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Analysis of the spleen colony-forming ability and lympho-myeloid repopulating ability of adherent marrow Henry, Elizabeth Anne 1984

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ANALYSIS OF THE SPLEEN COLONY-FORMING ABILITY AND LYMPHO-MYELOID REP OP UL ATING ABILITY OF ADHERENT MARROW by ELIZABETH ANNE HENRY B.Sc., The Univ e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Medical Genetics We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1984 ©Elizabeth Anne Henry, 1984 In 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 the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the 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 f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of 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 not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT In an attempt to characterize d i f f e r e n t populations of p r i m i t i v e hemopoietic stem c e l l s , the spleen colony forming and in vivo lympho-myeloid repopulating a b i l i t y of bone marrow c e l l s which adhere to p l a s t i c was assessed. The property of adherence was chosen f o r evaluation because i t had previously been reported that adherent stem c e l l s have greater self-renewal capacity than non-adherent stem c e l l s (1). When adherent marrow was assessed f o r spleen colony-forming a b i l i t y , i t contained 2 to 3 f o l d fewer day 10 CFU-S than fresh marrow but a r e l a t i v e l y increased myeloid repopulating a b i l i t y . These r e s u l t s are consistent with evidence that high self-renewal stem c e l l s form spleen colonies that do not become macroscopically v i s i b l e u n t i l 12 to 14 days post-transplantation (2). The r e s u l t s also suggested that adherence d i d not enrich f o r stem c e l l s with long-term T lymphoid repopulating a b i l i t y , although t h i s may have been generally compromised by minor h i s t o c o m p a t i b i l i t y differences between the donor and host s t r a i n s used. These studies thus provide further evidence of heterogeneity amongst p r i m i t i v e pluripotent marrow progenitor c e l l s . Adherence separation appears to enrich f o r a sub-population that does not form spleen colonies within 10 days, but has superior myeloid repopulating p o t e n t i a l . - i i i -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES V LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 1. Overview of Hemopoiesis 2 2. Spleen Colony Assay and the Concept of the Hemopoietic 3 Stem C e l l 3. Regulation of the Hemopoietic Stem C e l l 4 a. T r a n s i t i o n from Resting Phase to Active Cycle 4 b. Decision Between Self-renewal and D i f f e r e n t i a t i o n 6 c. Choice Between Alternate Pathways of D i f f e r e n t i a t i o n 9 4. ^n V i t r o Colony Assays 10 5. Present Studies 14 MATERIALS AND METHODS 16 1. Mice 16 2. Mouse I r r a d i a t i o n 16 3• Preparation of Bone Marrow C e l l Suspensions and 16 In V i t r o Adherence Separation 4. CFU-S Assay 19 5. Chimera Production and Assessment 19 - i v -Page 7. Antibody Staining 21 8. Antibody V a l i d a t i o n 24 RESULTS 26 1. General Experimental Design 26 2. Enumeration of CFU-S i n Adherent and Normal Bone Marrow 29 3. Hemopoietic Repopulation of L e t h a l l y Irradiated C3H Mice with B10.BR Adherent or Unseparated Bone Marrow 29 a. Repopulation of the Spleen 29 b. Repopulation of the Bone Marrow 34 c. Repopulation of the Thymus 38 DISCUSSION AND CONCLUSIONS 45 REFERENCES 48 - V -LIST OF TABLES Table Table I Murine Hemopoietic Progenitors and t h e i r In V i t r o Colony Stimulating Factors For Mouse. II Allotyope and C e l l Lineage S p e c i f i c i t i e s of Anti-Thy-1.2, Anti-Lyt-1, Anti-Lyt-2 and Anti-H9/25 Monclonal Antibodies. Page 12 22 Table I II Number of Fluorescent C e l l s i n A r t i f i c i a l Combinations of C3H and B10.BR C e l l s from the Thymus and Bone Marrow Stained with Strain S p e c i f i c Monoclonal Antibodies. 25 Table IV Comparison of the Mean Number of Spleen Colonies Produced i n Groups of 8 Mice Transplanted with Fresh or Adherent Marrow. 30 Table V Total Number of C e l l s i n the Bone Marrow i n Mice Trans-planted with Adherent and Fresh Marrow. 39 Table VI Total Number of C e l l s i n the Thymus i n Mice Transplanted with Adherent and Fresh Marrow. 44 - v i -LIST OF FIGURES Page Figure 1. Hierarchy of hemopoiesis. 13 Figure 2. Light micrograph of B10.BR adherent bone marrow c e l l s a f t e r 24 hours i n long term c u l t u r e . 18 Figure 3. General experimental design. 27 Figure 4. Experimental plan. 28 Figure 5. Gross appearance of the spleens of ^rradiated C3H mice ingected 10 days previously with 10 fresh (top) and 10 adherent (bottom) B10.BR marrow c e l l s . 31 Figure 6. K i n e t i c s of spleen repopulation a f t e r i r r a d i a t e d C3H mice ar^e i n j e c t e d with 10 B10.BR adherent (top panel) and 10 B10.BR fresh (bottom panel) marrow c e l l s . 32 Figure 7. K i n e t i c s of spleen repopulation a f t e r i r r a d i a t e d C3H mice are i n j e c t e d with 3 x 10 B10.BR adherent marrow c e l l s . 33 Figure 8. K i n e t i c s of H9/25 p o s i t i v e granulocyte repopulation of 2 femurs a f t e r i r r a d i a t e d C3H mice are i n j e c t e d with 10 B10.BR adherent (top panel) and 10 B10.BR fr e s h (bottom) marrow c e l l s . 35 Figure 9. K i n e t i c s of H9/25 p o s i t i v e granulocyte repopulation of 2 femurs a f t e r i r r a d i a t e d C3H mice are i n j e c t e d with 3 x 10 B10.BR adherent marrow c e l l s . 36 Figure 10. Phase contrast micrograph and fluoresence micrograph of a bone marrow c e l l stained with an^i-H9/25 from an i r r a d i a t e d C3H mouse i n j e c t e d with 10 B10.BR adherent 37 marrow c e l l s 5 weeks post-transplantation. Figure 11. K i n e t i c s of Lyt-1 (top panel) and Lyt-2 (bottom panel) p o s i t i v e donor (closed symbols) and r e c i p i e n t (open symbols) thymocyte repopulation a f t e r i r r a d i a t e d C3H mice are i n j e c t e d with 10 B10.BR adherent marrow c e l l s . 40 Figure 12. K i n e t i c s of Lyt-1 (top panel) and Lyt-2 (bottom panel) p o s i t i v e donor (closed symbols) and r e c i p i e n t (open symbols) thymocyte repopulation a f t e r i r r a d i a t e d C3H mice are i n j e c t e d with 10 B10.BR fresh marrow c e l l s . 41 - v i i -LIST OF FIGURES Page Figure 13. K i n e t i c s of Lyt-1 (top panel) and Lyt-2 (bottom panel) p o s i t i v e donor (closed symbols) and r e c i p i e n t (open symbols) thymocyte repopulation a f t e r i r r a d i a t e d C3H mice are i n j e c t e d with 3 x 10 B10.BR adherent marrow c e l l s . 42 Figure 14. Phase contrast micrograph (top) and fluoresence micrograph (bottom) of a thymocyte stained with a n t i - ^ Lyt-1.2 from an i r r a d i a t e d C3H mouse i n j e c t e d with 10 B10/BR adherent marrow c e l l s 5 weeks post-transplan-t a t i o n . 43 - v i i i -ACKNOWLEDG EMENTS I would l i k e to express my sincerest gratitude: to my supervisor, Dr. C.J. Eaves (Department of Medical Genetics and Terry Fox Lab, B.C. Cancer Research Centre), and her research c o l l a b o r a t o r , Dr. F. Takei (Department of Pathology and B.C. Cancer Research Centre), f o r t h e i r continual guidance and f a i t h f u l support throughout my project; to members of my advisory committee, Dr. P.A. Baird (Department of Medical Genetics), Dr. S. Wood (Department of Medical Genetics), Dr. F. Takei (Department of Pathology) f o r t h e i r h e l p f u l counsel and encouragement; to a post-doctoral fellow and research collaborator, Dr. D. Kerk f o r h e l p f u l discussions and p r a c t i c a l assistance e s p e c i a l l y i n the i n v i t r o studies described i n my project; to the s t a f f and students of the B r i t i s h Columbia Cancer Research Centre f o r providing an i n s p i r i n g s c i e n t i f i c environment i n which to t r a i n ; to Don Henkelman, f o r h i s guidance with the s t a t i s t i c a l analysis performed i n my project and L e s l i e Frego f o r her assistance i n computer typing of t h i s manuscript; and f i n a l l y to my family; e s p e c i a l l y to my brother David for his timeful assistance i n the preparation of the f i n a l d r a f t s of t h i s manuscript and to my parents f o r t h e i r l o v i n g support throughout my project. - 1 -INTRODUCTION 1. Overview of Hemopoiesis The hemopoietic system functions to maintain a constant supply of the various types of blood c e l l s . Mature blood c e l l s constitute the major portion of hemopoietic c e l l s and include red c e l l s , granulocytes, monocytes, p l a t e l e t s and lymphocytes. These c e l l s are highly d i f f e r e n t i a t e d f o r performance of s p e c i f i c functions as revealed by t h e i r c h a r a c t e r i s t i c morphology and s t a i n i n g . With the