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Studies of the role of mesenchymal cells in the regulation of hemopoiesis 1988

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STUDIES OF THE ROLE OF MESENCHYMAL CELLS IN THE REGULATION OF HEMOPOIESIS by LOUIS A. GABOURY M.D., U n i v e r s i t y of Montreal, 1978 D.E.S., U n i v e r s i t y of Montreal, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1988 © Louis A. Gaboury, 1988 ABSTRACT Hemopoiesis i s thought to be regulated i n part by s p e c i f i c , but as yet undefined, interactions between primitive hemopoietic c e l l s and fixed, non- hemopoietic marrow elements c o l l e c t i v e l y referred to as the stroma. Recently, a marrow culture system has been described that allows the maintenance of pr i m i t i v e human hemopoietic progenitor c e l l s for many weeks i n the absence of exogenously added hemopoietic growth factors. The formation of a heterogeneous adherent layer in which many stromal elements are found appears to be important to the maintenance of hemopoiesis i n this system. As part of the o v e r a l l goal of delineating the c e l l u l a r and molecular i n t e r a c t i o n s involved, my f i r s t objective was to develop an experimental system for assessing the hemopoiesis-sustaining function of the adherent layer of long- term human marrow cultures. This required the i d e n t i f i c a t i o n of a s u i t a b l e procedure for separating the hemopoietic and non-hemopoietic regulatory components so that the former could be used to quantitate the function of the l a t t e r . This was achieved using i r r a d i a t i o n to s e l e c t i v e l y i n a c t i v a t e r e s i d u a l hemopoietic c e l l s in long-term culture adherent layers, and using a medium containing cis-4-hydroxy-L-proline to s e l e c t i v e l y i n a c t i v a t e stromal c e l l s and the i r precursors present in suspensions of unseparated human marrow which were then added back i n co-culture experiments. My second objective was to develop a strategy for obtaining p u r i f i e d populations of c e l l s corresponding to the various mesenchymal c e l l types i n long-term adherent layers. I therefore prepared a high t i t r e SV-40 vi r u s stock and used i t to e s t a b l i s h permanent, cloned l i n e s from human marrow " f i b r o b l a s t " colonies, long-term culture adherent layers, and umb i l i c a l cord endothelial c e l l s . Characterization of the transformants generated showed i i i that they were a l l p o s i t i v e for SV-40, and i n general expressed the phenotypic c h a r a c t e r i s t i c s of the c e l l s o r i g i n a l l y infected. Functional studies showed that these transformants, l i k e their normal counterparts, respond to a c t i v a t i o n by producing two types of hemopoietic growth fac t o r s . These studies suggest that marrow mesenchymal c e l l s may regulate the growth and maintenance of primitive hemopoietic c e l l s by producing hemopoietic growth factors i n response to appropriate perturbation. The a v a i l a b i l i t y of permanent cloned l i n e s of human marrow stromal c e l l s should f a c i l i t a t e future analysis of these events at the molecular l e v e l . i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x Chapter I INTRODUCTION 1) Organization of the Hemopoietic System 1 A) Hemopoietic C e l l s 1 B) Stromal C e l l s of the Bone Marrow 7 C) Stromal C e l l Products 12 2) Regulation of the Hemopoietic System 18 A) C e l l u l a r I n t e r a c t i o n s 18 B) Humoral Regulation of the Hemopoietic System 22 3) Long-Term Bone Marrow Cu l t u r e s : An In V i t r o Model f o r Hemopoietic Stem C e l l Regulation 30 A) E a r l y Development of the Long-Term Marrow Cultures 30 B) Long-Term Human Marrow Cultures 31 C) Long-Term Marrow Cultures as a Model of In Vivo Regulation 36 4) Thesis Objectives 38 References 41 Chapter I I MATERIALS AND METHODS 1) C e l l s 55 A) Bone Marrow C e l l s 55 B) P e r i p h e r a l Blood 55 C) C e l l Lines Maintenance 57 D) I n f e c t i o n of Primary Human Mesenchymal C e l l s with SV-40 and I s o l a t i o n of Permanent C e l l Lines 57 E) C e l l P r o l i f e r a t i o n Measurements 59 2) Long-Term Cultures 60 A) Regular Long-Term Marrow Cultures 60 B) Long-Term P e r i p h e r a l Blood Cultures 61 C) Preparation of Long-Term Marrow Cultu r e Feeders 61 D) Cis-Hydroxy-L-Proline Experiments 62 3) Assays A) M e t h y l c e l l u l o s e Assay f o r Hemopoietic Colony-Forming Progenitors 62 B) Colony-Forming U n i t - F i b r o b l a s t (CFU-F) Assay 63 C) Assays f o r the Production of Hemopoietic Growth (HGF) 63 V 4) SV-40 Virus Preparation and Assay 65 A) Preparation of High T i t e r Virus Stock 65 B) Virus Plaque Assay 65 C) Assay for Large T Antigen 67 D) Transformation Assay of SV-40 Virus on NIH-3T3 C e l l s 67 5) Antisera and Immunofluorescence Measurements 67 6) Histochemical Analyses 70 7) Test for Anchorage-Independent Growth 70 8) Tumor Formation 70 9) I r r a d i a t i o n Procedures 71 10) Autoradiography 71 References 73 Chapter I II DIFFERENTIAL EFFECTS OF CIS-0H-L-PR0LINE ON THE PROLIFERATIVE AND STEM CELL REGULATORY FUNCTIONS OF HUMAN BONE MARROW MESENCHYMAL CELLS 1) Introduction 75 2) Results A) Cultures of Peripheral Blood C e l l s on Normal Marrow Adherent Layer 77 B) Ef f e c t of CHP on Marrow Mesenchymal C e l l P r o l i f e r a t i o n 81 C) Lack of a Direct E f f e c t of CHP on Hemopoietic Progenitors Function 85 3) Discussion 90 References 93 Chapter IV INDUCIBLE PRODUCTION OF HEMOPOIETIC GROWTH FACTORS BY SV-40 IMMORTALIZED MESENCHYMAL CELL LINES OF HUMAN BONE MARROW ORIGIN 1) Introduction 95 2) Results A) Transforming Potential of SV-40 Virus and i t s E f f e c t on DNA Synthesis of SV-40 Virus 97 B) Derivation and Immunological Characterization of C e l l Lines 97 C) C h a r a c t e r i s t i c s of Transformed C e l l Lines 102 D) Induction of Growth Factor Production 105 E) I r r a d i a t i o n Studies 115 3) Discussion 115 References 120 Chapter V SUMMARY AND FUTURE DIRECTIONS 123 References 130 LIST OF TABLES Recovery of Nucleated C e l l s and Progenitors from One Unit (500ml) of Human Peripheral Blood Culture Conditions for the Maintenance of C e l l Lines Origin, S p e c i f i c i t y and Source of Antibodies Used for Immunophenotypic Analyses Immunophenotypic Characterization of Lymphoblastoid C e l l s E f f e c t of CHP on Adherent Layer Formation Lack of Ef f e c t of Exposure of Hemopoietic Progenitors to CHP on their Subsequent Pla t i n g E f f i c i e n c y Lack of E f f e c t of 500 ug/ml of CHP on Hemopoiesis i n Long-Term Cultures I n i t i a t e d on Pre-Established Irradiated Adherent Layers (Feeders) Histochemical and Immunophenotypic Properties of SV-40 Transformed Human C e l l Lines Evidence for IL-lf3 Induced Production of Hemopoietic Colony-Stimulating A c t i v i t y by Representative SV-40 Transformed Human C e l l Lines E f f e c t s of SV-40 Immortalized Conditioned Medium on CFU-F Formation v i i L I S T OP F I G U R E S Page FIGURE 1 Major Sites of Hemopoiesis in the Human Embryo and Fetus 2 FIGURE 2 Schematic Representation of the Hemopoietic System as Defined by Clonogenic Assays for Pluripotent and Committed Progenitors 6 FIGURE 3 Schematic Representation of Bone Marrow Histology A) C e l l u l a r Arrangement Around a Marrow Sinus 9 B) Longitudinal Section of a Marrow Sinus 10 FIGURE 4 Diagrammatic Representation of the Various I n t r a c e l l u l a r and E x t r a c e l l u l a r Molecular Events Involved i n the Formation of a Collagen Molecule 14 FIGURE 5 Functional Domains of Fibronectin 16 FIGURE 6 Photomicrographs of a Formalin Fixed Long-Term Marrow Culture Adherent Layer Stained With Rabbit-Anti Factor VIII Antiserum and Developed With Peroxidase-Labelled Swine Anti-Rabbit Immunoglobulin Antibody A) Low Power View 33 B) High Power View 34 FIGURE 7 The C e l l u l a r i t y and Progenitor Content of the Adherent (Adh) and Nonadherent (NA) Fractions Assessed at Varying Incubation Times i n Human Long-Term Bone Marrow Cultures 35 FIGURE 8 T i t r a t i o n of SV-40 Virus Stock on BSC-1 C e l l s 66 FIGURE 9 Comparison of Total C e l l and Progenitor Content of Long- Term Normal Peripheral Blood Cultures I n i t i a t e d With or Without a Pre-Established Normal Marrow Feeder 78 FIGURE 10 E f f e c t of Increasing Doses of CHP on the Number of Colonies Obtained from CFU-F in Fresh Human Marrow 82 FIGURE 11 Photograph of Two Long-Term Marrow Cultures, One I n i t i a t e d and Maintained i n Regular Medium ( L e f t ) , and One I n i t i a t e d and Maintained in Proline-, and Lysine-Free Medium Containing 500 ug/ml of CHP (Right) 84 v i i i FIGURE 12 FIGURE 13 FIGURE 14 FIGURE 15 FIGURE 16 FIGURE 17 FIGURE 18 FIGURE 19 FIGURE 20 Ef f e c t s of 500 ug/ml of CHP on the Y i e l d of Hemopoietic Progenitors i n Long-Term Marrow Cultures Assessed 4-5 Weeks After I n i t i a t i o n 88 Transformed Focus of NIH-3T3 C e l l s 98 Autoradiograms of Confluent Marrow Adherent Layers Mock Infected Layer (Upper Panel) and SV-40 Infected Layer (Lower Panel) 99 FACS P r o f i l e of CFUST-CL16 C e l l s Stained With Monoclonal Antibody 6.19 (Panel A) and Anti-Leuk AH/T200 (Panel B) 103 FACS P r o f i l e of a Suspension of Spontaneously Immortalized Lymphoblastoid C e l l s (Panel A), and HUVE-EC-C C e l l s Before (Panel B), and After (Panel C) Transformation with SV-40 Virus Tritiated-Thymidine Uptake (Panel A) of SV-40 Infected (So l i d Lines) and Uninfected (Broken Lines) MH C e l l s Growth Rate (Panel B) of SV-40 Infected ( S o l i d Lines) (Broken Lines) MH C e l l s A Colony of Transformed C e l l s Generated i n a Methylcellulose Culture 14 Days After Seeding the Cultures with MH C e l l s Infected with SV-40 Virus (Panel A). Control (Uninfected) MH C e l l s Failed To Y i e l d Colonies (Panel B). Analysis of the Clonogenic Capacity of MH2SV-CL1 C e l l s Plated i n Methylcellulose Hemopoietic Colony-Stimulating A c t i v i t y of Media Conditioned for.24 Hours by CFUST-CL16 or MH2SV-CL1 C e l l s (or No C e l l s ) as a Function of the Concentration of IL-13 Used as a Stimulant 104 106 107 108 109 110 113 FIGURE 21 C e l l Survival Curves for CFUST-CL16, EC CL22, Ea.926 C e l l s and Normal Bone Marrow Fibroblasts 116 ix AC3W0VLEDGEMENTS I wish to express my sincere gratitude: To my supervisor Dr. Connie Eaves for her help and guidance throughout this project, To Dr. A l l e n Eaves, Dr. Johanne Cashman, Dr. R. Keith Humphries, Dr. Peter Lansdorp, Dr. Robert Kay, and Dr. A l i Turhan for h e l p f u l discussions and a c t i v e collaboration, To Dr. Heather Sutherland for her reviewing of the thesis To Mrs. Marjorie Hutchison, Mrs. Giovanna Cameron, Mrs. Karen Lambie, Ms. Judy P f e i f e r , and Ms. Dianne Reid for expert technical assistance, To Paule and Ariane for their patience, To Ms. Michele Coulombe for s e c r e t a r i a l assistance, To the National Cancer I n s t i t u t e of Canada for f i n a n c i a l support. 1 C H A P T E R I INTRODUCTION 1) ORGANIZATION OF THE HEMOPOIETIC SYSTEM (A) Hemopoietic Cells The hemopoietic system consists of a lymphoid and a myeloid arm, both of which are thought to originate from common ancestor cells that have undergone a series of migrations from their original location in the yolk sac to their fi n a l destination in the functional hemopoietic organs of the adult, i.e. the bone marrow, spleen, thymus and lymph nodes (1) (see Figure 1). A l l mature myeloid cells (red blood cells, granulocytes, monocytes and platelets) represent non-dividing "end" cells that survive for relatively short periods of time and are, therefore, being continuously replaced throughout adult l i f e (2). The production of new blood cells from more primitive proliferating precursors normally occurs in the bone marrow of the adult (3) . The structure of the hemopoietic system is currently viewed as consisting of four major c e l l compartments (4). The most primitive are the hemopoietic stem c e l l s , cells that have the potential both to self-renew and to differentiate into each of several lineages. Stem cells give rise to an intermediate, transient compartment of progenitors of various types that have undergone different degrees of lineage restriction but s t i l l have a considerable proliferative potential. Neither pluripotent stem cells nor  3 l i n e a g e - r e s t r i c t e d progenitors are uniquely d i s t i n g u i s h a b l e m o r p h o l o g i c a l l y , e i t h e r as a c l a s s , or from one another. The pr o g e n i t o r s , i n turn g i v e r i s e to a compartment of morphologically recognizable precursors that undergo a l i m i t e d number of d i v i s i o n s , 3-5, i n concert with the completion of t e r m i n a l maturation. The f u l l y d i f f e r e n t i a t e d , non-dividing blood c e l l s represent the l a s t compartment. Generally assumed i n t h i s model, i s that d i f f e r e n t i a t i v e t r a n s i t i o n s between compartments are u n i d i r e c t i o n a l ( c e l l s cannot increase t h e i r d i f f e r e n t i a t i v e p o t e n t i a l ) , and that there i s a progressive l o s s of p r o l i f e r a t i v e p o t e n t i a l as c e l l s become r e s t r i c t e d i n t h e i r d i f f e r e n t i a t i o n p o t e n t i a l (4,5). Assays for Hemopoietic Stem C e l l s . The f i r s t q u a n t i t a t i v e colony assay fo r p l u r i p o t e n t stem c e l l s was described f o r murine c e l l s i n 1961 ( 6 ) . I t i n v o l v e s i n j e c t i n g h e a v i l y i r r a d i a t e d histocompatible mice with an appropriate number of hemopoietic c e l l s and counting, 1-2 weeks l a t e r , the number of macroscopic nodules that have appeared on the surface of the spleen. C y t o l o g i c a l and chromosomal marker st u d i e s of the c e l l s w i t h i n such nodules revealed each to be a bonafide clone derived from a s i n g l e , p l u r i p o t e n t c e l l (7,8). I n j e c t i o n of c e l l s from primary spleen c o l o n i e s i n t o l e t h a l l y i r r a d i a t e d secondary r e c i p i e n t s was found to r e s u l t i n the formation of new spleen c o l o n i e s , again of m u l t i - l i n e a g e composition ( 9 ) . This was the f i r s t formal demonstration of the existence i n adult hemopoietic t i s s u e of a c e l l that can self-renew, and that a l s o has the p o t e n t i a l to undergo a l a r g e number of d i f f e r e n t i a t i v e d i v i s i o n s along several lineages. Further studies pointed out that the f i r s t appearing spleen c o l o n i e s (seen w i t h i n 7-9 days a f t e r t r a n s p l a n t a t i o n ) are derived from c e l l s that are n e i t h e r m u l t i p o t e n t i a l nor s e l f - m a i n t a i n i n g (10) i n contrast to the spleen c o l o n i e s v i s i b l e at l a t e r 4 times (12-14 days a f t e r t r a n s p l a n t a t i o n ) . There i s now much evidence to suggest some overlap between the progenitors of the l a t e appearing spleen c o l o n i e s and c e l l s capable of long-term repopulation i n mice (11). In a d d i t i o n , recent experiments with r e t r o v i r a l l y marked mouse marrow c e l l s have confirmed that s i n g l e c e l l s can repopulate the e n t i r e lymphoid and myeloid system of both primary and secondary r e c i p i e n t s (12,13). Obviously, a comparable i n v i v o assay f o r a p l u r i p o t e n t hemopoietic stem c e l l cannot be performed i n humans. However, s e v e r a l l i n e s of evidence i n d i c a t e the existence of hemopoietic stem c e l l populations analogous to those i d e n t i f i e d i n the murine system. F i r s t , i n chronic myelogenous leukemia (CML), d e t e c t i o n of a s p e c i f i c chromosomal marker, the P h i l a d e l p h i a chromosome (Ph^) i n a l l myeloid c e l l s and o c c a s i o n a l l y i n --lymphoid c e l l s , but never i n bone marrow f i b r o b l a s t s , has supported the notion of a p l u r i p o t e n t hemopoietic stem c e l l i n man that i s the target of n e o p l a s t i c transformation i n CML (14,15). Second, s t u d i e s of a number of women heterozygous f o r the X - l i n k e d enzyme glucose-6-phosphate dehydrogenase (G6PD), who a l s o had one of v a r i o u s m y e l o p r o l i f e r a t i v e d i s o r d e r s ( i n c l u d i n g CML) have a l s o traced back the o r i g i n of the expanded n e o p l a s t i c clone i n each case to a s i n g l e transformed p l u r i p o t e n t hemopoietic stem c e l l (16,17,18). More r e c e n t l y , probes f o r polymorphic regions i n the X-chromosome, that are d i f f e r e n t i a l l y methylated a f t e r X-chromosome i n a c t i v a t i o n i n females, have been used to confirm the o r i g i n of human m y e l o p r o l i f e r a t i v e disease clones i n p l u r i p o t e n t hemopoietic c e l l s (19). This l a t t e r approach has now a l s o been used to assess the c l o n a l i t y of populations i n human r e c i p i e n t s of normal, a l l o g e n e i c marrow t r a n s p l a n t s . In at l e a s t one case to date, long-term monoclonal hemopoiesis of donor o r i g i n has been documented (20). Thus, i t seems very l i k e l y that the 5 conceptual framework of hemopoietic c e l l d i f f e r e n t i a t i o n developed from murine studies w i l l apply to the human sytem. Assays for Hemopoietic Progenitors. Hemopoietic progenitor c e l l s are present at very low frequencies (10~3 to 10"^) in normal hemopoietic tissue and have no d i s t i n c t i v e morphological features. However, such c e l l s can be i d e n t i f i e d i n d i r e c t l y by their a b i l i t y to p r o l i f e r a t e and d i f f e r e n t i a t e i n v i t r o . Hemopoietic colony assays involve suspending the c e l l s to be tested in a semi-solid culture medium containing appropriate nutrients, serum (or the e s s e n t i a l components contained in serum) and a source of hemopoietic growth fact o r s , e i t h e r i n crude preparations or as highly p u r i f i e d (natural or recombinant) molecules. Depending on the nature and concentrations of the growth regulatory molecules present i n the cultures, s i n g l e , double, or multi- lineage colonies of daughter c e l l s can be obtained (21,22,23). Progenitors are defined by the types of colonies they produce, both in terms of the s i z e of the colony, and i t s time of maturation and ultimate composition. These parameters appear to be linked and invariant under most circumstances and therefore provide reproducible indicators of the p r o l i f e r a t i v e and d i f f e r e n t i a t i v e p o t e n t i a l of d i f f e r e n t types of progenitors (24,25). Progenitors categorized i n this way show differences i n surface antigen expression (26), and may d i f f e r i n th e i r responses to c e l l cycle s p e c i f i c agents (27) and to various growth regulatory stimulators (28). Such findings have helped to v a l i d a t e the assignment of the various types of progenitors to a s p e c i f i c l o c a t i o n and a r e l a t i v e rank order in the hemopoietic hierarchy (see Figure 2). C e l l s that form colonies consisting e x c l u s i v e l y of granulocytes, monocytes, or both, are named respectively: colony-forming u n i t - granulocyte (CFU-G), colony-forming unit-monocyte (CFU-M), and colony-forming ULTIMATE STEM CELL " » Lymphopoiesis ERYTHROID PROGENITORS Primitive BfU-E large erythroid colony or burst Mature BFU-E I © - CFU-E small erythroid colony Or burst erythroid cluster self -renewal MYELOID STEM CELL (C fU-S , C F U G E M M ) mixed colony MEGAKARYOCYTE PROGENITORS i CFU M arge megakaryocytic colony ft. small megakaryocytic colony GRANULOCYTE PROGENITORS © .; CFU C large granulocytic colony small granulocytic colony REO CELLS PLATELETS GRANULOCYTES 4 MACROPHAGES FIGURE 2. Schematic Representation of the Hemopoietic System as Defined by Clonogenic Assay for Pluripotent and Committed Progenitors. cn 7 unit-granulocyte/monocyte (CFU-GM). Erythroid l i n e a g e - r e s t r i c t e d progenitors have been subdivided into categories referred to as colony-forming u n i t - erythroid (CFU-E), and burst-forming unit-erythroid (BFU-E), according to the number of hemoglobinized c e l l c l u sters they generate, the l a t t e r often being further subdivided into p rimitive and mature BFU-E subclasses (21). The same p r i n c i p l e has led to the naming of progenitors of colonies of megakaryocytes (CFU-Mk), and of mixed colonies containing granulocytes, erythroid c e l l s , megakaryocytes and monocytes (CFU-GEMM). Although i t i s clear that CFU-GEMM represent a p r i m i t i v e pluripotent c e l l type, those detected i n normal marrow exhibit only a l i m i t e d capacity for self-renewal i n v i t r o . However, very recently a type of CFU-GEMM that shows delayed i n i t i a t i o n of p r o l i f e r a t i o n i n v i t r o leading to the production of small "blast colonies" when most other colonies" have already matured, has been described. These blast colonies eventually w i l l go on to form pure or mixed colonies, but by r e p l a t i n g can be shown to consist of CFU-GEMM, (and CFU-S i n the mouse) as well as other more r e s t r i c t e d progenitor types (29). Work i s now ongoing i n many laboratories to analyze the r e l a t i o n s h i p between the progenitors of these blast colonies (referred to as S - c e l l s or CFU-blast) and c e l l s capable of long-term repopulation in vivo. In summary, clonogenic assays have been developed that allow most classes of myeloid progenitors in both murine and human marrow to be detected, although there i s s t i l l controversy about the exact r e l a t i o n s h i p between stem c e l l s and progenitors of multi-lineage and "bl a s t " colonies. 