International Review of Cytology: A Survey of Cell Biology, Volume 153

international Review of

cyt0 I0gy VOLUME 153

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1 988 1949-1 984 19671984-1 992 1993-

ADVISORY EDITORS Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay Mark Hogarth M. Melkonian Keith E. Mostov Audrey Muggleton-Harris

Andreas Oksche Muriel J. Ord Vladimir R. Pantic M. V. Parthasarathy Lionel I. Rebhun L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Robin Wright Alexander L. Yudin

Edited by Kwang W. Jeon Department of Zoology University of Tennessee Knoxville, Tennessee

Jonathan Jarvik Department of Biological Sciences Carnegie Mellon University Pittsburgh, Pennsylvania

VOLUME 153

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Copyright 0 1994 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road. London NWI 7DX International Standard Serial Number: 0074-7696 International Standard Book Number: 0- 12-364556-5 PRINTED IN THE UNITED STATES OF AMERICA 94 95 9 6 9 7 98 9 9 B B 9 8 7 6 5

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CONTENTS

Contributors .......................................................................................

ix

Fluorescent in Sku Hybridization for the Diagnosis of Genetic Disease at Postnatal. Prenatal. and Preimplantation Stages Darren K. Griffin 1. I1. 111. IV. V. VI.

Introduction ................................................................................ Origin of Samples .......................................................................... Fluorescent in Situ Hybridization .......................................................... Sexing of Human Cells .................................................................... Further Fluorescent in Situ Hybridization Diagnoses ...................................... Concluding Remarks ....................................................................... References .................................................................................

1 3 6 21 25 33 35

Isolation and Function of Human Dendritic Cells Lisa A . Williams. William Egner. and Derek N. J. Hart 1. II. 111. IV. V. VI . VII . VIII. IX. X.

Introduction and Overview ................................................................. Blood and Bone Marrow Dendritic Cells ................................................... Nonlymphoid and Interstitial Dendritic Cells ............................................... Lymphoid Dendritic Cells .................................................................. Dendritic Cell Ontogeny: Differentiation and Migration .................................... Functional Properties of Human Dendritic Cells ........................................... Dendritic Cell Malignancies ................................................................ Role of Dendritic Cells in Transplantation. and Infectious and Autoimmune Diseases ... Clinical Applications ........................................................................ Future Dendritic Cell Research and Applications .......................................... References ................................................................................. V

41 47 53 64 68 73 85 86 90 90 91

vi

CONTENTS

Granulated Lymphoid Cells of the Pregnant Uterus: Morphological and Functional Features Chau-Ching Liu. Earl L. Parr. and John Ding-E Young I. I1. 111. IV. V. VI.

Introduction ................................................................................ Cells Associated with Decidual Tissue .................................................... Granulated Metrial Gland Cells of Rodents ................................................ Human Endometrial Granulocytes ......... .............................. Possible Functions of Uteri Concluding Remarks ...... References .................................................................................

105 106 110 117 122 126 127

The Replication Band of Ciliated Protozoa Donald E. Olins and Ada L. Olins I. Introduction ................................................................................ II. Early History: Prior to 1959 ................................................................

137 140

Functional Characteristics of Replication Bands in Cells .................................. Replication Band Ultrastructure ............................................................ Cytochemical and lmmunochemical Studies on Replication Bands ....................... Relevance to Current Models Conclusions and Speculations ..... References ......

143 148 154 161 166 168

111. IV. V. VI . VII.

Whole-Chromosome Hybridization S . D . Bouffler ......................................................................... I. rinciples of Whole-Chromosome Hybridization Techniques .......... II. Ill. Applications of Whole-Chromosome Hybridization ........................................ ............................................................ IV. ............................................................

171 174 197 219 220

Neuronal Modulation and Plasticity in Vitro Robert A . Smith and Zhi-Gang Jiang I. Introduction ................................................................................ 11. Neuronal Cell Cultures .....................................................................

233 235

CONTENTS

vii

Neurite Initiation and Elongation ............................ Synaptic Connections between Cultured Neurons ....................... Phenotypic Expression .... ....................................... Concluding Remarks ............... .................................... References ................................... ...........................

279 201

Index ..............................................................................................

297

111. IV. V. VI.

This Page Intentionally Left Blank

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin.

S. D. Bouffler (171), Biomedical Effects Department, National Radiological Protection Board, Chilton, Oxfordshire OX 1 1 ORQ, United Kingdom William Egner (41), Haematology/lmmunology Research Group, ChristchurchHospitall Christchurch, New Zealand Darren K. Griffin (1), Department of Genetics and Biometry, University College London, London NW1 2HE, United Kingdom Derek N. J. Hart (41), Haematology/lmmunology Research Group, ChristchurchHospital, Christchurch, New Zealand Zhi-Gang Jiang (233),Department of Anatomyl University of Glasgow, Glasgow, G 12 SQQ, Scotland, United Kingdom Chau-Ching Liu (105), Laboratory of Molecular Immunology and Cell Biology, The Rockefeller Universityl New York, New York 10021 Ada L. Olins (137), The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and The Biology Division, Oak Ridge National Laboratory, Oak Ridgel Tennessee 37831 Donald E. Olins (137), The University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and The Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Earl L. Parr (109, Department of Anatomy, School of Medicine, Southern Illinois University, Carbondale, Illinois 6290 1 Robert A. Smith (233),Department of Anatomy, University of Glasgow, Glasgow G12 8QQ1Scotland, United Kingdom ix

X

CONTRIBUTORS

Lisa A. Williams (41),Haematology//mmunol~yResearch Groupl ChistchurchHospital Chistchurchl New Zealand John Ding-E Young (105),Laboratory of Molecular lmmunology and Cell Biology, The Rockefeller Universityl New York, New York 10021

Fluorescent in Situ Hybridization for the Diagnosis of Genetic Disease a t Postnatal, Prenatal, and Preimplantation Stages Darren K. Griffin Department of Genetics and Biometry, University College London, London NWI 2HE, United Kingdom

I. Introduction Adapted from the pioneering work of Gall and Pardue, most significantly as described by Pinkel et al. (1986), fluorescent in situ hybridization (FISH) has become one of the most powerful techniques in modem genetic research. It is principally a tool for assigning particular nucleic acid clones (“probes”) to chromosome preparations in order to map those clones to a particular chromosome region, but it has proved to be a very powerful diagnostic technique also. FISH is now very much a complementary approach to classical cytogenetics because it can be used to illuminate whole chromosomes or certain regions of chromosomes at will, and because two or more chromosomal targets can be visualized in different colors simultaneously on the same preparation. It has allowed cytogenetic analysis of metaphases that are difficult (or impossible) to analyze because of poor chromosome preparations or when the chromosome anomaly is too complex or too small to see with classic chromosome banding techniques. It also allows certain types of analysis in interphase nuclei and is hence invaluable when metaphases cannot be prepared at all. Disorders accessible to FISH diagnosis range from simple numerical chromosome changes (e.g., Down’s syndrome), to tiny deletions (such as some cases of Duchenne’s muscular dystrophy-DMD), to complex chromosomal rearrangements. Diagnosis can be made after birth (postnatally), before birth (prenatally), or before implantation of the embryo (preimplantation diagnosis). Cytogenetic diagnosis following birth is particularly useful in deciding upon future treatment of an affected individual. Often a rapid cytogenetic International Review

of Cytology. Vol. 153

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Copyright 0 1994 by Academic Press, Inc.

All rights of reproduction in MY form reserved.

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diagnosis can be essential in deciding upon immediate treatment for a newborn child or indeed upon whether to sustain life at all. In the case of trisomy 13 for instance, often a decision is made to turn off life support systems upon diagnosis because the child would have no chance of treatment or survival. Furthermore, specific chromosomal anomalies in certain tissues of an individual (e.g., bone marrow) can be diagnostic for certain cancers; perhaps the most common example of this is the Philadelphia chromosome present in chronic myeloid leukemia cells. It is in prenatal diagnosis that there has been the most interest in diagnostic cytogenetics. Amniocentesis and/or chorionic villus sampling (CVS) in the first or second trimester of pregnancy reveals the chromosomal constitution of the unborn fetus and allows the family to decide (under genetic counseling)whether to continue with the pregnancy or to terminate it if the fetus is affected. Preimplantation diagnosis (PID) is carried out on an embryo prior to implantation into the mother’s uterus. Although considerable advances have been made in prenatal diagnosis, selective abortion of affected offspring is unacceptable in some cases. The need (at least initially)to develop PID hence is not as a working alternative or replacement for prenatal diagnosis but to alleviate the suffering of many special-case families: Penketh and McLaren (1987) report one thallasemia family who had one unaffected child after seven pregnancies, four of which were terminated. Another woman whose brother died of Duchenne’s muscular dystrophy has two teenage nephews with the condition and is a carrier herself. She had two terminations in her first marriage, which broke down as a result. In her second marriage, following second trimester prenatal diagnosis, she has had three terminations of male fetuses and consequently has been advised to refrain from further conception until research in DMD has reached a more advanced stage. In some families, selective abortion is morally or religiously unacceptable. For instance, the Roman Catholic, Islamic, and Orthodox Askenazi Jewish (Penketh and McLaren, 1987; Winston, 1987) faiths all prohibit terminations of pregnancy. Clearly, families such as these would benefit from PID. It should be noted, however, that the Roman Catholic faith has not embraced this approach. There have been a multitude of developments in FISH technology and literally tens of thousands of publications involving the technique and adaptations of it. It is certainly beyond the scope of this chapter to summarize all of them. There is a gap between what is technically possible in a high-flying research laboratory and what is technically feasible (and reproducible) in the average diagnostic laboratory. With this in mind, this chapter attempts to highlight how major advances in the field have been used in diagnostic situations to suggest how (with limited resources) a diagnostic cytogenetic laboratory might harness the technology to its own ends, and to speculate on what could be possible in the future.

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II. Origin of Samples

In this section, sampling and preparation of material for FISH studies are reviewed. Postnatal and prenatal sampling have been covered in great detail by many other authors and so are only briefly summarized here. However, since preimplantation diagnosis is a more recent development, it is covered in more detail. A. Postnatal Samples

Postnatal samples can be taken from blood, skin, bone marrow, lymph nodes, or directly from solid tumors. Blood samples are usually taken from peripheral veins and lymphocytes are stimulated into a dividing state using a mitogen. Metaphase arrest precedes hypotonic swelling of the cells and is followed by fixation and spreading onto glass slides. In the case of the other tissues, cells can be induced to divide (if necessary) by the culture techniques used to prepare chromosomes. The techniques used for hypotonic swelling, fixation, and spreading onto glass are similar to those for blood culture.

6. Prenatal Samples

Prenatal sampling involves removing samples of the chorionic villus, amniotic fluid, or fetal blood with a needle or catheter. In order to prepare chromosomes, cells from the amniotic fluid need to be cultured over 2 weeks. CVSs can also be cultured but an advantage of this sample is that chromosomes can be obtained directly without culturing, and hence a diagnosis can be achieved with speed. Direct CVS chromosome preparations are derived from the outer epithelial layer of the villus and, in general, give metaphases. Chromosome spreads can be difficult to analyze completely, however, and therefore in most diagnostic labs are merely homogeneously stained and analyzed for sex and aneuploidy alone. Cultured CVS preparations are derived from the inner mesenchymal core of the villus, and chromosome spreads prepared from this area are of much better quality. It is because of this that it is often recommended that laboratories use both methods so that a confident diagnosis can be made for each patient. CVS is usually routinely performed at 8-9 weeks of gestation whereas amniocentesis is routinely performed at 15- 16 weeks. Brambati and Lucia (1990) have reported CVS taken as early as the 6th week of gestation, and Smith et al. (1990) report that amniocentesis can be performed at

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DARREN K. GRIFFIN

11 weeks (bringing it into the first trimester). Rapid results mean that therapeutic abortion can be offered quickly, which ultimately leads to less trauma for families having to make the difficult decision of whether to proceed with a termination. Hence, CVS gained rapid popularity following its development. Various reports have appeared of more rapid approaches to both amniocentesis (as early as 11 weeks; Smith et al. (1990) and CVS (as early as the 6th week; Brambati and Lucia, 1990). Recent studies have, however, indicated that both early amniocentesis and early CVS may be potentially damaging to the fetus and most clinics have reverted to their original approaches. The great advantage of an earlier result associated with CVS is balanced by the fact that CVS reveals mosaic results more often than amniocentesis since amniocentesis analyzes cells shed directly from the fetus. Furthermore, because amniocyte chromosomes are analyzed more easily than those from direct CVS, the former are more likely to reveal more subtle chromosomal abnormalities (Lilford, 1991). As with postnatal material, cells taken for prenatal sampling are swelled in hypotonic solution, fixed, and spread onto glass slides. C. Preirnplantation Embryos

Prospectively, material from preimplantation embryos can be sampled in one of three ways. Each can involved either in vitro fertilized (IVF) embryos (Steptoe and Edwards, 1978)or embryos fertilized normally and flushed from the mother’s uterus (uterine lavage) (Buster et al., 1985): These methods are (1) removal and diagnosis of a polar body; (2) removal and diagnosis of trophectoderm (TE) material; (3) removal and diagnosis of single blastomeres at early cleavage stages. It is the latter technique that has aroused the most interest in this field (reviewed in Adinolfi and Polani, 1989). Polar-body (PB) biopsy involves physical removal of a polar body (usually the second polar body appearing postfertilization)from an IVF embryo at the 2-cell stage. This could be used for either DNA or chromosomal analyses. The technique of course only assesses the contribution of the maternal genome, but in the case of recessive disorders, the contribution of only one of the parents need be controlled. Genetic analysis is further clouded by the process of meiotic “crossing over” where the homologous chromosomes exchange genetic material at metaphase I. In the case of chromosome analysis, assessing the contribution of only one parent is obviously limiting despite the fact that 80% of all Down’s syndrome cases arise from an extra chromosome 21 donated by the mother. The second approach involves biopsy of material from the trophectoderm when the embryo is at the blastocyst stage. The first report of