exception of lymphocytes they have f i n i t e , r e l a t i v e l y short l i f e - s p a n s and are incapable of c e l l d i v i s i o n n e c e s s i t a t i n g t h e i r continual replacement from l e s s d i f f e r e n t i a t e d precursor c e l l s . The myeloid precursors of red c e l l s , granulocytes and p l a t e l e t s by d e f i n i t i o n are derived i n the adult bone marrow from progenitor c e l l s "committed" to d i f f e r e n t i a t e along each pathway. These committed progenitor c e l l s divide to a c e r t a i n extent but do not reproduce themselves and so also must ultimately be replaced. Evidence for a more p r i m i t i v e c e l l with extensive p r o l i f e r a t i v e a b i l i t y and self-renewal capacity came from studies where the l i v e s of l e t h a l l y i r r a d i a t e d mice were spared by the transplantation of a minimal number of bone marrow c e l l s (3,4). The c e l l s responsible for repopulating the i r r e p a r a b l y damaged hemopoietic system were therefore termed stem c e l l s . Such hemopoietic stem c e l l s have been shown i n chromosome and enzyme marker studies to have the p o t e n t i a l to d i f f e r e n t i a t e along lymphoid as well as myeloid pathways. In the mouse, evidence f o r a plu r i p o t e n t stem c e l l came from karyotype analysis of stem c e l l d e f i c i e n t (W/WV) mice transplanted with hemopoietic c e l l s c a r r y i n g unique radiation-induced chromosome markers. The same markers were found i n both myeloid c e l l s as well as T lymphocytes, - 2 -implying the existence of a lympho-myeloid stem c e l l (5). The existence of a p l u r i p o t e n t i a l hemopoietic stem c e l l has also been i n f e r r e d from i t s apparent transformation i n several diseases. In chronic myelogenous leukemia a l l transformed c e l l s i n c l u d i n g red c e l l s , granulocytes, p l a t e l e t s and some B 1 lymphocytes, bear the abnormal Ph (Philadelphia) chromosome (6,7). Female patients with t h i s disorder, as well as other myeloproliferative disorders, who are heterozygous for the X-linked glucose-6-phosphate (G-6-PD) A:B isozymes, show only a single isozyme type i n t h e i r hemopoietic c e l l s (8,9). Furthermore i n one case of s i d e r o b l a s t i c anemia only one isozyme type was demonstrated i n both B and T lymphoid c e l l s as well as myeloid c e l l s (10). These studies i n d i c a t e that i n both man and mouse there e x i s t s a pluripotent hemopoietic stem c e l l capable of d i f f e r e n t i a t i n g i n t o both the myeloid and lymphoid c e l l compartments. The hemopoietic system i s thus l i k e other c e l l renewal systems such as the skin and gut (11,12) i n i t s compartmentalization of stem c e l l s and t h e i r more d i f f e r e n t i a t e d progeny. The e s s e n t i a l difference i s that i n skin and gut c e l l development occurs i n an oriented structure so that the stem c e l l compartment can be morphologically v i s u a l i z e d i n s i t u . In the marrow the stem c e l l s are d i s t r i b u t e d throughout. The e s s e n t i a l s i m i l a r i t y i s that stem c e l l s i n both instances must r e t a i n an appropriate balance between self-renewal and d i f f e r e n t i a t i o n i n order to maintain a steady supply of the f u n c t i o n a l l y mature end c e l l s . 2. Spleen Colony Assay and the Concept of the Hemopoietic Stem C e l l The spleen colony assay was developed by T i l l and McCulloch i n 1961 (13), and represented the f i r s t q uantitative assay for hemopoietic stem c e l l s . Marrow c e l l s are i n j e c t e d intravenously i n t o l e t h a l l y i r r a d i a t e d mice. After - 3 -8 to 10 days, discrete macroscopic nodules composed of hemopoietic c e l l s can be seen on the spleen surface and a f t e r f i x a t i o n of the spleen can be r e a d i l y enumerated. That each spleen colony represented the c l o n a l growth of a single hemopoietic stem c e l l (CFU-S) was shown using c e l l s bearing unique r a d i a t i o n -induced chromosome markers (14). Spleen colonies were found to range i n s i z e 5 7 from 10 to 10 c e l l s showing that the CFU-S has extensive a b i l i t y to p r o l i f e r a t e (15). An a b i l i t y of the CFU-S to regenerate i t s e l f was shown by the demonstration of c e l l s i n spleen colonies that were able to generate secondary spleen colonies (16). The d i f f e r e n t i a t i o n p o t e n t i a l of the CFU-S was f i r s t studied by microscopic examination of the v a r i e t y of mature c e l l types i n i n d i v i d u a l spleen colonies. M u l t i l i n e a l d i f f e r e n t i a t i o n i n t o erythrocytes, granulocytes, monocytes and megakaryocytes demonstrated that the CFU-S was plur i p o t e n t f or a l l of the myeloid lineages (17,18,19). Mature lymphocytes have not been observed i n spleen colonies, although there has been evidence both f o r (20,21,22) and against (23) the presence of less mature lymphoid precursor c e l l s . The progeny of a single c e l l have been shown to be able to completely repopulate a l l of the hemopoietic organs of a l e t h a l l y i r r a d i a t e d animal i n c l u d i n g the lymphoid tissues (20). However, i t i s not yet c l e a r whether t h i s c e l l i s detected by the CFU-S assay. Nevertheless the properties of extensive p r o l i f e r a t i v e capacity, self-renewal capacity and m u l t i l i n e a l myeloid d i f f e r e n t i a t i o n capacity held by the CFU-S, have h i s t o r i c a l l y l e d to the acceptance of t h i s assay to enumerate hemopoietic stem c e l l s . The spleen colony assay has thus allowed many properties of hemopoietic stem c e l l s to be investigated, although i t i s not without some inherent problems. For instance unique chromosome markers have been found i n more than one spleen colony (24). In addition the number of spleen colonies found on - 4 -the spleen does not represent the t o t a l number of CFU-S i n j e c t e d since not a l l migrate to the spleen (16), and some might not remain i n the spleen (25). The number of CFU-S which migrate to the spleen has been termed the seeding f r a c t i o n ( f ) (16). Attempts to determine the value of (f) have yielded values ranging from 0.03 (26) to 0.24 (16). Thus there i s some inaccuracy i n the hemopoietic stem c e l l estimate depending on the value assumed for the seeding f r a c t i o n . F i n a l l y , v a r i a t i o n i n the value of (f) accompanies changes i n c y c l i n g status, as for example following treatment of animals with various drugs (19). 3. Regulation of the Hemopoietic Stem C e l l a. T r a n s i t i o n from Resting Phase to Active Cycle In 1961 , Becker demonstrated that a minimal proportion of the CFU-S i n normal mice were k i l l e d by a short exposure to high s p e c i f i c a c t i v i t y H-thymidine, a cytotoxic drug which i s taken up e x c l u s i v e l y by c e l l s i n the DNA synthetic phase, S, of the c e l l c ycle (27). This f i n d i n g was subsequently confirmed by others (19) using d i f f e r e n t S-phase s p e c i f i c drugs, such as hydroxyurea, and led to the conclusion that i n the normal mouse, most CFU-S are i n a r e s t i n g G Q phase. Major hemopoietic perturbations i n c l u d i n g sublethal i r r a d i a t i o n (27), bleeding (28), administration of cycle active cytotoxic drugs (29,30), or endotoxin (31) were shown to t r i g g e r most of the CFU-S into c y c l e . However not a l l manipulations e l i c i t e d t h i s response; for example, administration of erythropoietin which regulates red blood c e l l d i f f e r e n t i a t i o n f a i l e d to induce c y c l i n g of CFU-S (32). Evidence that these c e l l cycle t r a n s i t i o n s are c o n t r o l l e d by both l o c a l a c t i n g mechanisms and long range humoral factors has been obtained. - 5 -In a study with p a r t i a l l y i r r a d i a t e d mice, G i d a l i and Lajtha (33), found CFU-S i n the shielded areas were stimulated i n t o c e l l cycle 4 hours a f t e r i r r a d i a t i o n but l a t e r became quiescent even though CFU-S i n i r r a d i a t e d areas were low i n number and s t i l l c y c l i n g . I t was proposed that the protected CFU-S migrated (34,35,36) or d i f f e r e n t i a t e d at an increased rate (33) i n response to long range humoral factors r e s u l t i n g i n a depletion of CFU-S i n the shielded areas and tr a n s i e n t l o c a l stimulation of CFU-S p r o l i f e r a t i o n (37,38,39). Further attempts to i d e n t i f y the molecular mediators of the l o c a l marrow response l e d to the demonstration of two separable a c t i v i t i e s i n bone marrow conditioned media by Lord and co-workers (40,41,42). These a c t i v i t i e s could e i t h e r stimulate or i n h i b i t the d i v i s i o n of CFU-S i n v i t r o i