8 (B) Stromal C e l l s of the Bone Marrow In addition to precursors of c e l l s that are destined to c i r c u l a t e i n the blood, the bone marrow also comprises a v a r i e t y of c e l l s c o l l e c t i v e l y termed the marrow stroma (Figure 3A and 3B). U l t r a s t r u c t u r a l l y , several stromal c e l l types have been i d e n t i f i e d including endothelial c e l l s , f i b r o b l a s t s / a d v e n t i t i a l r e t i c u l a r c e l l s , fat-accumulating c e l l s or adipocytes, osteoblasts, and osteoclasts (30). Macrophages. Because of their putative hemopoietic supportive functions, fixed marrow macrophages have long been considered part of the stroma. However, marrow macrophages are derived from hemopoietic stem c e l l s and not from the mesenchyma l i k e a l l other marrow stromal c e l l s (31,32). Marrow macrophages are engaged i n active phagocytosis where they play a r o l e i n the disposal of p a r t i c u l a t e matter and c e l l debris. Numerous h y d r o l y t i c enzymes detectable by conventional cytochemical methods are present i n t h e i r cytoplasm in association with membrane-bound lysosomes (33). Macrophages are also associated with terminally d i f f e r e n t i a t i n g erythroid c e l l s i n the marrow and in this s i t u a t i o n are believed to play an important role in the d e l i v e r y of i r o n to the hemoglobin-producing erythroblasts. It has also been suggested that marrow macrophages in these e r y t h r o b l a s t i c i s l e t s may be involved i n nucleophagocytosis at the time when the erythroblast extrudes i t s nucleus to develop into a r e t i c u l o c y t e . Consistent with their hemopoietic o r i g i n , macrophages also express the hemopoietic markers T200 and the s p e c i f i c monocytic markers LeuMl and LeuM3, but are not involved i n collagen synthesis (34). 9 ADVENTITIAL RETICULAR CELL HEMOPOIETIC PROGENITOR FIGURE 3. Schematic Representation of Bone Marrow Histology. A) C e l l u l a r Arrangement Around a Marrow Sinus. The wall i s l i n e d by endothelial c e l l s which are separated from a d v e n t i t i a l c e l l s by a discontinuous basement membrane. Hemopoietic c e l l s are present i n the extra-vascular spaces. 10 B) Longitudinal Section of a Marrow Sinus. An a d v e n t i t i a l c e l l which has accumulated fat i s shown on the r i g h t . 11 Endothelial C e l l s . These c e l l s provide the i n t e r n a l l i n i n g of the marrow s i n u s o i d s and are a c t i v e l y involved i n r e g u l a t i n g the migration of newly formed blood c e l l s out of the bone marrow. In contrast to e n d o t h e l i a l c e l l s l o c a t e d elsewhere i n the body, e n d o t h e l i a l c e l l s i n the bone marrow are attached by loose overlapping j u n c t i o n s (35). They are supported by a t h i n basement membrane made up of a number of e x t r a c e l l u l a r matrix p r o t e i n s i n c l u d i n g l a m i n i n , f i b r o n e c t i n , and co l l a g e n type IV. In a d d i t i o n , they a c t i v e l y s ynthesize a number of molecules, one of which, the Factor V I I I - r e l a t e d antigen, serves to d i s t i n g u i s h e n d o t h e l i a l c e l l s from other mesenchymal c e l l types (36). F i b r o b l a s t s / A d v e n t i t i a l R e t i c u l a r C e l l s (ARC). A d v e n t i t i a l r e t i c u l a r c e l l s form a discontinuous c e l l l a y e r on the abluminal surface of marrow s i n u s o i d s . They have an abundant cytoplasm and numerous c e l l processes and are i n v o l v e d i n the production of a supportive meshwork of c o l l a g e n f i b e r s and r e t i c u l i n f i b e r s (an a r g e n t i p h i l l i c mixture of c o l l a g e n f i b e r s and proteoglycans) (37). The exact r e l a t i o n s h i p of f i b r o b l a s t s to ARC i s s t i l l debated but f i b r o b l a s t s l i k e l y represent ARC a c t i v e l y i nvolved i n the s e c r e t i o n of collagens (mostly types I and I I I ) . Fat-Storing Cells/Adipocytes. The pr e c i s e o r i g i n of these c e l l s i s a l s o somewhat c o n t r o v e r s i a l but they are c u r r e n t l y believed to be c l o s e l y r e l a t e d to ARC (38). They are not thought to be l i p i d - l a d e n macrophages as they f a i l to express any monocytic markers (39). They a l s o d i f f e r from adipocytes c h a r a c t e r i s t i c of other t i s s u e s since they accumulate l i p i d s i n the presence of hydrocortisone but not i n the presence of i n s u l i n (40). 12 Osteoblasts. Osteoblasts in the marrow are sometimes observed to be in close association with cancellous bone trabeculae. Osteoblasts are probably derived from cells categorized morphologically as preosteoblasts. Preosteoblasts are fibroblastic cells capable of proliferation located near osteoblasts. They are thought to play a role in the regeneration of the marrow stroma after depletion of the marrow cavity (41). Osteoclasts. Osteoclasts and their precursors are present near bone surfaces and within cavities in bones. There is now a large body of evidence to indicate that osteoclasts are derived from hemopoietic stem cells via blood-borne mononuclear cells (41). Studies with quail-chick chimeras in which quail cells can be recognized by their specific chromatin organization (32) have confirmed previous data suggesting that osteoclasts, monocytes and macrophages arise from a common ancestor cell. More recently, studies with beige mice (42) that have giant lysosomes in their granulocytes, monocytes and osteoclasts but not in their fibroblasts or in osteoblasts provided further evidence that osteoclasts and monocytes have a common origin. (C) Stromal Cell Products As mentioned, marrow stromal cells participate in the formation of a complex extracellular meshwork of fibrous and non-fibrous proteins. Various molecules are known to be present. Some of these have had their genes cloned and their amino acid sequences deciphered. Others have been purified to homogeneity, while some are s t i l l ill-characterized but may nonetheless be crucial to hemopoiesis. Collagens. Collagen i s the s i n g l e most abundant protein species i n the marrow stroma. It i s secreted by a number of mesenchymal c e l l s (ARC/marrow f i b r o b l a s t s , endothelial c e l l s ) and l a i d down e x t r a c e l l u l a r l y (Figure 4) . The collagen molecule has 3 important features: 1) It consists of 3 intertwined a chains forming a stable t r i p l e h e l i c a l structure. 2) Every t h i r d residue i n the a chain i s a glycine. 3) The content of proline and i t s hydroxylated form i s c h a r a c t e r i s t i c a l l y high. Collagens are further c l a s s i f i e d into d i f f e r e n t types according to the nature of t h e i r a chains. In the marrow, collagen type I predominates but collagens type III and IV are also present. There are i n d i c a t i o n s that the primary structure of the a chains determines the physical and chemical properties of the mature collagen molecule. This i s best exemplified by the important differences which e x i s t between collagen I and IV. Whereas collagen I self-assembles into fibrous proteins, collagen IV has no such propensity. Collagen biosynthetic pathways are now well characterized. The f i r s t step consists of the formation of i n d i v i d u a l a chains in the rough endoplasmic reticulum, a chains are next hydroxylated at proline and l y s i n e residues, glycosylated at selected hydroxylysine s i t e s , and converted into a stable t r i p l e h e l i x . The procollagen molecule i s then exported to the i n t e r s t i t i u m and trimmed of i t s non-helical amino and carboxy extensions by s p e c i f i c peptidases. L a s t l y , c r o s s - l i n k i n g of collagen I and I I I , catalyzed by the enzyme l y s y l oxydase helps to convert s i n g l e collagen molecules into tight bundles of collagen f i b e r s . R e t i c u l i n f i b e r s , putative ARC products, consist of a collagen type III core coated with proteoglycans. The f i b e r s are c h a r a c t e r i s t i c a l l y present in the i n t e r s t i t i a l tissue of the marrow where they form a t y p i c a l a r b o r i z i n g network upon impregnation with a reducible s i l v e r s a l t . Recently, using the matrix induced marrow model, evidence has been 14 — OH* OH n„ | OH OH # I OH HO OH <3g SYNTHESIS OF PRO-ALPHA CHAIN HYDROXYLATION AT SELECTED PROLINES AND LYSINES GLYCOSYLATION OF SELECTED HYDROXYLYSINES 3 PRO-ALPHA CHAINS TRIPLE HELIX FORMATION SECRETION CLEAVAGE OF EXTENSION PEPTIDES ASSEMBLY INTO MICROFIBRIL ASSEMBLY INTO MATURE COLLAGEN FIBRIL AGGREGATION OF COLLAGEN FIBRILS TO FORM A COLLAGEN FIBER FIGURE 4. Diagrammatic Representation of the Various I n t r a c e l l u l a r and E x t r a c e l l u l a r Molecular Events Involved in the Formati of a Collagen Molecule. 15 presented that collagen I I I may ac t u a l l y be surrounding nests of hemopoietic c e l l s (43). The s i g n i f i c a n c e of these findings i s not clear at present. Collagen type IV i s the major component of basement membranes. In the marrow, i t forms a fine woven meshwork i n close association with endothelial c e l l s . The presence of a number of sugar residues have been proposed to explain the conservation of i t s non-helical extensions e x t r a c e l l u l a r l y and thereby the lack of self-assembly into fibrous forms (44). Laminin. Laminin i s an extremely large glycoprotein (900,000 daltons), widely d i s t r i b u t e d amongst the various basement membranes (45). It has a peculiar crucifix-shaped configuration that allows s p e c i f i c portions of the molecule, also known as domains, to interact s p e c i f i c a l l y with adjacent c e l l s and the e x t r a c e l l u l a r matrix components proteoglycans and collagen IV. This molecule i s secreted by various e p i t h e l i a l c e l l s , endothelial c e l l s and some smooth muscle c e l l s (45). In addition to i t s binding a b i l i t y , laminin i s thought to play a ro l e i n regulating the types of macromolecules that pass across the basement membrane (46,47). F i b r o n e c t i n . Fibronectin i s also a major mesenchymal c e l l product (48). It has a high molecular weight (200,000-250,000 daltons) and i s normally present both i n the the serum and at the c e l l surface. The presence of a large number of free sulphydryl residues helps to explain the existence of dimeric or multimeric forms (49). This molecule has been studied i n t e n s i v e l y and a number of domains i d e n t i f i e d including a heparin binding s i te , a ce l l binding domain, and a collagen binding region (Figure 5) (50). A newly described family of i n t e g r a l transmembrane proteins known as int e g r i n s , has been shown to transduce f i b r o n e c t i n mediated e x t r a c e l l u l a r signals to actin DOMAINS OF FIBRONECTIN s/V 30< 40 K ?0K 75K 35K 60K 30K / V HEPARIN I COLLAGEN FIBRIN I FIBRIN II CELL HEPARIN I I FIBRIN I I I FIGURE 5. Functional Domains of Fibronectin. Each box i s a protease r e s i s t a n t functional domain. Size of the domains are indicated by the numbers, e.g. 75K = an apparent molecular weight of 75,000. The binding a c t i v i t i e s of each domain are l i s t e d underneath. The amino terminus portion of the molecule i s on the l e f t , and the carboxy terminus on the right, adjacent to the disulphide bridges. 17 filaments inside the c e l l (51). This suggests an a t t r a c t i v e mechanism by which various e x t r a c e l l u l a r matrices may interact with c e l l s and eventually a l t e r c a r d i n a l c e l l u l a r functions such as gene t r a n s c r i p t i o n . A novel function of the f i b r o n e c t i n molecule has been put forth by Patel et a l (52). These authors provided evidence that the release of mature red blood c e l l s from the i n t e r s t i t i a l matrix of the marrow i s mediated by the f i b r o n e c t i n molecule. A s i m i l a r model has recently been proposed for granulocytic c e l l s (53). Hemonectin. Hemonectin i s a very recently described marrow-specific adhesion molecule of a r e l a t i v e molecular mass of 60,000 daltons, that i s immunologically d i s t i n c t from any of the known e x t r a c e l l u l a r matrix components (53). Immature granulocytes bind firmly to hemonectin, i n contrast to mature granulocytes and granulocytic progenitors. This suggests that the loss of adhesiveness to this molecule may be part of the mechanism by which maturing neutrophils become able to leave the marrow. Proteoglycans. Proteoglycans are long molecular complexes composed of a protein core to which a number of repeating disaccharide units are attached (54,55). With the exception of hyaluronic acid, these complexes are i n v a r i a b l y a c i d i c and can transform their immediate environment into hydrated gels. Recently, i t has been shown that glycosaminoglycans (GAG's) produced by marrow c e l l s i n v i t r o can r e t a i n hemopoietic growth factor molecules (56). Further experiments are required to delineate the exact r o l e and s i g n i f i c a n c e of this i n t e r e s t i n g phenomenon to the regulation of hemopoiesis in vivo. 18 2) REGULATION OF THE HEMOPOIETIC SYSTEM Although s t i l l poorly understood, the regulation of many aspects of hemopoietic c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n appears to be mediated by a set of l o c a l environmental conditions and by the production of a number of hormone-like glycoprotein growth factors. (A) C e l l u l a r Interactions There are several l i n e s of evidence suggesting that stromal c e l l s of the bone marrow interact d i r e c t l y with hemopoietic c e l l s and play a ro l e i n t h e i r regulation. U l t r a s t r u c t u r a l Studies. Morphologic studies of the marrow have disclosed a number of consistent and s p e c i f i c interactions between hemopoietic c e l l s and the various stromal populations (38). For example, an intimate r e l a t i o n s h i p between a d v e n t i t i a l r e t i c u l a r c e l l s and hemopoietic c e l l s has been noted. This has suggested that some physiologic functions are mediated through these in t e r a c t i o n s , possibly including the release of granulocytes from the marrow. Another i n t r i g u i n g finding has been the t y p i c a l parasinal l o c a t i o n of megakaryocytes. This observation i s the basis of the proposal that endothelial c e l l s elaborate hemopoietic growth factors capable of regulating or supporting megakaryocytopoiesis (57). Regeneration Studies. A number of experimental strategies designed to disturb stromal c e l l support including l o c a l i r r a d i a t i o n (58,59) and 19 mechanical disruption (60) have demonstrated that such treatments r e s u l t i n loss of hemopoietic function. Studies of Spleen Colonies. It has been shown that the c e l l u l a r composition (erythroid vs granulopoietic) of i n d i v i d u a l spleen colonies varies with th e i r s p e c i f i c l o c a t i o n i n the spleen. Colonies a r i s i n g under the spleen capsule or i n the v i c i n i t y of septae are more often granulopoietic whereas i n a l l other locations colonies are mainly erythroid. In contrast, colonies in the marrow are almost excl u s i v e l y granulopoietic (E:G = 0.1). These observations suggest that l o c a l environments in these hemopoietic organs exert a profound influence on hemopoietic c e l l d i f f e r e n t i a t i o n (61). Further, when marrow fragments were implanted into the spleen of experimental animals, which were then used as spleen colony assay r e c i p i ents, the colonies produced i n the i n t r a - s p l e n i c a l l y implanted marrow stroma yielded a E:G r a t i o of 0.1 whereas those that developed i n the spleen i t s e l f yielded an E:G r a t i o of 2.9 or higher. Colonies that bridged the junction of the spleen and the implanted marrow stroma had th e i r erythroid portion in the splenic stroma and t h e i r granulopoietic portion i n the marrow stroma (62). Although these studies cannot be construed as evidence of an "inductive" microenvironment i n the det e r m i n i s t i c ( i . e commitment) sense of the word since they do not provide information as to the stage of hemopoietic c e l l development influenced, they do support the concept that fixed elements of hemopoietic tissues regulate some aspects of hemopoietic c e l l d i f f e r e n t i a t i o n . Transfer of the Hemopoietic Microenvironment. A series of studies undertaken by Friedenstein et a l . (63) have shed considerable l i g h t on the role fixed bone marrow populations play in providing an environment capable of 20 supporting hemopoiesis. The studies of this group have shown that heterotopic transplantation of bone marrow fragments under the kidney capsule of semi- syngeneic animals leads to the formation of bone which then rapidly becomes populated by hemopoietic c e l l s of recipient o r i g i n (63). In addition, transplantation of cloned, cultured f i b r o b l a s t s under the capsule of the kidney was shown to re s u l t i n the transfer of a microenvironment t y p i c a l of the hemopoietic tissue from which the f i b r o b l a s t s were obtained (64). Other groups have confirmed and expanded these observations and found that even subcutaneous implantation of a small amount of a c e l l u l a r diaphyseal bone extract can lead to the formation of a bone enclave which only then becomes populated with hemopoietic c e l l s (65). I r r a d i a t i o n of the subcutaneous area, however, prevents the formation of the bone enclave, i n d i c a t i n g that recruitment of subcutaneous c e l l s i s e s s e n t i a l l o r the creation of a stroma s u i t a b l e for hemopoietic stem c e l l invasion and support. Such studies have led to a model in which i t i s envisaged that undifferentiated " f i b r o b l a s t - l i k e " mesenchymal c e l l s present in a va r i e t y of tissues can regenerate the d i f f e r e n t i a t e d elements of the hemopoietic stroma under the influence of p a r t i c u l a r , but as yet undefined e x t r a c e l l u l a r matrix substances (66). Ad d i t i o n a l evidence in support of this i s provided by recent co-culture studies showing that even 3T3 c e l l s , which are undifferentiated cultured embryonic mouse f i b r o b l a s t s can support hemopoiesis in v i t r o for a l i m i t e d period of time (67). Studies of the Hemopoietic Microenvironmental Defect i n Mice Determined by the S l / S l ^ Genotype. Mice bearing a l t e r a t i o n s at the Steel locus exhibit a number of abnormalities including macrocytic anemia, hypopigmentation, abnormal s e n s i t i v i t y to ra d i a t i o n , and s t e r i l i t y (68). Mutants of the genotype S l / S l ^ have been the most thoroughly studied and experiments with these animals have revealed the nature of the defect that a f f e c t s t h e i r hemopoietic system. Hemopoietic stem c e l l s i n S l / S l ^ mice are i n t r i n s i c a l l y normal as judged by th e i r r a d i o s e n s i t i v i t y , their a b i l i t y to form spleen colonies in normal re c i p i e n t s and their a b i l i t y to cure anemic mice whose hemopoietic stem c e l l s are defective (69). However, when S l / S l ^ animals are i r r a d i a t e d and then injected with normal (+/+) hemopoietic c e l l s , they do not support hemopoietic recovery from the transplanted c e l l s . This can be seen by the f a i l u r e of the transplanted c e l l s to form spleen colonies i n S l / S l ^ r e c i p i e n t s (69). Since the hemopoietic defect can be cured by a graft of whole tissue from a +/+ mouse but not by an intravenous i n j e c t i o n of suspended +/+ c e l l s , the hemopoietic defect in the S l / S l ^ mouse i s thought to be due to a fixed c e l l of the marrow microenvironment that does not c i r c u l a t e and i s not derived from a c e l l that does c i r c u l a t e (70). Regulation of Stem C e l l Turnover i n P a r t i a l l y I r r a d i a t e d Mice. Under normal conditions in the adult, hemopoietic stem c e l l s are quiescent (G Q) or a l t e r n a t i v e l y , have entered a very long c e l l cycle (2). However, i n response to cytoreductive agents or radiation, a larger proportion of these c e l l s are triggered to enter the S phase of the c e l l cycle. To determine i f th e i r turnover i s regulated at a l o c a l l e v e l , experiments were performed i n which mice were given whole body radi a t i o n , except for one t i b i a which was shielded. Using the ^H-thymidine suicide assay (71), the c y c l i n g status of CFU-S was then followed both in the shielded and in the i r r a d i a t e d t i b i a s . After 5 days, CFU-S in the shielded areas had la r g e l y returned to a quiescent state (12% k i l l ) . In contrast, in the i r r a d i a t e d limb the proportion of c e l l s in S-phase was high (35 % k i l l ) and the CFU-S content low. This d i s p a r i t y 22 between the two limbs c l e a r l y indicates that the turnover of primitive hemopoietic c e l l s can be regulated l o c a l l y (72). (B) Humoral Regulation of the Hemopoietic System Hemopoietic Growth Factors. The development of culture techniques capable of supporting the c l o n a l growth of hemopoietic progenitors led to the i d e n t i f i c a t i o n and p u r i f i c a t i o n of a number of glycoproteins that are now c o l l e c t i v e l y referred to as hemopoietic growth factors (HGF) (73). At f i r s t , b i o l o g i c a l a c t i v i t i e s present in crude preparations were operationally defined i n terms of t h e i r a b i l i t y to support colony formation, and hence were referred to as colony stimulating a c t i v i t i e s (CSA). The related, and now more widely used term, colony stimulating factor (CSF), was coined to r e f e r to d i s c r e t e p u r i f i e d f a c t o r s . Only a f t e r such preparations were obtained could the f u l l range of a c t i v i t y of i n d i v i d u a l CSF's be rigorously defined. Recently, the genes for a number of human HGF's have been cloned and expressed. These include: Erythropoietin (Epo), GM-CSF, G-CSF, M-CSF, and Interleukin-3 (IL-3). The a v a i l a b i l i t y of large quantities of these pure recombinant molecules has made i t possible to investigate their in vivo b i o l o g i c a l a c t i v i t i e s i n both primates and in man (74,75,76,77)) and to investigate their mechanisms of actions, i n addition to defining the range of their a c t i v i t i e s on many d i f f e r e n t c e l l types. Several points of interest have emerged from such studies: 1) HGF are glycoproteins which s p e c i f i c a l l y interact with c e l l surface receptors present on target c e l l s and their b i o l o g i c a l e f f e c t s are often obtained even at a low (10%) receptor occupancy rates (78). 