Stem Cell Mobilization

METHODS IN MOLECULAR BIOLOGY™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfie...

Author: Mikhail G. Kolonin and Paul J. Simmons

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METHODS

IN

MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

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Stem Cell Mobilization Methods and Protocols

Edited by

Mikhail G. Kolonin and Paul J. Simmons Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA

Editors Mikhail G. Kolonin Institute of Molecular Medicine University of Texas Health Science Center at Houston Houston, TX, USA

Paul J. Simmons Institute of Molecular Medicine University of Texas Health Science Center at Houston Houston, TX, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-61779-942-6 ISBN 978-1-61779-943-3 (eBook) DOI 10.1007/978-1-61779-943-3 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2012943077 © Springer Science+Business Media, LLC 2012 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com)

Preface Initially noted as a phenomenon accompanying the recovery of patients from myelosuppressive chemotherapy, stem cell mobilization represents a transient increase in the levels of circulating stem and progenitor cells. Observation of progenitor cell mobilization following the administration of certain factors, such as G-CSF, heralded a new era in hematological cell therapies with “mobilized blood” now essentially replacing bone marrow as the tissue of choice for hematopoietic reconstitution in cancer therapy. There is considerable interest in the phenomenon of mobilization in terms of understanding the underlying molecular mechanisms that drive the process of cell egress from the bone marrow. Recent studies have also revealed mobilization of progenitor cells from organs other than the bone marrow, although the importance of this phenomenon and the extent to which extramedullary cells contribute to the mobilized pool are yet to be understood. The notion that progenitor cell mobilization results in systemic redistribution of several cell populations that may participate in repair and regeneration has considerable clinical implications. While recruitment of systemically circulating stem cells may be beneficial in bone marrow reconstitution or wound healing settings, progenitor trafficking to lesions in cancer or other fibrotic conditions could have adverse effects. Therefore, development of reliable methods to quantify trafficking and read out activity of individual precursor cell types is highly important. This book aims to overview the current standing in cell mobilization methodology and to outline recent developments in the field for basic and biomedical research community. Specifically, clinical hematopoietic progenitor cell mobilization protocols and the experimental techniques used in animal models are covered in Chapters 1–11. The remaining part of the book addresses the frontiers in mobilization and analysis of non-hematopoietic progenitors, with specific emphases on endothelial progenitor cells (Chapters 12–14), mesenchymal progenitor cells (Chapters 15 and 20), monocyte-derived fibroblast progenitors (Chapter 16), and very small embryonic-like cells (Chapter 17). Advanced methodologies to analyze physiological and pathological functions of these distinct progenitor populations are also described (Chapters 14–15, 18–19). Houston, TX, USA

Mikhail G. Kolonin Paul J. Simmons

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Contents Preface ..................................................................................................................... Contributors............................................................................................................. 1 Mobilization of Hematopoietic Stem/Progenitor Cells: General Principles and Molecular Mechanisms................................................... Halvard Bonig and Thalia Papayannopoulou 2 Quantifying Hematopoietic Stem and Progenitor Cell Mobilization .................. Shiri Gur-Cohen, Kfir Lapid, and Tsvee Lapidot 3 Hematopoietic Stem Cell Mobilization with G-CSF .......................................... Chitra Hosing 4 Hematopoietic Stem Cell Mobilization with Agents Other than G-CSF ............. Jonathan Hoggatt and Louis M. Pelus 5 Hematopoietic Stem Cell Mobilization: A Clinical Protocol .............................. Gina Pesek and Michele Cottler-Fox 6 Monitoring Blood for CD34+ Cells to Determine Timing of Hematopoietic Progenitor Cells Apheresis..................................................... M. Louette Vaughn and Edmund K. Waller 7 Hematopoietic Progenitor Cell Collection ........................................................ S. Darlene Marlow and Myra House 8 Managing Apheresis Complications During the Hematopoietic Stem Cell Collection ......................................................................................... S. Darlene Marlow and Myra House 9 Hematopoietic Progenitor Cell Apheresis Processing ......................................... Eleanor S. Hamilton and Edmund K. Waller 10 Toxicities of Mobilized Stem Cell Infusion ........................................................ Jonathan L. Kaufman 11 Mobilization of Hematopoietic Stem Cells by Depleting Bone Marrow Macrophages ....................................................................................... Valérie Barbier, Ingrid G. Winkler, and Jean-Pierre Lévesque 12 Combinatorial Stem Cell Mobilization in Animal Models .................................. Simon C. Pitchford and Sara M. Rankin 13 Vascular Progenitor Cell Mobilization ............................................................... Kirsten A. Kienstra and Karen K. Hirschi 14 Evaluation of Circulating Endothelial Precursor Cells in Cancer Patients ........... Francesco Bertolini, Patrizia Mancuso, Liat Benayoun, Svetlana Gingis-Velitski, and Yuval Shaked

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Contents

15 Tracking Inflammation-Induced Mobilization of Mesenchymal Stem Cells ........ Erika L. Spaeth, Shannon Kidd, and Frank C. Marini 16 Differentiation of Circulating Monocytes into Fibroblast-Like Cells .................. Darrell Pilling and Richard H. Gomer 17 Enumeration of Very Small Embryonic-Like Stem Cells in Peripheral Blood ...... Rui Liu and Mariusz Z. Ratajczak 18 Generation of a Vascular Niche for Studying Stem Cell Homeostasis ................. Jason M. Butler and Shahin Rafii 19 Studying Vascular Progenitor Cells in a Neonatal Mouse Model ........................ Kirsten A. Kienstra and Karen K. Hirschi 20 Progenitor Cell Mobilization from Extramedullary Organs ................................ Mikhail G. Kolonin

173 191 207 221 235 243

Index ................................................................................................................................ 253

Contributors VALÉRIE BARBIER • Mater Medical Research Institute, Aubigny Place, Raymond Terrace, South Brisbane, QLD, Australia LIAT BENAYOUN • Department of Molecular Pharmacology, Technion–Israel Institute of Technology, Haifa, Israel FRANCESCO BERTOLINI • Laboratory of Hematology-Oncology, European Institute of Oncology, Milan, Italy HALVARD BONIG • Department of Medicine/Hematology, University of Washington, Seattle, WA, USA JASON M. BUTLER • Weill Cornell Medical College and the Howard Hughes Medical Institute, Cornell University, New York, NY, USA SHIRI GUR-COHEN • Department of Immunology, Weizmann Institute of Science, Rehovot, Israel MICHELE COTTLER-FOX • Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA SVETLANA GINGIS-VELITSKI • Department of Molecular Pharmacology, Rappaport Faculty of Medicine, Technion–Israel Institute of Technology, Haifa, Israel RICHARD H. GOMER • Texas A&M University, College Station, TX, USA ELEANOR S. HAMILTON • Cellular Therapies Laboratory, Emory University Hospital, Atlanta, GA, USA KAREN K. HIRSCHI • Division of Neonatology, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA JONATHAN HOGGATT • Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA CHITRA HOSING • Department of Stem Cell Transplantation and Cell Therapy, M.D. Anderson Cancer Center, Houston, TX, USA MYRA HOUSE • Center for Transfusion and Cellular Therapies (CTCT), Emory University Hospital, Atlanta, GA, USA JONATHAN L. KAUFMAN • Hematology and Medical Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA SHANNON KIDD • Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA KIRSTEN A. KIENSTRA • Division of Neonatology, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA MIKHAIL G. KOLONIN • Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA KFIR LAPID • Department of Immunology, Weizmann Institute of Science, Rehovot, Israel TSVEE LAPIDOT • Department of Immunology, Weizmann Institute of Science, Rehovot, Israel JEAN-PIERRE LÉVESQUE • Mater Medical Research Institute, South Brisbane, QLD, Australia RUI LIU • Developmental Biology Program, James Graham Brown Cancer Center, University of Louisville Louisville, KY USA

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PATRIZIA MANCUSO • Laboratory of Hematology-Oncology, European Institute of Oncology, Milan, Italy FRANK C. MARINI • Institute for Regenerative Medicine, Comprehensive Cancer Center, Wake Forest University, Medical Center Blvd. Winston-Salem, NC S. DARLENE MARLOW • Center for Transfusion and Cellular Therapies, Emory University Hospital, Atlanta, GA, USA THALIA PAPAYANNOPOULOU • Department of Medicine/Hematology, University of Washington, Seattle, WA, USA LOUIS M. PELUS • Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA GINA PESEK • Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA DARRELL PILLING • Texas A&M University, College Station, TX, USA SIMON C. PITCHFORD • Leukocyte Biology Section, Faculty of Medicine National Heart and Lung Institute, Imperial College London, London, UK SHAHIN RAFII • Weill Cornell Medical College and the Howard Hughes Medical Institute, Cornell University, New York, NY, USA SARA M. RANKIN • Leukocyte Biology Section, Faculty of Medicine National Heart and Lung Institute, Imperial College London, London, UK MARIUSZ Z. RATAJCZAK • Developmental Biology Program, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA YUVAL SHAKED • Department of Molecular Pharmacology, Technion-Israel Institute of Technology, Haifa, Israel PAUL J. SIMMONS • Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA ERIKA L. SPAETH • Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA M. LOUETTE VAUGHN • Center for Transfusion and Cellular Therapies, Emory University Hospital, Atlanta, GA, USA EDMUND K. WALLER • Cellular Therapies Laboratory, Emory University Hospital, Atlanta, GA, USA INGRID G. WINKLER • Mater Medical Research Institute, Aubigny Place, Raymond Terrace, South Brisbane, QLD, Australia

Chapter 1 Mobilization of Hematopoietic Stem/Progenitor Cells: General Principles and Molecular Mechanisms Halvard Bonig and Thalia Papayannopoulou Abstract Hematopoietic stem/progenitor cell mobilization can be achieved by a variety of bone marrow niche modifications, although efficient mobilization requires simultaneous expansion of the stem/progenitor cell pool and niche modification. Many of the mechanisms involved in G-CSF-induced mobilization have been described. With regard to mobilization of hematopoietic stem/progenitor cells, challenges for the future include the analysis of genetic factors responsible for the great variability in mobilization responses, and the identification of predictors of mobilization efficiency, as well as the development of mobilizing schemes for poor mobilizers. Moreover, improved regimens for enhanced or even preferential mobilization of nonhematopoietic stem/progenitor cell types, and their therapeutic potential for endogenous tissue repair will be questions to be vigorously pursued in the near future. Key words: G-CSF, Mobilization, Hematopoietic stem/progenitor cell

1. Introduction Although mature hematopoietic cells are physiologically released from bone marrow to peripheral blood, their immature counterparts are found in circulation in very low frequencies. An enforced egress, referred to as “mobilization,” of a modest proportion of the latter cells from bone marrow to peripheral blood can be enacted by a variety of systemic “stressors.” Stem cell mobilization was uncovered mostly through empiric observations rather than rationally designed treatments. Why and how stem/progenitor cells physiologically escape the BM environment is not entirely clear, but it is very likely that the process of mobilization makes use of physiological molecular pathways leading to mobilization.

Mikhail G. Kolonin and Paul J. Simmons (eds.), Stem Cell Mobilization: Methods and Protocols, Methods in Molecular Biology, vol. 904, DOI 10.1007/978-1-61779-943-3_1, © Springer Science+Business Media, LLC 2012

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H. Bonig and T. Papayannopoulou

The considerable scientific interest in mobilization of immature cells is fuelled by its clinical relevance. Its importance in autologous repair mechanisms was demonstrated when after partial irradiation radiation-depleted marrow is repopulated from noncontiguous nonirradiated marrow sites, presumably by itinerant stem cells (1). Quantitatively, however, of greater clinical relevance at the current time, is the collection of mobilized cells by apheresis, enabling allogeneic transfer or temporary cryopreservation of autologous stem/progenitor cells for hematopoietic “stem cell” transplantation (2, 3). Protocols for several mobilization approaches are reported in this book and several recent comprehensive reviews have been published on clinical aspects or the cellular and molecular mechanisms of mobilization (4–8). This minireview focuses on issues relevant to G-CSF mobilization, because of its unique clinical importance and the plethora of studies on G-CSF mobilized cells. Mobilization by some other modalities is touched upon only because of their mechanistic insight and because they may display a synergistic or additive activity with G-CSF.

2. General Mobilization Principles

Under steady-state conditions, stem/progenitor cell location is almost exclusively restricted to the marrow, where these cells apparently reside in specific, supportive microenvironments (9–11). Environmental cues from stromal cells or matrix could influence cell fate, and are, under resting conditions, also responsible for their firm retention in the marrow. Active egress of stem/progenitor cells from bone marrow could be the default response when their restraining mechanisms are released, i.e., the HSPC could be inherently nomadic unless restrained. While this may appear to be a philosophical issue, the answer to this question could allow for a rational development of mobilizing agents. Currently available data on stem cell mobilization suggest that indeed the breakdown of retention mechanisms is sufficient for mobilization. Several common properties of mobilized hematopoietic cells have been emphasized irrespective of the mobilizing agent. Thus, mobilized immature cells are predominantly noncycling, in contrast to the cells left behind in the marrow (12–14), they express little VCAM-1, and low levels of many integrins (14–16). Specifically data generated with fast-acting mobilizing agents suggest that these phenotypic changes precede egress of cells from marrow, suggesting in turn that these properties are prerequisites for mobilization, rather than changes induced by the milieu in the peripheral blood (15). Likewise, gene expression patterns of mobilized immature subsets have been described; they differ markedly

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Mobilization of Hematopoietic Stem/Progenitor Cells…

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from their counterparts residing in unstimulated marrow (17, 18). Thus, in CD34+ cells from G-CSF mobilized blood, myeloid genes and cell cycle-associated genes were relatively up-regulated. These changes likely indicate differences in the heterogeneous mix of cells contained in the CD34+ fraction, entirely compatible with known effects of G-CSF, rather than necessarily pointing to molecular events involved in mobilization. In agreement with that, an extensive body of evidence has accumulated on differences in the ratio between primitive and more mature hematopoietic subsets, depending on the mobilizing agent. Thus, several publications have commented that AMD3100-alone mobilized immature cells are, on average, more functionally and phenotypically primitive than G-CSF- or G-CSF + AMD3100-mobilized ones (19, 20), resembling more closely the distribution in a steady-state marrow. This observation may be explained by the relative skewing of a G-CSF stimulated marrow towards less primitive (more mature) cells, i.e., the mobilized fractions are representative of marrow contents at the time of mobilization. As was reported many years ago, a G-CSF mobilized marrow is relatively depleted of immature hematopoietic subsets, and the marrow does not assume its normal cellular composition for several weeks after discontinuation of G-CSF (21). The precise locations from which mobilized immature cells originate, or the exact site of their egress, are not clear. A reasonable proposition is that egress into blood would require apposition to medullary blood vessels, most likely to medullary venous sinusoids. Mobilization by G-CSF is associated with a relative depletion of periosteal niches of hematopoietic stem cells, migration of stem cells to vascular niches where much of the proliferation occurs (5), followed by egress of both mature and immature subsets. With chemokine-induced mobilization the rapid kinetics likely do not allow for migration across significant distances, which may explain the relatively lower potency, and the synergism between G-CSF and AMD3100 (15). Of interest, data generated with the Gi protein inhibitor Pertussis toxin, which renders hematopoietic cells completely incapable of migration (22), clearly show that the ability to migrate is not a critical capacity of a mobilizable cell, i.e., that stem/progenitor cell pools might reside on the luminal side of medullary blood vessels. In aggregate, these data may indicate that although mobilized cells would at some time cross perivascular pools before they exit the bone marrow, they could initially originate from other bone marrow locations further away and that such movement to the perivascular space increases the number of mobilizable cells. How large is the fraction of stem cells that can be induced to leave the marrow? At first glance, extrapolation from the mouse model indicates that the efficiency of mobilization with G-CSF might be modest. After a similar mobilization regime as in humans

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(nine doses q12h) several thousand CFU-C per mL of blood, i.e., no more than 10,000 CFU-C in total, will be in circulation. The number of circulating CFU-C after G-CSF in the mouse (10,000 in a C57Bl/6 mouse, more in some other strains) must be compared against a CFU-C content of approximately 60,000 per steady-state (unmobilized) femur (15), which is estimated to represent 1/16 of the total marrow mass (23). Thus, the total number of CFU-C of the mouse is one million, 100 times the number that is found in circulation after G-CSF. However, this may not necessarily mean that only 1% of “stem cells” are mobilized by G-CSF, since other relevant variables in the equation are completely unfathomable. The transit time of mobilized cells is elusive (minutes to a few hours have been suggested) (24, 25), and their fate has not been completely elucidated. In other words, once the cells are in circulation, how long do they remain there, and when they leave the circulation, how many home back to marrow or are lost to other organs is unclear. Conceivably, many circulating cells could interact with and be siphoned off by nontarget organs. In that case the true number of mobilized cells would be much higher than the number in circulation suggests. What is the evidence for such “steal” effects? Experimental evidence has been provided that the spleen of a G-CSF mobilized mouse accumulates significant numbers of immature cells, so that mobilization of splenectomized mice is more pronounced (26). Trafficking of mobilized immature cells through the intestinal lymphoid system and to adipose tissue has also been shown (27, 28). The possibility that this likewise pertains to other organs must be entertained. Since unlike the spleen, other organs do not support immature hematopoietic cells, it is difficult to experimentally address this issue with currently available technology, but again, tracking experiments in transplanted animals demonstrate accumulation of progenitor cells in nonhematopoietic tissues (29). Thus, reliable estimates of the potency of G-CSF mediated mobilization cannot be given. How is mobilization quantified? Ultimately, the cell of interest in the context of hematopoietic cell mobilization is the stem cell. Yet the stem cell is defined functionally, as a cell capable of selfrenewal and long-term multilineage reconstitution in an appropriately conditioned host. It must be remembered that any other “stem cell” enumeration assay than the long-term engraftment assay (30) is measuring some surrogate parameter, so many caveats must be considered when interpreting the results of such assays. In addition to its tediousness, even a stem cell assay (limiting dilution transplantation and readout of long-term engraftment) has its limitations, since it tests at the same time stemness and transplantrelated properties like homing, niche-integration, retention, etc. Thus, if cells which would be capable of self-renewal and longterm repopulation in terms of their epigenetic status, i.e., are bona fide stem cells, are impaired in their ability to interact with the

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niche or to proliferate, the number of stem cells might be underestimated. In vitro colony assays have been used by most to quantify mobilization, or to compare mobilization efficiency (30–33). Since cells giving rise to colonies in colony assays are progenitor cells, i.e., more mature specimen, CFU-assays are not a true measure of stem cell mobilization. However, cumulative evidence indicates that during progenitor cell mobilization stem cells are always comobilized, but the relative frequency among the immature cells may vary, depending on the mobilizing agent. Thus, the CFU-C assay may be the most practicable assay for assessment of mobilization, but its shortcomings must be born in mind. Phenotypic analyses of “stem cells” using more or less complex surface marker panels have also been used. These assays are most problematic, because mobilizing agents can induce changes in surface phenotype (e.g., c-kit expression on immature cells is all but suppressed on G-CSF mobilized cells); thus, the stem cell phenotype of a stem cell in a steady-state marrow is likely different from that in mobilized peripheral blood (34, 35). With less complex surface marker panels (e.g., CD45/CD34), the relative mix between primitive and more mature subsets contained in this phenotypically defined, yet functionally heterogeneous population is not considered and can lead to misinterpretations of stem cell mobilization efficiency.

3. Mobilization by G-CSF The clinically most relevant mobilizing agent, G-CSF, expands the number of stem cells at the same time that it induces proliferation/ maturation towards the granulocytic lineage, and it causes marked alterations in the hematopoietic stroma in the marrow. Together these changes result in the release, or mobilization, of hematopoietic stem/progenitor cells. It seems clear that the summation of expansion and mobilization is responsible for the rather potent mobilization efficiency of G-CSF compared to other mobilizing agents. In humans, after a conventional course of G-CSF (5 μg/kg every 12 h, nine total doses) the number of circulating progenitor cells is increased approximately 60-fold, to 60–100 CD34+ cells/μL. Preliminary data indicate that other types of immature cells are comobilized alongside hematopoietic stem/progenitor cells, including endothelial and mesenchymal stroma cells (36–38). It is not unreasonable to hypothesize that the same changes which cause hematopoietic stem/progenitor cell mobilization are also involved in mobilization of these other stem cell specimen, but definitive data are lacking.

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Recently, significant advances have been made with respect to the molecular and cellular events involved in G-CSF mediated hematopoietic stem/progenitor cell mobilization. Informative data document that mobilization is not a direct effect of G-CSF on the stem cell proper. The receptor for G-CSF, G-CSFR, is conspicuously absent from hematopoietic stem cells (39). Indirect cues must therefore be responsible for numeric and spatial changes in the stem cell population. Recent data by the Link and Levesque laboratories suggest a chain of events starting with G-CSF mediated stimulation of certain marrow-resident macrophages which appear to relay signals to osteoblasts (also G-CSFR negative), which then downregulate SDF-1 gene transcripts (40–42). Proteolytic cleavage of SDF-1 off of stromal binding sites has also been demonstrated and functionally implicated, as truncation of SDF-1, resulting in nonfunctional SDF-1 molecules can compete with full-length SDF-1 for CXCR4 binding sites. The cellular and molecular architecture of a G-CSF treated bone marrow is significantly changed compared to a steady-state marrow. For instance, cleavage of a number of surface-bound chemokines, cytokines, receptors, etc. has been demonstrated. Some evidence has been provided that these changes are the work of proteases, which are elaborated during G-CSF mobilization, together with down regulation of protease-inhibitors during mobilization. However, the critical role of MMP9 emphasized in some studies (43) has not been confirmed, and even mice deficient in a whole panel of proteases responded to G-CSF with the expected efficiency (44). Further, deficiency in CD26 is associated with impaired mobilization by G-CSF (45). CD26 is a broad dipeptidase that (among many other putative target molecules) cleaves SDF-1 into a nonfunctional variant, which competes with SDF-1 for CXCR4 binding. It was proposed that the inability to cleave SDF-1 was responsible for the attenuated G-CSF responsiveness of the CD26deficient mice (45). At this point in time, a definitive contributory role of other proteases to mobilization cannot be pinpointed. As the marrow is exposed to G-CSF and the described profound changes in marrow architecture are happening, HSPC expand in regions located more centrally and closer to the blood vessel. Data from the Levesque laboratory suggest that this is at least in part a reflection of (a) greater oxygen needs of proliferating cells and (b) greater oxygen consumption in a proliferating marrow, i.e., during G-CSF stimulation, HSPC move towards higher oxygen concentrations (46). The potent mobilizing activity of certain chemotherapy drugs like cyclophosphamide has been solely attributed to endogenous G-CSF, since G-CSFR deficient mice treated with cyclophosphamide show the expected rebound proliferation in marrow, but egress of immature cells from marrow is virtually absent (47). The mechanisms involved in mobilization by cyclophosphamide would then likely be the same as during mobilization with exogenous G-CSF.

