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Cytogenetic analysis of single hemopoietic colonies Dubé, Ian David 1980

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CYTOGENETIC ANALYSIS OF SINGLE HEMOPOIETIC COLONIES by ,% IAN DAVID DUBE •Sc., The University of Br i t i s h Columbia, 1977 A THESIS SUBMITTED IN. PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF in THE FACULTY OF GRADUATE STUDIES Department of Medical Genetics We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1980 MASTER OF SCIENCE Ian David Dube, 1980 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of t h e r e q u i r e m e n t s for a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y of B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n for e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t n f Medical Genetics T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e September 10, 1980 - i i -ABSTRACT Cytogenetic a n a l y s i s of f r e s h marrow provides an assessment pr i m a r i l y of the d i s t r i b u t i o n of karyotypes i n the most d i f f e r e n t i a t e d compartments of p r o l i f e r a t i n g hemopoietic c e l l s . In theory, i t should be possible to obtain s i m i l a r information for more p r i m i t i v e c e l l types since, under appropriate conditions i n vitvo, these are stimulated to form colonies o f i d e n t i f i a b l e progeny. Although the importance of such an approach, p a r t i c u l a r l y i n studies of the hemopoietic malignancies, has been recognized f o r a number of years, colonies of more than 1 , 0 0 0 c e l l s are r a r e l y obtained, and severe t e c h n i c a l problems have hampered the a c q u i s i t i o n of r e l i a b l e data. In t h i s work, a microtechnique has been developed which enables high q u a l i t y chromosomes (suitable f o r G- or Q-banding) to be obtained from sin g l e e r y t h r o i d colonies grown from human blood or bone marrow i n standard methylcellulose c u l t u r e s . Thus, i t i s now p o s s i b l e to begin an analysis of chromosomal changes i n the progeny of i n d i v i d u a l e r y t h r o i d stem c e l l s . This provides a new approach to the cytogenetic study of the o r i g i n and progression of the human m y e l o p r o l i f e r a t i v e diseases. - i i i -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x INTRODUCTION 1. Chromosomal Involvement i n Cancer 1 a. Chromosomally Normal In d i v i d u a l s — Marker Chromosomes 2 and Karyotype Evolution as Features of Malignancy b. Chromosomally Normal In d i v i d u a l s — Association of 4 S p e c i f i c Chromosome Abnormalities With S p e c i f i c Malignancies c. Chromosomally Abnormal I n d i v i d u a l s 6 d. P r a c t i c a l Implications 8 2. The M y e l o p r o l i f e r a t i v e Disorders 9 a. Time Course 9 b. Single .Cell O r i g i n 10 c. Chromosome Studies 11 d. The MPD as Malignant Conditions 11 ; 3. The Hemopoietic System 12 a. Colony Assays For P r i m i t i v e C e l l Types 12 b. In Vitro Assays For Hemopoietic Progenitors 15 4. A p p l i c a t i o n of Chromosomal Analysis to Single Hemopoietic 17 Colonies a. Chronic Myeloid Leukemia (CML) 17 b. Polycythemia Vera (PV) 18 c. Present Objective 19 - i v -TABLE OF CONTENTS Page MATERIALS AND METHODS 1. Patient Specimens 21 2. Specimen Preparation 21 3. Chromosome Harvest 25 4. Chromosome Banding 28 5. D i f f e r e n t i a l S t a i n i n g of S i s t e r Chromatids 28 a. Rationale 28 b. Technique 29 i . Bone Marrow 29 i i . E r y t h r o p o i e t i c Colonies 30 i i i . D i f f e r e n t i a l Staining 30 RESULTS 1. Time Course Studies a. C e l l Recovery b. M i t o t i c Index i . Pooled Colonies i i . S ingle Colonies 2. S i s t e r Chromatid Exchange Studies a. Fresh Marrow b. E r y t h r o i d Colonies 3. Proof of the Single C e l l O r i g i n of Plucked Colonies 4. Karyotypes From Single Colonies 32 32 35 37 40 41 41 I n d i v i d u a l l y 47 49 TABLE OF CONTENTS DISCUSSION CONCLUSION REFERENCES APPENDIX 1A Procedure f o r the Preparation of Hemopoietic Stem C e l l s for P l a t i n g From Fresh Bone Marrow Aspirate APPENDIX IB Procedure f or the Preparation of Hemopoietic Stem C e l l s for P l a t i n g From Peripheral Blood APPENDIX IC Procedure f or the Preparation of the Culture Medium for Hemopoietic Stem C e l l s - v i -LIST OF TABLES Page Table I. S p e c i f i c Stimulating Factors for Myeloid 16 C e l l Lineages. Table I I . P a t i e n t Data. 22-24 Table I I I . Percentage of Recovered C e l l s i n Metaphase i n 36 Pooled E r y t h r o i d Colonies Harvested on D i f f e r e n t Days of Culture. Six Colonies Were Pooled to Give Each Value. Table IV. Average Number of S i s t e r Chromatid Exchanges 46 Per C e l l i n Pooled E r y t h r o i d Colonies From Four I n d i v i d u a l s . Table V. D i s t r i b u t i o n of Fluorescent Y-Body i n I n d i v i d u a l 48 Colonies Plucked From Dishes i n Which Equal Numbers of Male and Female C e l l s Were Plated. Table VI. Average Number of Analyzable Metaphases Per Colony f o r Six I n d i v i d u a l s . Ten Colonies Were Plucked i n Each Case and Q- or G-banded. 50 - v i i -LIST OF FIGURES Figure 1. The hemopoietic system.. A schematic i l l u s t r a t i o n of stem c e l l d i f f e r e n t i a t i o n showing lymphoid and myeloid development from a common p l u r i p o t e n t stem c e l l . Page 13 Figure 2. Colony plucking. Removal of a s i n g l e hemopoietic colony (estimated s i z e 1,500 c e l l s ) using a f i n e l y drawn out Pasteur p i p e t t e . 27 Figure 3. T o t a l recovery of c e l l s from a s i n g l e colony a f t e r hypotonic treatment, f i x a t i o n , and two washes, shown as a function of the day of colony harvest. (a) Cultures i n i t i a t e d from p e r i p h e r a l blood c e l l s from ID. (b) Cultures i n i t i a t e d from p e r i p h e r a l blood c e l l s from KH. 33/34 33 34 Figure 4. Percentage of recovered c e l l s i n metaphase. Ten colonies were plucked on each day for Days 9 to 13 of incubation. Also shown are the absolute number of metaphases observed per colony. (a) Cultures i n i t i a t e d from bone marrow c e l l s from AJ. (b) Cultures i n i t i a t e d from bone marrow c e l l s from HS. 38/39 38 39 Figure 5. C e l l cycle k i n e t i c s evaluation using 5BrdU incorporation and the d i f f e r e n t i a l s t a i n i n g technique. Metaphases a f t e r (a) one, (b) two, and (c) three syntheses i n 5BrdU. A l l three metaphases were taken from one bone marrow cult u r e from an i n d i v i d u a l with Bloom syndrome. 42/43 Figure 6. The number of c e l l s i n f i r s t , second, and t h i r d metaphases a f t e r various lengths of time i n incubation i n 50 UM 5BrdU. (a) Cultures i n i t i a t e d from bone marrow c e l l s from NF. (b) Cultures i n i t i a t e d from bone marrow c e l l s from HS. 44/45 44 45 - v i i i -LIST OF FIGURES Page Figure 7. Q-banded male karyotype obtained from a s i n g l e 51 e r y t h r o i d colony grown from p e r i p h e r a l blood c e l l s from WH. Figure 8. Q-banded female karyotype obtained from a s i n g l e 52 e r y t h r o i d colony grown from bone marrow c e l l s from EM. Figure 9. G-banded male karyotype obtained from a s i n g l e 53 e r y t h r o i d colony grown from bone marrow c e l l s from WS. Figure 10. G-banded female karyotype obtained from a s i n g l e e r y t h r o i d colony grown from p e r i p h e r a l blood c e l l s from EM. 54 - i x -ACKNOWLEDGEMENTS I would l i k e to express my sincer e . a p p r e c i a t i o n to the chairman of my t h e s i s committee, Dr. Connie Eaves (Medical Genetics), for her constant support and guidance during the past year. I would l i k e to thank the other members of my t h e s i s committee, Dr. Fred D i l l (Medical Genetics), Dr. P a t r i c i a Baird (Medical Genetics, Departmental Head), Dr. Tom G r i g l i a t t i (Zoology), and Dr. Tony G r i f f i t h s (Botany), for t h e i r guidance with t h i s work. I would also l i k e to thank Dr. James M i l l e r (Medical Genetics) f o r h i s c r i t i c a l reading of t h i s manuscript and for hi s help with the organization of my t h e s i s committees. I would espe-c i a l l y l i k e to thank Dr. Fred D i l l f o r providi n g me with the cytogenetic t r a i n i n g that made t h i s work possib l e , and Dr. Keith Humphries f or our stimulating d i s c u s s i o n s . I wish to thank the members of the B r i t i s h Columbia Cancer Research Centre for providing an environment i n which i t was t r u l y a pleasure to work. I am e s p e c i a l l y g r a t e f u l to Dr.. A l l e n Eaves f o r h i s help i n obtaining the patient materials, and to Dianne Reid, Cam Smith, and Ann Marie MacDougall, for t h e i r expert t e c h n i c a l assistance. I would a l s o l i k e to thank Sharon Masui and the s t a f f at the Cytogenetics Laboratory at the Vancouver General Hospital for t h e i r h e l p f u l suggestions, and Sharon B e l l f o r her p a t i e n t and expert a s s i s t a n c e . i n the preparation of t h i s manuscript. INTRODUCTION 1. Chromosomal Involvement i n Cancer Attention was f i r s t drawn to the s i g n i f i c a n c e of chromosomal involvement i n the transformation of c e l l s from a normal to a malignant state i n 1914 by Theodor Boveri (1). Today, the r o l e of chromosomal changes i n malignancy remains unclear. Advances i n human cytogenetics during the l a s t 20 years have not resolved the issue. We now know that the great majority of human cancers e x h i b i t v i s i b l e chromosome changes arid that every chromosome i n the human comple-ment has been described as being involved i n one or another type of karyo-ty p i c anomaly (tra n s l o c a t i o n , d e l e t i o n , d u p l i c a t i o n , trisomy, monosomy, etc.) i n human cancer (for a review, see Ref. 2). Some chromosomal changes are involved more frequently than others but the pr e c i s e r e l a t i o n -ship of these changes to the development of malignancy i s not known. In pr a c t i c e , i t has usu a l l y been impossible to obtain chromosome preparations from many tumor samples. In add i t i o n , the q u a l i t y of those preparations obtained has often been poor because of the condensed and fuzzy nature of the chromosomes i n many cases. In view of these t e c h n i c a l problems, i t i s not s u r p r i s i n g that the a p p l i c a t i o n of cytogenetic f i n d i n g s to the diagnosis and therapy of human cancers has not been f u l l y explored. There are a few human cancers with a c l e a r l y demonstrated and consistent chromosomal involvement. Investigators have emphasized these i n t h e i r attempts to answer a key question about the r o l e of chromosomes i n human cancer: i . e . whether chromosomal changes are causal or conse-quential to malignant transformation. -1--2-a. Chromosomally Normal I n d i v i d u a l s — Marker Chromosomes and Karyotype  Evolution as Features of Malignancy Most human tumors show de v i a t i o n s from the normal d i p l o i d number of chromosomes (2). In general, two s t r i k i n g features of tumor c e l l s are the presence of marker chromosomes, and a tendency towards a s p e c i f i c pattern of karyotype e v o l u t i o n . Chromosomal markers are the r e s u l t of rearrangements, which give r i s e to new chromosomes. In some cases, the marker chromosome can be i d e n t i f i e d i n terms of the chromosomes that give r i s e to i t , but i n others t h i s i s not p o s s i b l e . Markers are very commonly present i n tumors with hypodiploid chromosome numbers. Their frequency i s lower i n hyper-d i p l o i d and d i p l o i d tumors (2). In one study, markers were present i n each of 11 hypodiploid ovarian carcinomas, and i n 18 out of 21 with modal numbers of 46 or more; they were present i n each of 12 hypodiploid c e r v i c a l carcinomas but i n only eight of 18 tumors with modes of 46 or more (3). In a more recent report, karyotypic a n a l y s i s of 189 cases of carcinomas of the bladder revealed that there were marker chromosomes i n 65 out of 85 preparations that y i e l d e d analyzable metaphases (4). Karyotype evolution, a stepwise rearrangement of the karyotype occurring i n an apparently ordered fashion, i s a second feature of tumor karyotypes. Each i n d i v i d u a l tumor appears to vary with respect to the d i r e c t i o n and extent of any change i n karyotype. The tendency, however, may be c l e a r when the average extent to which chromosomes of each type are under- or overrepresented i n a s e r i e s of tumors i s c a l c u l a t e d (2). An analysis of