2) HGF are extremely potent molecules and are b i o l o g i c a l l y active at picomolar concentrations (78). 3) There are no 23 s t r i k i n g homologies between the various HGF and any oncogene products thus far i d e n t i f i e d . However, the receptor for M-CSF has been shown to be i d e n t i c a l to the product of the proto-oncogene, c-fms (79). 4) Most HGF have p l e i o t r o p i c e f f e c t s and can interact with a number of d i f f e r e n t target c e l l s . These include mature e f f e c t o r c e l l s which may have their functions augmented by the HGF's (80,81,82) as well as progenitor c e l l s on d i f f e r e n t lineages (83). Erythropoietin (Epo). Epo i s a glycosylated glycoprotein with a r e l a t i v e molecular mass of 39,000 daltons when f u l l y glycosylated. Native epo i s produced mainly by the kidney and has been p u r i f i e d to homogeneity from the urine of patients with a p l a s t i c anemia (84,85). Epo i s active on committed erythroid progenitors and enhances the s u r v i v a l and p r o l i f e r a t i o n of CFU-E and mature BFU-E (86,87). The gene for human Epo has been mapped to chromosome 7 (88). Recombinant Epo has recently been tested in patients s u f f e r i n g from anemia of renal f a i l u r e . The f u l l y glycosylated recombinant molecule retains i t s f u l l b i o l o g i c a l a c t i v i t y and shows great promise for the therapy of these patients (89). Macrophage Colony-Stimulating Factor (M-CSF). Human M-CSF i s a glycoprotein of a r e l a t i v e molecular mass of 47,000 to 76,000 daltons. This contrasts with the 70,000 dalton M-CSF p u r i f i e d from murine L c e l l s . Upon reduction, the l a t t e r species y i e l d s 2 i d e n t i c a l but b i o l o g i c a l l y i n a c t i v e subunits of about 35,000 daltons each (90). The gene has been cloned (91) and on the basis of i t s t r a n s c r i p t s , 2 d i s t i n c t forms have been postulated: a membrane-bound form and a secreted form, both of which are presumed to be b i o l o g i c a l l y active (92,93). The p r o l i f e r a t i v e e f f e c t of M-CSF on macrophage progenitors in v i t r o has been the best studied action of M-CSF (94). The human 24 gene for M-CSF is located on the long arm of chromosome 5 near the gene for the M-CSF receptor, c-fms. Granulocyte Colony-Stimulating Factor (G-CSF). G-CSF was f i r s t purified to homogeneity from medium conditioned by the human bladder carcinoma c e l l l ine 5637 (95). It has a relative molecular mass of 19,600 daltons. The gene for human G-CSF is located on chromosome 17 (96). It is produced by a variety of activated cel ls including lymphocytes and fibroblasts (97,98). Recombinant G-CSF has been shown to support granulocyte progenitor c e l l prol iferation in vi tro (99). Although i n i t i a l studies with purified natural human G-CSF suggested that this molecule could stimulate cel ls on other lineages, as well as pluripotent ce l l s , hence the original term "pluripoietin" (95), more recent data suggest that these latter effects were primarily indirect (100). Interestingly, murine G-CSF, unlike most other murine HGF's, is active on human as well as murine cel ls and enhances the formation of human neutrophil colonies (101). An in vivo effect of human G-CSF in stimulating granulocyte levels in chemotherapy treated patients has been demonstrated very recently (75). Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF). GM-CSF is a glycoprotein of a relative molecular mass of 22,000 daltons. The human gene has been mapped to the long arm of chromosome 5 near M-CSF, c-fms and adjacent to IL-3 (102). GM-CSF, l ike G-CSF and IL-3, is not known to be constitutively produced by any normal ce l l but is produced by a variety of activated cel ls including lymphocytes and fibroblasts (103,104). Purified natural and recombinant molecules share the same biological properties: they both stimulate granulocyte, macrophage, eosinophil and megakaryocyte progenitors, 25 probably d i r e c t l y (105,106). They can also enhance the p r o l i f e r a t i o n of prim i t i v e BFU-E and CFU-GEMM (107). The recombinant molecule has recently been tested c l i n i c a l l y and a stimulation of c i r c u l a t i n g granulocyte l e v e l s i n patients injected with GM-CSF has been demonstrated (76,77). In addition, GM-CSF can augment the function of mature e f f e c t o r c e l l s and can stimulate the expression of c e l l surface adhesion molecules Mol and LeuM5 (pl50,95) on mature granulocytes (81). Interleukin - 3 (IL - 3 ) , Multi-CSF. Human IL-3 i s a glycoprotein with a molecular mass of 14,000 to 28,000 daltons depending on the extent the f i n a l molecule has been glycosylated. Although the existence of human IL-3 was predicted from studies of murine IL-3, human IL-3 was f i r s t obtained as a p u r i f i e d molecule as a result of an expression cloning strategy (108). The molecule has a large spectrum of a c t i v i t i e s , as predicted from studies of murine IL-3 (109), which appears to extend from the pluripotent stem c e l l compartment to the various mature committed progenitors. In contrast to GM-CSF, IL-3 i s a more potent stimulator of erythroid bursts and mixed colonies and less potent stimulator of granulocyte-macrophage colonies (110). Sy n e r g i s t i c A c t i v i t i e s . There have been a serie s of reports describing factors that, alone, are devoid of i n t r i n s i c colony-stimulating a c t i v i t y but, nevertheless, are capable of synergizing with one or more HGF's to enhance hemopoietic colony formation. Listed below are several such recently described s y n e r g i s t i c factors. I n t e r l e u k i n - 1 ( I L - 1 ) . Two forms of IL-1 have been described: IL - l a and IL - i p . Both are very s i m i l a r , bind to the same receptor and, hence, share 26 most i f not a l l of th e i r b i o l o g i c a l e f f e c t s (111,112). 11-10 i s the predominant type expressed; however, an a c t i v i t y independently p u r i f i e d from 5637 conditioned medium on the basis of i t s a b i l i t y to synergize with other HGF's turned out to be I L-la (113,114,115). IL-16 i s a polypeptide of a r e l a t i v e molecular mass of 22,000 daltons. It i s produced mostly by activated monocytes (116) and TNF-oc stimulated endothelial c e l l s (117). It has a broad range of e f f e c t s i n vivo including the stimulation of secretion of some acute phase proteins (116), the acceleration of bone resorption (118), the enhancement of e x t r a c e l l u l a r matrix protein turnover, and the induction of a v a r i e t y of mesenchymal c e l l types (104,119,120) to secrete several growth factor molecules including: PDGF, nerve growth factor (NGF), and various CSF's (121,122,123,124,125,126). IL -1 has been shown to act on many c e l l types including keratinocytes, hypothalamic c e l l s , hepatocytes, lymphocytes and mature neutrophils (120). Interleukin - 4/B C e l l Stimulatory Factor - 1 (IL - 4)/(BCSF-l). A recently p u r i f i e d T c e l l product, murine IL - 4 , has been shown to stimulate a broad range of murine hemopoietic progenitors as well as B c e l l s and T c e l l s (127). However, these p r o l i f e r a t i v e e f f e c t s of IL -4 on myeloid c e l l s are only observed i n the presence of a d d i t i o n a l growth factor molecules. In concert with other factors murine IL -4 can enhance the in v i t r o p r o l i f e r a t i o n of CFU-GEMM, CFU-GM, BFU-E, CFU-E and CFU-Mk (128,129). Murine IL -4 has been cloned (130) but i t s e f f e c t on human myeloid c e l l s has not yet been established. Interleukin - 6 ( I L - 6 ) . Previously known as Interferon -32 (IFN-02) or hybridoma growth factor (131), this molecule has a molecular weight of 27 26,000 daltons. IL-6 i s produced by a variety of c e l l types including f i b r o b l a s t s and monocytes p a r t i c u l a r l y a f t e r their a c t i v a t i o n (132). It supports the growth of c e r t a i n B c e l l hybridomas and plasmacytomas and has recently been found to synergize with IL-3 to support the p r o l i f e r a t i o n of pr i m i t i v e multipotential murine progenitor c e l l s i n culture (133). IL-6 i s a weak stimulator of macrophage progenitors in v i t r o . IL-6, l i k e IL-1, also stimulates the secretion of acute phase proteins by hepatocytes (134). Negative Regulators of Hemopoiesis. There have been reports by several groups (135,136,137) suggesting that fractionated c e l l - f r e e extracts obtained from r e s t i n g murine hemopoietic tissues contain a murine hemopoietic stem c e l l i n h i b i t o r whose action i s r e v e r s i b l e . This a c t i v i t y i s associated with a molecular species whose mass appear to be in the 50,000 to 100,000 dalton range. It i s detected by i t s a b i l i t y to protect S-phase murine CFU-S from the l e t h a l e f f e c t s of exposure to high s p e c i f i c a c t i v i t y -^-thymidine (71). I n t e r e s t i n g l y , the e f f e c t s of this i n h i b i t o r can be competed o f f by a smaller s i z e (30,000 - 50,000 daltons) stimulator obtained from regenerating murine hemopoietic tissues (138,139). In a preliminary experiment the extracted i n h i b i t o r was found to be e f f e c t i v e i n vivo (140). TGF-|3 has also been shown to also act as a potent i n h i b i t o r of hemopoietic c e l l p r o l i f e r a t i o n (141,142). TGF-g i s a highly conserved homodimer of 25,000 daltons present in many tissues. It i s p a r t i c u l a r l y abundant in bone where i t i s present at a 100-fold higher concentration than elsewhere (142). Two very s i m i l a r forms of TGF-f3 have been i d e n t i f i e d : TGF-gl and TGF-{32 (142,143). Very l i t t l e i s known at present about the mechanism of action of TGF-gl or 2 at the molecular l e v e l , although the gene has been cloned (144). Recent reports have suggested that TGF-3 can increase the 28 incorporation of fi b r o n e c t i n and collagen into the e x t r a c e l l u l a r matrix and can enhance the expression of c e l l adhesion protein receptors (145). HGF-dependent murine hemopoietic c e l l l i n e s which express s p e c i f i c TGF -B c e l l surface receptors (K = 1-60 pM) are inh i b i t e d by TGF-61 (145). Recent experiments i n the Terry Fox Laboratory have further shown that TFG-01 can i n h i b i t the c y c l i n g of the most primitive myeloid progenitor c e l l types i n both mouse and human marrow in a d i r e c t , s e l e c t i v e and rev e r s i b l e fashion (146) . Mesenchymal C e l l A c t i v a t o r s . As mentioned above, many HGF's are now known to be produced by mesenchymal c e l l s following their a c t i v a t i o n by s p e c i f i c molecular mediators often produced i n inflammatory reactions. Some of these, such as IL-1 have been discussed above as they can also act as HGF's. Others with no known dir e c t HGF a c t i v i t y are reviewed b r i e f l y below. P l a t e l e t derived growth factor (PDGF). PDGF i s a c a t i o n i c glycoprotein of a molecular mass of 30,000 daltons. PDGF consists of 2 polypeptide chains, one of which i s i d e n t i c a l to the product of the c e l l u l a r proto-oncogene, c - s i s (147) . PDGF i s produced by both activated endothelial c e l l s and tissue macrophages (148,149). Induction of PDGF i s obtained with endotoxin, tumor necrosis factor-a, and phorbol esters. P l a t e l e t s contain the highest l e v e l s of PDGF, but release of PDGF from the p l a t e l e t i s conditional on the stimulation of p l a t e l e t adhesion (125). Connective t issue c e l l s ( f i b r o b l a s t s , smooth muscle c e l l s , g l i a l c e l l s and chondrocytes) which d i sp lay a large number of high a f f i n i t y c e l l surface PDGF receptors are thought to constitute the primary target of PDGF action. Upon binding to i t s receptor, PDGF triggers a cascade of events which culminate in the production of 29 e x t r a c e l l u l a r matrix substances, and the secretion of a range of b i o l o g i c a l a c t i v i t i e s , including GM-CSF (149,150). PDGF i s rapidly cleared from the c i r c u l a t i o n , and current evidence suggests that i t s turnover may be regulated l o c a l l y by i t s attachment to c e l l s or i n t e r c e l l u l a r matrix components (151). Tumor Necrosis Factor-a (TNF-a). Also known as cachectin, this molecule e l i c i t s hemorrhagic necrosis of tumors in recipient animals challenged with endotoxin (152). It i s produced p r i n c i p a l l y by activated monocytes which then secrete very large amounts of TNF-a (153 ). TNF-a i s r a p i d l y d i s t r i b u t e d v i a the c i r c u l a t i o n to normal target c e l l s : the monocytes and endothelial c e l l s (153). Human TNF-a has recently been p u r i f i e d , the gene cloned, expressed, and mapped to chromosome 6 (154). TNF-a has a wide spectrum of a c t i v i t i e s and mediates endotoxin-induced shock. TNF-a has also recently been shown to provide mitogenic signals for endothelial c e l l s in vivo, and to promote the formation of c a p i l l a r y tube-like structures in v i t r p . Like IL-13 and PDGF, TNF-a i s a potent inducer of mesenchymal c e l l s and stimulates them to produce and release G-CSF and GM-CSF (155,156). Summary. It i s clear from the above discussion that the bone marrow i s a highly heterogeneous organ. It consists of many c e l l types and a d i v e r s i t y of fibrous and non fibrous c e l l products. A large body of evidence suggests that the regulation of hemopoietic stem c e l l s i s mediated primarily by complicated l o c a l mechanisms involving stromal c e l l s and their secretory products. 30 3) LONG-TERM BONE MARROW CULTURES: AN IN VITRO MODEL FOR HEMOPOIETIC STEM CELL REGULATION Although i n vivo studies add weight to the phy s i o l o g i c a l relevance of any e f f e c t s observed following a given pertubation, they are usually poorly suited to the analysis of s p e c i f i c c e l l u l a r and molecular events that underlie p a r t i c u l a r responses. The establishment and v a l i d a t i o n of s u i t a b l e i n v i t r o models for many c e l l u l a r and tissue systems have therefore been a major objective of much research i n c e l l biology. In this respect the study of hemopoietic stem c e l l regulation i s no exception. The f i r s t d e s c r i p t i o n i n 1977 by Dexter et a l (157) of culture conditions which allow murine pluripotent stem c e l l s to be maintained for many months in the absence of exogenously provided HGF's represented a s i g n i f i c a n t advance in th i s regard. For the f i r s t time, i t became possible to investigate i n v i t r o , a system which supported stem c e l l self-renewal and d i f f e r e n t i a t i o n . Moreover, as revealed by subsequent studies discussed i n more d e t a i l below, this culture system has many features of the bone marrow as i t i s constituted i n vivo and appears to provide a useful model for analysis of the regulatory systems operative i n humans as well as mice. (A) Early Development of the Long-Term Marrow Cultures In i n i t i a l studies with this system (157) mouse marrow c e l l s were f i r s t placed i n culture at a concentration of 10^ cells/ml in a medium supplemented with horse serum to allow the formation of a confluent adherent layer which took 2-3 weeks. These cultures were then "recharged" with a second, s i m i l a r inoculum of mouse marrow c e l l s . Half the medium was then removed and replaced 31 each week. At the same time the c e l l s i n the non-adherent f r a c t i o n were simultaneously d i l u t e d in h a l f , and the c e l l s removed could be counted, stained and/or assayed for various types of progenitors. Under these conditions, i t was found that CFU-S, CFU-GM and granulopoiesis could be maintained for very extensive periods of time (158). An important feature of t h i s system was that no HGF's were added exogenously. When they were, the r e s u l t was not a p o s i t i v e one but rather an acceleration i n the rate at which hemopoiesis declined (157). This, together with the demonstration of the c r i t i c a l importance of the adherent layer (159), suggested that c e l l - c e l l i n t e r a c t i o n s , perhaps s i m i l a r to those occurring i n the marrow i n vivo had been re-established in this system. During the next several years, a number of s i g n i f i c a n t technical improvements were i d e n t i f i e d . The use of hydrocortisone as a supplement was p a r t i c u l a r l y important because i t made i t possible to use almost any batch of horse serum rather than only rare, selected batches. In addition, i t made i t possible to i n i t i a t e cultures with a single inoculum of marrow (40). These improvements, in turn, led to the successful establishment of long-term human marrow cultures (160,161). (B) Long-Term Human Marrow Cultures Long-term cultures in which granulopoiesis i s maintained for at least 8 weeks can now be routinely i n i t i a t e d from a single innoculum of normal human marrow. (For technical d e t a i l s , see Chapter I I ) . Within 3 weeks of placing the marrow c e l l s in culture, a confluent adherent layer i s established. In i t a variety of stromal c e l l types have been i d e n t i f i e d . These include c e l l s with properties of f i b r o b l a s t s (162), 32 endothelial c e l l s (see Figure 6A and 6B) (163), adipocytes (164), smooth- muscle c e l l s (165), and macrophages (158). Interspersed between these c e l l s , f o c i of developing hemopoietic c e l l s , d e s c r i p t i v e l y referred to as "cobblestone areas" (160), can be seen. The adherent layer also contains a number of e x t r a c e l l u l a r matrix components including: collagen I (162), collagen III (162), collagen IV (166), laminin (167), and f i b r o n e c t i n (168). Hovering over and derived from the adherent layer are loosely adherent and f r e e - f l o a t i n g c e l l s . Most of these are mature granulocytes and macrophages and th e i r immediate precursors (169). In the human system, some clonogenic progenitors can also be detected in the non-adherent f r a c t i o n , but the majority of such c e l l s , p a r t i c u l a r l y the more pri m i t i v e ones, are found in the adherent f r a c t i o n (169). To quantitate the hemopoietic progenitor content of the adherent layer requires s a c r i f i c i n g the culture to enzymatically d i s s o c i a t e the c e l l s i n this f r a c t i o n so that a si n g l e c e l l suspension s u i t a b l e for p l a t i n g i n semi-solid medium can be obtained. Suitable methods using either collagenase or trypsin have been developed for this purpose (169). Assessment of a series of cultures over time has shown that the hemopoietic progenitor content of the non-adherent f r a c t i o n s and the mature granulocytes and macrophages to which they give r i s e are maintained at roughly constant l e v e l s for up to 8 weeks in spite of the demi-depopulation of the non-adherent c e l l s at each weekly medium change (Figure 7). Note that although granulopoiesis i s supported to completion in this system, erythropoiesis i s not. Thus, only the most primitive erythroid progenitors, the BFU-E, are continuously detected and the more d i f f e r e n t i a t e d elements including the CFU-E rapidly disappear. This i s not s u r p r i s i n g since Epo i s not normally added to these cultures. In the murine system, a s i m i l a r s i t u a t i o n i s found and, although addition of Epo alone i s i n s u f f i c i e n t to 33 FIGURE 6. Photomicrographs of a Formalin Fixed Long-Term Marrow Culture Adherent Layer Stained With Rabbit-Anti Factor VIII Antiserum and Developed With Peroxidase-Labelled Swine Anti-Rabbit Immunoglobulin Antibody. A) Low Power View 34 FIGURE 6. Photomicrographs of a Formalin Fixed Long-Term Marrow Culture Adherent Layer Stained With Rabbit-Anti Factor VIII Antiserum and Developed With Peroxidase-Labelled Swine Anti-Rabbit Immunoglobulin Antibody. B) High Power View 35 I • r 10 7- Nucleated Cells 3 106 o i 10! 10* r 100 10 .01 0 1 2 3 4 5 6 7 8 Weeks in Culture 0 1 2 3 4 5 6 7 8 Weeks in Culture FIGURE 7. The C e l l u l a r i t y and Progenitor Content of the Adherent (Adh) and Non-adherent (NA) Fraction Assessed at Varying Incubation Times. Each point shown represents the geometric mean + 1 SEM of data from several experiments. Of the 15 experiments i n i t i a t e d , f i v e were terminated at 32 weeks, s i x at 4 weeks, and four maintained u n t i l 7 weeks and s a c r i f i c e d for assessment at that time. The downward arrows indicate maximum mean values i f one colony had been seen i n any of the assay dishes scored i n each i n d i v i d u a l experiment. 36 allow erythropoiesis to proceed, when other factors present in anemic serum are provided, massive production of mature red cel ls can be obtained (170). (C) Long-Term Marrow Cultures as a Model of In Vivo Regulation A number of studies in both the human and murine system have provided convincing evidence that the behaviour and regulation of hemopoietic cel ls in the long-term marrow culture system may reflect the operation of the same mechanisms that control hemopoiesis in vivo. This evidence may be summarized as follows: 1) In the murine system, where i t is possible to assay cel ls for marrow repopulating potential by transplantation into lethal ly irradiated recipients, i t has been shown that such cel ls are maintained in long-term marrow cultures (171). 2) When the mature differentiated granulocytes produced in long-term marrow cultures are compared with normal c irculat ing peripheral blood granulocytes no differences in any of a variety of physiological properties, including phagocytosis, degranulation, respiratory burst and bacterial k i l l i n g , are found (172). 