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4. G-CSFEnhancing and Alternative Activities in Mobilization


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Several cytokines (including GM-CSF, FLT3 ligand and SCF, the c-kit ligand) mobilize HSPC and synergize with G-CSF in stem cell mobilization (47–50). These modalities have in common similarly slow kinetics as G-CSF mobilization, suggesting a combined effect of proliferation and mobilization, as with G-CSF. Some clinical data with GM-CSF and SCF, the latter predominantly in combination with G-CSF, have been reported, but their clinical relevance is modest. The mechanisms of mobilization with these cytokines have not been studied in any detail. Considering the role of the coagulation/complement cascade in hematopoietic cell trafficking (51–53) and the strong activation of this system by GM-CSF (54), a contribution of this pathway is conceivable. A different group of mobilizing agents has gained a lot of attention in the last few years, namely, CXCR4 antagonists of various chemistries. The effectiveness of this intervention has been demonstrated in mice, monkeys, dogs and humans. One CXCR4 antagonist, the bicyclam AMD3100 (Mozobil, Plerixafor) is licensed for clinical mobilization in combination with G-CSF + chemotherapy for patients failing to adequately mobilize with G-CSF + chemotherapy alone (15, 31, 55–60). Preliminary data from the Di Persio laboratory, reported at the 2010 ISBT meeting, indicate that when given alone, as with G-CSF, other species of immature cells are also comobilized by CXCR4 antagonists, although their nature has not been definitively elucidated. The mechanism of action of CXCR4 antagonists appears to be interference between stromal SDF-1 and CXCR4 on the HSPC surface. The kinetics is rapid, quite unlike those of G-CSF, and no conclusive evidence has been provided that these CXCR4 inhibitors elicit changes in the hematopoietic niche. Proliferation is not a feature of mobilization with CXCR4 antagonists, which was thought to explain the relatively low potency of these inhibitors. Preliminary data from our group indicates, however, that novel, more potent CXCR4 inhibitors can exceed the mobilization achieved with a 5-day course of twice-daily G-CSF, at least in mice (unpublished data). The frequency of stem cells in CXCR4-antagonist mobilized grafts among the cells with an immature phenotype is greater than after G-CSF. It appears that this reflects the frequencies within a steady-state marrow as opposed to a G-CSF-treated marrow, so this observation should not be surprising. Clearly mobilization with CXCR4 antagonists argues against a hypothesis put forth about mechanisms of G-CSF mobilization, i.e., inversion of an SDF-1 gradient, where mobilization of HSPC would be in response to greater concentrations outside the marrow than inside (supposedly because SDF-1 is cleaved from the stroma, to circulate in blood and bone marrow fluid). Because

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of the very short half-life of SDF-1 in plasma, this seemed unlikely, but definitive evidence against this hypothesis comes from the following observation: since CXCR4 antagonists effectively block SDF-1 directed migration, yet potently synergize with (rather than antagonize) G-CSF mobilization (15), it is clear that in mobilization cells do not respond to SDF-1, but are on the contrary made temporarily unresponsive. Data about mobilization with CXCR4 agonists are in line with this hypothesis (61), since they lead to downregulation of CXCR4 surface expression on HSPC. Thus, the SDF-1-CXCR4 axis acts as a retention pathway, which is disturbed by various means in mobilization with G-CSF and CXCR4 antagonists or agonists. Data generated in mice transplanted with CXCR4 deficient hematopoietic cells are in agreement with these observations (62). Considering that several authors have proposed interference of the CXCR4/SDF-1 pathway as the mechanism of action of G-CSF mediated mobilization, the well-documented synergism between G-CSF and CXCR4 antagonists is surprising. The simplest explanation may be (1) that after G-CSF, this pathway is only partially obstructed and (2) that CXCR4 antagonists may find a larger population to mobilize in a G-CSF treated marrow than in an untreated one, because of expansion of the pool and of relocation to perivascular regions. The inhibitor of Gi protein signals, including of SDF-1/ CXCR4 signals, Pertussis toxin was reported to elicit potent and protracted HSPC mobilization. Specifically, Pertussis toxin also synergized with G-CSF induced mobilization (22). Why Gi protein blockade leads to mobilization is not clear. While irrelevant from a clinical perspective, the implications for mobilization mechanics are of interest. Pertussis toxin mobilized HSPC are incapable of migration. This suggests that activation of migratory signals may not be required for mobilization. Similarly, GRO-β induced mobilization is seen despite the fact that it inhibits migration in vitro (32). These data could be interpreted to indicate that mobilizable pools of HSPC reside not in the marrow immediately adjacent to bone surfaces, CAR cells and other stromal elements, but in regions adjacent to the venous sinuses in marrow. Such a location would be equally compatible with the rapid kinetics of IL-8, GRO-β and CXCR4 antagonists—in either case distant transmarrow migration might not be feasible within the relevant time frame. Fenestrae in the septum segregating the spaces between the marrow space and the venous sinuses have been described as the site of passage of mature cells into blood. Conceivably, these fenestrae could also be used by immature hematopoietic cells during mobilization. This hypothesis would also be compatible with the observation that HSPC in G-CSF treated marrow are preferentially located close to blood vessels (46).

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VLA4 is an adhesion molecule expressed on HSPC. It is normally present in its low-affinity conformation, but affinity can be induced by a number of cytokines, and it changes during cell cycle transit (14). Like CXCR4 and SDF-1, VLA4 finds cognate ligands in the marrow stroma, including, but potentially not limited to, VCAM1, fibronectin, and osteopontin, thus serving as a stem cell retention pathway. Studies in mice, nonhuman primates or humans, using genetic, small-molecule- or antibody-mediated ablation of VLA4 or of several of its ligands in marrow have demonstrated mobilization of HSPC into peripheral blood (31, 63–67). The kinetics follow an intermediate time course. Although reduced expression of VLA4 is also a feature of G-CSF mobilized HSPC (14–16), suggesting VLA4 downregulation as another mechanism of G-CSF mobilization, VLA4 inhibition or genetic deletion was synergistic or at least superadditive with G-CSF. Similarly to what we postulated for synergism between CXCR4 antagonists and G-CSF, interference of G-CSF induced events with VLA4-mediated adhesion is likely incomplete, while direct targeting of the molecule is complete. As we have shown, VLA4 blockade is effective at mobilizing HSPC in mice, monkeys, and humans, albeit with low potency (31, 64, 68). As an indication that VLA4-inhibition and CXCR4 blockade are mobilizing HSPC by independent mechanisms, we and others have demonstrated synergism of the two modalities in monkeys and mice (31, 67). Lower VLA4 expression was also observed on HSPC mobilized with CXCR4 antagonists and with a variety of other mobilizing agents (15). This could either indicate downregulation of VLA4 under the influence of mobilizing agents and preferential mobilization of these VLA4dim cell populations, or assumption of a VLA4-dim phenotype during the transition from marrow to blood. Two other chemokines, GRO-β and IL-8, mobilize HSPC with very rapid kinetics. With respect to GRO-β, it was shown that this mobilization was dependent on MMP9, suggesting that the target cell may be a mature neutrophil (32, 69). For IL-8, contradictory results about the role of MMP9 have been reported, yet a dependence on G-CSFR likewise suggests a role for mediators released from mature neutrophils (47, 70, 71). The proposed chain of events leading to mobilization for these two molecules is granulocyte degranulation, release of proteases, severing of retention factors, resulting in stem cell release. Several other examples of stroma or niche modification have also been reported which resulted in stem cell mobilization. Several of these involved modification of stromal ligands for established retention factors. Examples include very different substances, such as Fucoidan, which displays competitive displacement of chemokines, including SDF-1 (which is present in the stem cell niche as a surface-bound molecule) (72, 73), anti-VCAM-1 antibodies (74)

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and VCAM-1-deleted (75) or osteopontin-deleted (33) mice, which blocked/ablated relevant VLA4 ligands in the stroma. Other mobilizing agents likely exerted their effects indirectly, through induction of endogenous G-CSF (e.g., parathyroid hormone) (76). Moreover, a variety of mediators have been associated with mobilization that seemingly have very little in common, and where the molecular mechanics are sometimes poorly defined. These include sympathomimetics (77, 78), cannabinoid receptor agonists (79), complement (52, 53), elevated lipoprotein levels (80), defibrotide (81), glycosaminoglycans (82), and endotoxin (83). None of these have gained any clinical relevance, but the abundance of mobilizing agents indicates the precariousness of the equilibrium between marrow retention and egress, and may in the future support the rational development of mobilizing strategies for poorly mobilizing patients, or for individuals who are intolerant to G-CSF. References 1. Nothdurft W, Kreja L (1998) Hemopoietic progenitor cells in the blood as indicators of the functional status of the bone marrow after totalbody and partial-body irradiation: experiences from studies in dogs. Stem Cells 16(Suppl 1): 97–111 2. Holig K, Kramer M, Kroschinsky F, Bornhauser M, Mengling T, Schmidt AH et al (2009) Safety and efficacy of hematopoietic stem cell collection from mobilized peripheral blood in unrelated volunteers: 12 years of single-center experience in 3928 donors. Blood 114:3757–3763 3. Chao NJ, Schriber JR, Grimes K, Long GD, Negrin RS, Raimondi CM et al (1993) Granulocyte colony-stimulating factor “mobilized” peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy. Blood 81:2031–2035 4. Levesque JP, Winkler IG (2008) Mobilization of hematopoietic stem cells: state of the art. Curr Opin Organ Transplant 13:53–58 5. Levesque JP, Helwani FM, Winkler IG (2010) The endosteal ‘osteoblastic’ niche and its role in hematopoietic stem cell homing and mobilization. Leukemia [E-pub 2010 Sep 23] 6. Papayannopoulou T, Scadden DT (2008) Stem-cell ecology and stem cells in motion. Blood 111:3923–3930 7. Pelus LM (2008) Peripheral blood stem cell mobilization: new regimens, new cells, where do we stand. Curr Opin Hematol 15:285–292 8. Greenbaum AM, Link DC (2010) Mechanisms of G-CSF-mediated hematopoietic stem and progenitor mobilization. Leukemia [E-pub 2010 Nov 16]

9. Lymperi S, Ferraro F, Scadden DT (2010) The HSC niche concept has turned 31. Has our knowledge matured? Ann N Y Acad Sci 1192: 12–18 10. Oh IH, Kwon KR (2010) Concise review: multiple niches for hematopoietic stem cell regulations. Stem Cells 28:1243–1249 11. Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA et al (2010) Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466:829–834 12. Scott MA, Apperley JF, Bloxham DM, Jestice HK, John S, Marcus RE et al (1997) Biological properties of peripheral blood progenitor cells mobilized by cyclophosphamide and granulocyte colony-stimulating factor. Br J Haematol 97:474–480 13. Williams CD, Linch DC, Watts MJ, Thomas NS (1997) Characterization of cell cycle status and E2F complexes in mobilized CD34+ cells before and after cytokine stimulation. Blood 90:194–203 14. Yamaguchi M, Ikebuchi K, Hirayama F, Sato N, Mogi Y, Ohkawara J et al (1998) Different adhesive characteristics and VLA-4 expression of CD34(+) progenitors in G0/G1 versus S + G2/M phases of the cell cycle. Blood 92:842–848 15. Bonig H, Chudziak D, Priestley G, Papayannopoulou T (2009) Insights into the biology of mobilized hematopoietic stem/ progenitor cells through innovative treatment schedules of the CXCR4 antagonist AMD3100. Exp Hematol 37:402–415

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16. Prosper F, Stroncek D, McCarthy JB, Verfaillie CM (1998) Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function. J Clin Invest 101: 2456–2467 17. Graf L, Heimfeld S, Torok-Storb B (2001) Comparison of gene expression in CD34+ cells from bone marrow and G-CSF-mobilized peripheral blood by high-density oligonucleotide array analysis. Biol Blood Marrow Transplant 7:486–494 18. Steidl U, Kronenwett R, Rohr UP, Fenk R, Kliszewski S, Maercker C et al (2002) Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells. Blood 99:2037–2044 19. Fruehauf S, Veldwijk MR, Seeger T, Schubert M, Laufs S, Topaly J et al (2009) A combination of granulocyte-colony-stimulating factor (G-CSF) and plerixafor mobilizes more primitive peripheral blood progenitor cells than G-CSF alone: results of a European phase II study. Cytotherapy 11:992–1001 20. Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett PA et al (1995) Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 201:1307–1318 21. Drize N, Chertkov J, Samoilina N, Zander A (1996) Effect of cytokine treatment (granulocyte colony-stimulating factor and stem cell factor) on hematopoiesis and the circulating pool of hematopoietic stem cells in mice. Exp Hematol 24:816–822 22. Papayannopoulou T, Priestley GV, Bonig H, Nakamoto B (2003) The role of G-protein signaling in hematopoietic stem/progenitor cell mobilization. Blood 101:4739–4747 23. Katayama Y, Hidalgo A, Furie BC, Vestweber D, Furie B, Frenette PS (2003) PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and alpha4 integrin. Blood 102:2060–2067 24. Raghavachar A, Steinbach KH, Prummer O, Grilli G, Fliedner TM (1983) Survival of transfused cryopreserved granulocytic progenitor cells (CFU-C) in recipient circulation. Cell Tissue Kinet 16:303–311 25. Wright DE, Wagers AJ, Gulati AP, Johnson FL, Weissman IL (2001) Physiological migration of hematopoietic stem and progenitor cells. Science 294:1933–1936 26. Molineux G, Pojda Z, Dexter TM (1990) A comparison of hematopoiesis in normal and

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splenectomized mice treated with granulocyte colony-stimulating factor. Blood 75:563–569 27. Han J, Koh YJ, Moon HR, Ryoo HG, Cho CH, Kim I et al (2010) Adipose tissue is an extramedullary reservoir for functional hematopoietic stem and progenitor cells. Blood 115:957–964 28. Massberg S, Schaerli P, Knezevic-Maramica I, Kollnberger M, Tubo N, Moseman EA et al (2007) Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and peripheral tissues. Cell 131:994–1008 29. Watanabe T, Kajiume T, Takaue Y, Kawano Y, Kanamaru S, Okamura S et al (2001) Decrease in circulating hematopoietic progenitor cells by trapping in the pulmonary circulation. Cytotherapy 3:461–466 30. Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ (1990) Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc Natl Acad Sci USA 87:8736–8740 31. Bonig H, Watts KL, Chang KH, Kiem HP, Papayannopoulou T (2009) Concurrent blockade of alpha4-integrin and CXCR4 in hematopoietic stem/progenitor cell mobilization. Stem Cells 27:836–837 32. Fukuda S, Bian H, King AG, Pelus LM (2007) The chemokine GRObeta mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment. Blood 110:860–869 33. Grassinger J, Haylock DN, Storan MJ, Haines GO, Williams B, Whitty GA et al (2009) Thrombin-cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with alpha9beta1 and alpha4beta1 integrins. Blood 114:49–59 34. Randall TD, Weissman IL (1997) Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment. Blood 89:3596–3606 35. Rollini P, Faes-Van’t HE, Kaiser S, Kapp U, Leyvraz S (2007) Phenotypic and functional analysis of human fetal liver hematopoietic stem cells in culture. Stem Cells Dev 16:281–296 36. Kassis I, Zangi L, Rivkin R, Levdansky L, Samuel S, Marx G et al (2006) Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads. Bone Marrow Transplant 37:967–976 37. Tatsumi K, Otani H, Sato D, Enoki C, Iwasaka T, Imamura H et al (2008) Granulocyte-colony stimulating factor increases donor mesenchymal stem cells in bone marrow and their mobilization into peripheral circulation but does not repair dystrophic heart after bone marrow transplantation. Circ J 72:1351–1358

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38. Zubair AC, Malik S, Paulsen A, Ishikawa M, McCoy C, Adams PX et al (2010) Evaluation of mobilized peripheral blood CD34(+) cells from patients with severe coronary artery disease as a source of endothelial progenitor cells. Cytotherapy 12:178–189 39. Liu F, Poursine-Laurent J, Link DC (2000) Expression of the G-CSF receptor on hematopoietic progenitor cells is not required for their mobilization by G-CSF. Blood 95:3025–3031 40. Semerad CL, Christopher MJ, Liu F, Short B, Simmons PJ, Winkler I et al (2005) G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow. Blood 106:3020–3027 41. Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, et al (2010) Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSC. Blood [E-pub 2010 Aug 16] 42. Christopher MJ, Liu F, Hilton MJ, Long F, Link DC (2009) Suppression of CXCL12 production by bone marrow osteoblasts is a common and critical pathway for cytokine-induced mobilization. Blood 114:1331–1339 43. Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR et al (2002) Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 109:625–637 44. Levesque JP, Liu F, Simmons PJ, Betsuyaku T, Senior RM, Pham C et al (2004) Characterization of hematopoietic progenitor mobilization in protease-deficient mice. Blood 104:65–72 45. Christopherson KW, Cooper S, Hangoc G, Broxmeyer HE (2003) CD26 is essential for normal G-CSF-induced progenitor cell mobilization as determined by CD26−/− mice. Exp Hematol 31:1126–1134 46. Winkler IG, Barbier V, Wadley R, Zannettino AC, Williams S, Levesque JP (2010) Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: serially reconstituting hematopoietic stem cells reside in distinct nonperfused niches. Blood 116: 375–385 47. Liu F, Poursine-Laurent J, Link DC (1997) The granulocyte colony-stimulating factor receptor is required for the mobilization of murine hematopoietic progenitors into peripheral blood by cyclophosphamide or interleukin-8 but not flt-3 ligand. Blood 90:2522–2528 48. Brasel K, McKenna HJ, Charrier K, Morrissey PJ, Williams DE, Lyman SD (1997) Flt3 ligand synergizes with granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor to mobilize

hematopoietic progenitor cells into the peripheral blood of mice. Blood 90:3781–3788 49. Gianni AM, Siena S, Bregni M, Tarella C, Stern AC, Pileri A et al (1989) Granulocytemacrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet 2:580–585 50. Molineux G, Migdalska A, Szmitkowski M, Zsebo K, Dexter TM (1991) The effects on hematopoiesis of recombinant stem cell factor (ligand for c-kit) administered in vivo to mice either alone or in combination with granulocyte colony-stimulating factor. Blood 78:961–966 51. Ratajczak J, Reca R, Kucia M, Majka M, Allendorf DJ, Baran JT et al (2004) Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow. Blood 103:2071–2078 52. Wilschut IJ, Erkens-Versluis ME, Ploemacher RE, Benner R, Vos O (1979) Studies on the mechanism of haemopoietic stem cell (CFUs) mobilization. A role of the complement system. Cell Tissue Kinet 12:299–311 53. Molendijk WJ, van Oudenaren A, van Dijk H, Daha MR, Benner R (1986) Complement split product C5a mediates the lipopolysaccharideinduced mobilization of CFU-s and haemopoietic progenitor cells, but not the mobilization induced by proteolytic enzymes. Cell Tissue Kinet 19:407–417 54. Bonig H, Burdach S, Gobel U, Nurnberger W (2001) Growth factors and hemostasis: differential effects of GM-CSF and G-CSF on coagulation activation—laboratory and clinical evidence. Ann Hematol 80:525–530 55. Burroughs L, Mielcarek M, Little MT, Bridger G, Macfarland R, Fricker S et al (2005) Durable engraftment of AMD3100-mobilized autologous and allogeneic peripheral-blood mononuclear cells in a canine transplantation model. Blood 106:4002–4008 56. Larochelle A, Krouse A, Metzger M, Orlic D, Donahue RE, Fricker S et al (2006) AMD3100 mobilizes hematopoietic stem cells with longterm repopulating capacity in nonhuman primates. Blood 107:3772–3778 57. Devine SM, Vij R, Rettig M, Todt L, McGlauchlen K, Fisher N et al (2008) Rapid mobilization of functional donor hematopoietic cells without G-CSF using AMD3100, an antagonist of the CXCR4/SDF-1 interaction. Blood 112:990–998 58. Devine SM, Flomenberg N, Vesole DH, Liesveld J, Weisdorf D, Badel K et al (2004) Rapid mobilization of CD34+ cells following administration of the CXCR4 antagonist

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AMD3100 to patients with multiple myeloma and non-Hodgkin’s lymphoma. J Clin Oncol 22:1095–1102 59. DiPersio JF, Stadtmauer EA, Nademanee A, Micallef IN, Stiff PJ, Kaufman JL et al (2009) Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients with multiple myeloma. Blood 113:5720–5726 60. Liles WC, Broxmeyer HE, Rodger E, Wood B, Hubel K, Cooper S et al (2003) Mobilization of hematopoietic progenitor cells in healthy volunteers by AMD3100, a CXCR4 antagonist. Blood 102:2728–2730 61. Hattori K, Heissig B, Tashiro K, Honjo T, Tateno M, Shieh JH et al (2004) Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells. Blood 97:3354–3360 62. Foudi A, Jarrier P, Zhang Y, Wittner M, Geay JF, Lecluse Y et al (2006) Reduced retention of radioprotective hematopoietic cells within the bone marrow microenvironment in CXCR4−/− chimeric mice. Blood 107:2243–2251 63. Craddock CF, Nakamoto B, Elices M, Papayannopoulou T (1997) The role of CS1 moiety of fibronectin in VLA mediated haemopoietic progenitor trafficking. Br J Haematol 97:15–21 64. Craddock CF, Nakamoto B, Andrews RG, Priestley GV, Papayannopoulou T (1997) Antibodies to VLA4 integrin mobilize longterm repopulating cells and augment cytokineinduced mobilization in primates and mice. Blood 90:4779–4788 65. Papayannopoulou T, Craddock C, Nakamoto B, Priestley GV, Wolf NS (1995) The VLA4/ VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci USA 92:9647–9651 66. Papayannopoulou T, Priestley GV, Nakamoto B (1998) Anti-VLA4/VCAM-1-induced mobilization requires cooperative signaling through the kit/mkit ligand pathway. Blood 91:2231–2239 67. Ramirez P, Rettig MP, Uy GL, Deych E, Holt MS, Ritchey JK et al (2009) BIO5192, a small molecule inhibitor of VLA-4, mobilizes hematopoietic stem and progenitor cells. Blood 114:1340–1343 68. Bonig H, Wundes A, Chang KH, Lucas S, Papayannopoulou T (2008) Increased numbers of circulating hematopoietic stem/progenitor cells are chronically maintained in patients treated with the CD49d blocking antibody natalizumab. Blood 111:3439–3441