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5

preimplantation diagnosis (diagnosing sex in rabbits) used this approach (Gardner and Edwards, 1968). Micromanipulation techniques were used to remove the outer layer of TE. Sexing was achieved by detecting Barr bodies (inactive X chromosomes visible only in female interphases). Embryos were transferred into the mother’s uterus and sex was confirmed later in gestation or at birth. TE biopsy at the blastocyst stage has some attractive features: (1) The maximum number of cells are available for diagnosis, making any test more reliable than if fewer cells were available. (2) TE cells are extraembryonic and contribute only to the tissues surrounding the fetus (Handyside and Delhanty, 1993). The major problems with applying this strategy clinically (at least using IVF) are (1) the fact that only half the embryos survive to this stage in culture, (2) the unexpectedly low pregnancy rate reported in some (but not all) clinics following IVF and embryo transfer at this stage (Dawson et al., 1988; Bolton et al., 1991), and (3) the theory that removal of a substantial proportion of TE may affect implantation of the embryo. Cleavage-stage biopsy involves removal of one or more blastomeres when the embryo is at the 4-16-cell stage. Diagnosis can be made on the biopsied cell(s) and the remainder implanted into the mother’s uterus if necessary. At these early cleavage stages, mammalian blastomeres remain totipotent and preimplantation development is not adversely affected by biopsy at the &cell stage. This was confirmed by Hardy et al. (1990), who took embryos with one and two cells biopsied from them and found that glucose and pyruvate uptake only decreased in proportion to the reduction in cellular mass, and furthermore over half (the usual proportion) of the embryos hatched out of the zona pellucida in vitro. The technique (e.g., Handyside et al., 1990) involves placing the embryo in a drop of medium under oil and placing it under a dissecting microscope for micromanipulation. The embryo is immobilized by suction on a holding pipette; a small hole is drilled in the zona pellucida using a tiny pipette and a stream of acid tyrodes which dissolves the zona; and a second larger pipette is then pushed into the hole to remove one or two cells. The remaining embryo is quickly returned to culture and the biopsied cell($ are prepared for analysis. The technique for biopsy of a cleavage-stageembryo is illustrated in Fig. 1. Because of the problems associated with PB and TE biopsies, at present, cleavage-stage biopsy remains the only clinically applicable approach. Indeed, the first preimplantation diagnosis to be performed in a clinical situation used cleavage-stage biopsy followed by a polymerase chain reaction (PCR)-basedassay to determine the sex of the biopsied cell. Families at risk of transmitting sex-linked disorders to their male offspring were treated in this way and female live births have ensued (Handyside et al., 1990).

DARREN K. GRIFFIN

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CELL HUMAN EMBRYO

/

\\ /

HOLDINGPlPFlTE I STABILIZES EMBRYO

DRILLING P l m E MAKES A HOLE IN ZONA WITH A STREAM0F"ACID TYRODES"

ASPIRATION PlPmE TO REMOVE BLASTOMERE

/I

'/

\\

\\

FIG. 1 Biopsy of a human embryo at cleavage stage.

In order to prepare material biopsied from preimplantation embryos for FISH analysis, standard cytogenetic procedures need to be adapted. The cells need to be swelled, fixed, and spread onto glass slides as with other material, but because there is only one cell (or, in some cases, a few cells), the cell needs to be accurately watched when it is moved from one solution to the next and its position on the slide carefully noted (Griffin et al., 1991, 1992).

111. Fluorescent in Situ Hybridization A. Basic Principles

In situ hybridization (ISH) was first described by Gall and Pardue (1969) and involved "formation and detection of RNA-DNA hybrids in cytologi-

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cal preparations” to detect radioactively labeled rRNA genes in Xenupus tissue sections by autoradiographic means. Techniques incorporating DNA-DNA hybrids and using chromosomal preparations soon followed and the original approach remains remarkably unchanged to the present. Many cloned genes have been mapped directly to a particular chromosome band by using this approach (Malcolm et al., 1986). Isotopic ISH typically results in a scatter of silver grains over the chromosome spreads. These need to be analyzed statistically by examining 50-100 metaphases producing a mode of hybridization signals. Also, chromosomes need to be banded, either pre- or posthybridization, in order to achieve accurate cytogenetic assignment. Autoradiographic exposure takes about 2 weeks and the hazards associated with the handling of radioactive isotopes are well documented. The application of fluorescent ISH to chromosomes was first described by Rudkin and Stollar (1977), who detected rRNA genes in Drusuphilia by using a fluorescent antibody to DNA-RNA hybrids as the detection system. Van Prooijen-Knegt et al. (1982) first visualized 18s and 28s rRNA genes on human metaphase spreads, again by forming RNA-DNA hybrids and detecting via a fluorescent RNA-DNA antiserum. Detection using RNA-DNA hybrids, however, proved to be insensitive for most applications in comparison with isotopic ISH and it became clear that a totally different approach would have to be developed. Pinkel et al. (1986) developed a FISH system that involved directly labeling DNA probes with vitamin H (biotin) molecules. DNA can be labeled by nick labeling, random priming of oligonucleotides, or photoactivated labeling. Of the three methods, nick labeling (or nick translation as it is often referred to) is the most common. DNA-DNA hybrids are formed between the labeled probe and chromosomal DNA. Biotin detection is facilitated by fluorescently labeled avidin molecules which show an extremely high affinity for biotin. Furthermore, the fluorescent signal can be amplified by biotinylated antiavidin followed by a second layer of fluorescent avidin. This method has since proved to be the most widely applicable of all FISH approaches and hence the vast majority of advances in this field have incorporated it. Briefly, cytogenetic preparations are made by routine protocols. Exogenous RNA and protein are enzymatically removed. The chromosomal and labeled probe DNA is denatured and allowed to hybridize in situ. Fluorescence is detected with a fluorescein-avidin conjugate. Sequential layers of biotin-antiavidin conjugate followed by a second layer of avidinfluorescein facilitate signal amplification. This amplification step is referred to as the “Pinkel sandwich” (Trask, 1991). Finally, the chromosomes are stained with a fluorescent total DNA counterstain for relocation purposes. Figure 2 demonstrates how successive layers lead to signal

n

DARREN K. GRIFFIN

8

0 BIOTIN MOLECULE

CROSS SECTION THROC'QH 4 CHROMOSOME

A

CHROMOSOMAL DNA

@ FLUORESCEIN MOLECULE

ENZYMATIC REMOVAL OF CONTAMINATING RNA AND PROTEIN

9

DNA STRANDS

AVIDIN MOLECULE

,/

AVTN MOLECULE

APPLICATION

OF BIOTINYLATED

PROBE AND SEPARATION OF BOTH CHROMOSOMAL AND PROBE DNA STRANDS

.1

8

I

"f

IN-SITU HYBRIDAZATION OF PROBE FOLLOWED BY EXCESS BEING WASHED OFF

ADDITIONOF AVIDIN-FLUORESCEIN CONJUGATE

f

' I

1

ADDITION OF BIOTINANTI AVIDIN CONJUGATE

/

ADDITION OF AVIDIN-FLUORESCEIN CONJUGATE (2nd LAYER)

ADDITION OF FLUORESCENT COUNTERSTAW

FIG. 2 Diagrammatic representation of the FISH protocol. (Griffin. 1992.)

FLUORESCENT IN SITU HYBRIDIZATION FOR DIAGNOSING GENETIC DISEASE

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amplification. In fact, four biotin molecules bind to each avidin molecule. Figure 3 is a flow diagram of how a FISH protocol may be performed in the laboratory. The chromosomes appear in one color and the area of the chromosome to which the probe hybridizes (the target sequence) is marked by a second fluorescent color. A recent innovation in FISH technology has allowed Pinkel amplification in one detection layer. In this method, fluorescein-labeled avidin is preincubated with fluorescein-labeledantiavidin. The two conjugates form

CHROhfOSOhfES (AND INTERPHASES) PREPARED BY STANDARD ('YTOGENWIC MkTHODO1.OGY

* * * * *

(CIIROIIOSOMES BANDED AND PHOTOGRAPHED)

(1X:STAINI:D AND) DEHYDRATED RN APC TRtAThlENT PROBE LABELED .AND PLRIFTED

PROTbIN ASE TRE4TMENT

))r DISSOLVED IN HYBRIDIZATION MIX

FIXITION AND DEWDRATION

* * * * * * * * *

PROBE APPLIED TO SLIDE AND SEALED UNDER A COVERSLIP DENATLIRhTION OF PROBE .AND CHROMOSOMAL DNA

IN SITU HYBRIDIZATION POST HYBRIDIZATION WASHES FLLIOROCHROME BLOCKING STEP INCL'BATION WITH FLL'OROCHROME WASHES

(.AMPLIRC.ATION STEP WITH WASHES) MOI'NTED IN ANTI-FADE MEDILJMCONTAINING FLIIORESCENT CO1"TERSTAIN

\'IEWED I'NDER A . MICROSCOPE EQLIIPPED WITH APPROPRIATE FILTERS

FIG. 3 Flow diagram representing FISH methodology.

DARREN K. GRIFFIN

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a complex which is then applied to the biotinylated probe. This is illustrated in Fig. 4. When detecting probes hybridized in situ, two molecules are important-first the hapten and second, the fluorochrome. A hapten or “reporter molecule” is the name given to the molecule that labels the DNA. Biotin is not the only hapten available but is the most common. Also used are digoxigenin, 2-acetylaminofluorene (AAF), the sulfone radical, and mercury. Digoxigenin is a steroid derived from Digitalis plants and can be introduced into a probe in ways similar to biotin. It can be detected by fluorescent-conjugated antibodies to it or to digoxin, from which it is derived. Signal amplification can be achieved using secondary and tertiary fluorescent antibodies. AAF can be used to label a probe because it (a carcinogen) binds covalently in the C8 (8th carbon atom) position of guanosine residues in DNA or RNA. It is detected by fluorescent antibodies (Landegent et al., 1984; Tchen et al., 1984). Labeling with sulfone radicals (sulfonation/transamination)relies on the fact that bisulfite reacts reversibly with C5-C6 double bond of cytidine residues in DNA to give 5,6-dihydrocytidine-6-sulfonate. Detection can be achieved using fluorescent antibodies. Mercury modification is one of the oldest of the nonradioactive approaches. The probe is chemically mercurated at the C5 (5th carbon atom) position of the pyrimidines. To facilitate detection, a mercury binding ligand is attached that carries a sulfydryl group on one end and

CHROMOSOME

ADDITION OF AVIDIN-FLUORESCEIN CONJUGATE AND ANTI-AVIDIN-FLUORESCEIN CONJUGATES EXPOSED TO EACH OTHER

I CONJUGATES FORM

I

J.

ADDITION OF CONJUGATE COMPLEX

.1

ACoMPLEX

t

FIG. 4 Diagrammatic representation of one-step “Pinkel sandwich.”

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a hapten on the other. This secondary hapten can be trinitrophenol (TNP) or biotin, both of which can be detected by fluorescent conjugates, or a fluorescent material itself (Hopman et al., 1986; Raap et al., 1990). Of all these reporter molecules, biotin and digoxigenin are thought to be the most sensitive ( J. Wiegant, personal communication). Figure 5 shows immunocytochemical detection of digoxigenin. A fluorochrome is the fluorescent material which, when conjugated to a molecule which will bind to the hapten (such as avidin or an antibody), facilitates detection of the probe. By definition, a fluorochrome is a material which will become excited by light of one wavelength and then emit light of a higher wavelength. Thus, specially adapted microscopes can detect fluorochromes. Fluorescein isothiocyanate (FITC), which excites in the blue range and emits in the green, is the most commonly used. Also common is tetramethylrhodamine isothiocyanate (TRITC), which excites in the green range and emits in the red; Texas red (or sulforhodamine 101), which excites in the green and gives a very deep red color; and 7amino-4-methyl coumarin-3 acetic acid (AMCA) (Khalfan et al., 1986), which excites in the ultraviolet and emits in the blue range. Two fluorescent DNA counterstains for chromosome location are commonly used-6diamino phenylindole dihydrochloride (DAPI), which absorbs ultraviolet and emits blue, and PI, which absorbs in violet-green wavelengths and emits red. A combination of both counterstains can be used with FITC preparations (hence red chromosomes and yellow/green signals can be viewed simultaneously) but propidium iodide cannot be used with TRITC

I

ANT-DIGOSIGENIN ANTIBODY

PROBE

1 CHROMOSOME

FIG. 5

Fluorescent detection of a digoxigenin-labeledprobe.

DARREN K. GRIFFIN

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or Texas red because it drowns out the signal. Figure 6 shows the light path a fluorescent microscope when it is exciting and detecting FITC. Recently, labeling probes directly with the fluorochrome has become possible. The labeling is achieved by means similar to those for biotin and digoxigenin; signal amplification, if necessary, can be achieved by using antifluorochrome antibodies. The applicability of this approach has been assessed by Wiegant et al. (1991), who found that probes 50-100 kb in length could be detected when the signal was unamplified, whereas probes 1-5 kb could be detected when they were amplified. These figures

BARRIER. ALTER

RLTER COMBINATION BLCXX

DICHROIC MIRROR

MERCURY VAPOR

ExmER HLTER

+t

LAMP

LIGHT OF ALL WAVELENGTHS

II) +

BLUELIGHT GREWlLIGHr

-0BJEXTIVE

' - 4 4 4 ,d

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FIG. 6 Light path of a fluorescent microscope detecting fluorescein. 1. Light of all wavelengths emitted from lamp. 2. Exciter filter lets through only blue light. 3. Dichroic mirror reflects blue light. 4. Fluorescein is ecited by blue light and emits green. 5 . Dichroic mirror lets through green light. 6. Banier filter lets through only green light. 7. Green signal is viewed. (Grimn, 1992.)

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FLUORESCENT IN SITU HYBRIDIZATION FOR DIAGNOSING GENETIC DISEASE

are comparable to amplified and unamplified signals using the biotin-avidin-fluorochrome system. This strategy also has the advantage that a low background yield is obtained. Given that a number of probe labels and fluorochromes exist, the possibility of multiple labeling (i.e., simultaneous hybridization and detection of two or more probes on the same cytological preparation)arises. Applications of this are discussed in subsequent sections. Figure 7 illustrates the principle of dual labeling. DNA probes contain two elements-namely, the insert (i.e., the human DNA complementary to the target sequence on the chromosome) and the vector (i.e., unrelated DNA into which the insert is cloned and through which the DNA can be replicated). Four types of probes are common-plasmid probes, which have inserts ranging from 500 base pairs to around 6-kb and vectors of, on average 2 kb; phage clones with inserts of 3-20 kb and vectors of around 40 kb; cosmids with inserts of 40 kb and vectors of 5-6 kb; and finally, yeast artificial chromosomes (YACs) with inserts of 300+ kb and vectors of 7-8 kb. Throughout the whole genome, however, are interspersed repeated sequences of which the Alu elements are the most common. Many probes (invariable YACs and cosmids, frequently phages, and occasionally plasmids) contain these and other repeats between the unique sequences. If these probes were applied directly onto chromosomes via FISH, the whole chromosome complement would light up because these repeats would find Q biotin molecule

digoxignin molecule

w

A A

avidln molecule

dipxienin molecule

.......

ga

I FIG. 7 Dual detection of biotin and digoxigenin-labeledprobes in red and green.