3) The microenvironmental defect of the S l /S l^ mouse is reproduced in long-term cultures ini t iated with marrow from these mice (173). A normal appearing adherent layer is formed, but hemopoiesis is not maintained by comparison to cultures set up with marrow from +/+ littermates. Moreover, the defect can be overcome by seeding S l / S l ^ marrow onto pre-established normal stromal ce l l layers from W/Wv mice (who have deficient stem cel ls and cannot in i t ia te long-term hemopoiesis in vitro for this reason) (174). 4) Perhaps, the most compelling evidence comes from studies of the turnover of primitive hemopoietic cel ls in the long term marrow culture system. Assessment of the cycling status of different types of progenitors in the non-adherent and adherent fractions using the ^H-thymidine 37 s u i c i d e technique to measure the proportion of S-phase c e l l s (71) revealed a s i m i l a r (although not i d e n t i c a l ) pattern i n both murine and human c u l t u r e s . In the murine system, CFU-S were found to o s c i l l a t e between a c y c l i n g and non- c y c l i n g s t a t e d i c t a t e d by the timing of p e r t u r b a t i o n of the c u l t u r e s a s s o c i a t e d with each medium change. This r e s u l t e d i n an a c t i v a t i o n of a l l CFU-S w i t h i n 1-2 days, regardless of t h e i r s i t u a t i o n i n e i t h e r the adherent or non-adherent f r a c t i o n . I f the c u l t u r e s were then l e f t undisturbed, the CFU-S returned to a quiescent s t a t e w i t h i n the next 3 to 5 days (175). In contrast CFU-GM were found to remain continuous i n c y c l e as they do i n vivo even under normal homeostatic c o n d i t i o n s (175). In human marrow c u l t u r e s , a s i m i l a r p a t t e r n of a l t e r n a t i n g p r o l i f e r a t i o n and quiescence i n the most p r i m i t i v e progenitor compartments has a l s o been seen (176). However, t h i s behaviour i s e x c l u s i v e to the c e l l s contained w i t h i n the adherent l a y e r , suggesting a more important r o l e of d i r e c t c e l l contact i n t h i s species. This i s f u r t h e r suggested by the f i n d i n g that p h y s i c a l p e r t u r b a t i o n of human c u l t u r e s i s i n s u f f i c i e n t to achieve progenitor c e l l s t i m u l a t i o n . A d d i t i o n of some mesenchymal c e l l a c t i v a t o r appears to be required (177). Nevertheless, as i n the mouse, the c y c l i n g status of the various population of progenitors i n unperturbed c u l t u r e s c l o s e l y mimics that seen i n the marrow of the normal a d u l t . Those c l a s s e s of c e l l s that i n vivo are quiescent are those that i n the long-term c u l t u r e a l s o r e t u r n to a non-dividing s t a t e . S i m i l a r l y , those that i n v i v o are i n a s t a t e of constant turnover remain i n c y c l e i n the long- term c u l t u r e system, even when they are located i n the adherent l a y e r . 38 4) THESIS OBJECTIVES This research p r o j e c t was developed to explore the hypothesis that hemopoietic stem c e l l r e g u l a t i o n i s mediated by short-range i n t e r a c t i o n s with mesenchymal elements. To test t h i s hypothesis i n the human, i t would be advantageous to use an i n v i t r o model i n which the various components could be d i s s e c t e d out and t h e i r functions analysed separately. In the preceding s e c t i o n I have described an i n v i t r o system i n which the most p r i m i t i v e hemopoietic populations detectable by clonogenic assays can be demonstrated f o r periods of at l e a s t 8 weeks. A key feature of t h i s long-term marrow c u l t u r e system i s the development of a heterogeneous adherent l a y e r of c e l l s , i n c l u d i n g many of which are thought to resemble the various c o n s t i t u e n t s of the microenvironment of the marrow i n v i v o . The a v a i l a b i l i t y of clonogenic assays f o r the d e t e c t i o n of the most p r i m i t i v e c l a s s e s of progenitors c e l l s as w e l l as the p o s s i b i l i t y of using the long-term marrow c u l t u r e system as an i n v i t r o model of the stroma suggested that f u r t h e r a n a l y s i s of t h i s system might allow the c e l l u l a r and molecular mechanisms r e g u l a t i n g stem c e l l maintenance and turnover to be i d e n t i f i e d . However, the long-term marrow c u l t u r e model i s by no means a "simple" v e r s i o n of i n v i v o hemopoiesis. The heterogeneity of the c e l l u l a r composition of the adherent l a y e r and the vast array of molecular species produced by these c e l l s makes any proposed a n a l y s i s a r e a l challenge. The o v e r a l l o b j e c t i v e s of my research were, therefore, twofold. The f i r s t o b j e c t i v e was to develop a method for assaying the regulatory funct ion of the non-hemopoietic components of long-term marrow c u l t u r e adherent l a y e r s . Such an assay could then be used to evaluate the r o l e played by i n d i v i d u a l stromal c e l l types. P r e r e q u i s i t e for such an assay was the need to o b t a i n 39 pure target hemopoietic populations (or at l e a s t suspensions of hemopoietic c e l l s that were free of re g u l a t o r y c e l l s ) . I envisaged that t h i s might be achieved by appropriate p h y s i c a l separation techniques, or by the adoption of c u l t u r e c o n d i t i o n s that would s e l e c t i v e l y prevent contaminating accessory c e l l s from i n t e r f e r i n g with the proposed measurements. Both approaches were explored and the r e s u l t s are presented i n Chapter I I I . The second o b j e c t i v e of my research was to i s o l a t e homogeneous populations of c e l l s corresponding to the various mesenchymal components of the marrow stroma. The approach was to e s t a b l i s h permanent c e l l l i n e s that could be cloned and then e x t e n s i v e l y c h a r a c t e r i z e d . I t i s w e l l known that normal human mesenchymal c e l l s senesce r a p i d l y i n v i t r o (178) and, i n con t r a s t to t h e i r murine counterparts, r a r e l y become spontaneously immortalized. Therefore, an a l t e r n a t e s t r a t e g y had to be used to immortalize human marrow stromal c e l l s . Stimulated by reports of the a b i l i t y of SV-40 v i r u s to transform various types of human c e l l s without l o s s of t h e i r d i f f e r e n t i a t e d phenotype (179,180,181,182), I i n i t i a t e d a s e r i e s of experiments to t r y and o b t a i n a s e r i e s of human mesenchymal c e l l l i n e s that e x h i b i t e d p r o p e r t i e s of marrow stromal elements. The i n i t i a l work involved i n the pre p a r a t i o n and t i t r a t i o n of a s u i t a b l e SV-40 v i r u s preparation i s described i n Chapter I I ( M a t e r i a l s and Methods). The e f f e c t s of t h i s v i r u s p reparation on human marrow stromal c e l l s and the c h a r a c t e r i z a t i o n of the SV-40 transformed l i n e s I i s o l a t e d are described i n Chapter IV. 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A s i g n i f i c a n t part of macrophage derived growth factor consists of a least two forms of PDGF. C e l l 43: 277, 1985. 150. Delwiche F, Raines E, Powel J, Ross R, Adamson J. P l a t e l e t - d e r i v e d growth factor enhances in v i t r o erythropoiesis via stimulation of mesenchymal c e l l s . J C l i n Invest 76: 137, 1985. 151. Smith JC, Fugh JP, L i l l q u i s t JS, Goon DS, S t i l e s CD. Growth factors adherent to c e l l substrate are mitogenically active in s i t u . Nature 296: 154, 1982. 152. C a r s w i l l EA, Old LJ, Kassel SG, Green S , Fiore N, Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Nat Acad Sci USA 72: 3666, 1975. 52 153. Beutler B, Cerami A. Cachectin and tumor necrosis factor as two sides of the same b i o l o g i c a l coin. Nature 320: 584, 1986. 154. Nedwin GE, Naylor SL, Sakaguchi AY, Smith D, Jarett-Nedwin J, Pennica D, Goeddel DV, Gray PW. Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal l o c a l i z a t i o n . Nucleic Acid Research 13: 6361, 1985. 155. Munker R, Gasson J, Ogawa M, Ko e f f l e r HP. Recombinant human TNF induces production of granulocyte-monocyte colony-stimulating factor. Nature 323: 79, 1986. 156. Broudy VC, Kaushansky K, Segal GM, Harlan JM, Adamson JW. Tumor necrosis factor type a stimulates human endothelial c e l l s to produce granulocyte/macrophage colony-stimulating factor. Proc Natl Acad Sci USA 83: 7467, 1986. 157. Dexter TM, A l l e n TD, Lajtha LG. Conditions c o n t r o l l i n g the p r o l i f e r a t i o n of haemopoietic stem c e l l s i n v i t r o . J C e l l Physiol 91: 335, 1977. 158. Dexter TM, Spooncer E, Simmons P, A l l e n TD. Long-term cultures. An overview of techniques and experience. 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Long-term bone marrow cultures: An u l t r a s t r u c t u r a l review. In:"Scanning electron microscopy, SEM, Inc, AMF", 0'Hare, Chicago, IL, pp 1851, 1983. 165. Charbord P, Gown AM, Keating A, Singer JW. CGA-7 and HHF, two monoclonal antibodies that recognize muscle a c t i n and react with adherent c e l l s in human long-term bone marrow cultures. Blood 66: 1138, 1985. 166. Zuckerman KS, Wicha MS, Goodman DP, Patel VR, Mayo LA. Hematopoietic e x t r a c e l l u l a r matrix production in murine long-term bone marrow cultures Exp Hematol l l ( S u p p l 14): 124, 1983. 53 167 Zuckerman KS. Composition and function of the e x t r a - c e l l u l a r matrix i n the stroma of long-term bone marrow c e l l cultures. In: "Long-term Bone Marrow Cultures" Wright DG, Greenberger JS (eds), pp 157, 1984. 168. Zuckerman KS, Rhodes RK. The hematopoietic e x t r a c e l l u l a r matrix. 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In v i t r o d uplication and "cure" of haemopoietic defects i n g e n e t i c a l l y anaemic mice. Nature 269:412, 1977. 174. McCulloch EA, Siminovitch L, T i l l JE. Spleen-colony formation i n anemic mice of genotype W/Wv. Science 144: 844, 1964 175. Toksoz D, Dexter TM, Lord BI, Wright EG, Lajtha LG. The regulation of hemopoiesis i n long-term bone marrow cultures. I I . Stimulation and i n h i b i t i o n of stem c e l l p r o l i f e r a t i o n . Blood 55: 931, 1980. 176. Cashman J, Eaves AC, Eaves CJ. Regulated p r o l i f e r a t i o n of p r i m i t i v e hematopoietic progenitor c e l l s i n long-term human marrow cultures. Blood 66: 1002, 1985. 177. Eaves AC, Cashman JD, Kalousek DK, Eaves CJ. P o s i t i v e and negative regulators of primitive human hemopoietic progenitors. J C e l l Biochem ( i n press). 178. Hay f l i k L, Morehead PS. The s e r i a l c u l t i v a t i o n of human d i p l o i d s t r a i n s . Exp C e l l Res 25: 585, 1961. 179. Miranda AF, Babiss LE, Fisher PB. Transformation of human s k e l e t a l muscle c e l l s by simian virus 40. Proc Natl Acad Sci USA 80: 6581, 1983. 180. Pinet F, Corvol MT, Dench F, Bourguignon J, Feunteun J, Menard J, Corvol. I s o l a t i o n of renin-producing human c e l l s by transfection with three simian virus mutants. Proc Natl Acad Sci USA 82: 8503, 1985. 181. Coca-Prados M, Wax MB. Transformation of human c i l i a r y e p i t h e l i a l c e l l s by simian virus 40: Induction of c e l l p r o l i f e r a t i o n and retention of 62-adrenergic receptors. Proc Natl Acad Sci USA 83: 8754, 1986. 54 182. Brown KW, Gallimore PH. Malignant progression of an SV40-transformed human e p i t h e l i a l c e l l l i n e . Br J Cancer 56: 545, 1987. 55 C H A P T E R I I MATERIALS AND METHODS 1. CELLS ( A ) Bone Marrow C e l l s Marrow aspirate c e l l s were co l l e c t e d i n heparinized medium with informed consent from hematologically normal patients undergoing routine i n v e s t i g a t i o n s , or from normal allogeneic donors undergoing bone marrow harvests for transplantation. Light density mononuclear c e l l s were obtained by centr i f u g a t i o n on Ficoll-Hypaque (1.077 gm/cm3) and subsequently washed (3x) according to the di r e c t i o n s of the supplier (Pharmacia, Dorval P.Q.). (B) Peripheral Blood Normal peripheral blood was obtained through the courtesy of the Canadian Red Cross. To i s o l a t e T-lymphocyte depleted mononuclear c e l l s , a buffy coat preparation was f i r s t obtained by centrifuging whole units of peripheral blood d i l u t e d i n a c i d / c i t r a t e / dextrose at 800 x g for 5 minutes. Light density (<1.077 g/cm3) mononuclear c e l l s were then separated on Ficoll/Hypaque and T-lymphocytes then reduced to approximately 3-AX of t o t a l nucleated c e l l s by incubation with 2-aminoethylisothiouronium bromide (AET, Sigma, St-Louis, MO.) treated sheep erythrocytes and removal of the T c e l l rosettes by re- cent r i f u g a t i o n on Ficoll/Hypaque. This procedure yielded s u f f i c i e n t c e l l s from 1 unit (500 ml) of blood to i n i t i a t e . 3 to 5 cultures with 2 x 10? c e l l s each. Mean values from 14 experiments are shown on table I. Table I. Recovery of Nucleated C e l l s and Progenitors from One Unit of Blood (500 ml) Fraction Analysed Yield ( X ) Nucleated E CFU-E BFU-E BFU-E BFU-E BFU-E CFU-GEMM CFU-G/M Cel l s Rosettes (3-8) (8-16) (>16) Total Unseparated Blood 100 Buffy Coat 49 Light-Density C e l l s 23 74 100 100 100 100 100 100 100 T-Depleted C e l l s 4 5 92 68 53 99 59 72 59 T-Enriched C e l l s 7 81 2 1 2 2 1 3 3 57 (C) C e l l Line Maintenance A f r i c a n Green monkey kidney c e l l s (BSC-1), lung embryo f i b r o b l a s t s (VI-38), and the human umbilical cord endothelial c e l l l i n e HUV-EC-C were purchased from the American Type Culture C o l l e c t i o n (Rockville, MD). Skin f i b r o b l a s t s were i s o l a t e d and established in v i t r o by Dr. D. Hogge also in the Terry Fox Laboratory. EAhy.926 c e l l s were generously provided by Dr. C. J . Edgell (University of North Carolina, NC). The culture conditions used for the maintenance of these c e l l l i n e s are summarized in Table I I . (D) I n f e c t i o n of Primary Human Mesenchymal C e l l s with SV-40 and I s o l a t i o n of Permanent C e l l Lines Unless otherwise indicated, cultures to be infected were set up i n t r i p l i c a t e i n 60 mm tissue culture dishes, one of each containing 2 glass c o v e r s l i p s . The medium (a-10%) was removed and s u f f i c i e n t virus added to give 10 3 p f u / c e l l . In each case some cultures were not infected to serve as controls. A f t e r incubation at 37°C for 90 minutes to allow adsorption of the v i r u s , the medium was changed. Twenty four hours l a t e r some cov e r s l i p s were fixed and the proportion of adherent c e l l s expressing the large T antigen was determined by i n d i r e c t immunofluorescence (6). The remaining cultures were try p s i n i s e d , s e r i a l d i l u t i o n s prepared, and 4 or more new cultures seeded from each d i l u t i o n . Cultures were maintained at 37°C and fed twice weekly. One or two cultures were then reserved for i s o l a t i o n o f i n d i v i d u a l transformed f o c i and others stained with May-Grunwald Giemsa. Individual f o c i were harvested Table I I . Culture Conditions for the Maintenance of C e l l Lines CELL LINE BSC-1 (1) WI-38 (2) HUVE-EC-C (3) DH Fibroblasts (4) EBV Lymphoblastoid Line EAhy.926 (5) SV-40 Lines ORIGIN African Green Monkey Kidney Human Embryonic Lung Human Umbilical Cord Endothelium Skin Fibroblasts Human Marrow C e l l s in Long-Term Culture Factor VIII +ve Mouse-Human Hybrid (A549xHUVEC-C) Human Marrow and HUVE-EC-C c e l l s (see section ID) CULTURE MEDIUM a-102 a-10% F12 K Medium 20% FCS 100 ug/ml Heparin 50 ug/ml ECGS* a-10% a-10% a-10% a-10% A l l c e l l l i n e s were maintained i n an atmosphere of 5% C O 2 at 37°C. * Endothelial c e l l growth supplement. 59 using porcelain cylinders and the c e l l s transferred to f l a t bottom tissue culture multiwell plates (Linbro, McLean, VA). A few days l a t e r , the l i n e s were cloned by p l a t i n g at l i m i t i n g d i l u t i o n i n m i c r o t i t r e wells or by p l a t i n g at 10^ cells/35 mm dish i n methylcellulose (see Anchorage-independent growth assays, section 7 below). Three weeks l a t e r , c e l l s in p o s i t i v e wells or i s o l a t e d colonies were removed and transferred i n d i v i d u a l l y to 2 cm^ wells containing 2 ml of medium. Cloned l i n e s were then propagated by feeding twice a week and subculturing once a week at 10^ cell/cm^ i . e . 2.5 x 10^ cells/25cm2 f l a s k . (E) C e l l P r o l i f e r a t i o n Measurements The growth and population doubling times of SV-40 infected c e l l s and cloned l i n e s were determined from viable c e l l counts (nigrosin dye exclusion) or from measurements of ^H-thymidine incorporation. For measurements of population doubling time, c e l l s were resuspended to a f i n a l concentration of 4 x 10^ c e l l s / m l . 2.5 ml of this c e l l suspension ( i . e . 10^ c e l l s ) was then seeded into 35 mm tissue culture dishes. At d a i l y i n t e r v a l s thereafter, the c e l l s in a pair of dishes (one infected and one control) were trypsinized and via b l e c e l l counts performed. To measure ^H-thymidine incorporation, c e l l s were cultured i n Iscoves-10% i n f l a t bottomed microwells (Costar) (500 cells/100 u l / w e l l ) . At the times indicated, 1 yCi of 3H-thymidine (20 mCi/mmole, Amersham, Oakville, Ont) in 20 u l of growth medium was then added to each well. The c e l l s were incubated for an a d d i t i o n a l 4 hours at 37°C, harvested onto glass f i b e r f i l t e r paper, and the amount of ^H-thymidine incorporated determined by s c i n t i l l a t i o n counting. 60 2) LONG-TERM CULTURES (A) Regular Long-Term Marrow Cultures An aliquot of untreated marrow aspirate containing 2-2.5 x 10^ nucleated marrow c e l l s was placed in 8 ml of long-term culture growth medium in a 60 x 15 mm Falcon tissue culture dish. The growth medium was composed of a-medium supplemented with i n o s i t o l (40 mg/1), f o l i c acid (10 mg/1), extra glutamine (400 mg/ml), f e t a l c a l f serum (FCS, 12.5%), horse serum (HS, 12.5%), 2 mercaptoethanol (10 -^ M), and hydrocortisone sodium succinate (10~6 M). FCS and HS were pretested for their a b i l i t y to support maximal hemopoiesis i n this system. The cultures were incubated for 3 to 4 days at 37°C in an atmosphere of 5% C0£ i n a i r . After this i n i t i a l period of incubation, a l l nonadherent c e l l s were removed and layered over Ficoll/Hypaque 1.077 g/cm3 to remove remaining red blood c e l l s and mature granulocytes where necessary. The l i g h t density c e l l s were washed in a-medium supplemented with 2% FCS and returned to the o r i g i n a l dishes. The cultures were then fed on a weekly basis by removal of h a l f of the medium and ha l f of the nonadherent c e l l s . This was accomplished by p i p e t t i n g 2-3 ml of medium from the dish, and then gently s w i r l i n g the dish to ensure removal of a l l the nonadherent c e l l s with the remaining 5-6 ml of the medium. The culture medium was placed in a tube, vortexed to d i s t r i b u t e the c e l l s evenly, and 4 ml of this suspension was returned to the culture dish, along with 4ml of fresh culture medium. To harvest adherent layers, the cultures were f i r s t vigorously rinsed two or three times with serum free and Ca"1"1" and Mg + + free, Hanks balanced s a l t s o l u t i o n to remove a l l the nonadherent c e l l s (and any FCS l e f t i n dishes). The c e l l s i n the adherent layer were then exposed at 37°C to trypsin (0.25% in a 61 s o l u t i o n containing 5% c i t r a t e , 10% KCl and 1% glucose) for 10 minutes following the method of Coulombel et a l . (7). C e l l s that were s t i l l adherent at the end of the incubation period were gently removed by p i p e t t i n g , transfered to a test tube, centrifuged at 300 g x 10 minutes and washed twice i n 2% a-medium. (B) Long-Term Peripheral Blood Cultures To i n i t i a t e long-term peripheral blood cultures, 2 x 10? T-depleted l i g h t density mononuclear c e l l s were placed in 60mm tissue culture dishes. These were subsequently maintained using the same protocol established for long-term cultures with the exception that cultures were incubated d i r e c t l y at 33°C and the day 3-4 red c e l l and granulocyte separation step was omitted. After 4-5 weeks, non-adherent c e l l s were removed and then the adherent layer harvested by t r y p s i n i s a t i o n as described above. (C) Preparation of Long-Term Marrow Culture Feeders Long-term marrow cultures were i n i t i a t e d and maintained as described above with the following modifications. Hydrocortisone was omitted from the medium, the cultures were kept at 37°C, and at each medium change a l l of the nonadherent c e l l s were removed and new medium added. Such cultures were used as feeders a f t e r the adherent layer had reached confluence (approximately 2 weeks), or in some cases a f t e r subculture and re-attainment of confluence in the secondary dishes. A l l feeders were ir r a d i a t e d with 15 Gy (see i r r a d i a t i o n procedures, section 9, below) to ablate residual hemopoiesis, p r i o r to addition of " t e s t " c e l l s . Assays of such i r r a d i a t e d pre-established adherent layers 62 consistently showed that they contained no detectable colony-forming hemopoietic progenitor ce l l s . (D) Cis-Hydroxy-L-Proline (CHP) Experiments Long-term marrow cultures were init iated and maintained in the usual way, except that l ight density mononuclear cel ls rather than whole aspirate or marrow buffy coat cel ls were used to in i t ia te the cultures, the step to remove cel ls >1.077g/cm3 after 3-4 days was omitted, and cultures were placed from the beginning at 33°C. Cultures containing CHP (Sigma) were ini t iated and fed with a growth medium containing a-medium prepared without proline or lysine. Control cultures were maintained in the same medium but without CHP and with reconstituted levels of proline and lysine. After 4-5 weeks, nonadherent cel ls were removed and the adherent layer harvested by trypsinization. Some cultures were ini t iated by adding marrow cel ls into dishes containing pre-established irradiated feeders prepared as described above. These were handled in the same way as cultures set up in fresh dishes without feeders. 