [60] Tsoi MS, Storb R, Jones E, et al. Deposition of IgM and complement at the dermoepidermal junction in acute and chronic cutaneous graft-vs-host disease in man. J Immunol 1978;120:1485–92. [61] Corcione A, Benvenuto F, Ferretti E, et al. Human mesenchymal stem cells modulate B-cell functions. Blood 2006;107:367– 72. [62] Beyth S, Borovsky Z, Mevorach D, et al. Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness. Blood 2005;105:2214–9. [63] Rasmusson I, Le Blanc K, Sundberg B, Ringden O. Mesenchymal stem cells stimulate antibody secretion in human B cells. Scand J Immunol 2007;65:336–43. *[64] Ringden O, Uzunel M, Sundberg B, et al. Tissue repair using allogeneic mesenchymal stem cells for hemorrhagic cystitis, pneumomediastinum and perforated colon. Leukemia 2007;21:2271–6. [65] Hassan Z, Remberger M, Svenberg P, et al. Hemorrhagic cystitis: a retrospective single-center survey. Clin Transplant 2007;21:659–67. [66] Samuelsson H, Ringden O, Lonnies H, Le Blanc K. Optimizing in vitro conditions for immunomodulation and expansion of mesenchymal stromal cells. Cytotherapy 2009;11:129–36. [67] Ringden O, Le Blanc K. Pooled mesenchymal stem cells for treatment of severe hemorrhages. Bone Marrow Transplant, in press. [68] Ning H, Yang F, Jiang M, et al. The correlation between cotransplantation of mesenchymal stem cells and higher recurrence rate in hematologic malignancy patients: outcome of a pilot clinical study. Leukemia 2008;22:593–9. *[69] Karlsson H, Samarasinghe S, Ball LM, et al. Mesenchymal stem cells exert differential effects on alloantigen and virusspecific T-cell responses. Blood 2008;112:532–41.




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Ex vivo expansion of mesenchymal stromal cells Maria Ester Bernardo, MD, PhD a, Angela Maria Cometa, PhD a, Daria Pagliara, MD a, Luciana Vinti, MD a, Francesca Rossi, MD a, b, Rosaria Cristantielli, PhD a, Giuseppe Palumbo, MD, PhD c, Franco Locatelli, MD, PhD a, d, * a Dipartimento di Ematologia ed Oncologia Pediatrica, IRCCS Ospedale Pediatrico Bambino Gesù, P.le Sant’ Onofrio, 4, 00165 Roma, Italy b Seconda Università di Napoli, Italy c Clinica Pediatrica, Università degli Studi di Roma "Tor Vergata", Italy d Università degli Studi di Pavia, Italy

Keywords: mesenchymal stromal cells ex vivo expansion fetal calf serum platelet lysate malignant transformation cellular therapy

Mesenchymal stromal cells (MSCs) are adult multipotent cells that can be isolated from several human tissues. MSCs represent a novel and attractive tool in strategies of cellular therapy. For in vivo use, MSCs have to be ex vivo expanded in order to reach the numbers suitable for their clinical application. Despite being efficacious, the use of fetal calf serum for MSC ex vivo expansion for clinical purposes raises concerns related to immunization and transmission of zoonoses; the standardization of expansion methods, possibly devoid of animal components, such as those based on platelet lysate, are discussed in this paper. Moreover, this review focuses on the search of novel markers for the prospective identification/isolation of MSCs and on the potential risks connected with ex vivo expansion of MSCs, in particular that of their malignant transformation. Available tests to study the genetic stability of ex vivo expanded MSCs are also analyzed. Ó 2010 Elsevier Ltd. All rights reserved.

Introduction In addition to hematopoietic stem cells (HSCs), the bone marrow (BM) also contains mesenchymal stromal cells (MSCs). These cells were first recognized more than 40 years ago by Friedenstein et al. who described a population of adherent cells from the BM which were non-phagocytic, exhibited

* Corresponding author. Dipartimento di Ematologia ed Oncologia Pediatrica, IRCCS Ospedale Pediatrico Bambino Gesù, P.le Sant’ Onofrio, 4, 00165 Roma, Italy. Tel.: þ39 06 68592129; Fax: þ39 06 68592292. E-mail address: [email protected] (F. Locatelli). 1521-6926/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2010.11.002

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M.E. Bernardo et al. / Best Practice & Research Clinical Haematology 24 (2011) 73–81

a fibroblast-like appearance and could differentiate in vitro into bone, cartilage, adipose tissue, tendon and muscle [1]. Moreover, after transplantation under the kidney capsule, these cells gave rise to the different connective tissue lineages [2]. In general, MSCs represent a minor fraction in BM and their exact frequency is difficult to calculate because of the different methods of harvest and separation. However, the frequency in human BM has been estimated to be in the order of 0.001–0.01% of the total nucleated cells; moreover, this frequency declines with age, from 1/104 nucleated marrow cells in a newborn to about 1/2 106 nucleated marrow cells in a 80-year old person [3]. After their first identification in the BM [1], human MSCs (hMSCs) were isolated from a variety of other human tissues, including periosteum, muscle connective tissue, perichondrium, dental pulp, adipose tissue and fetal tissues, such as lung, BM, liver and spleen [4–7]. Amniotic fluid and placenta have been found to be rich sources of MSCs [8,9]; both fetal and maternal MSCs can be isolated from human placenta [8]. Although present at low frequency, MSCs have been also identified in umblical cord blood (UCB); in this respect, quality criteria for the selection of UCB units are considered critical for the successful isolation of MSCs from this source [10,11]. One of the hallmark of MSCs is their multipotency, defined as the ability to differentiate into several mesenchymal lineages, including bone, cartilage, tendon, muscle, marrow stroma and adipose tissue (AT) [7,12]. Usually, trilineage differentiation into bone, adipose tissue and cartilage is taken as a criterium for multipotentiality [12,13]. MSCs display unique immunological properties that modulate the responses, in vitro and in vivo, of all cells involved in the immune response, including T and B lymphocytes, dendrtic cells and natural killer (NK) cells [14–16]. However, the exact mechanisms by which MSCs exert their functions are still poorly understood. Whether MSCs mainly mediate their effect through soluble factors or cell-to-cell contact is still a matter of active investigation [14,17,18]. Moreover, the mechanisms by which MSCs display their immunosuppressive effect are largely restricted to in vitro studies; the in vivo biological relevance of the in vitro observations needs to be elucidated [14–16]. Thanks to their ability to home to inflamed sites and repair-injured tissues, and to their immunomodulatory properties, MSCs are today considered a promising tool in approaches of immunoregulatory and regenerative cell therapy [19]. Thus far, MSCs have been employed in phase I/II clinical trials [20–23], mainly addressing the issues of feasibility and safety of infusion; and to date no adverse effects have been registered after MSC administration. In particular, MSCs, usually harvested at passage 2 to 3 of in vitro culture, have been successfully employed in the clinic to enhance hematopoietic stem cell engraftment in HLA-haploidentical, T cell-depleted allografts [20] and UCB transplantation [21], as well as to treat the most severe and refractory forms of acute graft-versus-host disease [22]. Moreover, local injections of adipose tissue (AT)-derived MSCs have already been successfully employed in the clinical setting to treat complex perianal fistulas resistant to conventional treatments in Crohn’s Disease (CD) patients [23]. Ex vivo isolation and characterization of MSCs Most of the information available on MSC phenotypic and functional properties are derived from studies performed on cells cultured in vitro [13–15]. To date, MSC isolation/identification has mainly relied on their morphology and adherence to plastic, resulting in a heterogeneous population of cells, referred to as MSCs [13]. Immunephenotyping by flow cytometry is also applied to characterize ex vivo expanded MSCs and to define their purity. However, at present, no specific marker has been shown to specifically identify true MSCs and ex vivo expanded cells are currently stained with a combination of positive (CD105, CD73, CD90, CD166, CD44 CD29) and negative (CD14, CD31, CD34 and CD45) markers, at least in case of BM-derived cells (indeed, a proportion of AT-derived MSCs express CD34) [5,12,13]. Therefore, little is known about the characteristics of the primary precursor cells in vivo, since it has not yet been possible to isolate the most primitive mesenchymal cell from bulk cultures. One of the hurdles has been the inability to prospectively isolate MSCs because of their low frequency and the lack of specific markers. Recently, some groups have reported the identification and prospective isolation of the most primitive mesenchymal progenitors, both in murine and human adult BM, based on the expression of specific markers [24–34].