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DARREN K. GRIFFIN

complementary sequences all over the genome. In order to inactivate this and allow the unique portion of the probe to find its complementary sequence, the probe must be preannealed, with total unlabeled DNA in excess. Such an approach was first described for FISH by Lichter et al. (1988) and Cremer et al. (1988a) and is known as competitive in situ suppression or CISS. Chromosomal DNA is denatured separately; then the preannealed probe is applied, leaving unique sequences free to hybridize.

6. Types and Applications

1. Tandem Repetitive Probes Classical satellite, and a-satellite sequences have been studied using FISH. Both these types of DNA contain arrays of highly repeated DNA sequences. Hence probes for them have large areas of target sequence on the chromosome on which to hybridize and thus produce large and bright FISH signals. Classical satellite DNA sequences include the large C-bands on chromosomes 1,9, 16, and Y. The majority of chromosomespecific probes, however, consist of a-satellite or alphoid DNA. Alphoid repeats constitute a group of related, highly divergent sequences, each approximately 171 kb in length. These sequences show 20-40% divergence from one another. Arrays of alphoid repeats are found exclusively around the centromeric regions of all the chromosomes. Tandem arrays of these units show chromosome-specific, higher order repeat units; this is why probes for these regions generally recognize only one chromosome. Figure 8 shows this hierarchical order of repeat units for chromosomes X, 7, and 10. Since these tandem repeat sequences are largely chromosome specific and since they are generally cloned into plasmid vectors with inserts of around 2 kb, CISS is not necessary prior to FISH; however, high stringency conditions are often needed to maintain chromosomal specificity. It is in the exploitation of classical-satellite and a-satellite DNA that there has been much interest in FISH. When conditions are ideal, these probes brightly light up specific regions (usually centromeres) of human chromosomes. Signals are large enough to be seen and counted in interphase nuclei. Probes are now available commercially for nearly all the human chromosomes, including both sex chromosomes, although some probes detect more than one chromosome simultaneously. A notable example of this is chromosomes 21 and 13. Probe L1.26 (Devilee et al., 1986)recognizes the centromeres of chromosomes 13and 21. These probes have a multitude of applications, chiefly in fields where analyzable metaphases cannot always be obtained in cytogenetic preparations. Such fields

15

FLUORESCENT IN SlTU HYBRIDIZATION FOR DIAGNOSING GENETIC DISEASE

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FIG. 8 High- and low-order repeats in alphoid DNA. (A) X chromosome. (B) Chromosome 7. (A and B from Willard and Waye, 1985). (C) Chromosome 10 (from Devilee et al., 1988.)

include cancer cytogenetics and prenatal and preimplantation diagnoses. Detection of chromosomes in interphase nuclei is referred to as “interphase cytogenetics.” Figure 9 is a FISH preparation showing the detection of a centromeric probe specific for chromosome 18 in a metaphase and an interphase nucleus. The applications of these probes in postnatal, prenatal, and preimplantation diagnosis are discussed in subsequent sections. 2. Chromosome Painting

By using a collection of probes or a ‘‘library’’ specific for a particular chromosome, it is possible to light up or “paint” a whole chromosome along its length. Since many of the clones in the library contain inter-

16

DARREN K. GRIFFIN

FIG. 9 Detection of the alphoid probe for chromosome 18 using FISH.

spersed repeats, CISS generally needs to be applied. Julien et al. (1986) were the first to report the painting of chromosome 21 by creating chromosome 21 probes using a dual laser flow cytometer and subsequently detecting trisomy 21 in prenatal samples. Cremer et al. (1988a)and Lichter et al. (1988) (accompanying papers) describe the delineation of chromosomespecific libraries, subsequent chromosome painting, and detection of chromosome I , 4,7, 18, and 22 aberrations in tumor cells. Flow-sorted human chromosome libraries were used here also. Pinkel et al. (1988) detected trisomy 21 in interphase nuclei and chromosome 4 translocations using libraries obtained from the American Type Culture Collection which were cloned into “Bluescribe” plasmid vectors. An improvement in selecting library clones was demonstrated by Fuscoe et al. (1989), who subcloned a chromosome 21-specific library into “Bluescribe” and selectively picked unique sequence inserts. The result was more intense signals when the library of clones was put through CISS and FISH. Very intense chromosome painting was also reported by Jauch et al. (1990), who applied their studies to human sex chromosomes and amplified libraries in their original phage vectors. Currently, chromosome painting libraries are available for all human chromosomes. Recent innovations (e.g., Telenius et al., 1992) have generated chromosome painting probes by flow sorting whole human chromosomes and subsequently amplifying the resulting DNA by a PCR protocol designed to amplify total DNA [degenerate oligonucleotide-primed (DOP) PCR]. These paints tend to give brighter signals than those isolated from cloned libraries. The use of DOP-PCR to amplify the sorted chromosomes allows large quantities of DNA to be generated and these probes are now marketed commercially. Figure 10 shows the painting of chromosome 5 in a preparation of human lymphocytes.

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FIG. 10 Chromosome painting (chromosome 5).

3. Detection of a Single Locus In the field of gene mapping, it is in the direct visualization of large probes on chromosomes-namely phages, YACs, and more commonly cosmids-that most progress has been made. Mapping of longer sequences by FISH invariably requires the employment of CISS. However, as will be described in subsequent sections, such probes also have diagnostic uses. Lichter et al. (1990b) were the first to describe the FISH mapping of cosmids on chromosome 11. Biotin and digoxigenin labels were employed. Since the realization that cosmids could be mapped using FISH, most of the interest in this field has been in that direction. Signals are typically visible on both chromatids of both homologs. When conditions are optimal, a cosmid signal can be visualized in the interphase nucleus as well as on the metaphase chromosome. Using single-color FISH, signals between 25 and at least 250 kb can be resolved in interphase nuclei. Furthermore, the distance between two clones can be ascertained solely by measuring the distance between them in an interphase nucleus with an accuracy of within 40 kb (Trask et al., 1990; Bentley-Lawrence et al., 1990). Using dual-color fluorescence, signals 50 kb apart can be resolved at interphase and signals 3 megabases apart can be resolved at metaphase. Clones which are close together can be ordered with respect to one another by examining interphase nuclei. This is achieved by varying the hapten (usually biotin and digoxigenin are used) with which each of three or more probes is labeled (e.g., labeling two probes with biotin and the other with digoxigenin). The probes can be simultaneously hybridized on

DARREN K. GRIFFIN

18

the same preparation and then detected with red and green fluorochromes respectively. Simply scoring the order of red and green dots in a series of experiments where different combinations of haptens are used thus gives a physical order of clones. Such an approach is referred to as “interphase mapping” (Trask, 1991) and can be used not only for ordering genes but also, when the usual gene order is known, for detecting subtle rearrangements, deletions, or duplications that lead to genetic conditions. The mapping of yeast artificial chromosomes is also well documented in the literature. Being of very large insert size, YACs tend to give large signals; however, nonspecific background signals on unrelated chromosomes can be a problem when hybridizing if conditions are suboptimal (Rietman et al., 1989; Wada er al., 1990). YACs can be used to narrow down the molecular location of chromosomal breakpoints in translocations. A split YAC signal that is visible on derivative translocation chromosomes indicates that the breakpoint must be encompassed within the few hundred kilobases to which that YAC hybridizes (Rowley et al., 1990). YAC signals, like cosmids, are visible in the interphase nucleus. Figure 11 shows a YAC containing DNA from the gene adenomatous polyposis coli hybridizing to chromosome 5 . 4. Total Genomic Probes

The use of total genomic probes has been largely exploited in screening somatic cell hybrids. Total human DNA can be used as a probe on meta-

FIG. 11

FISH detection of a YAC containing the adenomatous polyposis coli gene.

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phases of hybrids to determine how many human pieces are present, whereas total hybrid DNA can be probed on to human spreads to determine the chromosomal nature of these pieces. As there are, to my knowledge, no living human-animal hybrids, it is difficult to envisage how total genomic probes could be used for diagnostic purposes and thus they will be discussed no further. 5. Further Developments

A number of areas of research involving FISH technology are rapidly becoming apparent.

MulticolorFISH Nederlof et al. (1989) were the first to achieve triple hybridization and detection using centromere-specific probes. Biotin, AAF, and mercury labeling were employed, coupled with FITC, TRITC, and AMCA fluorescent detection systems. Although it was a significant breakthrough, three-color fluorescence has limited usefulness because the DNA usually..needs to be counterstained fluorescently for location purposes, thus using up the spectra of one fluorochrome. Reid et al. (1992) demonstrated for the first time the simultaneous detection of probes for chromsomes 21, 18, 13, X and Y, each in a different color on the same preparation. The probes used were contig cosmid clones, and five-color detection was facilitated by labeling each probe with either one or a different combination of two labels, detecting them with appropriate fluorochromes, analyzing each digitally for the presence of one, two, or three colors and then assigning each a pseudocolor using computer software. For instance, with two fluorochromes, FITC and Texas red, a computer linked to an image analysis system could assign a Texas red image a red pseudocolor, an FITC image a green pseudocolor, and an image fluorescing both FITC and Texas red, a blue pseudocolor. Thus from a two-color system, three-color fluorescence can be obtained. Hence with a three-color detection system (red, green, and blue or infrared, red, and green), seven-color fluorescence could theoretically be achieved. Seven-colorfluorescencewould be invaluable in clarifyingcomplex karyotypes, whether using chromosome-specificcentromeric probes or chromosome painting. A further application would be the ability to order seven cosmids in relation to each other. This technology can be taken a step further by using “ratioing” of certain fluorochromes, for instance, distinguishing probes labeled with a I : 1 ratio of red to green from those labeled with a 2 : 1 ratio. In such a way, it is easy to envisage a large number of chromosomes or chromosome regions illuminated in different colors. Indeed, multiple-color chromosome painting has been described by Dauwerse et al. (1992), who performed 6-, Q.

DARREN K. GRIFFIN 20 7-, 9-, and 12-color chromosome detection on the same metaphase using

different ratios of red, green, and blue. Furthermore, pictures were generated by normal photography and not computer-aided image analysis.

b. Application of PCR Technology to FISH Another possible direction of investigation is to combine FISH and PCR technology. A small oligonucleotide could be annealed to a chromosome (as in PCR) and a string of nucleotides “zipped” on after it (as in PCR). This approach is referred to as “primed in situ DNA synthesis” or “PRINS.” Gosden et al. (1991) have described the use of this approach incorporatinga biotinylated deoxyuridine triphosphate (dUTP) in the nucleotide mix, followed by detection with fluorescent avidin. They detected human satellite sequences and Alu sequences and also telomere-specific sequences in Tetrahymena and Trypanosoma. I f these nucleotides were labeled with fluorescein dUTP, then this could be a very quick way to perform FISH and could be particularly useful in diagnostic applications when speed is important. Meltzer et al. (1992) have described the rapid (24-hr) generation of region-specific FISH probes and applying them to identify chromosomal rearrangements. The strategy they presented was to microdissect chromosomal regions and amplify them in uitro by PCR. PCR products were then labeled with biotin and used as probes onto metaphase preparations. Using such an approach, it is theoretically possible to generate probes (for FISH or other purposes) for any region of the genome and thus unequivocally identify most cytogenetically visible chromosomal rearrangements.

c. Three-Dimensional FISH A future application which is often mentioned in verbal presentations is the possibility of using FISH to map the physical position of genes in the intact interphase nucleus. If a method of preparing and fixing whole nuclei and hybridizing probes (e.g., cosmids) to them could be devised, then not only could the position of genes at interphase be mapped but also the communication between genes in the nucleus could be investigated. In such an application, a confocal microscope (a instrument which can visualize intact specimens in three dimensions) would be an invaluable tool. Trask et al. (1988) have described methods of preparing whole nuclei and performing FISH on them. They used total genomic probes on somatic cell hybrid nuclei and chromosomespecific satellite probes to reveal specific chromosomal domains in the interphase nucleus. Both groups of researchers are reportedly proceeding to the use of cosmid clones. d. Comparative Genome Hybridization This recent innovation in FISH technology was reported by Kallionemi et al. (1992) and du Manoir et al. (1993). It involves the simultaneous hybridization of normal DNA (labeled

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with one colored fluorochrome) and test DNA (labeled with another colored fluorochrome) on the same metaphase. The objective of the exercise is to compare the relative intensities of each color and thus elucidate gross chromosomal differences between the two DNAs. For instance, if the test DNA was from a patient with trisomy 18, the relative intensity of fluorescence from the test DNA would be brighter on chromosome 18 but of an equal ratio in the remaining chromosomes. This technique is still in its developmental stages but has already been used to analyze prenatal samples and tumor specimens.

e. “Ha1o”Preparations Wiegant et al. (1992) have reported a technique of preparing interphase nuclei which results in extended DNA loops arranged around the nucleus in a halo-like structure. Subsequent FISH with cosmid probes on these nuclei gives signals resembling beads on a string rather than discrete dots. It thus provides a simple, high-resolution approach for visualizing DNA strands. It is principally a method for mapping because it can detect the extent to which clones overlap, but it could feasibly be used to detect subtle DNA rearrangements that lead to clinical disorders.