the "chromosomal data obtained i n a s e r i e s of p a t i e n t s with chronic myeloid leukemia (CML), who also expressed a s p e c i f i c marker, the P h i l a d e l p h i a chromosome (Ph"1-) , suggested that the c l i n i c a l progression of CML i s u s u a l l y accompanied by concomitant progression of the karyo-t y p i c p i c t u r e , and that the l a t t e r may r e f l e c t the prognostic aspects of the disease (5). The Ph^-positive c o n d i t i o n i n which the Ph^ i s the only karyotypic aberration appears to be an e a r l y manifestation of CML. C l i n i c a l progression of the chronic phase of Ph^-positive CML i s accom-panied by gradual progression of the cytogenetic p i c t u r e , e s p e c i a l l y i n the more terminal b l a s t i c stage of the disease (6,7). Meningiomas provide a group of tumors i n which the phenomenon of karyotypic evolution has been r e l a t i v e l y w e l l studied. In h i s summary of the cytogenetic findings i n over 200 meningiomas, Sandberg (2) suggests that the i n i t i a l step i s u s u a l l y the l o s s or d e l e t i o n of a #22, and the subsequent steps a f f e c t chromosomes #8 and #9 i n group C ( i . e . #6 to #12) and #14 and #15 i n group D ( i . e . #13 to #15), i n p a r t i c u l a r , and also #1. A p o s s i b l e explanation for the evolution of karyotypic changes i n menin-gioma could be the following: Normal d i p l o i d stem l i n e ( a l l karyotypes normal) appearance of a new v a r i a n t stem l i n e with a growth advantage :and showing monosomy f o r chromosome #22 (mixture of normal karyotypes ;and karyotypes with monosomy 22 ->- increase i n the r e l a t i v e proportion of c e l l s with monosomy 22) -*- subsequent karyotypic deviations with accompanying increase i n growth advantage (mixture of monosomy 22 with and without a d d i t i o n a l chromosomal changes). b. Chromosomally Normal Indiv i d u a l s — Association of S p e c i f i c Chromosome  Abnormalities With S p e c i f i c Malignancies Chronic myeloid leukemia (CML), B u r k i t t lymphoma, meningioma, and retinoblastoma, are diseases i n which s p e c i f i c chromosomal aberrations have been described i n the malignant c e l l s . CML i s considered to represent a neoplastic disease of the marrow i n which the major c l i n i c a l manifesta-tions r e l a t e to abnormal, excessive, and apparently unrestrained overpro-duction of granulocytes. A more d e t a i l e d discussion of t h i s disease i s given below (section 2) . The P h i l a d e l p h i a chromosome (Ph"*") , f i r s t described by Nowell and Hungerford i n 1960 (8), i s formed by a t r a n s l o c a t i o n i n v o l v i n g the long arm of chromosome #22 (9). The majority (>80%) of CML patients have a hemopoietic c e l l l i n e which expresses the Ph"'". In the vast majority of cases, a l l other body t i s s u e s examined have turned out to be chromo-somally normal. In no other known neoplastic condition i s there a stronger case for the d i r e c t involvement of chromosomal change i n the development of the malignant state. This i s based on the consistency with which the Ph^ i s found i n the involved c e l l s very e a r l y i n the disease and, generally, i t s persistence throughout i t s course. In B u r k i t t lymphoma, n e o p l a s t i c c e l l s of lymphoreticular d e r i v a t i o n occur i n s o l i d tumors predominantly i n the f a c i a l (African type) or abdominal (non-African type) regions. In t h i s malignancy, there i s e v i -dence for a s p e c i f i c chromosomal defect i n the malignant c e l l s . In the most recent cytogenetic study of the non-African type of B u r k i t t lymphoma, a p a r t i c u l a r t r a n s l o c a t i o n between chromosomes #8 and #14 was found repeatedly (10). The t r a n s l o c a t i o n was found i n d i r e c t preparations of tumor material, or i n c e l l l i n e s derived from tumor c e l l s , i n 12 out of 18 unrelated p a t i e n t s . The same t r a n s l o c a t i o n has been frequently found i n -5-s i m i l a r preparations from the A f r i c a n type of B u r k i t t lymphoma (11). Meningiomas are tumors composed of s p e c i a l i z e d arachnoidal l i n i n g c e l l s . Chromosomal preparations of d i r e c t or cultured tumor material from over 200 meningiomas i n d i c a t e that these tumors have a remarkable tendency to develop hypodiploid stem l i n e s . The i n i t i a l step i s u s u a l l y a l o s s or del e t i o n of chromosome #22 (2). Retinoblastoma i s a malignant eye tumor of c h i l d r e n which i s gene-t i c a l l y determined i n some cases. One cytogenetic study of f i v e p a t i e n t s with nonhereditary retinoblastoma showed normal karyotypes i n mitoses from PHA stimulated p e r i p h e r a l blood, but revealed a s p e c i f i c chromosomal abnor-mality i n c e l l s derived from the tumor. Hashem and K h a l i f a (12) reported that i n a l l f i v e cases, abnormal chromosomes were discovered i n some of the mitoses prepared d i r e c t l y from the tumor c e l l s . In four of the f i v e patients, a consistent abnormality was the d e l e t i o n of the long arm of one of the D group chromosomes ( i . e . #13, #14, or #15). Recently, a more d e t a i l e d cytogenetic examination of tumor t i s s u e i n a patient with b i l a t e r a l retinoblastoma and chromosomally normal lymphocytes was performed. In t h i s case, Balaban-Malenbaum (in preparation and reported i n Ref. 13), found a small d e l e t i o n i n the long arm of chromosome #13. Apparently, the missing material was t r a n s l o c a t e d onto chromosome #20. These four examples (Ph^-positive. CML, B u r k i t t lymphoma, meningioma, and nonhereditary retinoblastoma) are the best examples, to.date, of human neoplastic, conditions i n which the malignant c e l l s have c h a r a c t e r i s t i c chromosomal markers. Such s p e c i f i c i t y has also been demonstrated i n murine thymomas. Chan and h i s colleagues used chromosome banding techniques to study the karyotype of tumor c e l l s from thymic lymphomas induced by three d i f f e r e n t carcinogens: X - i r r a d i a t i o n , p o l y c y c l i c aromatic hydrocarbons, and endogenous leukemogenic v i r u s (14). Newborn mice of two d i f f e r e n t s t r a i n s were used. Of a t o t a l of 89 tumors studied, 76 (85%) were c h a r a c t e r i z e d by trisomy for chromosome #15 (Thymus, spleen, and bone marrow c e l l s were k a r y o t y p i c a l l y normal i n these animals.) The remaining 13 tumors were k a r y o t y p i c a l l y normal. The r e s u l t s obtained were independent of the carcinogenic agents and the s t r a i n of mouse used. In a more recent report, cytogenetic studies on T c e l l lymphomas induced i n mice carrying t r a n s l o c a t i o n chromosomes in v o l v i n g centromeric fusion of chromosome #15 with chromosomes #1, #5, or #6, also suggested that d u p l i c a t i o n of the gene(s) located on chromosome #15 i s of c r i t i c a l impor-tance (15). Thus, i n these Robertsonian t r a n s l o c a t i o n s , even large chromo-somes such as #1 were forced i n t o a state of trisomy along with chromosome #15 i n a s i g n i f i c a n t number of cases. These five examples of conditions in which unique chromosomal defects appear to be associated with particular malignancies in otherwise chromo-somally normal individuals have been invoked as support for the hypothesis that a specific alteration in the genome, observable as a chromosomal change3 may be a primary event in transformation. c. Chromosomally Abnormal Indi v i d u a l s Hereditary r e n a l c e l l carcinoma, retinoblastoma, and Wilms tumor are examples of diseases i n which i n d i v i d u a l s with a s p e c i f i c germ c e l l mutation v i s i b l e at the chromosomal l e v e l i n a l l c e l l s , are predisposed to a s p e c i f i c type of malignancy. In addition, i n d i v i d u a l s with Down syndrome or any of the chromosomal breakage syndromes are predisposed to develop malignancies of a more general nature. Hereditary renal c e l l carcinoma i s a malignancy of the kidney. In a unique report, Cohen and h i s associates described a family i n which members with an i n h e r i t e d chromosomal t r a i t were predisposed to renal cancer (16). -7-An apparently balanced t r a n s l o c a t i o n between chromosomes #3 and #8 was found i n p e r i p h e r a l leukocytes i n a l l eight of the p a t i e n t s with r e n a l cancer who were karyotyped. No family member with a normal karyotype had renal cancer. The t r a n s l o c a t i o n was transmitted to approximately one h a l f of the l i v i n g o f f s p r i n g i n three generations. The development of r e n a l cancer i n such heterozygous persons with the t r a n s l o c a t i o n followed an autosomal dominant pattern of i n h e r i t a n c e . In the previous section, the f i n d i n g of a d e l e t i o n of the long arm of chromosome #13 i n tumor c e l l s from chromosomally normal i n d i v i d u a l s with retinoblastoma was noted. I n t e r e s t i n g l y , i n a minority of retinoblastoma patients, cytogenetic analysis of p e r i p h e r a l leukocytes and f i b r o b l a s t s has revealed i n these c e l l s a l s o the d e l e t i o n of a small but s p e c i f i c segment of the long arm of chromosome #13 (2,17). This f i n d i n g has l e d some authors to suggest that a p a r t i c u l a r region on chromosome #13 may c a r r y the dominant mutation i n some instances of f a m i l i a l retinoblastoma. (18), and that the genes involved at the somatic l e v e l are the same as those involved at the germ c e l l l e v e l (13). Wilms tumor i s a kidney cancer that a f f e c t s c h i l d r e n . I t i s sometimes associated with a n i r i d i a (lack of i r i s e s ) , and i n these cases, a consistent chromosomal abnormality has been reported. There are at l e a s t 18 cases i n the l i t e r a t u r e (19) i n which a d e l e t i o n of a s p e c i f i c band i n chromosome #11 has been reported to e x i s t i n nontumor t i s s u e (lymphocytes and/or f i b r o -blasts) , (20; Yunis and Ramsey, i n press and reported i n Ref. 13). These examples of hereditary retinoblastoma, h e r e d i t a r y renal c e l l carcinoma, and Wilms tumor, are the only known instances i n man i n which a s p e c i f i c chromosomal defect occurring p r e z y g o t i c a l l y appears to predispose these subjects to a s p e c i f i c tumor. The chromosomal breakage syndromes comprise a number of rare but d i s t i n c t c l i n i c a l e n t i t i e s i n c l u d i n g Fanconi 1s anemia, Bloom syndrome, ataxia t e l a n g i e c t a s i a , and xeroderma pigmentosum. These g e n e t i c a l l y determined syndromes have i n common an increased tendency to develop malig-nancies i n general (acute leukemia, adenocarcinoma, squamous c e l l carcinoma, etc.) and a pronounced d i s p o s i t i o n to spontaneous chromosomal breakage in vitro while r e t a i n i n g a d i p l o i d set of chromosomes (for a review see Ref. 21). Down syndrome i s a congenital disorder, due to trisomy of chromosome #21. The incidence of both.acute myeloblastic and acute lymphoblastic leukemia i n i n d i v i d u a l s with Down syndrome i s c l e a r l y higher than that observed i n the general population (22). These five examples of congenital predisposition to cancer lend further support to the hypothesis that genetic change may be essential for malignant transformation. They also serve to emphasize the possi-bility that the association of a particular chromosomal abnormality with malignant transformation does not necessarily reveal the primary event(s) that led to the development of a neoplastic state. d. P r a c t i c a l Implications In conclusion, abnormal chromosome c o n s t i t u t i o n s i n tumor t i s s u e are not a rare occurrence. Indeed, although t h e i r s i g n i f i c a n c e remains unknown, they are s u f f i c i e n t l y common to have provided a u s e f u l approach to the i d e n t i f i c a t i o n of c e l l s belonging to the malignant clone. Recently, some progress i n the c u l t u r i n g of c e l l s from human tumor s p e c i -mens has been reported (e.g. Refs. 23 and 24). The use of chromosome - 9 -a n a l y s i s has been p a r t i c u l a r l y important i n assessing the nature of the c e l l s p r o l i f e r a t i n g in vitro (25). Nevertheless, i t must be remembered that although karyotypic change may accompany or even correspond to the primary event i n some cases, i n others (probably the majority) the i n i t i a l mutation may be so small that i t i s not v i s i b l e by conventional cyto-genetic techniques. In such cases, changes v i s i b l e at the chromosomal l e v e l would represent l a t e r events. In t h i s s i t u a t i o n , apparently chromo-somally normal and abnormal members of the same malignant population might coexist, and the u t i l i t y of karyotype a n a l y s i s to d i s t i n g u i s h normal and malignant c e l l s would be correspondingly l i m i t e d . 