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Anjos-Afonso et al. have reported the identification, isolation and characterization of a novel multipotent cell population in murine BM, based on the expression of the stage-specific embryonic antigen-1 (SSEA-1). This primitive subset can be found both directly in the BM and in mesenchymal cell cultures and can give rise to SSEA-1þ MSCs [24]. In human cells, with the aim to prospectively isolate MSCs, surface markers such as SSEA-4, STRO-1 and the low affinity nerve growth factor receptor (CD271) [25–28], which enrich for MSCs, have been employed. Moreover, Battula et al. have recently isolated by flow cytometry MSCs from human BM, using antibodies directed against the surface antigens CD271, mesenchymal stem cell antigen-1 (MSCA-1), CD56 and SSEA-3, and identified novel MSC subsets with distinct phenotypic and functional properties [29,31]. In particular, CD271 has been reported to define a subset of MSCs with immunosuppressive and lymphohematopoietic engraftment-promoting properties in vivo [33]. Plateletderived growth factor receptor-beta (PDGF-RB; CD140b) has been also proposed as a marker for the isolation of clonogenic MSCs [29] and other reports have demonstrated a 9.5-fold enrichment of MSCs in human BM cells with prominent aldehyde dehydrogenase activity [30]. A STRO-4 monoclonal antibody has been also demonstrated to be specific for mesenchymal precursors cells from human and ovine tissues, being capable to enrich colony-forming fibroblasts when employed for MSC isolation from BM [34]. Table 1 details the published markers and/or combination of antigens identifying mesenchymal precursors. Despite the identification of these new MSC markers [24–34], none of the available has demonstrated to be singularly capable to identify the true mesenchymal progenitors. Indeed, MSCs may be composed by different cell subsets which might be responsible for specific functions and characterized by different cell surface markers. Therefore, further research in this field is warranted in order to identify an MSC-specific marker; this will hopefully allow to dissect the developmental hierarchy of MSCs and will facilitate the generation of homogenous cellular products. Novel techniques, such as proteomic approaches and microarray analysis, have also been proposed and might be useful for performing comparisons of expression profiles between different MSC populations (i.e. MSCs from different tissue sources, cultured in different conditions, at different in vitro passages) and, therefore, to define new MSC surface antigens that can be used to identify cell subsets [35–37]. For example, NOTCH3, JAG2 and ITGA11 transcripts have been recently demonstrated to be expressed on ex vivo expanded BM-MSCs through genome-wide, gene expression analysis [38]. Table 1 Antigens expressed on primary and culture-expanded MSCs. Antigen

Expanded/primary MSCs

Human/murine MSCs

CD105 (endoglin, SH2) [12,13] CD73 (ecto-5’ nucleotidase, SH3, SH4) [12,13] CD166 (ALCAM) [12,13] CD29 (b1-integrin) [12,13] CD44 (H-CAM) [12,13] CD90 (Thy-1) [12,13] TRA-1-81 Sca-1 STRO-1 [27] CD349 (frizzled-9) [27] SSEA-3; SSEA-4 [25,31] Oct-4

Expanded Expanded Expanded Expanded Expanded Expanded Expanded Expanded Primary Primary Primary Primary

Nanog-3 SSEA-1 [24] CD271 (low affinity nerve growth receptor) [30,33] MSCA-1 [30] CD140b (PDGF-RB) [29] STRO-4 [34] NOTCH3, JAG1, ITGA11 [38]

Primary Primary Primary

Human, murine Human Human Human, murine Human, murine Human Human (placenta) Murine Human (BM) Human (BM, placenta) Human þ/- (BM, placenta) Human þ/- (BM, placenta, fetal tissues) Human þ/- (BM, placenta) Murine (BM) Human

Primary Primary Primary Expanded

Human (BM) Human Human, ovine (BM) Human (BM)

Modified from Bernardo et al. Ann NY Acad Sci. 2009; 1176:101.

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Ex vivo expansion of MSCs Due to the low frequency of mesenchymal progenitors in human tissues, in vivo use of MSCs requires that the cells be extensively ex vivo manipulated to achieve the numbers that are necessary for their clinical application [20–22]. Indeed, MSCs can be expanded in vitro to hundreds of millions of cells from a 10 to 20 ml BM aspirate [12,13]. In this regard, the cells are mainly cultured, either under experimental or clinical-grade conditions, in the presence of 10% fetal calf serum (FCS) [12–14,19–22] and serum batches are routinely pre-screened to guarantee both the optimal growth of MSCs and the bio-safety of the cellular product. Most of the clinical experience that has been gained on MSCbased cellular therapies both in the context of hematopoietic stem cell transplantation (HSCT) with the aim to promote engraftment [20,21] and to treat acute GvHD [22], and in strategies of Regenerative Medicine [23], has been obtained with FCS-expanded cells. Differences in isolation methods, culture conditions, media additives greatly affect cell yield and possibly also the phenotype of the expanded cell product [39–45]. For these reasons, efforts have been made within the MSC Developmental Committee of the European Group for Blood and Marrow Transplantation (EBMT) for the standardization of MSC isolation and expansion procedures [20–22,46]. This organization, including European centers interested in the biology and clinical application of MSCs, has defined a common MSC expansion protocol based on the use of 10% pre-screened FCS, in order to facilitate comparisons between cell products generated at different sites and to run large-scale clinical studies. Nonetheless, the use of FCS raises concerns when utilized in clinical-grade preparations, because it might theoretically be responsible for the transmission of prions and still unidentified zoonoses or cause immune reactions in the host, especially if repeated infusions are needed, with consequent rejection of the transplanted cells [39–42]. In this regard, Horwitz et al. reported sensitization in a child with Osteogenesis Imperfecta treated with repeated infusions of MSCs [47]. This sensitization was characterized by the formation of alloantibodies directed against MSCs and was responsible for their rejection, already after the second infusion of ex vivo expanded allogeneic MSCs [47]. In view of these considerations, serum-free media, appropriate for extensive expansion and devoid of the risks connected with the use of animal products, are being investigated. Both autologous and allogeneic human serum have been tested for the in vitro expansion of MSCs and one group showed that autologous serum was superior to both FCS and allogeneic serum in terms of proliferative capacity of the expanded MSCs [39]. The reduction of bovine antigens by a final 48-hour incubation with medium supplemented with 20% human serum, to prepare hypoimmunogenic MSCs, has also been proposed [40]. Several serum-free media, based on the use of cytokines and growth/ attachment factors, such as basic fibroblast growth factor (b-FGF) and trasforming growth factor beta (TGF-b), have been also tested in experimental conditions [48,49]. Recently, platelet lysate (PL) has been demonstrated to be a powerful substitute for FCS in MSC expansion, offering a significative advantage in terms of proliferative capacity of MSCs, thanks to its high concentration of natural growth factors (GFs) [41–45]. Indeed, human PL contains all GFs that are secreted by platelets to initiate wound healing, including PDGFs, b-FGF, vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and TGF-b [41,42]; moreover, it can be easily obtained from apheresis products of healthy volunteers [42], as well as from buffy coats [45]. Immediately after apheresis, PLT products are frozen at 80 C, subsequently thawed to obtain the release of PLT-derived GFs and centrifuged to eliminate platelet bodies; PL preparations from several healthy donors are pooled to be used as a culture supplement for the generation of MSCs [42]. Doucet et al. first demonstrated that growth factors contained in PL are able to promote MSC expansion in a dosedependent manner [41]. This was further substantiated by the data published by our group and showing that a culture medium additioned with 5% PL is superior to 10% FCS in terms of clonogenic efficiency and proliferative capacity of MSCs, therefore providing more efficient expansion, together with a significant time saving [42]. Moreover, the in vitro immune regulatory properties of PLexpanded MSCs resulted to be comparable to those of MSCs cultured in the presence of FCS in terms of capacity to decrease alloantigen-induced cytotoxic activity, to promote differentiation of CD4þ T cell subsets expressing a Treg phenotype and to induce augmentation of IL-6 production in culture supernatant [42]. On the contrary, the suppressive effect in vitro on alloantigen-induced lymphocyte