3) ASSAYS (A) Methylcellulose Assay for Hemopoietic Colony-Forming Progenitors Erythropoietic (CFU-E and BFU-E), granulopoietic (CFU-GM) and pluripotent (CFU-GEMM) progenitors were assayed by standard procedures previously described (7,8). Unless specified otherwise, cel ls were plated at a f inal concentration of 10 5 ce l l s / 1.1 ml of methylcellulose assay culture medium. Colony counts were obtained 3 weeks after plating from a minimum of 2 assay replicates except 63 i n the case of fresh marrow and blood c e l l assays where CFU-E and mature BFU-E (erythroid colonies containing 1-2 and 3-8 clusters of erythroblasts, r e s p e c t i v e l y ) were scored a f t e r 1 1/2 weeks pr i o r to f i n a l counts and other progenitors classes a f t e r 3 weeks. (B) Colony-Forming Unit-Fibroblast (CFU-F) Assay E s s e n t i a l l y , the method described by Castro-Malaspina et a l (9) was followed except that the medium used was a-medium made up with or without pro l i n e or l y s i n e plus 10% f e t a l c a l f serum (FCS) to which varying concentrations of CHP or various growth factor preparations were then added, according to the experimental design. In b r i e f , marrow buffy coat or l i g h t density (< 1.077 g/cm3) c e l l s were plated in tissue culture dishes at 8.5 x 10^ cells/mm2/2.6 u l or or 2.6 x 10^ cells/mm2/2.6 y l respectively, and then incubated undisturbed for 12 days at 37°C in a humidified atmosphere of 5% C O 2 in a i r . At the end of this time, the medium was decanted, the cultures rinsed twice i n ice cold PBS, and adherent colonies fixed, a i r dried, stained with May-Griinwald-Giemsa. (C) Assays for Production of Hemopoietic Growth Factor (HGF) To test for Interleukin-16 (IL -10) induced production of HGF, 2 ml of suspension of 2 x 10^ c e l l s / m l were placed into single wells of 24 multi-well plates (Linbro, McLean, VA) and 24 hours l a t e r , a small amount of p u r i f i e d recombinant human IL -10 (Biogen, Geneva) or diluent was added to give the desired f i n a l concentration of IL-10 . Conditioned media were harvested a f t e r 64 a further 24 hours, spun at 1,200 rpm for 10 minutes and the supernatant stored at -20°C. To test i f IL-16 stimulated SV-40 immortalized c e l l l i n e s produced b i o l o g i c a l a c t i v i t y capable of stimulating marrow stromal c e l l progenitors I used the CFU-F assay with or without 10% IL-16 induced conditioned medium. As controls, we used growth medium alone (supplemented with 10% FCS), a crude source of hemopoietic growth factors (PHA-LCM 10%) and recombinant GM-CSF at a f i n a l concentration of 8 ng/ml. To i n i t i a t e CFU-F assays I used the method described above with the exception that CHP was omitted and only regular a-medium supplemented with 10% FCS was used. Colony-stimulating a c t i v i t y was tested by addition of conditioned media to standard methylcellulose cultures containing 3 to 5 x 10^ non-adherent human marrow c e l l s / m l . To reduce the background in negative controls (cultures containing no exogenous source of growth factor) and to obtain useful progenitor numbers at low c e l l concentrations, the target c e l l s were obtained from the l i g h t density (<1.077 gm/cm3) non-adherent c e l l f r a c t i o n of 1 week-old long-term human marrow cultures (10). P o s i t i v e controls included agar- stimulated leukocyte conditioned medium (LCM) and recombinant human GM-CSF (Biogen, Geneva). Unless s p e c i f i e d otherwise, a l l cultures contained 3 units/ml of p a r t i a l l y p u r i f i e d human urinary erythropoietin (>1,000 units/mg) (11). 65 4) SV-40 VIRUS PREPARATION AND ASSAY (A) Preparation of High T i t e r Virus Stock A freeze-dried preparation of SV-40 viru s , s t r a i n A2895, was obtained from ATCC (Rockville, MD), reconstituted and the t i t r e determined by plaque assay on confluent BSC-1 c e l l s (6). A 200-fold higher t i t r e stock was then prepared by i n f e c t i n g new, confluent BSC-1 monolayers at a low m u l t i p l i c i t y of i n f e c t i o n (0.01 p f u / c e l l ) with the di l u t e d lysate from a sing l e plaque. The t i t r e of the supernatant obtained from this i n f e c t i o n was 8 x 10*° pfu/ml (see Figure 8). It was aliquoted into multiple v i a l s and stored at -20°C. (B) Virus Plaque Assay The plaque assay described by Turler and Beard (6) was used. In b r i e f , s e r i a l 10-fold d i l u t i o n s of the virus were prepared i n Tris-Dulbecco's buffer (6) supplemented with 2% FCS and 100 y l was used to i n f e c t confluent monolayers of A f r i c a n Green Monkey Kidney C e l l s (BSC-1 c e l l s ) in 60 mm tissue culture dishes. As controls, some were mock infected with lOOul of Tris-Dulbecco's buffer-2% FCS. After a 90 minute adsorbtion period, fresh 10% DMEM medium was added and the dishes were returned to 37°C for the next 15 hours. The medium was then replaced with 5 ml of 0.9% agar made up in the same medium. After the agar had set, cultures were then returned to the 3 7 ° C incubator for another 7 days. Plaques were counted using an inverted phase contrast microscope and t i t e r s (pfu/ml) of o r i g i n a l virus stocks calculated. FIGURE 8. T i t r a t i o n of SV-40 Virus Stock on BSC-1 C e l l s . 67 (C) Assay For Large T Antigen The presence of intranuclear T-antigen was detected by immunofluorescence using the highly s p e c i f i c monoclonal Pab 1626 (12). The method i s f u l l y described i n section 5. (D) Transformation Assay of SV-40 Virus on Mouse NIH-3T3 C e l l s Freshly established NIH-3T3 c e l l s were seeded i n 60 mm dishes (Falcon) at a concentration of 2 x 10 4 c e l l s / m l i n 4.5 ml of Dulbecco's MEM with 10% FCS. Twenty four hours l a t e r 0.5 ml of s e r i a l l y dilu.ted virus was added with 20 ug of polybrene. The medium was changed the following day and plates examined for the presence of dense colonies of c e l l s a f t e r another 2 weeks. To score colonies, plates were fixed i n 10% formalin and stained with May-Grtinwald- Giemsa. 5) ANTISERA AND IMMUNOFLUORESCENCE MEASUREMENTS For immunofluorescence microscopy, c e l l s were grown to subconfluence on 22 x 22 mm co v e r s l i p s , fixed i n acetone/methanol 1:1 (v/v) for 5 minutes at -20°C, and then a i r - d r i e d for 10 minutes at room temperature. After r i n s i n g in Hanks balanced s a l t s o l u t i o n supplemented with 2% FCS and 0.1% sodium azide (HFN), c e l l s were covered with the f i r s t antibody ( l i s t e d i n Table III) for 1 hour. To control for non-specific binding, duplicates were incubated with HFN alone. After 3 successive washes in HFN, an appropriate second antibody (eit h e r FITC-conjugated goat anti-mouse immunoglobulin, (CBL, The Netherlands) Table I I I . Origin, S p e c i f i c i t y and Source of Antibodies Used for Inununophenotype Analyses Name Ce l l u l a r S p e c i f i c i t y Origin Source a n t i -laminin E p i t h e l i a l and endothelial c e l l s Rabbit* Dr. Furthmayr (Yale, New Haven) a n t i -collagen IV E p i t h e l i a l and endothelial c e l l s Rabbit* n n a n t i -collagen I Fibroblasts Goat* Southern Biotechnology Assoc. (Birmingham, AL) an t i -T200/LCA/anti-Leuk AH Nucleated Hemopoietic c e l l s Mousey- Becton-Dickinson Corp. (Mountain View, CA) an t i (CD -leuMl 15) Differentiated monocytes, granulocytes Mouse"1" n n a n t i -leuM3 (CD wl4) Monocytes, macrophages Mouse"*" n FI a n t i -6.1915 A variety of c e l l s including a l l mesenchymal c e l l types studied but excluding a l l hemopoietic c e l l s Mouse"1" Dr. Christopher Frantz (Rochester, NY) an t i -Factor VIII Factor V H I - r e l a t e d antigen Rabbit* DAKO corporation (Santa Barbara, CA) Pab 1626 21 SV-40 Large T antigen-positive c e l l s Mouse"1" Ciba-Geigy Ltd. (Basel, Switzerland) *Antisera. •Monoclonal antibodies. 69 or FITC-conjugated rabbit anti-goat immunoglobulin, (Nordic, The Netherlands) both at a f i n a l d i l u t i o n of 1/80 was added and the c e l l s then incubated for 30 minutes at room temperature. After 3 further washes i n HFN, c e l l s were mounted in buffered po l y v i n y l a l c o h o l (13) and examined with a Zeiss photomicroscope equipped with UV-epiillumination. For each antibody, a minimum of 400 c e l l s were evaluated. To detect surface antigens fres h l y trypsinized c e l l s were stained i n suspension using the appropriate i n d i r e c t procedure and a b r i e f exposure to propidium iodide (2 ug/ml) a f t e r the f i n a l wash, and then analysed with a Becton Dickinson FACS IV equipped with a log a m p l i f i e r . As negative controls, c e l l s were l a b e l l e d with the second FITC-labelled reagent only, or with an i r r e l e v a n t monoclonal antibody of the same isotype as the f i r s t reagent. To estimate the proportion of positive, c e l l s , the channel number where the negative control and the test sample curves crossed each other was f i r s t determined. The number of negative c e l l s to the righ t of this channel (higher fluorescence values) was then subtracted from the number of c e l l s i n the test sample that also f e l l to the right of the crossover point. For immunoperoxidase detection of Factor VIII related antigen p o s i t i v e c e l l s , long-term cultures were set up in Labteck ( N a p i e r v i l l e , IL) s l i d e dishes. However, equally good re s u l t s were obtained with Eahy.926 or HUV-EC-C c e l l l i n e s grown on regular 22 x 22 mm c o v e r s l i p s . Slides were l i g h t l y fixed (1 min.) i n acetone-methanol (1:1), endogeneous peroxidase a c t i v i t y suppressed by f i x a t i o n i n methanolic hydrogen peroxide for 20 minutes, and s l i d e s incubated sequentially with the following reagents (Dako Corp, Santa-Barbara, CA): Normal swine serum, polyclonal rabbit anti-Factor VIII anti-serum, swine a n t i - r a b b i t anti-serum, peroxidase-anti-peroxidase complex. The reaction was next revealed following incubation with a mixture of substrate, 3-amino- 9-ethylcarbazole, 0.3% hydrogen peroxide in water, and 0.1M acetate buffer, 70 pH 5.2. Slides were counterstained with G i l l ' s hematoxylin (Fisher, Orangeburg, N.Y.), diped several times i n ammonia water, rinsed, and mounted using g l y c e r o l g e l a t i n dip. 6) HTSTOCHEMICAL ANALYSES C e l l s were grown to subconfluence on coverslips, a i r dried and then stained for a l k a l i n e phosphatase and acid phosphatase by established histochemical methods (14,15). 7) TESTS FOR ANCHORAGE-INDEPENDENT GROWTH Trypsinized c e l l s were resuspended i n <x-2% to a f i n a l concentration of 3 x 10^ c e l l s / m l . Ten-fold d i l u t i o n s were prepared i n some cases down to concentrations of 30 c e l l s / m l . 0.3 ml of these c e l l suspensions were then added to 3 ml of a-10% made viscous by the addition of 0.8% methylcellulose (4,000 cps, Dow Chemical, Vancouver, B.C.). One ml cultures were plated i n 35 mm dishes (Greiner, FRG) and incubated at 37°C i n a f u l l y humidified atmosphere containing 5% C02- Colonies containing more than 50 c e l l s were scored 20 days l a t e r with an inverted microscope. 8) TUMOR FORMATION A t o t a l of 0.1 to 1 x 10 6 SV-40 immortalized c e l l s (MH2-SV) in 100 u l of s a l i n e were injected intradermally into 4 female nu/nu mice. As controls, 2 a d d i t i o n a l animals were injected with s a l i n e alone. 71 9) IRRADIATION PROCEDURES For c o c u l t i v a t i o n experiments, preestablished adherent layers were i r r a d i a t e d with 15-20 grays (60QO y rays or 250 KVp X-rays). To determine the r e l a t i v e r a d i o s e n s i t i v i t i e s of SV-40 immortalized l i n e s and normal marrow stromal c e l l s i n culture, freshly established layers were trypsinised and washed twice i n growth medium. Single c e l l suspensions were prepared and a fixed number (10^-10^) of c e l l s dispensed in test tubes (Falcon) and kept on ice except during i r r a d i a t i o n (<2 minutes). Radiation (250 KVp X-rays) was given at a sing l e dose at the rate of 5.2 Gy/min. C e l l s were then resuspended i n 7.5 ml of fresh a-20% tissue culture medium, dispensed i n 60mm tissue culture dishes and incubated at 37°C for 10-14 days . To assess the e f f e c t s of i r r a d i a t i o n on colony forming c e l l s the mean number of colonies formed was used to c a l c u l a t e the p l a t i n g e f f i c i e n c y and their surviving f r a c t i o n r e l a t i v e to the control cultures. 10) AUTORADIOGRAPHY To determine the e f f e c t s of SV-40 virus on contact-inhibited marrow adherent c e l l s , 10^ c e l l s from a trypsinized 4 week old normal long-term bone marrow culture were resuspended i n 2.5 ml of Dulbecco's MEM and seeded into 35 mm Falcon tissue culture dishes. Five days af t e r reaching confluence, the medium was removed and cultures incubated with or without 250 u l of SV-40 virus stock (10* u pfu/ml). Twenty four hours l a t e r dishes were r insed , covered with Iscove's medium supplemented with 15% FCS and returned to the incubator for one more day. C e l l layers were allowed to incorporate 3HTdR at a f i n a l concentration of 10uCi/ml and la b e l l e d nuclei subsequently v i s u a l i z e d by 72 autoradiography. C e l l s were rinsed with Tris-buffered s a l i n e , fixed i n 3.7% formaldehyde for 10 minutes, and washed sequentially with 5% t r i c h l o r o a c e t i c acid, water, and 70% ethanol. After drying, dishes were coated with Kodak NTB2 photographic emulsion, dried again, and l e f t i n the dark for 2 more days in a dessicating j a r . The emulsion was then developed and the c e l l s stained b r i e f l y with 2% methyl green. The percentage of l a b e l l e d nuclei was then calculated. 73 REFERENCES 1. Hopps HE, Bernheim BC, Nisalak A. B i o l o g i c c h a r a c t e r i s t i c s of a continuous kidney c e l l l i n e derived from the A f r i c a n green monkey. J Immun 91: 416, 1963. 2. H a y f l i c k L. The l i m i t e d in v i t r o l i f e t i m e of human d i p l o i d c e l l s t r a i n s . Exp C e l l Res 37: 614, 1965. 3. Hoshi H, McKeehan WL. Brain and l i v e r c e l l - d e r i v e d factors are required for growth of human endothelial c e l l s i n serum-free culture. Proc Natl Acad Sci USA 81: 6413, 1984. 4. Hogge DE, Coulombel L, Kalousek DK, Eaves CJ, Eaves AC. Nonclonal hemopoietic progenitors in a G6PD heterozygote with chronic myelogeneous leukemia revealed a f t e r long-term marrow culture. American Journal of Hematology 24: 389, 1987. 5. Edgell CJS, McDonald CC, Graham JB. Permanent c e l l l i n e expressing human factor V H I - r e l a t e d antigen established by h y b r i d i z a t i o n . Proc Natl Acad Sci USA 80: 3734, 1983. 6. Turler H, Beard P. Simian virus 40 and polyoma v i r u s : Growth, t i t r a t i o n , transformation and p u r i f i c a t i o n of v i r a l components. In: Maky BNJ (ed), "Virology a p r a c t i c a l approach", Oxford, IRL press, ppl69, 1985. 7. Coulombel L, Eaves AC, Eaves CJ. Enzymatic treatment of long-term human marrow culture reveals the p r e f e r e n t i a l location of p r i m i t i v e hemopoietic progenitors in the adherent layer. Blood 62: 291, 1983. 8. Gregory CJ, Eaves AC. Three stages of erythropoietic progenitor c e l l d i f f e r e n t i a t i o n distinguished by a number of physical and b i o l o g i c a l properties. Blood 51: 527, 1978. 9. Castro-Malaspina H, Gay RE, Resnick G, Kapoor N, Myers P, C h i a r i e r i D, McKenzie S, Broxmeyer HE, Moore MAS. Characterization of human bone marrow f i b r o b l a s t colony-forming c e l l s (CFU-F) and t h e i r progeny. Blood 56: 289, 1980. 10. Eaves CJ, Eaves AC. Erythropoietin (Ep) dose-response curves for three classes of erythroid progenitors in normal human marrow and in patient with polycythemia vera. Blood 52: 1196, 1978. 11. K r y s t a l G, Eaves CJ, Eaves AC. CM a f f i - g e l blue chromatography of human urine: A simple one-step procedure for obtaining erythropoietin s u i t a b l e for i n v i t r o erythropoietic progenitor assays. Br J Haematol 58: 533, 1984. 12. B a l l RK, S e i g l B, Quellhorst S, Brandner G, Braun DG. Monoclonal antibodies against simian virus 40 nuclear large T tumor antigen: Epitope mapping of papovavirus cross reaction and c e l l surface s t a i n i n g . Embo J 7: 1485, 1984. 74 13. Lenette DA. An improved mounting medium for immunofluorescence microscopy. Am J C l i n Pathol 69: 647, 1978. 14. Ackerman GA. Substituted naphtol AS phosphate derivatives for the l o c a l i z a t i o n of leukocyte a l k a l i n e phosphatase a c t i v i t y . Lab Invest 11: 563, 1962. 15. L i CY, Yam LT, Lam K.W. Acid phosphatase isoenzyme i n human leukocytes i n normal and pathological conditions. J Histochem Cytochem 18: 473, 1970. 75 C H A P T E R I I I DIFFERENTIAL EFFECTS OF CIS-OH-L-PROLINE ON THE PROLIFERATIVE AND STEM CELL REGULATORY FUNCTIONS OF HUMAN BONE MARROW MESENCHYMAL CELLS 1. INTRODUCTION There is now considerable evidence from both in vivo (1,2,3) and in vi tro (4,5,6) studies indicating that fixed, i . e . non-circulating, mesenchymal elements are involved in the regulation of hemopoiesis in the marrow. Such cel ls are a major component of the adherent layer of long-term marrow cultures, which serves as an attractive model for in vitro analyses since i t allows hemopoiesis to be maintained for extensive periods of time (months) in the absence of exogenously added hemopoietic growth factors. However, since the adherent layer of long-term marrow cultures consists of a diversity of c e l l types of both mesenchymal (e.g. fibroblasts, adipocytes and endothelial ce l ls ) and hemopoietic origin (e.g. macrophages and progenitors) (7,8,9) reconstruction experiments are needed to delineate both the nature and the ce l lu lar origin of the factors that needed for the maintenance and regulated turnover of primitive hemopoietic ce l l s . In the f i r s t part of my research, the main objective was to develop an experimental system that would allow direct measurements of the hemopoietic sustaining function of mesenchymal cel ls present in the adherent layer of long- term bone marrow cultures. A prerequisite for assaying such a function rests on the ava i lab i l i ty of a hemopoietic target population that is free of mesenchymal cel ls but nevertheless requires the establishment of close 76 int e r a c t i o n s with non-hemopoietic stromal c e l l s for long-term s u r v i v a l i n cult u r e . Several strategies to obtain such populations were explored: I s o l a t i o n of l i g h t - d e n s i t y non-adherent c e l l s from hemopoietically active long-term bone marrow cultures (10), physical separation of fresh bone marrow c e l l s on nylon wool columns (11), immunological purging of bone marrow c e l l s with a combination of monoclonal antibody 6.19 and complement (12) and, modification of the culture conditions by the addition of D-valine to the medium (13). None of these approaches could reduce to acceptable l e v e l s the p r o l i f e r a t i o n of re s i d u a l mesenchymal c e l l s present i n unseparated marrows. A more successful approach u t i l i z e d peripheral blood as a p o t e n t i a l target population. Peripheral blood represents an a t t r a c t i v e option since m u l t i p o t e n t i a l and high p r o l i f e r a t i v e p o t e n t i a l myeloid progenitor are present at a reasonable frequency whereas mesenchymal c e l l s are t o t a l l y absent (14,15). Thus, I investigated the p o s s i b i l i t y that progenitor c e l l s from this source could be enriched i n s u f f i c i e n t amount to i n i t i a t e cultures on i r r a d i a t e d pre- established adherent layers. In the second approach, I sought to determine i f Cis-4-hydroxy-L-proline (CHP), a r e l a t i v e l y s p e c i f i c i n h i b i t o r of collagen synthesis (16) could be used to s e l e c t i v e l y i n h i b i t the mesenchymal c e l l component of marrow c e l l suspensions and therefore allow marrow to be used as a simple source of hemopoietic progenitors. Although preliminary results with appropriate monoclonal antibodies have suggested that highly p u r i f i e d hemopoietic progenitors can now be obtained, and these might then be of some value for such experiments (17), the use of CHP had the a t t r a c t i o n that i t was t e c h n i c a l l y simpler and had the p o t e n t i a l to avoid the tremendous c e l l losses and considerable v a r i a b i l i t y t y p i c a l l y encountered in c e l l separation experiments. 77 Recently, CHP was reported to prevent the formation of an adherent layer i n murine long-term marrow cultures at doses that are not d i r e c t l y toxic to co- e x i s t i n g hemopoietic progenitor c e l l s (18). In the present study, I investigated the p o s s i b i l i t y that CHP might have a s i m i l a r d i f f e r e n t i a l e f f e c t on the p r o l i f e r a t i v e and stem c e l l regulatory functions of human marrow mesenchymal c e l l s . To address this question, I f i r s t evaluated the e f f e c t of CHP on the p r o l i f e r a t i o n of collagen-producing c e l l s from human marrow as assessed i n both CFU-F assays and in the long-term marrow culture system. CHP proved to be an e f f e c t i v e i n h i b i t o r of these c e l l s at concentrations that are not toxic to human hemopoietic c e l l s present i n the same cultures. CHP supplemented medium should therefore be useful to enable unseparated marrow to be used to assess the potential a b i l i t y of defined populations of mesenchymal c e l l s to support the long-term maintenance and p r o l i f e r a t i o n of human hemopoietic progenitor c e l l s . 