n a r e v e r s i b l e manner. I t has also been found that activated lymphocytes produce a fa c t o r (Interleukin-3 or 11-3, (43)) that can stimulate r e s t i n g CFU-S in t o S-phase (44). However whether any of these l a t t e r f a c t o r s have p h y s i o l o g i c a l s i g n i f i c a n c e i s not yet known. The f i r s t humoral type f a c t o r s shown to be able to t r i g g e r normal CFU-S into S-phase i n v i t r o were the -adrenergic agents (45,46). Subsequently histamine (47), c y c l i c nucleotides (48) and prostaglandin E (49) were shown to increase the number of CFU-S i n DNA synthesis i n c u l t u r e . The mechanism of action of these pharmacologic agents was postulated to be a c t i v a t i o n of the adenyl cyclase system v i a CFU-S c e l l - s u r f a c e receptors. b. Control of the Decision Between Self-renewal and D i f f e r e n t i a t i o n When a hemopoietic stem c e l l d ivides, i n theory i t may give r i s e ("birth"- 50) e i t h e r to two new stem c e l l s or to two daughters that are committed to d i f f e r e n t i a t e ultimately to "death", or to one of each. Two - 6 -models to explain how the choice between self-renewal and d i f f e r e n t i a t i o n i s regulated have been proposed. In the stochastic models the decision at the s i n g l e c e l l l e v e l has a random component that i s constrained i n a lax way independent of e x t r i n s i c f a c t ors such that the response of the c e l l can only be described by a f i n i t e p r o b a b i l i t y (50). In contrast according to deterministic models the choice i s e f f e c t i v e l y c o n t r o l l e d by the action of external s t i m u l i (51). Evidence f o r important factors both i n t r i n s i c to the stem c e l l and e x t r i n s i c to i t has come from studies of hereditary macrocytic anemias i n mice. In w/W mice stem c e l l s appear to have an i n t r i n s i c defect since when they are transplanted i n t o syngeneic W/WV or +/+ mice they are incapable of forming macroscopic spleen colonies (52). In contrast stem c e l l s from S l / S l d mice form normal spleen colonies, but S l / S l d mice cannot support the growth of CFU-S from +/+ mice (53) suggesting a defect i n the S l / S l ^ microenvironment. However, analysis of mechanisms that explain stem c e l l self-renewal behaviour i s complicated by evidence that normal stem c e l l s may be heterogeneous with respect to t h e i r self-renewal capacity. For example exposure of marrow to c e r t a i n cytotoxic drugs, has been found to leave a population of CFU-S that show an elevated self-renewal capacity (54). Thus, marrow c e l l s from mice treated with hydroxyurea or 5 - f l u o r o u r a c i l or both of these drugs, could outgrow normal c e l l s i n i r r a d i a t e d hosts. Such marrow c e l l s a lso had a greater capacity than normal c e l l s to repopulate the CFU-GM and megakaryocyte compartments i n spleens and marrows of i r r a d i a t e d hosts (55,56) and gave r i s e to i n d i v i d u a l spleen colonies containing more CFU-S than normal (57). In contrast, treatment of mice with the a l k y l a t i n g agent is o p r o p y l methane sulphate (IMS) appears to - 7 -s e l e c t for a low self-renewal CFU-S sub-population (58). In addition, self-renewal capacity has been shown to be higher i n bone marrow than i n blood (59), or spleen (60), and higher i n f e t a l l i v e r than i n bone marrow (61). Self-renewal of CFU-S also progressively declines a f t e r s e r i a l transplantation and can usually no longer be obtained a f t e r 6 s e r i a l i n  vivo t r a n s f e r s (62). These observations and the f a c t that high and low self-renewal CFU-S i n normal marrow can be p h y s i c a l l y separated (63) have suggested that hemopoietic stem c e l l s , i n c l u d i n g those assayable as CFU-S represent a much more heterogenous population than was o r i g i n a l l y appreciated. To account for the above observations, i t has been proposed that the p r o b a b i l i t y of self-renewal or d i f f e r e n t i a t i o n i s governed by the mitotic h i s t o r y of the stem c e l l and that the "youngest" stem c e l l s ( i . e . those who have divided the l e a s t ) are the most r e s i s t a n t to forces capable of r e c r u i t i n g stem c e l l s i n t o c y c l e . According to t h i s model, natural development would r e s u l t i n a hemopoietic stem c e l l compartment that i s comprised of a "continuum" of c e l l s with decreasing self-renewal capacity (64). In t h i s model, stem c e l l s having the greatest self-renewal capacity are the most i n a c t i v e . However recent transplantation experiments with Friend v i r u s - or s r c - i n f e c t e d c e l l s i n d i c a t e that CFU-S decline does not always occur (65,66) and t h i s r a i s e s questions about the general v a l i d i t y of the age-generation model. Heterogeneity i n the number of CFU-S i n i n d i v i d u a l spleen colonies was f i r s t observed by Siminovitch et a l . (16). This behavior could be explained by the stochastic model subsequently proposed by T i l l et a l . (50) although i t was postulated by Trentin and co-workers (51) that t h i s heterogeneity r e s u l t e d from stem c e l l s f a c i n g d i f f e r e n t microenvironments - 8 -within the spleen. Further data i n support of the stochastic model was provided more recently by the f i n d i n g that the number of CFU-S varied i n i n d i v i d u a l p l u r i p o t e n t i a l stem c e l l derived colonies generated i n d i l u t e semi-solid suspension cultures i n v i t r o (67). In addition, i t was further shown that the number of secondary stem c e l l s detectable i n v i t r o that were found i n primary i n v i t r o stem c e l l colonies also varied (68,69). This heterogeneity i n self-renewal capacity has been accounted f o r i n an expanded version of the stochastic model of self-renewal where hemopoiesis i s maintained by a succession of c e l l clones (70). According to the c l o n a l succession model, fewer clones of hemopoietic stem c e l l s contribute to the d i f f e r e n t i a t i n g population i n any ins t a n t of time compared to that predicted by the age-structure hypothesis. Such models do not however exclude the p o s s i b i l i t y that e x t r i n s i c factors may moderate self-renewal p r o b a b i l i t i e s . I n t e r e s t i n g l y when marrow c e l l s enriched f o r high self-renewal capacity stem c e l l s by treatment with 5 - f l u o r o u r a c i l were assayed for spleen colony forming a b i l i t y they appeared to have d i f f e r e n t growth k i n e t i c s i n the host spleen when compared to stem c e l l s from normal untreated mice (56). Whereas the number of spleen colonies generated from normal marrow remained steady between 8 and 14 days, the number of spleen colonies apparent i n the spleens of mice i n j e c t e d with 5 - f l u o r o u r a c i l marrow was shown to increase markedly i n t h i s i n t e r v a l . The l a g i n spleen colony formation by very p r i m i t i v e stem c e l l s has been interpreted i n two d i f f e r e n t ways. The reasons proposed why stem c e l l s s u r v i v i n g 5 - f l u o r o u r a c i l are unable to d i r e c t l y i n i t i a t e colony growth are e i t h e r , that they must f i r s t migrate to the bone marrow and undergo developmental maturation (71) or that they migrate d i r e c t l y to the spleen but remain quiescent i n G phase f o r the f i r s t several days (72). - 9 -c. Regulation of the Choice Between Alternate Pathways of D i f f e r e n t i a t i o n Deterministic and stochastic models have a l s o have been proposed to explain how p l u r i p o t e n t hemopoietic stem c e l l s eventually become r e s t r i c t e d to a s i n g l e d i f f e r e n t i a t i o n pathway. In 1970, Curry and Trentin published the hemopoietic inductive microenvironmental (HIM) model which postulated that the pathway of hemopoietic stem c e l l d i f f e r e n t i a t i o n i s determined by s p e c i f i c inductive microenvironments (73,74). The basis f o r the model was the observation that early 8 to 9 day spleen colonies that contained a l l three myeloid c e l l lineages were usually larger than those that i n which only a s i n g l e lineage was found. The early small spleen colonies were thought to be a f f e c t e d by one microenvironment but as they p r o l i f e r a t e d and were exposed to more microenvironments, they were direc t e d to d i f f e r e n t i a t e along other pathways. Evidence contrary to t h i s model came from the work done by Gregory and co-workers (75) who showed that spleen colonies containing mature c e l l s belonging to only a single lineage could also contain p r i m i t i v e progenitors committed to a d i f f e r e n t lineage. Time course spleen mapping studies have