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subset proliferation was greater in the presence of FCS-expanded MSCs, as compared to MSCs cultured in PL [42]. Moreover, gene expression changes in long term cultured PL-MSCs have been studied by microarray analysis and resulted to be similar to those of MSCs expanded in the presence of FCS, suggesting that replicative senescence modifications develop in both cases (in the absence of malignant transformation) and are independent of the culture conditions [50]. Altogether, these data suggest that PL could be a suitable substitute for FCS in the generation and expansion of MSCs; however, further studies are needed to better understand the characteristics and functional properties, in vitro and in vivo, of PL-expanded MSCs, as compared to those cultured in FCS-based medium. This information needs to be acquired before introducing this culture supplement in the routine preparation of cellular products to be employed for clinical application. Moreover, clinical data on the safety and efficacy of MSCs have been obtained, so far, mainly with cells expanded in the presence of FCS, whereas relatively little in vivo experience is available with MSCs cultured in PL. Other safety issues related to ex vivo expanded MSCs The utilization of ex vivo expanded MSCs for clinical application is associated not only with the potential risk of the immunogenicity of the cells or with the bio-ssafety of medium components, but also with the potential in vitro transformation of the cells during expansion. It has been shown by some groups that the ex vivo manipulation of both human and murine MSCs may alter the functional and biological properties of the cells, leading to the accumulation of genetic alterations [51–55]. In particular, the groups of Rubio and Røsland published that human AT- and BM-derived MSCs are prone to undergo malignant trasformation after long term ex vivo expansion; transformed cells showed an increased proliferation rate, displaied an altered morphology and immunephenotype and carried cytogenetic abnormalities. Moreover, these cells induced tumor formation when injected into immunideficient mice [51,54]. A high susceptibility to malignant transformation was also described in murine BM-derived MSCs by different groups [53,55]. These latter cells, probably due to their animal origin, display a high degree of chromosome instability, characterized by the development of both structural and numerical aberrations even at early culture passages [53], and induce tumor formation when infused into irradiated allogeneic recipients [55]. On the contrary, our own group and other laboratories did not confirm a propensity of human MSCs to develop morphological and genetic changes [40,42,56,57]. In particular, our group has investigated the potential susceptibility of human MSCs, derived from both BM and UCB and expanded in the presence of FCS or PL, to undergo transformation after in vitro culture prolonged to either passage 25 or cell senescence [40,42,56]. We found that both BM- and UCB-MSCs can be cultured for long term without loosing their usual phenotypical and functional characteristics. Using genetic studies, performed through both conventional and molecular (array-Comparative Genomic Hybridization, array-CGH) karyotyping, the absence of chromosomal abnormalities was documented [40,42,56]. Telomere length decreased along the different culture passages and telomerase activity was not found to be present. Moreover, in case of UCB-MSCs, p16ink4a , a protein regulating cell cycle and involved in the process of senescence, was found to be normally expressed on expanded cells and anchorage growth independence in soft agar was never observed [44]. Similar findings were obtained in BM-MSCs cultured until passage 3 following the FCS-based expansion protocol developed within the EBMT Developmental Committee in the perspective of being employed for clinical application [20–22]. Recently, French researchers have reported the presence of aneuploidy in a number of MSC preparations for clinical application, after cultivation both in the presence of FCS þ Fibroblast Growth factor2 (FGF-2) and PL [57]. To further characterize the genetic abnormalities, quantitative analysis of genes related to transformation and senescence was performed. Normal and stable expression of c-myc, p53 and p21 was demonstrated, whereas human telomerase reverse transcriptase (hTERT) was never expressed. Moreover, MSCs normally expressed p16ink4a and anchorage growth independence in soft agar was never obtained [57]. These data suggest that, although aneuploidy can occur during MSC

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expansion, it does not reflect cell transformation, but rather senescence of the cells. Based on these results, the French MSC clinical trials which were temporary interrupted due to the potential risks associated with the infusion of transformed cells, have been re-opened. Very recently three research groups, who previously reported the propensity of human MSCs to undergo malignant transformation after long term in vitro culture, have now discovered that the cancer-like cells they described were unrelated to the original MSCs and belonged to tumor cell lines that they were using for other projects [58,59]. The risk of cross-contamination and its consequences should always be considered when culturing concomitantly primary cells for long term and immortalized cells lines and it is highly recommended that cell line verification with DNA fingerprinting be performed to confirm experimental results before publication. In light of these discrepant observations, phenotypic, functional and genetic assays, although known to have limited sensitivity, should be routinely performed on MSCs before in vivo use to demonstrate whether their biological properties, after ex vivo expansion, remain suitable for clinical application. In particular, the absence of transformation potential in cultured cells has to be documented before infusion into patients, particularly into immune-compromised subjects where impairment of immune surveillance mechanisms might further favor the development of tumors in vivo. In particular, from a practical point of view, karyotyping should be performed on expanded cells and be included in the quality controls and release criteria for MSC administration into patients. Indeed, a precise characterization of the genetic profile of MSCs could allow to identify phenomena of senescence, developing in cells at the end of their life-span, versus transformation of cells, due to the occurrence of genetic alterations. Conclusions At present, MSCs are extensively expanded ex vivo before being used in the clinical setting [13,20–22]. The adoption of different isolation methods and culture conditions may lead to multiple MSC populations with slightly different biological and functional characteristics. For instance, differences in culture medium or supplements (FCS, human serum, PL, addition of GFs), plating density, level of confluency at cell detachment may influence their proliferative capacity, expression of surface markers or differentiation capacity, leading to the commitment towards a specific phenotype or tissue lineage [13,19,41,45]. This supports the need for the definition and validation of common isolation and expansion protocols for the preparation of MSCs both for experimental and clinical purposes. The use of a uniform expansion method, possibly devoid of animal components, facilitates the comparisons between cell products generated at different sites and allows to perform large multicenter collaborative studies. Platelet lysate has been recently proposed as alternative culture supplement for MSC ex vivo manipulation [41–45]; despite the fact that expansion procedures using PL have been implemented in different laboratories, definitive standards to produce clinical-grade PL-MSCs are lacking and need, therefore, to be defined. Moreover, it remains to be studied whether the clinical safety and efficacy profile of PL-expanded MSCs is similar to FCS-expanded MSCs. Given the reports of potential transformation of adult human MSCs after ex vivo culture [51– 55], phenotypic, functional and genetic assays should be routinely performed on MSCs before in vivo use to demonstrate whether, after ex vivo expansion, they remain suitable for clinical application. Moreover, surface markers and functional assays to specifically identify MSCs should be further investigated; these tools could allow for the prospective isolation of MSCs from human tissues, as well as to facilitate the generation of homogenous cell products in different laboratories. In particular, novel techniques, such as proteomic approaches and microarray analysis, might be of help for improving the knowledge on MSC expression profile and functional properties and be employed with the aim to identify the true and most primitive mesenchymal progenitors. Once more largely defined in their biological properties and culture-expanded state, MSCs could be properly employed as a therapeutic strategy in various areas of clinical application including modulation of alloimmune responses in the setting of allogeneic HSCT and organ transplantation and promotion of tissue repair in degenerative and inflammatory diseases.


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Practice points Ex vivo expanded MSCs are currently employed to promote hematopoietic engraftment and to treat steroid-refractory acute Graft-versus-Host Disease in patients given an allograft of hematopoietic progenitors, as well as to repair fistulas in patients with Crohn’s Disease. Usually MSCs to be employed in the clinic are cultured in the presence of 10% FCS and harvested at passage 2 to 3 of culture. PL is a suitable alternative culture supplement for MSC ex vivo expansion, able to abrogate the risk of sensitization to heterologous proteins and of transmission of known and unknown zoonoses. The proliferative capacity of MSCs expanded in the presence of PL is superior to that of FCScultured MSC. Characterization of genomic stability is a mandatory requirement for releasing MSC for clinical use.

Research agenda Biological and functional studies aimed at characterizing PL-expanded MSCs, in comparison to FCS-cultured MSCs, are warranted. Clinical trials comparing the safety and efficacy of MSCs obtained in the presence of either FCS or PL must be performed in order to better define the optimal culture medium supplement. Future studies should invest on the characterization of novel markers for the prospective identification/isolation of mesenchymal progenitors in the different human tissues. Extensive investigations on gene expression profile by microarray analyses and proteomic approaches could unravel peculiarities of MSC populations obtained with different culture methods.

Funding This work has been partly supported by grants from Istituto Superiore di Sanità (National Program on Stem Cells), MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca, Progetti di Rilevante Interesse Nazionale, PRIN), from Associazione Italiana per la Ricerca sul Cancro (AIRC) IG6071 and from the special project “5per mille” from AIRC to F.L.; and from AIRC IG9062 to M.E.B. Conflict of interest The authors have no conflict of interest to disclose.

References [1] Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 1968;6:230–47. [2] Friedenstein AJ, Deriglasova UF, Kulagina NN, et al. Precursors for fibroblast in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 1974;2:83–92. [3] Caplan AI. The mesengenic process. Clin Plast Surg 1994;21:429–35. [4] Arai F, Ohneda O, Miyamoto T, et al. Mesenchymal stem cells in perichondrium express activated leukocyte cell adhesion molecule and partecipate in bone marrow formation. J Exp Med 2002;195:1549–63. [5] Im G-I, Shin Y-W, Lee K-B. Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells? Osteoarthr Cartil 2005;13:845–53. [6] Campagnoli C, Roberts IA, Kumar S, et al. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001;98:2396–402.