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69. King AG, Horowitz D, Dillon SB, Levin R, Farese AM, MacVittie TJ et al (2001) Rapid mobilization of murine hematopoietic stem cells with enhanced engraftment properties and evaluation of hematopoietic progenitor cell mobilization in rhesus monkeys by a single injection of SB-251353, a specific truncated form of the human CXC chemokine GRObeta. Blood 97:1534–1542 70. Laterveer L, Lindley IJ, Hamilton MS, Willemze R, Fibbe WE (1995) Interleukin-8 induces rapid mobilization of hematopoietic stem cells with radioprotective capacity and long-term myelolymphoid repopulating ability. Blood 85:2269–2275 71. Laterveer L, Lindley IJ, Heemskerk DP, Camps JA, Pauwels EK, Willemze R et al (1996) Rapid mobilization of hematopoietic progenitor cells in rhesus monkeys by a single intravenous injection of interleukin-8. Blood 87:781–788 72. Sweeney EA, Papayannopoulou T (2001) Increase in circulating SDF-1 after treatment with sulfated glycans. The role of SDF-1 in mobilization. Ann N Y Acad Sci 938:48–52 73. Sweeney EA, Lortat-Jacob H, Priestley GV, Nakamoto B, Papayannopoulou T (2002) Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells. Blood 99:44–51 74. Kikuta T, Shimazaki C, Ashihara E, Sudo Y, Hirai H, Sumikuma T et al (2000) Mobilization of hematopoietic primitive and committed progenitor cells into blood in mice by anti-vascular adhesion molecule-1 antibody alone or in combination with granulocyte colony-stimulating factor. Exp Hematol 28:311–317 75. Ulyanova T, Priestley GV, Nakamoto B, Jiang Y, Papayannopoulou T (2007) VCAM-1 ablation in nonhematopoietic cells in MxCre + VCAM1f/f mice is variable and dictates their phenotype. Exp Hematol 35:565–571 76. Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC et al (2003) Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425:841–846 77. Maestroni GJ, Conti A (1994) Modulation of hematopoiesis via alpha 1-adrenergic receptors on bone marrow cells. Exp Hematol 22: 313–320 78. Katayama Y, Battista M, Kao WM, Hidalgo A, Peired AJ, Thomas SA et al (2006) Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell 124:407–421 79. Jiang S, Alberich-Jorda M, Zagozdzon R, Parmar K, Fu Y, Mauch P, et al (2010) Cannabinoid receptor 2 and its agonists mediate

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hematopoiesis and hematopoietic stem and progenitor cell mobilization. Blood [E-pub 2010 Nov 9] 80. Gomes AL, Carvalho T, Serpa J, Torre C, Dias S (2010) Hypercholesterolemia promotes bone marrow cell mobilization by perturbing the SDF-1:CXCR4 axis. Blood 115:3886–3894 81. Carlo-Stella C, Di NM, Magni M, Longoni P, Milanesi M, Stucchi C et al (2002) Defibrotide in combination with granulocyte colony-stimulating factor significantly enhances the mobilization of primitive and committed peripheral

blood progenitor cells in mice. Cancer Res 62:6152–6157 82. Albanese P, Caruelle D, Frescaline G, Delbe J, Petit-Cocault L, Huet E et al (2009) Glycosaminoglycan mimetics-induced mobilization of hematopoietic progenitors and stem cells into mouse peripheral blood: structure/ function insights. Exp Hematol 37:1072–1083 83. Cline MJ, Golde DW (1977) Mobilization of hematopoietic stem cells (CFU-C) into the peripheral blood of man by endotoxin. Exp Hematol 5:186–190

Chapter 2 Quantifying Hematopoietic Stem and Progenitor Cell Mobilization Shiri Gur-Cohen, Kfir Lapid, and Tsvee Lapidot Abstract Allogeneic donor blood cells and autologous peripheral blood leukocytes (PBL), obtained following clinical mobilization procedures, are routinely used as a major source of hematopoietic stem and progenitor cells (HSPC) for transplantation protocols. It is, therefore, essential to evaluate and to quantify the extent by which the HSPC are mobilized and enriched in the circulation in correlation with their longterm hematopoietic reconstitution capacity. In this chapter, we describe quantitative methods that measure the number of mobilized HSPC according to specific criteria, as well as their functional properties in vitro and in vivo. The described assays are useful for assessment of progenitor cell mobilization as applied to both human and murine HSPC. Key words: Mobilization, G-CSF, Hematopoietic stem and progenitor cells, SDF-1/CXCR-4

1. Introduction The process by which adult stem and progenitor cells are recruited from their supportive microenvironment during stress conditions and enter the blood stream is defined as mobilization. The major organ in which repopulating hematopoietic stem and progenitor cells (HSPC) reside is the bone marrow (BM); however, low levels of HSPC can also be localized in distinct organs, such as the spleen, liver, and muscle (reviewed in Schulz et al. (1)). Although the vast majority of progenitor cells reside in the BM reservoir of immature and maturing leukocytes, a small amount of HSPC continuously egress from the BM to the peripheral blood (PB). These small numbers of circulating HSPC can be dramatically increased during stress conditions, including injury, mild bleeding, physical exercise, inflammation due to bacterial and viral infections, and DNA


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damage (reviewed in Lapidot and Kollet (2)). Several clinical protocols mimicking stress conditions due to alarm situations were developed to enable the use of PB mobilized HSPC, rather than direct retrieval of the cells from the bones, in order to reconstitute hematopoiesis in ablated patients. A wide array of mobilizing agents have been discovered in the previous decades, including chemokines, cytokines, and chemotherapeutic agents, among which the most commonly used is the cytokine granulocyte-colony stimulating factor (G-CSF) either alone or following chemotherapy (3). The chemokine SDF-1 (also termed CXCL12) and its major receptor CXCR-4 play key roles in both maintenance and motility of HSPC. Of interest, the SDF-1/CXCR-4 signaling axis is extensively studied in the context of the mobilization process, HSPC homing to the BM and steady-state egress (4). The interactions between SDF-1 and CXCR-4 are crucial for balancing retained HSPC in a quiescent noncycling mode in the BM microenvironment (5, 6). More than two decades ago, the BM stromal microenvironment which robustly expresses SDF-1, was revealed as a negative regulator of HSPC proliferation and differentiation, believed to be a part of the mechanism to preserve quiescent HSPC pool in the BM (7). Maintenance of HSPC retention in the BM involves adhesion interactions and signals, which are mediated by BM stromal supporting cells (reviewed in Wilson et al. (8)). Tight regulation of SDF-1 in the BM promotes the expression of adhesion molecules such as VLA-4/5, LFA-1 (9), and CD44 (10), which mediate HSPC attachment to BM matrix components and stromal supporting cells. Breaking the fine-tuned SDF-1/CXCR-4 balance under stress conditions allows HSPC proliferation, differentiation and subsequent mobilization (11). The major roles of SDF-1 in HSPC mobilization can be inferred from the observation that, following G-CSF treatment, the levels of SDF-1 mRNA (12) and protein (11–13) are dramatically reduced in the BM, while the levels of CXCR-4 are increased, enabling motility of HSPC toward the blood (11). Indeed, increased plasma levels of SDF-1 attract HSPC and thereby increase cell mobilization to the circulation (14). Active disruption of SDF-1/CXCR-4 dynamic interactions by the CXCR-4 antagonist AMD3100 serves as another clinically used protocol to induce HSPC mobilization (15). While G-CSF induced mobilization requires repetitive daily stimulations, AMD3100 is a rapid mobilizing agent that triggers HSPC recruitment to the circulation within a few hours after administration (15, 16). Of interest, combined treatment of G-CSF and AMD3100 synergistically augment HSPC recruitment to the circulation (15, 17). Stress-induced mobilization is a multievent process that involves activation of bone-resorbing osteoclasts (18, 19), regulation of osteoblasts by β-adrenergic signals (20), neutrophil activation (11), proteolytic enzyme activity (21, 22), reactive oxygen species (ROS) signaling (23) and various cytokines and chemokines. All of these factors facilitate the detachment of HSPC from

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Quantifying Hematopoietic Stem and Progenitor Cell Mobilization

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their anchored microenvironment, their proliferation and differentiation, migration through the mechanical barrier of the bone-blood endothelial barrier and finally HSPC recruitment to the blood circulation (11, 24–26). Recently, interactions between SDF-1 and CXCR-4 were demonstrated by a rare mesenchymal stem cell (MSC) subpopulation identified by Nestin expression, which represent approximately 4% of the murine CD45− BM nonhematopoietic cells and 0.08% of total BM cells. Nestin+ MSC, functionally defined by exclusive colony-forming units-fibroblast (CFU-F) content in the murine BM, were found to express very high levels of SDF-1 and are physically associated with SLAM CD150+ CD41− CD48− hematopoietic stem cells (HSC). Interestingly, transplanted HSC rapidly home and lodge in proximity to mesenchymal Nestin+ stem cells, whereas in vivo depletion of Nestin+ cell significantly reduces the homing capacity of transplanted HSPC. Selective inhibition of SDF-1 transcription by Nestin+ MSC cells and suppression of their proliferation during G-CSF stimuli exemplifies the disruption of the SDF-1/CXCR-4 axis during HSPC mobilization (27). Not only HSPC undergo mobilization, but also other immature cell types, including endothelial progenitor cells (EPC) (28–30). Stromal progenitor cells (or CFU-F) were also found to undergo expansion as a consequence of increased bone turnover due to accelerated osteoclast activity during G-CSF induced mobilization (31). Notably, both BM derived HSPC and EPC have significant clinical benefits, share similarities and overlapping mobilization mechanisms, involving altered balance of the SDF-1/CXCR-4 interactions and RANKL induced osteoclast activation (28–30). In order to evaluate mobilization of immature leukocytes, several in vivo and in vitro assays have been developed for characterization and quantification of both progenitors and more primitive hematopoietic stem cell levels. Herein, we describe in vitro techniques and the use of functional in vivo models to assess murine, as well as a preclinical model for human HSPC mobilization in immune deficient NOD/SCID chimeric mice.

2. Materials 2.1. HSPC Marker Characteristics: Flow Cytometry (FACS) Assay

1. FACS buffer: DPBS−/− 10× (Dulbecco’s Phosphate Buffered Saline 10×, without calcium and magnesium) supplemented with heat-inactivated 5% fetal calf serum (FCS) and 0.1% sodium azide. 2. Antibodies: For murine SKL staining-FITC conjugated antilineage markers (CD4, NK, GR-1, B220, CD8a, and CD11b— additional markers can be included, such as Ter119 for erythrocytes), PE conjugated anti-Sca-1 and APC conjugated anti c-Kit (BioLegend). For human CD34+/CD38− staining,

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FITC conjugated anti-CD34 and PE conjugated anti-CD38 (BD Biosciences) (see Note 1). 3. FACS tubes (BD Biosciences). 4. Flow cytometry analyser (e.g., FACSCalibur with CellQuest software). 2.2. Cell Cycle Analysis

1. FACS buffer (see Subheading 2.1). 2. Antibodies: For murine SKL staining-FITC conjugated antilinage markers (CD4, NK, GR-1, B220, CD8a, and CD11b), PE conjugated anti-Sca-1 and APC conjugated anti-c-Kit (BioLegend). For human CD34+/CD38− staining, FITC conjugated anti-CD34 and PE conjugated anti-CD38 (BD Biosciences) (see Note 1). 3. FITC conjugated anti-KI-67 and 7-AAD (BD Biosciences). 4. Hematopoietic progenitor enrichment kit and BD IMagnet (BD Biosciences). The kit contains IMag buffer supplied as 10× stock, blocking antibody, cocktail of biotinylated anti mouse lineage depleting antibodies and streptavidin particles. 5. Fixation/permeabilization kit (BD Biosciences). The kit contains two reagents, fixation/permeabilization solution and the Perm/Wash Buffer supplied as 10× stock solution, which is diluted in FACS buffer to obtain 1× buffer. 6. FACS tubes (BD Biosciences). 7. Flow cytometry analyser (e.g., FACSCalibur with CellQuest software).

2.3. In Vitro Colony Assays (CFU-C) in Semi Solid Media (Methylcellulose)

1. Methylcellulose preparation: Boil 500 ml deionized water (ddH2O) and add 20 g of methylcellulose powder (SigmaAldrich), while stirring. Cool to RT and add 500 ml DMEM (concentrated 2×) with 1% penicillin and streptomycin antibiotics. Aliquots can be stored at −20°C. The mixture is stable at 4°C for up to 1 month. 2. Supplements: 30% FCS, 50 ng/ml SCF, 5 ng/ml IL-3, 5 ng/ ml GM-CSF (R&D Systems) and 2 μl/ml erythropoietin (Orto BioTech). 3. Tissue culture dishes of 35 × 10 mm and 100 × 20 mm, Nunclon Surface. 4. Syringe, 16 gauge blunt end needle is recommended.

2.4. In Vitro Transwell Migration Assays

1. Costar transwells (6.5 mm diameter, 5 μm pore). 2. Migration assay medium: RPMI supplemented with heat-inactivated 10% FCS, 2 mM L-glutamine, and 1% penicillin and streptomycin antibiotics. 3. SDF-1α 125 ng/ml (PeproTech), keep at −20°C and store at 4°C before use (stable at 4°C for up to 2 weeks).

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4. FACS tubes (BD Biosciences). 5. Flow cytometry analyser (e.g., FACSCalibur with CellQuest software). 2.5. Proteolytic Enzyme Activity

1. Plastic gel casting cassettes, 0.75 mm thick, 10 × 10 cm. 2. 30% Acrylamide. 3. 1% (w/v) gelatin: Dissolve 40 mg gelatin in 1 ml of ddH2O and heat the solution at 60°C in water bath for at least 20 min; mix well. Make sure that the gelatin is completely dissolved. Cool the gelatin solution to room temperature before use. Prepare it fresh. 4. 10% SDS. 5. 10% (w/v) ammonium persulfate. 6. N,N,N¢,N¢-Tetramethylethylenediamine (TEMED). 7. Running buffer stock (10×): Prepare 1 l of 0.25 M Tris base and 1.92 M glycine, pH 8.3. Adjustment of the pH is not required. Store at room temperature. Dilute the running buffer 10× stock with dH2O to make 1 l and supplement with 5 ml of 20% (w/v) SDS to a final concentration of 0.1% (w/v). Store solution at room temperature. All buffers are stable for months. 8. Sample buffer (4×): Prepare 10 ml of 250 mM Tris–HCl, pH 6.8, 40% (v/v) glycerol, 8% (w/v) SDS, and 0.01% (w/v) bromophenol blue. Store at −20°C. Before use, warm solution to dissolve the SDS. 9. Collagenase buffer stock (10×): Mix 60.6 g Tris base, 117 g NaCl, 5.5 g CaCl2, complete to 900 ml with ddH2O. Adjust to pH 7.6 with concentrated HCl, top up to 1 l with ddH2O and store at 4°C. For an 1× working solution, dilute the running buffer stock 10× with ddH2O to a volume of 1 l and add 670 μl 30% (w/v) Brij-35 (Sigma-Aldrich). 10. Developing buffer: Prepare 50 mM Tris–HCl, pH 8, 10 mM CaCl2, 1 mM ZnCl2 and 1% Triton X-100. 11. Coomassie Brilliant Blue staining R250. 12. Destaining solution: 5% Acetate, 10% methanol in dH2O. 13. Image analysis program (e.g., ImagJ or NIH Image processor) and a scanner.

2.6. Functional HSC Engraftment, Repopulation and Serial BM Transplantation

1. Mobilized mouse cell transplantation: (a) Recipient mice: B6.SJL (CD45.1) mice, 8–12 weeks old. (b) Cell donors: C57BL/6 (CD45.2) mice, 8–12 weeks old. 2. For mobilized human cell transplantation: (a) Mononuclear cells or CD34+ enriched cells collected from human mobilized blood. (b) Recipient immunodeficient mice: NOD/SCID or NOG mice, 8–12 weeks old.

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3. RPMI medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 1% penicillin and streptomycin antibiotics. 4. 16-Gauge, flat bore needles with syringe. 5. Phosphate-buffered saline (PBS 1×). Dilute DPBS−/− 10 × (-) CaCl2 (-)MgCl2. 6. Ficoll-Hypaque (light sensitive). 7. Anti-mouse antibodies: FITC conjugated anti-CD45.2, PE conjugated anti-CD45.1, and IgG isotype control (BioLegend). 8. Anti human antibody: FITC conjugated anti-CD34 (BD Biosciences). 9. FACS buffer: PBS−/− (DPBS−/− 10− (-)CaCl2 (-)MgCl2) supplemented with 5% heat-inactivated FCS and 0.1% sodium azide. 10. FACS tubes (BD Biosciences). 11. Ciprofloxacin antibiotic (Bayer). 12. Flow cytometry analyser (e.g., FACSCalibur with CellQuest software). 13. Irradiator. 14. Heat lamp (recommended).

3. Methods 3.1. Phenotypic Characterization of Mobilized Hematopoietic Stem and Progenitor Cells 3.1.1. HSPC Marker Characteristics: Flow Cytometry (FACS) Assay

HSPC respond to stress-induced signals, such as bleeding and inflammation, by detachment from their BM microenvironment and recruitment to the circulation. Clinical mobilization protocols mimic these stress signals by administration of mobilizing agents, such as repetitive G-CSF stimulations (3). Mobilized HSPC from the PB can be easily identified, characterized and quantified by a molecular signature of extracellular antigen sets. The heterogeneity of cell surface markers enables one to distinguish primitive stem cells from other uncommitted and lineage-restricted progenitors. Over the years, numerous flow cytometry based methods have been established to identify and distinguish human and murine stem cells (32). Enriched murine HSPC are identified as Sca1+Lineage−c-Kit+ (33, 34) cells (SKL) or CD34−Sca-1+Lineage−cKit+ (SKL/CD34−), while lineage + marker combinations identify differentiating leukocytes. Most SKL cells are considered as progenitor cells, while the SKL/CD34− cells are more primitive, retaining long-term repopulating potential (35). Recently, the expression of SLAM family antigens, CD150+ CD41− CD48−, has been reported as another tool to identify and characterize primitive

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HSC (36). Human HSPC are enriched in the CD34+ fraction, which can be further purified into CD34+/CD38− or CD34+/ CD38−/Lin−. Herein, we describe the use of a well-known set of markers for the identification and quantification of both murine and human HSPC. Beside those conventional sets of markers, others molecules that highly associate with mobilized HSPC function can be determined since their levels change and correlate with mobilization. These include CD44 (10), which is cleaved as part of the mobilization process, as well as the membrane-bound MT1MMP which its increased levels, correlate with clinical mobilization protocols (37). Both CD44 and MT1-MMP are also important for human CD34+ HSPC homing capacity and motile behavior (37, 38). Assessment of HSPC mobilization can be usually done by identifying murine SKL cells or human CD34+/CD38− in the PB. 1. For the detection of mobilized HSPC, murine or human PB mononuclear cells (MNC) (at least 1 × 106 cells, for the separation of PB MNC by ficoll see Subheading 3.4.1) should be centrifuged at 200 × g for 5 min. 2. Wash the cell pellet in 1 ml of FACS buffer (see Subheading 2), and centrifuge at 200 × g for 5 min; 4°C is recommended. 3. Discard supernatant and resuspend the cells in 100 μl of antibody mix. For detection of murine SKL cells, mix 0.5 μg from each of the lineage antigen marker FITC conjugated antibodies (CD4, NK, GR-1, B220, CD8a, and CD11b) and 0.5 μg from the PE conjugated anti-Sca-1 and APC conjugated antic-Kit antibodies for each sample. For detection of human HSPC, add 1 μg of anti-CD34 and anti-CD38 antibodies for each sample of 1 × 106 cells. 4. Mix the cells by gently vortexing and incubate for 30 min at 4°C. After incubation, wash in 1 ml of FACS buffer and centrifuge at 200 × g for 5 min. 5. Resuspend the cells in 300 μl FACS buffer and quantify mobilized HSPC percentage in comparison to controls by FACS. 3.1.2. Cell Cycle Analysis

HSC are maintained primarily in a homeostatic quiescent state, mostly due to a complex tight regulation by the BM microenvironment (5, 19, 39, 40). Following treatment with mobilizing agents, HSPC appear in the circulation in either the G0 or G1 phase of the cell cycle, entering the blood only after the M phase (41), while HSPC that reside in the BM and spleen are more actively cycling (41, 42). What dictates the noncycling state of mobilized HSPC in the PB of both mouse and human is not fully understood; however, it can be speculated that noncycling HSPC with condensed chromatin will migrate from the BM to the circulation more efficiently than cycling cells. Recent evidence suggests that cleavage of the potent cyclin-dependent kinase inhibitor p21 by

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proteinase 3 during stress-induced mobilization is responsible for cell cycle progression into the S phase in the BM and spleen as compared to noncycling circulating cells (43). The cell cycle status of mobilized HSPC can be assessed using DNA intercalating molecules, such as BrdU labeling, Ki-67 nuclear antigen staining, Pyronin Y, or 7-amino actinomycin D (7-AAD). This section describes analytical intracellular staining method for cell cycle marker Ki-67 and 7-AAD with membrane staining for multiple HSPC markers. Ki-67 is a nuclear antigen that is strictly associated with cell proliferation during all active phases of the cell cycle (G1, S, G2, M) but is absent from quiescent, noncycling cells (G0). 7-ADD is a nuclear acid stain with a strong affinity for GC-rich regions. The combination of Ki-67 and 7-ADD staining provides the ability to distinguish between the cell cycle stages in primitive subpopulations. 1. For the assessment of cell cycle stages of mobilized HSPC, centrifuge ficoll-separated MNC from murine or human PB (at least 5 × 106 cells, see Note 2) at 200 × g for 5 min (for the separation of PB MNC see Subheading 3.4.1). 2. Wash the pellet in 1 ml of FACS buffer (see Subheading 2), and centrifuge at 200 × g for 5 min; 4°C is recommended. 3. For murine cells, discard supernatant and continue with depletion of lineage positive cells using the hematopoietic progenitor enrichment kit accordingly to the instructions supplemented with the kit. 4. After the depletion procedure of murine cells or following the washing of human cells, discard supernatant and resuspend the pellet in 100 μl of antibody mix to identify murine or human HSPC, as depicted in Subheading 4.11. 5. This method combines both extracellular and intracellular staining; therefore, it is important to first stain the cell surface antigens and then fix and permeabilize the cells for intracellular staining of nuclear DNA content. After performing extracellular staining, wash the cells in 1 ml of FACS buffer and centrifuge at 200 × g for 5 min. Discard the supernatant, resuspend the cell pellet with 100 μl of fixation/permeabilization solution and incubate for 30 min at 4°C (this can be done also at room temperature). After incubation, wash the cells with 1 ml Perm/Wash buffer 1× and centrifuge at 200 × g for 5 min. It is important to use the Perm/Wash buffer in all subsequent washing steps in order to keep cells permeabilized for intracellular staining. 6. Add 20 μl of Ki-67 to each tube, mix the cells by gently vortexing and incubate for 30 min at 4°C. 7. Wash the cells with 1 ml Perm/Wash buffer 1× and centrifuge at 200 × g for 5 min.