IV. Sexing of Human Cells

Sexing of a human cell by FISH involves the detection of one or both of the sex chromosomes in an interphase or metaphase nucleus. A. Postnatal Samples Postnatal chromosomal diagnosis involving sex chromosomes has its uses when diagnosing XX males and XY females, when ascertaining the sex ratio of cells of an individual who is an XX/XY dispermic chimera, and also when monitoring the progress of a bone marrow transplant when donor and recipient are of opposite sexes. In each of these cases, FISH can obviate the need to prepare analyzable metaphases. B. Prenatal Samples Sexing is offered in the first or second trimester of pregnancy largely for the screening and selective termination of males in those families at risk of transmitting sex-linked recessive disorders. Selective termination of all

22

DARREN K. GRIFFIN

male pregnancies is only offered in the cases where specific diagnosis of the X-linked disorder is not available. FISH diagnosis of sex can be performed on the interphase nuclei of prenatal material when analyzable metaphases cannot be obtained. Sexing of prenatal samples has been cited by a number of authors using FISH probes specific for the Y chromosome (Kozma and Adinolfi, 1988; Guyot et al., 1988; Griffin et al., 1991). Use of a probe specific for the X chromosome on prenatal material (Griffin et al., 1991)eliminates the risk of failure of hybridization leading to misdiagnosis. Use of probes specific for both chromosomes, each detected in a different color (Griffin et al., 1992) is further advantageous though technically a little more difficult.

C. Sexing of Human Preimplantation Embryos

1. The Need to Research Sexing of Preimplantation Embryos Many families at risk of transmitting X-linked disorders (of which there are over 200) have already undergone one or more stressful abortions of male fetuses. Others disagree with terminations on moral or religious grounds. Hence, an effective means of preventing affected offspring would be to sex embryos fertilized in v i m and selectively implant females-socalled “preimplantation diagnosis.” Preimplantation diagnosis of sex has already been achieved using PCR to sex the embryo (Handyside et al., 1990; Handyside, 1991). This approach is, however, plagued with problems of contamination and amplification failure.

2. Early Work on Other in situ Hybridization Protocols Jones et al. (1987) were the first to report sexing of human embryonic nuclei and used both radioactive and nonradioactive ISH approaches. The nonisotopic technique employed enzymatic detection of a biotinylated probe. They reported good results using radioactivity but more ambiguous ones using biotin. West et af. (1987, 1988) using radioactive means attempted sexing on 14 morphologically normal and 9 apparently abnormal embryos. All 14 apparently normal embryos were sexed with confidence; however, clear diagnosis of sex for the 9 abnormal ones was not always possible. Angel1 et al. (1987) reported polyploidy in IVF embryos using an identical approach. Penketh et al. (1989) used an alkaline phosphatasebased detection system and biotinylated pHY2.1 and claimed a 66% success rate; that is, two-thirds of known male nuclei displayed a Y signal.

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3. FISH Studies

Studies using FISH on preimplantation embryos include those by Grifo et al. (1990), who apparently detected two Y chromosomes in a single human embryo, and Pieters et al. (1990), who detected chromosome 1 in a single human preimplantation embryo. However, Griffin et al. (1991) were the first to perform and evaluate FISH for X and Y chromosomespecific probes on human embryonic material. If one determines sex on the basis of number of hybridization signals in the majority of nuclei, the efficiencies with both probes (pHY2.1 and pBamX7) using FISH appeared to be higher in male embryonic nuclei than previously reported using other ISH methods with probe pHY2.1 (Jones et al., 1987; West et al., 1987, 1988;Penketh et al., 1989). For example, 85% of these nuclei gave positive signals, compared with 66% using an enzyme-based biotin detection system (Penketh et al., 1989). Figure 12 shows two X-chromosome signals on an interphase nucleus of a female human preimplantation embryo. Hybridization with probe pBamX7 had not been previously evaluated using any form of ISH on human embryos. There was a high incidence (18%)of nuclei displaying two (or more) signals in interphase nuclei classified as male and four signals in a nucleus designated as female. All of these embryos had more than 10 nuclei on day 5 postfertilization. In most cases, the nuclei with twice the number of expected signals were appreciably larger than those surrounding cells. This can be easily explained because of the occurrence of tetraploidy. Tetraploid nuclei with two Y chromosome signals have been already observed in human embryos (West et al., 1987, 1988), and it has been suggested that their occurrence in early preimplantation embryos may be a culture-induced phenomenon (West, 1990). Single-color FISH employing either probe had its disadvantages with

FIG. 12 Single nucleus from a human embryo displaying two X-chromosome signals.

24

DARREN K. GRIFFIN

regard to diagnostic accuracy. Use of the Y chromosome-specific probe pHY2.1 has an efficiency of around 85%. Thus, if two cells were biopsied (leaving six to be transferred), the probability of misdiagnosis would be more than 1%. Because of the frequent occurrence of twice the expected number of signals using the X probe alone, tetraploid male cells and female cells would be indistinguishable, again leaving the probability of misdiagnosisgreater than 1%. It was clear therefore that use of either probe alone was not an ideal approach to sexing human embryonic material. The problems associated with each of these probes alone were, however, alleviated when both were hybridized and detected simultaneously in different colors on the same nucleus (Griffin et af., 1992). Hybridization rates were high and, more important, the chance of misdiagnosis was reduced to a minimum. Positive diagnosis of sex could be made in 78% of interphase nuclei and 93% of metaphase nuclei (these were unsuitable for classical cytogenetic analysis), and efficiencies were greater than with other ISH techniques (Jones et al., 1987; West et af., 1987, 1988; Penketh et af., 1989; Grifo et al., 1990). This approach was an improvement on single labeling because the possibility of misdiagnosing a male as female is virtually eliminated since two clear X and no Y signals had to be seen before the embryo was identified as female.

4. Prospects for Clinical Application The biggest disadvantage of FISH methods for sexing preimplantation embryos compared with PCR protocols which had already been put into clinical applications (see Section 11) is that FISH methods generally take 24 hr to perform whereas the PCR-based sexing strategy takes only 5 hr. This is a disadvantage because, when performing preimplantation diagnosis, it is desirable to biopsy cells from the embryo, determine their sex, and selectively transfer female embryos all on day 3 postfertilization.This ensures maximum diagnostic efficiency and pregnancy potential. As a result, we developed a method of performing FISH sexing within one working day that involves simultaneous detection of X and Y chromosomes in green and red, respectively. Improvementto cytogeneticpreparation and the use of freshly prepared slides allowed the protocol to be followed with only a mild preproteinase digestion, high-stringency hybridization and washes, 90-120 min hybridization time, and no signal amplification. The whole procedure could be performed in 6-7 hr and high hybridization efficiencies were retained. On the basis of these results, it was decided to proceed with clinical application, treating families who were at risk of transmitting X-linked recessive disorders.

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5. Patient Strategies Thus far, 12 treatment cycles involving preimplantation sexing by FISH have been reported by our group (Griffin et al., 1993; Delhanty et af., 1993) and more are likely to be reported in the near future (Griffin et al., 1994). We can successfully sex over 80% of all our preimplantation embryos, and success rates are constantly increasing because of subtle changes in protocols. More important, our misdiagnosis rates are currently 0%. A number of live births have ensued, thus helping families who would have previously either have had to undergo stressful abortions or abstain from having children.

V. Further Fluorescent in Situ Hybridization Diagnoses A. Autosomal Imbalances

1. Chromosome-Specific Repeat Probes The probes most applicable for the detection of aneuploidy and polyploidy are the centromere-specificrepeat probes. When analyzable metaphases are unobtainable, these probes are visible in the interphase nucleus. There are probes available for all the human chromosomes; however, some detect more than one chromosome simultaneously. Use of these probes on live-born individuals includes confirmatory diagnosis and prenatal diagnosis of trisomy (e.g., trisomy 18), and investigations into the nonrandom nature of ploidy in the cells of certain cancers. Human trisomies that result in live births are those of chromosomes 21, 18, and 13. Cremer et al. (1986) described diagnosis of trisomy 18 in the interphase nuclei of prenatal samples using the probe L1.84 (specificfor the centromere of chromosome 18)and employing various ISH techniques. However, there is no current chromosome satellite-specific probe for chromosomes 21 or 13. As mentioned earlier, the probes available detect 21 and 13 together because the satellite DNAs are very similar (Willard, 1990).Furthermore, our own research has shown that the signal size varies on the acrocentric centromeres from individual to individual. Hence use of the 13/21 probe for the detection of trisomy can be problematic. Chromosome-specific repeat probes can also be of use in detecting the chromosomal nature of spontaneous abortions. Spontaneous abortions can arise as a result of polyploidy or aneuploidy in the fetus. The most common polyploidy in human abortuses is triploidy (Delhanty et al., 1961) and the most common aneuploidy is trisomy 16 (Hassold et al., 1984).

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DARREN K. GRIFFIN

Figure 13 shows the probe for chromosomes 13 and 21 detected on a normal lymphocyte chromosome preparation. Note that the signal on one of the chromosomes 21 is dimmer than the other three. Figure 14 shows the detection of trisomy 18 in interphase nuclei of an uncultured CVS sample. A number of individuals are at risk of transmitting aneuploidy because they carry balanced Robertsonian translocations of the acrocentric chromosomes. It is theoretically feasible to use probes specific for the centromeres of the acrocentric chromosomesto screen trisomy in the preimplantation embryos of these individuals. We are working on this but have run into problems because of the aforementioned differences from individual to individual and because overlying signals could lead to misdiagnosis. Most trisomies, however, do not arise as a result of balanced translocations involving the centromere in one of the parents. Hence the use of centromere-specific probes could be useful for preimplantation diagnosis only if a number of chromosomes were screened for and if the family ran a very high risk of transmitting trisomy for a reason other than a parent carrying a balanced translocation. Furthermore, given that many spontaneous abortions arise as a result of ploidy anomalies, it is theoretically possible to screen the embryos of individuals undergoing fertility treatment in in vitro fertilization clinics with a view to improving the success rate of the treatment. Both the above may be particularly applicable in older mothers who run a high risk of trisomic offspring (Morton et al., 1988).

FIG. 13 Detection of chromosomes 13 and 21 centromeres (alphoid probe).

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FIG. 14 Detection of trisomy 18 in interphase nuclei of an uncultured CVS.

2. Chromosome Painting and Locus-Specific Probes

a. Detection of Chromosome 22 Since trisomy 21 is the most common cause of mental retardation in man, much interest has been generated in using FISH to detect chromosome 21 prenatally, in postnatal samples (for instance to detect the level of mosaicism in live-born individuals), and also for preimplantation diagnosis. As mentioned, the centromere probe specific for chromosome 21 also detects chromosome 13 and its uses are limited. Lichter et al. (1990a) have reviewed the feasibility of various chromosome 21 probes, including 13/21 alphoid probes, chromosome painting libraries, and cosmid clones for detection in interphase nuclei. They conclude that use of a single cosmid clone for the detection of chromosome 21 at interphase is the most applicable. However, we find that single cosmids often give weak signals and can be difficult to detect

28

DARREN K. GRIFFIN

in interphase nuclei, Hence while a world-renowned laboratory such as Lichter’s may find no problem in detecting bright, single cosmid signals in interphase nuclei, ordinary diagnostic labs may not be as successful. It is possible to use a pool of overlapping cosmids to increase signal size. Such “contigs” (as they are referred to) are available commercially and we have looked at these in some detail. We find them to be of some use but find that YAC clones can give larger signals and hence are more applicable for the detection of chromosome 21 in interphase nuclei. Figure 15 shows adjacent metaphase and interphase nuclei: A, probed with the chromosome 21 contig; B, probed with a YAC for chromosome 21. Note that the YAC signals are brighter and more easily visible in the interphase nuclei.

6. Other ChromosomeAnomalies To detect chromosome translocations in the interphase nucleus, locus-specific probes such as cosmids, cosmid contigs, and YACs can be used. This was elegantly demonstrated by Arnoldus et af. (1990) and Tkaachuk et af.(1990), who reported detection of the “Philadelphia chromosome” in the interphases of the bone marrow of patients with chronic myeloid leukemia (CML). The Philadelphia chromosome is the der(22) from a reciprocal translocation t(9;22)(q34:qll) and causes the condition as result of fusion of the cancer genes bcr (on chromosome 22) and abl (on chromosome 9). Both groups used probes specific for these two genes in a dual-color detection strategy. Figure 16 illustrates diagrammaticallythe detection of these probes and the Philadelphia chromosome as reported by the two groups. These probes are currently available commercially and diagnostic laboratories interested in detecting the Philadelphia chromosome should perhaps consider using them when production of analyzable metaphases fails. Recently, Knight et al. (1992) and Joos et af. (1992) have used similar approaches to detect translocations in synovial sarcoma and Burkitt’s lymphoma cells respectively. It is theoretically possible to detect any chromosome translocation by these means in interphase nuclei (provided that the relevant probes are available) in postnatal, prenatal, or preimplantation material (Lichter and Ward, 1990). Chromosome painting is an invaluable technique when working with metaphases on which classic banding and analysis is difficult. Such instances arise when a rearrangement is very complex, when chromosomes are very short and/or clumped together, or when banding techniques fail. This can often be the case in some leukemias and also in some CVS preparations. Chromosome painting gives unequivocal identification of any one chromosome (or more if other colors are used). It is also useful in unequivocally determining the chromosomal origin of ring chromosomes

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FIG. 15 (A). Chromosome 21 cosmid contig (metaphase and interphase). (B). Chromosome

21 YAC (metaphase and interphase).

DARREN K. GRIFFIN

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00 00

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FIG. 16 Detection of the Philadelphia chromosome in interphase nuclei.

on which banding patterns are not always obvious (e.g., Svennevik and Hastings, 1993). Although about 90% of cells from human preimplantation embryos can be arrested in metaphase following overnight treatment with colchicine, analyzable chromosome spreads are universally very difficult to obtain because chromosomes are often short and clumped together (L. J. Wilton, personal communication). Figure 17 shows a typical chromosome preparation from a human embryo. Since karyotype analysis of CVS involves looking at 20-30 metaphases selected for optimal spreading, it is unlikely that preimplantation diagnosis by karyotyping alone is at present a feasible strategy. Hence chromosome painting approaches have been used to study the interphase nuclei of human preimplantation embryos. Figure 18 shows the detection of chromosome 5 (A) and chromosome 18 (B) on metaphase preparations of human preimplantation embryos.

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FIG. 17 Typical metaphase preparation of a human preimplantation embryo.

B. DNA Deletions

Several conditions arise as a result of DNA deletions. Two notable examples are muscular dystrophy and Angelman and Prader-Willi syndromes. Sixty percent of all males with Duchenne’s muscular dystrophy have a

FIG. 18 Chromosome painting of a human preimplantation metaphase. (A). Chromosome 5 . (B). Chromosome 18.

