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Studies of the role of mesenchymal cells in the regulation of hemopoiesis Gaboury, Louis A. 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 o f M o n t r e a l , 1978 D.E.S., U n i v e r s i t y o f M o n t r e a l , 1983  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Department o f P a t h o l o g y )  We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA February 1988 © L o u i s A. Gaboury, 1988  ABSTRACT  Hemopoiesis  i s thought  to be r e g u l a t e d i n part by s p e c i f i c , but as y e t  u n d e f i n e d , i n t e r a c t i o n s between p r i m i t i v e hemopoietic c e l l s and f i x e d , nonhemopoietic marrow elements  c o l l e c t i v e l y referred  to as the stroma.  Recently,  a marrow c u l t u r e system has been d e s c r i b e d that a l l o w s the maintenance o f p r i m i t i v e human hemopoietic p r o g e n i t o r c e l l s exogenously  f o r many weeks i n the absence o f  added hemopoietic growth f a c t o r s .  heterogeneous  adherent  to be important  The f o r m a t i o n o f a  l a y e r i n which many s t r o m a l elements  to the maintenance of hemopoiesis  the o v e r a l l g o a l o f d e l i n e a t i n g i n v o l v e d , my f i r s t  a r e found  i n t h i s system.  the c e l l u l a r and m o l e c u l a r  o b j e c t i v e was to develop an e x p e r i m e n t a l system f o r  term human marrow c u l t u r e s .  This required  the former c o u l d be used  T h i s was a c h i e v e d u s i n g i r r a d i a t i o n  r e s i d u a l hemopoietic c e l l s  layer of long-  the i d e n t i f i c a t i o n o f a s u i t a b l e  procedure f o r s e p a r a t i n g the hemopoietic and non-hemopoietic  latter.  As part o f  interactions  a s s e s s i n g the h e m o p o i e s i s - s u s t a i n i n g f u n c t i o n o f the adherent  components so that  appears  regulatory  to q u a n t i t a t e the f u n c t i o n o f the to s e l e c t i v e l y  i n long-term c u l t u r e adherent  medium c o n t a i n i n g c i s - 4 - h y d r o x y - L - p r o l i n e to s e l e c t i v e l y  inactivate  l a y e r s , and u s i n g a i n a c t i v a t e stromal  c e l l s and t h e i r p r e c u r s o r s present i n suspensions of unseparated human marrow which were then added back i n c o - c u l t u r e  experiments.  My second o b j e c t i v e was to develop a s t r a t e g y f o r o b t a i n i n g  purified  p o p u l a t i o n s o f c e l l s c o r r e s p o n d i n g to the v a r i o u s mesenchymal c e l l long-term adherent s t o c k and used  layers.  I t h e r e f o r e prepared a high t i t r e SV-40 v i r u s  i t to e s t a b l i s h permanent, cloned l i n e s  " f i b r o b l a s t " c o l o n i e s , long-term c u l t u r e adherent endothelial cells.  types i n  from human marrow  l a y e r s , and u m b i l i c a l c o r d  C h a r a c t e r i z a t i o n of the transformants generated showed  iii  that they were a l l p o s i t i v e f o r SV-40, and i n g e n e r a l expressed the p h e n o t y p i c c h a r a c t e r i s t i c s o f the c e l l s o r i g i n a l l y that  these t r a n s f o r m a n t s , l i k e  infected.  F u n c t i o n a l s t u d i e s showed  t h e i r normal c o u n t e r p a r t s , respond to  a c t i v a t i o n by p r o d u c i n g two types o f hemopoietic growth These s t u d i e s suggest  factors.  that marrow mesenchymal c e l l s may r e g u l a t e the  growth and maintenance o f p r i m i t i v e hemopoietic c e l l s by producing growth f a c t o r s i n response  to a p p r o p r i a t e p e r t u r b a t i o n .  hemopoietic  The a v a i l a b i l i t y o f  permanent c l o n e d l i n e s o f human marrow s t r o m a l c e l l s should f a c i l i t a t e a n a l y s i s o f these events a t the m o l e c u l a r  level.  future  iv TABLE OF CONTENTS  Page ABSTRACT  i i  LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENTS  Chapter I  v i i ix  INTRODUCTION 1)  O r g a n i z a t i o n o f the Hemopoietic System A) Hemopoietic C e l l s B) Stromal C e l l s o f the Bone Marrow C) S t r o m a l C e l l P r o d u c t s 2) R e g u l a t i o n o f the Hemopoietic System A) C e l l u l a r I n t e r a c t i o n s B) Humoral R e g u l a t i o n o f the Hemopoietic System 3) Long-Term Bone Marrow C u l t u r e s : An I n V i t r o Model f o r Hemopoietic Stem C e l l R e g u l a t i o n A) E a r l y Development o f the Long-Term Marrow C u l t u r e s B) Long-Term Human Marrow C u l t u r e s C) Long-Term Marrow C u l t u r e s as a Model of I n V i v o R e g u l a t i o n 4) T h e s i s O b j e c t i v e s References Chapter I I  vi  1 1 7 12 18 18 22 30 30 31 36 38 41  MATERIALS AND METHODS 1)  2)  3)  Cells  A) B) C) D)  Bone Marrow C e l l s P e r i p h e r a l Blood C e l l L i n e s Maintenance I n f e c t i o n o f Primary Human Mesenchymal C e l l s w i t h SV-40 and I s o l a t i o n o f Permanent C e l l L i n e s E) C e l l P r o l i f e r a t i o n Measurements Long-Term C u l t u r e s A) R e g u l a r Long-Term Marrow C u l t u r e s B) Long-Term P e r i p h e r a l Blood C u l t u r e s C) P r e p a r a t i o n o f Long-Term Marrow C u l t u r e Feeders D) C i s - H y d r o x y - L - P r o l i n e Experiments Assays A) M e t h y l c e l l u l o s e Assay f o r Hemopoietic Colony-Forming P r o g e n i t o r s B) Colony-Forming U n i t - F i b r o b l a s t (CFU-F) Assay C) Assays f o r the P r o d u c t i o n o f Hemopoietic Growth (HGF)  55 55 55 57 57 59 60 60 61 61 62 62 63 63  V  4)  SV-40 V i r u s P r e p a r a t i o n and Assay A) P r e p a r a t i o n o f High T i t e r V i r u s Stock B) V i r u s Plaque Assay C) Assay f o r Large T A n t i g e n D) T r a n s f o r m a t i o n Assay o f SV-40 V i r u s on NIH-3T3 C e l l s 5) A n t i s e r a and Immunofluorescence Measurements 6) H i s t o c h e m i c a l Analyses 7) T e s t f o r Anchorage-Independent Growth 8) Tumor Formation 9) I r r a d i a t i o n Procedures 10) Autoradiography References  Chapter I I I  1)  Introduction  2)  Results A)  C)  C u l t u r e s o f P e r i p h e r a l Blood C e l l s on Normal Marrow Adherent Layer E f f e c t o f CHP on Marrow Mesenchymal Cell Proliferation Lack o f a D i r e c t E f f e c t o f CHP on Hemopoietic P r o g e n i t o r s F u n c t i o n  3) D i s c u s s i o n References  75  77 81 85 90 93  INDUCIBLE PRODUCTION OF HEMOPOIETIC GROWTH FACTORS BY SV-40 IMMORTALIZED MESENCHYMAL CELL LINES OF HUMAN BONE MARROW ORIGIN 1) 2)  Chapter V  67 67 70 70 70 71 71 73  DIFFERENTIAL EFFECTS OF CIS-0H-L-PR0LINE ON THE PROLIFERATIVE AND STEM CELL REGULATORY FUNCTIONS OF HUMAN BONE MARROW MESENCHYMAL CELLS  B)  Chapter IV  65 65 65 67  Introduction Results A) Transforming P o t e n t i a l of SV-40 V i r u s and i t s E f f e c t on DNA S y n t h e s i s o f SV-40 V i r u s B) D e r i v a t i o n and Immunological C h a r a c t e r i z a t i o n of C e l l L i n e s 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 D) I n d u c t i o n of Growth F a c t o r P r o d u c t i o n E) I r r a d i a t i o n Studies 3) D i s c u s s i o n References  95  97 102 105 115 115 120  SUMMARY AND FUTURE DIRECTIONS References  123 130  97  LIST OF TABLES  Recovery o f Nucleated C e l l s and P r o g e n i t o r s from One Unit (500ml) o f Human P e r i p h e r a l Blood C u l t u r e C o n d i t i o n s f o r the Maintenance o f C e l l L i n e s O r i g i n , S p e c i f i c i t y and Source of A n t i b o d i e s Used f o r Immunophenotypic Analyses Immunophenotypic C h a r a c t e r i z a t i o n o f Lymphoblastoid Cells Effect  o f CHP on Adherent Layer Formation  Lack o f E f f e c t o f Exposure o f Hemopoietic P r o g e n i t o r s to CHP on t h e i r Subsequent P l a t i n g E f f i c i e n c y Lack o f E f f e c t of 500 ug/ml of CHP on Hemopoiesis i n Long-Term C u l t u r e s I n i t i a t e d on P r e - E s t a b l i s h e d I r r a d i a t e d Adherent Layers ( F e e d e r s ) H i s t o c h e m i c a l and Immunophenotypic P r o p e r t i e s o f SV-40 Transformed Human C e l l L i n e s Evidence f o r IL-lf3 Induced P r o d u c t i o n of Hemopoietic C o l o n y - S t i m u l a t i n g A c t i v i t y by R e p r e s e n t a t i v e SV-40 Transformed Human C e l l L i n e s E f f e c t s o f SV-40 Immortalized C o n d i t i o n e d Medium on CFU-F Formation  vii  LIST  OP F I G U R E S  Page FIGURE 1  FIGURE 2  FIGURE 3  FIGURE 4  Major S i t e s o f Hemopoiesis i n the Human Embryo and Fetus  2  Schematic R e p r e s e n t a t i o n o f the Hemopoietic System as Defined by Clonogenic Assays f o r P l u r i p o t e n t and Committed P r o g e n i t o r s  6  Schematic R e p r e s e n t a t i o n  of Bone Marrow H i s t o l o g y  A)  C e l l u l a r Arrangement Around a Marrow Sinus  B)  L o n g i t u d i n a l S e c t i o n of a Marrow Sinus  9 10  Diagrammatic R e p r e s e n t a t i o n of the V a r i o u s 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 M o l e c u l a r Events I n v o l v e d i n the Formation o f a C o l l a g e n Molecule  14  FIGURE 5  F u n c t i o n a l Domains of F i b r o n e c t i n  16  FIGURE 6  Photomicrographs o f a F o r m a l i n F i x e d Long-Term Marrow C u l t u r e Adherent Layer S t a i n e d With R a b b i t - A n t i F a c t o r V I I I Antiserum and Developed With P e r o x i d a s e - L a b e l l e d Swine A n t i - R a b b i t Immunoglobulin Antibody  FIGURE 7  A)  Low Power View  33  B)  High Power View  34  The C e l l u l a r i t y and P r o g e n i t o r Content of the Adherent (Adh) and Nonadherent (NA) F r a c t i o n s Assessed at V a r y i n g I n c u b a t i o n Times i n Human Long-Term Bone Marrow C u l t u r e s  35  FIGURE 8  T i t r a t i o n o f SV-40 V i r u s Stock on BSC-1 C e l l s  66  FIGURE 9  Comparison of T o t a l C e l l and P r o g e n i t o r Content of LongTerm Normal P e r i p h e r a l Blood C u l t u r e s I n i t i a t e d With or Without a P r e - E s t a b l i s h e d Normal Marrow Feeder  78  E f f e c t o f I n c r e a s i n g Doses of CHP on the Number o f C o l o n i e s Obtained from CFU-F i n Fresh Human Marrow  82  Photograph of Two Long-Term Marrow C u 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 Maintained i n P r o l i n e - , and L y s i n e - F r e e Medium C o n t a i n i n g 500 ug/ml o f CHP ( R i g h t )  84  FIGURE 10  FIGURE 11  viii  E f f e c t s o f 500 ug/ml of CHP on the Y i e l d of Hemopoietic P r o g e n i t o r s i n Long-Term Marrow C u l t u r e s Assessed 4-5 Weeks A f t e r I n i t i a t i o n  88  FIGURE 13  Transformed Focus of NIH-3T3 C e l l s  98  FIGURE 14  Autoradiograms o f C o n f l u e n t Marrow Adherent Layers Mock I n f e c t e d Layer (Upper Panel) and SV-40 I n f e c t e d Layer (Lower Panel)  99  FIGURE 12  FIGURE 15  FIGURE 16  FIGURE 17  FIGURE 18  FIGURE 19  FIGURE 20  FIGURE 21  FACS P r o f i l e o f CFUST-CL16 C e l l s S t a i n e d With Monoclonal Antibody 6.19 (Panel A) and Anti-Leuk AH/T200 ( P a n e l B)  103  FACS P r o f i l e of a Suspension o f Spontaneously Immortalized Lymphoblastoid C e l l s (Panel A ) , and HUVE-EC-C C e l l s Before (Panel B), and A f t e r ( P a n e l C) T r a n s f o r m a t i o n with SV-40 V i r u s  104  T r i t i a t e d - T h y m i d i n e Uptake (Panel A) o f SV-40 I n f e c t e d ( S o l i d L i n e s ) and U n i n f e c t e d (Broken L i n e s ) MH C e l l s  106  Growth Rate (Panel B) of SV-40 I n f e c t e d ( S o l i d (Broken L i n e s ) MH C e l l s  107  Lines)  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 C u l t u r e 14 Days A f t e r Seeding the C u l t u r e s 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 ) .  108  C o n t r o l ( U n i n f e c t e d ) MH C e l l s F a i l e d To Y i e l d (Panel B).  109  A n a l y s i s of the Clonogenic Plated i n Methylcellulose  Colonies  C a p a c i t y of MH2SV-CL1 C e l l s  Hemopoietic C o l o n y - S t i m u l a t i n g A c t i v i t y o f Media C o n d i t i o n e d for.24 Hours by CFUST-CL16 or MH2SV-CL1 C e l l s ( o r No C e l l s ) as a F u n c t i o n of the C o n c e n t r a t i o n of I L - 1 3 Used as a Stimulant C e l l S u r v i v a l Curves f o r CFUST-CL16, EC CL22, Ea.926 C e l l s and Normal Bone Marrow F i b r o b l a s t s  110  113  116  ix  AC3W0VLEDGEMENTS  I wish to express my s i n c e r e g r a t i t u d e :  this  To my s u p e r v i s o r Dr. Connie Eaves f o r her h e l p and guidance throughout project,  To Dr. A l l e n Eaves, Dr. Johanne Cashman, Dr. R. K e i t h Humphries, Dr. P e t e r Lansdorp, Dr. Robert Kay, and Dr. A l i Turhan f o r h e l p f u l d i s c u s s i o n s and a c t i v e c o l l a b o r a t i o n , To Dr. Heather S u t h e r l a n d f o r her r e v i e w i n g o f the t h e s i s To Mrs. M a r j o r i e H u t c h i s o n , Mrs. Giovanna Cameron, Mrs. Karen Lambie, Ms. Judy P f e i f e r , and Ms. Dianne Reid f o r expert t e c h n i c a l a s s i s t a n c e , To Paule and A r i a n e f o r t h e i r  patience,  To Ms. M i c h e l e Coulombe f o r s e c r e t a r i a l  assistance,  To the N a t i o n a l Cancer I n s t i t u t e o f Canada f o r f i n a n c i a l s u p p o r t .  1 CHAPTER  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 c e l l s that have undergone a series of migrations from their o r i g i n a l location i n the yolk sac to their f i n a l destination i n the functional hemopoietic organs of the adult, i . e . the bone marrow, spleen, thymus and lymph nodes (1) A l l mature myeloid  (see Figure  1).  c e l l s (red blood c e l l s , granulocytes, monocytes and  p l a t e l e t s ) represent non-dividing "end" c e l l s that survive for r e l a t i v e l y short periods of time and are, therefore, being continuously replaced throughout adult l i f e ( 2 ) .  The production of new blood c e l l s from more  primitive p r o l i f e r a t i n g precursors normally occurs i n the bone marrow of the adult  (3). The structure of the hemopoietic system i s 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 , c e l l s that have the potential both to self-renew and to d i f f e r e n t i a t e into each of several lineages. intermediate,  Stem c e l l s give r i s e to an  transient compartment of progenitors of various types that have  undergone d i f f e r e n t degrees of lineage r e s t r i c t i o n but s t i l l have a considerable p r o l i f e r a t i v e potential.  Neither pluripotent stem c e l l s 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 morphologically, e i t h e r as a c l a s s , or from one another.  The p r o g e n i t o r s , i n t u r n g i v e r i s e t o  a compartment of m o r p h o l o g i c a l l y r e c o g n i z a b l e p r e c u r s o r s  that undergo a  l i m i t e d number o f d i v i s i o n s , 3-5, i n c o n c e r t w i t h the c o m p l e t i o n maturation.  The f u l l y  l a s t compartment.  d i f f e r e n t i a t e d , non-dividing  of t e r m i n a l  blood c e l l s r e p r e s e n t the  G e n e r a l l y assumed i n t h i s model, i s t h a t 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 a r e 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 t h a t t h e r e i s a p r o g r e s s i v e l o s s o f 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 potential  (4,5).  Assays f o r Hemopoietic Stem C e l l s . for  differentiation  The f i r s t q u a n t i t a t i v e c o l o n y  assay  p l u r i p o t e n t stem c e l l s was d e s c r i b e d 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 h i s t o c o m p a t i b l e mice w i t h an a p p r o p r i a t e number o f h e m o p o i e t i c  c e l l s and c o u n t i n g , 1-2 weeks l a t e r , t h e number o f  m a c r o s c o p i c nodules that have appeared on the s u r f a c e o f the s p l e e n . C y t o l o g i c a l and chromosomal marker s t u d i e s o f the c e l l s w i t h i n such n o d u l e s r e v e a l e d each t o be a b o n a f i d e c l o n e d e r i v e d 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 o f c e l l s from primary  spleen colonies i n t o  lethally  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 f o r m a t i o n o f 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 formal demonstration  (9).  of the e x i s t e n c e i n a d u l t hemopoietic  T h i s was the f i r s t t i s s u e of a c e l l  t h a t can s e l f - r e n e w , and t h a t 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 t h a t the f i r s t a p p e a r i n g  s p l e e n 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 ) a r e d e r i v e d from c e l l s that a r e 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 c o n t r a s t to the spleen  colonies 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 e v i d e n c e to  suggest some o v e r l a p between the p r o g e n i t o r s of the l a t e a p p e a r i n g s p l e e n c o l o n i e s and c e l l s c a p a b l e of l o n g - t e r m r e p o p u l a t i o n i n mice ( 1 1 ) .  In  a d d i t i o n , r e c e n t experiments w i t h r e t r o v i r a l l y marked mouse marrow c e l l s have c o n f i r m e d that s i n g l e c e l l s can r e p o p u l a t e the e n t i r e lymphoid and m y e l o i d system of both p r i m a r y and secondary r e c i p i e n t s (12,13). O b v i o u s l y , a comparable c e l l cannot be performed  i n v i v o assay f o r a p l u r i p o t e n t h e m o p o i e t i c stem  i n humans.  However, s e v e r a l l i n e s of e v i d e n c e  i n d i c a t e the e x i s t e n c e of h e m o p o i e t i c stem c e l l p o p u l a t i o n s analogous i d e n t i f i e d i n the murine system.  to those  F i r s t , i n c h r o n i c myelogenous l e u k e m i a  (CML), d e t e c t i o n o f 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 m y e l o i d 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 n o t i o n of a p l u r i p o t e n t h e m o p o i e t i c stem c e l l i n man (14,15).  that i s the t a r g e t of n e o p l a s t i c t r a n s f o r m a t i o n i n CML  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  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) of  a l s o had one of v a r i o u s  have a l s o t r a c e d back the o r i g i n  the expanded n e o p l a s t i c c l o n e i n each case to a s i n g l e t r a n s f o r m e d  p l u r i p o t e n t h e m o p o i e t i c stem c e l l  (16,17,18).  More r e c e n t l y , probes f o r  p o l y m o r p h i c r e g i o n s i n the X-chromosome, that a r e d i f f e r e n t i a l l y m e t h y l a t e d 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 c o n f i r m the o r i g i n o f human m y e l o p r o l i f e r a t i v e d i s e a s e c l o n e s i n p l u r i p o t e n t c e l l s (19).  hemopoietic  T h i s l a t t e r approach has now a l s o been used to a s s e s s the  c l o n a l i t y o f p o p u l a t i o n s 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 transplants. of  I n at l e a s t one case to d a t e , long-term monoclonal  donor o r i g i n has been documented ( 2 0 ) .  