further shown that l a t e appearing m u l t i l i n e a l spleen colonies do not represent the continued growth of i n i t i a l l y predominately u n i l i n e a l spleen colonies since these a r i s e as separate e n t i t i e s (2). These experiments suggest that t i s s u e microenvironments vivo do not determine the fate of i n d i v i d u a l p l u r i p o t e n t stem c e l l s , although they do not r u l e out the p o s s i b i l i t y that the microenvironment may a l t e r the p r o b a b i l i t y of commitment to a p a r t i c u l a r pathway. Another p o s s i b i l i t y i s that s p e c i f i c humoral f a c t o r s , such as erythropoietin may influence stem c e l l commitment (76). However, convincing evidence that p l u r i p o t e n t stem c e l l s can respond to erythropoietin has not been obtained (77,78). Such - 10 -c e l l s do show a transient p r o l i f e r a t i v e response to granulocyte-macrophage colony stimulating factor (GM-CSF), but the c e l l s produced are not thereby r e s t r i c t e d to the granulocyte-macrophage pathway (79,80). More recently, analysis of the content of large numbers of i n d i v i d u a l mixed colonies generated under a f i x e d set of conditions i n v i t r o i n the absence of any other c e l l types has revealed marked v a r i a t i o n i n the commitment behavior of pluripotent stem c e l l s (81). Such studies have provided renewed support that r e s t r i c t i o n of d i f f e r e n t i a t i o n p o t e n t i a l l i k e self-renewal i s u l t i m a t e l y c o n t r o l l e d at the s i n g l e c e l l l e v e l by a set of v a r i a b l e parameters that are i n t r i n s i c to the c e l l i t s e l f . 4. In V i t r o Colony Assays The e a r l i e s t recognizable blood c e l l precursors were once thought to a r i s e d i r e c t l y from the hemopoietic stem c e l l (82). Recent evidence suggests instead that there are many intervening c e l l d i v i s i o n s with accompanying loss of self-renewal capacity and d i f f e r e n t i a t i o n p o t e n t i a l before c e l l s with the f i r s t morphological signs of maturity appear. This evidence i s based on the development of i n v i t r o c l o n a l techniques which allow the c h a r a c t e r i s t i c s of a s i n g l e precursor c e l l to be analyzed by inference from the type(s) of daughter c e l l s i t produces. This assay involves suspension of hemopoietic c e l l s i n immobilizing viscous or semi-solid media containing appropriate nutrients, serum and growth stimulatory f a c t o r s . The f i r s t i n v i t r o colony assay was described i n the 1960*s i n two independent studies (83,84). This assay detects c e l l s from the bone marrow or spleen of mice that are able to produce colonies containing granulocytes and macrophages. These progenitors were i n i t i a l l y named i n v i t r o colony forming c e l l s (CFU-C). Subsequently, i n v i t r o colony assays detecting c e l l s committed to d i f f e r e n t i a t e down every myeloid - 11 -pathway have been described (85). Names and abbreviations applied to the progenitor c e l l s and examples of the i n v i t r o stimulatory factors they require are provided i n Table I. These colonies contain c e l l s purely of one lineage and are considered to be descendents of committed progenitors since they a r i s e i n cultures i n which more than one colony type may be obtained. The following properties have distinguished CFU-C from CFU-S i n the mouse: c e l l volume (63), c y c l i n g k i n e t i c s (86), and r a d i a t i o n s e n s i t i v i t y (87). B i o l o g i c a l , p h y s i c a l and s t a t i s t i c a l comparisons of progenitors of unilineage i n v i t r o colonies have shown that committed progenitors i n a l l of the myeloid lineages comprise a s e r i e s of d i s t i n c t c e l l types of decreasing p r o l i f e r a t i v e capacity (88,89,90). As pi c t u r e d i n the lineage diagram (Figure 1) colonies become smaller as the p r o l i f e r a t i v e capacity of t h e i r progenitors decreases, hence the concept of a hierarchy of p r i m i t i v e p l u r i p o t e n t stem c e l l s , committed progenitors and maturing blood c e l l s . Many studies have been performed i n d i c a t i n g that colonies are derived from s i n g l e CFC, i n c l u d i n g : 1) Microculture of sin g l e c e l l s with subsequent observation of colonies (91), 2) In mixing experiments a l l colonies produced from mixtures of male and female marrow are e n t i r e l y of one karyotype (92), 3) In v i t r o colonies from the marrow of G6PD heterozygotes contain only one isozyme type (93). Clonal c e l l techniques f o r m u l t i p o t e n t i a l hemopoietic progenitors have recently been devised by several groups of inve s t i g a t o r s (67,68, 94-97). These colonies t y p i c a l l y reveal terminal d i f f e r e n t i a t i o n of c e l l s i n more than two lineages i . e . granulocyte-erythrocyte-macrophage-megacaryocyte (GEMM). The exact stage of CFU-GEMM i n the hierarchy of hemopoiesis i s not known. - 12 -Table I . Hemopoietic Progenitors and In V i t r o Colony  Stimulating Factors f o r Mouse.* Progenitor Progenitor Name ** Day Colonies C e l l Class (Abbreviation) Counted*** Source of Presumed Stimulatory Factor**** Neutrophil and Macrophage Progenitors Eosinophil Progenitors CFU-C CFU-GM Eo-CFU Medium conditioned by f i b r o b l a s t tumor c e l l monolayers Mitogen stimulated mouse spleen c e l l conditioned medium Megakaryocyte Progenitors Meg-CFU Mitogen stimulated mouse spleen c e l l conditioned medium Ery t h r o i d Progenitors Day 8 BFU-E Day 3 BFU-E CFU-E 8 3 2 Erythropoietin Erythropoietin Erythropoietin *For o r i g i n a l references, see review by Metcalf (85). **Shown are the most conmmonly used abbreviations. CFU f o r colony forming u n i t and CFC f o r colony forming c e l l are used interchangeably. ***Colonies are usually of maximal s i z e and contain mature progeny at the time chosen f o r counting. ****Sources l i s t e d are commonly used examples. - 13 -ULTIMATE STEM CELL ERYTHR0ID PROGENITORS Primitive BFU-E large erythroid colony or burst Mature BFU-E 1 © • CFU-E small erythroid colony or burst erythroid cluster Lymphopoiesis self-renewal MYELOID STEM CELL (CFU-S. CFU-GEMS) mixed colony MEGAKARYOCYTE PROGENITORS C F U M large megakaryocytic colony small megakaryocytic colony GRANULOCYTE PROGENITORS CFU-C large granulocytic colony small granulocytic colony RED CELLS PLATELETS GRANULOCYTES 4 MACROPHAGES F i g u r e I Hi e r a r c h y of Hemopoiesis,(110) . - 14 -S i m i l a r i t i e s i n the sedimentation v e l o c i t i e s and p r o l i f e r a t i v e states of CFU-S and CFU-GEMM and a s i g n i f i c a n t c o r r e l a t i o n between the number of spleen colonies and the number of multi-lineage colonies obtained from i n d i v i d u a l spleen colonies (97), the f i n d i n g of CFU-S within CFU-GEMM colonies (67), and the s i m i l a r i t y i n t h e i r self-renewal c a p a c i t i e s (67,68), suggests that these two types of progenitors are c l o s e l y r e l a t e d populations. Evidence f o r an i n v i t r o colony-forming stem c e l l ( S - c e l l ) more p r i m i t i v e than the CFU-GEMM came rece n t l y from the i d e n t i f i c a t i o n and r e p l a t i n g studies of unique l a t e appearing hemopoietic colonies c o n s i s t i n g only of und i f f e r e n t i a t e d b l a s t c e l l s (98). Replating of these b l a s t colonies produced a large number of secondary b l a s t c e l l and GEMM colonies while r e p l a t i n g of primary GEMM colonies produced no secondary b l a s t colonies and only a few secondary GEMM colonies. 