80


[7] in 't Anker PS, Noort WA, Scherjon SA, et al. Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica 2003;88:845–52. [8] in 't Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004;22:1338–45. [9] in 't Anker PS, Scherjon SA, Kleijburg-van der Keur C, et al. Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 2003;102:1548–9. [10] Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 2000;109: 235–42. [11] Bieback K, Kern S, Kluter H, et al. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 2004;22:625–34. [12] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284:143–7. [13] Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 2005;7:393–5. [14] Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood 2007;110:3499–506. [15] Locatelli F, Maccario R, Frassoni F. Mesenchymal stromal cells, from indifferent spectators to principal actors. Are we going to witness a revolution in the scenario of allograft and immune-mediated disorders? Haematologica 2007;92:872–7. [16] Uccelli A, Pistoia V, Moretta L. Mesenchymal stem cells: a new strategy for immunesuppression? Trends Immunol 2007; 28:219–26. [17] Meisel R, Zibert A, Laryea M, et al. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indolamine 2,3-dioxygenase-mediated tryptophan degradation. Blood 2004;103:4619–21. [18] Maccario R, Podestà M, Moretta A, et al. Interaction of human mesenchymal stem cells with cells involved in alloantigenspecific immune response favours the differentiation of CD4+ T-cell subsets expressing regulatory/suppressive phenotype. Haematologica 2005;90:516–25. [19] Bernardo ME, Locatelli F, Fibbe WE. Mesenchymal stromal cells: a novel treatment modality for tissue repair. Ann N Y Acad Sci 2009;1176:101–17. [20] Ball LM, Bernardo ME, Roelofs H, et al. Co-transplantation of ex-vivo expanded mesenchymal stem cells accelerates lymphocyte recovery and may reduce the risk of graft failure in haploidentical hematopoietic stem cell transplantation. Blood 2007;110:2764–7. [21] Bernardo ME, Ball LM, Cometa AM, et al. Co-infusion of ex vivo expanded, parental mesenchymal stromal cells prevents lifethreatening acute GvHD, but does not reduce the risk of graft failure in pediatric patients undergoing allogeneic umbilical cord blood transplantation. Bone Marrow Tranplant advance online publication; 19 April 2010; doi:10.1038/bmt.2010.87. [22] Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versushost disease: a phase II study. Lancet 2008;371:1579–86. [23] Garcia-Olmo D, Garcia-Arranz M, Herreros D, et al. A phase I clinical trial of the treatment of Crohn’s fistula by adipose mesenchymal stem cell transplantation. Dis Colon Rectum 2005;48:1416–23. [24] Anjos-Afonso F, Bonnet D. Nonhematopoietic/endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchymal compartment. Blood 2007;109:1298–306. [25] Gang EJ, Bosnakovski D, Figueiredo CA, et al. SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood 2007; 109:1743–51. [26] Battula VL, Treml S, Abele H, et al. Prospective isolation and characterization of mesenchymal stem cells from human placenta using a frizzled-9-specific monoclonal antibody. Differentiation 2008;76:326–36. [27] Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991;78:55–62. [28] Quirici N, Soligo D, Bossolasco P, et al. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol 2002;30:783–91. *[29] Buhring HJ, Battula VL, Treml S, et al. Novel markers for the prospective isolation of human MSC. Ann N Y Acad Sci 2007; 1106:262–71. *[30] Battula VL, Treml S, Bareiss PM, et al. Isolation of functionally distinct mesenchymal stem cells subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1 (MSCA-1). Haematologica 2009;94:173–84. [31] Buhring HJ, Treml S, Cerabona F, et al. Phenotypic characterization of distinct human bone marrow-derived MSC subsets. Ann N Y Acad Sci 2009;1176:124–34. [32] Gentry T, Foster S, Winstead L, et al. Simultaneous isolation of human BM hematopoietic, endothelial and mesenchymal progenitor cells by flow sorting based on aldehyde dehydrogenase activity: implications for cell therapy. Cytotherapy 2007;9:259–74. [33] Kuci S, Kuci Z, Kreyenberg H, et al. CD271 antigen defines a subset of multipotent stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. Haematologica 2010;95:651–9. [34] Gronthos S, McCarty R, Mrozik K, et al. Heat shoch protein-90 beta is expressed at the surface of multipotential mesenchymal precursor cells: generation of a novel monoclonal antibody, STRO-4, with specificity for mesenchymal precursor cells from human an ovine tissues. Stem Cells Dev 2009;18:1253–62. [35] Kubo H, Shimuzu M, Taya Y, et al. Identification of mesenchymal stem cell (MSC)-transcription factors by microarray and knockdown analyses, and signature molecule-marked MSC in bone marrow by immunehistochemistry. Genesis 2009; 14:407–24. [36] Song L, Webb NE, Song Y, et al. Identification and functional analysis of candidate genes regulating mesenchymal stem cell self-renewal and multi-potency. Stem Cells 2006;24:1707–18. [37] Wagner W, Wein F, Seckinger A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 2005;33:1402–16. [38] Kaltz N, Ringe J, Holzwarth C, et al. Novel markers of mesenchymal stem cells defined by genome-wide gene epression analysis of stromal cells from different sources. Exp Cell Res 2010;316:2609–17.


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[39] Shahdadfar A, Fronsdal K, Haug T, et al. In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptosome stability. Stem Cells 2005; 23:1357–66. [40] Spees JL, Gregory CA, Singh H, et al. Internalizwed antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy. Mol Ther 2004;9:747–56. *[41] Doucet C, Ernou I, Zhang Y, et al. Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol 2005;205:228–36. *[42] Bernardo ME, Avanzini MA, Perotti C, et al. Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute. J Cell Physiol 2007;211:121–30. [43] Bernardo ME, Avanzini MA, Ciccocioppo R, et al. Phenotypical/functional characterization of in vitro expanded mesenchymal stromal cells from Crohn’s disease patients. Cytotherapy 2009;11:825–36. *[44] Avanzini MA, Bernardo ME, Cometa AM, et al. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypical and functional comparison between umbilical cord blood- and bone marrow-derived progenitors. Haematologica 2009;94:1649–60. *[45] Schallmoser K, Bartmann C, Rohde E, et al. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 2007;47:1436–46. [46] Le Blanc K, Fibbe W. A new cell therapy registry coordinated by the European Group for Blood and Marrow Transplantation (EBMT). Bone Marrow Transplant 2008;41:319–21. [47] Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–13. [48] Jung S, Sen A, Rosenberg L, et al. Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells. Cytotherapy 2010;12:637–57. [49] Chase LG, Lakshmipathy U, Solchaga LA, et al. A novel serum-free medium for the expansion of human mesenchymal stem cells. Stem Cell Res Ther 2010;1:8–11. [50] Schallmoser K, Bartmann C, Rohde H, et al. Replicative senescence-associated gene expression changes in mesenchymal stromal cells are similar under different culture conditions. Haematologica 2010;95:867–72. *[51] Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res 2005; 65:3035–9. [52] Wang Y, Huso DL, Harrington J, et al. Outgrowth of a transformed cell population derived from normal human BM mesenchymal stem cell culture. Cytotherapy 2005;7:509–19. [53] Miura M, Miura Y, Padilla-Nash HM, et al. Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation. Stem Cells 2006;24:1095–103. *[54] Røsland GV, Svendsen A, Torsvik A, et al. Long-term cultures of bone marrow-derived human mesenchymal stem cells frequently undergo spontaneous malignant transformation. Cancer Res 2009;69:5331–9. *[55] Tolar J, Nauta AJ, Osborn MJ, et al. Sarcoma derived from cultured mesenchymal stem cells. Stem Cells 2007;25:371–9. *[56] Bernardo ME, Zaffaroni N, Novara F, et al. Human bone marrow-derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms. Cancer Res 2007; 67:9142–9. *[57] Tarte K, Gaillard J, Lataillade J, et al. Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation. Blood 2010;115:1549–53. *[58] Vogel G. To scientists’s dismay, mixed-up cell lines strike again. Science 2010;329:1004. [59] Torsvik A, Røsland GV, Svendsen A, et al. Spontaneous malignant transformation of human mesenchymal stem cells reflects cross-contamination: putting the research field on track - letter. Cancer Res 2010;70:6393–6.