hemopoiesis  Thus, i t seems v e r y l i k e l y  t h a t the  5  c o n c e p t u a l 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  s t u d i e s w i l l apply to the human sytem.  Assays f o r Hemopoietic P r o g e n i t o r s . present at very low f r e q u e n c i e s (10~3  Hemopoietic  to 10"^)  i n normal hemopoietic  and have no d i s t i n c t i v e m o r p h o l o g i c a l f e a t u r e s . identified vitro.  i n d i r e c t l y by t h e i r a b i l i t y  Hemopoietic  progenitor c e l l s  are  tissue  However, such c e l l s can  be  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  colony assays i n v o l v e suspending  the c e l l s  to be t e s t e d i n  a s e m i - s o l i d c u l t u r e medium c o n t a i n i n g a p p r o p r i a t e n u t r i e n t s , serum ( o r the e s s e n t i a l components c o n t a i n e d i n serum) and a source of hemopoietic growth f a c t o r s , e i t h e r i n crude p r e p a r a t i o n s or as h i g h l y p u r i f i e d recombinant)  molecules.  ( n a t u r a l or  Depending on the nature and c o n c e n t r a t i o n s of the  growth r e g u l a t o r y molecules present i n the c u l t u r e s , s i n g l e , double, or m u l t i l i n e a g e c o l o n i e s of daughter are of  c e l l s can be o b t a i n e d (21,22,23).  d e f i n e d by the types of c o l o n i e s they produce, the c o l o n y , and  parameters  appear  both i n terms o f the s i z e  i t s time of maturation and u l t i m a t e c o m p o s i t i o n .  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 P r o g e n i t o r s c a t e g o r i z e d i n t h i s way e x p r e s s i o n (26), and may (27) and  These  to be l i n k e d and i n v a r i a n t under most c i r c u m s t a n c e s and  t h e r e f o r e p r o v i d e r e p r o d u c i b l e i n d i c a t o r s of the p r o l i f e r a t i v e  agents  Progenitors  and  types of p r o g e n i t o r s (24,25).  show d i f f e r e n c e s i n s u r f a c e a n t i g e n  d i f f e r i n t h e i r responses  to c e l l c y c l e  to v a r i o u s growth r e g u l a t o r y s t i m u l a t o r s ( 2 8 ) .  have helped to v a l i d a t e the assignment  specific Such f i n d i n g s  of the v a r i o u s types of p r o g e n i t o r s 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 i n the hemopoietic h i e r a r c h y (see  Figure 2).  Cells  that form c o l o n i e s c o n s i s t i n g e x c l u s i v e l y of  g r a n u l o c y t e s , monocytes, or both, are named r e s p e c t i v e l y : c o l o n y - f o r m i n g u n i t g r a n u l o c y t e (CFU-G), c o l o n y - f o r m i n g unit-monocyte  (CFU-M), and c o l o n y - f o r m i n g  ULTIMATE  STEM  "  CELL  » Lymphopoiesis  self -renewal  MYELOID STEM CELL (CfU-S,  ERYTHROID  PROGENITORS  CFUGEMM)  MEGAKARYOCYTE  mixed colony  PROGENITORS  i Primitive BfU-E  ©  CFU M large erythroid colony or burst  GRANULOCYTE  PROGENITORS .;  CFU C arge  large granulocytic colony  megakaryocytic colony  ft. Mature BFU-E  I  small erythroid colony Or burst  small  small  megakaryocytic colony  granulocytic colony  ©CFU-E  erythroid cluster  REO C E L L S  PLATELETS  FIGURE 2.  GRANULOCYTES  4 MACROPHAGES  Schematic Representation of the Hemopoietic System as Defined by Clonogenic Assay for Pluripotent and Committed Progenitors.  cn  7  unit-granulocyte/monocyte have been s u b d i v i d e d erythroid  (CFU-GM).  progenitors  i n t o c a t e g o r i e s r e f e r r e d to as c o l o n y - f o r m i n g  (CFU-E), and  unit-  b u r s t - f o r m i n g u n i t - e r y t h r o i d (BFU-E), a c c o r d i n g  number of hemoglobinized further subdivided  Erythroid lineage-restricted  c e l l c l u s t e r s they generate,  i n t o p r i m i t i v e and  the l a t t e r o f t e n  mature BFU-E s u b c l a s s e s  (21).  to  the  being  The  same  p r i n c i p l e has  l e d to the naming of p r o g e n i t o r s of c o l o n i e s of megakaryocytes  (CFU-Mk), and  of mixed c o l o n i e s c o n t a i n i n g g r a n u l o c y t e s , e r y t h r o i d c e l l s ,  megakaryocytes and Although type,  monocytes (CFU-GEMM).  i t i s clear  those d e t e c t e d  i n normal marrow e x h i b i t o n l y a l i m i t e d c a p a c i t y f o r  self-renewal i n v i t r o . delayed  that CFU-GEMM represent a p r i m i t i v e p l u r i p o t e n t c e l l  However, very r e c e n t l y a type of CFU-GEMM that shows  i n i t i a t i o n of p r o l i f e r a t i o n  small "blast described.  i n v i t r o l e a d i n g to the p r o d u c t i o n  c o l o n i e s " when most other c o l o n i e s " have a l r e a d y matured, has These b l a s t  c o l o n i e s , but  c o l o n i e s e v e n t u a l l y w i l l go on  by r e p l a t i n g can be shown to c o n s i s t of CFU-GEMM, (and CFU-S i n p r o g e n i t o r types  now  the r e l a t i o n s h i p between  ongoing i n many l a b o r a t o r i e s to a n a l y z e  p r o g e n i t o r s of these b l a s t c o l o n i e s ( r e f e r r e d capable  of long-term  myeloid  although c e l l s and  Work i s the  to as S - c e l l s or C F U - b l a s t )  have been developed  and  that a l l o w most c l a s s e s  p r o g e n i t o r s i n both murine and human marrow to be  there i s s t i l l  (29).  repopulation in vivo.  In summary, c l o n o g e n i c assays of  been  to form pure or mixed  the mouse) as w e l l as other more r e s t r i c t e d  cells  of  c o n t r o v e r s y about the exact  p r o g e n i t o r s of m u l t i - l i n e a g e and  detected,  r e l a t i o n s h i p between stem  "blast" colonies.  8 (B)  Stromal C e l l s of the Bone Marrow  In a d d i t i o n to p r e c u r s o r s  of c e l l s  that are d e s t i n e d  to c i r c u l a t e i n  b l o o d , the bone marrow a l s o 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 the marrow stroma ( F i g u r e 3A and types have been i d e n t i f i e d  3B).  fibroblasts/adventitial reticular cells, osteoblasts,  and  Macrophages.  osteoclasts  termed  U l t r a s t r u c t u r a l l y , s e v e r a l stromal  including endothelial  fat-accumulating  c e l l s or  adipocytes,  (30).  Because of  their putative  However, marrow macrophages are d e r i v e d  hemopoietic s u p p o r t i v e part of the  functions,  stroma.  from hemopoietic stem c e l l s and  from the mesenchyma l i k e a l l other marrow s t r o m a l c e l l s  (31,32).  not  Marrow  macrophages are engaged i n a c t i v e p h a g o c y t o s i s where they p l a y a r o l e i n d i s p o s a l of p a r t i c u l a t e matter and by c o n v e n t i o n a l  c e l l debris.  in  c y t o c h e m i c a l methods are present i n t h e i r c y t o p l a s m Macrophages are  with t e r m i n a l l y d i f f e r e n t i a t i n g e r y t h r o i d c e l l s  t h i s s i t u a t i o n are  believed  to play an  important  i r o n to the hemoglobin-producing e r y t h r o b l a s t s .  the  I t has  time when the e r y t h r o b l a s t  develop i n t o a r e t i c u l o c y t e .  Consistent  (34).  LeuM3, but  i n the marrow  and  a l s o been suggested be  involved  in  extrudes i t s n u c l e u s  to  with t h e i r hemopoietic o r i g i n ,  macrophages a l s o express the hemopoietic markers T200 and monocytic markers LeuMl and  also  r o l e i n the d e l i v e r y of  that marrow macrophages i n these e r y t h r o b l a s t i c i s l e t s may n u c l e o p h a g o c y t o s i s at  the  Numerous h y d r o l y t i c enzymes  i n a s s o c i a t i o n w i t h membrane-bound lysosomes (33). associated  cell  cells,  f i x e d marrow macrophages have long been c o n s i d e r e d  detectable  the  are not  involved  in  the  specific  collagen  synthesis  9  ADVENTITIAL RETICULAR CELL  HEMOPOIETIC PROGENITOR  FIGURE 3.  Schematic R e p r e s e n t a t i o n A) C e l l u l a r i s l i n e d by adventitial Hemopoietic  o f Bone Marrow H i s t o l o g y .  Arrangement Around a Marrow Sinus. The w a l l e n d o t h e l i a l c e l l s which a r e separated from c e l l s by a d i s c o n t i n u o u s basement membrane. c e l l s a r e present i n the e x t r a - v a s c u l a r spaces.  10  B) L o n g i t u d i n a l S e c t i o n o f a Marrow Sinus. An a d v e n t i t i a l c e l l which has accumulated f a t i s shown on the r i g h t .  11 Endothelial Cells.  These c e l l s p r o v i d e the i n t e r n a l l i n i n g o f the marrow  s i n u s o i d s and a r e a c t i v e l y i n v o l v e d i n r e g u l a t i n g the m i g r a t i o n o f newly formed b l o o d c e l l s out o f the bone marrow. l o c a t e d elsewhere  I n c o n t r a s t to e n d o t h e l i a l c e l l s  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 a r e  a t t a c h e d by l o o s e o v e r l a p p i n g j u n c t i o n s ( 3 5 ) . They a r e supported  by a t h i n  basement membrane made up o f a number of e x t r a c e l l u l a r m a t r i x 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 c o l l a g e n type IV.  In addition,  a c t i v e l y s y n t h e s i z e a number o f m o l e c u l e s , one o f which,  they  the F a c t o r V I I I -  r e l a t e d a n t i g e n , s e r v e s 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 o t h e r mesenchymal c e l l  types ( 3 6 ) .  Fibroblasts/Adventitial  Reticular Cells  (ARC).  Adventitial reticular  c e l l s form a d i s c o n t i n u o u s c e l l l a y e r on the a b l u m i n a l s u r f a c e o f marrow sinusoids.  They have an abundant cytoplasm and numerous c e l l p r o c e s s e s and  a r e i n v o l v e d i n the p r o d u c t i o n o f a s u p p o r t i v e meshwork o f 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 m i x t u r e of c o l l a g e n f i b e r s and p r o t e o g l y c a n s ) ( 3 7 ) . The exact r e l a t i o n s h i p o f 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 r e p r e s e n t ARC a c t i v e l y i n v o l v e d i n the s e c r e t i o n o f c o l l a g e n s ( m o s t l y types I and I I I ) .  Fat-Storing Cells/Adipocytes.  The p r e c i s e o r i g i n o f 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 a r e c u r r e n t l y b e l i e v e d to be c l o s e l y to ARC ( 3 8 ) . They a r e not thought to e x p r e s s any monocytic  related  to be l i p i d - l a d e n macrophages as they  fail  markers ( 3 9 ) . They a l s o d i f f e r from a d i p o c y t e s  c h a r a c t e r i s t i c o f o t h e r t i s s u e s s i n c e they accumulate l i p i d s i n the presence of h y d r o c o r t i s o n e 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 c e l l .  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. molecules are known to be present.  Various  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  marrow stroma.  i s the s i n g l e most abundant p r o t e i n s p e c i e s  It i s secreted  by a number of mesenchymal c e l l s  f i b r o b l a s t s , e n d o t h e l i a l c e l l s ) and  3 important f e a t u r e s :  c o l l a g e n molecule has chains  l a i d down e x t r a c e l l u l a r l y 1)  i s a g l y c i n e . 3)  i s c h a r a c t e r i s t i c a l l y high. types a c c o r d i n g collagen  The  i n d i c a t i o n s that  collagens  ( F i g u r e 4).  residue  i t s hydroxylated  further c l a s s i f i e d  to the nature of t h e i r a c h a i n s .  type I predominates but  There are  are  (ARC/marrow  Every t h i r d  content of p r o l i n e and  Collagens  type I I I and  IV are a l s o  the primary s t r u c t u r e of the a chains  no such  This i s  the important d i f f e r e n c e s which e x i s t between c o l l a g e n  Whereas c o l l a g e n I s e l f - a s s e m b l e s  I  into fibrous proteins, collagen  IV  propensity.  Collagen  b i o s y n t h e t i c pathways are now  c o n s i s t s of  the formation  reticulum,  a chains  triple helix.  The  are next h y d r o x y l a t e d  procollagen  molecule i s then exported  bundles of c o l l a g e n f i b e r s . of a c o l l a g e n  to convert  lysine  residues,  to the i n t e r s t i t i u m  carboxy e x t e n s i o n s  by  specific  I I I , catalyzed  by  s i n g l e c o l l a g e n molecules i n t o  R e t i c u l i n f i b e r s , p u t a t i v e ARC  type I I I core coated with p r o t e o g l y c a n s .  c h a r a c t e r i s t i c a l l y present  i n the i n t e r s t i t i a l  step  converted i n t o a s t a b l e  L a s t l y , c r o s s - l i n k i n g of c o l l a g e n I and  enzyme l y s y l oxydase helps  first  i n the rough endoplasmic  at p r o l i n e and  s i t e s , and  trimmed of i t s n o n - h e l i c a l amino and  peptidases.  w e l l c h a r a c t e r i z e d . The  of i n d i v i d u a l a chains  g l y c o s y l a t e d at s e l e c t e d h y d r o x y l y s i n e  and  form  determines  by  has  in  present.  best  IV.  a  In the marrow,  chemical p r o p e r t i e s of the mature c o l l a g e n molecule.  and  The  into different  the p h y s i c a l and exemplified  the  I t c o n s i s t s of 3 i n t e r t w i n e d  forming a s t a b l e t r i p l e h e l i c a l s t r u c t u r e . 2)  the a c h a i n  in  t i s s u e of  The  the  tight  products,  consist  f i b e r s are  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 r e d u c i b l e  silver  salt.  been  Recently,  using  the matrix induced marrow model, evidence has  14  SYNTHESIS OF PRO-ALPHA CHAIN  HYDROXYLATION AT SELECTED PROLINES AND LYSINES  GLYCOSYLATION OF SELECTED HYDROXYLYSINES 3 PRO-ALPHA CHAINS —  TRIPLE HELIX FORMATION  OH* OH  SECRETION „ OH n  HO  #  | OH I OH  CLEAVAGE OF EXTENSION PEPTIDES  OH  ASSEMBLY INTO MICROFIBRIL  ASSEMBLY INTO MATURE COLLAGEN FIBRIL  <3g FIGURE 4.  AGGREGATION OF COLLAGEN FIBRILS TO FORM A COLLAGEN FIBER  Diagrammatic R e p r e s e n t a t i o n of the V a r i o u s 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 M o l e c u l a r Events I n v o l v e d i n the Formati of a C o l l a g e n M o l e c u l e .  15 presented  (43).  cells  Collagen it The  that c o l l a g e n I I I may a c t u a l l y be s u r r o u n d i n g n e s t s  o f hemopoietic  The s i g n i f i c a n c e o f these f i n d i n g s i s not c l e a r a t p r e s e n t .  type IV i s the major component o f basement membranes.  I n the marrow,  forms a f i n e woven meshwork i n c l o s e a s s o c i a t i o n w i t h e n d o t h e l i a l presence o f a number o f sugar r e s i d u e s  conservation  o f i t s n o n - h e l i c a l extensions  lack of self-assembly  Laminin.  cells.  have been proposed to e x p l a i n the e x t r a c e l l u l a r l y and thereby the  i n t o f i b r o u s forms (44).  Laminin i s an extremely l a r g e g l y c o p r o t e i n  w i d e l y d i s t r i b u t e d amongst the v a r i o u s peculiar crucifix-shaped  (900,000 d a l t o n s ) ,  basement membranes (45).  c o n f i g u r a t i o n that allows  I t has a  s p e c i f i c p o r t i o n s o f the  molecule, a l s o known as domains, to i n t e r a c t s p e c i f i c a l l y w i t h adjacent  cells  and  This  the e x t r a c e l l u l a r matrix components p r o t e o g l y c a n s and c o l l a g e n IV.  molecule i s s e c r e t e d  (45).  smooth muscle c e l l s thought across  epithelial cells,  I n a d d i t i o n to i t s b i n d i n g  the basement membrane  I t has a high  and  laminin i s  m o l e c u l a r weight (200,000-250,000 d a l t o n s )  both i n the the serum and a t the c e l l s u r f a c e .  or multimeric  forms (49).  helps  (48).  and i s n o r m a l l y The presence o f a  to e x p l a i n  the e x i s t e n c e o f  T h i s molecule has been s t u d i e d i n t e n s i v e l y including a heparin binding s i t e ,  domain, and a c o l l a g e n b i n d i n g  described  pass  F i b r o n e c t i n i s a l s o a major mesenchymal c e l l product  a number o f domains i d e n t i f i e d  binding  ability,  (46,47).  l a r g e number o f f r e e s u l p h y d r y l r e s i d u e s dimeric  e n d o t h e l i a l c e l l s and some  to p l a y a r o l e i n r e g u l a t i n g the types o f macromolecules that  Fibronectin.  present  by v a r i o u s  region  ( F i g u r e 5) (50).  a cell  A newly  f a m i l y o f i n t e g r a l transmembrane p r o t e i n s known as i n t 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 s i g n a l s to a c t i n  DOMAINS OF FIBRONECTIN  s/V 30<  40 K  ?0K  30K  35K  75K  60K V HEPARIN  I  COLLAGEN  FIBRIN  II  CELL  HEPARIN  II  FIBRIN I I I  FIBRIN I  FIGURE 5.  F u n c t i o n a l Domains of F i b r o n e c t i n . Each box i s a p r o t e a s e r e s i s t a n t f u n c t i o n a l domain. S i z e of the domains a r e i n d i c a t e d by the numbers, e.g. 75K = an apparent m o l e c u l a r weight of 75,000. The b i n d i n g a c t i v i t i e s of each domain a r e l i s t e d underneath. The amino terminus p o r t i o n of the molecule i s on the l e f t , and the carboxy terminus on the r i g h t , a d j a c e n t to the d i s u l p h i d e bridges.  /  17  filaments  i n s i d e the c e l l  (51).  T h i s suggests an a t t r a c t i v e mechanism  which v a r i o u s e x t r a c e l l u l a r m a t r i c e s alter cardinal cellular  may  f u n c t i o n s such as gene t r a n s c r i p t i o n .  f u n c t i o n of the f i b r o n e c t i n molecule has These authors p r o v i d e d from the i n t e r s t i t i a l  i n t e r a c t w i t h c e l l s and  been put  A  eventually novel  f o r t h by P a t e l et a l  evidence that the r e l e a s e of mature red blood matrix  molecule. A s i m i l a r model has  of the marrow i s mediated by  by  (52).  cells  the f i b r o n e c t i n  r e c e n t l y been proposed f o r g r a n u l o c y t i c  cells  (53).  Hemonectin.  Hemonectin i s a very  recently described  a d h e s i o n molecule of a r e l a t i v e molecular immunologically (53).  distinct  from any  Immature g r a n u l o c y t e s  granulocytes  and  bind  n e u t r o p h i l s become a b l e  Proteoglycans.  (54,55).  Further  f i r m l y to hemonectin, i n c o n t r a s t  to mature  T h i s suggests that  the l o s s of  be part of the mechanism by which maturing the marrow.  Proteoglycans  With the e x c e p t i o n  Recently,  marrow c e l l s  components  are long molecular  complexes composed of a  to which a number of r e p e a t i n g d i s a c c h a r i d e u n i t s are  i n v a r i a b l y a c i d i c and gels.  to l e a v e  60,000 d a l t o n s , that i s  of the known e x t r a c e l l u l a r matrix  granulocytic progenitors.  adhesiveness to t h i s molecule may  p r o t e i n core  mass of  marrow-specific  can  i t has  of h y a l u r o n i c a c i d , these complexes  transform  attached  are  t h e i r immediate environment i n t o  hydrated  been shown that g l y c o s a m i n o g l y c a n s (GAG's) produced  i n v i t r o can  r e t a i n hemopoietic growth f a c t o r molecules  experiments are r e q u i r e d  to d e l i n e a t e the  exact r o l e and  by  (56).  significance  of t h i s i n t e r e s t i n g phenomenon to the r e g u l a t i o n of hemopoiesis i n v i v o .  