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8. Discard the supernatant and add 20 μl of 7-AAD to each sample and incubate for 10 min at room temperature. 9. Resuspend the cells in 300 μl FACS buffer and quantify mobilized HSPC in comparison to control cells. After reading the samples by FACS, gate on the murine Sca-1 and c-Kit positive cells or the human CD34+/CD38− subpopulation to display the Ki-67 vs. 7-AAD dot plot. The lower left quadrant (cells negative for Ki67 and 7-AAD) represents cells in G0 and the upper right quadrant represents cells in the S, G2 and M phases (cells positive for Ki67 and 7-AAD). 3.2. In Vitro Assays 3.2.1. In Vitro Colony Assays (CFU-C) in Semi Solid Media (Methylcellulose), for Mobilized Human and Murine, as well as Enriched Human CD34+ Cells

Colony-forming cell assays are widely used as a functional method to identify myeloid lineage restricted progenitor cells. While these assays are easy to perform, they cannot identify true HSC and cannot be used to measure the rate of mobilization in real time. Colony-forming cells (CFC) or colony-forming unit cells (CFUC) are committed stem and early multipotent progenitor (MPP) cells that are able to proliferate and differentiate into more mature cells under appropriate conditions. The in vitro hematopoietic colony assay was developed at the beginning of the 1960s, making it possible to investigate and distinguish hematopoietic cell populations at different stages of differentiation. The multi-potent progenitors CFU-GEMM give rise to all the myeloid lineages; granulocyte–macrophage colony-forming cells (CFU-GM) are progenitor cells that can give rise to colonies containing a heterogeneous population of macrophages and granulocytes; lineagecommitted progenitors include burst-forming unit erythroid (BFU-E), colony-forming unit erythroid (CFU-E), which are primitive erythroid progenitors, and megakaryocyte precursors (CFU-Mk). In murine studies, assessment of mobilized progenitors can be performed with cells obtained from different sources, whole leukocytes or purified mononuclear cells (MNC) from the BM, PB or spleen. 1. Colony forming assays are performed by seeding hematopoietic cells (according to Table 1, while the use of PB and spleen MNC is enough for mobilization assessment) into a cell culture dish containing prewarmed semisolid matrix methylcellulose supplemented with 30% FCS, 50 ng/ml SCF, 5 ng/ml IL-3, 5 ng/ml GM-CSF, and 2 μl/ml erythropoietin. 2. The mixture should be vigorously vortexed before plating. Wait approximately 10 min until all air bubbles disappear from the vortexed cell suspension. 3. Collect the mixture with a syringe (a 16 gauge blunt end needle is recommended, since the solution is viscous) and plate 1 ml of the mixture in a 35 × 10 mm culture dish, spreading it out to cover the entire surface of the dish.

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Table 1 Number of cells to be seeded for colony formation assay Source

Cells per dish

Human PB-MNC Mobilized PB-MNC Mobilized PB CD34+ enriched cells

1–2 × 105 1 − 5 × 104 5 × 102 to 2 × 103

Murine Total crude BM cells Total crude spleen cells PB-MNC Mobilized PB-MNC

1.5 × 103 2 × 105 2 × 104 2 × 104

4. Duplicates of colony culture dishes should be incubated together with additional 35 × 10 mm dish containing sterile water but without a lid to maintain high humidity. The dishes are placed together in a 100 × 25 mm dish and incubated at 37°C in a humidified atmosphere (>96%), containing 5% CO2 for 7 days (murine colonies) or 14 days (human colonies). 5. In order to assess CFU-C frequency, place the dish containing cell colonies on a 60 mm gridded scoring dish, and count the colonies of interest using an inverted microscope. Moreover, the absolute number of CFU-C can be counted per 1 ml of blood by taking into account the frequency of CFU-C, the blood volume and the number of PB-MNCs after ficoll enrichment (e.g., (PBMNC number × 1 ml × CFU − C number) ). (blood volume (ml) × seeded cells) 3.2.2. In Vitro Transwell Migration Assays (Directional and Spontaneous Migration)

HSPC mobilization is modulated and controlled by multiple factors, including chemokines, cytokines, growth factors and hormones. Particularly, the SDF-1/CXCR-4 axis plays major roles in HSPC maintenance, homing and mobilization. During G-CSF induced mobilization, SDF-1/CXCR-4 signaling is altered, resulting in reduced SDF-1 levels in the BM and upregulation of CXCR-4 expression, leading to HSPC mobilization. In addition to the pivotal role played by SDF-1 and CXCR-4 in HSPC motility, the mobilization process was found to be tightly regulated by the cytokine hepatocyte growth factor (HGF), and its receptor c-Met, involving elevation in intracellular ROS signaling. Of interest, c-Met inhibition reduced HSPC mobilization following G-CSF treatment and interfered with their chemotactic migration towards SDF-1 (23). C-Met inhibition also reduced MT1-MMP elevation on mobilized human CD34+ HSPC (38). As SDF-1/CXCR-4 signaling is essential for directional HSPC migration and can predict clinical repopulation outcome in autologous transplantation (44).

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In vitro migratory capacity of CD34+ cells is related to hematopoietic recovery after autologous stem cell transplantation (44). An assay for SDF-1 induced transmigration evaluates the motility properties of mobilized HSPC. Recruitment of HSPC from the BM microenvironment to the blood stream and vice versa, is a multistep process that involves adhesion, directed migration and proteolytic enzymes, which degrade the extracellular matrix barrier. Coating the transwell filter with the barrier of interest, such as fibronectin, endothelial cells, or matrigel (which contains extracellular matrix proteins that partially mimic the extracellular basement membrane), can be applied to evaluate the ability of cells to penetrate through an artificial barrier under semi-physiological conditions. 1. For the transmigration assay, add 600 μl migration medium to the lower chamber supplemented with or without 125 ng/ml SDF-1α. The herein described method using human SDF-1 can be applied both to human and murine cells, because the SDF-1 sequence is highly conserved and there is a crossreactivity between human and mouse SDF-1. 2. Load 100 μl of 50,000–100,000 human enriched CD34+/ murine SKL cells or 100,000–200,000 BM/PB MNC gently into the upper chamber of each transwell (take special care to avoid air bubbles in the chamber and perforation of the membrane). 3. In parallel, add 100 μl of cells into a separate tube containing 500 μl of migration medium. This tube serves as the migration index (see Note 3). 4. After loading the cells, transfer the upper chamber into the lower chamber (avoid air bubbles that may interfere with the transmigration of cells). 5. Place the transmigration plate in a humidified 37°C containing 5% CO2 incubator for 2–4 h (see Note 4), and make sure it is not disturbed. 6. Stopping the transmigration experiment is done by transfer the upper filter chamber into an empty well very gently, avoiding media transfer between the upper and lower compartments of the transmigration wells. 7. Collect 300 μl of media from the lower chamber into a FACS tube and evaluate the migration capacity by counting the cells at high speed for 60 s using FACS. To calculate the percentage of cells migrating (the migration capacity), take into account the migration index (see Note 3). Migrating fractions can be also stained for murine Lin-c-Kit+, human CD34 (see Subheading 3.1.1), as well as examined for CFU-C potency (see Subheading 3.2.1). These last two assays are useful mainly for the detection of hematopoietic progenitor cells (e.g., Lin−cKit+, CD34+, and CFU-C) with high migration capacity.

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3.2.3. Proteolytic Activity (Zymography Assay)

A major group of proteases has been directly associated with HSPC mobilization from the BM microenvironment to the circulation following stress signals. Neutrophil accumulation and activation in the BM and other BM stromal cells play a pivotal role in HSPC motility, following G-CSF-induced mobilization, through the release of active neutrophil proteases, such as elastase, cathepsin G, and matrix metalloproteinase-9 (MMP-9) (21). Circulating human CD34+ cells (45), murine BM osteoclasts (46) and hematopoietic progenitor cells (45), secrete metalloproteinases MMP-2 or MMP-9. These active proteases are released into the BM cavity and selectively cleave chemokines, cytokines, and their receptors, as well as adhesion molecules, that are essential for HSPC retention in the BM microenvironment, such as VCAM-1(21), the receptor c-Kit (13, 21), and SDF-1 (21). Indeed, there is an accelerated induction of proteolytic activity in the BM following G-CSF administration, accompanied by a decrease in SDF-1 levels and subsequently HSPC mobilization to the circulation (11, 21). CD26/DPPIV is a murine progenitor and human CD34+ associated peptidase that has an important role in HSPC mobilization by facilitating SDF-1 cleavage in the BM (47). Notably, it has been shown that mice deficient either in MMP-9, neutrophil elastase, cathepsin-G, or CD26 are able to respond to G-CSF-induce mobilization normally, suggesting redundancy in the activity of different proteases (48). In addition, the membrane type 1–MMP (MT1-MMP) has been reported to be highly expressed on mobilized HSPC, regulating their motility and mobilization (21, 37, 49). Hereby, we provide a detailed protocol to measure MMP-9 activity in the mouse BM and plasma by gelatin zymography. The gelatin zymography assay is an easy yet powerful technique to detect the presence of MMP-9 (and MMP-2) in biological samples by identifying gelatindegrading activity. Protein extraction can be done from mouse BM supernatant. 1. For mouse BM MMP-9 expression, extract the BM from the femurs and tibias by cutting off the tips of the bones and flushing the marrow out of the bone cavity, using a 1 ml syringe containing PBS. 2. Centrifuge the samples at 180 × g for 10 min at 4°C and transfer the BM supernatant to a new Eppendorf tube. 3. For mouse plasma MMP-2 and MMP-9 expression, collect PB and centrifuge at 180 × g for 10 min at 4°C, and transfer plasma (the supernatant) into a new Eppendorf tube. 4. Samples can be maintained for long term storage at −20°C. 5. Prepare 10% SDS-polyacrylamide gels supplemented with 1 mg/ml gelatin. For the resolving gels (two gel preparations), mix 3 ml of dH2O, 3.3 ml of 40% acrylamide, 100 μl of 10% (w/v) SDS, 100 μl of ammonium per-sulfate and 2.5 ml of

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1.5 M Tris buffer pH 8.8 and 1 ml of gelatin (as mentioned in Subheading 2). Add 5 μl TEMED to the resolving gel solution to initiate polymerization and rapidly swirl the solutions, while avoiding air bubbles. Immediately, transfer 5 ml of the resolving gel solution into the running cassette, then overlay the separating gel solution with dH2O, and let the gel polymerize for at least 30 min at room temperature. After discarding the overlying water, add 5 μl TEMED to the stocking gel solution containing 3.15 ml of dH2O, 0.66 ml of 40% acrylamide, 50 μl of 10% (w/v) SDS, 100 μl of ammonium per-sulfate and 1.25 ml of 1.5 M Tris buffer pH 6.8. Gently swirl and rapidly transfer to the upper running cassette. Insert immediately the appropriate comb and let the stacking gel polymerize for 1 h at room temperature. 6. Mix volumes of BM supernatant equivalent to 2 μg protein or 5 μl of plasma with 7.5 μl nonreducing sample buffer 4× (without mercaptoethanol) and dilute to a final volume of 30 μl of collagenase buffer without heat treatment, loading 15–30 μl per lane (see Note 5). Use conditioned medium from HT1080 (human fibrosarcoma) cell line culture as a positive control for MMP-9 and MMP-2 expression. 7. Let the gels undergo electrophoresis at 300 V for 1 h and 40 min in running buffer. 8. Gently remove the gels from the running cassette, transfer the gels to a new container and wash them in a 2.5% Triton X-100 solution for 30 min (see Note 6). 9. Wash the gels 2–3 times in dH2O for 10–15 min. 10. Incubate the gels in developing buffer 1× with gentle shaking for 30 min at room temperature. 11. Discard used developing buffer 1× and incubate the gels in fresh developing buffer 1× over night in 37°C (see Note 7). Protease activity can be visualized as a clear zone with Coomassie Brilliant Blue staining within 3 min of incubation. 12. Add destaining solution with gentle shaking for 30 min. Discard used destaining solution and incubate the gels in ddH2O until clear bands appear on the gels. 13. Scan gels using a flat bed scanner and analyze gelatinase activity using an image processor. 3.3. Functional In Vivo Assays: HSC Engraftment, Repopulation and Serial Transplantation

Mobilized HSPC can be identified based on their in vivo functional capacity to repopulate host BM with high levels of maturing myeloid and lymphoid cells, while the “stemness” property is maintained by a small pool of undifferentiated stem cells with the potential to repeat the entire process in serially transplanted recipients. The gold standard for measuring HSC function following the mobilization process is the long-term repopulation assay. This assay

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determines the potential of HSC to fully differentiate into all blood lineage-restricted cells and their longevity by preserving the selfrenewal potential, which enables the preservation of the HSC pool for many years. The repopulation assay utilizes transplantation of mobilized blood MNC or sorted immature cells in preconditioned irradiated hosts. Short-term engraftment is initiated by differentiating progenitors (SKL Sca-1+c-Kit+Lin− cells in mouse or CD34+CD38+ cells in human), usually evaluated after four weeks up to a couple of months. Long-term multilineage engraftment is carried out by “true” stem cells (E-SLAM EPCR+CD48−CD150+ (50) cells or SKL CD34− cells in mouse and CD34+CD38− cells in human), evaluated after 4–6 months in mice and years in patients (51). HSPC transplantation requires preconditioning of the host with total body irradiation (TBI), thus the homing ability and engraftment potential of mobilized HSPC is not examined under physiological conditions. A more physiological relevant assay, using parabiotic mice with a shared blood circulation, has revealed that HSPC are constitutively circulating, trafficking from the BM to blood stream through the blood–bone barrier and return to the BM of the parabiont partner, and enabling functional engraftment of unconditioned BM (52–54). While the blood and spleens of these mice are equally repopulated by both parabionts, the BM is mostly host derived due to the blood–bone barrier. Of interest, HSPC mobilization by G-CSF (53) or AMD3100 (54) in parabiotic mice, which has been done in a more physiological context, is accompanied by a dramatic increased engraftment of the partner BM with primitive cells, revealing that mobilization and homing are sequential events with clinical relevance. Assessment of murine HSC engraftment and repopulation following stress induced mobilization requires congenic strains of donors and hosts, for example by identification of different alleles of the hematopoietic cell marker CD45. Assessment of human cell mobilization requires sublethally irradiated (300–375 cGy) immune-deficient recipient mice that are able to tolerate human xenografts, such as the NOD/SCID or the NOG strains (owing to the complete absence of T, B, and NK cell activity, and reduced function of innate immunity), allowing multilineage reconstitution of human hematopoiesis (55). Notably, some reports indicate that mobilized CD34+ cells, as compared to cord blood, can provide early multilineage reconstitution though nonsustained long-term multilineage engraftment of immunodeficient mice (56). The most stringent test for stemness is the serial transplantation assay, wherein only the most primitive HSC can yield longterm multilineage repopulation. In this assay, mobilized human MNC or enriched CD34+ cells are transplanted into primary recipients, then harvested after 4 weeks and transplanted again into secondary recipients. Obviously, subsequent serial transplantations can be done.

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1. Irradiate host mice 1 day prior to transplantation. Recipient mice for human grafts (NOD/SCID) should be irradiated with a sublethal dose of ~350 cGy, and recipient mice for mouse grafts (B6.SJL CD45.1) should be irradiated with a lethal dose of ~900 cGy, depending on the irradiation device. 2. Add 16 μg/ml Ciprofloxacin antibiotics to the drinking water after irradiation. Replace the antibiotic-containing water once a week for 3 weeks.

3.3.2. Donor Mobilized MNC Preparation

1. Once mobilization with a mobilizing agent has been assessed in human or in the mouse, dilutes mobilized cells at least 1:1 with sterile PBS and separate mononuclear cells by a ficoll gradient. 2. Prepare falcon tubes with 1 ml ficoll reagent. Load 2 ml of diluted mobilized blood cells slowly on the top of ficoll, using a 1 ml pipette at a 45 angle against the wall of the tube. 3. Carefully transfer the tubes to the centrifuge without mixing the layers. Centrifuge at 220 × g for 25 min. A fraction of enriched mononuclear cells is found at the middle of the tube arranged in an annular-like shape. 4. Discard the plasma from the top of the tube without disturbing the MNC fraction. 5. Transfer the MNC fraction to a new PBS containing tube. 6. Wash out ficoll reagent by centrifuging the tube at 200 × g for 5 min, discard the supernatant and resuspend the cell pellet in 1 ml of full-RPMI. 7. Prepare 20 million MNC (both for human or mouse cells) in 500 μl of RPMI per mouse and inject the cells immediately without incubation.

3.3.3. Transplantation

1. Mice are irradiated approximately 24 h prior to transplantation. 2. When ready, place the recipient mouse in a restrainer and inject 20 million cells per mouse intravenously. Placing recipient mice under a heating lamp for about 2 min might help to find the lateral tail veins more easily.

3.3.4. Evaluation of Engraftment

1. Engraftment is measured 1 month after transplantation for short-term engraftment and 6 month for long term engraftment. Assessment of the engraftment levels can be monitored from the PB by mild bleeding, while keeping the animal alive for further examination, or by sacrificing the animal and extracting total BM cells. Although both PB and the BM undergo engraftment, the best way to analyze reconstitution is by monitoring the level of engraftment in the BM of recipients. In addition, analysis of the BM rather than PB is recommended, since it has previously been demonstrated that

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bleeding results in accelerated hematopoiesis and mobilization of HSPC from the BM to the circulation (18). 2. Remove the femur and the tibia of the mice and extract total BM cells into 1 ml of PBS by cutting off the tips of the bones and flushing the marrow out of the bone cavity using a 1 ml syringe. 3. Transfer at least one million cells to a new FACS tube and resuspend the cells in 1 ml of FACS buffer. 4. Centrifuge the cells at 200 × g for 5 min, discard supernatant and resuspend the cell pellet in 100 μl of antibody mix containing CD34 for human or CD45.1 and CD45.2 for mouse. 5. Mix the cells by gently vortexing and incubate for 30 min at 4°C. After incubation, wash the cells in 1 ml of FACS buffer and centrifuge at 200 × g for 5 min. 6. Discard the supernatant, resuspend the cells in 300 μl FACS buffer and quantify the percentage of engrafted mobilized HSPC in the host. 3.4. Concluding Perspectives and Other Approaches to Analyzing HSPC Mobilization

Although an extensive body of literature describes the nature of HSPC mobilization, which is accelerated following additional stress conditions, still much has to be learned in order to better understand the molecular mechanisms by which HSPC are dynamically guided to navigate from their anchored microenvironment in the BM to the circulation. There is a constant demand for improvement of mobilization strategies, so as to overcome “poor mobilization” difficulties, by increasing the number of primitive stem cells, as well as their functional homing and engraftment efficacies for clinical transplantation. Numerous mobilization assays have been developed during the years, some of which have been detailed above, in order to evaluate the efficacy of novel mobilizing agents and to analyze the long-term repopulation potential of the mobilized stem cells. Despite the multiple established assays for mobilization assessment, critical distinction between the source organs for murine HSPC (e.g., BM vs. the spleen) remains obscure. However, recently, a novel technique of in situ perfusion, which is installed on the murine hind limb, enables a direct in vivo assessment of mobilization from the BM (57). This elegant approach contributes to the progress in the field by allowing detection of the ability of new mobilizing agents to induce HSPC recruitment directly from the BM cavity to the circulation, and thereby might assist in developing improved transplantation protocols. Extensive research in the field has uncovered key roles for microenvironmental cues in inducing mobilization and augmenting HSPC motility. For example, bone-resorbing osteoclasts, which are essential for homeostatic bone turnover, are activated in response to G-CSF stimulations, while bone-lining osteoblasts are suppressed. This dynamically coordinated and regulated microenvironment “clears the way out” for mobilized HSPC together with other myeloid

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cells, such as neutrophils. Osteoclast differentiation from monocyte precursors under RANK-ligand regulation was also associated with moderate HSPC mobilization (18). Of note, osteoclast activity releases active TGF-β1 from bone matrix, forming a gradient that recruits Sca-1+ MSC to the endosteum region, where they undergo osteoblast differentiation (58). G-CSF administration has been demonstrated to deplete a population of trophic endosteal macrophages (osteomacs) that support osteoblast function (59). Inhibition of osteoclast by bisphosphonates on the other hand, impaired HSC levels and quiescence and was demonstrated to induce delayed hematopoietic recovery following transplantation (60). Multiple studies have shed light on the putative role of BM stromal supporting cells in preserving the immature-primitive phenotype of HSC. Notably, osteoblast lineage cells robustly express the key molecule SDF-1, thus providing a unique supportive microenvironment for HSC maintenance, contributing to longterm hematopoiesis (61). SDF-1 is presented by Annexin-2, which is expressed by BM osteoblasts. Thus, CXCR4+ HSC are also anchored directly to the internal bone surface. Indeed, higher rates of G-CSF-induced mobilization of HSPC were observed in Annexin-2 deficient mice as compared to wild-type animals (62). HSPC mobilization also requires signals from the sympathetic nervous system, which plays a major role by affecting HSPC directly (e.g., catecholamines) and indirectly (e.g., suppression of osteoblasts or daily rhythmic regulation of SDF-1 synthesis) (20, 63–65). Neurotransmitters can be transmitted to HSPC through the blood stream or directly secreted from nerve endings in the BM. The catecholaminergic receptors were found to be dynamically expressed on human HSPC, while G-CSF stimulations enhance their expression on primitive human CD34+CD38−/low cells (65). Acute stress, mimicked by norepinephrine stimulation actively induced rapid release of SDF-1 and subsequently HSPC to the blood stream. Treatment with β2-adrenergic antagonist was demonstrated to inhibit HSPC mobilization in both steady-state and following AMD3100 administration (inducing SDF-1 release to the peripheral blood) (66). Recent evidence suggests that endocannabinoids secreted by BM stromal cells, signaling through the CB2 receptor that is functionally expressed by human and murine HSPC, induced mobilization of murine HSPC with short- and long-term repopulating abilities. Moreover, G-CSF-induced mobilization of HSPC was significantly decreased by CB2 antagonists (67, 68). Bone remodeling processes, including bone formation by osteoblasts and degradation by osteoclasts, and HSPC mobilization are sequential events which are both controlled by β2-adrenergic signals. In particular, Vitamin D receptor that is controlled by β2adrenergic signals was found to be essential for G-CSF induce mobilization, leading to suppression of osteoblast activity and upregulation of RANKL (69). This complex picture of the dynamic

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S. Gur-Cohen et al.

“brain-bone-blood” triad may be exploited in future designing of the exact timing of clinical mobilization protocols (70) and therapeutic transplantation procedures. Of note, it is yet unknown how to guide a particular progenitor cell type (e.g., hematopoietic or endothelial) egress and mobilization from the BM. Better understanding of the factors involved in cell mobilization will pave the way for better regenerative therapies, including the use of enriched EPC for neovascularization as part of regaining organ function, enriched MSC to restore skeletal and muscle tissue damage, as well as the use of enriched HSC for hematopoietic recovery following high dose radiotherapy and chemotherapy.