2. The M y e l o p r o l i f e r a t i v e Disorders A number of hematologic disorders appear to represent neoplastic conditions r e s u l t i n g i n the overproduction of one or more of the c e l l types that .constitute normal human blood. Three such disorders are chronic myeloid leukemia (CML), polycythemia vera (PV), and e s s e n t i a l thrombocytosis (ET). In each case, the c e l l type predominantly involved i n the overproduction i s d i f f e r e n t : granulocytes i n CML, erythrocytes i n PV, p l a t e l e t s i n ET. Since a l l three d i s o r d e r s appear to share a number of b i o l o g i c a l features, they have been grouped together under the term m y e l o p r o l i f e r a t i v e disorders (MPD) (26). a. Time Course A l l three MPD have i n common an e a r l y stage, which may l a s t f o r years, u s u a l l y characterized by a slow, p e r s i s t e n t increase i n the number and s e v e r i t y of symptoms, sometimes accompanied by periods of s t a b i l i t y . -10-During t h i s phase, abnormal p r o l i f e r a t i o n can us u a l l y be held i n check by various forms of therapy. Eventually, i n PV and ET, the disease may progress to an acute leukemic stage, and i n CML, t h i s i s the most common r e s u l t . This stage i s unresponsive to therapy and i s u s u a l l y f a t a l within three to si x months. b. Single C e l l O r i g i n Although the abnormal increase i n c i r c u l a t i n g blood c e l l s i s usually most pronounced f o r a s i n g l e c e l l type ( i . e . red c e l l s i n PV, granulocytes i n CML, p l a t e l e t s i n ET), there i s considerable evidence to indica t e that the primary defect l i e s i n a c e l l capable of gi v i n g r i s e to a l l three c e l l lineages. For example, i n CML the Ph i l a d e l p h i a chromosome (Ph'S has been found i n e r y t h r o i d , granulocytic, and megakaryocytic c e l l s i n i n d i v i d u a l s who are otherwise chromosomally normal (27), even though the predominant malignant c e l l type i s granulocytic. Subsequent studies of Phi^-positive CML pa t i e n t s who were heterozygous for the X-linked G6PD A and B a l l e l e s strongly support the hypothesis that these diseases, l i k e many other neoplasms, a r i s e from a si n g l e c e l l from which a clone of : c e l l s with superior growth or s u r v i v a l p o t e n t i a l p r o l i f e r a t e s (28,29). In addi-t i o n , they have confirmed the notion that t h i s c e l l i s a member of the plu r i p o t e n t stem c e l l compartment. PV has al s o been shown to be of u n i c e l l u l a r o r i g i n using female patients heterozygous for two G6PD a l l e l e s (30) and even though the pre-dominant c e l l type i s e r y t h r o i d , granulocytes and p l a t e l e t s also show a single enzyme marker. This suggests that i n PV, l i k e CML, the primary defect i s i n a sing l e c e l l capable of gi v i n g r i s e to a l l three c e l l types. -11-c. Chromosome Studies There i s a considerable amount of information a v a i l a b l e on the involvement of chromosomal changes i n the MPD. Mitelman estimates that t h i s group represents 70% of the 4,000 human tumors c y t o g e n e t i c a l l y i n v e s t i g a t e d to date (31) . The r o l e of the Ph"*" i n CML has already been mentioned. Up to 26% of untreated PV p a t i e n t s have some chromosomal abnor-mal i t y i n t h e i r bone marrow c e l l s at the time of diagnosis (32). The most common chromosomal abnormalities i n untreated PV appear to involve chromosomes i n group C ( i . e . #6 to #12) with an extra #8 or #9 being the most frequent abnormality (33,34,35). In treated PV, the most common abnormality appears to be a d e l e t i o n of the long arm of chromosome #20 (36). Rare cases of t h i s d e l e t i o n have been encountered i n untreated p a t i e n t s (37) . This chromosomal abnormality may be r e l a t e d to the genesis of PV (2). There i s no s p e c i f i c chromosomal abnormality yet known to be associated with ET. Among nearly 30 cases reported, the majority were found to be c y t o g e n e t i c a l l y normal (2,38,39,40). In a unique•report, Rajendra et a l . have studied one ET p a t i e n t with Ph"*" i n 90% of the bone marrow c e l l s (41). The s i g n i f i c a n c e of t h i s remains unclear. d. The MPD as Malignant Conditions Although the MPD have i n common a prolonged phase during which they can be f a i r l y well c o n t r o l l e d , they have features consistent with t h e i r being considered malignancies. They a r i s e from the transformation of a s i n g l e p l u r i p o t e n t stem c e l l (29,30) g i v i n g r i s e to a continuously -12-expanding clone of c e l l s r e l a t i v e l y i n s e n s i t i v e to normal re g u l a t i o n and capable of suppressing the growth of remaining normal c e l l s . The feature of a chronic stage, which may l a s t up to s i x years before the acute leukemic stage, make the MPD i d e a l model systems for the study of genetic features, i . e . chromosomal changes and a l t e r e d c e l l u l a r behaviour, that are part of an ongoing malignant transformation process. 3. The Hemopoietic System The majority of c e l l s that c i r c u l a t e i n the blood are highly d i f -f e r e n t i a t e d , incapable of p r o l i f e r a t i o n , and r e l a t i v e l y s h o r t - l i v e d (see Figure 1). Current concepts of hemopoiesis favour the existence of a hierarchy of committed progenitor c e l l types for each pathway with pro-g r e s s i v e l y decreasing capacity for p r o l i f e r a t i o n and l i m i t e d , i f any, self-renewal capacity. E r y t h r o i d , megakaryocytic, and granulopoietic progenitors are thought to have, i n turn, a common o r i g i n from a p l u r i -potent stem c e l l which i s capable of self-renewal but r e s t r i c t e d to myelo-p o i e s i s . There i s also now some evidence for the existence of a more "p r i m i t i v e " stem c e l l that can give r i s e to lymphoid as well as myeloid progeny. The experimental data supporting these concepts are discussed below. a. Colony Assays For P r i m i t i v e C e l l Types The f a c t that p r i m i t i v e hemopoietic c e l l s are rare and not morpho-l o g i c a l l y d i s t i n c t has led i n v e s t i g a t o r s to use clonogenic assays to i d e n t i f y them. In 1961, T i l l and McCulloch showed that mouse marrow c e l l s , when i n j e c t e d i n t o h e a v i l y i r r a d i a t e d syngenic mice, were capable Figure 1. The hemopoietic system. A schematic illustration of stem cell differentiation showing lymphoid and myeloid development from a common pluripotent stem c e l l . , -14-of forming d i s c r e t e nodules of hematopoietic c e l l s i n the spleens of these animals eight to ten days a f t e r i n j e c t i o n (42). These nodules were found to contain e r y t h r o i d , granulocytic, megakaryocytic, and undifferen-t i a t e d c e l l s , e i t h e r as pure populations or i n varying mixtures (43). D e f i n i t i v e proof of the c l o n a l nature of these nodules.was provided by studies i n which marrow c e l l s containing s p e c i f i c stable chromosomal markers were i n j e c t e d i n t o i r r a d i a t e d , but otherwise chromosomally normal r e c i p i e n t s . Some colonies showed uniquely abnormal karyotypes i n 95 to 99% of the metaphases (44). That the c e l l s that gave r i s e to these colonies had self-renewal p o t e n t i a l was demonstrated by i n j e c t i n g a sus-pension of c e l l s derived from a spleen colony into another l e t h a l l y i r r a d i a t e d mouse and observing new spleen colonies ten to 14 days l a t e r (45). Using t h i s method, c e l l s i n apparently pure spleen colonies ( i . e . only granulocytic or e r y t h r o i d c e l l s seen) were shown to be capable of giving r i s e to colonies of a l l c e l l types a f t e r r e t r a n s p l a n t a t i o n (46,47). These studies showed that one marrow c e l l could form a colony containing a l l three' myeloid c e l l types, and that t h i s colony f o r m i n g - c e l l could p r o l i f e r -ate and d i f f e r e n t i a t e extensively and undergo self-renewal. Studies by Wu and associates provided support f o r the existence of a p l u r i p o t e n t stem c e l l capable of g i v i n g r i s e to both myeloid and lymphoid c e l l s (48). These i n v e s t i g a t o r s showed that when i r r a d i a t e d mice were transplanted with marrow containing unique radiation-induced chromosomal, markers, the marker chromosomes were subsequently found i n the lymphoid as well as myeloid t i s s u e s . More re c e n t l y , P r c h a l and h i s colleagues reported a case of s i d e r o b l a s t i c anemia i n a human female heterozygous f o r G6PD A and B a l l e l e s who showed the same, s i n g l e isoenzyme in erythrocytes, granulocytes, and macrophages, as well as B and T -15-lymphocytes, suggesting an o r i g i n of both myeloid and lymphoid c e l l s from a s i n g l e c e l l (49). G6PD heterozygosity was shown by the presence of both isoenzymes i n her f i b r o b l a s t s and c e l l u l a r s a l i v a r y l y s a t e s . b. In Vitro Assays For Hemopoietic Progenitors In vitro colony assays have been developed that permit the growth and maturation of c e l l s that give r i s e to erythrocytes, granulocytes, and macrophages ( f o r a review see Ref. 50). The f a c t that these p r i m i t i v e pre-cursors are present at low frequency and do not possess d i s t i n c t morpho-l o g i c a l features precludes t h e i r d i r e c t r e c o g n i t i o n . The development of colonies containing only one c e l l lineage (e.g. e r y t h r o i d c e l l s ) i s dependent on the a d d i t i o n to the culture medium of s p e c i f i c stimulatory f a c t o r s . Such "pure" colonies are thought to represent the c l o n a l descendants of "committed" progenitors since they a r i s e i n cultures i n which more than one colony type i s obtained. (See Table I f o r a l i s t of the colony assays available.) The assays involve suspending hemopoietic c e l l s i n one of various types of s e m i s o l i d media containing the appropriate n u t r i e n t s , serum, and stimulatory f a c t o r s . A f t e r one to two weeks of incubation, e r y t h r o i d colonies can be i d e n t i f i e d by t h e i r red colour, i n d i c a t i v e of ongoing hemoglobin syn-th e s i s i n e r y t h r o i d c e l l s that have reached t h e i r f i n a l stage of d i f f e r e n t i a -t i o n . E r y t h r o i d colonies composed of 16 or more c l u s t e r s of c e l l s , each c l u s t e r containing about 50 c e l l s , are thought to represent the descendants of the most p r i m i t i v e c e l l s which are committed to the e r y t h r o i d lineage (66). The r e l a t i o n between the p r o l i f e r a t i v e capacity of the o r i g i n a l progenitor c e l l and the time taken to generate hemoglobin producing eryth r o b l a s t progeny, suggests that the s i z e of colony formed in vitro i s determined by the state of d i f f e r e n t i a t i o n of the colony forming c e l l in vivo (72). -16-Table I. S p e c i f i c Stimulating Factors f o r Myeloid C e l l Lineages. Colony Type Species Sources of Presumed Stimulating Factors Granulocyte-Macrophage Megakaryocyte Eosinophil Mouse (51,52) Man (55) Mouse (57) Man (59) Mouse (60) Man (56) Small E r y t h r o i d Mouse (61) Man (62) Large E r y t h r o i d Mouse (63,64) Man (66,67) Mixed Mouse (69,70) Man (71) Human urine (53) Mouse f i b r o b l a s t c e l l conditioned medium (54) Human leukocyte conditioned medium (56) Mitogen stimulated mouse spleen c e l l conditioned medium (58) Er y t h r o p o i e t i n from anemic sheep plasma Mitogen stimulated mouse spleen c e l l conditioned medium (58) Human leukocyte conditioned medium (56) Er y t h r o p o i e t i n from anemic sheep plasma E r y t h r o p o i e t i n from anemic sheep plasma E r y t h r o p o i e t i n and mitogen stimulated spleen c e l l conditioned medium (65) Er y t h r o p o i e t i n and human leukocyte conditioned medium (68) Er y t h r o p o i e t i n and mitogen stimulated mouse spleen c e l l conditioned medium Er y t h r o p o i e t i n and mitogen stimulated leukocyte conditioned medium This l i s t i s not meant to be a l l inclusive,- sources named are commonly used examples. Numbers i n brackets i n d i c a t e references. -17-Under i d e a l culture conditions, granulopoietic colonies can also be f a i r l y r e l i a b l y i d e n t i f i e d without f i x a t i o n and s t a i n i n g by v i r t u e of t h e i r c h a r a c t e r i s t i c morphology and lack of red colour (50). Several l i n e s of evidence i n d i c a t e that the colonies are clones derived from single colony forming c e l l s , and that the progenies of these c e l l s are i r r e v e r s i b l y committed to remain i n the same pathway of d i f f e r -e n t i a t i o n . These include the micromanipulation of s i n g l e colony forming c e l l s and the subsequent observation of colonies (73); time-lapse photog-raphy of the developing colonies. (74) ; and, p h y s i c a l i s o l a t i o n of the c e l l s i n the c u l t u r e d i s h by p l a s t i c r i n g s (75). 