18  2)  REGULATION OF THE HEMOPOIETIC SYSTEM  Although s t i l l hemopoietic c e l l set  p o o r l y understood,  the r e g u l a t i o n of many a s p e c t s o f  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  o f l o c a l environmental c o n d i t i o n s and by the p r o d u c t i o n o f a number o f  hormone-like  (A)  g l y c o p r o t e i n growth  Cellular  factors.  Interactions  There a r e s e v e r a l l i n e s o f evidence s u g g e s t i n g that s t r o m a l c e l l s o f the bone marrow i n t e r a c t d i r e c t l y w i t h hemopoietic c e l l s and p l a y a r o 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 s t u d i e s o f the marrow have  d i s c l o s e d a number o f c o n s i s t e n t and s p e c i f i c  i n t e r a c t i o n s between hemopoietic  c e l l s and the v a r i o u s s t r o m a l p o p u l a t i o n s (38).  For example, an i n t i m a t e  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. through from  T h i s has suggested  that some p h y s i o l o g i c f u n c t i o n s a r e mediated  these i n t e r a c t i o n s , p o s s i b l y i n c l u d i n g  the marrow.  the r e l e a s e o f g r a n u l o c y t e s  Another i n t r i g u i n g f i n d i n g has been the t y p i c a l  l o c a t i o n o f megakaryocytes.  parasinal  T h i s o b s e r v a t i o n i s the b a s i s o f the p r o p o s a l  that e n d o t h e l i a l c e l l s e l a b o r a t e hemopoietic growth f a c t o r s capable o f r e g u l a t i n g o r s u p p o r t i n g megakaryocytopoiesis ( 5 7 ) .  Regeneration Studies. d i s t u r b stromal c e l l  A number o f e x p e r i m e n t a l s t r a t e g i e s designed to  support i n c l u d i n g l o c a l  irradiation  (58,59) and  19 mechanical d i s r u p t i o n (60) have demonstrated l o s s of hemopoietic  function.  S t u d i e s o f Spleen C o l o n i e s . composition  that such treatments r e s u l t i n  I t has been shown that  the  cellular  ( e r y t h r o i d vs g r a n u l o p o i e t i c ) of i n d i v i d u a l s p l e e n c o l o n i e s  w i t h t h e i r s p e c i f i c l o c a t i o n i n the s p l e e n .  varies  C o l o n i e s a r i s i n g under the s p l e e n  c a p s u l e or i n the v i c i n i t y of septae are more o f t e n g r a n u l o p o i e t i c whereas i n all  o t h e r l o c a t i o n s c o l o n i e s are mainly e r y t h r o i d .  In c o n t r a s t , c o l o n i e s i n  the marrow a r e almost e x c l u s i v e l y g r a n u l o p o i e t i c (E:G = 0.1). o b s e r v a t i o n s suggest a profound  that l o c a l environments  i n these hemopoietic organs e x e r t  i n f l u e n c e on hemopoietic c e l l d i f f e r e n t i a t i o n  marrow fragments  These  (61).  F u r t h e r , when  were implanted i n t o the s p l e e n of e x p e r i m e n t a l animals, which  were then used as s p l e e n colony assay r e c i p i e n t s , the c o l o n i e s produced intra-splenically  implanted marrow stroma y i e l d e d a E:G  those that developed higher.  i n the s p l e e n i t s e l f y i e l d e d an E:G  (62).  Although  their  these s t u d i e s  be c o n s t r u e d as evidence of an " i n d u c t i v e " microenvironment  deterministic  or  the implanted  t h e i r e r y t h r o i d p o r t i o n i n the s p l e n i c stroma and  g r a n u l o p o i e t i c p o r t i o n i n the marrow stroma  whereas  r a t i o of 2.9  C o l o n i e s that b r i d g e d the j u n c t i o n of the s p l e e n and  marrow stroma had  cannot  r a t i o o f 0.1  i n the  ( i . e commitment) sense of the word s i n c e they do not p r o v i d e  i n f o r m a t i o n as to the stage of hemopoietic c e l l development i n f l u e n c e d , do support  the concept  that f i x e d elements  some a s p e c t s of hemopoietic c e l l  T r a n s f e r o f the Hemopoietic undertaken  i n the  of hemopoietic  they  tissues regulate  differentiation.  Microenvironment.  A s e r i e s of s t u d i e s  by F r i e d e n s t e i n et a l . (63) have shed c o n s i d e r a b l e l i g h t  r o l e f i x e d bone marrow p o p u l a t i o n s p l a y i n p r o v i d i n g an environment  on  the  c a p a b l e of  20 s u p p o r t i n g hemopoiesis.  The s t u d i e s of t h i s group have shown that h e t e r o t o p i c  t r a n s p l a n t a t i o n o f bone marrow fragments  under the kidney c a p s u l e of semi-  syngeneic animals l e a d s to the f o r m a t i o n of bone which then r a p i d l y becomes populated by hemopoietic c e l l s of r e c i p i e n t o r i g i n  (63).  In a d d i t i o n ,  t r a n s p l a n t a t i o n o f c l o n e d , c u l t u r e d f i b r o b l a s t s under the c a p s u l e o f the k i d n e y was  shown to r e s u l t  the hemopoietic  i n the t r a n s f e r of a microenvironment  t i s s u e from which the f i b r o b l a s t s were o b t a i n e d ( 6 4 ) .  groups have confirmed and expanded these o b s e r v a t i o n s and subcutaneous extract  Other  found that even  i m p l a n t a t i o n of a s m a l l amount of a c e l l u l a r d i a p h y s e a l bone  can l e a d  to the f o r m a t i o n of a bone e n c l a v e which o n l y then becomes  populated w i t h hemopoietic c e l l s  (65).  I r r a d i a t i o n of the subcutaneous  however, prevents the f o r m a t i o n of the bone e n c l a v e , i n d i c a t i n g r e c r u i t m e n t of subcutaneous  cells  s u i t a b l e f o r hemopoietic stem c e l l led  t y p i c a l of  that  i s e s s e n t i a l l o r the c r e a t i o n of a i n v a s i o n and support.  area,  stroma  Such s t u d i e s have  to a model i n which i t i s envisaged that u n d i f f e r e n t i a t e d  "fibroblast-  l i k e " mesenchymal c e l l s present i n a v a r i e t y of t i s s u e s can r e g e n e r a t e the differentiated  elements  of the hemopoietic stroma under the i n f l u e n c e of  p a r t i c u l a r , but as y e t undefined e x t r a c e l l u l a r matrix substances (66). A d d i t i o n a l evidence i n support of t h i s i s p r o v i d e d by r e c e n t c o - c u l t u r e s t u d i e s showing that even 3T3 c e l l s , which are u n d i f f e r e n t i a t e d embryonic  mouse f i b r o b l a s t s can support hemopoiesis  in vitro  cultured  for a limited  p e r i o d of time ( 6 7 ) .  S t u d i e s o f the Hemopoietic by the S l / S l ^ Genotype.  M i c r o e n v i r o n m e n t a l Defect i n Mice  Determined  Mice b e a r i n g a l t e r a t i o n s at the S t e e l l o c u s e x h i b i t  number of a b n o r m a l i t i e s i n c l u d i n g m a c r o c y t i c anemia,  hypopigmentation,  (68).  Mutants of the  abnormal s e n s i t i v i t y  to r a d i a t i o n , and s t e r i l i t y  a  genotype  S l / S l ^ have been the most thoroughly s t u d i e d and experiments  these animals have r e v e a l e d the nature of the d e f e c t hemopoietic system. normal  as judged  Hemopoietic  stem c e l l s  by t h e i r r a d i o s e n s i t i v i t y ,  c o l o n i e s i n normal  r e c i p i e n t s and  i r r a d i a t e d and  then i n j e c t e d w i t h normal  support hemopoietic the f a i l u r e  cells,  to form s p l e e n  to cure anemic mice whose  (+/+)  hemopoietic c e l l s ,  they do not  T h i s can be seen  mouse but not by an i n t r a v e n o u s i n j e c t i o n of  the hemopoietic d e f e c t  from a c e l l  i n the S l / S l ^ mouse i s thought  of suspended  to be due  that does not c i r c u l a t e and  that does c i r c u l a t e (70).  I r r a d i a t e d Mice.  Under or  Q  a l t e r n a t i v e l y , have e n t e r e d a very l o n g c e l l c y c l e ( 2 ) .  However, i n response  to c y t o r e d u c t i v e agents or r a d i a t i o n , a l a r g e r p r o p o r t i o n of these c e l l s to e n t e r the S phase of the c e l l c y c l e .  turnover i s r e g u l a t e d at a l o c a l  level,  U s i n g the ^H-thymidine  days, CFU-S i n the s h i e l d e d areas had  S-phase was  f o r one  were performed  i n which  t i b i a which was  i n the i r r a d i a t e d  shielded.  After  was 5  l a r g e l y r e t u r n e d to a q u i e s c e n t s t a t e  In c o n t r a s t , i n the i r r a d i a t e d h i g h (35 % k i l l ) and  tibias.  are  i f their  s u i c i d e assay (71), the c y c l i n g s t a t u s of CFU-S  then f o l l o w e d both i n the s h i e l d e d and  kill).  To determine  experiments  mice were g i v e n whole body r a d i a t i o n , except  (12%  to  i s not  c o n d i t i o n s i n the a d u l t , hemopoietic stem c e l l s are q u i e s c e n t ( G )  triggered  by  to form s p l e e n c o l o n i e s i n S l / S l ^  R e g u l a t i o n o f Stem C e l l Turnover i n P a r t i a l l y normal  intrinsically  However, when S l / S l ^ animals are  a f i x e d c e l l of the marrow microenvironment derived  their  S i n c e the hemopoietic d e f e c t can be cured by a g r a f t  whole t i s s u e from a +/+ +/+  their a b i l i t y  the t r a n s p l a n t e d c e l l s .  of the t r a n s p l a n t e d c e l l s (69).  recipients  r e c o v e r y from  i n S l / S l ^ mice are  their a b i l i t y  hemopoietic stem c e l l s are d e f e c t i v e (69).  that a f f e c t s  with  limb the p r o p o r t i o n of c e l l s i n  the CFU-S content low.  This disparity  22 between the  two  limbs c l e a r l y i n d i c a t e s that  h e m o p o i e t i c c e l l s can  (B)  be r e g u l a t e d  Humoral R e g u l a t i o n  i d e n t i f i c a t i o n and collectively  turnover of  (72).  The  development of c u l t u r e  terms of  p u r i f i c a t i o n of a number of g l y c o p r o t e i n s  r e f e r r e d to as hemopoietic growth f a c t o r s (HGF)  their a b i l i t y  term, c o l o n y s t i m u l a t i n g  purified  factors.  (CSA).  The  f a c t o r (CSF), was  Only a f t e r such p r e p a r a t i o n s  range of a c t i v i t y of i n d i v i d u a l CSF's be  The  coined  made i t p o s s i b l e primates and actions,  i n man  cell  Several  on  target  (10%) and  now  and  defined  more w i d e l y  were o b t a i n e d c o u l d  rigorously defined.  the  full  Recently,  expressed.  the  These  Interleukin-3  (IL-3).  these pure recombinant molecules  to i n v e s t i g a t e  to d e f i n i n g the range of  has  i n both  t h e i r mechanisms of  t h e i r a c t i v i t i e s on many  of i n t e r e s t have emerged from such s t u d i e s :  which s p e c i f i c a l l y  c e l l s and  receptor are  (74,75,76,77)) and  first,  types.  points  glycoproteins  At  to i n v e s t i g a t e t h e i r i n v i v o b i o l o g i c a l a c t i v i t i e s  in addition  different  (73).  now  to r e f e r to d i s c r e t e  (Epo), GM-CSF, G-CSF, M-CSF, and  a v a i l a b i l i t y of l a r g e q u a n t i t i e s of  that are  the  hence were r e f e r r e d  r e l a t e d , and  genes f o r a number of human HGF's have been cloned include: Erythropoietin  l e d to  were o p e r a t i o n a l l y  to support colony f o r m a t i o n , and  to as colony s t i m u l a t i n g a c t i v i t i e s used  techniques  the c l o n a l growth of hemopoietic p r o g e n i t o r s  b i o l o g i c a l a c t i v i t i e s present i n crude p r e p a r a t i o n s in  primitive  of the Hemopoietic System  Hemopoietic Growth F a c t o r s . c a p a b l e of s u p p o r t i n g  locally  the  i n t e r a c t with c e l l  1) HGF  surface receptors  are present  t h e i r b i o l o g i c a l e f f e c t s are o f t e n o b t a i n e d even at a  occupancy r a t e s  (78).  2) HGF  are extremely potent  b i o l o g i c a l l y a c t i v e at picomolar c o n c e n t r a t i o n s  (78).  low  molecules  3) There are  no  23 s t r i k i n g homologies between the v a r i o u s identified.  However, the r e c e p t o r  HGF  and  f o r M-CSF has  the product of the proto-oncogene, c-fms (79). e f f e c t s and  can  any  oncogene products thus f a r  been shown to be i d e n t i c a l 4) Most HGF  have p l e i o t r o p i c  i n t e r a c t with a number of d i f f e r e n t t a r g e t c e l l s .  i n c l u d e mature e f f e c t o r c e l l s which may HGF's (80,81,82) as w e l l as p r o g e n i t o r  E r y t h r o p o i e t i n (Epo).  Epo  produced mainly by  have t h e i r f u n c t i o n s augmented by c e l l s on d i f f e r e n t l i n e a g e s  when f u l l y g l y c o s y l a t e d .  the kidney and  has  been p u r i f i e d  u r i n e of p a t i e n t s with a p l a s t i c anemia (84,85). erythroid progenitors  and  These  Epo  enhances the s u r v i v a l and  epo  relative is  to homogeneity from i s a c t i v e on  the  committed  p r o l i f e r a t i o n of CFU-E  mature BFU-E (86,87).  The  gene f o r human Epo  (88).  has  r e c e n t l y been t e s t e d i n p a t i e n t s s u f f e r i n g from  Recombinant Epo  anemia of r e n a l f a i l u r e . its  full  The  shows great  been mapped to chromosome 7  promise f o r the  therapy of  Factor  (M-CSF).  Human M-CSF i s a  g l y c o p r o t e i n of a r e l a t i v e molecular mass of 47,000 to 76,000 d a l t o n s . c o n t r a s t s with the 70,000 d a l t o n M-CSF p u r i f i e d reduction,  on  these  (89).  Macrophage C o l o n y - S t i m u l a t i n g  subunits  and  f u l l y g l y c o s y l a t e d recombinant molecule r e t a i n s  b i o l o g i c a l a c t i v i t y and  patients  has  Native  the  (83).  i s a g l y c o s y l a t e d g l y c o p r o t e i n with a  m o l e c u l a r mass of 39,000 d a l t o n s  to  the l a t t e r s p e c i e s y i e l d s 2 i d e n t i c a l but  of about 35,000 d a l t o n s  each (90).  the b a s i s of i t s t r a n s c r i p t s , 2 d i s t i n c t  membrane-bound form and  a secreted  b i o l o g i c a l l y a c t i v e (92,93). progenitors  from murine L c e l l s .  i n v i t r o has  The  The  biologically  gene has  Upon  inactive  been cloned  (91)  forms have been p o s t u l a t e d :  form, both of which are  presumed  p r o l i f e r a t i v e e f f e c t of M-CSF on  been the best  This  to  and  a be  macrophage  s t u d i e d a c t i o n 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.  G r a n u l o c y t e C o l o n y - S t i m u l a t i n g F a c t o r (G-CSF).  G-CSF was  first  purified  to homogeneity from medium conditioned by the human bladder carcinoma c e l l l i n e 5637 (95).  It has a r e l a t i v e molecular mass of 19,600 daltons.  for human G-CSF is located on chromosome 17 (96).  The gene  It is produced by a variety  of activated c e l l s including lymphocytes and fibroblasts (97,98).  Recombinant  G-CSF has been shown to support granulocyte progenitor c e l l p r o l i f e r a t i o n in v i t r o (99).  Although i n i t i a l studies with purified natural human G-CSF  suggested that this molecule could stimulate c e l l s on other lineages, as pluripotent c e l l s ,  hence the o r i g i n a l term "pluripoietin" (95),  as well  more recent  data suggest that these l a t t e r effects were primarily indirect (100). Interestingly,  murine G-CSF, unlike most other murine HGF's,  is active on  human as well as murine c e l l s 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 C o l o n y - S t i m u l a t i n g F a c t o r (GM-CSF).  glycoprotein of a r e l a t i v e molecular mass of 22,000 daltons.  GM-CSF is  a  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 i k e G-CSF and I L - 3 , is not known to be c o n s t i t u t i v e l y  produced by any normal c e l l but is produced by a variety of activated including lymphocytes and fibroblasts (103,104).  cells  Purified natural and  recombinant molecules share the same b i o l o g i c a l properties: they both stimulate granulocyte, macrophage, eosinophil and megakaryocyte progenitors,  25  probably d i r e c t l y  (105,106).  They can a l s o enhance the p r o l i f e r a t i o n o f  p r i m i t i v e BFU-E and CFU-GEMM (107).  The recombinant molecule has r e c e n t l y  been t e s t e d c l i n i c a l l y and a s t i m u l a t i o n of c i r c u l a t i n g g r a n u l o c y t e p a t i e n t s i n j e c t e d with GM-CSF has been demonstrated (76,77).  levels in  In a d d i t i o n ,  GM-CSF can augment the f u n c t i o n o f mature e f f e c t o r c e l l s and can s t i m u l a t e the expression  o f c e l l s u r f a c e adhesion molecules Mol and LeuM5 (pl50,95) on  mature g r a n u l o c y t e s ( 8 1 ) .  Interleukin-3  (IL-3), Multi-CSF.  Human IL-3 i s a g l y c o p r o t e i n w i t h a  m o l e c u l a r mass o f 14,000 to 28,000 d a l t o n s molecule has been g l y c o s y l a t e d . predicted  depending on the extent  Although the e x i s t e n c e  of human IL-3 was  from s t u d i e s o f murine I L - 3 , human IL-3 was f i r s t  p u r i f i e d molecule as a r e s u l t o f an e x p r e s s i o n molecule has a l a r g e spectrum o f a c t i v i t i e s ,  the f i n a l  obtained  cloning strategy  as p r e d i c t e d  (108).  to the v a r i o u s  mature committed p r o g e n i t o r s .  The  from s t u d i e s o f  murine IL-3 (109), which appears to extend from the p l u r i p o t e n t stem compartment  as a  cell  In c o n t r a s t to  GM-CSF, IL-3 i s a more potent s t i m u l a t o r o f e r y t h r o i d b u r s t s and mixed c o l o n i e s and l e s s potent s t i m u l a t o r of granulocyte-macrophage c o l o n i e s  Synergistic Activities. factors  t h a t , alone,  nevertheless,  of i n t r i n s i c colony-stimulating  a c t i v i t y but,  formation.  L i s t e d below a r e s e v e r a l such  recently  synergistic factors.  Interleukin-1 IL-ip.  a r e devoid  describing  a r e capable o f s y n e r g i z i n g with one or more HGF's to enhance  hemopoietic colony described  There have been a s e r i e s o f r e p o r t s  (110).  (IL-1).  Both a r e very  Two forms o f IL-1 have been d e s c r i b e d :  s i m i l a r , bind  to the same r e c e p t o r  I L - l a and  and, hence, share  26 most i f not a l l o f t h e i r b i o l o g i c a l e f f e c t s (111,112). predominant  type expressed; however, an a c t i v i t y  11-10 i s the  independently p u r i f i e d  from  5637 c o n d i t i o n e d medium on the b a s i s of i t s a b i l i t y to s y n e r g i z e w i t h o t h e r HGF's turned out to be I L - l a (113,114,115). r e l a t i v e m o l e c u l a r mass of 22,000 d a l t o n s . monocytes (116)  IL-16  i s a p o l y p e p t i d e of a  I t i s produced  and TNF-oc s t i m u l a t e d e n d o t h e l i a l c e l l s  mostly by  (117).  activated  I t has a broad  range of e f f e c t s i n v i v o i n c l u d i n g the s t i m u l a t i o n of s e c r e t i o n of some acute phase p r o t e i n s (116),  the a c c e l e r a t i o n of bone r e s o r p t i o n (118),  enhancement of e x t r a c e l l u l a r matrix p r o t e i n turnover, and v a r i e t y o f mesenchymal c e l l  types (104,119,120)  the i n d u c t i o n of a  to s e c r e t e s e v e r a l growth  f a c t o r molecules i n c l u d i n g : PDGF, nerve growth f a c t o r (NGF), (121,122,123,124,125,126).  