4. Notes 1. Any other set of conjugated antibodies can be suitable for the HSPC quantification. 2. Since intracellular staining of DNA requires permeabilization, more cells per staining procedure are needed. 3. The migration index equals 100% migration capacity, and therefore it is a reference point for migrating cells in the transwell migration assay. 4. Duration time of the migration experiment should be considered based on cell-intrinsic motility properties. 2–4 h migration is recommended. 5. Do not heat the samples, since gelatin zymography examines proteolytic activity, and do not load more than 30 μg total protein per lane. 6. Do not exceed 30 min incubation, as prolonged incubation can damage the protease. 7. Cover the gel container to avoid vaporization of buffer. References 1. Schulz C, von Andrian UH, Massberg S (2009) Hematopoietic stem and progenitor cells: their mobilization and homing to bone marrow and peripheral tissue. Immunol Res 44:160–168 2. Lapidot T, Kollet O (2002) The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2m(null) mice. Leukemia 16:1992–2003 3. Metcalf D (1990) The colony stimulating factors. Discovery, development, and clinical applications. Cancer 65:2185–2195

4. Lapid, K., Vagima, Y., Kollet, O., Lapidot, T. (2009). Egress and mobilization of hematopoietic stem and progenitor cells. In: StemBook Cambridge (MA): Harvard Stem Cell Institute. 5. Sugiyama T, Kohara H, Noda M, Nagasawa T (2006) Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity 25:977–988 6. Nie Y, Han YC, Zou YR (2008) CXCR4 is required for the quiescence of primitive hematopoietic cells. J Exp Med 205:777–783

2


7. Zipori D, Sasson T (1980) Adherent cells from mouse bone marrow inhibit the formation of colony stimulating factor (CSF) induced myeloid colonies. Exp Hematol 8:816–817 8. Wilson A, Laurenti E, Trumpp A (2009) Balancing dormant and self-renewing hematopoietic stem cells. Curr Opin Genet Dev 19:461–468 9. Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Grabovsky V, Slav MM, Nagler A, Lider O, Alon R, Zipori D, Lapidot T (2000) The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 95:3289–3296 10. Avigdor A, Goichberg P, Shivtiel S, Dar A, Peled A, Samira S, Kollet O, Hershkoviz R, Alon R, Hardan I, Ben-Hur H, Naor D, Nagler A, Lapidot T (2004) CD44 and hyaluronic acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells to bone marrow. Blood 103:2981–2989 11. Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L, Ponomaryov T, Taichman RS, Arenzana-Seisdedos F, Fujii N, Sandbank J, Zipori D, Lapidot T (2002) G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 3:687–694 12. Semerad CL, Christopher MJ, Liu F, Short B, Simmons PJ, Winkler I, Levesque JP, Chappel J, Ross FP, Link DC (2005) G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow. Blood 106:3020–3027 13. Levesque JP, Hendy J, Winkler IG, Takamatsu Y, Simmons PJ (2003) Granulocyte colonystimulating factor induces the release in the bone marrow of proteases that cleave c-KIT receptor (CD117) from the surface of hematopoietic progenitor cells. Exp Hematol 31:109–117 14. Hattori K, Heissig B, Tashiro K, Honjo T, Tateno M, Shieh JH, Hackett NR, Quitoriano MS, Crystal RG, Rafii S, Moore MA (2001) Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells. Blood 97:3354–3360 15. Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett PA, Liles WC, Li X, Graham-Evans B, Campbell TB, Calandra G, Bridger G, Dale DC, Srour EF (2005) Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med 201: 1307–1318

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16. Pusic I, DiPersio JF (2010) Update on clinical experience with AMD3100, an SDF-1/ CXCL12-CXCR4 inhibitor, in mobilization of hematopoietic stem and progenitor cells. Curr Opin Hematol 17:319–326 17. Gazitt Y, Freytes CO, Akay C, Badel K, Calandra G (2007) Improved mobilization of peripheral blood CD34+ cells and dendritic cells by AMD3100 plus granulocyte-colony-stimulating factor in non-Hodgkin’s lymphoma patients. Stem Cells Dev 16:657–666 18. Kollet O, Dar A, Shivtiel S, Kalinkovich A, Lapid K, Sztainberg Y, Tesio M, Samstein RM, Goichberg P, Spiegel A, Elson A, Lapidot T (2006) Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells. Nat Med 12: 657–664 19. Kollet O, Dar A, Lapidot T (2007) The multiple roles of osteoclasts in host defense: bone remodeling and hematopoietic stem cell mobilization. Annu Rev Immunol 25:51–69 20. Spiegel A, Shivtiel S, Kalinkovich A, Ludin A, Netzer N, Goichberg P, Azaria Y, Resnick I, Hardan I, Ben-Hur H, Nagler A, Rubinstein M, Lapidot T (2007) Catecholaminergic neurotransmitters regulate migration and repopulation of immature human CD34+ cells through Wnt signaling. Nat Immunol 8:1123–1131 21. Levesque JP, Hendy J, Takamatsu Y, Williams B, Winkler IG, Simmons PJ (2002) Mobilization by either cyclophosphamide or granulocyte colony-stimulating factor transforms the bone marrow into a highly proteolytic environment. Exp Hematol 30:440–449 22. Levesque JP, Takamatsu Y, Nilsson SK, Haylock DN, Simmons PJ (2001) Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor. Blood 98:1289–1297 23. Tesio, M., Golan, K., Corso, S., Giordano, S., Schajnovitz, A., Vagima, Y., Shivtiel, S., Kalinkovich, A., Caione, L., Gammaitoni, L., Laurenti, E., Buss, E. C., Shezen, E., Itkin, T., Kollet, O., Petit, I., Trumpp, A., Christensen, J., Aglietta, M., Piacibello, W., Lapidot, T. (2010) Enhanced c-Met activity promotes G-CSF induced mobilization of hematopoietic progenitor cells via ROS signaling. Blood 24. Levesque JP, Winkler IG (2008) Mobilization of hematopoietic stem cells: state of the art. Curr Opin Organ Transplant 13:53–58 25. Kopp HG, Avecilla ST, Hooper AT, Rafii S (2005) The bone marrow vascular niche: home of HSC differentiation and mobilization. Physiology (Bethesda) 20:349–356

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26. Christopher MJ, Liu F, Hilton MJ, Long F, Link DC (2009) Suppression of CXCL12 production by bone marrow osteoblasts is a common and critical pathway for cytokine-induced mobilization. Blood 114:1331–1339 27. Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, Scadden DT, Ma’ayan A, Enikolopov GN, Frenette PS (2010) Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature 466:829–834 28. Pitchford SC, Furze RC, Jones CP, Wengner AM, Rankin SM (2009) Differential mobilization of subsets of progenitor cells from the bone marrow. Cell Stem Cell 4:62–72 29. Aicher A, Kollet O, Heeschen C, Liebner S, Urbich C, Ihling C, Orlandi A, Lapidot T, Zeiher AM, Dimmeler S (2008) The Wnt antagonist Dickkopf-1 mobilizes vasculogenic progenitor cells via activation of the bone marrow endosteal stem cell niche. Circ Res 103:796–803 30. Dimmeler S (2010) Regulation of bone marrow-derived vascular progenitor cell mobilization and maintenance. Arterioscler Thromb Vasc Biol 30:1088–1093 31. Brouard N, Driessen R, Short B, Simmons PJ (2010) G-CSF increases mesenchymal precursor cell numbers in the bone marrow via an indirect mechanism involving osteoclast-mediated bone resorption. Stem Cell Res 5:65–75 32. Purton LE, Scadden DT (2007) Limiting factors in murine hematopoietic stem cell assays. Cell Stem Cell 1:263–270 33. Spangrude GJ, Heimfeld S, Weissman IL (1988) Purification and characterization of mouse hematopoietic stem cells. Science 241: 58–62 34. Ikuta K, Weissman IL (1992) Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc Natl Acad Sci USA 89: 1502–1506 35. Yang L, Bryder D, Adolfsson J, Nygren J, Mansson R, Sigvardsson M, Jacobsen SE (2005) Identification of Lin(−)Sca1(+)kit(+)CD34(+) Flt3− short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. Blood 105:2717–2723 36. Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121:1109–1121 37. Vagima Y, Avigdor A, Goichberg P, Shivtiel S, Tesio M, Kalinkovich A, Golan K, Dar A, Kollet O, Petit I, Perl O, Rosenthal E, Resnick I,

Hardan I, Gellman YN, Naor D, Nagler A, Lapidot T (2009) MT1-MMP and RECK are involved in human CD34+ progenitor cell retention, egress, and mobilization. J Clin Invest 119:492–503 38. Jalili A, Shirvaikar N, Marquez-Curtis LA, Turner AR, Janowska-Wieczorek A (2010) The HGF/c-Met axis synergizes with G-CSF in the mobilization of hematopoietic stem/progenitor cells. Stem Cells Dev 19:1143–1151 39. Yin T, Li L (2006) The stem cell niches in bone. J Clin Invest 116:1195–1201 40. Roberts AW, Metcalf D (1995) Noncycling state of peripheral blood progenitor cells mobilized by granulocyte colony-stimulating factor and other cytokines. Blood 86:1600–1605 41. Wright DE, Cheshier SH, Wagers AJ, Randall TD, Christensen JL, Weissman IL (2001) Cyclophosphamide/granulocyte colonystimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle. Blood 97:2278–2285 42. Uchida N, He D, Friera AM, Reitsma M, Sasaki D, Chen B, Tsukamoto A (1997) The unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from peripheral blood. Blood 89:465–472 43. Witko-Sarsat V, Canteloup S, Durant S, Desdouets C, Chabernaud R, Lemarchand P, Descamps-Latscha B (2002) Cleavage of p21waf1 by proteinase-3, a myeloid-specific serine protease, potentiates cell proliferation. J Biol Chem 277:47338–47347 44. Voermans C, Kooi ML, Rodenhuis S, van der Lelie H, van der Schoot CE, Gerritsen WR (2001) In vitro migratory capacity of CD34+ cells is related to hematopoietic recovery after autologous stem cell transplantation. Blood 97:799–804 45. Janowska-Wieczorek, A., Matsuzaki, A., L, A. M. (2000) The Hematopoietic Microenvironment: Matrix Metalloproteinases in the Hematopoietic Microenvironment. Hematology 4:515–527. 46. Everts V, Korper W, Jansen DC, Steinfort J, Lammerse I, Heera S, Docherty AJ, Beertsen W (1999) Functional heterogeneity of osteoclasts: matrix metalloproteinases participate in osteoclastic resorption of calvarial bone but not in resorption of long bone. FASEB J 13:1219–1230 47. Christopherson KW 2nd, Cooper S, Broxmeyer HE (2003) Cell surface peptidase CD26/ DPPIV mediates G-CSF mobilization of mouse progenitor cells. Blood 101:4680–4686 48. Levesque JP, Liu F, Simmons PJ, Betsuyaku T, Senior RM, Pham C, Link DC (2004)

2


Characterization of hematopoietic progenitor mobilization in protease-deficient mice. Blood 104:65–72 49. Shirvaikar, N., Marquez-Curtis, L. A., Shaw, A. R., Turner, A. R., Janowska-Wieczorek, A. (2010) MT1-MMP association with membrane lipid rafts facilitates G-CSF-induced hematopoietic stem/progenitor cell mobilization. Exp Hematol. 50. Kent DG, Copley MR, Benz C, Wohrer S, Dykstra BJ, Ma E, Cheyne J, Zhao Y, Bowie MB, Zhao Y, Gasparetto M, Delaney A, Smith C, Marra M, Eaves CJ (2009) Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential. Blood 113:6342–6350 51. Lapidot T, Dar A, Kollet O (2005) How do stem cells find their way home? Blood 106: 1901–1910 52. Wright DE, Wagers AJ, Gulati AP, Johnson FL, Weissman IL (2001) Physiological migration of hematopoietic stem and progenitor cells. Science 294:1933–1936 53. Abkowitz JL, Robinson AE, Kale S, Long MW, Chen J (2003) Mobilization of hematopoietic stem cells during homeostasis and after cytokine exposure. Blood 102:1249–1253 54. Chen J, Larochelle A, Fricker S, Bridger G, Dunbar CE, Abkowitz JL (2006) Mobilization as a preparative regimen for hematopoietic stem cell transplantation. Blood 107:3764–3771 55. Hiramatsu H, Nishikomori R, Heike T, Ito M, Kobayashi K, Katamura K, Nakahata T (2003) Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/gammacnull mice model. Blood 102:873–880 56. Leung W, Ramirez M, Civin CI (1999) Quantity and quality of engrafting cells in cord blood and autologous mobilized peripheral blood. Biol Blood Marrow Transplant 5:69–76 57. Pitchford SC, Hahnel MJ, Jones CP, Rankin SM (2010) Troubleshooting: quantification of mobilization of progenitor cell subsets from bone marrow in vivo. J Pharmacol Toxicol Methods 61:113–121 58. Teitelbaum SL (2010) Stem cells and osteoporosis therapy. Cell Stem Cell 7:553–554 59. Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, Poulton IJ, van Rooijen N, Alexander KA, Raggatt LJ, Levesque JP (2010) Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood 116: 4815–4828 60. Lymperi, S., Ersek, A., Ferraro, F., Dazzi, F., Horwood, N. J. (2010) Inhibition of osteoclast

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function reduces hematopoietic stem cell numbers in vivo. Blood. 61. Levesque JP, Helwani FM, Winkler IG (2010) The endosteal ‘osteoblastic’ niche and its role in hematopoietic stem cell homing and mobilization. Leukemia 24:1979–1992 62. Jung, Y., Shiozawa, Y., Wang, J., Patel, L. R., Havens, A. M., Song, J., Krebsbach, P. H., Roodman, G. D., Taichman, R. S. (2010) Annexin-2 is a regulator of stromal cell-derived factor-1/CXCL12 function in the hematopoietic stem cell endosteal niche. Exp Hematol. 63. Kalinkovich A, Spiegel A, Shivtiel S, Kollet O, Jordaney N, Piacibello W, Lapidot T (2009) Blood-forming stem cells are nervous: direct and indirect regulation of immature human CD34+ cells by the nervous system. Brain Behav Immun 23:1059–1065 64. Mendez-Ferrer S, Lucas D, Battista M, Frenette PS (2008) Haematopoietic stem cell release is regulated by circadian oscillations. Nature 452:442–447 65. Lapidot, T., Kollet, O. (2010) The brain-boneblood triad: traffic lights for stem-cell homing and mobilization. Hematology 30th edition., 1–6. 66. Dar, A., Schajnovitz, A., Lapid, K., Kalinkovich, A., Itkin, T., Ludin, A., Kao, M., M., Battista, M., Tesio, M., Kollet, O., Netzer Cohen, N., Margalita, R., Buss, E., Baleux, F., Oishi, S., Fujii, N., Larochelle, A., Dunbar, C., Broxmeyer, H., Frenette, P., Lapidot, T. (2011) Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells. Leukemia (in press) 67. Jiang, S., Alberich-Jorda, M., Zagozdzon, R., Parmar, K., Fu, Y., Mauch, P., Banu, N., Makriyannis, A., Tenen, D. G., Avraham, S., Groopman, J. E., Avraham, H. K. (2010) Cannabinoid receptor 2 and its agonists mediate hematopoiesis and hematopoietic stem and progenitor cell mobilization. Blood 68. Hoggatt J, Pelus LM (2010) Eicosanoid regulation of hematopoiesis and hematopoietic stem and progenitor trafficking. Leukemia 24: 1993–2002 69. Kawamori Y, Katayama Y, Asada N, Minagawa K, Sato M, Okamura A, Shimoyama M, Nakagawa K, Okano T, Tanimoto M, Kato S, Matsui T (2010) Role for vitamin D receptor in the neuronal control of the hematopoietic stem cell niche. Blood 116:5528–5535 70. Lucas D, Battista M, Shi PA, Isola L, Frenette PS (2008) Mobilized hematopoietic stem cell yield depends on species-specific circadian timing. Cell Stem Cell 3:364–366

Chapter 3 Hematopoietic Stem Cell Mobilization with G-CSF Chitra Hosing Abstract Cytokine mobilized peripheral blood stem cells are the preferred source of stem cells in autologous stem cell transplantation and have virtually replaced bone marrow as the stem cell source. In recent years, a dramatic increase has been reported in the use of peripheral blood stem cells for allogeneic transplantation as well. The reason for this rise is that peripheral blood stem cell transplants when compared to bone marrow transplants are associated with a more rapid recovery of granulocytes and platelets after transplantation and a lower regimen-related and transplant-related mortality. Peripheral blood stem cells can be easily harvested on an outpatient basis without the need for general anesthesia. In most cases peripheral blood stem cells are collected after G-CSF administration. In this chapter we describe peripheral blood stem cell mobilization in autologous transplant patients and in allogeneic donors using G-CSF. Key words: Hematopoietic stem cell mobilization, G-CSF, Autologous stem cell transplantation, Allogeneic stem cell transplantation

1. Introduction High-dose chemotherapy followed by autologous or allogeneic stem cell transplantation is used in the management of a variety of hematological and nonhematological malignancies. Stem cells were first described in the peripheral blood of mice back in 1962 (1) and in humans in 1971 (2). The subsequent development of apheresis instruments made it possible to collect peripheral blood stem cells (PBSC) (3, 4). Goldman and colleagues and Korbling et al. were among the first to demonstrate that PBSC collected from patients could reestablish normal marrow hematopoiesis after high-dose chemotherapy (5, 6). Use of PBSC for allogeneic stem cell transplantation was first reported in 1989 when PBSC were collected


37

38

C. Hosing

from an “unstimulated” donor who required ten apheresis procedures, to collect adequate cells. The recipient experienced trilineage engraftment (7). In 1993, Dreger et al. reported successful engraftment using allogeneic PBSC collected after mobilization with granulocyte-colony stimulating factor (G-CSF) in a patient who had engraftment failure after bone marrow (BM) transplantation from the same donor (8). Since mid-1990s PBSCs mobilized with cytokines have virtually replaced BM as the source of stem cells for autologous transplantation and are been increasingly used in allogeneic transplantation. The advantages of PBSC rather than BM are ease of collection and rapid engraftment. PBSC can be harvested without the need of general anesthesia and the discomfort of multiple BM aspirations. Furthermore, some studies have shown that PBSC restore immune functions more rapidly than BM (9, 10). Other studies have reported a lower incidence of documented infections, fewer febrile days, a lower number of red cell and platelet transfusion, lower demand for antibiotics and intensive care requirements resulting in reduced costs of PBSC transplants compared with BM (11). The other advantages of G-CSF mobilization are the safe outpatient self-application and the fixed-day apheresis. Filgrastim (granulocyte colony-stimulating factor (G-CSF)) and sargramostim (granulocyte macrophage colony-stimulating factor (GM-CSF)) are currently the only FDA-approved colonystimulating factors for stem cell mobilization (12, 13). In a study of 1,306 normal donors 99% of donors were mobilized with G-CSF (14). Filgrastim is a granulocyte-colony stimulating factor analog used to stimulate the proliferation and differentiation of granulocytes. It is produced by recombinant DNA technology. The gene for human GCSF is inserted into E. coli and the G-CSF produced closely resembles naturally produced G-CSF in humans. G-CSF was originally used to treat neutropenia and is now the cytokine of choice for increasing the number of hematopoietic stem cells in the blood before collection by leukapheresis for use in allogeneic and autologous stem cell transplantation. G-CSF stimulates the expansion and activation of myeloid and granulocyte precursors within the bone marrow, resulting in PBSC mobilization after a few days. Pegylated filgrastim is a covalent conjugate of G-CSF and monomethoxypolyethylene glycol, with a half-life of about 33 h (15). As a result of this conjugation there is decrease in the renal elimination of pegylated filgrastim resulting in adequate levels of G-CSF for approximately 2 weeks. Pegylated filgrastim is currently approved for the treatment of chemotherapy-induced neutropenia, but has been also utilized for stem cell mobilization because of its convenience (16).

3

Hematopoietic Stem Cell Mobilization with G-CSF

39

2. Materials 1. Recombinant human G-CSF (Neupogen®, Amgen). 2. Pegylated filgrastim/pegfilgrastim (Neulasta®, Amgen).

3. Methods Grigg et al. evaluated the kinetics of mobilization by G-CSF in normal volunteers. G-CSF was injected subcutaneously at a dose of 3, 5, or 10 μg/kg/day. A subset of volunteers from each dose cohort underwent leukapheresis on study day 6 (after 5 days of G-CSF). Granulocyte-macrophage colony-forming cell (GM-CFC) numbers in the blood were maximal after 5 days of G-CSF, a broader peak was evident for CD34+ stem cells between days 4 and 6. The 95 % confidence intervals for mean number of PBSC per milliliter of blood in the three dose cohorts overlapped on each study day. However, on the peak day, CD34+ stem cells were significantly higher in the 10 μg/kg/day cohort than in a pool of the other two cohorts. Leukapheresis products obtained at the 10 μg/kg/day dose level contained a median GM-CFC number of 93 × 104/kg (range 50–172 × 104/kg). Collections from volunteers receiving lower doses of G-CSF contained a median GM-CFC number of 36 × 104/kg (range 5–204 × 104/kg). All leukapheresis products obtained at the 10 μg/kg/day dose level were potentially sufficient for allogeneic transplantation purposes. Thus, G-CSF 10 μg/kg/day for 5 days with a single leukapheresis on the following day is a highly effective regimen for PBSC mobilization and collection in normal donors (17). Molineux et al. studied the effects of single daily doses of pegfilgrastim (a PEGylated form of the recombinant human G-CSF) at 30, 60, 100, and 300 μg/kg (18). Successful CD34+ stem cell mobilization was observed at all doses. At the highest dose of 300 μg/kg a peak number of CD34+ cells/μl were seen at day 4. The numbers approached normal by day 12 or 13. In all other dose groups the peak was at the same time, but returned to normal range by day 9. In the same study mobilization of stem cells as measured by GM-CFC/ml was also noted with the maximum response seen at the 100 μg/kg dose level. There was no additional benefit to increasing the dose to 300 μg/kg (18–20). 3.1. Stem Cell Mobilization with G-CSF

1. G-CSF is generally given at a dose of 10 μg/kg/day subcutaneously and continued until the completion of apheresis. The half-life of G-CSF is only 3–4 h and therefore daily subcutaneous injections are required (11, 21–23) (see Note 1).

40

C. Hosing

2. The dose range for G-CSF that has been used varies from 10 μg/kg/day up to as high as 32 μg/kg/day, administered subcutaneously (24–29). 3. The optimal dose of G-CSF needed to collect enough CD34+ stem cells with minimal toxicity to the donors has not been established. 4. For G-CSF doses of up to 10 μg/kg/day, a dose–response relationship exists between the dose of G-CSF and the mobilization of CD34+ progenitor cells (30, 31). 5. G-CSF can be administered as a single daily injection or as twice-a-day injection (17, 32). 6. The total white blood cell count rises within 4–6 h but a substantial increase in circulating CD34+ stem cells does not occur until the third day after the first G-CSF injection (32). 7. Stem cell collection is usually started on the fourth or fifth day after the G-CSF injections begin. Most centers start collection when the circulating peripheral blood CD34 count is 10/μl (Fig. 1) (25–28). 8. The optimal stem cell dose for transplantation is not established, but most centers collect between 4 and 6 × 106 CD34+ cells/kg of the recipient’s body weight. The minimal acceptable dose is 2 × 106 CD34+ cells/kg of the recipient’s body weight (33). 9. G-CSF induces myeloid expansion, activation, and degranulation leading to release of neutrophil proteases in the marrow. The proteases cleave and inactivate some adhesive connections

Fig. 1. Efficacy of CD34+ mobilization and PBSC yield. Printed with permission from Holig et al. 2009 © American Society of Hematology.