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Mesenchymal stem cells in ex vivo cord blood expansion Simon N. Robinson, Ph.D., Senior Research Scientist, Department of Stem Cell Transplantation and Cellular Therapy a,1, Paul J. Simmons, Ph.D., Professor and Director, C. Harold and Lorine G. Wallace Distinguished Chair, Professor and Director, Center for Stem Cell Research, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston b, 2, Hong Yang, Ph.D., MD, Senior Research Scientist, Department of Stem Cell Transplantation and Cellular Therapy a, 3, Amin M. Alousi, MD, Assistant Professor, Department of Stem Cell Transplantation and Cellular Therapy a, 4, J. Marcos de Lima, MD, Associate Professor, Department of Stem Cell Transplantation and Cellular Therapy a, 5, Elizabeth J. Shpall, MD, Professor, Stem Cell Transplantation and Cellular Therapy; Medical Director, Cell Therapy Laboratory; Director, Cord Blood Bank a, * a

Department of Stem Cell Transplantation and Cellular Therapy, University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA The Centre for Stem Cell Research, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, 1825 Pressler Street, Houston, Texas 77030. USA b

Keywords: cord blood (CB) transplantation ex vivo expansion mesenchymal stem cells (MSC)

Umbilical cord blood (CB) is becoming an important source of haematopoietic support for transplant patients lacking human leukocyte antigen matched donors. The ethnic diversity, relative ease of collection, ready availability as cryopreserved units from CB banks, reduced incidence and severity of graft versus host disease and tolerance of higher degrees of HLA disparity between donor and recipient, are positive attributes when compared to bone

* Corresponding author. Tel.: þ1 713 745 2161; Fax: þ1 713 794 4902. E-mail addresses: [email protected] (S.N. Robinson), [email protected] (P.J. Simmons), [email protected] (H. Yang), [email protected] (A.M. Alousi), [email protected] (J. Marcos de Lima), [email protected] (E.J. Shpall). 1 Tel.: þ1 713 563 1897; Fax: þ1 713 563 1898. 2 Tel.: þ1 713 500 3427; Fax: þ1 713 500 2424. 3 Tel.: þ1 713 563 1885; Fax: þ1 713 563 1898. 4 Tel.: þ1 713 745 8613; Fax: þ1 713 794 4902. 5 Tel.: þ1 713 745 3219; Fax: þ1 713 794 4902. 1521-6926/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.beha.2010.11.001

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marrow or cytokine-mobilized peripheral blood. However, CB transplantation is associated with significantly delayed neutrophil and platelet engraftment and an elevated risk of graft failure. These hurdles are thought to be due, at least in part, to low total nucleated cell and CD34þ cell doses transplanted. Here, current strategies directed at improving TNC and CD34þ cell doses at transplant are discussed, with particular attention paid to the use of a mesenchymal stem cell (MSC)/CB mononuclear cell ex vivo coculture expansion system. Ó 2010 Elsevier Ltd. All rights reserved.

Background Since the first CB transplant (CBT) was performed by Gluckman et al.[1] in 1988, >20,000 patients have received this procedure to support treatment for a variety of malignant and non-malignant diseases.[2–16] The reported event-free survival rates for such patients are comparable with those achieved following the transplantation of unrelated allogeneic bone marrow (BM), or mobilized peripheral blood progenitor cells (PBPCs).[15] In addition, there are many reports of lower rates of graft versus host disease (GvHD) than are commonly observed with BM and PBPC transplantation, particularly in pediatric patients. This reduced incidence of GvHD is observed despite the use of CB grafts with greater donor-recipient human leukocyte antigen (HLA) mismatching than would be tolerated by recipients of BM,[4,5,9,12] or PBPC allografts.[17–19]

Challenges One major challenge associated with the use of CB for transplantation is the relatively low cell dose available. This is thought to contribute, at least in part, to the slower engraftment and an elevated risk of engraftment failure that is associated with CBT.[20–24] For example, the time required for a patient receiving CBT to achieve an absolute neutrophil count (ANC) of 0.5x109/L can range from 23 to 41 days. Similarly, the median time for a CBT patient to achieve a transfusion-independent platelet count of 20x109/L can range from 56 to >100 days. Further, while engraftment failure rates for CBT recipients as a whole (pediatric and adult CB recipients) can range from 12–20%,[5,9,13,14] those data for adult patients (>18 years old and/or >45 kg) are particularly poor with engraftment failure rates reaching of 20% or higher having been reported.

Evidence of threshold doses for effective transplantation Data from studies performed by Gluckman et al.[9] demonstrated that engraftment and survival were superior in CBT patients who received a transplant dose of 3.7x107 TNC/kg. These data suggest that there is a threshold CB total nucleated cell (TNC) dose above which time to engraftment is improved and graft failure rate reduced and below which time to engraftment is prolonged and graft failure rate increased. However, it is rare that a CBT cell dose of 3.7x107 TNC/kg is achieved. This is particularly true for CBT patients of >45 kg. Additional analyses of these data revealed that a lower, more readily realized target CBT dose ‘threshold’ of 1.0x107 TNC/kg was still associated with favorable engraftment rates and could be applied for this patient population.[2,9] However, analysis of data from patients who received myeloablative therapy and a single CB unit in North America or Europe through 2005 at the world’s three largest CBT registries: Center for International Blood and Marrow Transplant Research (CIBMTR), National Cord Blood Program (NCBP) and Eurocord,[25] revealed that a 100-day treatment-related mortality of approximately 44% was closely correlated with a CBT dose of 100 days. Further, while PBPC recipients have an engraftment failure rate of 400-fold increased TNCs and >20-fold increased CD34þ cells. It should be noted that in some cases, due to the upfront cell losses incurred by the immunomagnetic selection procedure, even marked ex vivo expansion only gives rise to a cell product whose numbers are not markedly different from that of the original CB unit. However, the argument can be made that exposure to the ex vivo expansion cytokine milieu generated a cell product that is different in ‘quality’ to the original CB unit with cells possibly ‘primed’ by the ex vivo exposure to growth factors and subsequently better able to home, engraft and proliferate when transplanted. Mesenchymal stem cell based cultures Ex vivo liquid culture removes the primitive haematopoietic cells from molecular cues provided by the haematopoietic microenvironment. As a consequence the addition of exogenous cytokines is required to prevent apoptosis and stimulate proliferation. An alternative approach is the ex vivo coculture of haematopoietic cells with components of their haematopoietic microenvironment. The haematopoietic microenvironment contains the putative stem cell ‘niche’ and is composed of haematopoietic and non-haematopoietic, cellular and extracellular components thought to provide the complex molecular cues that direct primitive haematopoietic progenitor self-renewal, proliferation and differentiation.[72–80] This would be consistent with the observation that ex vivo contact between primitive haematopoietic progenitors and stromal components of the haematopoietic microenvironment preserve stem cell activity.[81–87] Mesenchymal stem cells (MSC) are one component of the haematopoietic microenvironment and can be isolated from a variety of fetal and adult tissues.[89–92] Phenotypically MSC express CD73, CD90, CD105, CD16, and HLA-ABC(I) and do not express CD31, CD34, CD45, CD80 and HLA-DR(II). MSC can be grown and expanded as adherent, contact-inhibited monolayers in tissue culture flasks, although the primary nature of the cell-type limits the degree of expansion that can be achieved before senescence occurs. Ex vivo CB MNC/MSC co-culture The CB MNC/MSC ex vivo co-culture technique does not require the isolation of CD34þ (or CD133þ) cells from the CB, thereby minimizing the losses associated with this procedure. Using a co-culture strategy with bone marrow-derived MSCs and a supportive growth factor regimen which included Flt3-Ligand (FLT3-L), SCF, GCSF, and TPO, CB MNCs were cultured in 50 ml medium on stroma for 7 days at which time the non-adherent cells were removed in cultured in a larger volume (800 ml) for an additional 7 days. The flasks containing the adherent cells were also re-fed with 50 ml of media and the FLT3L-SCF-GCSF-TPO regimen and cultured for the subsequent 7 days. On day 14 all of the cells from the adherent and non-adherent cultures were pooled for evaluation. Using this strategy a 10–20 fold increase in TNCs, a 7–18-fold increase in committed progenitor cells (colony-forming units, CFU), a 2–5-fold increase in primitive haematopoietic progenitors (high proliferative potential