DARREN K. GRIFFIN 32 large deletion in the dystrophin gene on the X chromosome. In 60% of those individuals, the deletion is in exon 45 (Blonden et al., 1990). It is possible to detect exon 45 using an overlapping set of cosmids (a so-called “contig”). Hence it is possible to detect whether an individual is deleted for exon 45. Such an approach is useful postnatally when screening for female carriers (Reid et al., 1990) and prenatally when identifying affected fetuses. We are investigating the possibility of preimplantation diagnosis in couples at risk of transmittingthe deletion and hence Duchenne’s muscular dystrophy to their offspring. Figure 19 shows detection of the cosmid contig for exon 45 in a normal male. The large signal is the centromeric probe for the X chromosome and the two small dots above it are the cosmid signals. Also arising as a result of large deletions are certain cases of PraderWilli and Angelman syndrome. These are two distinct syndromes, both causing severe mental retardation, which can arise from deletions in the same region of chromosome 15 (15qll-12). If the deletion is in the maternal chromosome, the proband has Angelman syndrome and if the deletion is in the maternal chromosome, Prader-Willi syndrome. We have shown (R. J. Gardner, D. K. Griffin, and J. D. A. Delhanty, unpublished results) that it is possible to detect the deletion using FISH. Such a deletion, if relatively small, may easily be missed by classic cytogenetic analysis. Figure 20 shows a cosmid specific for this region detecting a deletion in a patient with Angelman syndrome in that only one chromosome 15 (top

FIG. 19 Detection of exon 45 of the dystrophin gene in a normal male.

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FIG. 20 Detection of a chromosome 15 deletion in a patient with Angelman syndrome.

left) is lit up. (This cosmid also cross-hybridizes to a region of chromosome 2.) Thus it is theoretically possible to detect any DNA deletion of above 40 kb provided the relevant cosmid is available.

C. DNA Duplications A condition arising from a DNA duplication is Charcot-Marie-Tooth disease type A. Lupski et al. (1991) used FISH to demonstrate this using a single cosmid probe on interphase nuclei of affected patients. This is particularly useful for a confirmatory diagnosis of a live-born individual or for prenatal diagnosis. Since preimplantation diagnosis is at present limited to one cell, detection of DNA duplications for clinical purposes would be unfeasible because overlying signals could lead to misdiagnosis. It is likely to be some time before detection of this disorder and ones like it becomes a possibility using FISH technology.

VI. Concluding Remarks

As mentioned in the introduction, there exists a gap in FISH technology between what is possible and what is practicable in a diagnostic laboratory.

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Also, almost inevitably, there is a time lag (often of 5 years or more) between the publication of a new approach and its use as a routine procedure. In the case of use of the chromosome-specific probes, companies (such as ONCOR) now sell these probes labeled with biotin or digoxigenin for all the human chromosomes. With a little training there is no reason why a moderately skilled technician could not perform a FISH experiment with these. Certain companies (such as CytoCell) even try and make things easier for the operator by sticking the probe to a coverslip. Chromosome painting can also be technically quite easy because companies such as ONCOR and CAMBIO produce paints labeled with biotin, digoxigenin, and/or fluorescein. Furthermore, since most of these companies also market probe detection kits, it is sometimes difficult to envisage how one could go wrong. FISH is certainly a much easier technique than it was 4 years ago. Hybridization and detection of locus-specific probes can, however, be more tricky. Because signals can be tiny and because competitive in situ suppression has to be used, experimental failure can often occur. Also, few such probes are marketed commercially. Exceptions to this include a cosmid contig for detection of chromosome 21 and bcrlabl probes for detection of the Philadelphia chromosome. It seems likely however that as the demand for these types of probes in the diagnostic situation increase, more and more will become commercially available. Whether diagnostic laboratories will keep pace with the rapidly moving FISH technology or simply consolidate the technology they are currently training themselves in remains to be seen. Certainly, techniques such as 12-colorchromosome painting and comparative genome hybridization would be useful tools. One could easily envisage it taking another 5 years at least for these to become routine protocols. In the case of preimplantation diagnosis, I cannot stress enough that it is an approach to be attempted only by labs with considerable experience in both embryo manipulation and research cytogenetics. It is a relatively new strategy, has aroused much attention in both the scientific and lay press, and hence could easily get a bad name if practized by the wrong hands. In terms of FISH technology, preimplantation diagnosis is a largely unexplored area. Only sexing has thus far been put into clinical practice. It may not be overoptimistic to hope that diagnosis of any chromosome abnormality or clinically significant DNA deletion could be possible by the end of the century. As for FISH technology itself, publications are constantly being produced announcing novel approaches to the FISH technique. Comparative genome hybridization is one such approach which looks certain to be exploited greatly. Also, multiple-color detection is very fashionable; one often hears mention of chromosomal bar codes where certain chromo-

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somes are banded in multiple fluorescent colors at will. There are also numerous cameras (cooled, charge-coupled device cameras) which can detect tiny signals invisible to the naked eye. Linked up to image-analysis setups, they can be extremely powerful both in grabbing and manipulating images and also in storing large numbers of them. It would be refreshing to see more research into the detection of DNA specimens in threedimensional specimens such as whole-mount embryos; the use of confocal microscopy (i.e., visualizing whole specimens in three dimensions) would help enormously here. The most interesting challenge to anyone involved in FISH research, I believe, would be the development of a FISH-based protocol which could distinguish between maternally and paternally derived homologs of any given chromosome.

Acknowledgments I express my thanks to Dr. Joy Delhanty for her critical reading of the manuscript, for initially giving me the opportunity to work on FISH, and for her help and encouragement throughout. I am also grateful to my friends and colleagues at the Hammersmith Hospital for providing material on which to work and ultimately making preimplantation diagnosis a reality.

References Adinofi, M., and Polani, P. E. (1989). Prenatal diagnosis of genetic disorders in preimplantation embryos: lnvasive and non-invasive approaches. Hum. Genet. 83, 16-19. Angell, R. R., Sumner, A. T., West, J. D., Thatcher, S. S., Glasier. A. F., and Baird, D. T. (1987). Post-fertilisation polyploidy in human preimplantation embryos fertilised invitro. Hum. Reprod. 2, 721-122. Arnoldus, E. P. J., Wiegant, J., Noordmeer, I. A., Wessels, J. W., Beverstock, G. C., Grosveld, G. C., van der Ploeg, M., and Raap, A. K. (1990). Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet. Cell Genet. 54, 108-1 11. Bentley-Lawrence, J., Singer, R. H., and McNeil, J. A. (1990). Interphase and metaphase resolution of different distances within the human dytrophin gene. Science 249,928-932. Blonden, L. A. J., Grootcholten, P. M., den Dunnen, J. T., Bakker, E., Abbs, S., Bobrow. M., van Broekhoven, C., Blaumbach, L., Chamberlain, J., Caskey, C. T., Denton, M., Felicetti, L., Galluzi, G.. Fishbeck. K. H., Franke, U . , Darras, B., Gilgenkrantz, H., Kaplan, J.-C., Henmann, F. H., Junien, C., Boileau, C., Lietchti-Gallati, S., Lindlof, M., Matsumoto, T., Niikawa. N., Muller, C. R., Poncin, J., Malcolm, S.,Robertson, E., Romeo, G., Covone, A. E., Scheffer, H., Schroder, E., Schwartz, M., Verellen, C., Walker, A., Worton, R., Gillard, E., and van Ommen, G. J. B. (1990). 242 breakpoints in the 200-kb deletion-prone P20 region of the DMD gene are widely spread. Genomics 10, 631-639.

Bolton, V. N., Wren, M.E., and Parsons, J. H. (1991). Pregnancies after in-uitrofertilization and transfer of human blastocysts. Fertil. Steril. 55, 830-832.

36

DARREN K. GRIFFIN

Brambati, B., and Lucia, T. (1990). Prenatal diagnosis before the 8th gestational week. Proc. Int. Con$ Early Fetal Diagn. 5th, Prague. Buster, J. E., Busillo, M., Rodi, I. A., Cohen, S. W., Hamilton, M., Simon, 3. A., Thorneycroft, I. H., and Marshall, S. R. (1985). Biologic and morphological development of donated human ova recovered by non-surgical uterine lavage. Am. J . Obstet. Gynecol. 153,211-217.

Cremer, T., Landegent, J . , Bruckner, A., Scholl, H. P., Shardin, M., Hager, H. D., Devilee, P., Pearson, P., and van der Ploeg, M. (1986). Detection of chromosome aberrations in the human interphase nucleus by visualisation of specific target DNAs with radioactive and non-radioactive in-situ hybridisation techniques. Hum.Genet. 14, 346-352. Cremer, T., Lichter, P., Borden, J . , Ward, D. C., and Mannuelidis. L. (1988a). Detection of chromosome aberrations in metaphase and interphase tumour cells by in-situ hybridisation using chromosome specific library probes. Hum. Genet. 80, 235-246. Cremer, T., Tesin, D., Hopman, A. H. N., and Mannuelidis, L. (1988b). Rapid interphase and metaphase assesement of specific chromosomal changes in neuroectodermal tumor cells by in-situ hybridisation with chemically modified DNA probes. Exp. Cell Res. 176, 199-220.

Dauwerse, I. G., Wiegant, J., Raap, A. K., Breuning, M. H., and van Ommen, G. J. B. (1992). Multiple colors by fluorescence in-situ hybridization using ratio-labelled probes create a molecular karyotype. Hum. Mol. Genet. 1, 593-598. Dawson, K. J., Rutherford, A. J., Winston, N. J., Subak-Sharpe, R., and Winston, R. M. L. (1988). Human blastocyst transfer, is it a feasible proposition? Hum.Reprod. 145(Suppl), 44-45.

Delhanty, J. D. A., Ellis, J. R., and Rowley, P. T. (1961). Triploid cells in a human embryo. Lancet 2, 1286.

Delhanty, J. D. A., Griffin, D. K., Handyside, A. H., Harper, J., Atkinson, G. H. G., Pieters, M. H. E. C., and Winston, R. M. L. (1993). Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplantation sex determination by fluorescent in-situ hybridisation. Hum. Mol. Genet. 2, 1183-1 185. Devilee, P., Cremer, T.. Slagboom, P., Bakker, E., Scholl, H. P., Hager, H. D., Stevenson, A. F. G., Hager, .I. Cornelisse, , C. J., and Pearson, P. L. (1986). Two subsets of human alphoid repetitive DNA show distinct preferential localisation to the pencentric regions of chromosomes 13, 18 and 21. Cytogenet. Cell Genet. 41, 193-201. Devilee, P., Kievits, T., Waye, J. S., Pearson, P. L., and Willard, H. F. (1988). Chromosome specific alpha satellite DNA: Isolation and mapping of a polymorphic alphoid repeat from human chromosome 10. Genomics 3, 1-7. du Manoir, S., Speicher, M. R., Joos, S., Schrock, E., Popp, S., Dohner, H., Kovacs, G., Robert-Nicoud, M.,Lichter, P., and Cremer, T. (1993). Detection of complete and partial gains and losses by comparative genome hybridization. Hum. Genet. 90, 590-610. Fuscoe, J. C., Collins, C. C., Pinkel, D., and Gray, J. W. (1989). An efficient method for selecting unique sequence clones from DNA libraries and its application to fluorescent staining of human chromosome 21 using in-situ hybridisation. Genomics 5, 100-109. Gall, J. G., and Pardue, M. L. (1969). Formation of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. U.S.A.63, 378-383. Gardner, R. J. (1992). Towards a diagnostic test for Angleman syndrome. Final year undergraduate research project. Department of Genetics and Biometry, University College, London. Gardner, R. L., and Edwards, R. G. (1%8). Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts. Nature (London) 218, 346-348. Gosden, J. R., Hanratty, D., Starling, J., Fantes. J., Mitchell, A., and Porteous, D. (1991). Oligonucleotide-primedin situ DNA synthesis (PRINS): A method for chromosome map-

FLUORESCENT IN SITU HYBRIDIZATION FOR DIAGNOSING GENETIC DISEASE

37

ping, banding and investigation of sequence organisation. Cytogenet. Cell Genet. 57, 100-104.

Griffin, D. K. (1992). Fluorescent molecular cytogenetics: Preimplantation diagnosis, colorectal cancer and the mapping of chromosome 9q. Ph.D. Thesis, University College, London. Griffin, D. K.,Handyside, A. H., Penketh, R. J. A.. Winston, R. M. L., and Delhanty, J. D. A. (1991). Fluorescent in-situ hybridisation to interphase nuclei of human preimplantation embryos with X and Y chromosome specific probes. Hum. Reprod. 6 , 101105.