the  and v a r i o u s CSF's  I L - 1 has been shown to a c t on many c e l l  i n c l u d i n g k e r a t i n o c y t e s , hypothalamic  types  c e l l s , hepatocytes, lymphocytes  and  mature n e u t r o p h i l s (120).  Interleukin-4/B C e l l Stimulatory Factor-1 (IL-4)/(BCSF-l). A recently purified T cell  product, murine I L - 4 ,  has been shown to s t i m u l a t e a broad  range of murine hemopoietic p r o g e n i t o r s as w e l l 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 I L - 4 on myeloid c e l l s are o n l y observed  i n the presence of a d d i t i o n a l growth f a c t o r molecules.  w i t h o t h e r f a c t o r s murine I L - 4  can enhance the i n 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). cloned  (130)  In c o n c e r t  Murine  I L - 4 has been  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 (IL-6).  P r e v i o u s l y known as I n t e r f e r o n - 3 2  hybridoma growth f a c t o r (131),  (IFN-02) or  t h i s molecule has a m o l e c u l a r weight  of  27 26,000 d a l t o n s .  IL-6 i s produced  by a v a r i e t y 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 t h e i r a c t i v a t i o n supports  (132).  It  the growth of c e r t a i n B c e l l hybridomas and plasmacytomas and  r e c e n t l y been found  to s y n e r g i z e with IL-3 to support  p r i m i t i v e m u l t i p o t e n t i a l murine p r o g e n i t o r c e l l s  the p r o l i f e r a t i o n of  i n c u l t u r e (133).  weak s t i m u l a t o r of macrophage p r o g e n i t o r s i n v i t r o .  IL-6 i s a  IL-6, l i k e IL-1,  s t i m u l a t e s the s e c r e t i o n of acute phase p r o t e i n s by hepatocytes  N e g a t i v e R e g u l a t o r s of Hemopoiesis.  has  also  (134).  There have been r e p o r t s by s e v e r a l  groups (135,136,137) s u g g e s t i n g that f r a c t i o n a t e d c e l l - f r e e e x t r a c t s o b t a i n e d from r e s t i n g murine hemopoietic  t i s s u e s c o n t a i n a murine hemopoietic  i n h i b i t o r whose a c t i o n i s r e v e r s i b l e .  This a c t i v i t y  stem  cell  i s associated with a  m o l e c u l a r s p e c i e s whose mass appear to be i n the 50,000 to 100,000 d a l t o n range.  I t i s d e t e c t e d by i t s a b i l i t y  to p r o t e c t S-phase murine CFU-S from  l e t h a l e f f e c t s of exposure to h i g h s p e c i f i c a c t i v i t y -^-thymidine Interestingly,  the  (71).  the e f f e c t s of t h i s i n h i b i t o r can be competed o f f by a s m a l l e r  s i z e (30,000 - 50,000 d a l t o n s ) s t i m u l a t o r obtained from r e g e n e r a t i n g murine hemopoietic  t i s s u e s (138,139).  i n h i b i t o r was  found  In a p r e l i m i n a r y experiment  to be e f f e c t i v e i n v i v o (140).  TGF-|3 has a l s o been shown to a l s o act as a potent hemopoietic  the e x t r a c t e d  cell proliferation  (141,142).  i n h i b i t o r of  TGF-g i s a h i g h l y conserved  homodimer of 25,000 d a l t o n s present i n many t i s s u e s .  It i s particularly  abundant i n bone where i t i s present at a 100-fold h i g h e r c o n c e n t r a t i o n than elsewhere  (142).  Two  and TGF-{32 (142,143). a c t i o n of TGF-gl cloned  (144).  very s i m i l a r Very l i t t l e  forms of TGF-f3 have been i d e n t i f i e d : i s known at present about  or 2 at the molecular  l e v e l , although  Recent r e p o r t s have suggested  TGF-gl  the mechanism of  the gene has  been  that TGF-3 can i n c r e a s e the  28 i n c o r p o r a t i o n o f f i b r o n e c t i n and c o l l a g e n i n t o the e x t r a c e l l u l a r m a t r i x and can enhance the e x p r e s s i o n o f c e l l adhesion p r o t e i n r e c e p t o r s (145). HGF-dependent murine hemopoietic c e l l  l i n e s which express s p e c i f i c T G F - B  s u r f a c e r e c e p t o r s (K = 1-60 pM) a r e i n h i b i t e d experiments inhibit  by TGF-61 (145).  cell  Recent  i n the T e r r y Fox L a b o r a t o r y have f u r t h e r shown that TFG-01 can  the c y c l i n g of the most p r i m i t i v e myeloid p r o g e n i t o r c e l l  types i n  both mouse and human marrow i n a d i r e c t , s e l e c t i v e and r e v 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  known to be produced by mesenchymal c e l l s  f o l l o w i n g t h e i r a c t i v a t i o n by  s p e c i f i c m o l e c u l a r mediators o f t e n produced of  Others w i t h no known d i r e c t HGF a c t i v i t y a r e reviewed b r i e f l y  P l a t e l e t d e r i v e d growth f a c t o r (PDGF). a m o l e c u l a r mass of 30,000 d a l t o n s .  one o f which (147) .  i s identical  PDGF i s produced  macrophages (148,149).  PDGF i s a c a t i o n i c  smooth muscle  glycoprotein  c-sis  by both a c t i v a t e d e n d o t h e l i a l c e l l s and t i s s u e I n d u c t i o n of PDGF i s o b t a i n e d w i t h endotoxin, tumor  adhesion (125).  cells, glial  number of high a f f i n i t y  below.  PDGF c o n s i s t s o f 2 p o l y p e p t i d e c h a i n s ,  P l a t e l e t s contain  PDGF, but r e l e a s e o f PDGF from the p l a t e l e t  s t i m u l a t i o n of p l a t e l e t  the  Some  to the product of the c e l l u l a r proto-oncogene,  n e c r o s i s f a c t o r - a , and phorbol e s t e r s . of  i n inflammatory r e a c t i o n s .  these, such as IL-1 have been d i s c u s s e d above as they can a l s o a c t as  HGF's.  of  above, many HGF's a r e now  the h i g h e s t l e v e l s  i s c o n d i t i o n a l on the  Connective  tissue c e l l s  (fibroblasts,  c e l l s and chondrocytes) which d i s p l a y  cell  s u r f a c e PDGF r e c e p t o r s are  primary target o f PDGF a c t i o n .  t r i g g e r s a cascade of events which  thought  a  large  to c o n s t i t u t e  Upon b i n d i n g to i t s r e c e p t o r , PDGF culminate i n the  production of  29  e x t r a c e l l u l a r matrix substances, and activities,  i n c l u d i n g GM-CSF (149,150).  c i r c u l a t i o n , and locally  by  current  evidence suggests that  Factor-a  hemorrhagic n e c r o s i s  e n d o t o x i n (152). secrete  PDGF i s r a p i d l y c l e a r e d  (TNF-a).  and  i t s turnover may  A l s o known as c a c h e c t i n ,  be  the  regulated (151).  t h i s molecule  of tumors i n r e c i p i e n t animals c h a l l e n g e d  with  I t i s produced p r i n c i p a l l y by a c t i v a t e d monocytes which  v e r y l a r g e amounts of TNF-a (153  the c i r c u l a t i o n to normal t a r g e t c e l l s : (153).  from  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  Tumor N e c r o s i s elicits  the s e c r e t i o n of a range of b i o l o g i c a l  Human TNF-a has  ).  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 monocytes and  endothelial  r e c e n t l y been p u r i f i e d , the gene c l o n e d ,  cells  expressed,  mapped to chromosome 6 (154).  TNF-a has  a wide spectrum of a c t i v i t i e s  mediates endotoxin-induced shock.  TNF-a has  a l s o r e c e n t l y been shown to  provide mitogenic s i g n a l s for e n d o t h e l i a l c e l l s f o r m a t i o n of c a p i l l a r y TNF-a i s a potent and  inducer  r e l e a s e G-CSF and  Summary.  tube-like structures  the  non  i n v i v o , and  in vitrp.  of mesenchymal c e l l s and  to promote  L i k e IL-13  stimulates  and  and  the  PDGF,  them to produce  GM-CSF (155,156).  I t i s c l e a r from the above d i s c u s s i o n  h i g h l y heterogeneous organ. f i b r o u s and  then  I t c o n s i s t s of many c e l l  f i b r o u s c e l l products.  r e g u l a t i o n of hemopoietic stem c e l l s  that  the bone marrow i s a  types and  a d i v e r s i t y of  A l a r g e body of e v i d e n c e suggests i s mediated p r i m a r i l y by  l o c a l mechanisms i n v o l v i n g s t r o m a l c e l l s and  their secretory  that  complicated  products.  30 3)  LONG-TERM BONE MARROW CULTURES: AN IN VITRO MODEL FOR HEMOPOIETIC STEM CELL REGULATION  Although i n v i v o s t u d i e s add weight to the p h y s i o l o g i c a l r e l e v a n c e e f f e c t s observed f o l l o w i n g a g i v e n p e r t u b a t i o n ,  they a r e u s u a l l y p o o r l y s u i t e d  to the a n a l y s i s o f s p e c i f i c c e l l u l a r and molecular p a r t i c u l a r responses.  The establishment  o f any  events that  underlie  and v a l i d a t i o n o f s u i t a b l e i n v i t r o  models f o r many c e l l u l a r and t i s s u e systems have t h e r e f o r e been a major o b j e c t i v e o f much r e s e a r c h h e m o p o i e t i c stem c e l l 1977  biology.  In t h i s r e s p e c t  r e g u l a t i o n i s no e x c e p t i o n .  The f i r s t  by Dexter e t a l (157) o f c u l t u r e c o n d i t i o n s which a l l o w  pluripotent  stem c e l l s  exogenously provided For  in cell  the f i r s t  the study o f description i n murine  to be maintained f o r many months i n the absence o f  HGF's represented  a significant  advance i n t h i s  time, i t became p o s s i b l e to i n v e s t i g a t e i n v i t r o ,  supported stem c e l l  s e l f - r e n e w a l and d i f f e r e n t i a t i o n .  by subsequent s t u d i e s d i s c u s s e d  regard.  a system which  Moreover, as r e v e a l e d  i n more d e t a i l below, t h i s c u l t u r e system has  many f e a t u r e s o f the bone marrow as i t i s c o n s t i t u t e d i n v i v o and appears to provide  a u s e f u l model f o r a n a l y s i s o f the r e g u l a t o r y systems o p e r a t i v e i n  humans as w e l l as mice.  (A)  E a r l y Development o f the Long-Term Marrow C u l t u r e s  In i n i t i a l s t u d i e s with placed  t h i s system (157) mouse marrow c e l l s were f i r s t  i n c u l t u r e at a c o n c e n t r a t i o n  w i t h horse serum to a l l o w took 2-3 weeks.  of 10^ c e l l s / m l i n a medium supplemented  the formation  of a c o n f l u e n t  adherent l a y e r which  These c u l t u r e s were then "recharged" with  inoculum o f mouse marrow c e l l s .  Half  a second, s i m i l a r  the medium was then removed and r e p l a c e d  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  s i m u l t a n e o u s l y d i l u t e d i n h a l f , and stained  and/or assayed f o r v a r i o u s  conditions,  i t was  r e s u l t was  not  a p o s i t i v e one (157).  importance of  interactions,  periods  but This,  feature  When they were,  The  than only  (For  a number of s i g n i f i c a n t use  of h y d r o c o r t i s o n e  technical  as a supplement  to use  almost any  In a d d i t i o n ,  was  b a t c h of  i t made i t These  l e d to the s u c c e s s f u l e s t a b l i s h m e n t of long-term human  Cultures  i n which g r a n u l o p o i e s i s  routinely initiated  technical details,  W i t h i n 3 weeks of p l a c i n g layer i s established. identified.  had  (160,161).  Long-term c u l t u r e s be  the  cell-cell  i n the marrow i n v i v o  rare, selected batches.  Long-Term Human Marrow  now  of  the  to i n i t i a t e c u l t u r e s with a s i n g l e inoculum of marrow (40).  marrow c u l t u r e s  marrow.  important  be  i n t h i s system.  improvements, i n t u r n ,  weeks can  could  together with the d e m o n s t r a t i o n of  important because i t made i t p o s s i b l e  h o r s e serum r a t h e r  Under these  the adherent l a y e r (159), suggested that  improvements were i d e n t i f i e d .  (B)  An  counted,  r a t h e r an a c c e l e r a t i o n i n the r a t e at which  D u r i n g the next s e v e r a l y e a r s ,  possible  time (158).  perhaps s i m i l a r to those o c c u r r i n g  been r e - e s t a b l i s h e d  particularly  of  be  granulopoiesis  that no HGF's were added exogenously.  hemopoiesis d e c l i n e d critical  types of p r o g e n i t o r s .  found that CFU-S, CFU-GM and  maintained f o r very e x t e n s i v e t h i s system was  the c e l l s removed c o u l d  see  i s maintained f o r at l e a s t 8  from a s i n g l e innoculum of normal human Chapter I I ) .  the marrow c e l l s  in culture, a confluent  In i t a v a r i e t y of s t r o m a l c e l l  These i n c l u d e  c e l l s with p r o p e r t i e s  adherent  types have been  of f i b r o b l a s t s (162),  32 endothelial cells muscle c e l l s  (see F i g u r e 6A and 6B)  (163), a d i p o c y t e s (164), smooth-  (165), and macrophages (158).  f o c i of d e v e l o p i n g hemopoietic  cells,  I n t e r s p e r s e d between these  descriptively referred  " c o b b l e s t o n e a r e a s " (160), can be seen.  The adherent  cells,  to as  layer also contains a  number of e x t r a c e l l u l a r matrix components i n c l u d i n g : c o l l a g e n I (162), c o l l a g e n I I I (162), c o l l a g e n IV (166), l a m i n i n (167), and Hovering over and d e r i v e d from free-floating cells. and  the adherent  fibronectin  l a y e r are l o o s e l y adherent  Most of these are mature g r a n u l o c y t e s and  t h e i r immediate p r e c u r s o r s (169).  In the human system,  adherent  fraction  the adherent dissociate  particularly  (169).  To q u a n t i t a t e the hemopoietic  but  the c u l t u r e  i n the  to e n z y m a t i c a l l y  i n t h i s f r a c t i o n so that a s i n g l e c e l l  u s i n g e i t h e r c o l l a g e n a s e or t r y p s i n have been developed  suspension S u i t a b l e methods  for this  purpose  Assessment of a s e r i e s of c u l t u r e s over time has shown that  hemopoietic  the  p r o g e n i t o r content of  s u i t a b l e f o r p l a t i n g i n s e m i - s o l i d medium can be o b t a i n e d .  (169).  macrophages  the more p r i m i t i v e ones, are found  layer requires s a c r i f i c i n g  the c e l l s  and  some c l o n o g e n i c  p r o g e n i t o r s can a l s o be d e t e c t e d i n the non-adherent f r a c t i o n , m a j o r i t y of such c e l l s ,  (168).  p r o g e n i t o r content of the non-adherent f r a c t i o n s and  the  the mature  g r a n u l o c y t e s and macrophages to which they g i v e r i s e are maintained  at r o u g h l y  constant l e v e l s  of the  f o r up  to 8 weeks i n s p i t e of the demi-depopulation  non-adherent c e l l s at each weekly medium change ( F i g u r e 7 ) . a l t h o u g h g r a n u l o p o i e s i s i s supported e r y t h r o p o i e s i s i s not.  to completion  i n c l u d i n g the CFU-E r a p i d l y d i s a p p e a r . not n o r m a l l y added to these c u l t u r e s . i s found and,  system,  Thus, o n l y the most p r i m i t i v e e r y t h r o i d p r o g e n i t o r s ,  the BFU-E, a r e c o n t i n u o u s l y d e t e c t e d and  situation  in this  Note that  the more d i f f e r e n t i a t e d  elements  T h i s i s not s u r p r i s i n g s i n c e Epo i s In the murine system,  a similar  although a d d i t i o n 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  Nucleated Cells  100  10 7  3  10  r  6  o  i  10  10  !  10*  .01 0  1  2  3  4  5  6  Weeks in Culture  FIGURE 7.  7  8  0  1  2  3  4  5  6  7  8  Weeks in Culture  The C e l l u l a r i t y and P r o g e n i t o r Content o f the Adherent (Adh) and Non-adherent (NA) F r a c t i o n Assessed at V a r y i n g I n c u b a t i o n Times. Each p o i n t shown r e p r e s e n t s the geometric mean + 1 SEM o f d a t a from s e v e r a l experiments. Of the 15 experiments i n i t i a t e d , f i v e were terminated a t 32 weeks, s i x at 4 weeks, and f o u r maintained u n t i l 7 weeks and s a c r i f i c e d f o r assessment at that time. The downward arrows i n d i c a t e maximum mean v a l u e s i f one c o l o n y had been seen i n any o f the assay d i s h e s 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 c e l l s 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 c e l l s in the long-term marrow culture system may reflect mechanisms that control hemopoiesis in vivo. as follows:  the operation of the same  This evidence may be summarized  1) In the murine system, where i t is possible  to assay c e l l s for  marrow repopulating potential by transplantation into l e t h a l l y i r r a d i a t e d recipients,  i t has been shown that such c e l l s 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 i r c u l a t i n g peripheral blood granulocytes no differences physiological properties,  in any of a variety of  including phagocytosis,  burst and b a c t e r i a l k i l l i n g , are found (172).  degranulation, respiratory  3) The microenvironmental  defect of the S l / S l ^ mouse is reproduced in long-term cultures i n i t i a t e d 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 c e l l layers from W/W mice (who v  have deficient  stem c e l l s and cannot i n i t i a t e long-term hemopoiesis in v i t r o  for this reason) (174).  4) Perhaps, the most compelling evidence comes from  studies of the turnover of primitive hemopoietic c e l l s 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 t e c h n i q u e t o measure the p r o p o r t i o n o f S-phase c e l l s (71) r e v e a l e d a s i m i l a r (although  not i d e n t i c a l ) p a t t e r n i n both murine and human c u l t u r e s .  I n 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 nonc y c l i n g s t a t e d i c t a t e d by the t i m i n g o f p e r t u r b a t i o n o f the c u l t u r e s a s s o c i a t e d w i t h each medium change.  T h i s r e s u l t e d i n an a c t i v a t i o n o f a l l  CFU-S w i t h i n 1-2 days, r e g a r d l e s s o f t h e i r s i t u a t i o n i n e i t h e r the adherent o r 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 u n d i s t u r b e d ,  returned  s t a t e w i t h i n the next 3 t o 5 days (175).  to a q u i e s c e n t  the CFU-S In contrast  CFU-GM were found t o remain c o n t i n u o u s i n c y c l e as they do i n v i v o even under normal h o m e o s t a t i c c o n d i t i o n s (175).  I n 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 o f 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 q u i e s c e n c e i n the most p r i m i t i v e progenitor  compartments has a l s o been seen (176).  e x c l u s i v e to the c e l l s c o n t a i n e d  However, t h i s b e h a v i o u r i s  w i t h i n the adherent l a y e r , s u g g e s t i n g  important r o l e of d i r e c t c e l l contact  i n this species.  a more  This i s further  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 o f human c u l t u r e s i s insufficient  to a c h i e v e p r o g e n i t o r  c e l l stimulation.  mesenchymal c e l l a c t i v a t o r appears t o be r e q u i r e d  A d d i t i o n o f some  (177).  the mouse, the c y c l i n g s t a t u s o f the v a r i o u s p o p u l a t i o n  Nevertheless,  as i n  of progenitors i n  u n p e r t u r b e d c u l t u r e s c l o s e l y mimics that seen i n the marrow o f the normal adult.  Those c l a s s e s o f c e l l s that i n v i v o a r e q u i e s c e n t  the l o n g - t e r m c u l t u r e a l s o r e t u r n t o a n o n - d i v i d i n g s t a t e . t h a t i n v i v o a r e i n a s t a t e o f constant  turnover  a r e those t h a t i n Similarly,  those  remain i n c y c l e i n the l o n g -  term c u l t u r e system, even when they a r e l o c a t e d i n the adherent l a y e r .  