5. Present Studies Of continuing major i n t e r e s t i s the cha r a c t e r i z a t i o n of the regulatory mechanisms determining the behavior (self-renewal) of p r i m i t i v e hemopoietic stem c e l l s . An i n v i t r o culture method which enriches f o r p r i m i t i v e hemopoietic stem c e l l s was devised i n 1980 by Mauch (1). Bone marrow c e l l s were cultured for 1 week i n Dexter-type cultures (99) and those CFU-S that adhered to the culture d i s h had a greater self-renewal capacity than the non-adherent c e l l s as measured by s e r i a l t r a n s f e r and p r o l i f e r a t i v e capacity (Rg)« Subsequently, the self-renewal capacity of 24 hour adherent marrow was studied by i t s r e p l a t i n g p o t e n t i a l using i n v i t r o multipotent colony assays. E n t i r e dishes of primary i n v i t r o colonies derived from adherent or fresh bone marrow were replated and approximately 3 times more CFU-GEMM were produced by adherent compared to fresh marrow c e l l s (D. Kerk, unpublished r e s u l t s ) . - 15 -In the Introduction, I have presented evidence that there e x i s t s i n the marrow a p r i m i t i v e hemopoietic stem c e l l that has extensive a b i l i t y to p r o l i f e r a t e , self-renew and d i f f e r e n t i a t e along lymphoid as well as myeloid pathways. On the basis of studies suggesting that day 12 spleen colonies are derived from plu r i p o t e n t CFU-S capable of self-renewal (whereas the majority of day 8 spleen colonies were not), and that adherence s e l e c t s f o r a population of high self-renewal CFU-S and CFU-GEMM (possibly equivalent to S-c e l l s (Ogawa) (98), I sought to examine the hypothesis that stem c e l l s with lymphoid and myeloid p o t e n t i a l would e x h i b i t properties s i m i l a r to the c e l l i n normal marrow that makes spleen colonies detected on day 12. The experiments described i n t h i s study were therefore designed to determine whether adherent marrow c e l l s were r e l a t i v e l y depleted of day 10 CFU-S, but enriched f o r c e l l s that could repopulate both the myeloid and lymphoid portions of the hemopoietic system. - 16 -MATERIALS AND METHODS 1. Mice Two s t r a i n s of mice i d e n t i c a l only at t h e i r major h i s t o c o m p a t i b i l i t y l o c i were used as donors and r e c i p i e n t s i n CFU-S assays and f o r production of chimeras. Recipients were C3H/He mice obtained from Charles River Breeders as supplied by the Canadian Breeding Farms, St. Constant, Quebec. Donors were B10.BR mice, a s t r a i n congenic with C57B1/10 but carrying the H-2 instead of the H-2b, a l l e l e . B10.BR i s also g e n e t i c a l l y very s i m i l a r to the C57B1/6 mouse which i s a close r e l a t i v e of the C57B1/10 mouse. Donor mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. The age of mice used as r e c i p i e n t s and donors was 2 to 5 months. 2. Mouse I r r a d i a t i o n C3H/He r e c i p i e n t s f o r CFU-S determinations and production of chimeras were given 9 to 10 Gray (Gy) of 280 KVp X-rays (1.13 to 1.35 Gy/min.) i n 2 equal f r a c t i o n s separated by a 6 hour i n t e r v a l . During the i r r a d i a t i o n the mice were supplied with a continuous flow of a i r from an a i r pump. 3. Preparation of Bone Marrow C e l l Suspensions and In V i t r o Adherence  Separation To obtain bone marrow c e l l s with high self-renewal capacity f o r CFU-S determinations and chimera production i n v i t r o , adherence separation was employed (1). B10.BR mice were s a c r i f i c e d by c e r v i c a l neck d i s l o c a t i o n and 7 the marrow content of two femurs (approximately 2.0-3.0 x 10 c e l l s ) was - 17 -flushed using a 21g needle i n t o 1 ml of alpha-medium supplemented with 2% f e t a l c a l f serum. Nucleated c e l l s were counted following d i l u t i o n i n 3% a c e t i c a c i d (to lyse a l l red c e l l s ) using a hemocytometer. Cultures were set up e s s e n t i a l l y according to L. Coulombel et a l . (100) by adding 3.0 x 10 7 B10.BR fr e s h femoral bone marrow c e l l s to 60 mm t i s s u e culture dishes containing 8 ml of alpha-medium supplemented with f e t a l c a l f serum ( FCS, 12.5%. Flow la b o r a t o r i e s , Lot 2901111), horse serum (HS, 12.5%, GIBCO, -4 Lot33P2824) and 2-mercaptoethanol (10 M). Culture dishes were each placed i n a 100 mm p e t r i dish and incubated at 33 degrees C i n a humidified atmosphere of 5% CC>2 i n a a i r for 24 hours. This period i s le s s than that used by Mauch et a l . (1) but s u f f i c i e n t for adherence separation of high s e l f -renewal CFU-GEMM (D. Kerk, personal communication). Adherent c e l l s were harvested by mechanical scraping with a rubber policeman or by enzymatic detachment. For enzymatic detachment, f i r s t non-adherent c e l l s and growth medium were removed from the cultures. Then the cultures were washed with 2 ml of alpha-medium and 2 ml of t r y p s i n (Difco, 1:250, 2.5 g/1 c i t r a t e s a l i n e ) s e q u e n t i a l l y . The remaining adherent c e l l s were layered over with 5 ml of t r y p s i n and incubated for 10 minutes at 37 degrees C. Immediately a f t e r , 2 ml of FCS was added to the culture to terminate the t r y p s i n action. Vigorous p i p e t t i n g with a Pasteur pipette back and f o r t h across the culture dish 3 times i n d i f f e r e n t d i r e c t i o n s was employed to detach the adherent c e l l s . The dish was washed once with fresh HBSS-Ca-Mg to c o l l e c t any remaining adherent c e l l s . The adherent c e l l s were then washed i n HBSS-Ca-Mg supplemented with 5% FCS. Assessment of c e l l v i a b i l i t y by Nigrosin dye exclusion showed l e s s than 15% of the c e l l s stained. Each culture y i e l d e d 1 to 5 x 10^ adherent c e l l s . The appearance of the B10/BR adherent c e l l s a f t e r the nonadherent c e l l s have been removed i s shown i n Figure 2. - 18 -•5 . ' •t- m - i • ° • 0 0 • " •• • • a • -5 * • » • " . • Figure 2 Light micrograph of B10.BR adherent bone marrow c e l l s a f t e r 24 hours i n long term c u l t u r e . - 19 -For mechanical harvesting the nonadherent c e l l s and growth medium were removed from the cultures, which were then washed with 5 ml of alpha-medium and 2 ml HBSS-Ca-Mg. To each culture was added 4 ml HBSS-Ca-Mg and 2 ml FCS i n t o which the adherent c e l l s were scraped with a rubber policeman. Vigorous and repeated f l u s h i n g of the c e l l s and media was done with a Pasteur p i p e t t e . Then detached c e l l s were aspirated from the dish and the dish was rinsed once with HBSS-Ca-Mg with 5% FCS. The r e s u l t i n g c e l l suspension was washed once with HBSS-Ca-Mg with 5% FCS, by c e n t r i f u g i n g at 1200 rpm f o r 10 minutes. 4. CFU-S Assay Recipients were i r r a d i a t e d as described above and i n j e c t e d intravenously with t e s t c e l l s . Mice were s a c r i f i c e d 10 days l a t e r and t h e i r spleens removed and placed i n Bouin's f i x a t i v e (101). Then v i s i b l e macroscopic spleen colonies were counted under a 2x magnifying lens. 5. Chimera Production and Assessment Recipient C3H/He mice were i r r a d i a t e d as described above and then i n j e c t e d 4 5 intravenously with 3 x 10 or 10 fresh or adherent bone marrow c e l l s from histocompatible (H-2 ) but allogeneic B10.BR donor mice. Lymphoid and myeloid repopulation of the r e s u l t i n g chimeras was evaluated a f t e r 15, 25, 35 and 125 days. Animals were s a c r i f i c e d by c e r v i c a l neck d i s l o c a t i o n , the thymus, spleen and femurs removed, and c e l l suspensions prepared. The thymus and spleen were each placed i n a 100 mm p e t r i dish containing 1 ml of antibody s t a i n i n g medium ( RPMI 1640 medium supplemented with 10%FCS, 110 mM HEPES buffer, 100 units/ml p e n i c i l l i n and 100 jig/ml streptomycin). The c e l l s were then teased out of the surrounding capsule using a rounded p a i r of s c i s s o r s . - 20 -Since the thymus was usually small the r e s u l t i n g medium and t i s s u e was repeatedly aspirated approximately 20 times using a 1 ml syringe (without any needle attached) to ensure that a l l c e l l s were freed from the capsule. A 21 g needle and 3 ml syringe was then used to transfe r the spleen or thymic c e l l suspensions to 100mm tes t tubes. I f the thymus was large, the thymic suspensions were made up to 2 ml volumes, otherwise the f i n a l volume was kept to 1 ml. A l l spleen c e l l suspensions were made up to 8 ml volumes. Femoral marrow plugs were flushed out of the bone i n t o a 100 mm t e s t tube using a syringe with a 21 g needle containing 1.0 ml of antibody s t a i n i n g medium. To determine the t o t a l number of c e l l s i n each t i s s u e , a sample of 25 y l of each c e l l suspension was tra n s f e r r e d to another t e s t tube containing 25 y l of 3% a c e t i c a c i d . The r e s u l t i n g concentration of f i x e d c e l l s were determined using a hemocytometer. The t o t a l number of c e l l s per organ ( i . e . c e l l u l a r i t y ) was then obtained simply by mu l t i p l y i n g the concentration by the volume of the t o t a l c e l l suspension. - 21 -6. Antibody Staining Thymocytes and bone marrow c e l l s from chimeras were subjected to s t a i n i n g with a l l o - s p e c i f i c monoclonal antibodies to determine the proportion of donor and host components. The allotype and c e l l lineage s p e c i f i t i e s of the monoclonal antibodies used are shown i n Table I I . The composition of the thymus was analyzed using an i n d i r e c t binding assay. Into each well of a f l e x i b l e round-bottom m i c r o t i t r e plate was added 10 target c e l l s and 50 y l of ei t h e r of the following: antibody-staining medium, anti-Thy-1.2 s p e c i f i c f o r B10.BR and C3H thymocytes, anti-Lyt-1.1 s p e c i f i c f o r C3H T helper c e l l s , anti-Lyt-1.2 s p e c i f i c for B10.BR T helper c e l l s , anti-Lyt-2.1 s p e c i f i c f o r C3H T k i l l e r and suppressor