Griffin, D. K., Wilton, L. J., Handyside, A. H., Winston, R. M. L., and Delhanty, J. D. A. (1992). Dual fluorescent in-situ hybridisation for simultaneous detection of X and Y chromosome specific probes for the sexing of human preimplantation embryonic nuclei. Hum. Genet. 89, 18-22. Griffin, D. K., Wilton, L. J., Handyside, A. H., Atkinson, G. H. G., Winston, R. M. L., and Delhanty, J. D. A. (1993). Diagnosis of sex in preimplantation embryos by fluorescent in-situ hybridisation. Br. Med. J . 306, 1382. Griffin, D. K., Handyside, A. H., Harper, J., Wilton, L. J., Atkinson, G. H. G., Wells, D., Kontogianni, E. H., Tarin, J., Geber, S., Ao, A., Winston, R. M. L., and Delhanty, J. D. A. (1984). Results of a clinical trial involving preimplantation diagnosis of sex by dual fluorescent in-situ hybridization. In preparation. Grifo, J. A., Boyle, A., Fischer, E., Lavy, G., DeCherney, A. H., Ward, D. C., and Sanyal, M. K. (1990). Preembryo biopsy and analysis of blastomeres by in-situ hybridisation. A m . J . Obstet. Gynecol. 6 , 2013-2019. Guyot, B., Bezin, A., Sole, Y., Julien, C., Daffos, F., and Forrestier, R. (1988). Prenatal diagnosis with biotinylated chromosome specific probes. Prenaral Diagn. 8, 485-493. Handyside, A. H. (1991). Biospy of human cleavage stage embryos and sexing by DNA amplification. I n “Proceedings of the First Symposium on Preimplantation Genetics” (Y. Verlinksy and J. Strom, eds.), pp. 75-83. Plenum, New York. Handyside, A. H., and Delhanty, J. D. A. (1993). Cleavage stage biopsy of human embryos and diagnosis of X-linked recessive disease. I n “Preimplantation Diagnosis of Human Genetic Disease” (R. G. Edwards, ed.). Cambridge Univ. Press, Cambridge. Handyside, A. H., Kontogianni, E. H., Hardy, K., and Winston, R. M. L . (1990). Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature (London) 344, 768-770. Hardy, K., Martin, K. L., Leese, H. J., Winston, R. M. L., and Handyside, A. H. (1990). Human preimplantation development in-uitro is not adversely affected by biopsy at the 8-cell stage. Hum. Reprod. 5 , 708-714. Hassold, T., Chiu, D., and Yamane, J. A. (1984). Parental origin of autosomal trisomies. A m . Hum. Genet. 48, 129-144. Hopman, A. H. N., Wiegant, J., and R a p , A. K. (1986). Bi-color detection of two target DNAs by non-radioactive in-sifu hybridisation. Histochemistry 85, 1-4. Hopman, A. H. N., Ramaekers, F. C. S.. Raap, A. K.. Beck, J. L. M., Devilee, P., van der Ploeg, M., and Voojs, G. P. (1988). In-situ hybridisation as a tool to study numerical chromosome aberrations in solid bladder tumours. Histochemistry 89, 307-3 16. Jalech, A., Daumer, C., Lichter, P., Murken, J., Schroeder-Kurth, T., and Cremer, T. (1990). Chromosomal in-situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics. Hum. Genet. 85, 145-150. Jones, K. W., Singh, L., and Edwards, R. G. (1987). The use of probes for the Y chromosome in preimplantation embryo cells. Hum. Reprod. 2, 439-445. Joos, S., Falk, M. H., Lichter, P., Hakuska, F. G., Henglein, B., Lenoir, G. M.. and Bornkamm. G. W. (1992). Variable breakpoints in Burkitt lymphoma cells with chromo-

38

DARREN K. GRIFFIN

soma1 t(8;14) translocation separate c-myc and the IgH locus up to several hundred kb. Hum. Mol. Genet. 1, 625-632. Julien, C., Bazin, A., Guyot, B., Forrestier, F., and Daffos, F. (1986). Rapid prenatal diagnosis of Down’s Syndrome with in-situ hybridisation of fluorescent DNA probes. Lancet 2, 863-864. Kallionemi, A., Kallionemi, 0.-P., Sudar, D., Rutovitz, D., Gray, J. W., Waldman, F., and Pinkel, D. (1992). Comparative genome hybridization for molecular cytogenetic analysis of solid tumours. Science 258, 818-821. Khalfan, H., Abuknesha, R., Randweaver, M., Prid, R. G., and Robinson, D. (1986).Aminomethyl coumarin acetic acid: A new fluorescent labelling agent for proteins. Histochern. J. 18,497-499. Knight, J., Reeves, B. R., Kearney, L., Monaco, A. P., Lerach, H., and Cooper, C. S. (1992). Localisation of the synovial sarcoma t(X;l8)(pl1.2;qlI .2) breakpoint by in-situ hybridisation. Hum. Mol. Genet. 1, 633-637. Kozma. R., and Adinolfi, M. (1988). In-situ fluorescence hybridisation of Y translocations: Cytogenetic analysis using probes Y190-Y431. Clin. Genet. 33, 156-161. Landegent, J. E., Jansen, D., Wal, N., Baan, R. A., Hoeijmakers, J. H. J., and van der Ploeg, M. (1984). 2-acetylaminofluorene modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences. Exp. Cell Res. lS3, 61-72. Lichter, P., and Ward, D. C. (1990). Is non-isotopic in-situ hybridisation finally coming of age? Nature (London) 345, 93-95. Lichter. P., Cremer, T., Borden, J., Mannuelidis, L., and Ward, D. C. (1988). Delineation of individual human chromosomes in metaphase and interphase cells by in-situ suppression hybridisation using recombinant DNA libraries. Hum. Genet. 80, 224-234. Lichter, P., Jauch, A., Cremer T., and Ward, D. C. (1990a). Detection of Down Syndrome by in-situ hybridisation with chromosome specific DNA probes. In “Molecular Genetics of Chromosome 21 and Down Syndrome” (D. Patterson and C. J. Epstein, eds.). pp. 69-78. Wiley-Liss, New York. Lichter, P., Chang-Tang, C.-J., Call, K., Hermanson, G . , Evans, G. A., Housrnan, D., and Ward, D. C. (1990b). High resolution mapping of human chromosome 11 by in-situ hybridisation with cosmid clones. Science 247, 64-69. Lilford, R. J. (1991). The rise and fall of chorionic villus sampling. Br. Med. J . 303,936-937. Lupski. J. R., Montes de Oca-Luna, R., Slaugenhaupt, S., Penatao, I., Guzzeta, V.. Trask, B. J., Saucedo-Cardenas, O., Barker, D. F., Killian, J. K.. Garcia. C. A., Chakravarti. A., and Patel, P. I. (1991). DNA duplication associated with Charcot-Marie Tooth Disease Type I A . Cell (Cambridge, Mass.) 66, 219-232. Malcolm, S., Cowell, J. K., and Young, B. D. (1986). Specialist techniques in research and diagnostic clinical cytogenetics. I n “Human Cytogenetics: A Practical Approach” (D. Rooney and B. H. Czepulkowski, eds.), Chapter 7. IRL Press, Oxford. Meltzer, P. S.. Guan, X.-Y., Burgess, A.. and Trent, J. F. (1992). Rapid generation of region specific probes by chromosome microdissection and their application. Nut. Genet. 1, 24-28. Morton, N . E., Jacobs, P. A., Hassold, T.. and Wu, D. (1988). Maternal age in trisomy. Ann. Hum. Genet. 52,227-235. Nederlof, P. M., Robinson, D., Abuknesha, R., Wiegant, J., Hopman, A. H. N., Tanke, H. J., and Raap, A. K. (1989). Three colour fluorescence in-situ hybridisation for the simultaneous detection of multiple nucleic acid sequences. Cytometry 10. 20-27. Penketh, R. J. A., and McLaren, A. (1987). Prospects for prenatal diagnosis duringpreimplantation human development. Ballieres Clin. Obstet. Gynecol. 1, 747-764. Penketh, R.J. A., Delhanty, J. D. A., van den Bergh, J. A., Finklestone, E. M., Handyside, A. H.,Malcolm. S., and Winston, R. M. L. (1989). Rapid sexing of human embryos by

FLUORESCENT I N

siru HYBRIDIZATION

FOR DIAGNOSING GENETIC DISEASE

39

non-radioactive in-situ hybridisation, potential for preimplantation diagnosis of X-linked disorders. Prenatal Diagn. 9, 489-500. Pieters, M. H. E. C.. Geraedts, J. P. M.. Meyer. H., Dumoulin, J. C. M., Evers, J. L. H.. Jongbloed, R. J. E., Nederlof, P. M., amd van der Flier, S. (1990). Human gametes and zygotes studied by non-radioactive in-situ hybridisation. Cytogenet. Cell Genet. 53, 15- 19. Pinkel, D., Straume, T.. and Gray, J. (1986). Cytogenetic analysis using quantitative high sensitivity fluorescence hybridisation. Proc. Natl. Acad. Sci. U . S . A . 83, 2934-2938. Pinkel, D.. Landegent. J., Collins, C., Fuscoe, J., Segraves, R.. Lucas, J.. and Gray. J. (1988). Fluorescence in-situ hybridisation with human chromosome specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Nut/. Acad. Sci. U.S.A. 85,9138-9142. Raap, A. K.. Dirks, R. W., Jiwa, N. M., Nederlof, P. M., and van der Ploeg, M. (1990). In-situ hybridisation with hapten modified DNA probes. I n “Modern Pathology of AIDS and Other Retroviral Infections” (P.Raez. A. T. Haase, and J. C. Glucman. eds.), pp. 17-28. Karger, Basel. Reid, T.. Mahler. V., Vogt, P., Blonden, L., van Ommen, G. J. B.. Cremer, T., and Cremer, M. (1990). Direct carrier detection by in situ suppression hybridization with cosmid clones of the Duchenne/Becker muscular dystrophy locus. Hum. Genet. 85, 581-586. Reid. T.. Landes, G., Dackowski, W., Klinger, K., and Ward, D. C. (1992). Multicolour fluorescence in-situ hybridization for the simultaneous detection of probe sets for chromosomes 13, 18.21, X and Y in uncultured amniotic fluid cells. Hum. Mol. Genet. 1,307-313. Rietman, H. C., Moyzis, R. K., Meyne, J.. Burke, D. T., and Olson. M. V. (1989). Cloning human telomeric DNA fragments into Sacchoromyces cereuisiae using a yeast artificial chromosome vector. Proc. Natl. Acad. Sci. U.S.A. 84, 6240-6244. Rowley, J. D., Diaz, M. O., Espinosa, R., Ill, Patel, Y. D., van Melle, E., Ziemin, S.. Taillon-Miller. P.. Lichter, P., Evans, G. A., Kersey, J. H., Ward, D. C., Domer, P. H.. and le Beau, M. (1990). Mapping chromosome llq23 in human acute leukemia with biotinylated probes: Identification of I lq23 translocation breakpoints with a yeast artificial chromosome. Proc. Natl. Acud. Sci. U.S.A. 87, 9358-9361. Rudkin. G. T., and Stollar, B. D. (1977). High resolution detection of DNA-RNA hybrids in-situ by direct immunofluorescence. Nature (London) 265, 472-473. Smith, J.. Rebello. M.. Gray, C., Rooney, D., Leffler, F.. andColeman, D. (1990). Amniocentesis at 11-14 weeks gestation. Proc. In:. Symp. Early Fetal Diagn. 5th, Prague. Steptoe, P. C., and Edwards, R. G. (1978). Birth after implantation of a human embryo. Lancet 2, 366. Svennevik, E. C., and Hastings, R. J. (1993). A translocation ring chromosome 21 and fragment in a child with Down syndrome. Assoc. Clin. Cytogenet. Bull. Z7), 48. Tchen, P., Fuchs, R. P. P., Sage, E., and Leng. (1984). Chemical modified nucleic acids a s immunodetectable probes in hybridisation experiments. Proc. Natl. Acad. Sci. U . S . A . 81, 3466-3470. Telenius, H.. Pelmear. A. H., Tunnacliffe, A., Carter, N . P., Behmel, A., Ferguson-Smith, M.A.. Nordenskjold, M.. Pfrangner, R., and Ponder, B. A. J. (1992). Cytogeneticanalysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes. Chromosomes, Cancer 4, 257-263. Tkaachuk, D. C., Westbrook, C. A.. Andreelf, M.. Donlon,T. A.,Clearly, M. L.. Suryanarayan, K.. Homge, M., Redner, A., Gray, J., and Pinkel, D. (1990). Detection of bcr-abl fusion in chronic myelogenous leukemia by in-situ hybridisation. Science 250, 559-562. Trask, B. J. (1991). Fluorescence in-situ hybridisation applications in cytogenetics and gene mapping. Trends Genet. 7 , 149-154. Trask, B. J., van den Engh, G., Pinkel, D., Mullikin, J., Waldman, F., van Dekken, H., and Gray, J. (1988). Fluorescence in situ hybridisation to interphase nuclei in suspension

40

DARREN K. GRIFFIN

allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organisation. Hum. Genet. 78, 251-259. Trask, B. J., Pinkel, D., and van den Engh, G. (1990). The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs. Genomics 5, 710-717. Trask, B. J., Massa, H., Kenwrick, S., and Gitschier, J. (1991). Mapping of human chromosome Xq28 by two-colour fluorescence in-situ hybridisation of DNA sequences to interphase cell nuclei. Am. J. Hum. Genet. 48, 1-15. Van Prooijen-Knegt, A. C., van Hoek, J. F. M., Bauman, J. G. J., van Duijn, P., Wool, I. G., and van der Ploeg, M. (1982). In-situ hybridisation of DNA sequences in human metaphase chromosomes by an indirect fluorescent immunocytochemicalprocedure. Exp. Cell Res. 141, 397-407. Wada, M., Little, R. D., Abidi, F., Porta, G., Labella, T., Cooper, T., Della-Valle, G., Diloso, M., and Schlessinger, D. (1990). Human Xq24-q28: Approaches to mapping with yeast artificial chromosomes. Am. J. Hum. Genet. 46,95-106. West, J. D. (1990). Sexing the human conceptus by in-situ hybridisation. I n “In-sifu Hybridisation and the Study of Development and Differentiation” (N. Hams and D. Wilkinson, eds.). West, J. D., Gosden, J. R., Angell, R. R., Hastie, N. D., Thatcher, S. S., Glasier, A. F., and Baird, D. T. (1987). Sexing the human pre-embryo by in-situ hybridisation. Lancet 1, 1344-1347. West, J. D., Gosden, J. R., Angell, R. R., West, K. M., Glasier, A. F., Thatcher, S. S., and Baird, D. T. (1988). Sexing whole human pre-embryos by in-situ hybridisation to a Y chromosome specific DNA probe. Hum. Reprod. 3, 1010-1019. Wiegant, J., Reid, T.. Nederlof, P. M., van der Ploeg, M., Tanke, H. J., and Raap, A. K. (1991).In-situ hybridisation with fluoresceinated DNA. Nucleic Acids Res. 19,3237-3241. Wiegant, J., Kalle, W., Mullenders, L., Brookes, S., Hoovers, J. M. N., Dauwerse, J. G., van Ommen, G. J. B., and Raap, A. K. (1992). High resolution in-situ hybridisation using DNA halo preparations. Hum. Mol. Genet. 1, 587-591. Willard, H.F. (1990). Alpha and beta satellite sequences on chromosome 21: The possible role of centomere and chromosome structure in non-disjunction. I n “Molecular Genetics of Chromosome 21 and Down Syndrome” (D. Patterson and C. J. Epstein, eds.), pp. 39-52. Wiley-Liss, New York. Willard, H. F., and Waye, J. S. (1985). Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genet. 3, 192-198. Winston, R. M. L. (1987). Why a ban on embryo research would be such a tragedy. Br. Med. J . 295, 1501-1502.