3


41

like CXCR4/SDF-1α, VCAM-1/VLA-4 and facilitate stem cell mobilization (34). The down regulation of SDF-1α during G-CSF administration is beside the proteolytic activity caused also by the decrease of CXCL 12 transcription in stromal cells (35). G-CSF with chemotherapy mobilization is further associated with down regulation of expression of serine-proteinase inhibitors (α1antitrypsin, etc.), which under normal conditions block activity of serine proteases released by BM neutrophils. Together with this fact the down regulation facilitates an accumulation of proteases within the marrow microenvironment (36). 10. Almost all patients report some bone pain after growth factor administration. In most cases, the pain can be relieved by acetaminophen, and very rarely narcotics are required. The side effects are dose related and resolve within a few days of G-CSF discontinuation (31, 37, 38), see Table 1. 11. As summarized in Table 2, uncommon but severe side effects requiring discontinuation of G-CSF have been reported in 1–3% of donors (31, 38). G-CSF administration has been reported to precipitate serious and even life-threatening sickle cell crises in donors with hemoglobin SS, hemoglobin S ± beta

Table 1 Commonly reported symptoms associated with G-CSF administration Reference

Grigg (17) Anderlini (38) Bishop (26) Stroncek (51) Stroncek (51)

Dose of G-CSF (μg/kg/day) 10

12

5

5

10

41

19

21

–

53

76

83

32/11

68/0

44

74

67

0

27

–

–

No. of patients

15

341

Bone pain

87

84

Myalgias/arthralgias

27

Headache

33

Fever/flu-like symptoms

7

– 54

Chills/rigors

–

–

22

5

14

Body aches

–

–

–

–

–

Fatigue

47

31

–

37

43

Nausea/vomiting

–

13

–

16

24

Insomnia

–

–

–

16

24

Paresthesia

–

–

–

16

38

Diarrhea

–

–

–

11

5

Rash

–

–

–

11

5

42

C. Hosing

Table 2 Unusual and/or major adverse events reported during (or shortly after) G-CSF administration in normal donors Reference

Side effect

Nuamah et al. (52), Becker et al. (53)

Splenic rupture

de Azevedo and Tabak (54), Arimura et al. (55)

Capillary leak syndrome

Vij et al. (56)

Unstable angina

Bensinger et al. (57)

Myocardial infarctiona

Parkkali et al. (58), Huhn et al. (59)

Iritis, episcleritis

Storek et al. (60)

Flare-up of rheumatoid arthritis; ankylosing spondylitis

Spitzer et al. (61)

Acute gouty arthritis

Pei et al. (62)

Intracranial hemorrhage

Adkins (63)

Anaphylactoid reaction

Abboud et al. (39), Adler et al. (40), Grigg (41)

Sickle cell crisis in patients with hemoglobin SS, hemoglobin SC, or hemoglobin S ± β thalassemia

a

Donor had a prior history of coronary artery disease and myocardial infarction

thalassemia, or hemoglobin SC (39–41). This complication has not been reported in donors with the sickle cell trait (42) (see Note 2). 12. There is an increase in the white blood cell count after G-CSF administration, mostly due to the increase in the absolute neutrophil counts. The total white blood cell count may be as high as 70 or 80 × 109/l after 5 days of administration (43–45) (see Note 3). 13. There is a slight, but significant, decrease of platelet counts and hemoglobin levels after G-CSF administration, which persists for approximately 30 days after PBSC donation. 14. Transient elevations in the levels of lactate dehydrogenase, alkaline phosphatase, and alanine aminotransferase are seen after 4–5 days of G-CSF administration (31, 46). Levels of serum potassium, magnesium, and blood urea nitrogen decline minimally (31). Holig et al. found that leukocyte counts 4 weeks after PBSC collection were significantly lower than the white blood count on day 0 (P < .001). After 6 months, 1 year, and 5 years after PBSC donation, the white blood counts were higher than at 4 weeks but never completely returned to baseline values. Changes in absolute neutrophil counts resembled those in white blood count.

3


43

Lymphocyte counts were significantly diminished up to 1 year after PBSC donation and were slightly elevated after 2–5 years. Platelet counts reached pretreatment values 6 months after apheresis and remained stable thereafter (47). Pulsipher et al. reviewed the NMDP follow-up data regarding the incidence of development of malignancies in this cohort of 2,408 donors. Annual attempts at follow-up were made for all donors (median follow-up, 49 months; range, 2 days to 99 months). No cases of acute myelogenous leukemia or myelodysplasia were reported. Twenty-five nonhematologic cancers of various types occurred along with one case of chronic lymphocytic leukemia. Comparison of the incidence of these cancers to expected rates according to the SEER database showed no evidence of increased cancer risk in the donor cohort (47). Others have reported similar results (14, 45, 48, 49). 3.2. Stem Cell Mobilization with Pegfilgrastim

In a prospective, phase II study of pegfilgrastim, administered as a single injection to mobilize autologous PBSC in patients with multiple myeloma, 19 patients received 12 mg pegfilgrastim. A median of 8.4 (range 4.1–15.8) × 106 CD34+ cells/kg could be collected. Sustained hematological recovery occurred in all the patients who underwent high-dose chemotherapy followed by autologous PBSC transplantation with pegfilgrastim-mobilized cells (20). Use of single-dose pegfilgrastim for the mobilization of allogeneic peripheral blood stem cells in healthy family and unrelated donors was described by Kroschinsky et al. (50). In their study 25 related or unrelated healthy donors received a single-dose of 12 mg pegfilgrastim for mobilization of allogeneic peripheral blood progenitor cells. In 80% of donors only a single apheresis procedure was necessary to reach the target progenitor cell dose. 1. Pegfilgrastim is also effective for mobilization of PBSC for collection by apheresis and is given as a single subcutaneous injection of 6 or 12 mg. 2. The efficacy and toxicity profile of pegfilgrastim is similar to that described with G-CSF treatment (20). 3. Bone pain, headaches and transient elevations of liver enzymes were the main adverse events (50).

3.3. Overcoming Inefficient Stem Cell Mobilization

There are no established mobilization strategies for poorly mobilizing patients. Some approaches that have been used include: 1. Increasing the dose of G-CSF to 32 μg/kg/day in two divided doses. 2. Adding plerixafor, GM-CSF, or other chemokines if available. 3. If failure to mobilize with cytokines alone, then remobilize with chemotherapy priming and cytokines.

44

C. Hosing

4. If any evidence for viral infection during mobilization, then remobilize after infection has resolved (viral infection may exert a myelosuppressive effect on the marrow). 5. Bone marrow harvest or in some instances G-CSF primed marrow can be used.

4. Notes 1. Most physicians will round the dose of G-CSF to the nearest vial size. G-CSF is available in the US in prefilled syringes of 300 and 480 μg. 2. G-CSF administration has been reported to precipitate lifethreatening sickle cell crises in donors with hemoglobin SS, hemoglobin S ± beta thalassemia, or hemoglobin SC. Therefore, G-CSF should be administered with great caution (if at all) to normal donors with any demonstrable hemoglobin S. 3. Although leukostasis has never been reported in donors, most physicians decrease the G-CSF dose when the WBC is higher than 70 or 75 × 109/l. References 1. Goodman JW, Hodgson GS (1962) Evidence for stem cells in the peripheral blood of mice. Blood 19:702–714 2. McCredie KB, Hersh EM, Freireich EJ (1971) Cells capable of colony formation in the peripheral blood of man. Science 171:293–294 3. Weiner RS, Richman CM, Yankee RA (1977) Semicontinuous flow centrifugation for the pheresis of immunocompetent cells and stem cells. Blood 49:391–397 4. Hillyer CD, Tiegerman KO, Berkman EM (1991) Increase in circulating colony-forming units-granulocyte-macrophage during largevolume leukapheresis: evaluation of a new cell separator. Transfusion 31:327–332 5. Korbling M, Dorken B, Ho AD, Pezzutto A, Hunstein W, Fliedner TM (1986) Autologous transplantation of blood-derived hemopoietic stem cells after myeloablative therapy in a patient with Burkitt’s lymphoma. Blood 67:529–532 6. Goldman JM (1979) Autografting cryopreserved buffy coat cells for chronic granulocytic leukaemia in transformation. Exp Hematol 7(Suppl 5):389–397 7. Kessinger A, Smith DM, Strandjord SE, Landmark JD, Dooley DC, Law P, Coccia PF, Warkentin PI, Weisenburger DD, Armitage JO

8.

9.

10.

11.

(1989) Allogeneic transplantation of bloodderived, T cell-depleted hemopoietic stem cells after myeloablative treatment in a patient with acute lymphoblastic leukemia. Bone Marrow Transplant 4:643–646 Dreger P, Suttorp M, Haferlach T, Loffler H, Schmitz N, Schroyens W (1993) Allogeneic granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells for treatment of engraftment failure after bone marrow transplantation. Blood 81:1404–1407 Storek J, Dawson MA, Storer B, Stevens-Ayers T, Maloney DG, Marr KA, Witherspoon RP, Bensinger W, Flowers ME, Martin P, Storb R, Appelbaum FR, Boeckh M (2001) Immune reconstitution after allogeneic marrow transplantation compared with blood stem cell transplantation. Blood 97:3380–3389 Bensinger WI, Martin PJ, Storer B, Clift R, Forman SJ, Negrin R, Kashyap A, Flowers ME, Lilleby K, Chauncey TR, Storb R, Appelbaum FR (2001) Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med 344:175–181 Schmitz N, Linch DC, Dreger P, Goldstone AH, Boogaerts MA, Ferrant A, Demuynck

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12. 13. 14.

15. 16.

17.

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19.

20.


HM, Link H, Zander A, Barge A (1996) Randomised trial of filgrastim-mobilised peripheral blood progenitor cell transplantation versus autologous bone-marrow transplantation in lymphoma patients. Lancet 347:353–357 Product information. Leukine (sargrastim). W.B.H. and Pharmaceuticals, A, Seattle Product information. Neupagen (filgrastim). Amgen Inc., Thousand Oaks, CA Anderlini P, Rizzo JD, Nugent ML, Schmitz N, Champlin RE, Horowitz MM (2001) Peripheral blood stem cell donation: an analysis from the International Bone Marrow Transplant Registry (IBMTR) and European Group for Blood and Marrow Transplant (EBMT) databases. Bone Marrow Transplant 27:689–692 Curran MP, Goa KL (2002) Pegfilgrastim. Drugs 62:1207–1213, discussion 1214–1205 Isidori A, Tani M, Bonifazi F, Zinzani P, Curti A, Motta MR, Rizzi S, Giudice V, Farese O, Rovito M, Alinari L, Conte R, Baccarani M, Lemoli RM (2005) Phase II study of a single pegfilgrastim injection as an adjunct to chemotherapy to mobilize stem cells into the peripheral blood of pretreated lymphoma patients. Haematologica 90:225–231 Grigg AP, Roberts AW, Raunow H, Houghton S, Layton JE, Boyd AW, McGrath KM, Maher D (1995) Optimizing dose and scheduling of filgrastim (granulocyte colony-stimulating factor) for mobilization and collection of peripheral blood progenitor cells in normal volunteers. Blood 86:4437–4445 Molineux G, Kinstler O, Briddell B, Hartley C, McElroy P, Kerzic P, Sutherland W, Stoney G, Kern B, Fletcher FA, Cohen A, Korach E, Ulich T, McNiece I, Lockbaum P, MillerMessana MA, Gardner S, Hunt T, Schwab G (1999) A new form of filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans. Exp Hematol 27:1724–1734 Johnston E, Crawford J, Blackwell S, Bjurstrom T, Lockbaum P, Roskos L, Yang BB, Gardner S, Miller-Messana MA, Shoemaker D, Garst J, Schwab G (2000) Randomized, dose-escalation study of SD/01 compared with daily filgrastim in patients receiving chemotherapy. J Clin Oncol 18:2522–2528 Hosing C, Qazilbash MH, Kebriaei P, Giralt S, Davis MS, Popat U, Anderlini P, Shpall EJ, McMannis J, Korbling M, Champlin RE (2006) Fixed-dose single agent pegfilgrastim for peripheral blood progenitor cell mobilisation in patients with multiple myeloma. Br J Haematol 133:533–537

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21. Nademanee A, Sniecinski I, Schmidt GM, Dagis AC, O’Donnell MR, Snyder DS, Parker PM, Stein AS, Smith EP, Molina A et al (1994) High-dose therapy followed by autologous peripheral-blood stem-cell transplantation for patients with Hodgkin’s disease and nonHodgkin’s lymphoma using unprimed and granulocyte colony-stimulating factor-mobilized peripheral-blood stem cells. J Clin Oncol 12:2176–2186 22. Gazitt Y, Freytes CO, Callander N, Tsai TW, Alsina M, Anderson J, Holle L, Cruz J, Devore P, McGrath M, West G, Alvarez R, Montgomery W (1999) Successful PBSC mobilization with highdose G-CSF for patients failing a first round of mobilization. J Hematother 8:173–183 23. Bensinger WI, Price TH, Dale DC, Appelbaum FR, Clift R, Lilleby K, Williams B, Storb R, Thomas ED, Buckner CD (1993) The effects of daily recombinant human granulocyte colony-stimulating factor administration on normal granulocyte donors undergoing leukapheresis. Blood 81:1883–1888 24. Anderlini P, Przepiorka D, Champlin R, Korbling M (1996) Peripheral blood stem cell apheresis in normal donors: the neglected side. Blood 88:3663–3664 25. Waller CF, Bertz H, Wenger MK, Fetscher S, Hardung M, Engelhardt M, Behringer D, Lange W, Mertelsmann R, Finke J (1996) Mobilization of peripheral blood progenitor cells for allogeneic transplantation: efficacy and toxicity of a high-dose rhG-CSF regimen. Bone Marrow Transplant 18:279–283 26. Bishop MR, Tarantolo SR, Jackson JD, Anderson JR, Schmit-Pokorny K, Zacharias D, Pavletic ZS, Pirruccello SJ, Vose JM, Bierman PJ, Warkentin PI, Armitage JO, Kessinger A (1997) Allogeneic-blood stem-cell collection following mobilization with low-dose granulocyte colony-stimulating factor. J Clin Oncol 15:1601–1607 27. Sato N, Sawada K, Takahashi TA, Mogi Y, Asano S, Koike T, Sekiguchi S (1994) A time course study for optimal harvest of peripheral blood progenitor cells by granulocyte colonystimulating factor in healthy volunteers. Exp Hematol 22:973–978 28. Bensinger WI, Weaver CH, Appelbaum FR, Rowley S, Demirer T, Sanders J, Storb R, Buckner CD (1995) Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. Blood 85:1655–1658 29. Kroger N, Zeller W, Fehse N, Hassan HT, Kruger W, Gutensohn K, Lolliger C, Zander AR (1998) Mobilizing peripheral blood stem cells with high-dose G-CSF alone is as effective

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31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

C. Hosing as with Dexa-BEAM plus G-CSF in lymphoma patients. Br J Haematol 102:1101–1106 Hoglund M, Smedmyr B, Simonsson B, Totterman T, Bengtsson M (1996) Dosedependent mobilisation of haematopoietic progenitor cells in healthy volunteers receiving glycosylated rHuG-CSF. Bone Marrow Transplant 18:19–27 Stroncek DF, Clay ME, Petzoldt ML, Smith J, Jaszcz W, Oldham FB, McCullough J (1996) Treatment of normal individuals with granulocyte-colony-stimulating factor: donor experiences and the effects on peripheral blood CD34+ cell counts and on the collection of peripheral blood stem cells. Transfusion 36:601–610 Link H, Arseniev L, Bahre O, Berenson RJ, Battmer K, Kadar JG, Jacobs R, Casper J, Kuhl J, Schubert J, Diedrich H, Poliwoda H (1995) Combined transplantation of allogeneic bone marrow and CD34+ blood cells. Blood 86:2500–2508 Wuchter P, Ran D, Bruckner T, Schmitt T, Witzens-Harig M, Neben K, Goldschmidt H, Ho AD (2010) Poor mobilization of hematopoietic stem cells-definitions, incidence, risk factors, and impact on outcome of autologous transplantation. Biol Blood Marrow Transplant 16:490–499 Winkler IG, Levesque JP (2006) Mechanisms of hematopoietic stem cell mobilization: when innate immunity assails the cells that make blood and bone. Exp Hematol 34:996–1009 Semerad CL, Christopher MJ, Liu F, Short B, Simmons PJ, Winkler I, Levesque JP, Chappel J, Ross FP, Link DC (2005) G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow. Blood 106:3020–3027 Winkler IG, Hendy J, Coughlin P, Horvath A, Levesque JP (2005) Serine protease inhibitors serpina1 and serpina3 are down-regulated in bone marrow during hematopoietic progenitor mobilization. J Exp Med 201:1077–1088 Anderlini P, Przepiorka D, Champlin R, Korbling M (1996) Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood 88:2819–2825 Anderlini P, Donato M, Chan KW, Huh YO, Gee AP, Lauppe MJ, Champlin RE, Korbling M (1999) Allogeneic blood progenitor cell collection in normal donors after mobilization with filgrastim: the M.D. Anderson Cancer Center experience. Transfusion 39:555–560 Abboud M, Laver J, Blau CA (1998) Granulocytosis causing sickle-cell crisis. Lancet 351:959 Adler BK, Salzman DE, Carabasi MH, Vaughan WP, Reddy VV, Prchal JT (2001) Fatal sickle

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cell crisis after granulocyte colony-stimulating factor administration. Blood 97:3313–3314 Grigg AP (2001) Granulocyte colony-stimulating factor-induced sickle cell crisis and multiorgan dysfunction in a patient with compound heterozygous sickle cell/beta + thalassemia. Blood 97:3998–3999 Kang EM, Areman EM, David-Ocampo V, Fitzhugh C, Link ME, Read EJ, Leitman SF, Rodgers GP, Tisdale JF (2002) Mobilization, collection, and processing of peripheral blood stem cells in individuals with sickle cell trait. Blood 99:850–855 Anderlini P, Korbling M, Dale D, Gratwohl A, Schmitz N, Stroncek D, Howe C, Leitman S, Horowitz M, Gluckman E, Rowley S, Przepiorka D, Champlin R (1997) Allogeneic blood stem cell transplantation: considerations for donors. Blood 90:903–908 Stroncek DF, Clay ME, Herr G, Smith J, Ilstrup S, McCullough J (1997) Blood counts in healthy donors 1 year after the collection of granulocyte-colony-stimulating factor-mobilized progenitor cells and the results of a second mobilization and collection. Transfusion 37:304–308 Pulsipher MA, Chitphakdithai P, Miller JP, Logan BR, King RJ, Rizzo JD, Leitman SF, Anderlini P, Haagenson MD, Kurian S, Klein JP, Horowitz MM, Confer DL (2009) Adverse events among 2408 unrelated donors of peripheral blood stem cells: results of a prospective trial from the National Marrow Donor Program. Blood 113:3604–3611 Anderlini P, Przepiorka D, Seong D, Miller P, Sundberg J, Lichtiger B, Norfleet F, Chan KW, Champlin R, Korbling M (1996) Clinical toxicity and laboratory effects of granulocyte-colony-stimulating factor (filgrastim) mobilization and blood stem cell apheresis from normal donors, and analysis of charges for the procedures. Transfusion 36:590–595 Holig K, Kramer M, Kroschinsky F, Bornhauser M, Mengling T, Schmidt AH, Rutt C, Ehninger G (2009) Safety and efficacy of hematopoietic stem cell collection from mobilized peripheral blood in unrelated volunteers: 12 years of single-center experience in 3928 donors. Blood 114:3757–3763 Miflin G, Charley C, Stainer C, Anderson S, Hunter A, Russell N (1996) Stem cell mobilization in normal donors for allogeneic transplantation: analysis of safety and factors affecting efficacy. Br J Haematol 95:345–348 Cavallaro AM, Lilleby K, Majolino I, Storb R, Appelbaum FR, Rowley SD, Bensinger WI (2000) Three to six year follow-up of normal donors who received recombinant human

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granulocyte colony-stimulating factor. Bone Marrow Transplant 25:85–89 Kroschinsky F, Holig K, Poppe-Thiede K, Zimmer K, Ordemann R, Blechschmidt M, Oelschlaegel U, Bornhauser M, Rall G, Rutt C, Ehninger G (2005) Single-dose pegfilgrastim for the mobilization of allogeneic CD34+ peripheral blood progenitor cells in healthy family and unrelated donors. Haematologica 90:1665–1671 Stroncek DF, Clay ME, Jaszcz W, Lennon S, Smith J, McCullough J (1999) Collection of two peripheral blood stem cell concentrates from healthy donors. Transfus Med 9:37–50 Nuamah NM, Goker H, Kilic YA, Dagmoura H, Cakmak A (2006) Spontaneous splenic rupture in a healthy allogeneic donor of peripheral-blood stem cell following the administration of granulocyte colony-stimulating factor (g-csf). A case report and review of the literature. Haematologica 91:ECR08 Becker PS, Wagle M, Matous S, Swanson RS, Pihan G, Lowry PA, Stewart FM, Heard SO (1997) Spontaneous splenic rupture following administration of granulocyte colony-stimulating factor (G-CSF): occurrence in an allogeneic donor of peripheral blood stem cells. Biol Blood Marrow Transplant 3:45–49 de Azevedo AM, Goldberg Tabak D (2001) Life-threatening capillary leak syndrome after G-CSF mobilization and collection of peripheral blood progenitor cells for allogeneic transplantation. Bone Marrow Transplant 28:311–312 Arimura K, Inoue H, Kukita T, Matsushita K, Akimot M, Kawamata N, Yamaguchi A, Kawada H, Ozak A, Arima N, Te C (2005) Acute lung injury in a healthy donor during mobilization of peripheral blood stem cells using granulocyte-colony stimulating factor alone. Haematologica 90:ECR10 Vij R, Adkins DR, Brown RA, Khoury H, DiPersio JF, Goodnough T (1999) Unstable