c e l l s , and a n t i -Lyt-2 .2 s p e c i f i c f o r B10.BR T k i l l e r and suppressor c e l l s (104). The Lyt antibodies were purchased from Cedarlane Laboratories (Hornby, Ontario, Canada). Antibody-cell suspensions were incubated at 4 degrees C for 1 hour. The d i l u t i o n s of antibodies determined by t i t r a t i o n were as follows: 1/20 for anti-Thy-1.2, 1/5 for anti-Lyt-1.1, 1/80 for anti-Lyt-1.2, 1/5 for a n t i - L y t -2.1, 1/7 for anti-Lyt-2.2, and 1/16 for FITC-conjugated anti-H9/25. C e l l s were then washed 3 times by c e n t r i f u g i n g f o r 5 minutes at 1200 rpm and resuspended i n 150 y l of antibody s t a i n i n g medium three times. After the l a s t wash the c e l l s were incubated with 50 y l per well of the (Fab') 2 f r a c t i o n s of f l u o r e s c e i n isothyocynate-conjugated goat anti-mouse Ig antibodies (Fab'^-FITC-Gt-anti-MIg) purchased from Cappel Laboratories (Philadelphia, Pa.); previously absorbed by incubation f o r 2 hours at 4 degrees C with B6C3F^ (C57Bl/6xC3H)F^ spleen c e l l s . The antibody-cell suspensions were again incubated at 4 degrees C for 1 hour. - 22 -Table I I . Allotype and Lineage S p e c i f i t i e s of Anti-Thy-1.2,Anti-Lyt-1,  Anti-Lyt2 and Anti-H9/25 Monoclonal Antibodies Monoclonal Antibody S t r a i n S p e c i f i c i t y C e l l S p e c i f i c i t y anti-Thy-1.2 anti-Lyt-1.1 anti-Lyt-1.2 anti-Lyt-2.1 B10.BR C3H B10 .BR C3H a l l T lymphocytes mostly T helper c e l l s mostly T helper c e l l s mostly T k i l l e r and suppressor c e l l s anti-Lyt-2.2 B10 .BR mostly T k i l l e r and suppressor c e l l s anti-H9/25 B10 .BR granulocytes and some T k i l l e r and B plasma c e l l s - 23 -In the l a s t minute of the incubation 50 y l of propidium iodide (Sigma chemicals) (25 yg/ml) was added to each well to s t a i n the nonviable c e l l s . (Propidium iodide enters nonviable c e l l s and binds to t h e i r DNA so that when exposed to u l t r a - v i o l e t l i g h t these c e l l s e x h i b i t an orange fluorescence). A f t e r incubation, the c e l l s were washed 3 times and resuspended i n a f i n a l volume of 50 y l . The composition of the femoral bone marrow was analyzed using d i r e c t fluoresence. The procedure was the same as that just described except that only one incubation f o r antibody s t a i n i n g was needed since the f l u o r e s c e i n -isothyocyanate was conjugated d i r e c t l y to the f i r s t antibody, anti-H9/25 ( g i f t from F. Takei). This antibody i s s p e c i f i c f o r B10.BR granulocytes and plasma c e l l s and T k i l l e r c e l l s (105). S l i d e s f or immunofluoresence analysis were prepared by adding 25 y l of c e l l suspension to a glass s l i d e which was then covered by a coverglass and mounted with p a r a f f i n . Fluorescent c e l l s were counted using a Zeiss photomicroscope I I I with an epifluorescence attachment. This included a 2 00 watt mercury vapour lamp and f i l t e r s . C e l l s were examined with a 40x planapochromatic objective. Approximately 100 c e l l s were counted under phase contrast and then scored under epifluorescence to give the percentage of v i a b l e fluorescent c e l l s . Photographs were taken with 400 ASA Kodak color f i l m . 8. Antibody V a l i d a t i o n To determine the expected number of antibody-positive c e l l s i n the marrow and thymus of normal mice and to evaluate the accuracy with which the antibodies would detect d i f f e r e n t combinations of donor and host c e l l s , the following experiment was performed. Anti-Lyt and anti-Thy-1.2 antibodies were - 24 -used to s t a i n the following a r t i f i c i a l combinations of thymic c e l l s : 100%C3H; 100%B10.BR; 90%C3H and 10%B10.BR; 50%C3H and 50%B10.BR. The same combinations of bone marrow c e l l s were stained with FITC-H9/25. The r e s u l t s of 2 such experiments are shown i n Table I I I . A l l monoclonal antibodies stained 100 percent of the c e l l s except f o r anti-Lyt-2 and anti-H9/25 which stained approximately 87 and 50 percent of the c e l l s . These expected percentages of antibody-positive c e l l s are presented as control values i n the Results (Figures 6-9,11-13). - 25 -Table I I I . Number of Fluorescent C e l l s i n A r t i f c i a l Combinations  of C3H and B10.BR C e l l s From the Thymus and Bone Marrow  Stained With S t r a i n - S p e c i f i c Monoclonal Antibodies Monoclonal Antibody Mouse S t r a i n C 3H B10.BR 50/50 C3H/B10.BR 90/10 C3H/B10.BR a) Thymocytes Ly1 .2 0,0* (53),(64)** 100 ,100 (84),(69) 47,48 (131),(98) 7 , 1 3 ( 9 7 ) , ( 1 0 1 ) Ly2 .2 0,0 (61),(50) 80,95 ( 107),(77) 40 (100) 29,4 (94),(72) Ly1 .1 100,100 (122),(66) 2,0 (102),(50) 47,53 (104),(95) 80,92 (-103),( 105) Ly2.1 96,98 (11 1),(101) 0,0 (57),(50) 44,45 ( 100),( 100) 77,99 (97),(96) Thy1.2 100,100 (60),(55) 100,100 (61),(53) 100,100 (51),(53) 100,100 (48),(50) Media 0,0 (63),(57) 0 ,0 (71),(57) 0,1 (53),(97) 0,0 (60),(65) b) Bone marrow c e l l s FITC-H9/25 0 (103) 47 ( 106) 25 ( 135) 2 ( 100) * Percent fluorescent c e l l s . ** No. c e l l s counted. - 26 -RESULTS 1. General Experimental Design The purpose of these experiments was to determine the r e l a t i v e spleen colony forming a b i l i t y and i n vivo repopulating a b i l i t y of adherent versus unseparated bone marrow. In each experiment groups of l e t h a l l y i r r a d i a t e d C3H 4 5 '' r e c i p i e n t mice were i n j e c t e d with 3 x 10 or 10 adherent or unseparated B10.BR donor bone marrow (Figure 3 and 4). An a d d i t i o n a l 8 C3H mice were i r r a d i a t e d but received no c e l l s to monitor endogenous CFU-S s u r v i v a l i n each experiment. The number of spleen colonies produced by each c e l l suspension was determined 10 days a f t e r transplantation (8 recipients/group). The c e l l u l a r i t y of the regenerating spleen, thymus and bone marrow was evaluated at 15, 25, 35, and 125 days a f t e r transplantation on the basis of values obtained from 3 mice i n each group. Pools of thymus and bone marrow c e l l s were then made and studied with myeloid and T lymphocyte a l l o - s p e c i f i c antibodies to allow donor and host components to be distinguished and quantitated. The absolute number of antibody-positive donor c e l l s i n the marrow of one femur and i n the thymus was then obtained by m u l t i p l y i n g the percent p o s i t i v e c e l l s by the t o t a l number of c e l l s per t i s s u e ( c e l l u l a r i t y measurements). A t o t a l of three experiments were performed. In two of these both CFU-S and l a t e r repopulation determinations were made. In the t h i r d only CFU-S measurements were made. - 27 -DONOR B 1 Oi>B R 0 0 MARROW 4.5 , 4.5 GRAY 1 CO 15. 25 , 3 5 . and 125 DAYS THYMUS RECIPIENT C ( 3 j H DONOR SPECIF IC McAb 's FITC-GTaMlg F L U O R E S E N C E M I C R O S C O P Y DAY 10 C O L O N I E S 24hr A D H E R E N T C E L L S No. DONOR C E L L S Figure 3 General Experimental Design. - 28 -90 C3H (4.5 + 4.5)Gray - 6 hr interval +10J BIO.BR ABM 10 8 CFU-S DAY10 + 3X10H BIO. BR ABM NO 12 CHIMERAS % DONOR CELLS DAY .15,25,35,120 8 CFU-S DAY10 12 CHIMERAS % DONOR CELLS DAY 15,25,35,120 .20 +105 BIO.BR NBM + 3X10^ B10.BR NBM CELLS 8 CFU-S DAY10 8 CFU-S DAY 10 12 CHIMERAS % DONOR CELLS DAY 15,25,35 ,120 12 CHIMERAS % DONOR CELLS DAY 15 ,25 ,35 ,120 CONTROL 8 CFU-S DAY 10 Figure 4 Experimental plan - 29 -2. Enumeration of CFU-S i n Adherent and Normal Bone Marrow The average numbers of spleen colonies produced by adherent and normal bone marrow are compared i n Table IV. Groups of con t r o l mice i r r a d i a t e d and not i n j e c t e d with any c e l l s showed an average of les s than one colony per spleen, i n d i c a t i n g e f f e c t i v e elimination of hemopoietic stem c e l l s by the i r r a d i a t i o n procedure used. In a l l three experiments, adherent marrow c e l l s produced fewer day 10 spleen colonies than normal marrow c e l l s by a factor of 2 to 3 (p < 0.5, Student t - t e s t ) . Representative spleens from two groups are shown i n Figure 5. 3. Hemopoietic Repopulation of L e t h a l l y I r r a d i a t e d C3H Mice with B10/BR  Adherent or Unseparated Bone Marrow a. Repopulation of the Spleen Figure 6 compares the repopulation of the spleen of C3H mice re c e i v i n g 5 10 donor B10.BR adherent (top panel) or fresh (bottom panel) bone marrow c e l l s . In both groups the spleen had recovered to i t s normal s i z e Q (approximately 1.5 x 10 c e l l s ) by day 15 post-transplantation (the f i r s t time point evaluated) and t h i s c e l l u l a r i t y was r e l a t i v e l y well maintained thereafter. Figure 7 shows the repopulation of the spleen of C3H mice r e c e i v i n g 4 only 3 x 10 donor B10.BR fresh or adherent bone marrow c e l l s . In a l l of these the spleen was s t i l l recovering at 2 weeks. However, t h i s recovery appeared 4 f a s t e r i n the mice r e c e i v i n g 3 x 10 adherent marrow c e l l s ( s o l i d symbols) by comparison to the mice who received an equivalent number of unseparated marrow - 30 -Table IV. Comparison of the Mean Number of Spleen Colonies Produced  In Groups of 8 Mice Transplanted with Fresh or Adherent Marrow Marrow Injected Expt. Number of C e l l s Injected control 3 x 10 10" Fresh 1 . 