Isolation and Function of Human Dendritic Cells Lisa A. Williams, William Egner, and Derek N. J. Hart Haematology/Immunology Research Group, Christchurch Hospital, Christchurch, New Zealand

1. Introduction and Overview

A. General Understanding of the cellular costimulatory requirements for primary and secondary T-lymphocyte responses has improved markedly since the identification of potent primary antigen-presentingcells (APCs) in the mouse, collectively termed dendritic cells (DCs) (Steinman and Cohn, 1973). These cells were clearly distinct from typical monocyte-macrophages and B lymphocytes. A similar cell population was subsequently confirmed in human subjects (Hart et al., 1981) and DCs have now been delineated in practically every human organ (Daar et al., 1983; Hancock and Atkins, 1984; Hart et al., 1989; Steinman, 1991) with the exception of the brain and central cornea. These tissue-resident DCs (such as the Langerhans cell, LC) are thought to provide an extensive network of sentinel APCs capable of antigen capture, processing, and subsequent migration to local lymphoid tissue where efficient primary T-lymphocyte stimulation occurs. A DC may be defined primarily by the possession of a more potent ability to initiate primary T-lymphocyte proliferation than monocytes or B cells, and an immunophenotype which distinguishes it from these and other leukocyte populations. Other criteria which are considered to support the identification of DCs are either not restricted to DCs alone-such as high-density major histocompatibility complex (MHC) class I1 expression, the presence of dendritiform morphology in uitro, and lack of phagocytosis in vim-or are relative in comparison with other myeloid cells, for example, the presence of lower levels of cytoplasmic enzymes which may be found in a distinctive perinuclear distribution. The morphological identification of DCs is difficult, and the subdivision of DCs into three subtypes on this basis after exposure to metrizamide during preparation lnternafional Review 11fCylo/ogy. Vol. 153

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Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(Knight et al., 1986) is most controversial. Critical interpretation of all studies on DCs must be made with these points in mind, while recognizing that further work may identify DC subpopulations. It is also important to note that there are considerable differences between human DCs and those from other species with regard to such properties as adherence and the expression of certain leukocyte differentiation antigens. Recent work on human DCs in this and other laboratories has emphasized the close relationship of DCs with other myeloid cells, despite their distinctive morphological and functional properties. The precise relationships between the DCs found in different tissues and their relationship to other myeloid cells remain to be conclusively established. Identification of DC precursors in human bone marrow and understanding the regulation of tissue migration and differentiationis still in its infancy, but important advances are being made and we will outline these later. DCs are phenotypically quite different from, and should not be confused with, follicular dendritic cells (FDC), which are B-lymphocyte-associated cells that are involved in the maintenance of B-cell memory and are probably not bone marrow derived. To present antigen effectively, the DCs must be able to acquire intact antigen, process it into small immunogenic peptide fragments, present this fragment on the surface of an MHC class I1 molecule to a T lymphocyte (signal l), and then provide a number of appropriate “accessory” signals (signal 2) sufficient to costimulate proliferation of naive T cells. This multistep process is being dissected in uitro. Intimate membrane contact between DCs and T lymphocytes in cellular aggregates or clusters is essential. Rapid progress is also being made in defining the cellular attributes (cytokines and adhesion or costimulator molecules) responsible for their extremely potent functional activity in presentation of primary antigen to T lymphocytes (Steinman and Young, 1991) as discussed in Section V1,D. Work on DC in uitro has always been hampered by the low yield of purified cells obtained from a cell population present in very small numbers. It is hoped that this situation will be improved by new isolation methods developed by our laboratory and others. Improved yields will be especially important given the interest in the use of DCs as a potent in uiuo immunizing agent for presentation of vaccines against both malignant cell antigens and infective microorganisms. There is also considerable interest in the possible role of DCs in the dissemination of HIV infection within the human host and the role of impaired DC function in the immunological abnormalities of AIDS. An understanding of the signals which activate DCs may allow their already powerful APC properties to be enhanced or suppressed, both attractive propositions which may have many therapeutic applications.

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6. Dendritic Cells: An Overview

DCs were first identified in 1973 as a weakly and transiently adherent cell population in murine spleen (Steinman and Cohn, 1973) which displayed stellate morphology, a paucity of intracellular organelles, and poor phagocytic capabilities in comparison with monocyte/macrophages (Mo/Mp). Cells displaying similar characteristics were isolated from other rodent tissues (Steinman, 1991) and from other species (Hart and McKenzie, 1990). These DCs lacked most of the lineage-associated surface antigens of Mo/Mp or lymphocytes, but expressed MHC class I and class I1 molecules in high density. Human interstitial DCs were first described in 1981 (Hart et al., 1981) and were isolated from peripheral blood (Van Voorhis et al., 1982) using a modification of the method for purifying murine splenic DCs. Subtle differences between the properties of murine and human DCs, however, made alteration of the techniques desirable to maximize yields. DCs occur in a number of different tissues and can be subdivided on the basis of tissue location as shown in Fig. 1. Minor phenotypic and functional differences among these cell populations have led to the hypothesis that these cells may represent different stages of activation, maturation, or tissuespecific differentiation. Human DCs, in situ,are characterized by long cytoplasmic extensions. Most reports describe an irregularly shaped nucleus and a lack of phagolysosomes evident in classical macrophages. DCs also lack, or express lower levels of many myeloid enzymes such as nonspecific esterases (NSE) and acid phosphatase (AP) when assayed by various techniques. Immunophenotypic analysis typically reveals abundant expression of human leukocyte antigen (HLA) locus products and expression of the leukocyte common (CD45) antigen. Other important surface molecules described on DC include the adhesion or costimulator molecules leukocyte function-associated antigen-1 (LFA-1) (CD18/CD1la), LFA-3 (CD58), intercellular adhesion molecule-1 (ICAM-1) (CD54), ICAM-2; and more recently, ICAM-3 (Prickett et al., 1988; Thomas et al., 1993; Starling et al., 1994), CD40 (Hart and McKenzie, 1988), and B7/BB1 (Hart et al., 1993; Young et al., 1992). High levels of adhesion molecule expression account in part for the ability of these cells to establish strong initial contact with surroundingT lymphocytes. While DCs were initially thought to constitutively express MHC class I1 products, adhesion molecules, and important costimulator ligands in high density, the role of in uiuo and in uitro differentiation or activation in upregulating these surface antigens is only now being appreciated. Molecules associated with myeloid and lymphoid lineages are generally absent from DC, although weak CD4 staining has been observed (Landry

Q

Stemcell

@@ (DCprecursor)

Afferent lymph (veiled cells)

Lymph node (interdigitationgcells)

Interstitial DC (Liver, kidney, pancreas, etc)

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et al., 1988; Hart and McKenzie, 1988) both in situ and in uitro. Weak

CD13, CD14, and CD33 expression has also been observed on freshly isolated human blood DCs (Thomas et al., 1993; Egner et al., 1994). Murine DCs have been shown to express an aminopeptidase in common with macrophages which may represent a murine CD13 analog (Leenen et al., 1992). Mature DCs have historically been characterized by a lack of complement or Fc receptors (FcR) (Hart and McKenzie, 1988; Van Voorhis et al., 1982;Steinman and Cohn, 1973).However, some variations have been observed. LCs express low levels of C3bi receptors (CDI lb), FcRyII (CD32), and show functional complement and FcR activity (Teunissen et al., 1990; Romani et al., 1989; Cohen and Katz, 1992). CD32 (FcRyII); also, lesser amounts of CD64 ( FcRyI) have been detected on fresh blood DCs (Thomas et al., 1993). Low-level CD32 expression has certainly been detected on other in situ DCs using sensitive techniques (Prickett et al., 1988; Buckley et al., 1987). The presence of low-level FcR expression has been inferred from the ability of DCs to function in FcR-dependent assays such as CD3 mitogenesis. The most important identifying feature of DCs is their powerful activity in primary T-cell stimulation. The most commonly used assay for this activity is the allogenic mixed leukocyte reaction (MLR), although primary antigen-specific T-Iymphocyte proliferation and autologous MLR are also used. DCs have been shown to exhibit between 10 and 100 times more potent allostimulatory activity than other leukocytes (Van Voorhis et al., 1982). C. Assessment of Purity of Dendritic Cell Preparations

Purities of >90% have been claimed with many isolation methods. One of the more confusing and difficult aspects of the DC literature is the varied criteria used to assess the purity of the final DC population. 1. Morphology

DCs have traditionally been defined morphologically and indeed murine DCs were named because of their distinctive appearance in situ in the mouse (Steinman and Cohn, 1973). Lately, however, it has become clear that morphological identification of human DCs in uitro is extremely diffi-

FIG. 1 Dendritic cell migration and differentiation. Dendritic cells derived from different tissues are suggested to be related via a pathway such as this. Cytokines such as IL-I, TNFa,and GM-CSF appear to influence DC behavior at the various stages indicated.

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

cult. Dendriform morphology can be demonstrated for numerous cell types in uitro, including B lymphocytes (Corradi e l al., 1987; Hart and McKenzie, 1988).DC preparations obtained by plastic adherence and metrizamide contain contaminating low-density, strongly CD 14-positiveMo/Mp, which also display MHC class 11-positiveveiled morphology (Knight et al., 1986). The majority of these cells express low levels of the ectoenzyme 5'-nucleotidase, despite two-thirds clearly expressing Mo/Mp markers and active phagocytosis. 2. Dendritic CeU-Specific Monoclonal Antibodies

Easy identification of DCs has been hampered by the lack of DC-specific monoclonal antibodies (mAb). In rodents, NLDC 145 (Kraal et al., 1986), M342 (Agger et al., 1992),OX-62 (Brenan and Puklavec, 1992), and 33DI (Nussenzweig et al., 1982) have been used to identify certain subtypes of DCs, but they are not restricted to DCs alone and react with macrophage subpopulations (Kraal et al., 1986)and B cells (Agger et al., 1992). Some also appear to act as activation or differentiation antigens with variable expression. The new antihuman mAbs, CMRF44 (Hock et al., submitted) and HBl5a (Zhou et al., 1992) have shown promise and appear to label activated human blood DCs, but like RFDl (Poulter et al., 1986), are not restricted to DCs. The histiocytosis X (an LC malignancy)-specificmAb, Lagl, may prove an important cytoplasmic marker in the future (Ishikawa et al., 1992), but has yet to be evaluated.

3. Cytochemical Characteristics Cytochemical characteristics have been used to differentiate DCs from other myeloid cells. DCs are reported to lack many of the enzymes associated with classic Mo/Mps in certain staining techniques (e.g., myeloperoxidase, MPO) (Buckley et al., 1987; Steinman and Cohn, 1973; Hart and Fabre, 1981). Other reports demonstrate that they do in fact express 5'nucleotidase, dipeptidyl peptidase-I (DPPl) and cathepsin B activity in low levels (Knight et al., 1986; Thomas et al., 1993; Romani and Schuler, 1992). Other intracellular enzymes or lysosomal antigens (CD68) may be present in an unusual intracellular distribution (NSE, AP, CD68). Staining with AP, for example, displays perinuclear dot cytoplasmic staining in contrast to the diffuse appearance of Mo-Mps (Hart and McKenzie, 1988), while staining and CD68 reveals perinuclear distribution in DCs (Prickett et al., 1988; Betjes et al., 1991; Arkema et al., 1991). Significant phenotypic changes may result from certain isolation techniques. Loss of weak FcR expression (Thomas et al., 1993), or changes in antigen expression or morphology may further complicate the identification of DCs or DC precursors. Identification of DCs on the basis of single

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parameters is thus unreliable and the demonstration of potent primary APC activity should be paramount. Criteria such as distinctive enzyme cytochemistry, dendritiform morphology, and the presence of DCassociated antibodies such as CMRF44 or HBlSa may be regarded as confirmatory, but should not be used alone to define DCs. The interrelationship of lymphoid and nonlymphoid DCs (Fig. 1) has been inferred principally from the behavior of murine Langerhans cells in uitro and in uiuo (Rowden et al., 1992), and from the homing of DCs when injected into recipients. DCs move from the bone marrow (BM) via the blood to become resident in tissues. Following antigen acquisition, they migrate to the lymph nodes as veiled cells (Larsen et al., 1990b). Freshly isolated LCs are initially capable of acquiring antigen and processing it in uitro (or in uiuo during contact sensitization), but are weak stimulators of primary T-cell responses. They rapidly become incapable of antigen acquisition and processing, yet become powerful stimulators of primary antigen-specific responses. During this process many of the characteristic features of LCs are altered (Romani et al., 1989; Stossel et al., 1990), as discussed in Section 111,A,2. Antigen-bearing DCs derived from the tissues subsequently migrate centrally to the draining lymph node to become resident antigen-bearing interdigitating DCs (IDCs) capable of initiating a central T-lymphocyte response (Larsen et al., 1990b). DCs are thus part of a sophisticated mechanism for immunological surveillance. They are probably also capable of initiating and perpetuating a local immune response in situ in the periphery, which might be particularly important in certain autoimmune diseases. DCs are distinctive cells in both morphology and function, but their precise relationship to other myeloid cells is unclear, although it appears likely that these cell types share a common BM progenitor at some early stage. While it has been claimed that DCs derive from classic mature monocytes under certain circumstances, most evidence suggests that DCs are a distinctive cellular population with functional properties different from the majority of Mo/Mps, and diverge at an earlier stage of differentiation in the BM. In the next section we discuss the isolation and properties of individual DC types, and highlight the evidence for the interrelationships among them.

II. Blood and Bone Marrow Dendritic Cells A. Bone Marrow Dendritic Cells

DCs bear the CD45 (leukocyte common) antigen and as such are undoubtedly hemopoietic cells derived from the bone marrow. Observations on

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

the repopulation of DCs in bone marrow transplant recipients also demonstrate this clearly in mice (Frelinger et al., 1979), rats (Hart et al., 1981), and humans (Katz et al., 1979; Murphy et al., 1985). DC precursors are also thought to circulate in peripheral blood pending entry into the tissues. The study of both cell types is therefore of great benefit in understanding DC interrelationships and ontogeny. Putative precursors of LC-type DCs have been identified in human BM (Reid et al., 1990; de Frassinette et al., 1988) and derived in uitro from human cord blood cells (Caux et al., 1992).This is controversial, however, since one of the principal identifying features of these LC precursors was CDla expression. This antigen appears to be widely inducible on both immature and mature myeloid cells. Furthermore, studies of fetal tissue have shown that in situ LCs are initially CDla negative (Foster et al., 1986). CDla is also virtually undetectable in human cord blood or adult blood (Gothelf et al., 1988) and is not found in fresh human blood DCs (Thomas et al., 1993) or BM DCs (Egner et al., 1993a). This is discussed in more detail in Section VI.