2) RESULTS (A) Cultures of Peripheral Blood C e l l s on Normal Marrow Adherent Layer Light density (<1.077 gm/cm3) peripheral blood c e l l s were depleted of T c e l l s (to enrich for myeloid progenitors) and then used to i n i t i a t e long-term cultures by seeding the c e l l s into dishes containing a confluent, i r r a d i a t e d adherent layer from a previously established long-term marrow culture, or into new dishes without such a "feeder" as controls. The t o t a l numbers of nucleated c e l l s and clonogenic erythroid and granulopoietic progenitors present i n these cultures are shown on Figure 9. In the presence of a marrow adherent layer the number of non-adherent c e l l s (panel a) and clonogenic progenitors 78 I 10"! i i i i i i i i I 1 I i i i i i i i 1—I 1 I 1 1 1 1 1 1 1—J- 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Weeks in Culture Weeks in Culture Weeks in Culture I i FIGURE 9. • Comparison of Total C e l l and Progenitor Content of Long-Term Normal PBL Cultures I n i t i a t e d With ( S o l i d Symbols) or Without (Open Triangles) a Pre-Established Normal Marrow Feeder. C i r c l e s - data for non-adherent fracti o n s of cultures with feeders assessed weekly. Squares - data for adherent fr a c t i o n s of cultures with feeders. Triangles - data for non-adherent f r a c t i o n s of cultures without feeders (no adherent f r a c t i o n obtained), from which no c e l l s were removed at each weekly medium change u n t i l the culture was s a c r i f i c e d and which were used for p a r a l l e l c y c l i n g measurements (19). Values shown are the geometric means + 1 SEM from data of 5 experiments. 79 (panels b and c) sharply declined during the f i r s t 2-3 weeks, but a f t e r 3-4 weeks a plateau phase was reached and thereafter the numbers remained r e l a t i v e l y constant for the duration of the cultures. When these cultures were harvested at 3 weeks, the majority of the clonogenic progenitors were found i n the adherent layer and values were s u f f i c i e n t to allow their p r o l i f e r a t i v e status to be determined by Dr. J. Cashman (19). These studies revealed an a l t e r n a t i n g pattern of p r o l i f e r a t i o n and quiescence of pr i m i t i v e but not mature progenitor c e l l types i n the adherent layer as a function of the feeding schedule. Cultures that were i n i t i a t e d i n the absence of a pre-established marrow adherent layer also contained s u f f i c i e n t progenitors at 3 weeks to allow c y c l i n g measurements to be performed. In these cultures a l l progenitors were non-adherent and in contrast to cultures with adherent layers a l l progenitors, in c l u d i n g those c l a s s i f i e d as primitive were p r o l i f e r a t i n g , i r r e s p e c t i v e of the feeding pattern. However, since the feeders had no e f f e c t on the number of progenitors present a f t e r 3 weeks, cultures had to be maintained for at le a s t 5 weeks to enable a d i f f e r e n t i a l e f f e c t of the marrow adherent layers to be observed. This became impractical for routine experiments due to the emergence in a number of cultures of polyclonal B-lymphoblastoid populations o r i g i n a t i n g from c e l l s l a t e n t l y infected with Epstein-Barr vi r u s , a f i n d i n g also reported by others (20). These c e l l s f i r s t became noticeable i n methylcellulose cultures where they formed large (>10,000 c e l l s ) , d i f f u s e colonies which sometimes overgrew the ent i r e culture. Microscopic examination of plucked colonies revealed a homogeneous population of lymphoblastoid c e l l s with a high nucleo-cytoplasmic r a t i o , prominent n u c l e o l i , and a small rim of basophilic cytoplasm. C e l l markers studies were also performed on these colonies (see Table IV) and their B - c e l l o r i g i n confirmed. Because of this phenomenon I explored a l t e r n a t i v e sources of hemopoietic progenitors. Table IV. Immunophenotypic Characterization of Lymphoblastoid C e l l s Marker Assessed X P o s i t i v e T-Cells E-Rosettes 0 Leu-4 1 a-Thy 1 B-Cells Leu-12 72 B4 59 X 9 K 31 Pan-Hemopoietic T-200 81 81 (B) E f f e c t of CHP on Marrow Mesenchymal C e l l P r o l i f e r a t i o n This was evaluated i n two d i f f e r e n t types of experiments, one using the CFU-F assay and one using the long-term marrow culture system. These d i f f e r both i n the number of c e l l s i n i t i a l l y used to seed the cultures, and in the duration of time p r i o r to assessment of the CHP e f f e c t . The experiments employing the CFU-F assay demonstrated a reproducible dose-dependent i n h i b i t i o n of f i b r o b l a s t colony formation i n cultures containing CHP (Figure 10). Ad d i t i o n a l treatment groups included i n these experiments demonstrated that omission of proline and ly s i n e from the medium had, on i t s own, no e f f e c t on the number or s i z e of f i b r o b l a s t colonies obtained i n the absence of CHP and, conversely, that the restoration of the proline, and l y s i n e content of the medium to "normal" l e v e l s completely abrogated the CHP e f f e c t (data not shown). Concentrations of CHP above 50 ug/ml reduced marrow f i b r o b l a s t s colony numbers to <50X of control values and at concentrations above 250 ug/ml, no f i b r o b l a s t colonies were seen. In the second set of experiments, the ef f e c t of CHP on adherent layer formation i n the long-term marrow culture system was assessed. Preliminary experiments showed that CHP concentrations below 100 yg/ml, although s i g n i f i c a n t l y i n h i b i t o r y i n CFU-F assays, s t i l l allowed s i g n i f i c a n t adherent layer formation i n the long-term culture system when this was assessed a f t e r 4-5 weeks. However, when the concentration of CHP was increased to 500 yg/ml, the formation of an adherent layer was severely and i r r e v e r s i b l y impaired. Table V shows the res u l t s of c e l l counts performed on adherent layers fromparallel cultures i n i t i a t e d and maintained i n the presence or absence of CHP harvested a f t e r 4-5 weeks. The dramatic difference i n the appearance of these cultures i s i l l u s t r a t e d i n Figure 11. 82 FIGURE 10. E f f e c t of Increasing Doses of CHP on the Number of Colonies Obtained from CFU-F i n Fresh Human Marrow. Values represent the mean + 1 SEM for normalized data (% of number of colonies seen i n the absence of CHP) from 5 d i f f e r e n t experiments ( d i f f e r e n t marrow samples). Table V. Ef f e c t of CHP on Adherent Layer Formation No. of c e l l s i n the adherent layer at 4-5 weeks (x 10 -*) Exp. no. - CHP + CHP (X of control without CHP) 1 38.0 3.1 (8.2) 2 24.0 0.4 (1.6) 3 12.0 0.8 (6.7) 4 8.4 1.8 (21.4) Mean + SEM 9.5 + 4.2 The differ e n c e between - CHP and + CHP i s s i g n i f i c a n t (p<0.05 using paired t-test (27)). Photograph of Two Long-Term Marrow Cu l t u r e s , One I n i t i a t e d and Maintained i n Regular Medium ( L e f t ) , and One I n i t i a t e d and f CHP ( R i h" P r ° l i n e _ ' a n d L y s i n e - p r e e Medium Containing 500 ug/ml 85 (C) Lack of a Direct Effect of CHP on Hemopoietic Progenitor Function To investigate possible CHP effects on hemopoietic ce l l s , marrow cel l s were f i r s t incubated for 3 hours in proline-, lysine-free medium with or without added CHP, and then washed and plated in methylcellulose assays. As shown in Table VI, such short-term exposure of marrow cel ls to even 500 ug/ml CHP had no effect on the number, size or composition of the colonies produced by any of the progenitor classes assessed. The effect of a longer exposure of hemopoietic cel ls to CHP in the long- term marrow culture system was then evaluated. Since the presence of CHP in the medium was known to inhibit adherent layer formation, i t was anticipated that hemopoiesis might also be reduced even i f there were no direct effect of CHP on the hemopoietic cel ls themselves, since previous studies have indicatedthat the long-term support of hemopoiesis in this system requires an intact adherent layer (4,6,17,18). An additional group was therefore included to allow any direct effects of CHP on the production or survival of hemopoietic cel ls to be distinguished from potential indirect effects caused by CHP inhibit ion of mesenchymal cel ls that might be essential for the maintenance of the hemopoietic ce l l s . Thus, the following three conditions were tested in each experiment. The f irs t two compared the presence and absence of CHP in long-term marrow cultures init iated using the standard protocol, i . e . marrow cel l s were seeded into new dishes in medium with or without 500 ug/ml CHP. In the third group, marrow cells were maintained in medium containing 500 ug/ml of CHP, but the dishes into which they were seeded already contained a preestablished marrow adherent layer. This had been obtained by subculturing a 86 Table VI. Lack of E f f e c t of Exposure of Hemopoietic Progenitors to CHP on Their Subsequent P l a t i n g E f f i c i e n c y CHP (ug/ml) Progenitor Exp no. 0 25 500 CFU-E 1 218 209 266 2 163 125 91 3 256 230 265 BFU-E 1 164 177 140 2 88 87 55 3 176 186 140 CFU-G/M 1 168 144 146 2 80 93 82 3 151 154 158 CFU-G/E 1 10 16 10 2 4 3 4 3 7 2 7 Each value shown represents the mean value from 2 r e p l i c a t e assay cultures. Light density mononuclear marrow c e l l s were incubated for 3 hours at 37°C i n the presence or absence of CHP in l y s i n e - , p r o line-free medium. At the end of the incubation period, c e l l s were washed once and the equivalent of 10-> of the o r i g i n a l c e l l s were plated i n standard 1.1 ml methylcellulose assays. 87 previously established, regular long-term marrow culture and then i r r a d i a t i n g the secondary adherent layer produced with 15 Gy to in a c t i v a t e any r e s i d u a l hemopoietic progenitors s t i l l present (19). It has been our experience at the Terry Fox Laboratory that such subcultured adherent layers are equivalent to primary adherent layers i n terms of the i r a b i l i t y to support and regulate long- term human hemopoiesis, but are not associated with the same problems of adherent layer detachment frequently encountered when such co-cultures are set up using primary adherent layers. The number of progenitors present i n each of these three types of long-term cultures was assessed a f t e r 4-5 weeks. The e f f e c t of CHP on progenitor maintenance i n cultures established using the standard protocol ( i . e . no feeders) i s shown in Figure 12. It can be seen that, i n the absence of a feeder, hemopoiesis was markedly i n h i b i t e d i n cultures containing CHP. However, when a pre-existing feeder was provided, hemopoiesis was equivalent to that observed i n control cultures (no CHP, no feeders) (Table VII). Thus, the continuous presence of even 500 ug/ml of CHP does not appear to be d i r e c t l y detrimental to the s u r v i v a l , p r o l i f e r a t i o n or d i f f e r e n t i a t i o n of hemopoietic c e l l s . 88 100 8 0 6 0 O DC Z o o 20 0 B F U - E C F U - G / M C F U - G / E FIGURE 12. The E f f e c t s of 500 ug/ml of CHP on the Yi e l d of Hemopoietic Progenitors i n Long-Term Marrow Cultures Assessed 4-5 Weeks After I n i t i a t i o n . Values shown represent the mean + 1 SEM for normalized data (% of number of progenitors from both adherent and non-adherent fracti o n s detected i n control cultures without CHP for 3 d i f f e r e n t experiments ( d i f f e r e n t marrow samples). Data are from the same experiments shown in Table V. Table VII. Lack, of Ef f e c t of 500 Ug/ml of CHP on Hemopoiesis i n Long-Term Cultures I n i t i a t e d on Pre-Established, Irradiated Adherent Layers (Feeders) No. of progenitors per 10' c e l l s i n i t i a l l y seeded Progenitor Exp. + CHP - CHP type no. + Feeder - Feeder CFU-G/M 1 2 3 BFU-E CFU-GE 7,324 903 7,212 17,792 598 1,668 1,400 141 808 768 69 26 132 24 120 480 15 14 Values shown are the mean of 2 r e p l i c a t e assays from 1 culture group. The diffe r e n c e between + CHP + Feeder and - CHP - Feeder i s not s i g n i f i c a n t (p>0.05 using f a c t o r i a l analysis of variance (26) on log transformed values). 90 3) DISCUSSION A number of l i n e s of evidence suggest that non-circulating elements of the marrow constitute an e s s e n t i a l component of the long-term marrow culture system. In addition, they suggest that d i r e c t interactions between stromal c e l l s and primitive hemopoietic c e l l s may be an important part of the mechanisms which allow hemopoiesis to be maintained for many weeks in v i t r o in a medium to which no hemopoietic growth factors are added (4-7,21). The present studies provide a d d i t i o n a l evidence i n support of t h i s model in the human system. In a f i r s t series of experiments in which c i r c u l a t i n g myeloid progenitors were used, maintenance was present in cultures i n i t i a t e d i n the presence of an adherent layer and to a ce r t a i n extent in i t s absence. However, c y c l i n g data from a c o l l a b o r a t i v e experiment (19) revealed profound differences between the 2 types of cultures. Primitive progenitors in the adherent layer were o s c i l l a t i n g between p r o l i f e r a t i o n (40-47% k i l l ) and quiescence (0-18% k i l l ) as a function of the feeding pattern. In contrast, p r i m i t i v e progenitors in the absence of an adherent layer were continuously c y c l i n g ( 44-58% k i l l ) , i r r e s p e c t i v e of the feeding schedule. However, persistence of progenitors for many weeks in the absence of a feeder layer together with the s e l e c t i v e advantage of endogeneous lymphoblastoid c e l l s in v i t r o rendered this approach impractical. Fortunately, an alternate approach could be used. CHP, a proline analogue that i s s e l e c t i v e l y toxic for collagen-producing c e l l s , was shown to i n h i b i t the establishment of long-term hemopoiesis by human marrow c e l l s by v i r t u e of i t s s e l e c t i v e i n h i b i t o r y action on c e l l s that allow development of an adherent layer, at least some of which are detected by the CFU-F assay. This conclusion i s based on the demonstration that the f a i l u r e of hemopoiesis seen in long-term human marrow cultures i n i t i a t e d and maintained in 91 the presence of CHP at doses that inhibited the formation of an adherent layer, could be completely abrogated i f a pre-established adherent layer was provided. Both the progeny of CFU-F and a significant proportion of the f ibroblast- l ike cel ls in the adherent layer are collagen-synthesizing cel ls (22,23). It is therefore not surprising that CHP has a particularly toxic effect on the prol i feration and establishment of these cel ls in v i tro . The fact that the same concentration of CHP did not inhibit hemopoiesis in the presence of a pre- established adherent layer suggests that collagen synthesis does not continue at high levels after the adherent layer becomes confluent or that i ts turnover is not crucial to hemopoiesis once a feeder layer is established. Further i t indicates that such concentrations of CHP do not adversely affect the v i a b i l i t y or regulatory function of human marrow adherent layer cel ls once they have become established in v i tro . This confirms similar findings in the murine system (18), and suggests the usefulness of CHP supplemented medium as a way of exploiting unseparated marrow as a substitute for purified hemopoietic progenitors in future experiments designed to assess the regulatory potential of defined mesenchymal populations. This approach offers the advantage that there is no c e l l loss, as is usually the case during most hemopoietic c e l l purif ication procedures. In addition, other non-mesenchymal c e l l types, such as macrophages and T-lymphocytes, which may play a role in the long-term marrow culture system, are not eliminated from the test inoculum. It is important to note that any procedure which seeks to provide target cel ls to assay for long-term hemopoietic support in the human system needs to meet the requirements of a 4-5 week endpoint. This time can be deduced from two types of experiments. The f irs t involves the assessment of the rate of progenitor decline in long-term cultures init iated with peripheral blood cel ls described above in which a di f ferent ia l effect is seen only after several weeks 92 at a time when EBV-transformed lymphoblastoid c e l l s show a s e l e c t i v e advantage i n cultures. S i m i l a r l y when suspensions of marrow c e l l s are exposed to 4-hydroperoxycyclyphosphamide and thereby s e l e c t i v e l y depleted of their content of clonogenic hemopoietic c e l l s , but not of the more primitive hemopoietic c e l l s from which clonogenic c e l l s are generated i n the long-term marrow culture system, regeneration of the clonogenic compartment under these conditions can be seen to require approximately 4-5 weeks (24,25). Thus any test hemopoietic c e l l suspension to be used to assay the hemopoietic supportive function of a candidate regulatory c e l l type must be able to generate clonogenic c e l l s for at least this length of time and also show a f a i l u r e of endogenous supportive function for an equivalent period. This places an a d d i t i o n a l stringent requirement on the purity of the test c e l l s , since a very small number of stromal c e l l precursors over 4 to 5 weeks can reconstitute an adherent layer. In my experience this also precludes the use of non-adherent f r a c t i o n s of fresh or 1 week old long-term culture c e l l s of human marrow o r i g i n for such assays. On the other hand, i t appears that the use of CHP supplemented media may o f f e r a simple and reproducible method for s e l e c t i v e l y i n a c t i v a t i n g the stromal c e l l component of fresh human marrow samples. 93 REFERENCES 1. McCulloch EA, Siminovitch L, T i l l JE, Russell ES, Bernstein SE. The c e l l u l a r basis of the g e n e t i c a l l y determined hemopoietic defect i n anaemic mice of genotype S l / S l d . Blood 26: 399, 1965. 2. Bernstein SE. Tissue transplantation as an an a l y t i c and therapeutic tool i n hereditary anemias. Am J Surg 119: 448, 1970. 3. G i d a l i J, and Lajtha LG. Regulation of haemopoietic stem c e l l turnover in p a r t i a l l y i r r a d i a t e d mice. C e l l Tissue Kinet. 5: 147, 1972. 4. Dexter TM, Spooncer E, Toksoz D, Lajtha LG. The role of c e l l s and the i r products i n the regulation of in v i t r o stem c e l l p r o l i f e r a t i o n and granulocyte development. J Supramolec Structure. 13: 513, 1980. 5. Cashman JD, Eaves AC, and Eaves CJ. Regulated p r o l i f e r a t i o n of pr i m i t i v e hematopoietic progenitor c e l l s i n long-term marrow cultures. Blood 66: 1002, 1985. 6. Roberts RA, Spooncer E, Parkinson EK, Lord BI, Al l e n TD, Dexter TM. Metabolically i n a c t i v e 3T3 c e l l s can substitute for marrow stromal c e l l s to promote the p r o l i f e r a t i o n and development of multipotent haemopoietic stem c e l l s . J C e l l Physiol. 132: 203, 1987. 7. Castro-Malaspina H, Saletan S, Gay RE, Oettgen B, Gay S, Moore MAS. Immunocytochemical i d e n t i f i c a t i o n of c e l l s comprising the adherent layer of long-term human bone marrow cultures. Blood'58(Suppl 1): 107a, 1981. 8. A l l e n TD, and Dexter TM. The e s s e n t i a l c e l l s of the hemopoietic microenvironment. Exp Hematol. 12: 517, 1984. 9. Eaves AC, Cashman JD, Gaboury LA, and Eaves CJ. C l i n i c a l s i g n i f i c a n c e of long-term cultures of myeloid blood c e l l s . CRC Reviews i n Oncol/Hematol, 7: 125, 1987. 10. Anklesaria P, Klassen V, Sakakeeny MA, FitzGerald TJ, Harrison D, Rybak ME, Greenberger JS. B i o l o g i c a l characterization of cl o n a l permanent stromal c e l l l i n e s from anemic S l / S l d mice and +/+ litter m a t e s . Exp Hematol. 15: 636, 1987. 11. J u l i u s MH, Simpson E, Herzenberg LA. A rapid method for the i s o l a t i o n of functional thymus-derived murine lymphocytes. Eur J Immunol. 3: 645, 1973. 12. Abboud CM, Duerst RE, Frantz CN, Ryan D H , Liesveld, JL, Brennan JK. Lysis of human f i b r o b l a s t colony-forming c e l l s and endothelial c e l l s by monoclonal antibody (6-19) and complement. Blood 68: 1169, 1986.. 13. G i l b e r t SF, Migeon BR. D-valine as a s e l e c t i v e agent for normal human and rodent e p i t h e l i a l c e l l s in culture. C e l l 5: 11, 1975. 14. Chertkov JL, Drize NJ, Gurevitch OA, Samoylova RS. Origin of hemopoietic stromal progenitor c e l l s in chimeras. Exp hematol. 13: 1217, 1985. 94 15. H o l l i n s PE, FitzGerald PH, Heaton DC, Beard MEJ. Host o r i g i n of i n v i t r o bone marrow f i b r o b l a s t s a f t e r marrow transplantation i n man. International Journal of C e l l Cloning 2: 348, 1984. 16. Kao WW-Y, Prockop DJ. Can proline analogues be used to prevent f i b r o b l a s t s from overgrowing cultures of e p i t h e l i a l c e l l s ? B i r t h Defects: O r i g i n a l A r t i c l e Series, XVI(Number 2): 53, 1980. 17. Andrews RG, Takahashi M, Segal GM, Powell JS, Bernstein, ID, Singer, JW. The L4F3 antigen i s expressed by unipotent and multipotent colony-forming c e l l s but not by th e i r precursors. Blood 68: 1030,1986. 18. Zuckerman KS, Rhodes RK, Goodrum DD, Patel VR, Sparks B, Wells J, Wicha MS, Mayo LA. I n h i b i t i o n of collagen deposition in the e x t r a c e l l u l a r matrix prevents the establishment of a stroma supportive of hematopoiesis in long-term murine bone marrow cultures. J C l i n Invest. 75: 970, 1985. 19. Eaves AC, Cashman JD, Gaboury LA, Kalousek, DK, Eaves CJ. Unregulated p r o l i f e r a t i o n of primitive chronic myeloid leukemia progenitors i n the presence of normal marrow adherent c e l l s . Proc Natl Acad Sci USA. 83: 5306, 1986. 20. Gartner S, Kaplan HS. Long-term culture of human bone marrow c e l l s . Proc Natl Acad Sci USA. 77: 4756, 1980. 21. Simmons PJ, Przepiorka D, Thomas ED, Torok-Storb B. Host o r i g i n of marrow stromal c e l l s following allogeneic bone marrow transplantation. Nature 328: 429, 1987. 22. Castro-Malaspina H, Gay RE, Resnick G, Kapoor N, Myers P, C h i a r i e r i , D. McKenzie S, Broxmeyer HE, and Moore MAS. Characterization of human bone marrow f i b r o b l a s t colony-forming c e l l s (CFU-F) and their progeny. Blood 56: 289, 1980. 23. McCarthy KF, Wientroub S, Hale M, and Reddi AH. Establishment of the hematopoietic microenvironment in the marrow of matrix-induced endochondral bone. Exp Hematol 12: 131, 1984. 24. Siena S, Castro-Malaspina H, Gulati SC, Lu L, Colvin M0, Clarkson BC, O'Reilly RJ, and Moore MAS, E f f e c t s of in v i t r o purging with 4-hydroperoxycyclophosphamide on the hematopoietic and microenvironmental elements of human bone marrow. Blood 65: 655, 1985. 