2. 3 . Combined 0.4 + 0.2* 0.3 + 0.3 0.5 + 0.4 0.4 + 0.2 6.6 + 1 .2 6.3 + 1.1 7.0 + 1.8 6.6 + 0.8 14.8 + 1.6 N.D. 12.8 + 1 .2 13 .6 + 1 .0 Adherent 1 . 2. 3 . Combined 0.2 + 0.2 0.0 + 0.0 0.6 + 0.2 0.4 + 0.1 3.2 + 1.1 2.0 + 1.0 2.5 + 0.5 2.6 + 0.5 5.8 + 0.9 N.D. 6.3 + 1.4 6.1 + 0.9 * Mean + 1 S.E.M. - 32 -i—//—r 0) O o 2 •p 1 0 ! 10 8 10> Normal No. ol Spleen Cells 1 Normal No. ol Spleen Cel ls 30 40 120 130 Time P o s t - t r a n s p l a n t a t i o n (day s ) Figure 6 Ki n e t i c s of spleen c e l l repopulation i n i r r a d i a t e d C3H mice i n j e c t e d wi|h 10 B10.BR adherent marrow c e l l s (closed symbols) and with 10 B10.BR fresh marrow c e l l s (open symbols). The r e s u l t s of 2 experiments are shown separately (squares-Experiment 1, and circles-Experiment 2). Each datum point represents the geometric mean of the number of spleen c e l l s i n 3 mice. Curves are drawn through the mean of the values of the 2 experiments. - 33 -CD O 6 «5 o 20 30 40 120 Time P o s t - t r a n s p l a n t a t i o n (days) Figure 7 K i n e t i c s of spleen c g l l repopulation i n i r r a d i a t e d C3H mice i n j e c t e d with 3 x 10 B10. BR adherent marrow c e l l s . The r e s u l t s of 2 experiments are shown separately (squares-Experiment 1, and circles-Experiment 2). Each datum point represents the geometric mean of the t o t a l number of spleen c e l l s i n 3 mice. Curves are drawn through the mean of the values of the 2 experiments. - 34 -c e l l s (open symbols) since at 2 weeks the mean of the adherent c e l l s was s i g n i f i c a n t l y greater than the mean of the fresh marrow (p <0.02, student t -4 t e s t ) . Moreover, no r e c i p i e n t s of 3 x 10 normal marrow c e l l s survived beyond 25 days, providing further evidence that the adherent f r a c t i o n had superior p r o t e c t i v e capacity. b. Repopulation of the Bone Marrow The k i n e t i c s of repopulation of the femoral marrow granulocyte compart-ment with donor B10.BR c e l l s as a function of the number of adherent or unseparated marrow c e l l s i n j e c t e d , i s shown i n Figure 8 (10^ c e l l g rafts) and 4 Figure 9 (3 x 10 c e l l g r a f t s ) . The data obtained again supports the hypothesis that adherence separation enriched f o r a stem c e l l with i n vivo myeloid repopulating p o t e n t i a l . I t can be seen i n Figure 8 that the femoral 5 marrow content of donor-derived H9/25 p o s i t i v e c e l l s rose from <10 c e l l s per 2 femurs on day 15 to approximately 3 to 4 x 10 c e l l s per 2 femurs by day 25. However, although marked differences between mice r e c e i v i n g 10~* adherent and unseparated marrow c e l l s were not seen p r i o r to day 25, thereafter the mice r e c e i v i n g the adherent c e l l s showed continuing recovery to 50% of normal values by day 120 whereas r e c i p i e n t s of the same number of normal marrow c e l l s showed a decline i n donor-derived granulocytes to approximately 10% of normal values by day 120. The appearance of a marrow c e l l stained with anti-H9/25 from and 5 i r r a d i a t e d C3H mouse i n j e c t e d with 10 B10.BR marrow c e l l s 5 weeks a f t e r transplantation i s seen i n Figure 10. Figure 9 shows the repopulation of C3H bone marrow with H9/25 p o s i t i v e 4 granulocytes grafted with 3 x 10 adherent marrow c e l l s . The k i n e t i c s of repopulation i s s i m i l a r to the curves shown i n Figure 6 but plateaus e a r l i e r - 35 -1 1 / / — r No. Normal B10.BR Femoral Marrow Cella No. H9/2S Positive Cells In Normal BIO. BR * 20 30 4 0 1 2 0 130 Time P o s t - t r a n s p l a n t a t i o n ( d a y s ) Figure 8 K i n e t i c s of granulocyte repopulation i n 2 femurs i n i r r a d i a t e d C3H mice i n j e c t e d wijjh 1° B10.BR adherent marrow c e l l s (closed symbols) and with 10 B10.BR fresh marrow c e l l s (open symbols). Donor c e l l s are s p e c i f i c a l l y stained by anti-H9/25. The r e s u l t s of 2 experiments are shown separately (squares-Experiment 1, and circles-Experiment 2). Each datum point represents the geometric mean of the t o t a l number of H9/25 p o s i t i v e c e l l s i n 3 mice. Curves are drawn through the mean of the values of the 2 experiments. - 36 -Time Post - t ransp lantat ion (days) Figure 9 K i n e t i c s of granulocyte repopulation i n 2 femursin i r r a d i a t e d C3H mice i n j e c t e d with 3 x 10 B10.BR adherent marrow c e l l s . Donor c e l l s are s p e c i f i c a l l y stained by anti-H9/25. The r e s u l t s of 2 experiments are shown separately (squares-Experiment 1, and circles-Experiment 2). Each datum point represents the geometric mean of the t o t a l number of H9/25 p o s i t i v e c e l l s i n 3 mice. Curves are drawn through the mean of the values of the 2 experiments. - 37 -F i g u r e 10 Phase c o n t r a s t m i c r o g r a p h ( t o p ) and f l u o r e s e n c e m i c r e g r a p h (bottom) o f a bone marrow c e l l s t a i n e d w i t h a n t i - H 9 / 2 5 from an i r r a d i a t e d C3H mouse i n j e c t e d w i t h 10 B10.BR a d h e r e n t marrow c e l l s . - 38 -(at 25 days a f t e r grafting) at a lower value of 1.5 x 10^ c e l l s (approximately 7% of the normal value). Since the t o t a l c e l l u l a r i t y of the marrow of a l l these mice (Table V) recovered eventually (between 1 and 4 months a f t e r transplantation)the most l i k e l y explanation f o r the l e v e l s of chimerism observed are var i a b l e degrees of host-derived myelopoietic recovery according to the competitive repopulating a b i l i t y of the transplanted stem c e l l s (106). c. Repopulation of the Thymus The k i n e t i c s of thymic repopulation with donor and host derived T c e l l s (Lyt 1-and Lyt 2-positive c e l l s ) i n the same mice grafted with adherent or 5 unseparated marrow c e l l s i s shown i n Figure 11 (10 adherent marrow c e l l s ) , 5 4 Figure 12 (10 unseparated c e l l s ) and Figure 13 (3 x 10 adherent marrow c e l l s ) . The t o t a l c e l l u l a r i t y of the thymus i s shown i n Table VI. A l l groups showed a tr a n s i e n t repopulation of the thymus with donor c e l l s reaching a maximum at 35 days. However, t h i s donor derived T c e l l population was not sustained and was displaced to varying extents by T c e l l s of host o r i g i n i n a l l groups. There were no obvious differences between the mice transplanted with 5 4 10 adherent or unseparated marrow c e l l s . Mice transplanted with 3 x 10 adherent marrow c e l l s showed poorer o v e r a l l recovery of the thymus. A generally lower donor derived population was present on days 25 and 35 and t h i s had completely disappeared by day 120. The appearance of a thymocyte stained 5 with anti-Lytl-1.2 from an i r r a d i a t e d C3H mouse grafted with 10 B10.BR adherent marrow c e l l s 5 weeks a f t e r transplantation i s shown i n Figure 14. - 39 -Table V. T o t a l Number of C e l l s i n the Bone Marrow i n Mice Transplanted with Adherent and Fresh Marrow No. c e l l s Marrow Time Post-transplantation (days) Injected Injected Exp. 15 25 35 125 10 Adherent 1. 5* (2-9) 2. 17 (6-49 ) Fresh 1 . 7 (5-11) 2. 9 (8-11) 3X10 Adherent 1. 8 (6-11) 2. 6 (5-6) Fresh 1 . 3 (2-4) 107 109 205 (96-119) (104-116) (193-217) 87 205 N.D. (57-131) (177-237) 84 146 116 (80-89) (129-165) (87-154) 122 84 N.D. (100-149) (41-175) 107 72 26 (59-195) (57-91) (18-29) 14 N.D. N.D (9-2 3) 136 N.D. N.D. (96-193) N.D. N.D. N.D. * Geometric mean x 10 ( Range defined by + or - 1S.E.M. ) - 40 -FIGURE 11 K i n e t i c s of thymocyte repopulation i n i r r a d i a t e d C3H mice i n j e c t e d with 10 B10.BR adherent marrow c e l l s . Donor c e l l s (closed symbols) were stained with anti-Lyt-1.2 (top panel) and anti-Lyt-2.2 (bottom panel). Recipient c e l l s (open symbols) were stained with anti-Lyt-1.1 (top panel) and anti-Lyt-2.1 (bottom panel). Recipient c e l l s are stained s p e c i f i c a l l y by anti-Ly1.1 (top panel) anti-Lyt-2.1. The r e s u l t s of 2 experiments are shown separately (squares-Experiment 1, and circles-Experiment 2). Each datum point represents the geometric mean of the t o t a l number of Lyt p o s i t i v e c e l l s i n 3 mice. Curves are drawn through the mean of the values of the 2 experiments. - 41 -o o c o Q 1 0 1 0 1 0 6 o <D 1 0 8 o w .Q < 1 0 ' 1 0 ' Y / r N o . o l C e l l s in 8 - 1 5 W e e k B I O ^ B R T h y m u s ( 1 0 0 % L y t P o s i t i v e ) — T — ' — N o . ot C e l l s in 6 - 1 5 W e e k B 1 0 / B R T h y m u s ( 6 7 % L y 2 P o s i t i v e ) 2 0 3 0 4 0 1 2 0 1 3 0 T ime P o s t - t r a n s p l a n t a t i o n ( d a y s ) Figure 12 K i n e t i c s of thymocyte repopulation i n i r r a d i a t e d C3H mice i n j e c t e d with 10 B10.BR fresh marrow c e l l s . Donor c e l l s (closed symbols) were stained with a n t i - L y t - 1 . 