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colony-forming cells) and a 16–37-fold increase in CD34þ cells was achieved.[88] Building on this experience, a CB MNC/MSC co-culture strategy was developed to maximize the available expanded cells dose for transplant.[89] In the research laboratory, ex vivo expansion culture was characterized by a 6-fold increase in TNC, 30-fold increase in CD133þ cells, 8-fold increase in CD34þ cells, >200-fold increase in CFU, 50-fold increase in cobblestone area-forming cells persisting in culture for 2 weeks (CAFCwk2) and thought to be representative of more mature haematopoietic progenitors, and a reduction in (0.05-fold) cobblestone area-forming cells persisting in culture for 6 weeks (CAFCwk6) and thought to be representative of more primitive haematopoietic progenitors. These CAFCwk6 data provide evidence of the expansion of the more mature haematopoietic progenitors at the expense of the more primitive haematopoietic progenitor cell population. The fold increases cited are over those numbers originally present in the CB unit prior to ex vivo expansion and therefore represent true expansion. Ex vivo CB MNC/MSC co-culture expansion trial at the M. D. Anderson Cancer Center Clinical-scale CB MNC-MSC ex vivo expansion procedures were developed[89] and validated.[90] A trial was subsequently designed to test the clinical feasibility of transplanting the expansion product from CB MNC/MSC co-cultures into patients with haematologic malignancies. The trial, approved by the MD Anderson Institutional Review Board (IRB Protocol 05-0781) and the U.S. Food and Drug Administration (FDA) (IND 13,034), details that patients will receive two CB units matched in at least 4/ 6 HLA antigens, with a minimum dose of 1x107 TNC/kg from each unit. For the initial cohort of 12 patients, a family member (matched at 2/6 antigens) serves as the third party haploidentical MSC donor.[90] Approximately 100 ml of marrow is aspirated from the donor and MSC isolated by plastic adherence. In preparation for co-culture with CB MNC, the MSC are grown to >70% confluence in 10 x T175 culture flasks. The CB unit with the lowest TNC dose is then thawed and divided equally between each of the 10 MSC layers, each in 50 ml of ex vivo expansion media containing 100 ng/ml each of SCF, Flt-3-ligand (Flt-3L), G-CSF and TPO. After 7 days of co-culture at 37 C in a 5% CO2-in-air fully humidified atmosphere, non-adherent cells are removed from each flask and each transferred into individual 10 x 1-liter Teflon-coated culture bags (American Fluoroseal) with fresh ex vivo expansion medium added to generate 800 ml. This liquid culture step (in the absence of MSC) is performed for an additional 7 days (14 days total). The original co-culture flasks also receive 50 mls of fresh medium. At the end of the ex vivo expansion procedure, all non-adherent cells from the culture bags and culture flasks are pooled, washed and prepared for transplantation. Samples are removed for prospective flow cytometric analysis and quality assurance testing. Preliminary clinical results with family member-derived MSCs Patients were admitted on day -9 for hydration and received the designated preparative regimen on days -8 through -2. On day 0, the unmanipulated CB unit was thawed and infused, followed by infusion of the expanded CB cells. Median TNC and CD34þ cell expansions were 12-fold (range, 1–13) and 12fold (range, 1-27), respectively. The mean expanded doses of 5.7x107 TNC/kg and 3.8x105 CD34þ cells/ kg representing important increases compared to those achieved in our previous expansion studies. Furthermore, when the second unmanipulated CB unit is considered, patients on this trial received a total of 9.5x107 TNC/kg and 8.2x105 CD34þ cells/kg. Recipients of myeloablative therapy, engrafted neutrophils in a median of 14.5 days (range, 12–23) and platelets in 30 days (range, 25–51). Rationale for the use of “Off-The-Shelf” MSC for the clinical protocol The complex logistics of generating MSC from a patient’s family member have limited accrual to the clinical trial. Such limitations include: (a) an appropriate family member was not always be available to donate marrow

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(b) disease progression is rapid for selected patients with for example, acute leukemia who were relapsing during the 3 weeks it took to generate sufficient MSCs and 2 weeks to perform the CB MNC/MSC co-culture expansion (delaying transplant for 5 weeks total after patient enrolled on the trial). We postulated that the availability of an “off-the-shelf” source of Good Manufacturing Practice (GMP)-compliant, allogeneic MSC would alleviate this logistical problem with MSC essentially available for immediate use. The development of master cell banks from young, healthy volunteers provides an optimal source of MSC and standardization of selection and isolation procedures ensures a reproducible, readily available MSC product. The Stro-1 antibody, developed by Dr. Paul Simmons,[91] allows prospective isolation of human bone marrow MSC without plastic adherence. Angioblast Systems, Inc., acquired the technology and developed the allogeneic MSC product RevascorÔ for clinical use primarily in the treatment of ischaemic cardiovascular disease.[92–95] To date, more than 20 congestive heart failure patients have received injections of RevascorÔ with no adverse events related to the cells (Dr. Silviu Itescu, Angioblast Systems, Inc., personal communication). Angioblast has agreed to supply RevascorÔ as an ‘off-the-shelf’ product for the M. D. Anderson CB MNC/MSC co-culture expansion trial. Pre-clinical studies of Angioblast-MSC-CB expansion Pre-clinical studies have confirmed that the 10 flasks of MSC required for the ex vivo CB expansion protocol can be routinely generated in 4 days from a single vial (107 cells) of the Angioblast MSC product. Multiple experiments performed to compare the performance of the Angioblast MSC product with that of normal donor-derived MSC have revealed no difference in the expanded CB product generated.[96] Preliminary clinical results with Angioblast-derived MSCs With the pre-clinical Angioblast data described above, the M.D. Anderson CB MNC/MSC ex vivo expansion Protocol 05-0781 and FDA IND 13,034 were amended to include a separate cohort of patients who would be treated identically to the first cohort, but who would receive CB cells expanded on the Angioblast product. Accrual to that cohort has been initiated. During CB MNC-MSC ex vivo expansion, a median expansion of 14-fold (range 1–30) for TNC and 40-fold (range 4-140) for the CD34þ cells was achieved. At transplant, the contribution of the unmanipulated CB included 2.35x107 (range 0.2–8.2) TNC/kg and 0.95 X 105 (range 0–4) CD34þ cells/kg, while the contribution of the ex vivo expanded CB unit was 5.8x107 (range, 0.3–14.4) TNC/kg and 8.7x105 (range, 0-93.4) CD34þ cells/kg. These were higher doses than we have ever infused into any of our recipients of unmanipulated double CBT, or CB expanded with our liquid culture system. As with the family member-derived MSCs, median time to neutrophil engraftment (500/ml) was 15 days (range 9-42) and platelet engraftment (>20,000/ ml) was 38 days (range 13–62) with 26 patients (81%) of patients becoming platelet transfusion independant. On transplant dayþ21, the chimerism assays revealed that the MSC-expanded unit contributed to engraftment with a mean of 19% of the mononuclear cell, 16% of the T cell, and 14% of the myeloid fractions due to the expanded unit. Subsequently, haematopoiesis was increasingly derived from the unexpanded unit with long-term engraftment provided exclusively by the unexpanded unit by six months post-transplant in the vast majority of patients. Summary Our initial CB expansion protocol (#02-407, IND#7166) involved culture of CD133 þ CB cells in teflon bags for 14 days with media containing 100 ng/ml SCF, G-CSF and TPO. In this system the CD133 þ CB cells are initially cultured in bags with 50 ml of media/growth factors for 7 days and then transferred to a bag with 800 ml of fresh media and growth factors for another 7 days at which time they are washed and infused. With this strategy our patients experienced a modest improvement in engraftment of 20 days for neutrophils and 65 days for platelets compared to recipients of double