4. A p p l i c a t i o n of Chromosomal A n a l y s i s to Single Hemopoietic Colonies a. Chronic Myeloid Leukemia (CML) New techniques are making i t e a s i e r to obtain banded karyotypes from human bone marrow (e.g. Ref. 76), but these give no information regarding the state of d i f f e r e n t i a t i o n of the c e l l karyotyped. Based on current concepts of hemopoiesis, one would expect the majority of karyotypes obtained from a bone marrow aspi r a t e to be derived from r e l a -t i v e l y mature erythrocytes. Analysis of c e l l s at t h i s end stage of d i f f e r e n t i a t i o n may give an incomplete p i c t u r e i n the case of the MPD where karyotype evolution i s known to accompany disease progression. The s p e c i f i c i t y of chromosomal abnormalities i n myeloid c e l l s amongst the MPD, and the f a c t that these diseases have i n common a neo-p l a s t i c transformation event probably occurring i n a myeloid stem c e l l , have already been mentioned. Karyotypic a n a l y s i s of f r e s h l y i s o l a t e d bone marrow c e l l s i n these diseases i s a recognized t o o l i n t h e i r -18-d i f f e r e n t i a l diagnosis and treatment (77) , and current information suggests that the percentage of chromosomally abnormal marrow c e l l s increases as the disease progresses. However, t h i s type of analysis w i l l f a i l to detect even a large, but quiescent or suppressed, population of chromosomally normal stem c e l l s . Analysis of chromosomes obtained from sing l e c o l o n i e s of hemopoietic c e l l s provides an approach to the question of r e s i d u a l normal stem c e l l s i n the MPD. Such a f i n d i n g could be of s i g n i f i c a n t importance i n determining the treatment of choice i n a given patient. There i s some evidence to support the hypothesis that chromosomally normal, nonmalignant, stem c e l l s may p e r s i s t i n p a t i e n t s with MPD. Chervenick and associates reported f i n d i n g chromosomally normal hemo-p o i e t i c c o l o n i e s i n Ph^-positive CML (78) . More r e c e n t l y , Singer and h i s colleagues studied a Ph^-positive CML pa t i e n t , who was al s o heterozygous for the A and B forms of G6PD, before and a f t e r i n t e n s i v e chemotherapy (30) . P r i o r to treatment, only one G6PD isoenzyme was present i n red blood c e l l s , p l a t e l e t s , and granulocytes, and only a small percentage of marrow meta-phases were Ph^-hegative. A f t e r three months of i n t e n s i v e chemotherapy, both isoenzymes were detected i n the blood c e l l s , and the majority of the marrow metaphases were Ph^-negative. These p r e l i m i n a r y data indicated that with conversion to Ph"*" n e g a t i v i t y a f t e r i n t e n s i v e chemotherapy, nonclonal and, presumably, nonneoplastic hematopoiesis was restored. b. Polycythemia Vera (PV) Cytogenetic a n a l y s i s of s i n g l e hemopoietic c o l o n i e s from i n d i v i d u a l s with PV provides a unique opportunity to compare the chromosomal findings i n -19-two apparent populations of stem c e l l s i n a disease i n which the malig-nant transformation i s thought to occur i n a s i n g l e myeloid stem c e l l . E r y t h r o p o i e t i n (epo) i s a hormone required for the maturation of e r y t h r o i d colonies from normal i n d i v i d u a l s . Recent reports have shown that PV patients can be characterized by having a high proportion of e r y t h r o i d colonies that do not require normal l e v e l s of epo f o r maturation i n methylcellulose cultures (66,79,80,81). By comparing chromosomal findings i n e r y t h r o i d c o l o n i e s obtained i n c u l t u r e s with and without epo, the relevance of the percentage of epo-dependent e r y t h r o i d colonies may be determined. I f , f o r example, abnormal chromosomes were found only i n the epo-independent e r y t h r o i d colonies, then t h i s would suggest that epo-independence i s a marker f o r the malignant clone, and that the observation of epo-dependent e r y t h r o i d colonies should be i n d i c a t i v e of the existence of normal myeloid stem c e l l s . In PV, as i n CML, the existence of normal myeloid stem c e l l s has important therapeutic i m p l i c a t i o n s . I f chromosomal studies support the hypothesis that the epo-dependent colonies are derived from normal stem c e l l s , then by simply counting the numbers of epo-dependent colonies derived from a PV patient, one can obtain a q u a n t i t a t i v e measure of the normal myeloid stem c e l l s . c. Present Objective In the preceding sections, arguments were presented that i n d i c a t e the importance of being able to analyze the chromosomal p i c t u r e at the l e v e l of p r i m i t i v e hemopoietic c e l l d i f f e r e n t i a t i o n . Since t h i s type of c e l l can only be i d e n t i f i e d by assays that permit c l o n a l expansion -20-and maturation, the main o b j e c t i v e i n t h i s work was to develop a r e l i a b l e method for obtaining high q u a l i t y G- and Q-banded karyotypes from s i n g l e hemopoietic colonies. -21-MATERIALS AND METHODS 1. Patient Specimens Pe r i p h e r a l blood and bone marrow samples were obtained through the courtesy of physicians at the following i n s t i t u t i o n s : Vancouver General Hospital, Prince George General H o s p i t a l , Royal Columbian H o s p i t a l (New Westminster), Royal Jubilee Hospital ( V i c t o r i a ) , Island Medical Labora-t o r i e s ( V i c t o r i a ) , and Lions Gate H o s p i t a l (North Vancouver). Relevant patient information, i n c l u d i n g c l i n i c a l diagnosis and treatment, the type of specimen obtained, and the experiment f o r which i t was used, are shown i n Table I I . A l l samples were l e f t o v e r specimens taken with informed consent f o r the purpose of c l i n i c a l diagnosis, staging, or follow-up. Five a d d i t i o n a l normal adult p e r i p h e r a l blood specimens were c o l l e c t e d from l o c a l volunteers. 2. Specimen Preparation Bone marrow aspirate from the superior p o s t e r i o r i l i a c c r e s t , and perip h e r a l blood taken by venipuncture, were c o l l e c t e d i n pr e s e r v a t i v e -free heparin (Connaught Laboratories), 400 units/marrow specimen and 50 units/ml blood to prevent c l o t formation. The volume of the bone . marrow a s p i r a t e v a r i e d from 1 to 5.5 ml. Peripheral blood specimens were c o l l e c t e d i n volumes of 10 ml. Marrow c e l l s were prepared f o r p l a t i n g using the method of Gregory and Eaves (66). Marrow buffy c e l l s were obtained r e l a t i v e l y free of red c e l l s using a two-step procedure of c e n t r i f u g a t i o n at 1,000 rpm for two to three minutes, followed by sedimentation of the buffy coat at u n i t gravity f o r ten to 20 minutes. The c e l l r i c h plasma was then recentrifuged and the plasma discarded. F i n a l l y , c e l l s were washed once i n a-medium Table II. Patient Data; S O U J C C S of * Subject Sex Age Diagnosis . Treatment Experiment ID M 23 Normal KH M 31 Normal HS M 31 Normal AJ M 31 CML IH F 52 Acute myelofibrosis MD M 27 Lymphoma TS F ' 43 CML PC M 61 Stress erythrocytosis NF F 65 PV HS M 31 Normal QL M 58 Plasmacytoma WK. M 59 2° Erythrocytosis DL F 60 Myeloma JB ' F 9 Bloom syndrome GK M 35 Normal NN F 45 Normal KN M . 41 PV LD F 8 Trisomy 8 JM F 62 PV HK M 56 ET FN M 58 Myeloma PB N i l Time course studies PB N i l M N i l M C&R PB N i l M C&R M N i l PB • . R M SCE studies M N i l M. N i l M N i l M N i l PB&M N i i PB N i l PB N i l PB N i l Karyotypic studies PB N i l PB&M N i l M N i l M N i l (continued...) (continued) Table II. Patient Data. Source of „ , * „ . Subiect Sex Age Diagnosis „ . Treatment Experiment J - 0 - 3 Specimen RR M 40 CML . PB C GH F 52 ET M Ni l IM F 18 CML PB C CF F 64 2° Erythrocytosis M Nil WH M 30 PV PB Nil CF M 63 CML M Nil RH M 77 PV PB&M Nil RAl F 52 Lymphoma M N i l WK M 59 2° Erythrocytosis PB&M Nil EM F 56 Normal PB&M Nil SH F 37 PV PB&M Nil SL F CML PB C KS M 47 Lung cancer PB&M R NF F 65 PV PB&M Nil WC M 74 2° Polycythemia M C RA2 M 43 .  Erythrocytosis PB&M Nil AE M 38 Normal PB Nil PD M 64 . PV M Nil HW M 26 CML PB Nil HC F 71 ET M N i l Karyotypic studies (continued...) (continued) Table II. Patient Data. Subject Sex Age Diagnosis Source of Specimen * Treatment Experiment NF F 65 PV PB&M Nil Karyotypic studies WS M 24 Hodgkins lymphoma M R AM M 91 Myeloma M Nil II OD F 52 PV PB C II CD M 61 Myeloma M Nil it Abbreviations used: PB, peripheral blood; M, bone marrow; C, chemotherapy; R, radiotherapy; CML, chronic myeloid leukemia; PV, polycythemia vera; ET, essential thrombocytosis. Within 6 months. (Connaught Laboratories 1403-01-07) containing f e t a l c a l f serum at a f i n a l concentration of 2% (2% FCS) and resuspended i n the same f o r f i n a l p l a t i n g . (For the exact d e t a i l s of the preparation procedure, see Appendix 1A.) P e r i p h e r a l blood specimens were processed using Ficoll-hypaque (LSM B i o n e t i c s B410-01) density c e n t r i f u g a t i o n to i s o l a t e the mononuclear f r a c t i o n according to the manufacturer's d i r e c t i o n s . C e l l s were washed twice i n 2% FCS and then f i n a l l y resuspended i n the same for p l a t i n g (see Appendix IB). C e l l s were plated i n 35 x10 mm standard nontissue c u l t u r e p e t r i dishes (Lux #5221-R) i n 1.1 ml of c u l t u r e medium. The f i n a l medium consisted of 0.8% methylcellulose, 30% f e t a l c a l f serum (the. same l o t was used for a l l specimens), 0.1% deionized bovine serum albumin, 9% human leukocyte condi--4 tioned medium (66), 10 M 2-mercaptoethanol, 2.5 units/ml of erythropoietin (epo, Step I I I , Connaught Laboratories), and 10% of the f i n a l c e l l suspension i n 2% FCS (see Appendix 1C). Bone marrow preparations were plated at a f i n a l c e l l concentration of • 2 x 10.^  c e l l s per .dish,-while, p e r i p h e r a l blood preparations were plated at 4 x10^ c e l l s per d i s h . Cultures were incubated at 37°C i n a 5% C0 2 i n a i r environment and maintained at high humidity. Dishes were examined v i s u a l l y , using an inverted microscope, a f t e r eight or nine days, f o r the presence of e r y t h r o p o i e t i c c o l o n i e s . These can be r e a d i l y and s p e c i f i c a l l y d i s t i n g u i s h e d from other types of colonies present i n l i v i n g c u l t u r e s only a f t e r the onset of hemo-globin synthesis. When t h i s occurs, e r y t h r o i d colonies acquire a d i s t i n c t reddish tinge (72). The day of harvest was chosen to maximize colony s i z e and m i t o t i c index. 