25. Winton EF, and Colenda KW. Use of long-term human marrow cultures to demonstrate progenitor c e l l precursors in marrow treated with 4-hydroperoxycyclophosphamide. Exp Hematol 15: 710, 1987. 26. Sokal RR, Rohlf FJ. Biometry, The p r i n c i p l e s and practice of s t a t i s t i c s in b i o l o g i c a l research, second e d i t i o n , U.H. Freeman and Company, San Francisco, 1981. 27. Sokal RR, Rohlf FJ. Biometry, The p r i n c i p l e s and practice of s t a t i s t i c s in b i o l o g i c a l research, second e d i t i o n , W.H. Freeman and Company, San Francisco, 1981. 95 C H A P T E R IV INDUCIBLE PRODUCTION OF HEMOPOIETIC GROWTH FACTORS BY SV-40 IMMORTALIZED MESENCHYMAL CELL LINES OF HUMAN MARROW ORIGIN 1. INTRODUCTION Current evidence suggests that the control of many aspects of hemopoiesis i s regulated l o c a l l y within the marrow (1) and may involve the a c t i v a t i o n of hemopoietic growth factor gene expression by resident non-hemopoietic c e l l s that constitute the marrow stroma (2,3). Although some controversy e x i s t s as to the o r i g i n of these c e l l s and their ontological r e l a t i o n to the hemopoietic hierarchy, studies i n animal models indicate an early embryological divergence in the mesenchymal and hemopoietic components of the marrow ( 4 ) . More recent experiments have established that mesenchymal c e l l s ' o f donor o r i g i n are not normally detectable i n the marrow of transplant r e c i p i e n t s (5,6,7) confirming e a r l i e r data i n mice i n d i c a t i n g that these c e l l s and thei r precursors do not c i r c u l a t e ( 8 , 9 ) . On the other hand c e l l s with the phenotypic markers of f i b r o b l a s t s , adipocytes and/or endothelial c e l l s can be re a d i l y obtained as expanded populations i n various types of human marrow culture systems. These include cultures i n i t i a t e d by incubating marrow c e l l s at r e l a t i v e l y low concentrations s u i t a b l e for obtaining i s o l a t e d f i b r o b l a s t - l i k e colonies (CFU-F ( 1 0 ) , CFU-RF (6) or CFU-ST (11) assays), as well as cultures i n i t i a t e d with somewhat higher concentrations of marrow c e l l s . The l a t t e r result in the formation of a complex adherent layer of mesenchymal c e l l s that are able to support p r i m i t i v e human hemopoietic progenitor p r o l i f e r a t i o n and d i f f e r e n t i a t i o n for periods of many weeks (1 2 ) . 96 More de t a i l e d studies of the cy c l i n g status of very pr i m i t i v e hemopoietic progenitors i n long-term human marrow cultures have shown that the adherent layer plays an important role i n the mechanism that regulates whether the majority of these c e l l s are quiescent, as they are in the marrow i n vivo, or whether they are p r o l i f e r a t i n g i n response to some perturbation (13,14). Thus for the f i r s t time, i t i s possible to analyze i n v i t r o both p o s i t i v e and negative loops i n a regulatory network that controls the behaviour of very pri m i t i v e hemopoietic c e l l s . Since long-term marrow culture adherent layers contain a v a r i e t y of c e l l s expressing d i f f e r e n t mesenchymal c e l l phenotypes, including 6.19-antigen-positive c e l l s (15,16), l i p i d - l a d e n adipocytes and Factor V H I - p o s i t i v e c e l l s arranged i n c a p i l l a r y - l i k e structures (1), a l l of which are normal constituents of the marrow stroma, i t i s d i f f i c u l t to u t i l i z e the long-term marrow culture system to investigate the role of s p e c i f i c stromal components. Because a l l of these mesenchymal c e l l types are known to exhibit only a l i m i t e d p o t e n t i a l for expansion in v i t r o (17), novel st r a t e g i e s for thei r i s o l a t i o n are required. We describe the use of SV-40 for this purpose. A high t i t e r v irus stock was generated, tested for b i o l o g i c a l a c t i v i t y and used for the immortalization of human marrow mesenchymal c e l l s i n v i t r o . A large number of immortalized, cloned c e l l l i n e s expressing the d i f f e r e n t i a t e d phenotype of fixed bone marrow stromal c e l l s have been obtained and p a r t i a l l y characterized. Of p a r t i c u l a r i n t e r e s t , i s the finding that these are capable, upon stimulation, of secreting regulatory molecules that are act i v e on pri m i t i v e hemopoietic progenitors. These c e l l l i n e s should be useful for the further d e l i n e a t i o n of the c e l l u l a r and molecular basis of hemopoietic stem c e l l regulation. 97 2. RESULTS (A) Transforming Potential of SV-40 Virus ans i t s E f f e c t on DNA Synthesis The transforming p o t e n t i a l of SV-40 virus was f i r s t tested on murine NIH- 3T3 c e l l s which are known to be readily infected and transformed i n v i t r o (18). Mock infected cultures did not show any transformed f o c i whereas dishes infected with SV-40 virus yielded transformed f o c i (Figure 13) at a frequency of 1 per 4 x l 0 3 c e l l s exposed. From these experiments the transformation t i t e r of this v irus stock on NIH-3T3 was 2.7 x 10^ focus forming unit/ml. To demonstrate a d i r e c t b i o l o g i c a l e f f e c t of SV-40 that would v a l i d a t e i t s intended use in immortalizing human marrow stromal c e l l s , we examined i t s e f f e c t on c e l l u l a r DNA synthesis following addition of virus to contact i n h i b i t e d c e l l s subcultured from regular long-term marrow cultures adherent layers. Whereas the majority of the c e l l s i n mock infected cultures remained quiescent (Figure 14, panel A), infected cultures showed a dramatic increase in ^H-Thymidine incorporation 48 hours l a t e r (Figure 14 panel B). Since these data provided an i n d i c a t i o n that SV-40 virus may be useful for the immortalization of human marrow stromal c e l l s , this preparation was use to generate cloned human continuous l i n e s . (B) Derivation and Immunological Characterization of C e l l Lines Cloned l i n e s of SV-40 immortalized c e l l s were is o l a t e d from a v a r i e t y of primary cultures. Both primary and f i r s t passage adherent layers of 3-4 98 FIGURE 13. Transformed Focus of NIH-3T3 C e l l s . 99 FIGURE 14. Autoradiograms of Confluent Marrow Adherent Layers. Mock, i n f e c t e d l a y e r (upper panel) and SV-40 i n f e c t e d l a y e r (lower panel). 100 week old long-term human marrow cultures were used for i n f e c t i o n s since these are i n d i s t i n g u i s h a b l e i n their a b i l i t y to regulate p r i m i t i v e hemopoietic progenitor c y c l i n g i n the long-term culture system (14). Lines were also obtained following i n f e c t i o n of " f i b r o b l a s t " monolayers established from pooled colonies of human marrow o r i g i n , and HUV-EC-C endothelial c e l l monolayers o r i g i n a l l y derived from umbilical vein endothelium. Twenty-four hours a f t e r i n f e c t i o n , expression of the SV-40 large T antigen was detected i n 45 + 23 % of the c e l l s i n the adherent layer of long-term marrow cultures (with higher values for infected f i b r o b l a s t c u ltures), whereas mock-infected c e l l s were consi s t e n t l y negative. Two to 3 weeks a f t e r i n f e c t i o n , low density subcultures could be seen to contain f o c i of morphologically altered c e l l s at a frequency of ~1 focus per 100 i n i t i a l l y T Ag-positive marrow c e l l s . The c e l l s i n these f o c i were more elongated and r e f r a c t i l e than normal and grew i n a disordered fashion t y p i c a l of c e l l s whose growth i s not contact i n h i b i t e d . This morphology was exhibited by a l l transformed f o c i and was independent of the type of population i n i t i a l l y exposed to the v i r u s . Lines derived by i n f e c t i n g long-term marrow adherent layers are designated as MH, l i n e s derived from marrow f i b r o b l a s t s as CFUST, and l i n e s derived from endothelial c e l l s as EC. From these three types of c e l l s , a t o t a l of 13, 13, and 5 such l i n e s were independently i s o l a t e d . After cloning, each l i n e was expanded and then frozen i n DMSO and stored at -70°C u n t i l required for further study. Many l i n e s have been maintained continuously for periods of months and, in one case, (CFUST-16) for over a year. The r e s u l t s of immunophenotyping studies are summarized in Table VIII. A l l c e l l s i n a l l l i n e s were p o s i t i v e for the SV-40 large T antigen i n d i c a t i n g a functional integrated SV-40 genome consistent with their immortalized state. A l l l i n e s tested (5/5) were also p o s i t i v e for the surface marker 6.19 Table VIII. Histochemical and Immunophenotypic Properties of SV-40 Transformed C e l l Lines* Line Origin Long-Term BM BM Fibroblast Umbilical Cord Adherent Layer C e l l s Colony C e l l s Endothelial C e l l s Marker Assessed (MH2SV-C11) (CFUST-C116) (EC22) Membrane 6.19 + + + T200 - - + LEU-MI - - - LEU-M3 - - Cytoplasmic Acid Phosphatase + + - Alkaline Phosphatase - - - Factor VIII - - + Collagen I + + - Collagen IV + + + Laminin + ' + + Nuclear SV-40 large T Ag + + + *Data shown are for representative l i n e s , for which the most complete documentation was obtained. Positive means that more than 30% of the c e l l s were p o s i t i v e . Negative means no positive c e l l s could be detected. 102 (Figure 15) which appears to react only with mesechymal c e l l s i n the marrow (15,16). Marrow-derived l i n e s were phenotypically s i m i l a r , regardless of the type of culture i n i t i a l l y infected and were consistently p o s i t i v e for acid phosphatase, laminin and collagens type I and IV. A s i g n i f i c a n t proportion of c e l l s i n the adherent layer of long-term human marrow cultures also show these features as do c e l l s produced by CFU-RF (6)). Although I showed that c e l l s p o s i t i v e for Factor V H I - r e l a t e d antigen can also be found i n the adherent layer of long-term marrow cultures (1), none of the marrow-derived transformants showed this property. This could not, however, be att r i b u t e d to an i n a b i l i t y of SV-40 to immortalize Factor V H I - p o s i t i v e c e l l s as shown by the continued expression of this antigen i n l i n e s derived following i n f e c t i o n of HUV-EC-C c e l l s . None of the l i n e s , including representatives from a l l three categories (by o r i g i n ) contained detectable LeuMl or LeuM3 p o s i t i v e c e l l s . Marrow-derived l i n e s were also consistently negative for T200 (Figure 15). However, SV-40 transformation of HUV-EC-C c e l l s did activa t e weak T200 p o s i t i v i t y ( i n the one l i n e tested), whereas untreated HUV-EC-C c e l l s were con s i s t e n t l y negative (Figure 16). (C) C h a r a c t e r i s t i c s of Transformed c e l l l i n e s The SV-40 large T antigen, a multifunctional 90-100 Kd oncogenic protein, i s expressed i n a l l SV-40 transformed c e l l s (19). In addition to i t s r o l e in virus r e p l i c a t i o n , i t exerts numerous e f f e c t s on susceptible host c e l l s including the stimulation of c e l l u l a r DNA synthesis even in quiescent c e l l s (20). A longer l a s t i n g e f f e c t of SV-40 on these same c e l l s has a lso now been documented. This can be seen as a decreased population doubling time 103 10-1 10° 101 102 10 3 Fluorescence Intensity (arbitrary units) FIGURE 15. FACS P r o f i l e of CFUST-CL16 C e l l s Stained With Monoclonal Antibody 6.19 (Panel A) and Anti-LeukVT200 (Panel B). The s o l i d l i n e shows the p r o f i l e of the test sample i n each case. The dotted l i n e shows the p r o f i l e of the corresponding negative control sample. 104 FIGURE 16. FACS Profile of a Suspension of Spontaneously Immortalized Lymphoblastoid Cells (Panel A), and HUVE-EC-E Cells Before (Panel B), and After (Panel C) Transformation With SV-40 Virus. 105 (Figure 17 B), or an increase in ^H-thymidine incorporation (Figure 17 A) of infected as compared to non-infected ce l l s . Capacity for anchorage-independent growth was tested by plating transformed and uninfected cel ls in semi-solid medium. SV-40 transformed lines routinely formed dist inct colonies containing more than 100 cel ls within 20 days in culture (Figure 18 A). In contrast, cel ls from non-infected control cultures yielded only a few small clusters of 4 to 8 cel ls and did not form any colonies containing more than 50 cel ls (Figure 18 B). After cloning, the plating efficiency of the 3 lines tested remained high (>5%) and was independent of c e l l concentration (Figure 19), although some var iab i l i ty in the colony-forming efficiency of different lines was seen (data not shown). (D) Induction of Growth Factor Production Recent reports have shown that fibroblasts and endothelial cel ls of various tissue origins constitutively produce low or undetectable levels of hemopoietic growth factors, but upon stimulation with various secretory products of macrophages, including IL-1, hemopoietic growth factor production is rapidly and markedly enhanced (2,3,21,22). This response thus appears to be a tightly regulated part of the functional program of a variety of mesenchymal c e l l populations. Recent data from the Terry Fox Lab have shown that I L - l g also stimulates the production of hemopoietic growth factor production by cel ls in the adherent layer of long-term human marrow cultures (23). It was therefore of interest to evaluate the act ivity of media conditioned by the various transformed ce l l lines before and after exposure to IL-1(3. The results of two representative experiments are shown in Table IX. It can be seen that addition of 12 units/ml IL-16 greatly enhanced the production and release of 106 FIGURE 17. Tritiated-Thymidine Uptake (Panel A) of SV-40 Infected ( S o l i d Lines) and Uninfected (Broken Lines) MH C e l l s . 107 FIGURE 17. . Growth Rate (Panel B) of SV-40 Infected ( S o l i d Lines) and Uninfected (Broken Lines) MH C e l l s . 108 FIGURE 18. A Colony of Transformed C e l l s Generated i n a M e t h y l c e l l u l o s e Culture 14 Days A f t e r Seeding the Cultures with MH C e l l s I n f e c t e d With SV-40 V i r u s (Panel A). 109 FIGURE 18. Co n t r o l (Uninfected) MH C e l l s F a i l e d To Y i e l d Colonies (Panel B). 110 . Analysis of the Clonogenic Capacity of MH2SV-CL1 C e l l s Plated in Methylcellulose. Each data point represents the mean + 1 SEM of values obtained in each of two d i f f e r e n t experiments ~ (shown separately as c i r c l e s and t r i a n g l e s ) . I l l Table IX. Evidence for IL-16 Induced Production of Hemopoietic Colony-Stimulating A c t i v i t y by Representative SV-40 Transformed Human C e l l Lines Erythroid Colonies Granulocyte-Macrophage Colonies Addition to Methylcellulose Assay Exp 1 Exp 2 Exp 1 Exp 2 MH2SV-C11 1 3 17 19 MH2SV-C11 + IL-16 7 7 111 124 CFUST-C116 3 2 32 8 CFUST-C116 + IL-lf3 8 4 108 99 EC22 0 2 9 2 EC22 + IL-16 9 4 97 26 Skin Fibroblasts 1 - 6 - Skin Fibroblasts + IL-16 2 - 56 - No addition 0 0 0 0 IL-16 (1.2 units/ml) 3 4 47 23 M2-10B4 (mouse marrow f i b r o b l a s t l i n e ) - 3 - 12 M2-10B4 (mouse marrow f i b r o b l a s t l i n e ) + IL-16 - 3 - 25 Human LCM (10%) 18 9 126 115 GM-CSF (8 ng/ml) 5 3 168 86 * C e l l s were incubated with or without 12 units/ml of IL-16 for 24 hours as described i n the Materials and Methods. Conditioned media were added to methylcellulose assays at a f i n a l concentration of 10% (v/v). Erythroid colonies include a l l categories (CFU-E plus BFU-E derived). M2-10B4 c e l l s are a spontaneously immortalized cloned l i n e of c e l l s phenotypically s i m i l a r to the MH and CFUST l i n e s described here but derived i n this laboratory from a cul t u r e of adherent mouse marrow c e l l s . 112 colony-stimulating factor(s) active on primitive human hemopoietic progenitor classes on two separate differentiation lineages. A total of 15 lines were tested in this way. A l l showed this response. Although there was some variation in the f inal act ivi ty of the conditioned media from different lines exposed to IL-10, an act ivi ty equivalent to or greater than 80 ng/ml of GM-CSF was often obtained. In contrast, s imilarly prepared conditioned media obtained from IL-16 stimulated marrow stromal lines of murine origin had no act iv i ty on human progenitors (Table IX). This suggests that the weak act iv i ty sometimes seen in controls given IL-16 alone was due to endogenous production of colony- stimulating act iv i ty by co-existing cel ls in the methylcellulose assay and that residual IL-16 concentrations in the test conditioned media were considerably reduced (below 1.2 units/ml). Dose response studies established that 12 units of IL-16/ml was well above the minimal dose required to attain maximal growth factor release (Figure 20). Conditioned media were also assayed (Dr. P . Lansdorp, Terry Fox Laboratory) for IL-6 act ivi ty as measured by their a b i l i t y to stimulate ^H-thymidine incorporation into B13.29 ce l l s , a murine hybridoma c e l l l ine that is specif ical ly responsive to IL-6 (24). Results from these studies have indicated that the production of IL-6 bioactivity by the SV-40 transformed l ines, l ike the production of colony-stimulating bioactivity was consistently found to be markedly increased by the stimulation of the lines with IL-16. Since there is now evidence that hemopoietic growth factors may act not only on hemopoietic cel ls but also on target populations of widely diverse embryonic origin (25), I also investigated the possibi l i ty that medium conditioned by SV-40 marrow immortalized lines might have biological act iv i ty on normal mesenchymal ce l l s . As shown in Table X, conditioned media from one 113 FIGURE 20. Hemopoietic Colony-Stimulating A c t i v i t y of Media Conditioned for 24 Hours by CFUST-CL16 or MH2SV-CL1 C e l l s (or No C e l l s ) as a Function of the Concentration of IL-16 Used as a Stimulant. The maximal number of colonies obtainable from the progenitors present i n the assay as indicated by stimulation of these with an optimal concentration of recombinant human GM-CSF (8 ng/ml) i s also shown for comparison. C e l l l i n e conditioned media were present i n the methylcellulose assays at a concentration of 10% (v/v). 114 Table X. E f f e c t s of Conditioned Medium of an IL-1 Stimulated, SV-40 Immortalized Marrow Stromal C e l l Line (CFUST-16) on CFU-F Formation Addi tion ( f i n a l concentration in CFU-F assay) No. of CFU-F/Dish Exp. 1 Exp. 2 Exp. 3 (Day 12) 1 (Day 5) (Day 10) (Day 6) Medium (a-10%) Human PHA Stimulated Leukocyte CM (10%) CFUST-16 with IL-1 CM (10 % ) 2 CFUST-16 without IL-1 CM (10%) GM-CSF (8 ng/ml) 13 29 28 29 10 24 24 58 62 95 8 22 100 14 7 24 iNo. of days of CFU-F colony growth p r i o r to f i x a t i o n and st a i n i n g . ^Conditioned medium from confluent CFU-STC116 c e l l monolayers incubated for 24 hours with or without 12 U/ml recombinant human IL-16 (Biogen). 115 l i n e tested (CFUST-16) shared with GM-CSF the a b i l i t y to stimulate the p l a t i n g e f f i c i e n c y of fresh normal bone marrow f i b r o b l a s t s . (E) I r r a d i a t i o n studies To examine the p o s s i b i l i t y that SV-40 could transform human marrow stromal c e l l s without a l t e r i n g their r a d i o b i o l o g i c a l properties, c e l l s u r v i v a l curves were obtained from normal and SV-40 transformed l i n e s a f t e r s i n g l e dose i r r a d i a t i o n . In both cases, s u r v i v a l curves are exponential and shouldered with a DQ of 1.3 Gy (Figure 21) which i s in accord with previously reported data on cultured bone marrow f i b r o b l a s t s (26). 3. DISCUSSION In humans, hemopoiesis i s normally r e s t r i c t e d to the bone marrow where i t proceeds in close association with as yet poorly characterized fixed mesenchymal elements. Recently, an v i t r o model has been developed in which the long-term maintenance and regulated turnover of primitive hemopoietic progenitors occurs in the absence of exogenously provided growth factors. However, a key component of this culture system i s a complex adherent c e l l layer containing a variety of mesenchymal c e l l types of marrow o r i g i n . To f a c i l i t a t e analysis of the i n d i v i d u a l roles of these various mesenchymal c e l l types, I used SV-40 virus as an immortalizing agent. A high t i t e r v irus stock was prepared and i t s b i o l o g i c a l a c t i v i t y tested on both murine and human mesenchymal c e l l s . Lines could be re a d i l y obtained from human long-term marrow culture adherent layers, and marrow f i b r o b l a s t cultures. Both of these contain c e l l s with properties s i m i l a r to those exhibited by a l l marrow-derived c e l l 116 FIGURE 21. C e l l Survival Curve for SV-40 Immortalized Marrow F i b r o b l a s t s . CFUST-CL16 (open and s o l i d t r i a n g l e s ) , SV-40 immortalized endothelial c e l l s EC CL22 (open diamond), Factor VIII p o s i t i v e c e l l hybrid Ea.926 ( s o l i d c i r c l e s ) , and normal bone marrow f i b r o b l a s t s ( s o l i d squares). 