38 4)  THESIS OBJECTIVES  T h i s r e s e a r c h p r o j e c t was hemopoietic  developed  to e x p l o r e the h y p o t h e s i s  stem c e l l r e g u l a t i o n i s mediated by s h o r t - r a n g e  mesenchymal elements.  To  that  interactions with  t e s t t h i s h y p o t h e s i s i n the human, i t would  be  advantageous to use an i n v i t r o model i n which the v a r i o u s components c o u l d d i s s e c t e d out and In  be  t h e i r f u n c t i o n s analysed s e p a r a t e l y .  the p r e c e d i n g s e c t i o n I have d e s c r i b e d an i n v i t r o system i n which the  most p r i m i t i v e hemopoietic  p o p u l a t i o n s d e t e c t a b l e by c l o n o g e n i c assays can  demonstrated f o r p e r i o d s of a t l e a s t 8 weeks.  A key f e a t u r e of t h i s  be  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 a r e thought to resemble the v a r i o u s c o n s t i t u e n t s of the microenvironment of the marrow i n v i v o . of c l o n o g e n i c assays  The  availability  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  p r o g e n i t o r s c e l l s as w e l l as the p o s s i b i l i t y of u s i n g the long-term 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 a l l o w the c e l l u l a r and m o l e c u l a r r e g u l a t i n g stem c e l l maintenance and long-term  t u r n o v e r to be i d e n t i f i e d .  mechanisms However, the  marrow c u l t u r e model i s by no means a " s i m p l e " v e r s i o n of i n v i v o  hemopoiesis. l a y e r and  marrow  The h e t e r o g e n e i t y of the c e l l u l a r c o m p o s i t i o n of the adherent  the v a s t a r r a y of m o l e c u l a r s p e c i e s produced by these c e l l s makes  any proposed a n a l y s i s a r e a l c h a l l e n g e . The  o v e r a l l o b j e c t i v e s of my  f i r s t o b j e c t i v e was  to develop  r e s e a r c h were, t h e r e f o r e , t w o f o l d .  a method for  assaying the  of the non-hemopoietic components of long-term  types.  function  marrow c u l t u r e adherent  Such an assay c o u l d then be used to e v a l u a t e the stromal c e l l  regulatory  The  r o l e p l a y e d by  P r e r e q u i s i t e f o r such an assay was  layers.  individual  the need to o b t a i n  39 pure t a r g e t h e m o p o i e t i c  populations (or at l e a s t suspensions  c e l l s t h a t were f r e e o f r e g u l a t o r y c e l l s ) .  I envisaged  of  hemopoietic  t h a t t h i s might be  a c h i e v e d by a p p r o p r i a t e p h y s i c a l s e p a r a t i o n t e c h n i q u e s , o r by the a d o p t i o n o f 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  c e l l s from i n t e r f e r i n g w i t h the proposed measurements. e x p l o r e d and the r e s u l t s a r e presented  Both approaches were  i n Chapter I I I .  The second o b j e c t i v e of my r e s e a r c h was to i s o l a t e populations of c e l l s corresponding the marrow stroma.  accessory  homogeneous  to the v a r i o u s mesenchymal components o f  The approach was to e s t a b l i s h permanent c e l l l i n e s that  c o u l d be c l o n e d 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 t h a t  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 c o n t r a s t to  t h e i r murine c o u n t e r p a r t s , r a r e l y become spontaneously  immortalized.  T h e r e f o r e , an a l t e r n a t e s t r a t e g y had to be used to i m m o r t a l i z e human marrow stromal c e l l s .  S t i m u l a t e d by r e p o r t s of the a b i l i t y of SV-40 v i r u s to  t r a n s f o r m v a r i o u s types of human c e l l s w i t h o u t l o s s of t h e i r  differentiated  phenotype (179,180,181,182), I i n i t i a t e d a s e r i e s o f e x p e r i m e n t s to t r y and o b t a i n a s e r i e s o f 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 s t r o m a l elements.  The i n i t i a l work i n v o l v e d i n the p r e p a r a t i o n and  t i t r a t i o n o f a s u i t a b l e SV-40 v i r u s p r e p a r a t i o n i s d e s c r i b e d 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 r e p a r a t i o n on human  marrow s t r o m a l 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  lines I  i s o l a t e d a r e d e s c r i b e d i n Chapter IV. L i k e most r e s e a r c h p r o j e c t s , the achievement of these i n i t i a l o b j e c t i v e s turned out to r e p r e s e n t d e f i n i t i v e but r e l a t i v e l y modest f i r s t steps the o v e r a l l g o a l . how next  towards  N e v e r t h e l e s s , they made p o s s i b l e a c l e a r e r d e s c r i p t i o n of  to proceed i n u n r a v e l i n g the r o l e of the marrow stroma.  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T r a n s f o r m a t i o n 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 s i m i a n v i r u s 40: I n d u c t i o n of c e l l p r o l i f e r a t i o n and r e t e n t i o n of 6 2 - a d r e n e r g i c r e c e p t o r s . Proc N a t l Acad S c i USA 83: 8754, 1986.  strains.  54 182. Brown KW, G a l l i m o r e PH. Malignant p r o g r e s s i o n o f 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  CHAPTER  I I  MATERIALS AND METHODS  1.  CELLS  (A)  Bone Marrow C e l l s  Marrow a s p i r a t e c e l l s were c o l l e c t e d consent  i n h e p a r i n i z e d medium w i t h  from h e m a t o l o g i c a l l y normal p a t i e n t s undergoing  or from normal a l l o g e n e i c donors undergoing transplantation.  bone marrow h a r v e s t s f o r  (1.077 gm/cm ) and subsequently 3  a c c o r d i n g to the d i r e c t i o n s o f the s u p p l i e r (Pharmacia,  Cross.  D o r v a l P.Q.).  through  the c o u r t e s y o f the Canadian  To i s o l a t e T-lymphocyte d e p l e t e d mononuclear c e l l s , a b u f f y coat  p r e p a r a t i o n was f i r s t diluted  washed (3x)  P e r i p h e r a l Blood  Normal p e r i p h e r a l blood was o b t a i n e d Red  routine investigations,  L i g h t d e n s i t y mononuclear c e l l s were o b t a i n e d by  c e n t r i f u g a t i o n on F i c o l l - H y p a q u e  (B)  informed  o b t a i n e d by c e n t r i f u g i n g whole u n i t s o f p e r i p h e r a l blood  i n a c i d / c i t r a t e / dextrose at 800 x g f o r 5 minutes.  Light density  (<1.077 g/cm ) mononuclear c e l l s were then separated on F i c o l l / H y p a q u e and 3  T-lymphocytes then reduced  to approximately  incubation with 2-aminoethylisothiouronium  3-AX o f t o t a l bromide (AET,  t r e a t e d sheep e r y t h r o c y t e s and removal of the T c e l l c e n t r i f u g a t i o n on F i c o l l / H y p a q u e . 1 unit  (500 ml) o f blood  T h i s procedure  n u c l e a t e d c e l l s by Sigma, S t - L o u i s , MO.)  r o s e t t e s by r e -  yielded sufficient  cells  to i n i t i a t e . 3 to 5 c u l t u r e s with 2 x 10? c e l l s  Mean v a l u e s from 14 experiments a r e shown on t a b l e I .  from  each.  Table I.  Recovery of Nucleated C e l l s and P r o g e n i t o r s from One U n i t o f Blood  F r a c t i o n Analysed  Yield ( X )  Nucleated Cells  Unseparated  Blood  B u f f y Coat  (500 ml)  E  CFU-E  Rosettes  BFU-E  BFU-E  BFU-E  BFU-E  (3-8)  (8-16)  (>16)  Total  CFU-GEMM  CFU-G/M  100 49  Light-Density  Cells  23  74  100  100  100  100  100  100  100  T-Depleted  Cells  4  5  92  68  53  99  59  72  59  T-Enriched  Cells  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 (VI-38), and  the human u m b i l i c a l cord  endothelial c e l l  purchased from the American Type C u l t u r e f i b r o b l a s t s were i s o l a t e d and Terry  Fox  Laboratory.  (BSC-1), lung embryo f i b r o b l a s t s  C o l l e c t i o n ( R o c k v i l l e , MD).  established  i n v i t r o by Dr.  (D)  these c e l l  i n 60 mm  c o v e r s l i p s . The 3  pfu/cell.  controls. virus,  by Dr.  culture conditions  C.  the  J.  used f o r  the  Lines  medium (a-10%) was  one  removed and  i n f e c t e d were set up of each c o n t a i n i n g  the p r o p o r t i o n  give  as  adsorption  of  the  Twenty four hours l a t e r some c o v e r s l i p s were  of adherent c e l l s e x p r e s s i n g  i n d i r e c t immunofluorescence ( 6 ) .  t r y 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 each d i l u t i o n . C u l t u r e s  2 glass  i n f e c t e d to s e r v e  at 37°C f o r 90 minutes to a l l o w  changed.  in  s u f f i c i e n t v i r u s added to  In each case some c u l t u r e s were not  A f t e r incubation  determined by  to be  tissue culture dishes,  the medium was  f i x e d and  Hogge a l s o i n  I n f e c t i o n of Primary Human Mesenchymal C e l l s with SV-40 and  Unless otherwise i n d i c a t e d , c u l t u r e s  10  The  Skin  l i n e s are summarized i n T a b l e I I .  I s o l a t i o n of Permanent C e l l  triplicate  D.  EAhy.926 c e l l s were g e n e r o u s l y provided  E d g e l l ( U n i v e r s i t y of North C a r o l i n a , NC). maintenance of  l i n e HUV-EC-C were  The  c u l t u r e s were then reserved  for i s o l a t i o n  and  o t h e r s s t a i n e d with May-Grunwald Giemsa.  of  was  remaining c u l t u r e s were  4 or more new  were maintained at 37°C and  two  the l a r g e T a n t i g e n  fed  c u l t u r e s seeded twice weekly.  i n d i v i d u a l  transformed  One  from or  foci  I n d i v i d u a l f o c i were h a r v e s t e d  Table I I .  Culture Conditions  Lines  CELL LINE  ORIGIN  CULTURE MEDIUM  BSC-1  A f r i c a n Green Monkey Kidney  a-102  WI-38 (2)  Human Embryonic Lung  a-10%  HUVE-EC-C (3)  Human U m b i l i c a l Cord Endothelium  F12 K Medium 20% FCS 100 ug/ml Heparin 50 ug/ml ECGS*  DH F i b r o b l a s t s (4)  Skin F i b r o b l a s t s  a-10%  EBV Lymphoblastoid Line  Human Marrow C e l l s i n Long-Term C u l t u r e  a-10%  (1)  EAhy.926 (5)  SV-40 L i n e s  All  f o r the Maintenance o f C e l l  cell  F a c t o r V I I I +ve Mouse-Human Hybrid (A549xHUVEC-C) Human Marrow and HUVE-EC-C c e l l s (see s e c t i o n ID)  l i n e s were maintained  a-10%  a-10%  i n an atmosphere o f 5% C O 2 at 37°C.  * E n d o t h e l i a l c e l l growth supplement.  59 using  porcelain  c y l i n d e r s and the c e l l s  culture multiwell  plates  (Linbro,  transferred  McLean, VA).  to f l a t  bottom  A few days l a t e r ,  were c l o n e d 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 w e l l s at  10^ c e l l s / 3 5 mm d i s h  i n methylcellulose  a s s a y s , s e c t i o n 7 below). isolated colonies containing  were removed and t r a n s f e r r e d  a week and s u b c u l t u r i n g  the l i n e s  o r by p l a t i n g  (see Anchorage-independent  Three weeks l a t e r ,  2 ml of medium.  tissue  cells  growth  i n p o s i t i v e w e l l s or  i n d i v i d u a l l y to 2 cm^  wells  Cloned l i n e s were then propagated by f e e d i n g  once a week at 10^ c e l l / c m ^  twice  i . e . 2.5 x 10^ cells/25cm2  flask.  (E)  C e l l P r o l i f e r a t i o n Measurements  The growth and p o p u l a t i o n  d o u b l i n g times of SV-40 i n f e c t e d c e l l s and  c l o n e d l i n e s were determined from v i a b l e c e l l counts ( n i g r o s i n dye e x c l u s i o n ) or  from measurements of ^H-thymidine  population  d o u b l i n g time, c e l l s were resuspended  4 x 10^ c e l l s / m l . seeded i n t o 35 mm cells  incorporation.  For measurements of  to a f i n a l  concentration  of  2.5 ml of t h i s c e l l suspension ( i . e . 10^ c e l l s ) was then tissue culture dishes.  At d a i l y i n t e r v a l s t h e r e a f t e r , the  i n a p a i r of d i s h e s (one i n f e c t e d and one c o n t r o l ) were t r y p s i n i z e d and  v i a b l e c e l l counts performed. were c u l t u r e d  To measure ^H-thymidine  i n Iscoves-10% i n f l a t  (500 c e l l s / 1 0 0  ul/well).  bottomed  incorporation,  microwells  At the times i n d i c a t e d ,  cells  (Costar)  1 yCi of H-thymidine 3  (20 mCi/mmole, Amersham, O a k v i l l e , Ont) i n 20 u l of growth medium was then added  to each w e l l .  The c e l l s were incubated f o r an a d d i t i o n a l 4 hours at  37°C, h a r v e s t e d onto g l a s s incorporated  fiber f i l t e r  determined by s c i n t i l l a t i o n  paper, and the amount of ^H-thymidine counting.  60 2)  LONG-TERM CULTURES  (A)  Regular Long-Term Marrow C u l t u r e s  An a l i q u o t of u n t r e a t e d marrow a s p i r a t e c o n t a i n i n g 2-2.5 marrow c e l l s was a 60 x 15  mm  p l a c e d i n 8 ml of long-term c u l t u r e growth medium i n  Falcon tissue culture dish.  a-medium supplemented glutamine  with i n o s i t o l  (400 mg/ml), f e t a l c a l f  2 mercaptoethanol  of  5% C0£  The growth medium was  (40 mg/1), f o l i c a c i d  serum (FCS,  composed of  (10 mg/1), e x t r a  12.5%), horse serum (HS,  to support maximal hemopoiesis  in a i r .  After  this i n i t i a l  p e r i o d of i n c u b a t i o n , a l l nonadherent  l a y e r e d over F i c o l l / H y p a q u e 1.077  d e n s i t y c e l l s were washed i n a-medium supplemented the o r i g i n a l d i s h e s .  g/cm  3  to remove  by p i p e t t i n g 2-3 to ensure removal The  with 2% FCS and  The  ml of medium from the d i s h , and of a l l the nonadherent  c u l t u r e medium was  e v e n l y , and 4 ml of  cells.  T h i s was  light  r e t u r n e d to  The c u l t u r e s were then fed on a weekly b a s i s by  h a l f of the medium and h a l f of the nonadherent  cells  in this  The c u l t u r e s were incubated f o r 3 to 4 days at 37°C i n an atmosphere  remaining red blood c e l l s and mature g r a n u l o c y t e s where n e c e s s a r y .  medium.  FCS  -  c e l l s were removed and  of  12.5%),  ( 1 0 ^ M), and h y d r o c o r t i s o n e sodium s u c c i n a t e (10~6 M).  and HS were p r e t e s t e d f o r t h e i r a b i l i t y system.  x 10^ n u c l e a t e d  removal  accomplished  then g e n t l y s w i r l i n g  c e l l s with the remaining 5-6  the d i s h ml of the  p l a c e d i n a tube, v o r t e x e d to d i s t r i b u t e  t h i s suspension was  r e t u r n e d to the c u l t u r e  the  dish,  a l o n g w i t h 4ml of f r e s h c u l t u r e medium. To h a r v e s t adherent or  layers,  the c u l t u r e s were f i r s t  t h r e e times w i t h serum f r e e and Ca""" and M g 1  solution cells  to remove a l l the nonadherent  i n the adherent  1  cells  l a y e r were then exposed  ++  vigorously rinsed  f r e e , Hanks balanced  (and any FCS  left  two  salt  in dishes).  at 37°C to t r y p s i n  (0.25% i n a  The  61 s o l u t i o n c o n t a i n i n g 5% c i t r a t e ,  10% KCl and  1% g l u c o s e ) f o r 10 minutes  f o l l o w i n g the method o f Coulombel et a l . ( 7 ) . at  C e l l s that were s t i l l  the end of the i n c u b a t i o n p e r i o d were g e n t l y removed by  transfered  to a t e s t  adherent  pipetting,  tube, c e n t r i f u g e d at 300 g x 10 minutes and washed  twice  i n 2% a-medium.  (B)  Long-Term P e r i p h e r a l Blood C u l t u r e s  To i n i t i a t e long-term p e r i p h e r a l blood c u l t u r e s , 2 x 10? T - d e p l e t e d d e n s i t y mononuclear c e l l s were p l a c e d i n 60mm t i s s u e c u l t u r e d i s h e s . were subsequently maintained u s i n g the same p r o t o c o l e s t a b l i s h e d  light  These  f o r long-term  c u l t u r e s with the e x c e p t i o n that c u l t u r e s were incubated d i r e c t l y at 33°C and the day  3-4  red c e l l and g r a n u l o c y t e s e p a r a t i o n s t e p was  weeks, non-adherent c e l l s were removed and  omitted.  then the adherent  After  4-5  layer harvested  by  t r y p s i n i s a t i o n as d e s c r i b e d above.  (C)  P r e p a r a t i o n of Long-Term Marrow C u l t u r e Feeders  Long-term marrow c u l t u r e s were i n i t i a t e d and maintained as d e s c r i b e d above w i t h the f o l l o w i n g m o d i f i c a t i o n s .  H y d r o c o r t i s o n e was  omitted from  the medium,  the c u l t u r e s 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 as f e e d e r s a f t e r  the adherent  l a y e r had  medium added. reached  weeks), or i n some cases a f t e r s u b c u l t u r e and the secondary procedures,  dishes.  Such c u l t u r e s were used  confluence (approximately 2  re-attainment of c o n f l u e n c e i n  A l l f e e d e r s were i r r a d i a t e d with 15  Gy  s e c t i o n 9, below) to a b l a t e r e s i d u a l hemopoiesis,  of " t e s t " c e l l s .  Assays  of such i r r a d i a t e d  (see prior  p r e - e s t a b l i s h e d adherent  irradiation to a d d i t i o n layers  62 consistently  showed that they contained no detectable colony-forming  hemopoietic progenitor  (D)  cells.  Cis-Hydroxy-L-Proline (CHP) Experiments  Long-term marrow cultures were i n i t i a t e d and maintained in the usual way, except that l i g h t density mononuclear c e l l s rather than whole aspirate or marrow buffy coat c e l l s were used to i n i t i a t e  the cultures,  the step to remove  c e l l s >1.077g/cm after 3-4 days was omitted, and cultures were placed from the 3  beginning at 33°C.  Cultures containing CHP (Sigma) were i n i t i a t e d and fed with  a growth medium containing a-medium prepared without proline or l y s i n e . Control cultures were maintained in the same medium but without CHP and with reconstituted levels of proline and l y s i n e .  After 4-5 weeks, nonadherent c e l l s  were removed and the adherent layer harvested by t r y p s i n i z a t i o n . were i n i t i a t e d by adding marrow c e l l s into dishes containing irradiated feeders prepared as described above. way as cultures set up in fresh dishes without  3)  Some cultures  pre-established  These were handled in the same feeders.  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). of  10  5  Unless specified otherwise, cells/  c e l l s were plated at a f i n a l concentration  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  in  the case of f r e s h marrow and  blood  ( e r y t h r o i d c o l o n i e s c o n t a i n i n g 1-2  c e l l assays where CFU-E and  and  r e s p e c t i v e l y ) were scored a f t e r 1 1/2  3-8  mature BFU-E  c l u s t e r s of e r y t h r o b l a s t s ,  weeks p r i o r  to f i n a l counts and  other  p r o g e n i t o r s c l a s s e s a f t e r 3 weeks.  (B)  Colony-Forming U n i t - F i b r o b l a s t (CFU-F) Assay  Essentially, f o l l o w e d except  the method d e s c r i b e d by C a s t r o - M a l a s p i n a  that the medium used was  p r o l i n e or l y s i n e p l u s 10% concentrations according  of CHP  in a i r . twice  without  to which v a r y i n g  design.  