3. Chromosome Harvest The method of Moorhead and h i s colleagues (82) was applied to the hemopoietic colonies. One hour p r i o r to the end of the incubation period, -26-c e l l d i v i s i o n was arrested at metaphase by the ad d i t i o n of 0.12 ml colcemid (Gibco #120-5210) to each 1.1 ml c u l t u r e ( f i n a l concentration, 0.1 ug/ml). The colcemid was d i l u t e d to 1 yg/ml i n Hanks BSS and then applied using a 26-gauge syringe needle to ensure even a p p l i c a t i o n over the surface of the methylcellulose. Single colonies were plucked i n t o a f i n e l y drawn out Pasteur p i p e t t e ( i n t e r n a l diameter of approximately 0.4 mm) using an inverted microscope and f i n a l magnification 75x (see Figure 2). The colony, suspended i n approximately 0.01 ml of growth media, was then t r a n s f e r r e d to a c o n i c a l polypropylene centrifuge tube (capacity 0.5 ml, Evergreen 3403) containing 0.4 ml of 0.075 M KC1 at room temperature. Clumps i n the colony were broken up by gently bubbling a i r through the hypotonic s o l u t i o n with a f i n e l y drawn out p i p e t t e . The c e l l suspension was then incubated for 20 minutes at room temperature before c e n t r i f u g a t i o n . To f a c i l i t a t e c e n t r i f u g a t i o n , the small c o n i c a l tubes were placed into larger tubes (17x100 mm p l a s t i c disposable tubes, Falcon 2057), and then spun at 800 rpm (~200 g) for eig h t minutes. At the end of ce n t r i f u g a -t i o n , a l l but 0.05 ml of the supernatant was removed by a s p i r a t i o n and the c e l l s were resuspended i n the remaining KC1 by gently bubbling with a i r . 0.4 ml of f i x a t i v e (3:1 absolute methyl a l c o h o l : g l a c i a l a c e t i c acid) was then added to the small centrifuge tube and the e n t i r e contents mixed by gently bubbling a i r . A f t e r at l e a s t 20 minutes, the c e l l s were spun down as before and a l l but 0.05 ml of the supernatant was discarded. C e l l s were washed i n 0.4 ml of fresh f i x a t i v e , centrifuged, and the supernatant d i s -carded as before. This washing procedure was repeated before the c e l l s were f i n a l l y suspended i n 0.05 ml of f i x a t i v e . The f i n a l c e l l suspension was c a r e f u l l y drawn up in t o a f i n e l y drawn out pipe t t e and dropped from a height of about 1 cm onto a clean, dry, 22 mm square No. 1 cover glass (Corning). The c e l l s were spread by gently blowing, -27-(C) Figure 2. Colony plucking. Removal of a s i n g l e hemopoietic colony (estimated s i z e of 1,500 c e l l s ) u s i n g a f i n e l y drawn out Pasteur p i p e t t e . and the cover glass was d r i e d by holding i t 5 cm from a 150 watt grow lamp (Sylvania spot-grow) f or 30 seconds. 4. Chromosome Banding The G-banding technique used was a modi f i c a t i o n of that of Chandhuri and associates (83). A i r dried, unstained cover glasses, at l e a s t 24 hours old, were immersed i n pH 6.8 phosphate bu f f e r (BDR Chemicals 702002) at 65°C for one hour, and then stained immediately i n 30% Wrights s t a i n (Matheson, Coleman, and B e l l Inc.) i n pH 6.8 phosphate bu f f e r f o r 45 seconds. The cover glasses were mounted on standard microscope s l i d e s i n 25% E u k i t t mounting media (Otto C. Watzka and Co.) i n xylene. G-banded metaphases were photo-graphed under high power (lOOx objective) using a Zeiss photomicroscope I I I with automatic l i g h t metering on Kodak high contrast copy f i l m . Films were developed i n Kodak D19 developer and p r i n t e d on grade 3 paper. The method used for producing.Q-bands was a modification of that of Caspersson (84). Cover glasses were immersed i n a t e b r i n (Gurr 2900) for 20 minutes. The cover glasses were mounted on standard microscope s l i d e s i n a 5% sucrose s o l u t i o n and sealed around the edges with rubber cement. Metaphases were photographed using a Z e i s s photomicroscope I I I with I V F l -epi-fluorescence and a Zeiss 487709 e x c i t e r / b a r r i e r f i l t e r combination. Photographs were taken using spot metering on Kodak plus X f i l m pushed to ASA 300, and developed i n Dia f i n e developer. Photographs were p r i n t e d on grade 4 paper with the negative s l i g h t l y out of focus to reduce gr a i n i n e s s . 5. D i f f e r e n t i a l Staining of S i s t e r Chromatids a. Rationale S i s t e r chromatid exchanges are thought to represent the interchange of DNA r e p l i c a t i o n products at apparently homologous chromosomal l o c i . These exchanges presumably involve DNA breakage and reunion. Analysis of the frequency of such exchanges provides a s e n s i t i v e c y t o l o g i c a l method to detect DNA interchanges following DNA damage (85). Some authors (e.g. Ref. 2) question the v a l i d i t y of karyotypes obtained from c e l l s that have undergone more than a few d i v i s i o n s i n culture because of the undefined r o l e of poten-t i a l in vitro agents that may cause chromosomal damage. In order to evaluate p o s s i b l e c l a s t o g e n i c ( i . e . chromosome damaging) e f f e c t s of the system used i n the c u l t u r e of the hemopoietic c e l l s used throughout t h i s work, the frequency of s i s t e r chromatid exchanges i n such c e l l s was determined and compared to that obtained from c e l l s grown in vitro f o r much shorter periods of time. b. Technique i . Bone Marrow 0.1 ml of fresh bone marrow aspirate was incubated i n 5 ml of a-medium (Connaught Laboratories 1403-01-07) with 20% f e t a l c a l f serum and 0.25 units of e r y t h r o p o i e t i n per ml (epo, Step I I I , Connaught Laboratories) i n a s t e r i l e 15 ml glass t e s t tube with a loosely f i t t i n g screw cap. A f t e r eight hours of incubation at 37°C i n a 5% CO^ i n a i r environment, 10 3 M 5BrdU (Sigma B-5002) was added to give a f i n a l concentration of 50 yM 5BrdU. Cultures were kept i n the dark u n t i l the time of harvest. One hour p r i o r to harvest, c e l l d i v i -s ion was a r r e s t e d at metaphase by the add i t i o n of colcemid (Gibco #120-5210) at a f i n a l concentration of 0.1 yg/ml. Cultures were harvested 24 to 91 hours a f t e r the a d d i t i o n of 5BrdU. At the end of t h i s incubation period, the c e l l s were gently resuspended i n the growth tubes and then emptied i n t o covered centr i f u g e tubes and spun down at 800 rpm f o r eight minutes. A l l but 0.05 ml of the supernatant was discarded. The c e l l s were c a r e f u l l y resuspended i n 5 ml of hypotonic KC1 (0.075 M). A f t e r standing for 20 minutes, the c e l l s were ce n t r i f u g e d at 800 rpm for eight minutes, and again, a l l but a few drops -30-of the supernatant was discarded. The c e l l s were resuspended i n 5 ml of cold f i x a t i v e (3:1 absolute methyl a l c o h o l : g l a c i a l a c e t i c a c i d ) . To reduce clumping of the c e l l s , the f i r s t amount of f i x a t i v e was added slowly and the c e l l s were immediately gently agitated with a Pasteur p i p e t t e . The remainder o f the f i x a t i v e was then added. The tubes were allowed to stand at room temperature for 20 minutes. The c e l l s were then centrifuged at 800 rpm for eight minutes and the supernatant was discarded. The c e l l s were washed two to three times i n cold f i x a t i v e a f t e r the i n i t i a l f i x a t i o n . A f t e r the f i n a l wash, the supernatant was removed and the c e l l s were resuspended i n f i v e or s i x drops of fresh f i x a t i v e . About three drops of t h i s suspension were dropped onto three drops of a 30% g l a c i a l a c e t i c a c i d s o l u t i o n on a precleaned s l i d e from a height of about 8 cm to f a c i l i t a t e spreading of the chromosomes. The s l i d e was gently blown on and then allowed to a i r dry overnight. i i . E r y t h r o p o i e t i c Colonies To obtain d i f f e r e n t i a l s t a i n i n g of s i s t e r chromatids from chromosomes obtained from e r y t h r o i d colonies, 5BrdU were added to each c u l t u r e on Day 9 of growth and at a f i n a l concentration of 50 yM. Forty-eight hours a f t e r the addition of 5BrdU, the cultures were harvested as described i n section 3. i i i . D i f f e r e n t i a l S t a i n i n g A f t e r at l e a s t 24 hours of a i r drying, the s l i d e s or cover glasses were immersed i n a 0.5 ug/ml aqueous s o l u t i o n of Bisbenzimid Hoechst 33258 (Riedel-De Haen Ag Seelze Hannover #33217) f o r 13 minutes and then ri n s e d i n water. The fluorescence plus Giemsa technique used was that of Goto and associates (86), with the following modifications: s l i d e s or cover glasses -31-stained with Hoechst 33258 were placed i n p e t r i dishes and covered with about 2 cm of phosphate c i t r a t e b u f f e r pH 7.0 (0.16 M sodium phosphate-0.04 M sodium c i t r a t e ) . The p e t r i dishes were then covered to minimize evaporation, and the dish and i t s contents exposed to 24 hours of continuous l i g h t from a 150 watt grow lamp (Sylvania spot-grow). The dishes were kept at a distance of 35 cm from the l i g h t source. A f t e r l i g h t exposure, the s l i d e s or cover glasses were r i n s e d b r i e f l y i n d i s t i l l e d water and then stained i n 5% Giemsa (Harleco No. 620) i n 2:3 phosphate b u f f e r pH 6.8: d i s t i l l e d water, f o r eight minutes. Cover glasses were mounted i n 25% E u k i t t mounting media i n xylene, and metaphases with chromosomes showing d i f f e r e n t i a l s t a i n i n g of s i s t e r chromatids were photographed, using a Zeiss photomicroscope I I I with auto-matic l i g h t metering, on Kodak high contrast copy f i l m . Films were developed i n Kodak D19 developer and p r i n t e d on grade 4 paper. Frequencies of s i s t e r chromatid exchanges were determined by the an a l y s i s of the photo-graphic p r i n t s . For c e l l c ycle k i n e t i c s experiments, number of c e l l s i n f i r s t , second, and t h i r d d i v i s i o n metaphase were scored v i s u a l l y . -32-RESULTS 1. Time Course Studies a. C e l l Recovery Before determining the day(s) when maximal numbers of metaphases could be 'obtained from e r y t h r o i d colonies, time course studies of the t o t a l recoverable number of c e l l s from i n d i v i d u a l c o l o n i e s were run. Colonies were harvested as i f for chromosome a n a l y s i s and t o t a l c e l l counts performed on each cover g l a s s . On Day 8 of c u l t u r e , the s i x largest e r y t h r o i d colonies that could be found on four c u l t u r e dishes were plucked and harvested independently. This procedure was repeated every day, using d i f f e r e n t culture dishes, for the next seven days. The colo n i e s . s e l e c t e d were v i s u a l l y estimated as containing several hundred to a few thousand c e l l s . Two separate experiments were performed using d i f f e r e n t p e r i p h e r a l blood .samples. The r e s u l t s are shown g r a p h i c a l l y i n Figure 3a,b. A maximum recovery of approximately 500 c e l l s could be obtained a f t e r 11 to 12 days incubation. The data agree with previous observations on the growth k i n e t i c s of erythroid colonies, i . e . , most of the l a r g e s t e r y t h r o i d colonies reach t h e i r maximum s i z e by two weeks. By t h i s time, v i r t u a l l y a l l of the c e l l s have reached the nondividing erythroblast stage of maturation and soon a f t e r , the c e l l s e x h i b i t the f r a g i l e c h a r a c t e r i s t i c s of maturing red c e l l s (72). The present r e s u l t s suggest that some colony d i s i n t e g r a t i o n begins around Day 11 or 12 i n many normal er y t h r o i d c o l o n i e s and thus maximum numbers of metaphases would not usu a l l y be a n t i c i p a t e d beyond Day 12. -33-_L 1 t r w Number of Days in Culture Before Colony Harvest Figure 3a. T o t a l recovery of c e l l s from a s i n g l e colony a f t e r hypotonic treatment, f i x a t i o n , and two washes, shown as a function of the day of colony harvest. Each point represents the mean recovery of s i x colonies harvested independently. Bars i n d i c a t e the standard erro r . Cultures i n i t i a t e d from p e r i p h e r a l blood c e l l s from ID. -34-Figure 3b. T o t a l recovery of c e l l s from a s i n g l e colony a f t e r hypotonic treatment, f i x a t i o n , and two washes, shown as a function of the' day of colony harvest. Each point represents the mean recovery of s i x colonies harvested independently. Bars i n d i c a t e the standard erro r . Cultures i n i t i a t e d from p e r i p h e r a l blood c e l l s from KH. -35-b. M i t o t i c Index Previous studies on the growth c h a r a c t e r i s t i c s of e r y t h r o p o i e t i c colonies have suggested that p r o l i f e r a t i o n and maturation are c l o s e l y associated (66). Thus, most c e l l s i n a given colony reach the f i n a l states of d i f f e r e n t i a t i o n at approximately the same time. At t h i s point, the production of hemoglobin becomes v i s i b l e i n the l i v i n g c e l l s . There-a f t e r , the c e l l s continue to produce hemoglobin and become much redder and, hence, ea s i e r to recognize, but the c o l o n i e s do not increase further i n s i z e . I t was a n t i c i p a t e d , therefore, that the m i t o t i c index would begin to f a l l d ramatically j u s t before c o l o n i e s reached t h e i r maximum s i z e . Experiments were then undertaken to determine the time dependence of m i t o t i c index. This was done f i r s t using pooled colonies and then using s i n g l e c o l o n i e s . i . Pooled Colonies Five cultures from d i f f e r e n t donors were set up for e r y t h r o i d colonies. On Day 10, the s i x l a r g e s t e r y t h r o i d colonies that could be found on four c u l t u r e dishes were pooled before the hypotonic step of the chromosome harvest procedure. This procedure was repeated every day, using d i f f e r e n t culture dishes, f o r the next nine days. Following routine chromosome harvest, the t o t a l number of metaphases, as well as the t o t a l number of c e l l s recovered per cover glass, were scored. The r e s u l t s , summarized i n Table I I I , i n d i c a t e that there was no consistent change i n the m i t o t i c index on d i f f e r e n t days of harvest, between Days 6 and 15, when pooled colonies were used. O v e r a l l , the m i t o t i c index averaged about 1.5%. Table III. Percentage of Recovered Cells in Metaphase in Pooled Erythroid Colonies Harvested On Different Days of Culture. Six Colonies Were Pooled to Give Each Value. Percentage of Cells in Metaphase Specimen — : ^ ^ * ID HI MD TS PC Mean Day of Harvest 6 No recovery 0 0 1.23 0.46 0.4 ± 0.3 7 0.23 0 0 3.15 0 0.7. ±0.6 8 1.23 0.79 0.50 1.88 1.76 1.2 ± 0.3 9 0.87 0 ' 0.12 3.02 3.06 1.4 ±0.7 10 3.99 6.70 1.23 1.07 2.82 3.2 ±1.0 11 1.07 1.03 1.25 0.33 1.05 1.0 ±0.2 12 1.59 0.16 . 