B. Blood DC and Dendritic Cell Precursors

1. Isolation Protocols for Blood Dendritic Cells Human peripheral blood DCs were first isolated by Van Voorhis et al. in 1982, using methods based on those for the isolation of murine splenic DCs. DCs are present in very low numbers in blood and are difficult to isolate in quantity. Isolation protocols have exploited the DC characteristics of nonadherence to plastic, lack of Fc receptors, low buoyant density and lack of myeloid, T- or B-lymphocyte-specificsurface markers. Various protocols have used different combinations of techniques to maximize yield, as illustrated in Fig. 2. The techniques for producing DCs that are based mainly on adherence depletion, density gradients, or removal of residual contaminating cells from other lineages are considered in the FIG. 2 Preparation of human blood DCs. The various methods which have been developed for (A) conventional DC preparation and (B) fresh DC isolation are shown here in diagrammatic form. AD, adherent cells; NAD, nonadherent cells; LD, low-density cells; HD, highdensity cells; non-T, non-T cells; C’, complement; LLME, L-leucyl L-leucine 0-methyl ester; Hu Ig, human immunoglobulin; RAM Ig, rabbit antimouse immunoglobulin. Superscript numbers indicate references used. I. Van Voorhis ef al., 1982; 2. Kuntz-Crow and Kunkel, 1982; 3. Van Voorhis er al., 1983; 4. Knight er al., 1986; 5. Vakkila el al., 1987; 6. Young and Steinman, 1988; 7. Brooks and Moore, 1988; 8. Freudenthal and Steinman, 1990; 9. Xu ef al., 1992; 10. Karhumaki er al., 1993; I I. Thomas ef al., 1993; 12. Markowicz and Engleman. 1990. 13. Egner er al., 1993c.

A PBMC

PBMC

l 6 h r culture

I

16hr culture

-1 carbonyl

gradimt

panning

I

Dc

(non-adherent)

1

50 following paragraphs. Other methods for obtaining less-activated DC preparations are also discussed. a. Adherence Depletion The original techniques for adherence depletion used differential adherence of peripheral blood mononuclear cells (PBMCs) to plastic surfaces (Van Voorhis et al., 1982; Kuntz-Crow and Kunkel, 1982). DCs were transiently adherent during short incubations (60 min), became nonadherent after overnight culture, and could be enriched by readherence cycles, which depleted firmly adherent Mo/Mps. Subsequent separation into low-density (DCs) and high-density fractions (B cells) over a bovine serum albumin (BSA) gradient and a further cycle of adherence enriched the DCs to 20-60%. Depletion of T and B cells using mAb and complement increased this to 70-78% (Van Voorhis et al., 1983). A similar method was followed by Kuntz-Crow and Kunkel(l982) using repeated adherence cycles, but the density gradient was omitted. Nonadherent Mo/Mps were later depleted from nonphagocytic DCs by ingestion of carbonyl iron and magnetic removal. Subsequent rosetting with immunoglobulin-sensitized ox red blood cells (RBC) removed FcRpositive Mo/Mp and B cells, the latter being further depleted by panning with antihuman immunoglobulin. Purities of greater than 90% were claimed on the basis of class I1 positivity and lack of staining for myeloid intracellular enzymes. All of these initial studies estimated the frequency of DCs in the initial PBMC population to be 0.1-1.0% and consistently monitored potent allostimulatory activity in the transiently adherent, lowdensity, FcR fraction. Human DC adherence differs significantly from that of murine DCs. In man, active allostimulatory cells can be detected in both nonadherent and adherent fractions after short (90-min) incubations, which are sufficient to separate murine DCs (Knight et al., 1986). Early work in our own laboratory led us to similar conclusions (Hart and Calder, 1993). Recent work with purified blood DCs also demonstrates considerable adherence in a subpopulation of human DCs (Thomas et al., 1993).These differences increase the difficulty of obtaining purified human DCs, particularly since transient weak adherence is one of the cornerstones of DC purification in the mouse model. Teflon cultureware (which is assumed to preclude adhesion of all but the most strongly adherent macrophages) has been used by some workers (Markowicz and Engleman, 1990; Xu et al., 1992) in the light of growing uncertainty whether adherence alters the phenotype or activity of DC populations. b. Density-Gradient Separations Most DCs, particularly those isolated from solid organs, are of low buoyant density. Careful use of sophisticated

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density gradients has enabled purification of DC from low-density Mo/Mps. These physical separations may isolate only a portion of the DCs and are heavily dependent on density changes induced by a period of 37°C incubation (Youngand Steinman, 1988;Xu et al., 1992;Markowicz and Engleman, 1990).DC precursors in particular may be of higher density than other DCs and density gradients may be less efficient in their isolation. Culture times have been increased to 36 hr to improve separation (Young and Steinman, 1988; Freudenthal and Steinman, 1990; Markowicz and Engleman, 1990;Xu et al., 1992),or omitted altogether to avoid phenotypic changes which may occur in uitro (Karhumaki et al., 1993; Egner et al., 1993b; Thomas et al., 1993). Various gradient media are used but the original BSA gradient (Van Voorhis et al., 1982) has generally been replaced by metrizamide (Knight et al., 1986; Brooks and Moore, 1988; Freudenthal and Steinman, 1990) or Percoll (Young and Steinman, 1988; Xu et al., 1992; Vakkila et al., 1987; Markowicz and Engleman, 1990). Markowicz and Engleman (1990) substituted a new and complex 4-step discontinuous Percoll gradient system and omitted plastic adherence. We have found a Nycodenz gradient to be useful as a final purification step for removing residual contaminating small lymphoid cells (Hart et al., 1993). The gradient media basically perform similar functions, although there is growing concern about the phenotypic or functional changes which cells may undergo when exposed to these materials. For example, Kabel et al. (1989) have suggested that interaction with metrizamide may alter monocytoid cells so that they resemble DCs. Density-gradient separation may be unreliable as a primary method of isolating human DCs for the reasons outlined earlier. Using metrizamide for example, enrichment varied between 2 and 78% (Knight et al., 1986), prompting attempts by some workers to remove density gradients from their protocols altogether. c. Removal of Contaminating Cells i. Panning Techniques Nonadherent surface immunoglobulin (sIg+) and FcR+ cells can be removed by panning (Brooks and Moore, 1988). This has been extensively used prior to, or following density-gradient separation (Freudenthal and Steinman, 1990, Vakkila et af., 1987; Xu et af., 1992; Young and Steinman, 1988). There is also a potential problem with anti-aFcR panning because some DCs express low amounts of FcRyII (CD32) and FcRyI (CD64). CD45RA panning as used by Freudenthal and Steinman (1990) is not widely applied and is based on the unconfirmed observation that DCs lack a particular CD45 epitope (Wood et al., 1991). ii. Depletion ofPhagocytes This is accomplished by the use of carbony1 iron (Kuntz-Crow and Kunkel, 1982; Vakkila et d., 1987) or L-

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

leucyl L-leucine 0-methyl ester (LLME) (Thomas et al., 1993). LLME is metabolized to a toxic by-product and has been used to deplete esterasepositive natural killer (NK) as well as monocytoid cells (Thomas et al., 1993). While the resulting DCs are clearly similar to those prepared by conventional techniques, the functional effects that LLME will have on DCs remain to be determined, as this compound is clearly toxic to LCs (Simon et al., 1992). iii. Zmmunophenotypic Cell Separations Negative selection using an mAb cocktail to remove unwanted cell populations has latterly become a mainstay of DC preparation protocols, although not all studies have demonstrated that these mAbs do not react with the stimulatory cells, a procedure which is in our view absolutely essential (Egner et al., 1993b; Thomas et al., 1993). Depletion is either accomplished by flow cytometry (Freudenthal and Steinman, 1990) where purity is high but yields often low, or by immunomagneticbead depletion (Karhumakiet al., 1993)where yields may be improved and purity approaches that of flow cytometrybased methods. Both of these methods are routinely used in our laboratory with considerable success (Egner et al., 1993b; Hart et al., 1993). d. Isolution ofFresh Dendritic Cells Conventional methods of DC preparation rely on the use of extended culture periods to allow density and adherence changes which enable DCs to be separated from other myeloid cells (Young and Steinman, 1988; Xu et al., 1992; Markowicz and Engleman, 1990). Phenotypic and functional changes may arise as a result, perhaps inducing DC activation. This may make examination of the unactivated DC or DC precursors impossible by these methods (Egner et al., 1993b; Thomas et al., 1993). We have developed an isolation protocol for human blood and BM DCs which enriches for minimally manipulated DCs (Egner et al., 1993a,b) (Figure 2). Processing the PBMC at 4°C throughout the isolation procedure, we deplete T lymphocytes by rosetting with sheep red blood cells (SRBCs), and negatively select by flow cytometry using a mixture of mAbs. Each mAb has been tested individually to confirm that the allostimulatory activity resides in the mAb-negative fraction. Prior depletion with immunomagnetic beads reduces sorting time and can improve yields. These populations of putative DCs are minimally activated, and provide the best example of a constitutive blood DC phenotype to date. Recent data (Hart et al., 1993; Thomas et al., 1993) confirm that these cells have phenotypic and morphologicaldifferences from their counterparts isolated by conventional methods involving prolonged culture. Fresh DCs can acquire an activated immunophenotype upon culture, including the expression of BBl/B7 (Hart et al., 1993; Thomas et al., 1993), or activation and interdigitating cell markers such as CMRF44 and HB15a (G. Starling,

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personal communication). Adhesion molecule expression may also be altered (Thomas et al., 1993; Young et al., 1992).

2. Properties of Human Blood Dendritic Cells Human blood DCs are assumed to be precursors of tissue DCs (Fig. 1) but direct evidence for this is lacking in man (see Section V for detailed discussion). They are clearly potent allostimulatory cells in contrast to other lineages (Van Voorhis et al., 1982; Kuntz-Crow and Kunkel, 1982; Thomas et al., 1993)and present specific antigen in primary systems (Hart and McKenzie, 1988;Thomas et al., 1993).DC-enriched populations were 10-100-fold more efficient than other cell populations and induced significant T-cell proliferation at stimulator/responder ratios as low as 1 : 100. Unlike murine DCs, human blood DCs appear to display low levels of CD13, CD14, and CD33 as detected by sensitive flow cytometry (Thomas et al., 1993; Egner et al., 1994). Detection of these antigens may be quite epitope dependent and some may not be detected by less sensitive techniques such as immunoperoxidase staining. Blood DCs undergo maturational activation changes similar to other DCs upon in uitro culture. As a result, they phenotypically converge with tissue DCs (Hart et al., 1993; Thomas et al., 1993) (Fig. 2). BBl/B7 expression on human blood DC is induced by cellular activation during isolation rather than being constitutively expressed (Hart et al., 1993). Murine organ allograft experiments demonstrate that some DCs can recirculate from nonlymphoid tissue to lymphoid organs via the bloodstream (Larsen et al., 1990a). It is therefore possible that human blood may also contain recirculating DC subsets. There is no direct evidence for this at present, however, and allograft DC behavior may not parallel that of autologous DCs. Human tonsil DCs appear to express lower levels of CD43 (leukosialin) than most Mo/Mps (Egner et al., 1993c), and blood allostimulatory cells show heterogeneous CD43 expression. However, CD43 detection may be epitope and mAb dependent; therefore any attempts to relate CD43 expression to potential DC subsets or activation or differentiation state requires further study.

111. Nonlymphoid and Interstitial Dendritic Cells

Highly MHC class I1 positive cells with morphological similarities to the DCs have been identified in most human tissues (Daar et al., 1983; Hancock and Atkins, 1984; Hart et d.,1989). These cells probably develop from precursor DCs in the blood and BM. DCs from different tissues

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LISA A. WILLIAMS, WILLIAM EGNER, AND DEREK N. J. HART

display many similarities and are collectively known as interstitial DCs. The most extensively studied tissue DC is the Langerhans cell and relatively few studies have been undertaken on DCs from other tissues. A. Langerhans Cells

Epidermal LCs, initially thought to be part of the nervous system, are an important part of the skin immune system (Katz et al., 1979; Frelinger et al., 1979).Skin transplantation and BM reconstitution experiments in mice have shown that these cells are not a static component of the skin, but derive from BM precursors and bear the surface MHC molecules of the donor animal (Katz et al., 1979; Frelinger et al., 1979). They also seem to be subject to neuroendocrine control and are intimately associated with nerve endings (Hosoi et al., 1993). LCs express high-density MHC class I1 antigens, CDla, and possess typical stellate morphology in situ (Davis et al., 1988; Takezaki et al., 1982; Rowden et af., 1977). CDla is an MHC class I-like molecule which is useful for the immunohistological detection of LCs. CDla may have an MHC class I-like function in human LC antigen presentation (Moulon et al., 1991). It is interesting that no other “mature” member of the DC family has been shown to express this molecule, with the possible exception of thymic DCs. Early mAb studies revealed an extensive suprabasal network of epidermal LCs, with branched cytoplasmic processes extending threedimensionally to make physical contact with large numbers of adjacent LCs. Another distinctive feature of LCs is the presence of intracytoplasmic Birbeck granules of uncertain function but which are believed to be associated with endocytosis and phagocytosis (Romani and Schuler, 1992). LCs have proved invaluable as a model to investigate the phagocytic and antigen-processingcapabilities of DCs at different stages of differentiation. This is discussed further in Section III,A,2. CDla-positive LCs are also found deeper in the dermis (Davis et al., 1988) but have not been extensively studied. They may represent LCs that are moving to the afferent lymphatic system. 1. Isolation of Human Langerhans Cells Isolation of DCs from solid organs is more difficult than from blood. LCs have provided an important model for the study of interstitial DCs, and also provide the bulk of the evidence for the migration and differentiation pathway of resident tissue DCs. Most of this work has been done in the

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mouse but an increasing number of human studies suggest that the two systems are very similar. To prepare LCs (Fig. 31, epidermal sheets are separated from the dermis of skin sections (usually operative specimens in humans) by short (