In b r i e f , marrow b u f f y coat or  light  g/cm ) c e l l s were p l a t e d i n t i s s u e c u l t u r e d i s h e s at 8.5  the end  x  3  undisturbed At  was  or v a r i o u s growth f a c t o r p r e p a r a t i o n s were then added,  cells/mm2/2.6 u l or or 2.6 incubated  a-medium made up w i t h or  f e t a l c a l f serum (FCS)  to the experimental  d e n s i t y (< 1.077  et a l (9)  x 10^  cells/mm2/2.6 y l r e s p e c t i v e l y , and  then  f o r 12 days at 37°C i n a h u m i d i f i e d atmosphere of 5%  of t h i s  i n i c e c o l d PBS,  and  time, the medium was  10^  CO2  decanted, the c u l t u r e s r i n s e d  adherent c o l o n i e s f i x e d , a i r d r i e d , s t a i n e d  with  May-Griinwald-Giemsa.  (C)  To  Assays f o r P r o d u c t i o n  test  suspension  f o r Interleukin-16 (IL-10)  of 2 x 10^  induced  c e l l s / m l were placed  p l a t e s ( L i n b r o , McLean, VA) recombinant human I L - 1 0 desired  of Hemopoietic Growth F a c t o r  and  (Biogen,  production  (HGF)  of HGF,  i n t o s i n g l e w e l l s of 24  24 hours l a t e r , a s m a l l amount of Geneva) or d i l u e n t was  f i n a l c o n c e n t r a t i o n of IL-10  .  Conditioned  2 ml  of  multi-well purified  added to g i v e  the  media were h a r v e s t e d  after  64 a f u r t h e r 24 hours, spun a t 1,200 rpm f o r 10 minutes and the supernatant at  stored  -20°C. To  test  i f IL-16 s t i m u l a t e d  SV-40 immortalized  cell  b i o l o g i c a l a c t i v i t y capable o f s t i m u l a t i n g marrow stromal used the CFU-F assay with or without  lines cell  produced progenitors  10% IL-16 induced c o n d i t i o n e d  medium.  I As  c o n t r o l s , we used growth medium alone (supplemented with 10% FCS), a crude source o f hemopoietic growth f a c t o r s (PHA-LCM 10%) and recombinant GM-CSF a t a final  concentration  described  o f 8 ng/ml.  above with the  To i n i t i a t e CFU-F assays I used the method  exception  that CHP was omitted and only  regular  a-medium supplemented with 10% FCS was used. Colony-stimulating standard  a c t i v i t y was t e s t e d by a d d i t i o n o f c o n d i t i o n e d  methylcellulose  marrow c e l l s / m l .  cultures containing  3 to 5 x 10^ non-adherent human  To reduce the background i n n e g a t i v e  controls  c o n t a i n i n g no exogenous source o f growth f a c t o r ) and to o b t a i n progenitor  numbers at low c e l l c o n c e n t r a t i o n s ,  from the l i g h t  density  long-term human marrow c u l t u r e s (10). stimulated  leukocyte  (Biogen, Geneva). 3 units/ml (11).  conditioned  (cultures useful  the t a r g e t c e l l s were  (<1.077 gm/cm ) non-adherent c e l l 3  media to  obtained  f r a c t i o n o f 1 week-old  P o s i t i v e controls included  agar-  medium (LCM) and recombinant human GM-CSF  Unless s p e c i f i e d otherwise, a l l c u l t u r e s  contained  o f p a r t i a l l y p u r i f i e d human u r i n a r y e r y t h r o p o i e t i n (>1,000 units/mg)  65  4)  SV-40 VIRUS PREPARATION AND ASSAY  (A)  P r e p a r a t i o n o f High T i t e r V i r u s  Stock  A f r e e z e - d r i e d p r e p a r a t i o n o f SV-40 v i r u s , s t r a i n A2895, was o b t a i n e d from ATCC ( R o c k v i l l e , MD), r e c o n s t i t u t e d and the t i t r e determined by plaque assay on c o n f l u e n t BSC-1 c e l l s  (6).  A 2 0 0 - f o l d h i g h e r t i t r e s t o c k was then  prepared by i n f e c t i n g new, c o n f l u e n t BSC-1 monolayers infection  (0.01 p f u / c e l l ) with the d i l u t e d  a t a low m u l t i p l i c i t y o f  l y s a t e from a s i n g l e plaque.  The  t i t r e o f the supernatant o b t a i n e d from t h i s i n f e c t i o n was 8 x 10*° pfu/ml (see Figure 8).  (B)  I t was a l i q u o t e d  i n t o m u l t i p l e v i a l s and s t o r e d a t -20°C.  V i r u s Plaque Assay  The plaque assay d e s c r i b e d by T u r l e r and Beard (6) was used. serial  1 0 - f o l d d i l u t i o n s o f the v i r u s were prepared i n T r i s - D u l b e c c o ' s b u f f e r  (6) supplemented  w i t h 2% FCS and 100 y l was used to i n f e c t  of A f r i c a n Green Monkey Kidney C e l l s dishes.  In b r i e f ,  (BSC-1 c e l l s ) i n 60 mm  confluent tissue  monolayers  culture  As c o n t r o l s , some were mock i n f e c t e d with lOOul o f T r i s - D u l b e c c o ' s  b u f f e r - 2 % FCS.  A f t e r a 90 minute a d s o r b t i o n p e r i o d , f r e s h 10% DMEM medium was  added and the d i s h e s were r e t u r n e d to 37°C f o r the next 15 hours.  The medium  was then r e p l a c e d with 5 ml of 0.9% agar made up i n the same medium.  After  the  agar had s e t , c u l t u r e s were then r e t u r n e d to the 3 7 ° C i n c u b a t o r f o r another 7 days.  Plaques were counted u s i n g an i n v e r t e d  phase c o n t r a s t microscope and  titers  (pfu/ml) o f o r i g i n a l v i r u s s t o c k s c a l c u l a t e d .  FIGURE 8.  T i t r a t i o n of SV-40 V i r u s Stock on BSC-1 C e l l s .  67  (C)  Assay F o r Large T A n t i g e n  The presence o f i n t r a n u c l e a r T - a n t i g e n was d e t e c t e d by u s i n g the h i g h l y s p e c i f i c monoclonal described  immunofluorescence  Pab 1626 ( 1 2 ) . The method i s f u l l y  i n s e c t i o n 5.  (D)  T r a n s f o r m a t i o n Assay o f SV-40 V i r u s on Mouse NIH-3T3  F r e s h l y e s t a b l i s h e d NIH-3T3 c e l l s were seeded a concentration of 2 x 1 0  4  Cells  i n 60 mm d i s h e s ( F a l c o n ) a t  c e l l s / m l i n 4.5 ml of Dulbecco's MEM w i t h 10% FCS.  Twenty f o u r hours l a t e r 0.5 ml o f s e r i a l l y dilu.ted v i r u s was added w i t h 20 ug of p o l y b r e n e .  The medium was changed the f o l l o w i n g day and p l a t e s examined f o r  the presence o f dense c o l o n i e s of c e l l s a f t e r another 2 weeks. c o l o n i e s , p l a t e s were f i x e d i n 10% f o r m a l i n and s t a i n e d w i t h  To s c o r e  May-Grtinwald-  Giemsa.  5)  ANTISERA AND IMMUNOFLUORESCENCE  For immunofluorescence 22 x 22 mm c o v e r s l i p s , -20°C,  Hanks balanced s a l t (HFN), 1 hour.  microscopy, c e l l s were grown to s u b c o n f l u e n c e on  fixed  and then a i r - d r i e d  MEASUREMENTS  i n acetone/methanol  f o r 10 minutes  at room temperature.  s o l u t i o n supplemented  c e l l s were covered with the f i r s t  1:1 ( v / v ) f o r 5 minutes at After rinsing i n  with 2% FCS and 0.1% sodium antibody ( l i s t e d  azide  i n Table I I I ) f o r  To c o n t r o l f o r n o n - s p e c i f i c b i n d i n g , d u p l i c a t e s were i n c u b a t e d w i t h  HFN a l o n e .  A f t e r 3 s u c c e s s i v e washes i n HFN, an a p p r o p r i a t e second a n t i b o d y  ( e i t h e r FITC-conjugated goat anti-mouse  immunoglobulin,  (CBL, The N e t h e r l a n d s )  Table I I I .  O r i g i n , S p e c i f i c i t y and Source of A n t i b o d i e s Used f o r Inununophenotype Analyses  Cellular  Name  Specificity  Origin  Source  Dr. Furthmayr ( Y a l e , New Haven)  anti -laminin  Epithelial  and e n d o t h e l i a l  cells  Rabbit*  a n t i - c o l l a g e n IV  Epithelial  and e n d o t h e l i a l  cells  Rabbit*  anti -collagen I  Fibroblasts  a n t i -T200/LCA/anti-Leuk AH  Nucleated Hemopoietic  a n t i -leuMl (CD 15)  D i f f e r e n t i a t e d monocytes, granulocytes  Mouse" "  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 s t u d i e d but e x c l u d i n g a l l hemopoietic c e l l s  Mouse" "  Dr. C h r i s t o p h e r F r a n t z (Rochester, NY)  a n t i -Factor VIII  Factor V H I - r e l a t e d a n t i g e n  Rabbit*  DAKO c o r p o r a t i o n (Santa Barbara, CA)  SV-40 Large T a n t i g e n - p o s i t i v e c e l l s  Mouse" "  Ciba-Geigy L t d . (Basel, Switzerland)  Pab  1626 1 2  *Antisera. •Monoclonal  antibodies.  cells  n  n  Goat*  Southern B i o t e c h n o l o g y Assoc. (Birmingham, AL)  Mousey-  Becton-Dickinson Corp. (Mountain View, CA)  1  1  1  69 or FITC-conjugated  r a b b i t a n t i - g o a t immunoglobulin,  both a t a f i n a l d i l u t i o n of 1/80 minutes at room temperature.  was  added and  ( N o r d i c , The  the c e l l s  then i n c u b a t e d f o r 30  A f t e r 3 f u r t h e r washes i n HFN,  i n b u f f e r e d p o l y v i n y l a l c o h o l (13) and examined with a Z e i s s equipped  with U V - e p i i l l u m i n a t i o n .  were e v a l u a t e d . stained  c e l l s were mounted photomicroscope  For each antibody, a minimum of 400  To d e t e c t s u r f a c e a n t i g e n s f r e s h l y  w i t h a Becton  i o d i d e (2 ug/ml) a f t e r  D i c k i n s o n FACS IV equipped  and a b r i e f  the f i n a l wash, and  with a l o g a m p l i f i e r .  c o n t r o l s , c e l l s were l a b e l l e d with the second  then As  To e s t i m a t e the p r o p o r t i o n of p o s i t i v e , c e l l s ,  where the n e g a t i v e c o n t r o l and first  determined.  ( h i g h e r f l u o r e s c e n c e v a l u e s ) was  first  the channel  long-term  dishes. cell  then s u b t r a c t e d from  to the r i g h t  channel  the number of c e l l s i n  ( N a p i e r v i l l e , IL)  positive slide  However, e q u a l l y good r e s u l t s were obtained with Eahy.926 or HUV-EC-C coverslips.  S l i d e s were l i g h t l y  i n acetone-methanol (1:1), endogeneous peroxidase a c t i v i t y  by f i x a t i o n i n methanolic  hydrogen peroxide f o r 20 minutes,  i n c u b a t e d s e q u e n t i a l l y with the f o l l o w i n g reagents  and  (Dako Corp,  anti-rabbit  anti-serum,  p e r o x i d a s e - a n t i - p e r o x i d a s e complex.  r e v e a l e d f o l l o w i n g i n c u b a t i o n with a mixture  9 - e t h y l c a r b a z o l e , 0.3%  The  fixed  suppressed  slides Santa-Barbara,  CA): Normal swine serum, p o l y c l o n a l r a b b i t a n t i - F a c t o r V I I I anti-serum,  next  was  of the c r o s s o v e r p o i n t .  c u l t u r e s were set up i n Labteck  l i n e s grown on r e g u l a r 22 x 22 mm  (1 min.)  number  to the r i g h t of t h i s  For immunoperoxidase d e t e c t i o n of F a c t o r V I I I r e l a t e d a n t i g e n cells,  o n l y , or  the t e s t sample curves c r o s s e d each o t h e r  The number of n e g a t i v e c e l l s  the t e s t sample that a l s o f e l l  analysed  negative  F I T C - l a b e l l e d reagent  w i t h an i r r e l e v a n t monoclonal antibody of the same i s o t y p e as the reagent.  cells  t r y p s i n i z e d c e l l s were  i n suspension u s i n g the a p p r o p r i a t e i n d i r e c t procedure  exposure to propidium  Netherlands)  swine  reaction  was  of s u b s t r a t e , 3-amino-  hydrogen peroxide i n water, and 0.1M  acetate buffer,  70 pH 5.2.  S l i d e s were c o u n t e r s t a i n e d w i t h G i l l ' s hematoxylin  Orangeburg, N.Y.), diped s e v e r a l times i n ammonia water,  (Fisher,  r i n s e d , 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 c o v e r s l i p s , a i r d r i e d and then stained  f o r a l k a l i n e phosphatase  h i s t o c h e m i c a l methods  7)  and a c i d phosphatase  by e s t a b l i s h e d  (14,15).  TESTS FOR ANCHORAGE-INDEPENDENT GROWTH  T r y p s i n i z e d c e l l s were resuspended 3 x 10^ c e l l s / m l .  i n <x-2% to a f i n a l c o n c e n t r a t i o n o f  T e n - f o l d d i l u t i o n s were prepared i n some cases down to  c o n c e n t r a t i o n s 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 o f a-10% made v i s c o u s by the a d d i t i o n of 0.8% m e t h y l c e l l u l o s e (4,000 cps, Dow Chemical, Vancouver,  B.C.).  One ml c u l t u r e s were p l a t e d i n  35 mm d i s h e s ( G r e i n e r , FRG) and incubated at 37°C i n a f u l l y h u m i d i f i e d atmosphere c o n t a i n i n g 5% C02-  C o l o n i e s c o n t a i n i n g more than 50 c e l l s were  s c o r e d 20 days l a t e r w i t h an i n v e r t e d  8)  microscope.  TUMOR FORMATION  A t o t a l o f 0.1 to 1 x 1 0 s a l i n e were i n j e c t e d  6  SV-40 immortalized c e l l s  (MH2-SV) i n 100 u l of  i n t r a d e r m a l l y i n t o 4 female nu/nu mice.  a d d i t i o n a l animals were i n j e c t e d w i t h s a l i n e a l o n e .  As c o n t r o l s , 2  71  9)  IRRADIATION PROCEDURES  For c o c u l t i v a t i o n experiments, i r r a d i a t e d with 15-20 grays  (60Q  O  p r e e s t a b l i s h e d adherent  l a y e r s were  y rays or 250 KVp X - r a y s ) .  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 o f SV-40 immortalized l i n e s and normal marrow stromal c e l l s  i n culture,  f r e s h l y e s t a b l i s h e d l a y e r s were t r y p s i n i s e d and  washed twice i n growth medium.  Single c e l l  f i x e d number (10^-10^) o f c e l l s dispensed i c e except  during i r r a d i a t i o n  suspensions  i n test  (<2 minutes).  tubes  were prepared  ( F a l c o n ) and kept on  R a d i a t i o n (250 KVp X-rays)  g i v e n at a s i n g l e dose at the r a t e o f 5.2 Gy/min.  C e l l s were then  i n 7.5 ml o f f r e s h a-20% t i s s u e c u l t u r e medium, dispensed c u l t u r e d i s h e s and incubated a t 37°C f o r 10-14 days . i r r a d i a t i o n on colony forming c e l l s to c a l c u l a t e the c o n t r o l  10)  and a  was  resuspended  i n 60mm t i s s u e  To a s s e s s the e f f e c t s o f  the mean number o f c o l o n i e s formed was  used  the p l a t i n g e f f i c i e n c y and t h e i r s u r v i v i n g f r a c t i o n r e l a t i v e to cultures.  AUTORADIOGRAPHY  To determine adherent  cells,  the e f f e c t s o f SV-40 v i r u s on c o n t a c t - i n h i b i t e d marrow  10^ c e l l s from a t r y p s i n i z e d 4 week o l d normal long-term  marrow c u l t u r e were resuspended  i n 2.5 ml of Dulbecco's  35 mm F a l c o n t i s s u e c u l t u r e d i s h e s .  MEM and seeded  u  250 u l of SV-40 v i r u s  Twenty four hours l a t e r d i s h e s were r i n s e d ,  I s c o v e ' s medium supplemented with 15% FCS and returned to the more day.  Cell  l a y e r s were allowed  into  F i v e days a f t e r r e a c h i n g c o n f l u e n c e , the  medium was removed and c u l t u r e s incubated with or without s t o c k ( 1 0 * pfu/ml).  bone  to i n c o r p o r a t e HTdR at 3  c o n c e n t r a t i o n of 10uCi/ml and l a b e l l e d n u c l e i subsequently  a  covered with  i n c u b a t o r f o r one final  v i s u a l i z e d by  72  autoradiography.  C e l l s were r i n s e d w i t h T r i s - b u f f e r e d  3.7% formaldehyde  f o r 10 minutes, and washed s e q u e n t i a l l y w i t h 5%  t r i c h l o r o a c e t i c a c i d , water, and 70% e t h a n o l .  saline, fixed i n  After drying,  w i t h Kodak NTB2 photographic emulsion, d r i e d a g a i n , and l e f t 2 more days i n a d e s s i c a t i n g j a r . c e l l s stained was then  i n the dark f o r  The emulsion was then developed and the  b r i e f l y w i t h 2% methyl green.  calculated.  d i s h e s were coated  The percentage of l a b e l l e d n u c l e i  73  REFERENCES 1.  Hopps HE, Bernheim BC, N i s a l a k 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 d e r i v e d 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. Exp C e l l Res  3.  Hoshi H, McKeehan WL. B r a i n and l i v e r c e l l - d e r i v e d f a c t o r s are r e q u i r e d f o r growth of human e n d o t h e l i a l c e l l s i n serum-free c u l t u r e . Proc N a t l Acad S c i USA 81: 6413, 1984.  4.  Hogge DE, Coulombel L, Kalousek DK, Eaves CJ, Eaves AC. Nonclonal hemopoietic p r o g e n i t o r s i n a G6PD h e t e r o z y g o t e with c h r o n i c myelogeneous leukemia r e v e a l e d a f t e r long-term marrow c u l t u r e . American J o u r n a l of Hematology 24: 389, 1987.  5.  E d g e l l CJS, McDonald CC, Graham JB. Permanent c e l l l i n e e x p r e s s i n g human f a c t o r V H I - r e l a t e d a n t i g e n e s t a b l i s h e d by h y b r i d i z a t i o n . Proc N a t l Acad S c i USA 80: 3734, 1983.  6.  T u r l e r H, Beard P. Simian v i r u s 40 and polyoma v i r u s : Growth, t i t r a t i o n , t r a n s f o r m a t i o n and p u r i f i c a t i o n of v i r a l components. In: Maky BNJ ( e d ) , " V i r o l o g y a p r a c t i c a l approach", Oxford, IRL p r e s s , ppl69, 1985.  7.  Coulombel L, Eaves AC, Eaves CJ. Enzymatic treatment of long-term human marrow c u l t u r e r e v e a l s the p r e f e r e n t i a l l o c a t i o n of p r i m i t i v e h e m o p o i e t i c p r o g e n i t o r s i n the adherent l a y e r . Blood 62: 291, 1983.  8.  Gregory CJ, Eaves AC. Three stages of e r y t h r o p o i e t i c p r o g e n i t o r cell d i f f e r e n t i a t i o n d i s t i n g u i s h e d by a number of p h y s i c a l and b i o l o g i c a l properties. Blood 51: 527, 1978.  9.  C a s t r o - M a l a s p i n a 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. C h a r a c t e r i z a t i o n of human bone marrow f i b r o b l a s t c o l o n y - f o r m i n g c e l l s (CFU-F) and t h e i r progeny. Blood 56: 289, 1980.  10.  Eaves CJ, Eaves AC. E r y t h r o p o i e t i n (Ep) dose-response curves f o r three c l a s s e s of e r y t h r o i d p r o g e n i t o r s i n normal human marrow and i n p a t i e n t 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 u r i n e : A simple one-step procedure f o r o b t a i n i n g e r y t h r o p o i e t i n s u i t a b l e f o r i n v i t r o e r y t h r o p o i e t i c p r o g e n i t o r assays. Br J Haematol 58: 533, 1984.  12.  B a l l RK, S e i g l B, Q u e l l h o r s t S, Brandner G, Braun DG. Monoclonal a n t i b o d i e s a g a i n s t s i m i a n v i r u s 40 n u c l e a r l a r g e T tumor a n t i g e n : E p i t o p e mapping of papovavirus c r o s s r e a c t i o n and c e l l s u r f a c e s t a i n i n g . Embo J 7: 1485, 1984.  The 37:  l i m i t e d i n 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 . 614, 1965.  74 13.  L e n e t t e DA. microscopy.  An improved mounting medium f o r immunofluorescence Am J C l i n P a t h o l 69: 647, 1978.  14.  Ackerman GA. S u b s t i t u t e d n a p h t o l AS phosphate d e r i v a t i v e s f o r the l o c a l i z a t i o n o f l e u k o c y t e 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. A c i d phosphatase isoenzyme i n human l e u k o c y t e s i n normal and p a t h o l o g i c a l c o n d i t i o n s . J Histochem Cytochem 18: 473, 1970.  75  CHAPTER  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 v i t r o (4,5,6) studies indicating that fixed, i . e .  non-circulating,  elements are involved in the regulation of hemopoiesis  mesenchymal  in the marrow.  Such  c e l l s are a major component of the adherent layer of long-term marrow cultures, which serves as an a t t r a c t i v e model for in v i t r o analyses since i t hemopoiesis  allows  to be maintained for extensive periods of time (months) i n the  absence of exogenously added hemopoietic growth factors.  However, since  the  adherent layer of long-term marrow cultures consists of a d i v e r s i t y of c e l l types of both mesenchymal (e.g. and hemopoietic o r i g i n (e.g.  fibroblasts, adipocytes and endothelial  macrophages and progenitors)  cells)  (7,8,9)  reconstruction experiments are needed to delineate both the nature and the c e l l u l a r o r i g i n of the factors that needed for the maintenance and regulated turnover of primitive hemopoietic  cells.  