0.60 0.87 1.00 0.8 ± 0.2 13 0.71 0.25 ' 0.75 6.56 0.63 1.8 ±1.2 14 No recovery 0.92 2.41 0 0.21 0.9 ± 0.6 15 No recovery 0.71 0.71 0 0.98 0.6 ± 0.2 16 No recovery No recovery No recovery 0 No recovery 0 Mean 1.4 ±0.5 1.0±0.7 0.7±0.2 1.7±0.6 1.2±0.4 Overall Mean 1.2 ± 0. 3 * Colonies initi a t e d from peripheral blood. ** " -v  Colonies initi a t e d from bone marrow. -37-The p o s s i b i l i t y that within each colony the c e l l s were r e l a t i v e l y synchronized, but that d i f f e r e n t c o l o n i e s were at d i f f e r e n t stages of the c e l l cycle at any given time, could not be excluded. A more rigorous examination of p o s s i b l e i n t e r c o l o n y v a r i a t i o n s i n m i t o t i c index was made by repeating the experiment using s i n g l e e r y t h r o i d colonies. i i . Single Colonies Ten erythroid colonies were plucked independently and harvested for chromosomes on each day from Day 9 to Day.13 of c u l t u r e . The m i t o t i c index was estimated as a percentage of the recovered c e l l s i n metaphase. Two experiments were run using d i f f e r e n t specimens. The data are shown gr a p h i c a l l y i n Figures 4a and 4b. There was a large v a r i a t i o n i n the m i t o t i c index for d i f f e r e n t colonies from the same i n d i v i d u a l harvested at the same time. The l a r g e s t range on any given day was from ,0 to 6% (Fig. 4a, Day 10). However, there was, again, no consistent trend during the time i n t e r v a l studied. The absolute number of metaphases recovered per colony i s a l s o shown i n Figures 4a and 4b. These ranged from zero to f i v e , with occa-si o n a l colonies y i e l d i n g up to 21 metaphases. On average, more metaphases were obtained from 9- to 11-day-old c o l o n i e s than from 12- to 13-day-old colonies. In most cases, the number of metaphases per colony p a r a l l e l s the m i t o t i c indices.. This i n d i c a t e s that m i t o t i c c e l l s were not p r e f e r -e n t i a l l y gained or l o s t during the harvesting procedure. The single colony data, s i m i l a r to those for the pooled colonies, suggested the average m i t o t i c index does not vary markedly with the day of harvest between Days 9 and 13. However, marked intercolony v a r i a t i o n does -38-Day of Colony Harvest 20 j » 10 11 12 13 ihas r-| 15 c -IT of Cells o - • Numbe • -0 'IT M r l l f f ir • r l f l l 9 10 11 12 13 F A. j-r F : nrrr 1 rf r f 1 . r f f l f Colon I M Figure 4a. Percentage of recovered c e l l s i n metaphase. Ten colonies were plucked on each day for Days 9 to 13 of incubation. Also shown i s the absolute number of metaphases observed per corresponding colony. The average number of c e l l s recovered per colony was 268 ±169. The range was from 27 to 752. Cultures i n i t i a t e d from bone marrow c e l l s from AJ. -39-Day ol Colony Harvest 9 10 11 12 J3 20 cn CO SZ of Cells in Metap -JD 5 E z rf - F-fh-n rrfTrrr n r r r . 0 9 10 i ; > 13 5 0) cn ro SZ Q. £ 4 0) 5 c « -Percentage of Ci pf! rf rf" Colonial rftl j-T nTff -r Figure 4b. Percentage of recovered c e l l s i n metaphase. Ten colonies were plucked on each day f o r Days 9 to 13 of incubation. Also shown are the absolute number of metaphases observed per colony. Cultures i n i t i a t e d from bone marrow c e l l s from HS. -40-occur and i s present throughout t h i s period. In subsequent experiments, colonies were harvested between Days 9.and 11. Colony s i z e and colour were also considered when estimating the best day for chromosome harvest. .2. S i s t e r Chromatid Exchange Studies The complexity of karyotypic changes i n some human c e l l s grown i n long-term cultures has given some authors reason to exclude such data from consideration (2). The s i s t e r chromatid exchange (SCE) assay i s considered to be much more s e n s i t i v e to clastogens ( i . e . agents that cause chromosomal breakage) than the chromosomal aberration assay (87). In order to deter-mine whether the culture conditions used for the growth of e r y t h r o i d colonies were clastogenic, the SCE frequency i n 1- to' 2-week-old e r y t h r o i d colonies was compared to that of erythropoietin-stimulated f r e s h marrow c e l l s . The technique used to produce d i f f e r e n t i a l s t a i n i n g of s i s t e r chromatids e n t a i l s two rounds of. DNA r e p l i c a t i o n i n the presence of the base analogue, 5BrdU. This time, period i s approximately 24 hours f o r d i v i d i n g lymphocytes (88), 48 hours f o r d i v i d i n g f i b r o b l a s t s (89), and 48 hours for fresh marrow preparations (90). There i s no published data on t h i s aspect of e r y t h r o i d colony growth k i n e t i c s . To get an approximate estimate of the time required f or e r y t h r o p o i e t i c c e l l s to progress from synthesis to metaphase of the second cy c l e , a time course experiment was run using fresh marrow c e l l s . In order to enhance the proportion of erythroid c e l l s i n the d i v i d i n g population analyzed, e r y t h r o p o i e t i n was added. In the absence of t h i s hormone, the v i a b i l i t y of e r y t h r o i d c e l l s r a p i d l y decreases i n short-term c u l t u r e (91). -41-a. Fresh Marrow Myeloid c e l l s were c u l t u r e d from 24 to 91 hours i n the presence of 5BrdU,. and then harvested f o r chromosomes and stained f o r d i f f e r e n t i a l l a b e l l i n g of s i s t e r chromatids. One thousand c e l l s were analyzed f o r each of five, independent harvests and the number of c e l l s i n f i r s t , second, and t h i r d d i v i s i o n metaphase were scored. Metaphases were i d e n t i f i e d as being e i t h e r f i r s t , second, or t h i r d d i v i s i o n by the d i s t r i b u t i o n of 5BrdU, v i s u a l l y discernable as the l i g h t e r s t a i n i n g regions, i n the chromatid (see F i g . 5). Duplicate experiments were run using two d i f f e r e n t specimens. The r e s u l t s are shown g r a p h i c a l l y i n Figures 6a and 6b, and confirm that the maximum number of second d i v i s i o n metaphases could be obtained by incubating fresh marrow f o r 48 hours. b. Erythroid Colonies The information gained by the previous experiment was used to obtain d i f f e r e n t i a l s t a i n i n g of s i s t e r chromatids i n metaphases from pooled erythroid colonies. F o rty-eight hours p r i o r to harvest, 5BrdU was added. For each of four specimens, groups of s i x to 12 e r y t h r o i d colonies ; pooled p r i o r to the hypotonic step were processed. A f t e r routine chromo-some harvest, and s t a i n i n g procedure, metaphases showing d i f f e r e n t i a l l y s t a i n i n g chromatids were observed r e g u l a r l y . One coverglass containing at l e a s t 30 d i f f e r e n t i a l l y stained metaphases was selected f o r each specimen and scored f o r s i s t e r chromatid exchanges. The mean frequency of SCE ranged from 3.1 to 5.3 per c e l l . The r e s u l t s are shown i n Table IV. These values f a l l within the range observed for fresh marrow treated i n the same way. (The value shown i n Table IV i s taken from the data i n -42-Figure 5. C e l l c y c l e k i n e t i c s evaluation using 5BrdU incorporation and t h e . d i f f e r e n t i a l s t a i n i n g technique. Metaphases a f t e r (a) one, (b) two, and (c) three syntheses i n 5 BrdU. A l l three metaphases were taken from one bone marrow cul t u r e from an i n d i v i d u a l with Bloom syndrome. - 4 3 --44-co O CD X I E 80 70 60 5 0 H 40 30&-20 10 2nd Division Metaphases 1st Division Metaphases 1 \ \ \ 3rd Division Metaphases 20 40 60 Al T T i . 80 100 Incubation Period in 5 BrdU (Hours) Figure 6a. The number of c e l l s i n f i r s t , second, and t h i r d metaphase .after various lengths of time i n incubation i n 50 ]XM 5BrdU. One thousand c e l l s were scored at each harvest. Bars i n d i c a t e the standard e r r o r . Cultures i n i t i a t e d from bone marrow c e l l s from NF. -45-80 70 6 0 * -50 CD o 40 0) E Z 30 20 10 1st Division Metaphases 5 ? MS , T 2nd Di vision Metaphases T ' v •V J\ \ 3rd Division / / * I \/ Metaphases • » ^'*»* 40 60 80 : 1 0 0 Incubation Period in 5 BrdU (Hours) Figure 6b. The number of c e l l s i n f i r s t , second, and t h i r d metaphase a f t e r various lengths of time i n incubation i n 50 yM 5BrdU. One thousand c e l l s were scored at each harvest. Bars i n d i c a t e the standard e r r o r . Cultures i n i t i a t e d from bone marrow c e l l s from HS. Table IV. Average Number of Sister Chromatid Exchanges Per Cell in Pooled Erythroid Colonies From •Four Individuals. For comparison, the SCE frequency is shown for normal.lymphocytes, normal fibroblasts, normal marrow, and lymphocytes and marrow from an individual with Bloom syndrome. In a l l cases, the fi n a l concentration of 5BrdU was 50 uM. Experiment ^. . „ n n m No. Cells SCE/Metaphase „ Diagnosis Cell Type ^ . , „„ No. ^ . Counted ± SE WK DL GK Myeloma Erythrocytosis Myeloma Normal Erythroid colonies from marrow Erythroid colonies from marrow Erythroid colonies from marrow Erythroid colonies from peripheral blood 30 30 30 30 3.6±0.7 4.8 ± 0.5 3.1±0.6 5.3 ± 0.5 HS NN i HT12 i HT21 i HT31 HT41* * FT1 Normal Normal Normal Normal Normal Normal Normal Epo-stimulated fresh marrow Epo-stimulated fresh marrow PHA-stimulated peripheral blood PHA-stimulated peripheral blood PHA-stimulated peripheral blood PHA-stimulated peripheral blood Skin fibroblasts-passage six 30 15 30 50 46 79 30 3.810.4 4.5 1 0.6 3.110.4 4.6 1 0.3 3.910.3 3.7 1 0.2 4.210.4 . JB JB Bloom syndrome Bloom syndrome Epo-stimulated fresh marrow PHA-stimulated peripheral blood 5 10 115 126 97 1 22 * Values taken from a previous study (92). -47-section 2a.) For comparison, the SCE frequency i n normal human lympho-cytes i s shown for four i n d i v i d u a l s . (These values were obtained i n a previous study i n which the same s t a i n i n g conditions were used (92)). SCE frequency determined i n a sample of cultured f i b r o b l a s t s from normal human skin i s also given. To obtain a p o s i t i v e c o n t r o l , fresh marrow and PHA stimulated p e r i p h e r a l blood c e l l s from a patient with Bloom syndrome were analyzed, and the g r e a t l y enhanced frequency of SCE c h a r a c t e r i s t i c of t h i s disease i s shown i n Table IV. These r e s u l t s suggest that under the c u l t u r e conditions used, the erythroid c e l l s produced after-one to. two weeks..incubation are cytogene-t i c a l l y s table. 