117 lines studied. This phenotype is characterized by the expression at the surface of the antigen 6.19 and in the cytoplasm of laminin and collagen IV, with a lack of expression of Factor VIII. Two other reports of SV-40 immortalized human marrow cel ls have been published. In one, phenotypic characterization suggested a fibroblastic origin of the l ine (27). In the other, the presence of "round" cells that reacted both with the pan-hemopoietic c e l l determinant, T200, and the monocytic antigen, LeuM3 was a consistent finding (28). In contrast, I did not observe a subpopulation of more spherical ce l l s in any of my lines and a search for evidence of LeuM3 or T200 expression gave consistently negative results with one exception. This was the expression of detectable T200 surface antigen after (but not before) transformation of HUV-EC-C endothelial cel ls with SV-40 suggesting that SV-40 may cause some phenotypic alteration of mesenchymal ce l l antigen expression when used as a transforming agent. This was not surprising since preliminary data have indicated that SV-40 transformed marrow cells were also grossly abnormal cytogenetically. On the other hand, I have found in a collaborative study that SV-40 transformed cells are stimulated by GM-CSF (29) as was also noted by Singer et a l (28). As mentioned earl ier such responsiveness has further been demonstrated to be a feature of a variety of developmentally unrelated ce l l s of non-hemopoietic origin (25). GM-CSF responsiveness can therefore not be used to indicate a close relationship to hemopoietic ce l l s . SV-40 transformed cel ls grew at a faster rate than non-infected cel ls and showed other properties of transformed cells such as loss of contact inhibit ion and the acquisition of anchorage-independence. Interestingly, this could not be correlated with tumorigenicity since at least one line when injected into 4 nude mice failed to generate any tumors (data not shown), a finding also reported by others for SV-40 transformed cells (30). This could not be 118 correlated either with increased radiosensitivity since both SV-infected and non-infected cel ls had similar response to ionizing radiation. The ab i l i t y of transformants to form colonies in semi-solid medium, particularly at very low c e l l concentrations was consistent with the l ike ly single c e l l origin of the colonies obtained under these conditions. This was confirmed by assessment of a methylation sensitive restr ict ion fragment length polymorphism in the X-linked HPRT gene (31) in the DNA of a clone isolated from a female heterozygote (data not shown, obtained by Dr. A l i Turhan, Terry Fox Laboratory). Thus, these studies have demonstrated the feas ib i l i ty of generating a large number of clonal lines and subclones by exploiting their ab i l i t y to form colonies in semi-solid medium. In addition, our results indicate that upon stimulation with as l i t t l e as 3 units/ml of IL-16, a l l SV-40 transformed lines displayed the a b i l i t y of normal mesenchymal ce l l populations to show a marked induction of hemopoietic colony-stimulating factor production, including the.production of at least GM-CSF and IL -6 . This has been confirmed at the molecular level by Northern Blotting and Sl mapping analyses (Dr. RK. Humphries, Dr. R. Kay, Terry Fox Laboratory). Recently, i t was demonstrated that primitive progenitors in the adherent layer of long-term marrow cultures can be activated into cycle following the addition of IL-16 (32). In addition, data suggesting that IL-6 may synergize with other colony-stimulating factors to stimulate the prol i ferat ion of very primitive hemopoietic cel ls in methylcellulose has been reported (33). Lastly, the finding that medium conditioned by IL-16 stimulated SV-40 transformed lines stimulates the clonal growth of fresh normal f ibroblast ic cel ls is intriguing. It raises the tantalizing poss ibi l i ty that inflammatory ce l l s , monocytes for instance, may recruit mesenchymal cel ls local ly to produce hemopoietic growth factors which could in turn participate 119 i n various physiopathologic processes including wound healing, a t h e r o s c l e r o s i s , myelofibrosis and possibly desmoplastic reaction of tumors. The a v a i l a b i l i t y of permanent cloned l i n e s of c e l l s with a stromal phenotype that exhibit the regulated a b i l i t y to produce such factors should f a c i l i t a t e future studies of the role that these c e l l s may play in the control of hemopoietic stem c e l l turnover. 120 REFERENCES 1. Eaves AC, Cashman JD, Gaboury LA, Eaves CJ. C l i n i c a l s i g n i f i c a n c e of long-term cultures of myeloid blood c e l l s . CRC C r i t i c a l Reviews in Oncology/Hematology 7: 125, 1987. 2. Zucali JR, D i n a r e l l o CA, Obion DJ, Gross MA, Anderson L, Weiner RS. I n t e r l e u k i n - l stimulates f i b r o b l a s t s to produce granulocyte-macrophage colony-stimulating a c t i v i t y and prostaglandin E2. J C l i n Invest 77: 1857, 1986. 3. Lee M, Segal GM, Bagby GC. I n t e r l e u k i n - l induces human bone marrow- derived f i b r o b l a s t s to produce multilineage hematopoietic growth fac t o r s . Exp Hematol 15: 983, 1987. 4. Moore MAS. 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Singer JW, Charbord P, Keating A, Nemunaitis J, Raugi G, Wignt TN, Lopez JA, Roth GJ, Dow LW, Fialkow PJ: Simian virus 40-transformed adherent c e l l s from human long-term marrow cultures: Cloned c e l l l i n e s produce c e l l s with stromal and hematopoietic c h a r a c t e r i s t i c s . Blood 70:464, 1987 29. Dedhar S, Gaboury L, Galloway P & Eaves CJ. Human GM-CSF i s a growth factor active on a variety of c e l l types of non-hemopoietic o r i g i n . Science (submitted) 30. S t i l e s CD, Desmond W, Sato GE, Sauer MH: F a i l u r e of human c e l l s transformed by simian virus 40 to form tumors in athymic mice. Proc Natl Acad Sci USA 72:4971, 1975 31. Vogelstein B, Fearon ER, Hamilton SR, Preisenger AC, Willard FW, Michelson AM, Riggs AD, Orkin SH: Clonal analysis using recombinant DNA probes from the X-chromosome. Cancer Res 47:4806, 1987 32. Eaves CJ, Cashman JD, Ross R, Raines E, Eaves AC: Factors that a c t i v a t e quiescent hemopoietic progenitors in normal long-term human marrow cultures. Blood 68 (Suppl 1): 141a, 1986 " 33. Ikebuchi K, Wong GG, Clark SC, Ihle JN, H i r a i Y, Ogawa M. Interleukin-6 enhancement of Interleukin-3 dependent p r o l i f e r a t i o n of m u l t i p o t e n t i a l hemopoietic progenitors. Blood 70 (Suppl 1):173a, 1987 123 C H A P T E R V SUMMARY AND FUTURE DIRECTIONS In humans, hemopoiesis i s maintained for the l i f e t i m e of the i n d i v i d u a l through the d i f f e r e n t i a t i v e d i v i s i o n s of a l i m i t e d number of hemopoietic stem c e l l s (1). These c e l l s originate in the yolk sac mesoderm and then undergo a s e r i e s of migrations early in ontogeny to eventually e s t a b l i s h themselves in the extravascular spaces of the bone marrow of the adult. There they are found in close proximity to a number of fixed bone marrow elements which c o l l e c t i v e l y constitute a hemopoietic "microenvironment" (2). Very l i t t l e i s known about the molecular events involved i n the commitment of pluripotent hemopoietic stem c e l l s . However, over the past several years evidence has accumulated to support the idea that the p r o l i f e r a t i v e state of hemopoietic stem c e l l s i s e x t r i n s i c a l l y regulated through short-range interactions with non-circulating bone marrow c e l l s (3). While the exact nature of the signals involved in these in t e r a c t i o n s awaits f u l l biochemical characterization, an a t t r a c t i v e hypothesis i s that marrow stromal c e l l s respond to perturbations a f f e c t i n g mature blood c e l l s which r e s u l t s i n the l o c a l secretion of hormone-like hemopoietic growth factors or the expression by stromal c e l l s of surface bound forms of such growth f a c t o r s . P r i m i t i v e hemopoietic c e l l s cannot be recognized morphologically. Studies on the e a r l i e s t p r o l i f e r a t i v e events occurring in hemopoietic d i f f e r e n t i a t i o n have therefore come to r e l y mainly on in v i t r o culture techniques that detect these c e l l s by measuring their developmental p o t e n t i a l under defined conditions. In v i t r o colony assays have been invaluable in providing a conceptual framework about the h i e r a r c h i c a l organization of the 124 hemopoietic system. They have also led to the i n i t i a l discovery and i d e n t i f i c a t i o n of a number of d i s t i n c t hemopoietic growth factors (4). However, colony assays have ce r t a i n i n t r i n s i c l i m i t a t i o n s . F i r s t , they only support progenitor c e l l l i m i t e d self-renewal. Second, by the i r very nature, they minimize c e l l u l a r interactions with stromal c e l l s which are suspected to play a c r i t i c a l r ole i n the regulation of stem c e l l s i n vivo ( 5 ) . Thus, the de s c r i p t i o n by Dexter et a l . (6) of an al t e r n a t i v e i n v i t r o system which allows the long-term maintenance of high p r o l i f e r a t i v e p o t e n t i a l progenitor c e l l s was nothing less than revolutionary. For the f i r s t time, i t became possible to study the turnover of hemopoietic stem c e l l s under i n v i t r o conditions which allow, and l i k e l y require, close interactions between stem c e l l s and marrow stromal c e l l types. U t i l i z a t i o n of the long-term marrow culture system by many d i f f e r e n t groups has since provided considerable evidence that this i s the case. However, many important questions are s t i l l unanswered. For example, the nature of the c e l l u l a r regulatory signals that regulate stem c e l l turnover i n this system has not yet been established nor has t h e i r o r i g i n . These issues cannot be resolved without f i r s t d i s s e c t i n g out the i n d i v i d u a l components of the long-term marrow culture system and then developing experimental approaches to reconstruct the function provided by the adherent layer. The purpose of my research was to i n i t i a t e studies along these l i n e s , based on the assumption that the mesenchymal c e l l s of the marrow are important i n the regulation of hemopoiesis. To achieve this goal, I set myself the task of i s o l a t i n g and then analyzing these elements from the long- term marrow culture system. The f i r s t goal was to develop an assay that would allow quantitation of the hemopoietic supportive function of long-term culture adherent layers which 125 might then later be applied to assess the essential ce l lular components within these cultures once such individual c e l l populations had been isolated. Ideally, such an assay had to be reproducible and easily set-up using commonly available sources of hemopoietic cel ls (e.g. regular marrow aspirates or peripheral blood). Thus, I wished to avoid or minimize the use of lengthy and expensive c e l l separation procedures i f possible. Since contamination by mesenchymal cel ls would markedly reduce the sensit ivity of the assay, selection of a suitable target population was the major concern. Results of cocultivation experiments init iated with T-depleted l ight density peripheral blood cel ls indicated that progenitors from that source could be maintained on pre-established adherent layers for at least 3 weeks. This finding was exploited in a collaborative study to analyze the prol i ferat ive status of progenitors maintained in the presence or absence of an adherent layer. These experiments revealed that primitive progenitors were down regulated only when close interactions with the adherent layer were maintained and that peripheral blood i t se l f did not contain cel ls able to establish an adherent layer (7). These results validated the feas ib i l i ty and usefulness of reconstitution experiments to study the regulation of hemopoiesis by non-hemopoietic stromal ce l l s . However, they also revealed that this strategy was impractical for routine longterm assays of hemopoietic supporting functions. I therefore next turned my attention to bone marrow as an alternative source of hemopoietic progenitor ce l l s . However, because bone marrow aspirates contain mesenchymal progenitor cel ls i t was necessary to develop a strategy to adequately eliminate or suppress these cel ls to reduce the background in control dishes to an acceptable level . Although many investigators have described potential methods for this (8,9,10,11), most 126 proved unsatisfactory. One which did work however was based on experimental evidence in the murine system which suggested that Cis-4-Hydroxy-L-Proline (CHP) could be used to s e l e c t i v e l y i n h i b i t stromal c e l l p r o l i f e r a t i o n . CHP i s a r e l a t i v e l y s p e c i f i c i n h i b i t o r of collagen synthesis. When I tested i t s e f f e c t s on the stromal and hemopoietic components of normal human bone marrow a s i g n i f i c a n t l y d i f f e r e n t i a l e f f e c t on these two c e l l types was revealed. These studies showed that CHP could be used to completely block marrow mesenchymal c e l l p r o l i f e r a t i o n at doses that had no e f f e c t on the stem c e l l regulatory functions of such c e l l s once they had already been established as a confluent layer in v i t r o . These findings provided further evidence that collagen-producing mesenchymal c e l l s of marrow o r i g i n play an important r o l e i n promoting hemopoiesis in long-term marrow cultures. Moreover they showed that supplementation of long-term marrow culture medium with CHP makes i t possible to use unseparated marrow c e l l suspensions to investigate and define mesenchymal c e l l types that may have stem c e l l regulatory properties. To further characterize the c e l l populations present in long-term marrow cultures and th e i r respective functions as part of the hemopoietic environment, a major e f f o r t was then directed at obtaining cloned l i n e s of i n d i v i d u a l marrow stromal c e l l s . Since human mesenchymal c e l l s senesce in v i t r o , some immortalization procedure was needed. Recently, several groups have reported that SV-40 virus can be used to transform various human c e l l s type without a l t e r a t i o n of the o r i g i n a l d i f f e r e n t i a t e d phenotype of the c e l l s . I therefore set out to generate a high t i t e r preparation of SV-40 and then use i t to i s o l a t e a large number of a continuous c e l l l i n e s of human marrow and u m b i l i c a l cord endothelial c e l l o r i g i n . Lines were derived from long-term culture adherent layers and marrow f i b r o b l a s t s . These a l l expressed the same phenotype as c e l l s present in the 127 non-infected population. A l l were p o s i t i v e for collagen I, collagen IV, and laminin, but negative for factor VIII. None expressed any of the d i f f e r e n t i a t i o n markers of hemopoietic c e l l s that were examined. Several immortalized endothelial l i n e s were also obtained. These too resembled the parent c e l l s phenotypically, with the one exception that weak a c t i v a t i o n of T200 expression was noted in one l i n e examined. However, a l l of these l i n e s did e x h i bit altered properties that are generally c h a r a c t e r i s t i c of transformed c e l l s . These include a reduced doubling time, a b i l i t y to grow to a higher c e l l density, and anchorage independence. This l a t t e r property was sometimes exploited to clone the l i n e s . A major conclusion from the f i r s t functional studies performed with these l i n e s was that they retained the a b i l i t y of normal parent mesenchymal c e l l s present i n long-term marrow cultures to show a marked production of hemopoietic colony stimulating factors upon induction with IL-1. A d d i t i o n a l information about the probable i d e n t i t i e s of the factors produced was obtained in a c o l l a b o r a t i v e study with Dr R.K. Humphries and Dr R. Kay who demonstrated a s u b s t a n t i a l increase in GM-CSF and IL-6 mRNA le v e l s following induction using Northern b l o t t i n g and S-l protection analysis techniques. The presence of IL-6 i t s e l f was further confirmed by s p e c i f i c bioassays using an IL-6 dependent c e l l l i n e . The enhanced production of r e a d i l y detectable l e v e l s of hemopoietic growth factors such as GM-CSF and IL-6, both of which can act on p r i m i t i v e hemopoietic c e l l s (12) i s a t a n t a l i z i n g finding. However, I have presented evidence that regulation of primitive hemopoietic c e l l s i s l i k e l y to operate at the l o c a l l e v e l rather than v i a the c i r c u l a t i o n of released f a c t o r s . Obviously, one p o s s i b i l i t y i s that the l o c a l i z a t i o n of responses apparent i n vivo i s simply due to the d i l u t i o n of released growth factors to i n e f f e c t u a l 128 l e v e l s at more distant regions. Recently, evidence was presented to indic a t e that GM-CSF can also bind to glycosaminoglycans of the marrow e x t r a c e l l u l a r matrix. This suggests an i n t r i g u i n g mechanism by which released growth factors might be l o c a l l y concentrated in tissues. Another p o s s i b i l i t y i s that enhanced production of secreted forms of growth factors might be accompanied by a b i o l o g i c a l l y more important enhanced production of membrane-bound growth factor molecules. Such a p o s s i b i l i t y has a precedent i n the case of M-CSF (13). However, these findings do not address the question of how negative regulation of primitive hemopoietic c e l l s i s achieved ei t h e r in v i t r o or i n vivo. One p o s s i b i l i t y i s that negative regulation occurs at the l e v e l of the regulatory c e l l s i . e . i n the absence of appropriate stimulation of the stromal c e l l s hemopoietic growth factor production declines to l e v e l s that are i n s u f f i c i e n t to activate nearby hemopoietic progenitors and these then enter a GQ state. A l t e r n a t i v e l y , i t i s possible that stromal c e l l s may also release s p e c i f i c i n h i b i t o r y substances i n a fashion that i s subject to regulation. Recent data from the Terry Fox Laboratory have indicated that TGF-61 can override the stimulation of hemopoietic progenitor c e l l s that occurs i n long- term marrow cultures following the addition of IL-1. Whether TGF-61 i s made by stromal c e l l s and i t s possible involvement in the regulation of hemopoiesis by marrow c e l l s w i l l be very i n t e r e s t i n g to e s t a b l i s h . In summary, my studies have i d e n t i f i e d collagen-producing c e l l s of the adherent layer as l i k e l y to be an important population for the maintenance and turnover of primitive hemopoietic c e l l s . In addition, phenotypic and func t i o n a l characterization of human marrow mesenchymal l i n e s have suggested that production of hemopoietic growth factors may well be involved i n the regulation of primitive hemopoietic c e l l p r o l i f e r a t i o n . However, other molecules may also be involved. For example, further work i s needed to 129 e s t a b l i s h whether s p e c i f i c c e l l surface determinants e x i s t that allow p r i m i t i v e c e l l s to "home" to s p e c i f i c tissues. S i m i l a r l y i t i s not known whether molecules that f a c i l i t a t e i n t e r - c e l l interactions and thereby contribute to cell-mediated growth factor responses e x i s t . The a v a i l a b i l i t y of several cloned human marrow mesenchymal l i n e s that e x h i b i t the phenotype and regulated a b i l i t y of cultured marrow c e l l s to produce hemopoietic growth factors and s y n e r g i s t i c a c t i v i t i e s should f a c i l i t a t e further studies of this type. Of p a r t i c u l a r importance w i l l be future tests of the a b i l i t y of these l i n e s alone and/or in combination to reconstitute the hemopoietic supportive function of intact long-term marrow culture adherent layers. 130 REFERENCES 1. Dexter TM, Simmons P, Purnell RA, Spooncer E, Schofield R. The regulation of hemopoietic c e l l development by the stromal c e l l environment and d i f f u s i b l e regulatory molecules. In: A p l a s t i c anemia: Stem c e l l biology and advances i n treatment. Alan R L i s s , New York, pp 131, 1984. 2. Trentin J J . Influence of hematopoietic organ stroma (hematopoietic inductive microenvironment) on stem c e l l d i f f e r e n t i a t i o n . In: "Regulation of hematopoiesis", Appleton-Century Crofts, educational d i v i s i o n , Meridith Corp, pp 61, 1970. 3. Dexter TM, Spooncer E, Toksoz D, Lajtha LG. The ro l e of c e l l s and t h e i r products i n the regulation of in v i t r o stem c e l l p r o l i f e r a t i o n and granulocyte development. Journal of Supramolecular Structure 13: 513-524, 1980. 4. Metcalf D. Hemopoietic colonies i n v i t r o : Cloning of normal and leukaemic c e l l s , Springer Verlag, B e r l i n , pp227, 1977. 5. Eaves AC, Cashman JD, Gaboury LA, Eaves CJ. C l i n i c a l s i g n i f i c a n c e of long-term cultures of myeloid blood c e l l s . CRC C r i t i c a l Reviews i n Oncology/Hematology 7: 125, 1987. 6. Dexter TM, A l l e n TD, Lajtha LG. Conditions c o n t r o l l i n g the p r o l i f e r a t i o n of haemopoietic stem c e l l s i n v i t r o . J C e l l Physiol 91: 335, 1977. 7. Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves CJ. Unregulated p r o l i f e r a t i o n of primitive chronic myeloid leukemia progenitors i n the presence of normal marrow adherent c e l l s . Proc Natl Acad Sci USA 83: 5306, 1986. 8. G i l b e r t SF, Migeon BR. D-valine as a s e l e c t i v e agent for normal human and rodent e p i t h e l i a l c e l l s , in culture. C e l l 5: 11, 1975. 9. E b e l l W, Castro-Malaspina H, Moore MAS, O'Reilly RJ. Depletion of stromal c e l l elements i n human marrow graft s separated by soybean ag g l u t i n i n . Blood 65: 1105, 1985. 10. Paraskeva C, Buckle B.G, Thorpe PE. Selective k i l l i n g of contaminating human f i b r o b l a s t s in e p i t h e l i a l cultures derived from c o l o r e c t a l tumours using an anti-Thy-1 antibody-ricin cojugate. Br J Cancer 51: 131, 1985. 11. Abboud CM, Duerst RE, Frantz CN, Ryan DH, Liesveld JL, Brennan JK. Lysis of human f i b r o b l a s t colony-forming c e l l s and endothelial c e l l s by monoclonal antibody (6-19) and complement. Blood 68: 1169, 1986. 131 12. Ikebuchi K, Wong GG, Clark SC, Ihle JN, H i r a i Y, Ogawa M. Interleukin-6 enhancement of interleukin-3 dependent p r o l i f e r a t i o n of mu l t i p o t e n t i a l hemopoietic progenitors. Blood 70 (Suppll):173a, 1987. 13. Rettenmier CW, Roussel MF, Ashmun RA, Ralph P, Price K, Sherr CJ. Synthesis of membrane-bound colony-stimulating Factor-1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 c e l l s transformed by cotransfection of the human CSF-1 and c-FMS (CSF-1 receptor) genes. Molecular and C e l l u l a r Biology 7: 2378, 1987.

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