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angina in a peripheral blood stem and progenitor cell donor given granulocyte-colony-stimulating factor. Transfusion 39:542–543 Bensinger WI, Buckner CD, Rowley S, Storb R, Appelbaum FR (1996) Treatment of normal donors with recombinant growth factors for transplantation of allogeneic blood stem cells. Bone Marrow Transplant 17(Suppl 2):S19–S21 Parkkali T, Volin L, Siren MK, Ruutu T (1996) Acute iritis induced by granulocyte colonystimulating factor used for mobilization in a volunteer unrelated peripheral blood progenitor cell donor. Bone Marrow Transplant 17:433–434 Huhn RD, Yurkow EJ, Tushinski R, Clarke L, Sturgill MG, Hoffman R, Sheay W, Cody R, Philipp C, Resta D, George M (1996) Recombinant human interleukin-3 (rhIL-3) enhances the mobilization of peripheral blood progenitor cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF) in normal volunteers. Exp Hematol 24:839–847 Storek J, Glaspy JA, Grody WW, Susi E, Slater ED (1993) Adult-onset cyclic neutropenia responsive to cyclosporine therapy in a patient with ankylosing spondylitis. Am J Hematol 43:139–143 Spitzer T, McAfee S, Poliquin C, Colby C (1998) Acute gouty arthritis following recombinant human granulocyte colonystimulating factor therapy in an allogeneic blood stem cell donor. Bone Marrow Transplant 21:966–967 Pei RZ, Ma JX, Zhang PS, Liu XH, Cao JJ, Du XH (2008) Intracranial hemorrhage caused by cerebrovascular malformation after donation of rhG-CSF-primed allogeneic PBSC. Bone Marrow Transplant 42:61–62 Adkins DR (1998) Anaphylactoid reaction in a normal donor given granulocyte colonystimulating factor. J Clin Oncol 16:812–813

Chapter 4 Hematopoietic Stem Cell Mobilization with Agents Other than G-CSF Jonathan Hoggatt and Louis M. Pelus Abstract Hematopoietic stem and progenitor mobilization has revolutionized the field of hematopoietic transplantation. Currently, hematopoietic grafts acquired from the peripheral blood of patients or donors treated with granulocyte-colony stimulating factor (G-CSF) are the preferred source for transplantation. G-CSF mobilization regimens, however, are associated with known morbidities and a significant number of normal donors and patient populations fail to mobilize sufficient numbers of hematopoietic stem and progenitor cells for transplantation, necessitating the need for non-G-CSF mobilization strategies. Mechanistic studies evaluating hematopoietic bone marrow niche interactions have uncovered novel agents with the capacity for hematopoietic mobilization. This chapter provides a comprehensive overview of mobilizing agents, other than G-CSF, and experimental procedures and technical aspects important to evaluate and define their hematopoietic mobilizing activities alone and in combination. Key words: Mobilization, Hematopoietic stem cells, AMD3100, GRO beta, VLA-4 inhibitor, Fucoidan, BIO5192, CXCR4, SDF-1, G-CSF

1. Introduction Allogeneic hematopoietic stem cell transplantation (HCT) is a curative option for many patients with hematological malignancies. The source of stem cells used for transplant can have a significant impact on patient outcome. In spite of a higher incidence of chronic graft-versus-host disease (GVHD) observed with mobilized peripheral blood hematopoietic stem cell grafts (1–3), studies comparing granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) to bone marrow have shown that PBSC are associated with more rapid engraftment, reduction in infectious complications, and in patients with advanced


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malignancies, lower regimen-related mortality (4–6). In many centers PBSC are now the preferred hematopoietic stem cell source used for human leukocyte antigen (HLA)-identical sibling as well as matched related and unrelated donor transplantation (7, 8). G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), and more recently plerixafor (AMD3100), for patients who fail to mobilize with a G- or GM-CSF, are the only FDA approved agents for mobilizing autologous PBSC, and G-CSF is the preferred mobilizing agent in the allogeneic setting. G-CSF, however, is associated with morbidity in the form of bone pain that may result in absence from work and disruption of lifestyle for the donor during the mobilization process. Furthermore, G-CSF has also been associated with serious, albeit rare, toxicity, including splenic rupture, in normal donors (9–12). Methods of mobilizing PBSC that avoid the use of growth factors such as G-CSF are, therefore, of great interest.

2. The Hematopoietic Niche Control of hematopoietic proliferation and differentiation is highly complex, and homeostatic balance is likely maintained by both intrinsic and genetic cues within individual cells and extrinsic cues from the supportive microenvironment in which hematopoietic stem cells (HSC) reside. HSC reside in very defined and limited microenvironments, or “niches” (13), and signals within these niches direct HSC maintenance. In mammals, the primary HSC niche is contained within the bone marrow, which is comprised of stromal cells and an extracellular matrix of collagens, fibronectin, and proteoglycans (14). Recent studies have shown that osteoblasts within the endosteal bone marrow niche are a significant regulatory component of hematopoiesis (15–18). Within the niche, HSCs are thought to be “tethered” to osteoblasts, other stromal cells, and the extracellular matrix through a variety of adhesion molecule interactions, many of which are likely redundant systems. Early studies exploring the role of osteoblasts in maintaining HSCs suggested that N-cadherin interactions mediated the positive effects on HSCs (16); however, more recent studies have contradicted these findings (19, 20). Numerous other adhesion molecules have been implicated as contributing to HSC and HPC tethering, including, but not limited to, the integrins α4β1—very late antigen-4 (VLA-4) (21–26), α5β1—very late antigen-5 (VLA5) (22, 23, 25, 27), α4β7—lymphocyte Peyer’s patch adhesion molecule-1 (LPAM-1) (28), the alpha 6 integrins (Laminins) (29, 30), CD44 (22, 31), E-selectins (32–34), the angiopoietin receptor tyrosine kinase with immunoglobulin-like and EGF-like

4

Hematopoietic Stem Cell Mobilization with Agents Other than G-CSF

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domains-2 (Tie-2) (18), osteopontin (OPN) (35, 36), endolyn (CD164) (37), and the calcium-sensing receptor (CaR) (38). The most explored niche interaction, and perhaps the most important in regulating HSC and HPC trafficking to and from the marrow niche, is the interaction between the CXC chemokine receptor 4 (CXCR4) and its ligand stromal cell-derived factor-1α (SDF-1α). SDF-1α is produced by osteoblasts (39), and has also been found on endothelial cells and within bone itself (40, 41). HSC and hematopoietic progenitor cells (HPC) express CXCR4 and are chemo-attracted to and retained within the bone marrow by SDF-1α (42–44). Under steady state conditions, HSC and HPC normally reside within the bone marrow niches, while the mature cells ultimately exit the marrow and enter the peripheral blood. However, considerable evidence over the last several decades demonstrates that HSC and HPC also traffic to the peripheral blood (45–50), and this steady state trafficking leaves open niche spaces that can be repopulated by transplanted HSC (51). Based on observations that increased HPC were found in patients after chemotherapy (52, 53), we now know that this natural egress of HSC and HPC into the periphery can be enhanced, allowing for “mobilization” of these cells to the peripheral blood (47, 48). Mobilized adult HSC and HPC are widely used for autologous and allogeneic transplantation and have improved patient outcomes compared to bone marrow. Mobilization can be achieved through administration of chemotherapy (52–54), or hematopoietic growth factors, chemokines, or small molecule inhibitors or antibodies against chemokine receptors and integrins. Procedures to monitor hematopoietic mobilization induced by G-CSF have been described in other chapters in this book. Many of the methods and procedures to monitor hematopoietic stem and progenitor cells are not unique to specific mobilizing agents and can be applied to mobilization experiments exploring novel agents. This remainder of this chapter will describe current compounds and procedures for non-G-CSF mediated PBSC mobilization, with a focus on unique aspects and procedures relevant to these agents and rapid mobilizers.

3. Mobilization by Agents Other Than G-CSF 3.1. Agents That Disrupt CXCR4/ SDF-1a

Many agents capable of mobilizing HSC and HPC act through mechanisms that disrupt the CXCR4/SDF-1α axis. Most notably, the CXCR4 antagonist AMD3100 (Plerixafor; Mozobil™) mobilizes HSC and HPC (55–60) and received FDA approval in December 2008 for use in combination with G-CSF for patients with Non-Hodgkin’s lymphoma and multiple myeloma. In addition to AMD3100, the CXCR4 antagonists T140 (61) and T134

52


(62) are both capable of mobilization. Hematopoietic mobilization has also been reported after administration of CXCR4 partial agonists, including (met)-SDF-1β (63), CTCE-0214 (64), and CTCE-0021 (60), through desensitization and reduced surface expression of the CXCR4 receptor. A number of polymeric compounds have been reported to mobilize HSC and HPC including Betafectin (65, 66), sulfated polysaccharides (Fucoidan) (67–69), sulfated colominic acid (70), and the smaller glycosaminoglycan (GAG) mimetics (71), which appear to alter plasma SDF-1α levels (69–71), enhance matrix metalloproteinase-9 (MMP-9) production (65, 68, 71), increase CXCR4 receptor function on HPC (70), and perhaps affect selectin and other adhesion molecules through undefined mechanisms. The complement system, particularly the C3a peptide fragment of the third complement system, has been reported to increase sensitivity of HSC and HPC to SDF-1α (72), which acts to counteract normal mobilization responses that reduce marrow SDF-1α. An inhibitor of the C3a receptor, SB 290517, when used in combination with G-CSF reduces this increased sensitivity to SDF-1α and results in an enhancement in mobilization, marked by a reduced requirement for G-CSF (73). 3.2. Other Mobilization Agents

As previously described, HSC and HPC are tethered within the bone marrow through adhesion interactions within the niche. The mobilization agents described thus far mechanistically function by disrupting one of these interactions, the CXCR4/SDF-1α axis; however, disruption of other HSC/niche interactions presents further targets for hematopoietic mobilization strategies. Targeting the interaction between VLA-4 and VCAM-1 with either antibodies against VLA-4 (24, 74), antibodies against VCAM-1 (75, 76), or a small molecule inhibitor of VLA-4 (BIO5192) (77), results in hematopoietic mobilization. Recently, signaling through the Ephephrin A3 axis was shown to increase adhesion to fibronectin and VCAM-1, and disruption of this signaling axis in vivo with a soluble EphA3-Fc fusion protein results in hematopoietic mobilization (78). Defibrotide, an adenosine receptor agonist, has been reported to reduce expression of the adhesion molecules P-selectin (79) and intercellular adhesion molecule-1 (ICAM-1) (80), and in vivo administration along with G-CSF enhances mobilization (81). The CXCR2 agonist GROβΔ4 has been shown to mobilize HSC and HPC, with peak mobilization occurring 15 min post administration (82–84), due primarily to a rapid increase in MMP-9 activity. GROβΔ4 can synergistically increase mobilization by G-CSF, reduce the requirement for G-CSF, and mobilizes HSC with enhanced engraftment and long-term repopulating abilities. In contrast to CXCR4, CXCR2 is not expressed on HSC and HPC, rather mobilization is mediated via protease release from

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neutrophils (84, 85), demonstrating that mobilization agents can target nonhematopoietic cells to illicit mobilization responses. While the agents discussed thus far have targeted receptors or ligands at the cell surface, particularly the CXCR4/SDF-1α pathway, mobilization strategies have also targeted intracellular signaling pathways downstream of CXCR4 and other receptors. Pertussis toxin (Ptx) is an inhibitor of the Gαi G-protein, which is coupled to the CXCR4 receptor, and in vivo administration results in abrogation of response to SDF-1α, and mobilization (86). Similarly, the Rho GTPase, Rac1, is a mediator of the downstream signaling pathways of CXCR4 and β integrins, and administration of a small molecule inhibitor of Rac1, NSC23766, has been reported to mobilize HSC and HPC (87).

4. Mobilization Agent Administration and Blood Collection 4.1. Dosing and Kinetics of Administration 4.2. Evaluating Combinations of Agents

While mobilization by G-CSF typically requires 4–6 days of administration to achieve optimal mobilization, a common characteristic of non-G-CSF mobilization agents is that they are considerably more rapid. Table 1 represents the optimal dose and route of administration for agents reported to mobilize hematopoietic progenitor cells, and the time post administration that has been reported to result in peak mobilization. The rapid kinetics of response and variation in mobilization mechanisms make exploration of combination treatment regimens highly attractive to evaluate for potential synergy in response. However, when exploring combination treatment, several different dosing regimens should be attempted to fully explore possible synergistic activity. As an example, we explored the combination of AMD3100 with GROβΔ4. The peak mobilization with AMD3100 is at 60 min post administration, while the peak for GROβΔ4 is 15 min. Therefore, we hypothesized that if GROβΔ4 was given 45 min post administration of AMD3100, and blood was collected 15 min later, allowing for blood collection at the peak time for each agent on its own, that the maximum mobilization of the combination would be achieved. However, this dosing regimen did not show any synergy and at best resulted in only additive mobilization of HPC (Fig. 1). However, if both AMD3100 and GROβΔ4 are given at the same time, and blood is collected 15 min post administration, a significant synergy in mobilization was observed, that persisted for >2 h. Thus, optimum kinetics of a mobilization agent on its own may not be the optimal kinetics when used in combination with other agents. Similarly, some compounds, based on molecular mechanism of action, may be theorized to enhance mobilization; however, the compounds may only work in combination with

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Table 1 Mobilization agent dosing and kinetics of administration Mobilization agent

Regimen

Route

Bleeding time (post last dose)

References

AMD3100

5 mg/kg

SC

1h

(55, 56)

T140

5 mg/kg

SC

2h

(61)

T134

10 mg/kg

SC

1h

(62)

(met)-SDF-1β

300 μg/mouse

IV

48 h

(63)

CTCE-0214

75 μg/mouse

IV

4h

(64)

CTCE-0021

25 mg/kg

SC

1h

(60)

Betafectin (PGG-β glucan) Fucoidan

2 mg/kg 9.6 mg/kg 100 mg/kg × 3 days 25 mg/kg × 6 doses in 48 h

IV IV IV IP

30 min 24 h 3h 2h

(66) (65) (68, 69) (67)

Sulfated colominic acid

100 mg/kg

IV

30 min

(70)

GAG mimetics

50 mg/kg

IP

3h

(71)

SB290157*

500 ng/mouse × 3 days

IP

6h

(73)

Anti-CD49d(VLA-4)

2 mg/kg × 3 days

IV

~24 h

(24, 74)

Anti-VCAM-1

5 mg/kg × 2 days 2 mg/kg × 3 days

IV IV

6h ~24 h

(75) (76)

BIO5192

1 mg/kg

IV

1h

(77)

300 μg/mouse

IP

30 min

(78)

Defibrotide

15 mg/mouse × 5 days

IP

2h

(81)

GROβΔ4

2.5 mg/kg

SC

15 min

(82, 83)

Pertussis toxin

100 ng i.v.

IV

96 h

(86)

NSC23766

2.5 mg/kg

IP

6h

(87)

EphA3-Fc a

SC subcutaneous, IV intravenous, IP intraperitoneal a Mobilization response only seen when used in combination with a G-CSF regimen

another mobilizing agent (as is the case with Defibrotide and SB290157). In some circumstances, it may be advantageous to perform mobilization assays in combination with G-CSF or another agent, even if an experimental agent failed to mobilize on its own. 4.3. Peripheral Blood Collection

Peripheral blood should be collected at the peak of mobilization. There are two important parameters to consider when designing and performing mobilization experiments with rapid mobilizing agents: (1) it has been reported that circadian rhythms can affect

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55

Fig. 1. Evaluation of AMD3100 and GROβΔ4 combination treatment. Mice were treated with AMD3100 (5 mg/kg, SC), GROβΔ4 (2.5 mg/kg, SC), or the combination of the two agents, either staggered (as shown), or simultaneously, and peripheral blood was assayed for CFU-GM content at the indicated time points post treatment.

hematopoietic trafficking and mobilization (88, 89). In our laboratory, we typically time all of our mobilization experiments so that bleeding begins at about 10:00 a.m., which in our facility is ~4 h after initiation of light. Variations in bleeding time, if not controlled, can make interpretation of results from experiment to experiment difficult. (2) Many of the agents described, particularly GROβΔ4, exhibit rapid peaks (15 min) in peripheral HSC and HPC that return to baseline within an hour post administration. Therefore, in experiments with many mice, it is important to stagger the dosing of the mobilization agent and the acquisition of peripheral blood, such that blood collection can be performed at the peak mobilization time. In the case of GROβΔ4 we typically treat a cage of 3–5 mice approximately every 10 min, allowing for enough time in-between cages for blood collection and injections in the next set of mice. In addition, a permanent marker can be used to mark the tail of each mouse in a cage to keep track of the order of injections. Our laboratory normally collects peripheral blood using a cardiac puncture technique; however, other techniques may be suitable (i.e., retroorbital). After collection of blood, a complete blood count is performed using either a veterinary cell counter (Hemavet 950FS, Drew Scientific; or similar

56


instrument), or white blood cell count determined manually with a hemocytometer. For analysis of HSC and HPC mobilization, we isolate low density mononuclear cells (LDMC) before additional assays using the Lympholyte®-Mammal (Cedarlane Labs, Burlington, NC, #CL5120) gradient separation technique.

5. Determination of HPC and HSC Mobilization 5.1. Progenitor Cell Analysis: Colony Assays

5.2. Competitive Transplantation Assays

Numerous in vitro colony-forming cell assays are available to identify populations of HPC with distinct lineage-restricted differentiation patterns and can be characterized by the type of colonies they form in semi-solid agar, methylcellulose or plasma clot. HPC can be identified as colony-forming unit-granulocyte (CFU-G), colonyforming unit-monocyte/macrophage (CFU-M), colony forming unit-granulocyte/macrophage (CFU-GM), burst-forming uniterythroid (BFU-E) or the colony-forming unit-erythroid (CFUE). Additionally, megakaryocyte progenitor cells (CFU-Mk or CFU-Meg), and progenitors with multipotential have been described, the most common one assayed today referred to as a colony-forming unit granulocyte/erythrocyte/monocyte/megakaryocyte (CFU-GEMM) (90–94). Typically our laboratory utilizes the semi-solid agar CFU-GM assay to screen mobilization agents and regimens as we have previously described (83, 95). In some experiments, multiple colony types, including CFU-GM, BFU-E and CFU-GEMM are enumerated in 1% methylcellulose containing erythropoietin (EPO), GM-CSF and stem cell factor (SCF) (82, 96). The reader is referred to Chapter 3 of this book for further details on progenitor cell assays. Initially, the colony forming unit-spleen (CFU-S) assay (97) was believed to measure HSC, and is still used by many investigators today as a surrogate HSC assay. Other surrogate assays commonly used to imply HSC function include the cobblestone area-forming cell (CAFC) assay (98–100) and the long-term culture-initiating cell (LTC-IC) assay (101–103). While these assays may certainly be indicative of more immature populations of cells than detected in CFU assays, they are not definitive assays for HSC function (104, 105). The only true measure of HSC function is the ability to fully repopulate a lethally irradiated host. By this definition, the “presence” of HSC could be determined just by monitoring survival of lethally irradiated transplant recipients. If the mice live (longer than 16 weeks), with reconstitution of all blood lineages, then by definition, the graft is considered to have contained HSC. However, this strict “survival” method does not allow for the ability to quantify HSC number or function, and limits the ability to

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57

compare HSC grafts, and therefore the HSC mobilizing capacity of a given mobilizer or combination of agents. To address this problem, various types of long-term repopulation assays, which assess long-term repopulating cells (LTRC), an HSC synonym, with comparison against a “competitor” graft were developed. The standard competitive HSC repopulation assay was first described by Harrison (106) followed by description of a calculation for competing repopulating units (RU) (107) that are one measure used to enumerate HSC. In this assay, a donor HSC graft is admixed with a competing bone marrow graft from a congenic, wild-type mouse, and the mixture is transplanted into a lethally irradiated recipient. Markers distinct for the donor graft and the competitor graft are then used to distinguish blood production from each source of cells, allowing for a comparison of the repopulating ability of each. The standard method of employing this technique today uses the C57Bl/6 (CD45.2) mouse and the B6.SJLPtrcAPep3B/BoyJ (BOYJ) (CD45.1) mouse. These congenic strains of mice only differ at the CD45 antigen, and can be distinguished with specific monoclonal antibodies, allowing for assessment of chimerism in recipient animals (108). A variation of this assay is the limiting-dilution competitive repopulation assay, in which a series of dilutions of the donor, or “test” graft, is compared to a standard number of competing cells (normally 2 × 105 whole bone marrow cells). A minimum threshold of peripheral blood cell (or bone marrow) reconstitution is set (~2–5 %) and the number of mice that do not reconstitute with the test graft is determined and the frequency of competitive repopulating units (CRU), or HSC, contained within the test graft determined by Poisson statistics (109–111). It has recently been suggested by Drs. Purton and Scadden that a nomenclature distinction between RU and CRU should be made to describe the above transplantation assays (112); however, to date, CRU is still commonly used in both instances. 5.3. Transplantation Assays with Mobilized Blood

Our laboratory utilizes these competitive transplantation assays to validate the presence of LTRC in a mobilized peripheral blood product and assess the relative quantity and function of HSC contained in the graft. We use LDMC from a Lympholyte®-Mammal separation and compare these cells in ratios to 2 × 105 whole bone marrow competitor cells. It should be noted that LDMC from mobilized mice, depending on the mobilization regimen used, are considerably less competitive than bone marrow. Therefore, higher ratios of cells should be used, particularly in limiting dilution analysis. Smaller pilot studies evaluating the relative competitiveness of LDMC from mobilized donors is often advantageous to establish appropriate donor–competitor ratios. Typically, mobilized LDMC– competitor ratios of 1:1 to 5:1 are evaluated.

58


5.4. Serial Transplantation

Competitive transplantation assays are routinely analyzed 12–16 weeks post transplant, and if multilineage peripheral blood reconstitution is seen at this time point, it is assumed that HSC were transplanted. However, it is becoming increasingly clear that HSCs are a heterogeneous cell population with varying capacities for self-renewal, and as a consequence, varying capacities for extended repopulation. Early studies analyzing CFU-S after serial transplantation hinted at a reduction in self-renewal ability following multiple transplants (113), and serial transplantation was used by others to assess the potential of “younger” HSC (114–116). It was found that in normal mice, the ability of HSC to self-renew is lost after four or five serial transplantations (115). Recently, experimental evidence indicates the presence of three classes of HSC that differ in the ability to self-renew and the capacity for multipotent differentiation into all blood lineages: short-term HSC (ST-HSC) capable of full reconstitution for up to 16 weeks, intermediateterm HSC (IT-HSC) capable of full reconstitution for up to 32 weeks, and long-term HSC (LT-HSC) capable of reconstitution for longer than 32 weeks and/or through serial transplantation (117). In light of these various potentials for self-renewal, the most stringent test of HSC potential, specifically the LT-HSC, is serial transplantation from primary recipients into secondary recipients, or beyond.