3. Proof of the Single C e l l O r i g i n of I n d i v i d u a l l y Plucked Colonies In order to show that a s i n g l e colony of c e l l s containing about several hundred c e l l s could be plucked from a 35 mm d i s h containing at l e a s t 30 s i m i l a r colonies, the f o l l o w i n g mixing experiment was performed. Peripheral blood samples were taken from a male and a female volunteer and prepared i n the usual manner. Equal numbers- of c e l l s from the two. samples were mixed and p l a t e d at 1 x 10^ c e l l s of each type per d i s h . F i f t y - s i x e r y t h r o i d colonies were harvested for chromosomes on Days 11 to 13 of growth, and from these, 32 preparations y i e l d e d metaphases. These were then Q-banded and scored f o r the number of c e l l s that could be p o s i t i v e l y i d e n t i f i e d as male or female. Inconclusive metaphases, l a r g e l y due to- poor spreading of the metaphases or missing chromosomes, were also recorded. The r e s u l t s are shown i n Table V. Of 32 i n d i v i d u a l colonies i n which metaphases were obtained, 16 were i d e n t i f i e d as male and -48-Table V. D i s t r i b u t i o n of Fluorescent Y-Body i n In d i v i d u a l Colonies Plucked From Dishes i n Which Equal Numbers of Male and Female C e l l s Were Plated. Number of Metaphases Colony Conclusion Y-Body P o s i t i v e Y-Body Negative Inconclusive 1 0 24 . 1 6 Female 2 0 3 12 Female 3 0 38 20 Female 4 0 1 1 Female 5 0 4 3 Female 6 0 7 1 Female 7 0 8 13 Female 8 0 49 12 Female 9 0 18 21 Female 10 0 1 3 Female 11 0 8 4 Female 12 0 17 18 Female 13 0 6 4 Female 14 0 24 12 Female 15 3 0 0 Male 16 15 0 3 Male 17 8 0 4 Male 18 34 0 10 Male 19 31 0 2 Male 20 10 0 4 Male 21 4 0 2 Male 22 13 0 8 Male 23 61 0 18 Male 24 49 0 2 Male 25 26 0 1 Male 26 11 0 0 Male 27 11 0 0 Male 28 34 0 2 Male 29 22 0 1 Male 30 1 0 1 Male 31 0 0 2 Inconclusive 32 0 0 2 Inconclusive -49-14 as female. In no instance were both male and female metaphases found i n the c e l l s recovered from a s i n g l e colony. Two colonies y i e l d e d meta-phases that gave inconclusive r e s u l t s . 4. Karyotypes From Single Colonies Extensive chromosome a n a l y s i s was performed on s i n g l e e r y t h r o i d colonies from s i x i n d i v i d u a l s . In each case, ten colonies were selected on the basis of s i z e and colour. A l l colonies were harvested between Days 9 and 14 of incubation. Metaphases were G- or Q-banded and analyzed under the microscope. At l e a s t one G-banded and one Q-banded karyotype were prepared f o r each i n d i v i d u a l . Table VI shows the average number of analyzable metaphases obtained per colony i n each case. Figures 7-10 show the q u a l i t y of G- and Q-banded karyotypes obtainable from s i n g l e erythroid colonies. -50-Table VI. Average Number of Analyzable Metaphases Per Colony for Six I n d i v i d u a l s . Ten Colonies Were Plucked i n Each Case and Q- or G-banded. „ , . ^ Number of Metaphases per Colony Sub Dect (Ave.iSE) Karyotype WH 3.6±1.0 46, XY RA2 4.211.1 46, XY EM 5.4 ± 0.9 46, XX KS 3.3 ±1.1 46, XY PD 6.4 ± 1.2 46, XY WS 3.9 ± 0.7 46, XY -51-% > « / J: i l (( II II )> ^ 11 14 15 a i f i\ II ii * » ii II I t i l l H 17 11 1 J? n JO Figure 7. Q-banded male karyotype obtained from a sin g l e e r y t h r o i d colony grown from per i p h e r a l blood c e l l s from WH. -52-I 1 II i i i i i i i i t i 4 . II i t 5 i t i t 7 I » I 1 10 11 .1 i i i • n it 14 15 JO I t 17 >i a it i i X X • Figure 8. Q-banded female karyotype obtained from a single erythroid c o l o n y g r o w n f r o m b o n e m a r r o w c e l l s f r o m E M . -53-h • • 41 n II - T • A 7 | fl 14 1 1 i 7 u •1 * 0 i l r i 12 i i t. 22 Figure 9. G-banded male karyotype obtained from a sin g l e e r y t h r o i d colony grown from bone marrow c e l l s from WS. -54-Figure 1 0 . G-banded female karyotype obtained from a single erythroid colony grown from peripheral blood cells from EM. -55-DISCUSSION There are only a few reports i n the l i t e r a t u r e of cytogenetic analysis of s i n g l e hemopoietic co l o n i e s (93,94,95,96). None of these include a n a l y s i s of banded chromosomes, and i n no instance has a karyotype been published. In the present study, a technique has been developed that enables high q u a l i t y chromosomes (suitable for G- or Q-banding) to be obtained from si n g l e e r y t h r o i d colonies grown from human blood or bone marrow. Variables that a f f e c t the y i e l d of analyzable metaphases have been pre-sented. For the f i r s t time, the s i s t e r chromatid exchange t e s t for chromo-somal breaking agents has been applied to e r y t h r o i d colonies growing i n methylcellulose cultures for one to two weeks. The major findings are as follows: (a) On average, 9- to 11-day-old erythroid colonies gave the best r e s u l t s with respect to y i e l d of analyz-able metaphases; (b) S i s t e r chromatid exchange studies showed no evidence of a c l a s t o g e n i c e f f e c t throughout the p e r i o d required to obtain d i f f e r -entiated colonies •in vitro; (c) Results of a n a l y s i s of mixed male-female cultures were consistent with the s i n g l e c e l l o r i g i n of colonies and showed that s i n g l e colonies were, i n f a c t , removed by the plucking procedure used; (d) On average, between four and s i x high q u a l i t y karyotypes, s u i t a b l e f o r G- or Q-banding, were obtained from s i n g l e e r y t h r o i d colonies. There are two major d i r e c t i o n s i n which the technique may be f u r t h e r r e f i n e d : c e l l recovery and m i t o t i c index. Preliminary experiments have i n d i c a t e d that the problem of c e l l recovery may be t o t a l l y overcome by u t i l i z i n g the p o l y l y s i n e treatment technique of Rajendra (97). This simple technique involves the pretreatment of microscope s l i d e s with -56-the c a t i o n i c p o l y l y s i n e (Sigma, P1886). The e n t i r e colony can then be dispersed i n t o a drop of hypotonic s o l u t i o n on the treated s l i d e . The negatively charged c e l l s adhere to the p o l y l y s i n e coating, and at the end of the treatment period, the hypotonic s o l u t i o n may be removed and the c e l l s f i x e d immediately. Using t h i s method, close to 100%. recovery has been obtained. Increasing the m i t o t i c index by the a d d i t i o n and removal of con-ventional c e l l c y c l e blocking agents, such as methotrexate (98), i s not f e a s i b l e since an agent, once added to the semisolid c u l t u r e medium, cannot be removed without d i s r u p t i n g the colonies. Experiments i n which attempts to release methotrexate blocks by the a d d i t i o n of excess thymidine and dihydrofolate reductase have been unsuccessful so f a r . Synchronization of cultures by c o l d shock (99) has also been t r i e d but proved not to be f e a s i b l e since methylcellulose has the p e c u l i a r property of becoming less, viscous upon cooling. Under such conditions, the colonies are no longer i d e n t i f i a b l e as d i s t i n c t e n t i t i e s . • Recent.results suggest that the r e v e r s i b l e microtubule i n h i b i t o r , nocodazole ( A l d r i c h ) , may give better r e s u l t s than colcemid i n terms of accumulating m i t o t i c c e l l s (100). Whether or not t h i s agent w i l l be u s e f u l i n i n c r e a s i n g the y i e l d of analyzable metaphases from a s i n g l e colony remains to be established. -57-CONCLUSION A microtechnique has been developed that permits the routine cyto-genetic a n a l y s i s of s i n g l e hemopoietic colonies grown i n standard methyl-c e l l u l o s e c u l t u r e s . I t i s now po s s i b l e to begin a systematic study of chromosomal involvement e x c l u s i v e l y i n the progeny of i n d i v i d u a l e r y t h r o i d stem c e l l s i n a s e r i e s of newly diagnosed p a t i e n t s with m y e l o p r o l i f e r a t i v e disease. Using t h i s method, i t would be p o s s i b l e to look for chromosomally normal stem c e l l s i n Ph^-positive CML, and for s p e c i f i c marker chromosomes i n stem c e l l s of p a t i e n t s with polycythemia vera and e s s e n t i a l thrombo-c y t o s i s . In polycythemia vera, two types of colony populations may be c y t o g e n e t i c a l l y i n v e s t i g a t e d : those which have l o s t t h e i r dependence on erythropoietin f o r growth, and those which require i t . The value of the microtechnique developed here w i l l only be appre-ci a t e d when the r e s u l t s . o b t a i n e d by i t s routine use are compared with the cytogenetic p i c t u r e obtained from d i r e c t bone marrow preparations, and attempts are made to c o r r e l a t e these chromosome findings with the c l i n i c a l p i c t u r e , response to therapy, disease progression, and prognosis. -58-REFERENCES 1. Boveri, T-. : Zur Frage der Entstehung Maligner Tumoren. 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Wu, A.M., Siminovitch, L., T i l l , J.E., and McCulloch, E.A.: Evidence fo r a r e l a t i o n s h i p between mouse hemopoietic stem c e l l s and c e l l s forming colonies i n c u l t u r e . Proc. Natl. Acad. S c i . USA _59, 1209-1215, 1968. 49. Prchal, J.T., Throckmorton, D.W., C a r r o l l , I I I , A.J., Fuson, E.W., Gams, R.A., and Prchal, J.F. : A common progenitor f o r human myeloid and lymphoid c e l l s . Nature 274, 590-591, 1978. 50. Metcalf; D.: Hemopoietic Colonies. In Vitro Cloning of Normal and Leukemic C e l l s , Recent Results i n Cancer Research. New York, Springer-Verlag, 1977, pp. 1-227. 51. Bradley, T.R., and Metcalf, D.: The growth of mouse bone marrow c e l l s in vitro. Aust. J . Exp.. B i o l . Med. S c i . 44_, 287-300, 1966. 52. Pluznik, D.H., and Sachs, L. : The induction of clones of normal mast c e l l s by a substance from conditioned medium. Exp. C e l l Res. 43, 553-563, 1966. 53. Stanley, E.R., and Metcalf, D.: P a r t i a l p u r i f i c a t i o n and some pr o p e r t i e s of the f a c t o r i n normal and leukaemic human urine capable of stimulating mouse bone marrow colony growth in vitro. Aust. J . Exp. B i o l . Med. S c i . 47_, 467-483, 1969. 54. Austin, P.E., McCulloch, E^A., and T i l l , J.E.: C h a r a c t e r i z a t i o n of the f a c t o r i n L - c e l l conditioned medium capable of stimulating colony formation by mouse marrow c e l l s i n cul t u r e . J . C e l l . P h y s i o l . 77, 121-133, 1971. -62-55. Senn, J.S., McCulloch, E.A., and T i l l . , J.E.: Comparison of colony-forming a b i l i t y of normal and leukaemic human marrow i n c e l l c u l t u r e . Lancet ijL, 597-598, 1967. 56. Iscove, N.N., Senn, J.S., T i l l , J.E., and McCulloch, E.A.: Colony formation by normal and leukaemic human marrow c e l l s i n cu l t u r e : E f f e c t of conditioned medium from human leukocytes. Blood 3J7, 1-5, 1971. 57. Metcalf, D., MacDonald, H.R., Odartchenko, N., and Sordat, B.: Growth of mouse, megakaryocyte colonies in vitro. Proc. N a t l . Acad. S c i . USA 7_2, 1744-1748, 1975. 58. Parker, J.W. , and Metcalf, D.: Production of colony stimulating f a c t o r i n mixed leucocyte c u l t u r e s . Immunology 26, 1039-1049, 1974. 59. Vainchenker, W., Guichard, J . , and Breton-Gorius, J . : Growth of human megakaryocyte colonies i n c u l t u r e from f e t a l , neonatal, and adult p e r i p h e r a l blood c e l l s : U l t r a s t r u c t u r a l a n a l y s i s . Blood C e l l s 5_, 25-42, 1979. 60. Metcalf, D.., Parker, J . , Chester, H.M., and Kincade,' P.W.: Formation of e o s i n o p h i l i c - l i k e granulocytic colonies by mouse bone marrow c e l l s in vitro. J . C e l l . P h y s i o l . 84_, 275-290, 1974. 61. Stephenson, J.R.., Axelrad, A.A. > McLeod, D.L., and Shreeve, M.M. : Induction of colonies of hemoglobin-synthesising c e l l s by erythro-p o i e t i n in vitro. Proc. N a t l . Acad. S c i . USA 68, 1542-1546, 1971. 62. Tepperman, A.D., C u r t i s , J.E., and McCulloch, E.A.: .'Erythropoietic colonies i n cultures of human marrow. Blood 44, 659-669, 1974. 63. Axelrad, A.A., McLeod, • D.L., Shreeve, M.M., and Heath, D.S.: Properties of c e l l s that produce e r y t h r o c y t i c colonies in vitro. In Robinson, W.A. (Ed.): Hemopoiesis i n Culture. Washington, D.C., U.S. Government P r i n t i n g O f f i c e , 1974, pp. 226-234. 64. Gregory, C.J.: E r y t h r o p o i e t i n s e n s i t i v i t y as a d i f f e r e n t i a t i o n marker i n hemopoietic systems: Studies of three e r y t h r o p o i e t i c colony responses i n c u l t u r e . J . C e l l . P h y s i o l . 8_9, 289-302, 1976. 65. Axelrad, A.A., McLeod, D.C., Suzuki, S., and Shreeve, M.M.: Regulation of the population s i z e of e r y t h r o p o i e t i c progenitor c e l l s . In Clarkson, B., Marks, P.A.., and T i l l , J.E. (Eds.): D i f f e r e n t i a t i o n of Normal and Neoplastic Hematopoietic C e l l s . Cold Spring Harbor Conferences on C e l l P r o l i f e r a t i o n _5,. Cold Spring Harbor Laboratory, 1978, pp. 155-162. 