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 c e l l s present in the adherent layer of longterm bone marrow cultures.  A prerequisite for assaying such a function rests  on the a v a i l a b i l i t y of a hemopoietic target population that is free of mesenchymal c e l l s but nevertheless requires the establishment  of close  76  i n t e r a c t i o n s w i t h non-hemopoietic s t r o m a l  cells  f o r long-term s u r v i v a l i n  culture. S e v e r a l s t r a t e g i e s to o b t a i n such p o p u l a t i o n s were e x p l o r e d : l i g h t - d e n s i t y non-adherent c e l l s  Isolation of  from h e m o p o i e t i c a l l y a c t i v e long-term bone  marrow c u l t u r e s ( 1 0 ) , p h y s i c a l s e p a r a t i o n of f r e s h bone marrow c e l l s on n y l o n wool columns ( 1 1 ) , immunological purging combination of monoclonal antibody  of bone marrow c e l l s w i t h a  6.19 and complement (12) and, m o d i f i c a t i o n  of the c u l t u r e c o n d i t i o n s by the a d d i t i o n of D - v a l i n e  to the medium ( 1 3 ) . None  of these approaches c o u l d reduce to a c c e p t a b l e  the p r o l i f e r a t i o n of  r e s i d u a l mesenchymal c e l l s present approach u t i l i z e d P e r i p h e r a l blood  represents  A more s u c c e s s f u l  population.  an a t t r a c t i v e o p t i o n s i n c e m u l t i p o t e n t i a l and h i g h p r o g e n i t o r a r e present  at a  reasonable  whereas mesenchymal c e l l s a r e t o t a l l y absent (14,15).  investigated enriched  i n unseparated marrows.  p e r i p h e r a l blood as a p o t e n t i a l t a r g e t  p r o l i f e r a t i v e p o t e n t i a l myeloid frequency  levels  the p o s s i b i l i t y  in sufficient  amount  that p r o g e n i t o r c e l l s  Thus, I  from t h i s source  c o u l d be  to i n i t i a t e c u l t u r e s on i r r a d i a t e d p r e -  e s t a b l i s h e d adherent l a y e r s . In the second approach, I sought (CHP), a r e l a t i v e l y to  selectively  specific  inhibit  to determine i f C i s - 4 - h y d r o x y - L - p r o l i n e  i n h i b i t o r of c o l l a g e n s y n t h e s i s  the mesenchymal c e l l  suspensions and t h e r e f o r e a l l o w marrow hemopoietic progenitors.  component of marrow  to be used as a simple  Although p r e l i m i n a r y r e s u l t s with  monoclonal a n t i b o d i e s have suggested p r o g e n i t o r s can now be o b t a i n e d ,  that h i g h l y p u r i f i e d  and these might  and had the p o t e n t i a l to a v o i d  considerable v a r i a b i l i t y  source o f  appropriate  hemopoietic  that i t was  the tremendous c e l l  t y p i c a l l y encountered  cell  then be of some value  experiments ( 1 7 ) , the use of CHP had the a t t r a c t i o n simpler  (16) c o u l d be used  in c e l l  for  such  technically  l o s s e s and  separation  experiments.  77  Recently,  CHP was r e p o r t e d  i n murine long-term  on  progenitor c e l l s  the p o s s i b i l i t y  (18).  In the present  To address  i n both CFU-F assays  study, I  this question, I f i r s t cells  and i n the long-term  evaluated  toxic  to human hemopoietic  supplemented medium should be used to a s s e s s cells  to support  hemopoietic  2)  from human marrow as  marrow c u l t u r e system.  c e l l s present  i n the same c u l t u r e s .  t h e r e f o r e be u s e f u l to enable  CHP  that a r e CHP  unseparated  marrow to  the p o t e n t i a l a b i l i t y of d e f i n e d p o p u l a t i o n s o f mesenchymal the long-term  progenitor  maintenance and p r o l i f e r a t i o n of human  cells.  RESULTS  (A)  C u l t u r e s o f P e r i p h e r a l Blood C e l l s on Normal Marrow Adherent  L i g h t d e n s i t y (<1.077 gm/cm ) p e r i p h e r a l blood 3  cells  effect  the e f f e c t o f  proved to be an e f f e c t i v e i n h i b i t o r o f these c e l l s at c o n c e n t r a t i o n s not  to c o -  r e g u l a t o r y f u n c t i o n s o f human marrow  CHP on the p r o l i f e r a t i o n o f c o l l a g e n - p r o d u c i n g assessed  toxic  that CHP might have a s i m i l a r d i f f e r e n t i a l  the p r o l i f e r a t i v e and stem c e l l  mesenchymal c e l l s .  the formation o f an adherent l a y e r  marrow c u l t u r e s at doses that a r e not d i r e c t l y  e x i s t i n g hemopoietic investigated  to prevent  ( t o e n r i c h f o r myeloid  c u l t u r e s by s e e d i n g  the c e l l s  c e l l s were d e p l e t e d o f T  p r o g e n i t o r s ) and then used to i n i t i a t e into dishes containing a confluent,  adherent l a y e r from a p r e v i o u s l y e s t a b l i s h e d long-term new d i s h e s without  such a " f e e d e r " as c o n t r o l s .  number o f non-adherent c e l l s  long-term  irradiated  marrow c u l t u r e , or i n t o  The t o t a l numbers o f n u c l e a t e d  c e l l s and c l o n o g e n i c e r y t h r o i d and g r a n u l o p o i e t i c p r o g e n i t o r s present c u l t u r e s a r e shown on F i g u r e 9.  Layer  i n these  In the presence of a marrow adherent l a y e r the  ( p a n e l a) and c l o n o g e n i c  progenitors  78  I  10"!  i  i  i  1  2  3  i 4  i 5  i 6  i 7  Weeks in Culture  i I 8  1 I  i  i  i  1  2  3  i 4  i 5  i 6  i 7  Weeks in Culture  8  1—I  1I  1  1  1  1  2  1  3  4  1  1  5  6  1—J-  7  8  Weeks in Culture  I  i  FIGURE 9.  • Comparison of T o t a l C e l l and P r o g e n i t o r Content of Long-Term Normal PBL C u l t u r e s I n i t i a t e d With ( S o l i d Symbols) or Without (Open T r i a n g l e s ) a P r e - E s t a b l i s h e d Normal Marrow Feeder. C i r c l e s - data f o r non-adherent f r a c t i o n s of c u l t u r e s with f e e d e r s assessed weekly. Squares - d a t a f o r adherent f r a c t i o n s of c u l t u r e s with f e e d e r s . T r i a n g l e s - d a t a f o r non-adherent f r a c t i o n s of c u l t u r e s without f e e d e r s (no adherent f r a c t i o n o b t a i n e d ) , from which no c e l l s were removed at each weekly medium change u n t i l the c u l t u r e was s a c r i f i c e d and which were used f o r p a r a l l e l c y c l i n g measurements (19). V a l u e s shown a r e the geometric means + 1 SEM from d a t a of 5 experiments.  79  ( p a n e l s b and c) s h a r p l y d e c l i n e d d u r i n g  the f i r s t  2-3 weeks, but a f t e r  3-4  weeks a p l a t e a u phase was reached and t h e r e a f t e r the numbers remained r e l a t i v e l y constant harvested  f o r the d u r a t i o n o f the c u l t u r e s .  a t 3 weeks, the m a j o r i t y o f the c l o n o g e n i c  the adherent l a y e r and v a l u e s were s u f f i c i e n t  When these  c u l t u r e s were  p r o g e n i t o r s were found i n  to a l l o w  their  proliferative  s t a t u s to be determined by Dr. J . Cashman ( 1 9 ) . These s t u d i e s r e v e a l e d an a l t e r n a t i n g p a t t e r n o f p r o l i f e r a t i o n and quiescence progenitor c e l l schedule.  types  Cultures  o f p r i m i t i v e but not mature  i n the adherent l a y e r as a f u n c t i o n o f the f e e d i n g  that were i n i t i a t e d  marrow adherent l a y e r a l s o contained  i n the absence o f a p r e - e s t a b l i s h e d  sufficient  c y c l i n g measurements to be performed.  p r o g e n i t o r s at 3 weeks to a l l o w  In these c u l t u r e s a l l p r o g e n i t o r s were  non-adherent and i n c o n t r a s t to c u l t u r e s with adherent l a y e r s a l l p r o g e n i t o r s , i n c l u d i n g those  c l a s s i f i e d as p r i m i t i v e were p r o l i f e r a t i n g ,  feeding pattern.  However, s i n c e the f e e d e r s had no e f f e c t  p r o g e n i t o r s present  on the number o f  a f t e r 3 weeks, c u l t u r e s had to be maintained  weeks to enable a d i f f e r e n t i a l e f f e c t observed.  i r r e s p e c t i v e o f the  of the marrow adherent l a y e r s to be  T h i s became i m p r a c t i c a l f o r r o u t i n e experiments due to the emergence  i n a number o f c u l t u r e s o f p o l y c l o n a l B-lymphoblastoid from c e l l s l a t e n t l y by o t h e r s  i n f e c t e d with E p s t e i n - B a r r  ( 2 0 ) . These c e l l s  c u l t u r e s where they  first  populations o r i g i n a t i n g  virus, a finding also  formed l a r g e (>10,000 c e l l s ) ,  d i f f u s e c o l o n i e s which  Microscopic  examination of plucked  c o l o n i e s r e v e a l e d a homogeneous p o p u l a t i o n of lymphoblastoid nucleo-cytoplasmic cytoplasm.  ratio,  c e l l s with a high  prominent n u c l e o l i , and a s m a l l rim o f b a s o p h i l i c  C e l l markers s t u d i e s were a l s o performed on these  IV) and t h e i r B - c e l l o r i g i n confirmed.  explored  reported  became n o t i c e a b l e i n m e t h y l c e l l u l o s e  sometimes overgrew the e n t i r e c u l t u r e .  Table  f o r at l e a s t 5  a l t e r n a t i v e sources  o f hemopoietic  c o l o n i e s (see  Because o f t h i s phenomenon I progenitors.  T a b l e IV.  Immunophenotypic C h a r a c t e r i z a t i o n of Lymphoblastoid  Marker Assessed  X Positive  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  Cells  81  (B)  E f f e c t o f CHP on Marrow Mesenchymal C e l l  T h i s was e v a l u a t e d i n two d i f f e r e n t  Proliferation  types o f experiments, one u s i n g the  CFU-F assay and one u s i n g the long-term marrow c u l t u r e system. both i n the number o f c e l l s d u r a t i o n o f time p r i o r employing of  initially  to assessment  differ  used to seed the c u l t u r e s , and i n the o f the CHP e f f e c t .  the CFU-F assay demonstrated  fibroblast  These  The experiments  a r e p r o d u c i b l e dose-dependent  inhibition  c o l o n y f o r m a t i o n i n c u l t u r e s c o n t a i n i n g CHP ( F i g u r e 10).  A d d i t i o n a l treatment groups i n c l u d e d  i n these experiments demonstrated  that  o m i s s i o n o f p r o l i n e and l y 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 o f f i b r o b l a s t  conversely, medium  that  c o l o n i e s o b t a i n e d i n the absence o f CHP and,  the r e s t o r a t i o n o f the p r o l i n e , and l y s i n e content o f the  to "normal" l e v e l s completely abrogated the CHP e f f e c t  ( d a t a not shown).  C o n c e n t r a t i o n s o f CHP above 50 ug/ml reduced marrow f i b r o b l a s t s c o l o n y numbers to  <50X o f c o n t r o l v a l u e s and at c o n c e n t r a t i o n s above 250 ug/ml, no f i b r o b l a s t  c o l o n i e s were seen. In  the second s e t of experiments, the e f f e c t o f CHP on adherent  f o r m a t i o n i n the long-term marrow c u l t u r e system was a s s e s s e d . experiments showed that CHP c o n c e n t r a t i o n s below 100 yg/ml, significantly  inhibitory  i n CFU-F assays, s t i l l  layer  Preliminary  although  allowed s i g n i f i c a n t  adherent  l a y e r f o r m a t i o n i n the long-term c u l t u r e system when t h i s was a s s e s s e d a f t e r 4-5 weeks. the  However, when the c o n c e n t r a t i o n of CHP was i n c r e a s e d  to 500 yg/ml,  f o r m a t i o n o f an adherent l a y e r was s e v e r e l y and i r r e v e r s i b l y i m p a i r e d .  T a b l e V shows the r e s u l t s of c e l l  counts performed on adherent  layers  f r o m p a r a l l e l c u l t u r e s i n i t i a t e d and maintained i n the presence o r absence o f CHP h a r v e s t e d a f t e r 4-5 weeks. these c u l t u r e s i s i l l u s t r a t e d  The dramatic d i f f e r e n c e i n the appearance o f i n F i g u r e 11.  82  FIGURE 10.  E f f e c t o f I n c r e a s i n g Doses o f CHP on the Number o f C o l o n i e s Obtained from CFU-F i n F r e s h Human Marrow. Values r e p r e s e n t the mean + 1 SEM f o r n o r m a l i z e d data (% of number o f c o l o n i e s 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).  T a b l e V.  E f f e c t o f CHP on Adherent  Layer Formation  No. of c e l l s i n the adherent at 4-5 weeks (x 1 0 * )  layer  -  Exp. no.  - CHP  + CHP (X of c o n t r o l without  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  CHP)  9.5 + 4.2  The d i f f e r e n c e between - CHP and + CHP i s s i g n i f i c a n t (p<0.05 u s i n g paired t-test (27)).  Photograph o f Two Long-Term Marrow C u l t u r e s , One I n i t i a t e d and M a i n t a i n e d i n R e g u l a r Medium ( L e f t ) , and One I n i t i a t e d and f CHP ( R i h" ° ' y - r e e Medium C o n t a i n i n g 500 ug/ml P  r  l  i  n  e  _  a n d  L  s i n e  p  85 (C)  Lack of a Direct Effect of CHP on Hemopoietic Progenitor Function  To investigate possible CHP effects on hemopoietic c e l l s ,  marrow c e l l s  were f i r s t incubated for 3 hours in p r o l i n e - , 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 c e l l s 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 c e l l s to CHP in the longterm marrow culture system was then evaluated.  Since the presence of CHP in  the medium was known to i n h i b i t 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 c e l l s themselves, since previous studies have indicatedthat  the long-term support of hemopoiesis  intact adherent layer (4,6,17,18).  in this system requires an  An additional group was therefore included  to allow any direct effects of CHP on the production or s u r v i v a l of hemopoietic c e l l s to be distinguished from potential indirect effects caused by CHP i n h i b i t i o n of mesenchymal c e l l s that might be essential the hemopoietic c e l l s . each experiment.  for the maintenance of  Thus, the following three conditions were tested in  The f i r s t two compared the presence and absence of CHP in  long-term marrow cultures i n i t i a t e d using the standard protocol, i . e . marrow c e l l s were seeded into new dishes in medium with or without 500 ug/ml CHP.  In  the third group, marrow c e l l s 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 o f E f f e c t o f Exposure o f Hemopoietic P r o g e n i t o r s Subsequent  Plating Efficiency  CHP Progenitor  CFU-E  BFU-E  CFU-G/M  CFU-G/E  to CHP on T h e i r  Exp no.  0  1  (ug/ml) 25  500  218  209  266  2  163  125  91  3  256  230  265  1  164  177  140  2  88  87  55  3  176  186  140  1  168  144  146  2  80  93  82  3  151  154  158  1  10  16  10  2  4  3  4  3  7  2  7  Each v a l u e shown r e p r e s e n t s the mean value from 2 r e p l i c a t e assay c u l t u r e s . L i g h t d e n s i t y mononuclear marrow c e l l s were incubated f o r 3 hours at 37°C i n the presence or absence o f CHP i n l y s i n e - , p r o l i n e - f r e e medium. At the end of the i n c u b a t i o n p e r i o d , c e l l s were washed once and the e q u i v a l e n t o f 10-> o f the o r i g i n a l c e l l s were p l a t e d i n standard 1.1 ml m e t h y l c e l l u l o s e a s s a y s .  87 p r e v i o u s l y e s t a b l i s h e d , r e g u l a r long-term marrow c u l t u r e and then the  irradiating  secondary adherent l a y e r produced w i t h 15 Gy to i n 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 ( 1 9 ) . I t has been our e x p e r i e n c e at the  T e r r y Fox L a b o r a t o r y that such s u b c u l t u r e d adherent l a y e r s a r e e q u i v a l e n t to primary adherent l a y e r s i n terms o f t h e i r a b i l i t y  to support and r e g u l a t e  long-  term human hemopoiesis, but a r e not a s s o c i a t e d w i t h the same problems of adherent l a y e r detachment f r e q u e n t l y encountered when such c o - c u l t u r e s a r e s e t up u s i n g primary adherent l a y e r s .  The number of p r o g e n i t o r s present i n each of  these t h r e e types o f long-term c u l t u r e s was assessed a f t e r 4-5 weeks. e f f e c t o f CHP on p r o g e n i t o r maintenance  i n c u l t u r e s e s t a b l i s h e d u s i n g the  s t a n d a r d p r o t o c o l ( i . e . no f e e d e r s ) i s shown i n F i g u r e 12. t h a t , i n the absence o f a f e e d e r , hemopoiesis was markedly c u l t u r e s c o n t a i n i n g CHP. hemopoiesis was e q u i v a l e n t feeders) (Table V I I ) . does not appear  The  I t can be seen inhibited in  However, when a p r e - e x i s t i n g feeder was p r o v i d e d , to that observed i n c o n t r o l c u l t u r e s (no CHP, no  Thus, the continuous presence o f even 500 ug/ml o f CHP  to be d i r e c t l y d e t r i m e n t a l to the s u r v i v a l ,  d i f f e r e n t i a t i o n o f hemopoietic  cells.  p r o l i f e r a t i o n or  88  100  80  O DC  60  Z  o o  20  0  BFU-E  FIGURE 12.  CFU-G/M  CFU-G/E  The E f f e c t s o f 500 ug/ml o f CHP on the Y i e l d of Hemopoietic P r o g e n i t o r s i n Long-Term Marrow C u l t u r e s Assessed 4-5 Weeks A f t e r I n i t i a t i o n . Values shown r e p r e s e n t the mean + 1 SEM f o r n o r m a l i z e d d a t a (% o f number o f p r o g e n i t o r s from both adherent and non-adherent f r a c t i o n s d e t e c t e d i n c o n t r o l c u l t u r e s without CHP f o r 3 d i f f e r e n t experiments ( d i f f e r e n t marrow samples). Data a r e from the same experiments shown i n T a b l e V.  Table VII.  Lack, o f E f f e c t o f 500 Ug/ml o f CHP on Hemopoiesis  C u l t u r e s I n i t i a t e d on P r e - E s t a b l i s h e d , I r r a d i a t e d Adherent  No. o f p r o g e n i t o r s per 10' c e l l s Progenitor type  CFU-G/M  BFU-E  CFU-GE  Exp. no.  i n Long-Term  Layers ( F e e d e r s )  initially  + CHP + Feeder  - CHP - Feeder  1  7,324  17,792  2  903  598  3  7,212  1,668  1,400  768  141  69  808  26  132  480  24  15  120  14  V a l u e s shown a r e the mean of 2 r e p l i c a t e assays from 1 c u l t u r e  seeded  group.  The d i f f e 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 u s i n g f a c t o r i a l a n a l y s i s o f v a r i a n c e (26) on l o g transformed v a l u e s ) .  90  3)  DISCUSSION  A number of l i n e s of evidence suggest  that n o n - c i r c u l a t i n g elements  o f the  marrow c o n s t i t u t e an e s s e n t i a l component of the long-term marrow c u l t u r e system.  In a d d i t i o n ,  c e l l s and  they suggest  that d i r e c t  p r i m i t i v e hemopoietic c e l l s may  mechanisms which a l l o w hemopoiesis  i n t e r a c t i o n s between s t r o m a l  be an important p a r t of the  to be maintained f o r many weeks i n v i t r o i n  a medium to which no hemopoietic growth f a c t o r s are added (4-7,21).  The  present s t u d i e s p r o v i d e a d d i t i o n a l evidence i n support of t h i s model i n the human system.  