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unmanipulated CB units who engrafted neutrophils in 22 days and platelets in 100 days. However, we experienced a loss of >70% of the CB CD34þ cells following the CD133-selection procedure, which stimulated us to investigate the MSC-based co-culture system, where we could culture the entire CB unit without need for positive selection. The use of the MSC-based CB expansion protocol (#05-0781) used a similar 14-day strategy where for the first 7 days the cells are cultured in 50 ml, but this time in 10 flasks containing 10% of the CB unit plus MSCs that are 70% or more confluent (rather than the entire CD133þ fraction in one bag with the liquid culture system). The growth factor regimen for this trial included the addition of Flt3-ligand to the SCF-GCSF-TPO regimen. On day 7, the non-adherent cells in each of the 10 flasks are transferred to 10 teflon-coated bags with 800 ml of media and the four growth factors (rather than transfer to a single bag in the liquid culture system with three growth factors). Fifty ml of media/growth factors are added to the 10 flasks containing the adherent layers of MSC-CB, and both those flasks and the 10 bags are cultured for another 7 days. On day 14, all of the cells in the bags and flasks are pooled, washed and infused. This strategy has shown more promising results providing markedly higher TNC and CD34þ cell doses than have ever before been achieved. The improvments are likely due to the use of MSCs to recapitulate the haematopoietic milieu, the ability to culture the whole CB unit rather than the CD133þ fraction, minimizing the large upfront CD34þ cell losses due to the positive selection procedure, and possibly the addition of FLT3-ligand to the cultures. The improvement in median times to engraftment of neutrophils (14 days) and platelets (35 days) are encouraging and accrual to the trial continues. Conflict of interest statement None. Acknowledgements This work was supported in part by NIH Grant #RO1-CA061508-15 and Cancer Prevention & Research Institute of Texas (CPRIT) Grant # RP100469. The authors would like to acknowledge the dedicated researchers, clinicians and nurses of the M. D. Anderson Cancer Center. References *[1] Gluckman E, Devergie A, Bourdeau-Esperou H, et al. Transplantation of umbilical cord blood in Fanconi’s anemia. Nouv Rev Fr Hematol 1990;32:423–5. [2] Wagner JE. Umbilical cord blood stem cell transplantation. Am J Pediatr Hematol Oncol 1993;15:169–74. [3] Wagner JE, Kernan NA, Steinbuch M, et al. Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 1995;346:214–9. [4] Wagner JE, Rosenthal J, Sweetman R, et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood 1996;88:795–802. *[5] Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996;335:157–66. [6] Laporte JP, Gorin NC, Rubinstein P, et al. Cord-blood transplantation from an unrelated donor in an adult with chronic myelogenous leukemia. N Engl J Med 1996;335:167–70. [7] Laporte JP, Lesage S, Portnoi MF, et al. Unrelated mismatched cord blood transplantation in patients with hematological malignancies: a single institution experience. Bone Marrow Transplant 1998;(Suppl 1):S76–7. [8] Cairo MS, Wagner JE. Placental and/or umbilical cord blood: an alternative source of hematopoietic stem cells for transplantation. Blood 1997;90:4665–78. *[9] Gluckman E, Rocha V, Boyer-Chammard A, et al. Outcome of cord-blood transplantation from related and unrelated donors. Eurocord Transplant Group and the European Blood and Marrow Transplantation Group. N Engl J Med 1997;337: 373–81. [10] Locatelli F, Rocha V, Chastang C, et al. Cord blood transplantation for children with acute leukemia. Eurocord Transplant Group. Bone Marrow Transplant 1998;21(Suppl. 3):S63–5. [11] Locatelli F, Rocha V, Chastang C, et al. Factors associated with outcome after cord blood transplantation in children with acute leukemia. Eurocord-Cord Blood Transplant Group. Blood 1999;93:3662–71. [12] Rocha V, Wagner Jr JE, Sobocinski KA, et al. Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. N Engl J Med 2000;342:1846–54. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. [13] Long GD, Laughlin M, Madan B, et al. Unrelated umbilical cord blood transplantation in adult patients. Biol Blood Marrow Transplant 2003;9:772–80.

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[14] Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001;344:1815–22. [15] Barker JN, Davies SM, DeFor T, et al. Survival after transplantation of unrelated donor umbilical cord blood is comparable to that of human leukocyte antigen-matched unrelated donor bone marrow: results of a matched-pair analysis. Blood 2001;97:2957–61. [16] Eapen M, Rubinstein P, Zhang MJ, et al. Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study. Lancet 2007;369:1947–54. [17] Bensinger WI, Clift R, Martin P, et al. Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation. Blood 1996;88:2794–800. [18] Bensinger WI, Weaver CH, Appelbaum FR, et al. Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. Blood 1995;85:1655–8. [19] Bensinger WI, Martin PJ, Storer B, et al. Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med 2001;344:175–81. [20] McGlave P, Bartsch G, Anasetti C, et al. Unrelated donor marrow transplantation therapy for chronic myelogenous leukemia: initial experience of the National Marrow Donor Program. Blood 1993;81:543–50. [21] Kernan NA, Bartsch G, Ash RC, et al. Analysis of 462 transplantations from unrelated donors facilitated by the National Marrow Donor Program. N Engl J Med 1993;328:593–602. [22] Schiller G, Feig SA, Territo M, et al. Treatment of advanced acute leukaemia with allogeneic bone marrow transplantation from unrelated donors. Br J Haematol 1994;88:72–8. [23] Szydlo R, Goldman JM, Klein JP, et al. Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings. J Clin Oncol 1997;15:1767–77. [24] Berthou C, Legros-Maida S, Soulie A, et al. Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes. Blood 1995;85:1540–6. [25] Cohen YC, Scaradavou A, Stevens CE, et al. Factors affecting mortality following myeloablative cord blood transplantation in adults: a pooled analysis of three international registries. Bone Marrow Transplant; 2010. Epub doi:10.1038/bmt.2010.83. [26] Delaney C, Varnum-Finney B, Aoyama K, et al. Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood 2005;106:2693–9. [27] de Lima M, McMannis J, Komanduri K, et al. Randomized study of double cord blood transplantation (CBT) with versus without ex-vivo expansion. Blood 2007;110:2014a. [28] Brunstein CG, Weisdorf DJ, DeFor T, et al. Marked increased risk of Epstein-Barr virus-related complications with the addition of antithymocyte globulin to a nonmyeloablative conditioning prior to unrelated umbilical cord blood transplantation. Blood 2006;108:2874–80. [29] Barker JN, Weisdorf DJ, Wagner JE. Creation of a double chimera after the transplantation of umbilical-cord blood from two partially matched unrelated donors. N Engl J Med 2001;344:1870–1. [30] Barker J, Defor T, Davies S. Impact of multiple unit unrelated donor umbilical cord blood transplantation in adults: Preliminary analysis of safety and efficacy. Blood 2001;98:666a. [31] Kurtzberg J, Balber A, Mendizabal A. Preliminary results of a pilot trial of unrelated umbilical cord blood transplantation (UCBT) augmented with cytokine-primed aldehyde dehydrogenase-bright cells (ALDHbr). Blood 2006;108:3641a. [32] Shpall E, de Lima M, Chan K. Transplantation of cord blood expanded ex vivo with copper chelator. Blood 2004;104: 281a. *[33] Barker JN, Weisdorf DJ, DeFor TE, et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 2005;105:1343–7. [34] Broxmeyer HE, Hangoc G, Cooper S, et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 1992;89:4109–13. [35] Haylock DN, To LB, Dowse TL, et al. Ex vivo expansion and maturation of peripheral blood CD34þ cells into the myeloid lineage. Blood 1992;80:1405–12. [36] Haylock DN, To LB, Dowse TL, et al. Ex vivo expansion of hematopoietic stem cells. The Stem Cell Manual. Alpha Med Press; 1994. pp291–301. [37] Simmons PJ, Haylock DN. Use of hematopoietic growth factors for in vitro expansion of precursor cell populations. Curr Opin Hematol 1995;2:189–95. [38] Haylock DN, Horsfall MJ, Dowse TL, et al. Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand. Blood 1997;90:2260–72. [39] Makino S, Haylock DN, Dowse T, et al. Ex vivo culture of peripheral blood CD34þ cells: effects of hematopoietic growth factors on production of neutrophilic precursors. J Hematother 1997;6:475–89. *[40] Prince HM, Simmons PJ, Whitty G, et al. Improved haematopoietic recovery following transplantation with ex vivoexpanded mobilized blood cells. Br J Haematol 2004;126:536–45. [41] Andrews RG, Briddell RA, Hill R, et al. Engraftment of primates with G-CSF mobilized peripheral blood CD34þ progenitor cells expanded in G-CSF, SCF and MGDF decreases the duration and severity of neutropenia. Stem Cells 1999;17:210–8. [42] Boiron JM, Dazey B, Cailliot C, et al. Large-scale expansion and transplantation of CD34(þ) hematopoietic cells: in vitro and in vivo confirmation of neutropenia abrogation related to the expansion process without impairment of the longterm engraftment capacity. Transfusion 2006;46:1934–42. [43] McAlister IB, Teepe M, Gillis S, Williams DE. Ex vivo expansion of peripheral blood progenitor cells with recombinant cytokines. Exp Hematol 1992;20:626–8. [44] Purdy MH, Hogan CJ, Hami L, et al. Large volume ex vivo expansion of CD34-positive hematopoietic progenitor cells for transplantation. J Hematother 1995;4:515–25. [45] Dick JE, Guenechea G, Gan OI, et al. In vivo dynamics of human stem cell repopulation in NOD/SCID mice. Ann N Y Acad Sci 2001;938:184–90. [46] Guenechea G, Gan OI, Dorrell C, et al. Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat Immunol 2001;2:75–82.