66. Gregory, C.J., and Eaves, A.C.: Human marrow c e l l s capable of e r y t h r o p o i e t i c d i f f e r e n t i a t i o n in vitro. D e f i n i t i o n of three e r y t h r o i d colony responses. Blood 49, 855-864, 1977. -63-67. Clarke, B.J., and Housman, D.: Characterization of an e r y t h r o i d precursor c e l l of high p r o l i f e r a t i v e a c t i v i t y i n normal human per i p h e r a l blood. Proc. Natl. Acad. S c i . USA 74, 1104-1109, 1977. 68. Aye, M.T.: I d e n t i f i c a t i o n of the factor i n leukocyte conditioned medium able to enhance human erythroid colony growth. (Abst.) Blood 50 (Suppl. 1), 122, 1977. 69. Johnson, G.R., and Metcalf, D..: Pure and mixed e r y t h r o i d colony formation in vitro stimulated by spleen c e l l conditioned medium with no detectable e r y t h r o p o i e t i n . Proc. Natl. Acad. S c i . USA 74_, 3879-3882, 1977. 70. Humphries, R.K., Eaves, A.C. , and Eaves, C.J.: C h a r a c t e r i z a t i o n of a p r i m i t i v e e r y t h r o p o i e t i c progenitor found i n mouse marrow before and a f t e r several weeks i n c u l t u r e . Blood 53_, 746-763, 1979. 71. Fauser, A.A., and Messner, H.A.: Granulo-erythropoietic colonies i n human bone marrow, p e r i p h e r a l blood, and cord blood. Blood 52, 1243-1247, 1978. 72. Eaves, C.J., Humphries, R.K. , and Eaves, A.C: In vitro character-i z a t i o n of e r y t h r o i d precursor c e l l s and the e r y t h r o p o i e t i c d i f f e r -e n t i a t i o n process. In.Stamatoyannopoulos, G., and Nienhuis, A.W. (Eds.): C e l l u l a r and Molecular Regulation of Hemoglobin Switching. New York, Grune and Stratton, 1979, pp. 251-273. 73. Moore,, M.A.S., Williams, N., and Metcalf, D.: P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of the in vitro colony forming c e l l in.monkey hemopoietic t i s s u e . J . C e l l . P h y s i o l . 7_9, 283-292, 1972. 74. Cormack, D.H. : Time-lapse c h a r a c t e r i z a t i o n ..of e r y t h r o c y t i c colony-forming c e l l s i n plasma c u l t u r e s . Exp. Hemat. 4, 319-327, 1976. 75. Pluznik, D.H., and Sachs, L. : The cloning of normal "mast" c e l l s i n t i s s u e c u l t u r e . J . C e l l . Comp. Physiol. 66, 319-324, 1965. 76. Hozier, J.C., and Lindquist, L.L.: Banded karyotypes from bone marrow: A c l i n i c a l u s e f u l approach. Hum. Genet. 53, 205-209, 1980. 77. Rowley, J.D.: Nonrandom chromosome changes i n leukemia. Presented at the 33rd Annual Symposium on Fundamental Cancer Research, U n i v e r s i t y of Texas System Cancer Center and the M.D. Anderson H o s p i t a l and Tumor I n s t i t u t e , 1980. 78. Chervenick, P.A., E l l i s , L.D., Pan, S.F., and Lawson, A.L.: Human leukemic c e l l s : In vitro growth of colonies containing the P h i l a d e l p h i a (Ph 1) chromosome. Science 174, 1134-1136, 1971. 79. Prchal, J.F., and Axelrad, A.A.: Bone marrow response i n polycythemia vera. N. Engl. J . Med. 289, 1382, 1974. -64-80. Eaves, A.C., and Eaves, C.J.: Erythropoietin-independent colony formation i n 50 patients diagnosed as primary polycythemia and i n 50 p a t i e n t s diagnosed as secondary polycythemia. In preparation, 1980. 81. Eaves, A.C., and Eaves, C.J.: Erythropoietin-independent colony formation i n patients diagnosed as chronic myelogenous leukemia, e s s e n t i a l thrombocytosis or u n s p e c i f i e d chronic m y e l o p r o l i f e r a t i v e disease. In preparation, 1980. 82. Moorhead, P.S., Nowell, P.W., Mellman, W.J., Batipps, D.M., and Hungerford, D.A. :- Chromosome preparations of leucocyte cultured from human peripheral blood. Exp. C e l l . Res. 20_, 613-616, 1960. 83. Chandhuri, J.P., Vogel, W., Voiculescu, I., and Wolf, U.: A s i m p l i -f i e d method of demonstrating Giemsa band patterns i n human chromo-somes. Humangenetik 14_, 83-84, 1971. 84. Caspersson, T., Zech, L., and Johanssen, C.: D i f f e r e n t i a l binding of a l k y l a t i n g fluorochromes i n human chromosomes. Exp. C e l l . Res. 60, 315-319, 1970. 85. Latt, S.A., and Schreck, R.R.: S i s t e r chromatid exchange a n a l y s i s . Am. J . Hum. Genet. 32./ 297-313, 1980. 86. Goto, K., Akematsu, T., Shimazu, H., and Sugiyama,. T.: Simple d i f f e r e n t i a l Giemsa s t a i n i n g of s i s t e r chromatids a f t e r treatment with photosensitive dyes and exposure to l i g h t and the mechanism of s t a i n i n g . Chromosoma 53^ , 223-230, 1975. 87. Kato, H.: Mechanisms f o r s i s t e r chromatid exchanges and t h e i r r e l a -t i o n to the. production of chromosomal aberrations. .Chromosoma 59, 179-191, 1977. " 88. C a r s t a i r s , K.: Transformation of the small lymphocyte i n c u l t u r e . Lancet i i , 984, 1961. 89. Schneider, E.L., and Gilman, B.: S i s t e r chromatid exchanges and aging. I I I . The e f f e c t of donor age on mutagen-induced s i s t e r chromatid exchange i n human d i p l o i d f i b r o b l a s t s . . Hum. Genet. 46, 57-63, 1979. 90. Kakati, S., Abe, S., and Sandberg, A.A.: S i s t e r chromatid exchange i n P h i l a d e l p h i a chromosome ( P h 1 ) - p o s i t i v e leukemia. Cancer Res. 38, 2918-2921, 1978. 91. Krantz, S.B., and Jacobson, L.O.: E r y t h r o p o i e t i n and the Regulation of E r y t h r o p o i e s i s . Chicago, U n i v e r s i t y of Chicago Press, 1970. 92. Dube, I.D.: The incidence of s i s t e r chromatid exchange i n cultured human lymphocytes. A t h e s i s submitted i n p a r t i a l f u l f i l m e n t of the requirements for the degree of Bachelor of Science i n Zoology. The U n i v e r s i t y of B r i t i s h Columbia, 1977, pp. 1-31. -65-93. Aye, M.T., T i l l , J.E., and McCulloch, E.A.: C y t o l o g i c a l studies of granulopoietic colonies from two patients with chronic myelogenous leukemia. Exp. Hemat. 1, 115-118, 1973. 94. Chervenick, P.A., E l l i s , L.D., Pan, S.F., and Lawson, A.L.: Human leukemic c e l l s : In vitro growth of colonies containing the P h i l a d e l p h i a (Ph 1) chromosome. Science 174, 1134-1136, 1971. 95. Moore, M.A.S., and Metcalf, D.: Cytogenetic a n a l y s i s of human acute and chronic myeloid leukemic c e l l s cloned i n agar c u l t u r e . Int. J . Cancer 11, 143-152, 1973. 96. B u l l , J . : Cytogenetic studies of marrow and p e r i p h e r a l blood granulo-cyte c o l o n i e s i n treated chronic myelogenous leukemia. Blood C e l l s 1^, 161-162, 1975. 97. Rajendra, B.: Personal communication. 98. Yunis, J . J . , and Chandler, M.E.: High-resolution chromosome a n a l y s i s i n c l i n i c a l medicine. In S t e f a n i n i , M., and Hossaini, A. (Eds.): Progress i n C l i n i c a l Pathology, V o l. VII. New York, Grune and Stratton, 1977, pp. 267-288. 99. Rjz5nne, M. , Vang Nielsen, K. , and Mogens, E. : E f f e c t of c o n t r o l l e d colcemid exposure on human metaphase chromosome stru c t u r e . Hereditas 91, 49-52, 1979. . • -100. Zieve, G.W., Turnbull, D., Mullins, J.M., and Mcintosh, I.R.: Production of large numbers of m i t o t i c mammalian c e l l s by the use of the r e v e r s i b l e microtubule i n h i b i t o r hocodazole. Exp. C e l l . Res. 126, 397-405, 1980. -66-APPENDIX 1A Procedure for the Preparation of Hemopoietic Stem C e l l s f o r P l a t i n g  From Fresh Bone Marrow Aspirate A l l manipulations of human c e l l s were c a r r i e d out i n a laminar flow Biogard hood under s t e r i l e conditions. A l l apparatus which came into d i r e c t contact with human c e l l s were autoclaved before they were discarded. Procedure: 1) Measure the t o t a l volume of marrow using a s t e r i l e c a l i b r a t e d p i p e t t e . 2) Remove 0.1 ml of as p i r a t e and use t h i s to do a s t a r t i n g nucleated c e l l count on the hemocytometer (magnification:' lOOx). 3) Using a s t e r i l e Pasteur p i p e t t e , t r a n s f e r the e n t i r e sample to a 17 x100 mm p l a s t i c tube (Falcon 2057) and spin the specimen at 1,000 rpm for 4 minutes at room temperature (LEC HN-S c e n t r i f u g e ) . 4) Remove the buffy coat with a s t e r i l e Pasteur p i p e t t e and t r a n s f e r to another Falcon 2057 tube. 5) Let the tube stand at room temperature f o r 10-20 minutes to f a c i l i -t ate sedimentation of r e s i d u a l red c e l l s . 6) - Transfer the e n t i r e contents of the tube, except for the red c e l l button, to another Falcon 2057 tube, and add enough 2% f e t a l c a l f serum i n a-medium (2% FCS) to b r i n g the volume up to 10 ml. 7) Spin the specimen at 950 rpm f o r 10 minutes at 4°C (Sorvall RC-3 automatic, r e f r i g e r a t e d c e n t r i f u g e ) . 8) Pour o f f the supernatant and resuspend i n 2% FCS to give a volume of 10 ml. 9) Centrifuge as i n step 7. 10) Pour o f f the supernatant and resuspend the specimen i n 2% FCS to give a f i n a l volume of 3 ml. 11) Remove 0.1 ml of the c e l l suspension and use t h i s to do a f i n a l c e l l count on the hemocytometer. 12) D i l u t e the c e l l s i n 2% FCS ( f i n a l concentration of 2x 10 6 c e l l s / m l ) . -67-APPENDIX IB Procedure f o r the Preparation of Hemopoietic Stem C e l l s f o r P l a t i n g  From P e r i p h e r a l Blood A l l manipulations of human c e l l s were c a r r i e d out i n a laminar flow Biogard hood under s t e r i l e conditions. A l l apparatus which came into d i r e c t contact with human c e l l s were autoclaved before they were discarded. Procedure: 1) Measure the t o t a l volume of blood using a s t e r i l e c a l i b r a t e d p i p e t t e . 2) Remove 0.1 ml of blood and use t h i s to do a s t a r t i n g nucleated c e l l count on the hemocytometer (magnification: lOOx). 3) Gently layer 10 ml of blood on top of 15 ml of lymphocyte separa-t i o n medium (Bionetics.8410-01) i n a s t e r i l e 50 ml c o n i c a l tube (Falcon 2070). 4) Centrifuge the specimen at 1,700 rpm f o r 30 minutes at room temperature (LEC HN-S c e n t r i f u g e ) . 5) Using a Pasteur p i p e t t e , c a r e f u l l y a s p i r a t e o f f and di s c a r d the plasma l a y e r down to 2 mm above the lymphocyte la y e r . 6) Transfer the lymphocyte layer (3 to 5 ml) to a Falcon 2057 tube and add 2% FCS to give a f i n a l volume of 10 ml. 7) Spin the specimen at 950 rpm f o r 10 minutes at 4°C (So r v a l l RC-3 automatic, r e f r i g e r a t e d c e n t r i f u g e ) . 8) Pour o f f the supernatant and resuspend the p e l l e t i n 10 ml 2% FCS. 9) Spin as i n step 7. 10) Pour o f f the supernatant and resuspend the p e l l e t i n 1 ml of 2% FCS. 11) Remove 0.1 ml of the c e l l suspension and use t h i s to do a f i n a l , c e l l count on the hemocytometer. 12) D i l u t e the c e l l s i n 2% FCS to give a f i n a l concentration of 4 x 10 6 c e l l s / m l . -68-APPENDIX 1C Procedure for the Preparation of the Culture Medium f o r Hemopoietic Stem C e l l s Medium was made up i n batches of 102 ml. Medium was frozen i n 17x100 mm Falcon 2057 s t e r i l e disposable p l a s t i c tubes. 2.7 ml of medium was aliquoted per tube. For p l a t i n g , the tubes were thawed, and 0.3 ml of the f i n a l c e l l suspension was added to each tube. Af t e r mixing.by vortex, 1.1 ml of the medium containing c e l l s were plated per 35x10 mm standard dish (Lux #5221-R) using p l a t i n g needles (Monojet 202, 15 gauge, lh", blunt, on standard 3 cc syringes). To prepare 102 ml of culture medium: * 40 ml 2.2% methylcellulose (Dow Chemicals) 1 ml L-glutamine 29.2 mg/ml (General Biochemicals 10510) 1 ml 10~ 2 M mercaptoethanol (Baker Chemical Co. 08865) 30 ml f e t a l c a l f serum (one l o t used throughout) 10 ml bovine serum albumin (Sigma A 4503)^ 10 ml human leukocyte conditioned medium 10 ml e r y t h r o p o i e t i n at 27.5 units/ml (epo, Step I I I , Connaught Laboratories) * 2.2% methylcellulose was made i n batches of 2 l i t r e s according to the routine protocol used i n the laboratory of.Dr. Connie Eaves (70). ** Prepared according to Gregory and Eaves (34). Procedure: J 1) Mix 22 grams of Dow standard grade methylcellulose with 22 grams of Dow premium - grade m e t h y l c e l l u l o s e . Autoclave. 2) Add the mixture of methylcellulose grades to 1 l i t r e of autoclaved double d i s t i l l e d water at 80°C i n a 2 - l i t r e Erlenmeyer f l a s k . Use continuous s t i r r i n g by magnetic s t i r r e r while adding the powder. 3) B o i l mixture for 1 minute and l e t cool to room temperature. 4) Add 1 l i t r e of 2 x a-medium at 37°C (Connaught Laboratories). 5) S t i r at 4°C overnight to complete c l a r i f i c a t i o n process. 6) Dispense 40 ml a l i q u o t s and l e t stand at 20°C f o r 2 weeks as a s t e r i l i t y check. 7) 8) Freeze the a l i q u o t s f or 24 hours, then thaw i n the r e f r i g e r a t o r . Store at 4°C. 

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