5.5. Flow Cytometric Methods

So far, the discussion on stem and progenitor identity has focused on experimental assays to determine HSC and HPC that are all direct or indirect measures of the functional ability of these cells; whether the ability to form lineage specific colonies in media, or repopulation of lethally irradiated recipients. In addition to these functional assays, immunophenotypic analysis is commonly used to determine the number or frequency of HSC and HPC, and used as a means to “sort” specific populations for further experimentation. Immunophenotypic analysis utilizes antigen specific antibodies coupled with fluorescent labels and fluorescence-activated cell sorting (FACS) that is able to rapidly enumerate and/or collect specific cell populations. Early work on immunophenotyping hematopoietic cell populations demonstrated that mature B cells and their immediate precursors could be defined by a specific antibody (118), which has lead to a set of lineage markers (Lin) to define mature blood cells including erythrocytes, granulocytes, macrophages, T-cells, B-cells, natural killer (NK) cells and megakaryocytes, and lineage negative cells that are enriched for earlier stem and progenitor populations (119). Later, it was demonstrated that repopulating cells could be further defined by the absence of lineage markers (Linneg) with expression of stem cell antigen-1 (Sca1) and low expression of Thy1.1 (120). An additional marker for the stem cell factor (SCF) receptor (c-kit) (121–123) was later added to further define the murine HSC population. These cells

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59

will be referred to as Sca-1+ c-kit+ Linneg (SKL) cells. Although enriched for HSC function, SKL cells are still heterogeneous. Additional markers have recently been identified to further enrich for HSCs, including CD34 (124), and fms-related tyrosine kinase-3 (Flt3) (125, 126), which allow for the characterization of LT-HSC (CD34− Flt3− SKL), ST-HSC (CD34+ Flt3− SKL) and multipotent progenitors (MPPs) (CD34+ Flt3+ SKL) (125). While CD34 may be an appropriate marker to distinguish short-term and long-term HSC at steady state, G-CSF mobilized HSC express CD34, and revert to CD34− when back at steady state (127), perhaps reducing the reliability of CD34 when evaluating other mobilization regimens. Several additional markers have now been identified that further refine HSC identity, including Endoglin (CD105) (128, 129), Tie2 (CD202) (18), endothelial protein C receptor (CD201) (130), CD49b (117), and notably the signaling lymphocyte activation molecule (SLAM) family of receptors CD150, CD48, and CD244 (131, 132). Defining an HSC population as CD150+ CD48− SKL, which is highly enriched for LT-HSC (133), has been routinely used by our laboratory to evaluate a wide array of mobilization agents with success. However, interpretation of phenotypic analysis for HSC content in mobilized grafts should always be cautious in the absence of transplantation data to verify HSC function. For detailed methods on flow cytometry techniques to identify HSC, the reader is referred to (134–136).

6. The Need for Transplantation Assays

Without HSC activity in a mobilized graft, the graft will fail to fully reconstitute a myeloablated host long-term, ultimately leading to graft failure and mortality. In most cases, in vitro progenitor assays or flow cytometric analysis are appropriate for broad characterization and optimization of mobilization regimens and correlate with HSC mobilization. However, we have observed two specific instances where this has not held true. We evaluated the ability of 2 × 106 peripheral blood LDMC from mice mobilized with G-CSF, GROβΔ4, an alternate CXCR2 ligand GROγ, and the CXCR4 partial agonist CTCE-0021 to rescue a lethally irradiated recipient, all of which were able to significantly mobilize CFU-GM to peripheral blood compared to vehicle control (Table 2). However, even though both CTCE-0021 and GROγ mobilized equivalent amounts of CFU-GM compared to GROβΔ4, they failed to mobilize sufficient numbers of HSC to rescue lethally irradiated recipients, demonstrating the importance of transplantation assays to validate HSC content in mobilized grafts. Thus, one cannot rely solely on in vitro progenitor cell assays or flow cytometry to predict HSC mobilization. While mobilized products containing HPC but

60


Table 2 Mobilization of CFU-GM versus Survival in Lethally Irradiated Host Mobilizing agent

CFU-GM/ml Blood

Survival

Vehicle G-CSF GROβΔ4 GROg

34±2 1749±711 386±48 421±72

0/5 5/5 5/5 1/5

CTCE-0021

386±161

0/5

Mice were treated with G-CSF (50µg/mouse, bid, SC, 4 days), GROβΔ4 (2.5 mg/kg, SC), GROg (2.5 mg/kg, SC), or CTCE-0021 (25 mg/kg, SC), and LDMC and femur flushes collected. Whole bone marrow from femur flushes were plated in semi-solid agar for CFU-GM colony assays, and 2x106 LDMC were transplanted into 5 lethally irradiated (1100 cGy, split dose) mice. Shown are the number of mice, out of 5, which survived >16 weeks post-transplant

not HSC may have clinical utility in some cases, validation of HSC content is imperative if the graft is intended for hematopoietic reconstitution in a myeloablated host.

7. Conclusion While G-CSF mobilized hematopoietic grafts have revolutionized hematopoietic cell transplantation, there still remains a need for alternatives and improvements. The procoagulant effects of G-CSF increase the risk of myocardial infarction and cerebral ischemia in high-risk individuals (137, 138). G-CSF is contraindicated in patients with Sickle Cell Disease, owing to its potential to precipitate sickle crisis (139, 140), which has a negative impact on the potential utility of using G-CSF mobilized blood for adult HSC gene therapy for these patients. Poor mobilization in response to G-CSF occurs in 25 % of patients, particularly those with lymphoma and multiple myeloma (141) and 15 % of normal donors (142), requiring extended aphereses (143). In addition, the incidence of chronic GVHD is higher (1–3) for G-CSF-mobilized PBSC than bone marrow. Hence, there continues to be a need to search for additional safe and effective mobilizing agents to expand the use of hematopoietic grafts and PBSC transplantation. Multiple agents already identified and others to be identified in the future may provide these alternatives. Their potential use, however, depends on exacting characterization of their effects and function. The procedures we have described are a guide we have found useful to evaluate HPC and HSC mobilization.

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Chapter 5 Hematopoietic Stem Cell Mobilization: A Clinical Protocol Gina Pesek and Michele Cottler-Fox Abstract Autologous hematopoietic stem cell transplantation is the standard treatment for a wide variety of malignancies. At present, most hematopoietic progenitor/stem cell (HPC) collections are collected from the peripheral blood via leukapheresis following chemotherapy and/or growth factor-mediated mobilization. Most mobilization regimens consist of chemotherapy followed by one or more growth factors such as G-CSF, GM-CSF, or plerixafor. Occasionally a subset of patients will prove unable to mobilize effectively and will not collect at least 2.0 × 106 CD34+ cells/kg, the number of HPC currently considered to be appropriate for transplant in order to achieve timely engraftment and recovery of hematopoiesis. When this occurs it may be necessary to either remobilize, possibly with a different method, or to do a marrow harvest. Recent research has explored the benefits of using HPC outside of the oncology arena, notably in the area of cardiac regeneration following infarction, making the subject of mobilization potentially important to many areas of medicine. Key words: Mobilization, HPC, Apheresis, Growth factors, Clinical practice

1. Introduction Harvest of hematopoietic progenitor cells is primarily accomplished today via leukapheresis due to increased cell yields and improved patient comfort over bone marrow harvest, although it is possible this may change as other progenitor cell populations more prevalent in marrow than in blood (endothelial progenitor cells and mesenchymal stromal cells) become clinically important. Mobilization of HPC from marrow into blood is influenced by many factors but may best be predicted in the myeloma population by age, prior treatment, and platelet count (1, 2). Mobilization can be accomplished with a variety of agents;


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however, chemotherapy used in conjunction with growth factors has been shown to be more effective than either alone (3). Granulocyte colony-stimulating factor (G-CSF), granulocytemacrophage colony-stimulating factor (GM-CSF), and plerixafor are currently the agents most commonly used in the USA. Other mobilizing growth factors include erythropoietin (mobilizes CD34+ endothelial progenitors), GROb/CXCL2 (under investigation) and stem cell factor (not approved for use in the USA). The three clinical protocols used most commonly at our institution, chemotherapy plus G-CSF, G-CSF alone or with GM-CSF, and G-CSF plus plerixafor with or without preceding chemotherapy, are detailed within this chapter.

2. Materials 2.1. Administration of Growth Factors

1. Syringe. 2. Sub-q needle. 3. Alcohol swabs. 4. Nonsterile gloves. 5. Band-Aid. 6. Sharps container. 7. Ambulatory pump tubing with 0.22 μm inline filter.

2.2. Mobilization with G-CSF With or Without GM-CSF

1. G-CSF dosed at 5 mcg/kg body weight BID (see Note 1).

2.3. Mobilization with VDT-PACE Chemotherapy Plus G-CSF

1. G-CSF dosed at 5 mcg/kg body weight BID.

2. GM-CSF dosed at 250 mcg QD.

2. Cisplatin (P), Cyclophosphamide (C), and Etoposide (E). 1 L prepared in Normal Saline (NS). 3. Dexamethasone (D) and Thalidomide (T)—outpatient oral prescriptions. 4. Doxorubicin (A) Prepared in NS. 5. Bortezomib (V).

2.4. Mobilization with G-CSF Plus Plerixafor

1. G-CSF dosed at 5 mcg/kg body weight BID. 2. Plerixafor dosed at 240 mcg/kg/day, not to exceed 40 mg/ day (see Note 2).

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3. Methods The choice of mobilization method is dependent on multiple factors, including whether the donor is autologous or allogeneic, the disease state, and results of previous attempts at mobilization. For example, a patient who has failed to mobilize with chemotherapy plus G-CSF may obtain adequate HPC with the addition of plerixafor to the regimen. A discussion of mobilization would not be complete without addressing the question “Is the regimen working?” Beginning collection on a fixed day after administration of chemotherapy and/or growth factors is possible, but optimal timing of collection can be predicted based on enumeration of circulating CD34+ cells in the donor’s peripheral blood (4). Peripheral blood CD34+ cells may be monitored daily by flow cytometry using one of two commercially available single platform systems, ProCount (Becton-Dickinson, Mt. View, CA) Note (3) or StemKit (Beckman-Coulter, Fullerton, CA), or by using the ISHAGE method and an automated cell counter (a dual platform system) which evaluates cells based on surface expression of CD34. The single platform tests allow direct comparison of results between institutions, a feature not always possible with standard flow cytometry (5). While some centers use this result as the primary criterion for when to start collection (usually set at between 5 and 20 CD34+ cells/μL), other institutions use the result as part of a predictive formula which gives an estimation of expected number of CD34+ cells which may be collected: Blood volume processed (L) × (CD34+ cells/μL × machine collection efficiency)/Patient weight (kg). Currently a common goal of apheresis is to provide a graft for transplant of 2–4 × 106 CD34+ cells/kg in a single apheresis (6, 7). Another method of enumeration now in use is the Sysmex automated cell counter (Kobe, Japan), which uses an HPC window (the Immature Information channel or IMI) based on cell size, density, and lysis resistance to predict optimal collection times (8). Studies comparing the HPC counts of the Sysmex and flow cytometry for CD34 expression have shown predominantly favorable results using the Sysmex system (8, 9), although the Sysmex HPC number does not work in the predictive formula above ( Cottler-Fox, personal observation). Finally, since the above methods are based on cell surface marker expression, and since it is recognized that not all HPC express surface CD34 (10), the intracellular enzymatic activity of HPC has become a target of interest. A recently developed commercially available system (Aldagen, Durham, NC) employs staining with Aldecount® reagent to assess intracytosolic aldehyde dehydrogenase (ALDH) activity, present in both CD34+ and CD34− cells (11).

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3.1. Administration of Growth Factors

1. Verify order on the patient medication record, including dosage, frequency, and route of administration. 2. Verify the patient’s identity by asking name and date of birth. 3. Explain procedure and potential side effects to patient (see Note 4). 4. Gather materials and medication needed. 5. Inspect medication, ensuring that there is no cloudiness or discoloration. 6. Wash hands. 7. Aseptically draw up medication according to dosage ordered. Change to Sub-q needle. 8. Choose injection site. 9. Put on gloves. 10. Cleanse site with alcohol and allow to dry. 11. Remove needle cap ensuring that needle remains sterile. 12. Administer growth factor. 13. Grasp skin around injection site with nondominant hand, forming a 1 in. fat fold. 14. Position needle with bevel up. 15. Tell the patient that he/she will feel a prick. 16. Insert needle quickly in one motion at 45 or 90° angle and depress plunger. 17. Remove needle gently and quickly at same angle used for insertion. 18. Dispose of needle in sharps container. 19. If oozing, place Band-Aid over injection site. 20. Remove the gloves and wash hands.

3.2. Mobilization Using G-CSF With or Without GM-CSF (see Note 5)

Autologous HPC Collection: 1. Informed consent is obtained by the physician (or designee) prior to the administration of growth factor (GF). 2. Patients undergoing autologous HPC mobilization have a thorough physical, laboratory, radiologic, and serologic screening prior to receiving GF for mobilization. Donor qualification and infectious disease testing results must be documented according to the institutional SOP and per AABB/FACT/JACIE standards (FACT Standard C6 Donor, Evaluation, and Management; AABB Standard 5.10 Donor evaluation). 3. Pregnancy test must be done on all female donors who are of childbearing potential. Contraception and/or barrier protection should be used while patient is undergoing this process.

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4. The initial dose of G-CSF for individuals who have not undergone prior attempts at HPC mobilization is 5 mcg/kg BID (see Note 1). Patients who have mobilized poorly previously or patients who are at significant risk for poor mobilization may receive G-CSF at a higher dose (approximately 8 mcg/ kg/day) at the discretion of the physician (see Note 1). 5. A decision may be made at this time about use of GM-CSF in addition to G-CSF. Our institutional protocol uses 250 mcg of GM-CSF QD × 3 days prior to initiation of G-CSF. 6. G-CSF is supplied by the manufacturer in 300 and 480 mcg vials. The dose of G-CSF is rounded to the nearest vial size. 7. The initial dose of GF is typically administered in the outpatient clinic area. Outpatient nursing staff educate the donor in selfadministration of the growth factor if insurance allows this. 8. Donors undergo vascular access device placement if their veins are not adequate to support collection. Our apheresis protocol is for large volume leukapheresis, which means that the vein or catheter must tolerate a flow rate of 125–150 mL/ min. (see Note 6). On the 5th day of G-CSF administration, a complete blood count, blood chemistry panel, ionized calcium and HPC blood CD34 quantification are obtained on autologous donors. For best results using the Terumo (formerly Cobe) Spectra apheresis device, hematocrit should be at least 27% (personal observation, M. Cottler-Fox). Lab work will be reviewed on day 5 of G-CSF and daily thereafter to determine start date of HPC collection based upon predictive formula. HPC mobilization may be interrupted at the discretion of the physician if the yield is poor. If mobilization with G-CSF alone is poor, the autologous donor is assessed for collection with alternate growth factors or chemotherapy. 3.3. Mobilization with VDT-PACE Chemotherapy Plus G-CSF 3.3.1. Autologous HPC Collection

1. Informed consent is obtained and insurance screening is performed prior to the administration of VTD-PACE for HPC mobilization. 2. Patients undergoing autologous HPC mobilization undergo a thorough physical, laboratory, radiologic, and serologic screening prior to receiving GF for mobilization. Donor qualification and infectious disease testing results must be documented according to the institutional SOP and per AABB/FACT/ JACIE standards (FACT Standard C6 Donor, Evaluation, and Management; AABB Standard 5.10 Donor evaluation). 3. Pregnancy test must be done on all female donors who are of childbearing potential. Contraception and/or barrier protection should be used while patient is undergoing this process.

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3.3.2. Eligibility Criteria

1. Patients with Multiple Myeloma ((MM) responsive to chemotherapy, primary refractory disease, or patients with recurrent disease), selected patients with plasmacytomas, Waldenstrom macroglobulinemia, light-chain amyloid (AL), and non-Hodgkin lymphoma (NHL) that meet published criteria for treatment of MM and/or HPC mobilization. 2. Capacity to provide informed consent. 3. Physiologic age >18 years and 1.2 mL are administered as two subcutaneous injections. 6. Patients undergo vascular device placement on day 4 of G-CSF. 7. On Day 5 of G-CSF administration, patient reports for HPC collection. A complete blood count, blood chemistry panel, ionized calcium and HPC blood CD34 quantification are obtained on the patient. 8. Mobilization of HPC may be interrupted at the discretion of the physician if HPC mobilization is poor.

4. Notes 1. Standards may vary among institutions. 2. Dosing of plerixafor during clinical trials occurred with a 10h interval between dose and collection (10 p.m. dose for 8 a.m. collection); however, we have shown improved collection with a 15h dosing interval (12) (5 p.m. dose for 8 a.m. collection). 3. We use the ProCount test (Becton Dickinson, San Jose, California) and the test is not validated for a WBC less than 2.0. 4. The most commonly reported side effect with colony stimulating factors is bone pain, and other adverse effects include headache, fever, myalgia and nausea. Serious but rare events include splenic rupture, acute respiratory distress syndrome, exacerbation of autoimmune conditions and sickle cell crisis (13, 14). The most commonly reported adverse effects of plerixafor include diarrhea, nausea/vomiting, fatigue, headache, dizziness, arthralgia, and injection site reactions (15). 5. We do not use GM-CSF in amyloid patients as we have seen significant adverse reactions to the drug in this population. 6. Choice of central venous catheter is based on the flow rate needed for the procedure. Institutions not performing large volume leukapheresis may use slower inlet flow rates than we use and choose their catheters accordingly.

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References 1. Tricot G, Jagannath S, Vesole D et al (1995) Peripheral blood stem cell transplants for multiple myeloma: identification of favorable variables for rapid engraftment in 225 patients. Blood 85:588–596 2. Morris CL, Siegel E, Barlogie B et al (2003) Mobilization of CD34+ cells in elderly patients (>/= 70 years) with multiple myeloma: influence of age, prior therapy, platelet count and mobilization regimen. Br J Haematol 120:413–423 3. Bensinger W, Appelbaum F, Rowley S et al (1995) Factors that influence collection and engraftment of autologous peripheral-blood stem cells. J Clin Oncol 13:2547–2555 4. Rosenbaum ER, O’Connell B, Cottler-Fox M (2012) Validation of a formula for predicting daily CD34(+) cell collection by leukapheresis. Cytotherapy 14(4):461–466 5. Rivadeneyra-Espinoza L, Perez-Romano B, Gonzalez-Flores A et al (2006) Instrumentand protocol-dependent variation in the enumeration of CD34+ cells by flow cytometry. Transfusion 46:530–536 6. Bender JG, To LB, Williams S et al (1992) Defining a therapeutic dose of peripheral blood stem cells. J Hematother 1:329–341 7. Weaver CH, Hazelton B, Birch R et al (1995) An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 86:3961–3969

8. Suh C, Kim S, Kim SH et al (2004) Initiation of peripheral blood progenitor cell harvest based on peripheral blood hematopoietic progenitor cell counts enumerated by the Sysmex SE9000. Transfusion 44:1762–1768 9. Park KU, Kim SH, Suh C et al (2001) Correlation of hematopoietic progenitor cell count determined by the SE-automated hematology analyzer with CD34(+) cell count by flow cytometry in leukapheresis products. Am J Hematol 67:42–47 10. Dao MA, Arevelo J, Nolta JA (2003) Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution. Blood 101:112–118 11. Hess DA, Wirthlin L, Craft TP et al (2006) Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells. Blood 107:2162–2169 12. Rosenbaum ER, Nakagawa M, Pesek G et al (2009) A 15 hour extended dosing-collection interval for Plerixafor is at least as effective as the standard 10 hour interval. Blood 114:2152 13. Product information. Leukine (sargramostim). Seattle, WA: Bayer Healthcare Pharmaceuticals, April 2008 14. Product information. Neupogen (filgrastim). Thousand Oaks, CA: Amgen Inc., 1991–1996. 15. Product information. Mozobil (plerixafor). Cambridge, MA: Genzyme Corporation, December 2008

Chapter 6 Monitoring Blood for CD34+ Cells to Determine Timing of Hematopoietic Progenitor Cells Apheresis M. Louette Vaughn and Edmund K. Waller Abstract Hematopoietic Progenitor Cell (HPC) Apheresis generally results in a mononuclear cell product that is highly enriched for hematopoietic stem and progenitor cells when performed on autologous patients in whom autologous stem cell transplant is planned who have been mobilized with cytotoxic chemotherapy and exogenous hematopoietic growth factors (cytokines) and possibly CXCR4 antagonists. Alternatively, patients scheduled for autologous transplants may be mobilized with cytokines only or a combination of cytokines and CXCR4 antagonists. Allogeneic Donors, either matched related donors (MRD) or matched unrelated donors (MUD), are typically mobilized with cytokines only. The HPC Apheresis product, enriched for hematopoietic progenitor cells collected from the patient/donor’s peripheral blood via an apheresis system, is used for restoring hematopoiesis in the patient/recipient who has received myeloablative therapy. Timing of the collection of an HPC Apheresis product from allogeneic donors is based on the schedule of the recipient’s myeloablative regime. However, the optimal timing of collection on HPC Apheresis product from a patient scheduled for an autologous stem cell transplant can be complex. Key words: Peripheral blood progenitor cells (PBPC), Cytokines, Granulocyte colony stimulating factor (G-CSF), CD34+ cells, Plerixarfor (Mozobil™), COBE® Spectra Apheresis System™, Sysmex XE-2100L® automated hematology analyzer

1. Introduction Granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSC) have become the major source of hematopoietic stem cells for autologous transplant (1). The most commonly used method of stem cell mobilization involves daily or twice injections of G-CSF with or without chemotherapy. (2). In 2009 plerixafor (Mozobil™), an antagonist of the α-chemokine receptor CXCR4, was approved by Food and Drug Administration (FDA) for use in combination with G-CSF to mobilize PBPC in Mikhail G. Kolonin and Paul J. Simmons (eds.), Stem Cell Mobilization: Methods and Protocols, Methods in Molecular Biology, vol. 904, DOI 10.1007/978-1-61779-943-3_6, © Springer Science+Business Media, LLC 2012

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lymphoma and myeloma patients anticipating autologous transplant (3). Several clinical trials have demonstrated that plerixafor (Mozobil™) can dramatically increase peripheral blood CD34+ cell counts, increasing the efficiency of stem cell collection in lymphoma and myeloma patients (4–6). Successful collection of the HPC graft by apheresis involves coordinating the administration and timing of the CXCR4 antagonist and (in the case of autologous transplant recipients) cytotoxic chemotherapy with the apheresis team and treating physician. Autologous donors respond differently to mobilization regimes based on diagnosis, age, and amount and type of prior treatment (chemotherapy and/or radiation) (7). 1.1. Chemotherapy and Cytokine Mobilization Initial Follow-Up

Determining the timing of HPC Apheresis after chemotherapy and G-CSF mobilization can be complex. Generally the Autologous donor is scheduled for outpatient lab tests, including a CBC to be drawn 7–10 days after the administration of cytotoxic chemotherapy that is used for mobilization in conjunction with once daily G-CSF injections. Follow-up Standard of Care Labs include Complete Blood Count (CBC), Automated Differential, Magnesium, and basic Biochemical Profile with a turnaround time of approximately 60 min. After the labs are resulted the Autologous donor is seen and evaluated by a mid-level Practitioner. At this point in time, it is not unusual for the Autologous donor’s absolute WBC count to be

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