91

[47] Lemischka IR, Jordan CT. The return of clonal marking sheds new light on human hematopoietic stem cells. Nat Immunol 2001;2:11–2. [48] Hogan CJ, Shpall EJ, Keller G. Differential long-term and multilineage engraftment potential from subfractions of human CD34þ cord blood cells transplanted into NOD/SCID mice. Proc Natl Acad Sci U S A 2002;99:413–8. [49] Williams DA. Ex vivo expansion of hematopoietic stem and progenitor cells–robbing Peter to pay Paul? Blood 1993;81: 3169–72. [50] McNiece IK, Almeida-Porada G, Shpall EJ, Zanjani E. Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential. Exp Hematol 2002;30:612–6. *[51] McNiece I, Jones R, Bearman SI, Cagnoni P, et al. Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer. Blood 2000;96:3001–7. [52] Shimizu Y, Ogawa M, Kobayashi M, et al. Engraftment of cultured human hematopoietic cells in sheep. Blood 1998;91: 3688–92. [53] Tisdale JF, Hanazono Y, Sellers SE, et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 1998;92:1131–41. [54] Abkowitz JL, Taboada MR, Sabo KM, et al. The ex vivo expansion of feline marrow cells leads to increased numbers of BFU-E and CFU-GM but a loss of reconstituting ability. Stem Cells 1998;16:288–93. [55] Von Drygalski A, Alespeiti G, Ren L, et al. Murine bone marrow cells cultured ex vivo in the presence of multiple cytokine combinations lose radioprotective and long-term engraftment potential. Stem Cells Dev 2004;13:101–11. [56] Peters SO, Kittler EL, Ramshaw HS, et al. Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts. Blood 1996;87:30–7. [57] Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ. Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts. Exp Hematol 1995;23:461–9. [58] Holyoake TL, Alcorn MJ, Richmond L, et al. CD34 positive PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens. Bone Marrow Transplant 1997;19:1095–101. [59] Piacibello W, Sanavio F, Severino A, et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(þ) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999;93:3736–49. [60] Lewis ID, Almeida-Porada G, Du J, Lemischka IR, et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood 2001;97: 3441–9. [61] Guenechea G, Segovia JC, Albella B, Lamana M, et al. Delayed engraftment of nonobese diabetic/severe combined immunodeficient mice transplanted with ex vivo-expanded human CD34(þ) cord blood cells. Blood 1999;93:1097–105. [62] Traycoff CM, Cornetta K, Yoder MC, et al. Ex vivo expansion of murine hematopoietic progenitor cells generates classes of expanded cells possessing different levels of bone marrow repopulating potential. Exp Hematol 1996;24:299–306. [63] Muench MO, Moore MA. Accelerated recovery of peripheral blood cell counts in mice transplanted with in vitro cytokineexpanded hematopoietic progenitors. Exp Hematol 1992;20:611–8. [64] Holyoake TL, Freshney MG, McNair L, et al. Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow. Blood 1996;87:4589–95. [65] Zhai QL, Qiu LG, Li Q, et al. Short-term ex vivo expansion sustains the homing-related properties of umbilical cord blood hematopoietic stem and progenitor cells. Haematologica 2004;89:265–73. [66] Tian H, Huang S, Gong F, et al. Karyotyping, immunophenotyping, and apoptosis analyses on human hematopoietic precursor cells derived from umbilical cord blood following long-term ex vivo expansion. Cancer Genet Cytogenet 2005; 157:33–6. [67] Briddell RA, Kern BP, Zilm KL, et al. Purification of CD34þ cells is essential for optimal ex vivo expansion of umbilical cord blood cells. J Hematother 1997;6:145–50. [68] McNiece IK, Stoney GB, Kern BP, et al. CD34þ cell selection from frozen cord blood products using the Isolex 300i and CliniMACS CD34 selection devices. J Hematother 1998;7:457–61. [69] de Lima M, McMannis J, Gee A, et al. Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial. Bone Marrow Transplant 2008;41:771–8. *[70] Shpall EJ, Quinones R, Giller R, et al. Transplantation of ex vivo expanded cord blood. Biol Blood Marrow Transplant 2002; 8:368–76. [71] McNiece I, Kubegov D, Kerzic P, et al. Increased expansion and differentiation of cord blood products using a two-step expansion culture. Exp Hematol 2000;28:1181–6. [72] Lemischka IR, Moore KA. Stem cells: interactive niches. Nature 2003;425:778–9. [73] Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stem cells and their niche. Cell 2004;116:769–78. [74] Hackney JA, Charbord P, Brunk BP, et al. A molecular profile of a hematopoietic stem cell niche. Proc Natl Acad Sci U S A 2002;99:13061–6. [75] Etheridge SL, Spencer GJ, Heath DJ, et al. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells 2004;22:849–60. [76] Kadereit S, Deeds LS, Haynesworth SE, et al. Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(þ)/CD38(-) early progenitors cultured over human MSCs as a feeder layer. Stem Cells 2002;20:573–82. [77] Rattis FM, Voermans C, Reya T. Wnt signaling in the stem cell niche. Curr Opin Hematol 2004;11:88–94. [78] Zhang J, Niu C, Ye L, et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003; 425:836–41. [79] Majumdar MK, Keane-Moore M, Buyaner D, et al. Characterization and functionality of cell surface molecules on human mesenchymal stem cells. J Biomed Sci 2003;10:228–41. [80] Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells. J Cell Physiol 1998;176:57–66.

92


[81] Yildirim S, Boehmler AM, Kanz L, Mohle R. Expansion of cord blood CD34þ hematopoietic progenitor cells in coculture with autologous umbilical vein endothelial cells (HUVEC) is superior to cytokine-supplemented liquid culture. Bone Marrow Transplant 2005;36:71–9. [82] Breems DA, Blokland EA, Siebel KE, et al. Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34þ mobilized peripheral blood stem cells. Blood 1998;91:111–7. [83] Brandt JE, Galy AH, Luens KM, et al. Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line. Exp Hematol 1998;26:950–61. [84] Rafii S, Shapiro F, Rimarachin J, et al. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 1994;84:10–9. [85] Rafii S, Shapiro F, Pettengell R, et al. Human bone marrow microvascular endothelial cells support long-term proliferation and differentiation of myeloid and megakaryocytic progenitors. Blood 1995;86:3353–63. [86] Chute JP, Saini AA, Chute DJ, et al. Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow. Blood 2002;100:4433–9. [87] Davis TA, Robinson DH, Lee KP, et al. Porcine brain microvascular endothelial cells support the in vitro expansion of human primitive hematopoietic bone marrow progenitor cells with a high replating potential: requirement for cell-tocell interactions and colony-stimulating factors. Blood 1995;85:1751–61. *[88] McNiece I, Harrington J, Turney J, et al. Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells. Cytotherapy 2004;6:311–7. *[89] Robinson SN, Ng J, Niu T, et al. Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells. Bone Marrow Transplant 2006;37:359–66. [90] de Lima M, McMannis JD, Hosing CM, et al. Rapid engraftment of neutrophils and platelets with mesenchymal stem cell based cord blood expansion. Blood 2009;114:3394a. *[91] Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991;78:55–62. [92] Martens TP, See F, Schuster MD, et al. Mesenchymal lineage precursor cells induce vascular network formation in ischemic myocardium. Nat Clin Pract Cardiovasc Med 2006;3(Suppl 1):S18–22. [93] Hasnat AK. van d, V, Hon JK, Yacoub MH. Reproducible model of post-infarction left ventricular dysfunction: haemodynamic characterization by conductance catheter. Eur J Cardiothorac Surg 2003;24:98–104. [94] Moainie SL, Gorman III JH, Guy TS, et al. An ovine model of postinfarction dilated cardiomyopathy. Ann Thorac Surg 2002; 74:753–60. [95] Charles CJ, Elliott JM, Nicholls MG, et al. Myocardial infarction with and without reperfusion in sheep: early cardiac and neurohumoral changes. Clin Sci 2000;98:703–11. [96] Robinson SN, Simmons PJ, Brouard N, et al. Efficacy of off-the-shelf, commercially-available, third-party mesenchymal stem cells (MSC) in ex vivo cord blood (CB) co-culture expansion. Blood 2007;110:4106a.

Best Practice & Research Clinical Haematology Vol. 24, No. 1, pp. i1, 2011

Keyword Index autoimmune diseases, 49 Bone marrow stromal cell, 37 cell therapy, 49 cellular therapy, 73 central nervous system (CNS): neurons, 59 cord blood (CB) transplantation, 83 experimental autoimmune encephalomyelitis (EAE), 59 ex vivo expansion, 73, 83

Mesenchymal stem cell, 37 mesenchymal stem cells (MSC), 59, 83 Mesenchymal stem cells, 49, 65 mesenchymal stroma cell (MSC), 13 mesenchymal stromal cells, 73 mesenchymal/stromal stem cells, 25 microglia, 59 MSC subsets, 25 MSC, 25 multipotent differentiation, 13 neuroprotection, 59

fetal calf serum, 73

oligodendrocytes, 59

Graft-versus-host disease, 65

pericyte adventitial reticular cell, 13 platelet lysate, 73 pluripotency, 3 prospective isolation, 25

Hematopoietic niche, 37 Hematopoietic stem cell transplantation, 65 Hematopoietic stem cell, 37 Hemorrhagic cystitis, 65 inflammation, 49 malignant transformation, 73 MAPC, 3 mesenchymal progenitor cell (MPC), 13

reprogramming, 3 sca-1, 13 surface antigens, 25 Tissue toxicity, 65 vascular smooth muscle cell (VSMC), 13

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