2 (top panel) and a n t i - L y t -2 . 2 (bottom panel). Recipient c e l l s (open symbols) were stained with a n t i - L y t - 1 . 1 (top panel) and a n t i - L y t - 2 . 1 (bottom panel). The r e s u l t s of 2 experiments are shown separately (squares-Experiment 1, and circles-Experiment 2 ) . Each datum point represents the geometric mean of the t o t a l number of Lyt p o s i t i v e c e l l s i n 3 mice. Curves are drawn through the mean of the values of the 2 experiments. - 42. -Time Post-transplantation (days) Figure 13 K i n e t i c s of thymocyte^ repopulation i n i r r a d i a t e d C3H mice i n j e c t e d with 3 x 1 0 adherent marrow c e l l s . Donor c e l l s (closed symbols) were stained with anti-Lyt-1.2 (top panel) and a n t i - L y t -2.2 (bottom panel). Recipient c e l l s (open symbols) were stained with anti-Lyt-1.2 (top panel) and anti-Lyt-2.2 (bottom panel). The r e s u l t s of 2 experiments are shown separately (squares-Experiment 1, and circles-Experiment 2). Each datum point represents the geometric mean of the t o t a l number of Lyt p o s i t i v e c e l l s i n 3 mice. Curves are drawn through the mean of the values of the 2 experiments. - 43 Figure 14 Phase contrast micrograph (top) and fluoresence micrograph (bottom) of a thumocyte stained with anti-Lyt-1.2 from an i r r a d i a t e d C3H mouse i n j e c t e d with 10 B10.BR adherent marrow c e l l s 5 weeks post-transplantation. - 44 -Table VI. Total Number of C e l l s i n the Thymus i n Mice Transplanted with Adherent and Fresh Marrow No. C e l l s Injected Marrow Injected Exp. Time Post-transplantation (days) 15 25 35 120 10' Adherent 1 . 15* (4-54) 209 (95-463) 404 (328-497) 109 (37-316) 2. 20 47 441 (5-72) (18-118) (287-676) N.D. Fresh 112 (83-152) 33 (18-60) 19 (8-48) 23 (5-115) 2. 8 ( 1-50) 130 (63-27) 433 (296-633) N.D. 3X10 Adherent 1 . 26 (9-8 0) 53 (46-60) 111 (73-167) 6 ( 1-44) 2 . 6 (3-11) 2 (1 - 7 ) N.D, N.D. Fresh 1 . 1 ( 1-2) 37 (21-66) N.D. N.D. 2 . N.D. N.D. N.D. N.D. * Geometric mean x 10 ( Range defined by + or - 1S.E.M. ) - 45 -DISCUSSION AND CONCLUSIONS The objective of the present work was to t e s t the day 10 spleen colony-forming a b i l i t y and the _in vivo lympho-myeloid repopulating a b i l i t y of adherent marrow. The r e s u l t s showed that adherence does not s e l e c t f o r day 10 CFU-S (Table IV). Previously Magli has reported that l e s s than 50% of the spleen colonies v i s i b l e before 10 (8 to 9) days post-transplantation are derived from CFU-S with high self-renewal capacity. High self-renewal CFU-S y i e l d spleen colonies that appear only a f t e r 10 (12 to 14) days post-transplantation (2). Subsequently evidence that high self-renewal CFU-S are adherent i n long- term cultures was obtained (1). The present r e s u l t s extend these findings by showing that e a r l y appearing CFU-S (with low self-renewal p o t e n t i a l - 2) are not adherent. Adherence also appears to s e l e c t f o r a c e l l with greater myeloid (granulocyte) repopulating p o t e n t i a l . Repopulation of the spleen occurred e a r l i e r with adherent marrow grafts than by the same number of fr e s h marrow c e l l s (Figure 7). Also by 4 months the l e v e l of repopulation of the bone marrow by adherent c e l l s had reached a higher l e v e l of repopulation than that achieved with the same number of unseparated marrow c e l l s (Figure 8). A high myeloid repopulating stem c e l l i s also obtained following treatment of marrow with 5 - f l u o r o u r a c i l (55,56). This drug, which s e l e c t i v e l y eliminates p r o l i f e r a t i n g c e l l s , l i k e adherence, has been shown to sel e c t against day 8 to 10 CFU-S. The r e s u l t s do not in d i c a t e that adherence also s e l e c t s f o r a c e l l with T lymphocyte repopulating p o t e n t i a l . Donor repopulation of the thymus d i d occur but was not sustained a f t e r 4 months (Figures 11,12 and 13). The disappearance - 46 -of donor-derived T lymphocytes may be explained by a lack of, or an inappropriate stimulation of donor adherent c e l l s with T lymphoid repopulating p o t e n t i a l , or by minor histocompatability differences between B10.BR and C3H causing deleterious or at l e a s t confounding e f f e c t s on T lymphopoietic recovery i n the chimeras. Support f o r the l a t t e r i s provided by the demonstration that long-term repopulation of the T-lymphocyte compartment was not achieved by fre s h marrow gr a f t s that are usually successful when other non-syngeneic s t r a i n combinations are used (107). No attempt was made i n t h i s project to investigate the B lymphocyte repopulating p o t e n t i a l of adherent marrow. However, Dorshkind and P h i l l i p s demonstrated that colony-forming B c e l l s were generated more r a p i d l y and to higher l e v e l s i n animals grafted with adherent compared to nonadherent c e l l s from established long-term cultures (108). The number of CFU-S innoculated i n each type of suspension was s i m i l a r , i n d i c a t i n g that CFU-S and B c e l l repopulating c e l l numbers do not c o r r e l a t e . These r e s u l t s together with those presented i n t h i s t h e s i s support the idea that the stem c e l l with granulocyte and B c e l l repopulating a b i l i t y i s not the day 10 CFU-S. The exact stage where adherent hemopoietic progenitors may f i t i n the hierarchy of hemopoietic stem c e l l d i f f e r e n t i a t i o n events has not been determined. Adherence has been shown to enrich f o r a progenitor of the CFU-GEMM but not f o r CFU-GEMM i t s e l f (D. Kerk, unpublished r e s u l t s ) . The frequency of CFU-GEMM found i n adherent marrow was approximately the same as that f o r o r i g i n a l marrow, but the production of secondary CFU-GEMM i n assays of adherent marrow approximately 3 f o l d higher than that obtained i n assays of fresh marrow. Since there i s evidence that the CFU-GEMM population overlaps with the 8 to 10 day CFU-S population (67), the adherent CFU-GEMM progenitor i s probably more c l o s e l y r e l a t e d to the high self-renewal day 12+ CFU-S, and the high s e l f -- 47 -renewal S - c e l l of Ogawa (98). Whether the adherent progenitor and S c e l l a lso have lymphoid repopulating p o t e n t i a l i s s t i l l to be discovered. To elucidate further the precise r e l a t i o n s h i p s between more p r i m i t i v e hemopoietic stem c e l l s tissue-repopulation studies with si n g l e colonies of various p r i m i t i v e stem c e l l o r i g i n s could be performed i n the future. F i n a l l y , the p o s s i b i l i t y that the gain or loss of the property of adherence that i s associated with a high self-renewal capacity and delayed spleen colony forming a b i l i t y may be r e v e r s i b l e deserves consideration. Recent evidence suggests that the p r o b a b i l i t y of self-renewal i s not f i x e d , but may be subject to manipulation. Stem c e l l s (CFU-S) from long term cultures i n f e c t e d with src-(MoMULV), a molecular recombinant of Rous sarcoma v i r u s and a murine amphotropic leukemia v i r u s , u n l i k e normal CFU-S showed sustained in vivo s e r i a l t r a n s p l a n t a b i l i t y and an increased mean CFU-S per spleen colony (66). The extended self-renewal of stem c e l l s was demonstrated to be d i r e c t l y due to s r c -(MoMuLV) i n f e c t i o n and expression by the continuous generation of mixed stem c e l l clones i n the absence of stromal c e l l s required for long term culture (109). These r e s u l t s i n d i c a t e that the p r o b a b i l i t y of self-renewal i s not f i x e d but can be modified. Whether Friend v i r u s - and s r c - i n f e c t e d stem c e l l s are a l s o adherent and whether they s t i l l have T-lymphoid repopulating p o t e n t i a l are questions s t i l l r e q u i r i n g i n v e s t i g a t i o n . Also r e q u i r i n g study are the pharmacologic agents (previously shown to be capable of a c t i v a t i n g stem c e l l p r o l i f e r a t i o n (46)), which might have the a b i l i t y to a l t e r the p r o b a b i l i t y of self-renewal. For a l l of these questions, the model system developed i n t h i s study may o f f e r a system f o r obtaining the relevant answers. - 48 -REFERENCES 1. Mauch, P., Greenberger, J.S., Botnick, L., Mannon, E., and Hellman, S.: Evidence f o r structured v a r i a t i o n i n self-renewal capacity within long-term bone marrow cultures. Proc. N a t l . Acad. S c i . 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