In a f i r s t  s e r i e s of experiments  p r o g e n i t o r s were used, maintenance was presence of an adherent  l a y e r and  (19) r e v e a l e d profound  of an adherent  advantage  CHP, c e l l s , was  CFU-F assay. hemopoiesis  i n v i t r o rendered c o u l d be  that i s s e l e c t i v e l y  this  approach  used.  toxic for collagen-producing  the e s t a b l i s h m e n t of long-term hemopoiesis  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 development of an adherent  ( 44-58% k i l l ) ,  together w i t h the s e l e c t i v e  F o r t u n a t e l y , an a l t e r n a t e approach  shown to i n h i b i t  (0-18%  However, p e r s i s t e n c e of p r o g e n i t o r s f o r  of a feeder l a y e r  a p r o l i n e analogue  layer  In c o n t r a s t , p r i m i t i v e p r o g e n i t o r s  of endogeneous l y m p h o b l a s t o i d c e l l s  impractical.  differences  (40-47% k i l l ) and q u i e s c e n c e  l a y e r were c o n t i n u o u s l y c y c l i n g  i r r e s p e c t i v e of the f e e d i n g s c h e d u l e . many weeks i n the absence  However,  P r i m i t i v e p r o g e n i t o r s i n the adherent  k i l l ) as a f u n c t i o n of the f e e d i n g p a t t e r n . the absence  i n the  to a c e r t a i n extent i n i t s absence.  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  in  myeloid  present i n c u l t u r e s i n i t i a t e d  c y c l i n g d a t a from a c o l l a b o r a t i v e experiment between the 2 types of c u l t u r e s .  i n which c i r c u l a t i n g  i n h i b i t o r y a c t i o n on c e l l s  l a y e r , at l e a s t  by human  that  some of which are d e t e c t e d by  T h i s c o n c l u s i o n i s based on the demonstration seen i n long-term human marrow c u l t u r e s  that  initiated  allow the  the f a i l u r e  of  and maintained i n  91 the presence of CHP at doses that inhibited the formation of an adherent l a y e r , 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 i b r o b l a s t l i k e c e l l s in the adherent layer are collagen-synthesizing c e l l s (22,23).  It  is therefore not surprising that CHP has a p a r t i c u l a r l y toxic effect on the p r o l i f e r a t i o n and establishment of these c e l l s in v i t r o .  The fact that the  same concentration of CHP did not i n h i b i t hemopoiesis in the presence of a preestablished adherent layer suggests that collagen synthesis does not continue at high levels after the adherent layer becomes confluent or that i t s turnover is not c r u c i a l to hemopoiesis once a feeder layer is established. indicates that such concentrations of CHP do not adversely affect  Further i t the v i a b i l i t y  or regulatory function of human marrow adherent layer c e l l s once they have become established in v i t r o . system (18),  This confirms similar findings in the murine  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 p u r i f i c a t i o n 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 c e l l s to assay for long-term hemopoietic support in the human system needs to meet the requirements of a 4-5 week endpoint. two types of experiments.  This time can be deduced from  The f i r s t involves the assessment of the rate of  progenitor decline in long-term cultures i n i t i a t e d with peripheral blood c e l l s described above in which a d i f f e r e n t i a l effect is seen only after several weeks  92  at  a time when EBV-transformed  in cultures.  S i m i l a r l y when suspensions of marrow c e l l s a r e exposed  4-hydroperoxycyclyphosphamide of  l y m p h o b l a s t o i d c e l l s show a s e l e c t i v e  and  c l o n o g e n i c hemopoietic c e l l s ,  c e l l s from which  advantage to  thereby s e l e c t i v e l y d e p l e t e d of t h e i r content but not of the more p r i m i t i v e  hemopoietic  c l o n o g e n i c c e l l s are generated i n the long-term marrow c u l t u r e  system,  r e g e n e r a t i o n of the c l o n o g e n i c compartment under these c o n d i t i o n s can  be seen  to r e q u i r e approximately 4-5  c e l l suspension to be used candidate regulatory c e l l least  weeks (24,25).  Thus any  test  hemopoietic  to assay the hemopoietic s u p p o r t i v e f u n c t i o n of a type must be a b l e to generate c l o n o g e n i c c e l l s  f o r at  t h i s l e n g t h of time and a l s o show a f a i l u r e of endogenous s u p p o r t i v e  f u n c t i o n f o r an e q u i v a l e n t p e r i o d . requirement  T h i s p l a c e s an a d d i t i o n a l  on the p u r i t y of the t e s t c e l l s ,  stringent  s i n c e a very s m a l l number o f  s t r o m a l c e l l p r e c u r s o r s over 4 to 5 weeks can r e c o n s t i t u t e an adherent e x p e r i e n c e t h i s a l s o p r e c l u d e s the use of non-adherent  layer.  In  my  or  1 week o l d long-term c u l t u r e c e l l s of human marrow o r i g i n f o r such a s s a y s .  On  the other hand, i t appears  a  simple and r e p r o d u c i b l e method f o r s e l e c t i v e l y  that the use of CHP  component of f r e s h human marrow  samples.  f r a c t i o n s of f r e s h  supplemented  media may  offer  inactivating  the s t r o m a l  cell  93  REFERENCES 1.  McCulloch EA, S i m i n o v i t c h L, T i l l J E , R u s s e l l ES, B e r n s t e i n SE. The c e l l u l a r b a s i s o f the g e n e t i c a l l y determined hemopoietic d e f e c t i n anaemic mice o f genotype S l / S l . Blood 26: 399, 1965. d  2.  B e r n s t e i n SE. T i s s u e t r a n s p l a n t a t i o n as an a n a l y t i c and t h e r a p e u t i c i n h e r e d i t a r y anemias. Am J Surg 119: 448, 1970.  tool  3.  G i d a l i J , and L a j t h a LG. R e g u l a t i o n of haemopoietic stem c e l l t u r n o v e r i n p a r t i a l l y i r r a d i a t e d mice. C e l l T i s s u e K i n e t . 5: 147, 1972.  4.  Dexter TM, Spooncer E, Toksoz D, L a j t h a LG. 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McCarthy KF, Wientroub S, Hale M, and Reddi AH. E s t a b l i s h m e n t of h e m a t o p o i e t i c microenvironment i n the marrow of m a t r i x - i n d u c e d endochondral bone. Exp Hematol 12: 131, 1984.  24.  Siena S, C a s t r o - M a l a s p i n a H, G u l a t i SC, Lu L, C o l v i n M0, C l a r k s o n BC, O ' R e i l l y RJ, and Moore MAS, E f f e c t s of i n v i t r o p u r g i n g with 4-hydroperoxycyclophosphamide on the h e m a t o p o i e t i c and m i c r o e n v i r o n m e n t a l elements of human bone marrow. Blood 65: 655, 1985.  25.  Winton EF, and Colenda KW. Use of long-term human marrow c u l t u r e s to demonstrate p r o g e n i t o r c e l l p r e c u r s o r s i n marrow t r e a t e d with 4-hydroperoxycyclophosphamide. Exp Hematol 15: 710, 1987.  26.  Sokal RR, R o h l f F J . Biometry, The p r i n c i p l e s and p r a c t i c e of s t a t i s t i c s i n b i o l o g i c a l r e s e a r c h , second e d i t i o n , U.H. Freeman and Company, San F r a n c i s c o , 1981.  27.  Sokal RR, R o h l f F J . Biometry, The p r i n c i p l e s and p r a c t i c e of s t a t i s t i c s i n b i o l o g i c a l r e s e a r c h , second e d i t i o n , W.H. Freeman and Company, San F r a n c i s c o , 1981.  the  95 CHAPTER  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 c o n t r o l o f many a s p e c t s o f hemopoiesis  i s r e g u l a t e d l o c a l l y w i t h i n the marrow ( 1 ) and may i n v o l v e the a c t i v a t i o n o f hemopoietic  growth f a c t o r gene e x p r e s s i o n by r e s i d e n t non-hemopoietic  that c o n s t i t u t e the marrow stroma ( 2 , 3 ) .  Although  some c o n t r o v e r s y  to the o r i g i n o f these c e l l s and t h e i r o n t o l o g i c a l r e l a t i o n h i e r a r c h y , s t u d i e s i n animal in  e x i s t s as  to the hemopoietic  models i n d i c a t e an e a r l y e m b r y o l o g i c a l  the mesenchymal and hemopoietic  cells  components of the marrow ( 4 ) .  divergence  More r e c e n t  experiments have e s t a b l i s h e d that mesenchymal c e l l s ' o f donor o r i g i n a r e not normally  d e t e c t a b l e i n the marrow o f t r a n s p l a n t r e c i p i e n t s  e a r l i e r data circulate  i n mice i n d i c a t i n g  (8,9).  (5,6,7)  confirming  that these c e l l s and t h e i r p r e c u r s o r s do not  On the other hand c e l l s with  the phenotypic  markers o f  f i b r o b l a s t s , a d i p o c y t e s and/or e n d o t h e l i a l c e l l s can be r e a d i l y o b t a i n e d as expanded p o p u l a t i o n s  i n v a r i o u s types of human marrow c u l t u r e systems.  These  i n c l u d e c u l t u r e s i n i t i a t e d by i n c u b a t i n g marrow c e l l s a t r e l a t i v e l y low concentrations s u i t a b l e f o r obtaining isolated  f i b r o b l a s t - l i k e c o l o n i e s (CFU-F  ( 1 0 ) , CFU-RF ( 6 ) o r CFU-ST ( 1 1 ) a s s a y s ) , as w e l l as c u l t u r e s i n i t i a t e d somewhat h i g h e r c o n c e n t r a t i o n s of marrow c e l l s . formation support  The l a t t e r  o f a complex adherent l a y e r of mesenchymal c e l l s  p r i m i t i v e human hemopoietic  differentiation  result  i n the  that a r e a b l e  p r o g e n i t o r p r o l i f e r a t i o n and  f o r p e r i o d s of many weeks ( 1 2 ) .  with  to  96  More d e t a i l e d s t u d i e s o f the c y c l i n g s t a t u s o f very p r i m i t i v e p r o g e n i t o r s i n long-term human marrow c u l t u r e s have shown that  hemopoietic  the adherent  l a y e r p l a y s an important r o l e i n the mechanism that r e g u l a t e s whether the m a j o r i t y o f these c e l l s a r e q u i e s c e n t , as they a r e i n the marrow i n v i v o , or whether they a r e p r o l i f e r a t i n g i n response f o r the f i r s t  time, i t i s p o s s i b l e  to some p e r t u r b a t i o n (13,14).  to a n a l y z e i n v i t r o both p o s i t i v e and  n e g a t i v e l o o p s i n a r e g u l a t o r y network that c o n t r o l s p r i m i t i v e hemopoietic c e l l s .  Thus  the behaviour of very  Since long-term marrow c u l t u r e adherent  c o n t a i n a v a r i e t y o f c e l l s e x p r e s s i n g d i f f e r e n t mesenchymal c e l l including 6.19-antigen-positive c e l l s  layers  phenotypes,  (15,16), l i p i d - l a d e n a d i p o c y t e s and  F a c t o r 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 s t r u c t u r e s ( 1 ) , a l l o f which a r e normal  c o n s t i t u e n t s of the marrow stroma,  the long-term marrow c u l t u r e system components.  Because a l l o f these mesenchymal c e l l  i s o l a t i o n are required.  in vitro  types a r e known to e x h i b i t  (17), n o v e l s t r a t e g i e s f o r  We d e s c r i b e the use o f SV-40 f o r t h i s  A h i g h t i t e r v i r u s s t o c k was generated, for  to u t i l i z e  to i n v e s t i g a t e the r o l e o f s p e c i f i c s t r o m a l  o n l y a l i m i t e d p o t e n t i a l f o r expansion their  i t is difficult  tested  f o r b i o l o g i c a l a c t i v i t y and used  the i m m o r t a l i z a t i o n o f human marrow mesenchymal c e l l s  number o f i m m o r t a l i z e d , c l o n e d c e l l  purpose.  in vitro.  A large  l i n e s e x p r e s s i n g the d i f f e r e n t i a t e d  phenotype o f f i x e d bone marrow s t r o m a l c e l l s have been o b t a i n e d 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 f i n d i n g that  upon s t i m u l a t i o n , of s e c r e t i n g r e g u l a t o r y molecules p r i m i t i v e hemopoietic p r o g e n i t o r s .  These c e l l  these a r e c a p a b l e ,  that a r e a c t i v e on  l i n e s should be u s e f u l  f u r t h e r d e l i n e a t i o n o f the c e l l u l a r and molecular b a s i s of hemopoietic cell  regulation.  f o r the stem  97  2.  RESULTS  (A)  Transforming  The 3T3  P o t e n t i a l o f SV-40 V i r u s ans i t s E f f e c t on DNA  t r a n s f o r m i n g p o t e n t i a l o f SV-40 v i r u s was f i r s t  c e l l s which a r e known to be r e a d i l y  i n f e c t e d w i t h SV-40 v i r u s y i e l d e d transformed  foci  ( F i g u r e 13) at a  1 per 4 x l 0  of  t h i s v i r u s s t o c k on NIH-3T3 was 2.7 x 10^ focus forming To demonstrate a d i r e c t  intended effect  i n v i t r o (18).  f o c i whereas d i s h e s  of  3  c e l l s exposed.  t e s t e d on murine NIH-  i n f e c t e d and transformed  Mock i n f e c t e d c u l t u r e s d i d not show any transformed  Synthesis  frequency  From these experiments the t r a n s f o r m a t i o n  titer  unit/ml.  b i o l o g i c a l e f f e c t o f SV-40 that would v a l i d a t e i t s  use i n i m m o r t a l i z i n g human marrow stromal c e l l s , we examined i t s  on c e l l u l a r DNA s y n t h e s i s f o l l o w i n g a d d i t i o n o f v i r u s to c o n t a c t  inhibited layers.  c e l l s subcultured  from r e g u l a r long-term  Whereas the m a j o r i t y of the c e l l s  quiescent  i n mock i n f e c t e d  d a t a p r o v i d e d an i n d i c a t i o n  ( F i g u r e 14 panel B ) .  cloned human continuous  primary  these  lines.  l i n e s of SV-40 immortalized  cultures.  Since  t h i s p r e p a r a t i o n was use to  D e r i v a t i o n and Immunological C h a r a c t e r i z a t i o n of C e l l  Cloned  increase i n  that SV-40 v i r u s may be u s e f u l f o r the  i m m o r t a l i z a t i o n o f human marrow s t r o m a l c e l l s ,  (B)  c u l t u r e s remained  ( F i g u r e 14, panel A ) , i n f e c t e d c u l t u r e s showed a dramatic  ^H-Thymidine i n c o r p o r a t i o n 48 hours l a t e r  generate  marrow c u l t u r e s adherent  Both primary  c e l l s were i s o l a t e d  and f i r s t  Lines  from a v a r i e t y o f  passage adherent l a y e r s o f 3-4  98  FIGURE 13.  Transformed Focus of NIH-3T3 C e l l s .  99  FIGURE 14.  Autoradiograms o f C o n f l u e n t Marrow Adherent L a y e r s . Mock, i n f e c t e d l a y e r (upper p a n e l ) and SV-40 i n f e c t e d (lower panel).  layer  100  week o l d long-term human marrow c u l t u r e s were used f o r i n f e c t i o n s s i n c e are i n d i s t i n g u i s h a b l e i n t h e i r a b i l i t y progenitor obtained  to r e g u l a t e p r i m i t i v e hemopoietic  c y c l i n g i n the long-term c u l t u r e system (14).  L i n e s were a l s o  f o l l o w i n g i n f e c t i o n of " f i b r o b l a s t " monolayers e s t a b l i s h e d from  c o l o n i e s of human marrow o r i g i n , and HUV-EC-C e n d o t h e l i a l c e l l o r i g i n a l l y derived  from u m b i l i c a l v e i n endothelium.  i n f e c t i o n , expression the c e l l s values  monolayers  i n 45 + 23 % o f  i n the adherent l a y e r of long-term marrow c u l t u r e s (with  c o n s i s t e n t l y negative. c o u l d be seen to c o n t a i n  higher  c u l t u r e s ) , whereas mock-infected c e l l s were  Two to 3 weeks a f t e r i n f e c t i o n , f o c i of morphologically  low d e n s i t y  and r e f r a c t i l e  subcultures  a l t e r e d c e l l s at a frequency  of ~1 focus per 100 i n i t i a l l y T A g - p o s i t i v e marrow c e l l s . f o c i were more elongated  pooled  Twenty-four hours a f t e r  of the SV-40 l a r g e T a n t i g e n was d e t e c t e d  for infected fibroblast  fashion  these  The c e l l s  i n these  than normal and grew i n a d i s o r d e r e d  t y p i c a l o f c e l l s whose growth i s not contact  inhibited.  This  morphology was e x h i b i t e d by a l l transformed f o c i and was independent o f the type o f p o p u l a t i o n  initially  exposed to the v i r u s .  long-term marrow adherent l a y e r s a r e designated marrow f i b r o b l a s t s as CFUST, and l i n e s d e r i v e d From these three independently  types o f c e l l s ,  isolated.  been maintained c o n t i n u o u s l y  from  from e n d o t h e l i a l c e l l s as EC.  a t o t a l o f 13, 13, and 5 such l i n e s were  required  f o r periods  f o r f u r t h e r study.  frozen  Many l i n e s have  of months and, i n one case, (CFUST-16)  cells  r e s u l t s o f immunophenotyping s t u d i e s a r e summarized  lines  i n Table  i n a l l l i n e s were p o s i t i v e f o r the SV-40 l a r g e T a n t i g e n  f u n c t i o n a l i n t e g r a t e d SV-40 genome c o n s i s t e n t with All  l i n e s derived  over a year. The  All  as MH,  A f t e r c l o n i n g , each l i n e was expanded and then  i n DMSO and s t o r e d a t -70°C u n t i l  for  L i n e s d e r i v e d by i n f e c t i n g  VIII.  indicating a  t h e i r immortalized  t e s t e d (5/5) were a l s o p o s i t i v e f o r the s u r f a c e marker 6.19  state.  Table VIII.  H i s t o c h e m i c a l and Immunophenotypic P r o p e r t i e s o f SV-40 Transformed C e l l  Lines*  Line O r i g i n  Long-Term BM Adherent Layer C e l l s Marker Assessed  (MH2SV-C11)  BM F i b r o b l a s t Colony C e l l s  U m b i l i c a l Cord Endothelial Cells  (CFUST-C116)  (EC22)  Membrane 6.19 T200 LEU-MI LEU-M3  + -  + -  + + -  Cytoplasmic A c i d Phosphatase A l k a l i n e Phosphatase Factor VIII Collagen I C o l l a g e n IV Laminin  + + + +  + + + +  + + +  Nuclear SV-40 l a r g e T Ag  +  +  +  '  *Data shown a r e f o r r e p r e s e n t a t i v e l i n e s , f o r which the most complete documentation was o b t a i n e d . P o s i t i v e means that more than 30% of the c e l l s were p o s i t i v e . Negative means no p o s i t i v e c e l l s could be detected.  102  ( F i g u r e 15) which appears to r e a c t o n l y with mesechymal c e l l s (15,16).  Marrow-derived l i n e s were p h e n o t y p i c a l l y s i m i l a r ,  type o f c u l t u r e i n i t i a l l y  regardless  o f the  i n f e c t e d and were c o n s i s t e n t l y p o s i t i v e f o r a c i d  phosphatase, l a m i n i n and c o l l a g e n s cells  i n the marrow  type I and IV.  A significant  proportion of  i n the adherent l a y e r o f long-term human marrow c u l t u r e s a l s o show these  f e a t u r e s as do c e l l s produced by CFU-RF ( 6 ) ) . positive  f o r Factor V H I - r e l a t e d  antigen  Although I showed that  cells  can a l s o be found i n the adherent  l a y e r o f long-term marrow c u l t u r e s ( 1 ) , none of the marrow-derived transformants an  showed t h i s p r o p e r t y .  i n a b i l i t y o f SV-40 to immortalize  continued  expression  HUV-EC-C c e l l s . categories  of t h i s a n t i g e n  None o f the l i n e s ,  (by o r i g i n ) contained  T h i s c o u l d not, however, be a t t r i b u t e d to Factor V H I - p o s i t i v e i n l i n e s derived  positivity  ( i n the one l i n e  c o n s i s t e n t l y negative  (C)  The  detectable  t e s t e d ) , whereas u n t r e a t e d  lines  a m u l t i f u n c t i o n a l 90-100 Kd oncogenic p r o t e i n , (19).  In a d d i t i o n to i t s r o l e i n  i t e x e r t s numerous e f f e c t s on s u s c e p t i b l e host  i n c l u d i n g the s t i m u l a t i o n o f c e l l u l a r DNA s y n t h e s i s  documented.  HUV-EC-C c e l l s were  ( F i g u r e 16).  SV-40 l a r g e T a n t i g e n ,  A longer  f o r T200 ( F i g u r e 15).  o f HUV-EC-C c e l l s d i d a c t i v a t e weak T200  i s expressed i n a l l SV-40 transformed c e l l s  (20).  from a l l three  LeuMl or LeuM3 p o s i t i v e c e l l s .  C h a r a c t e r i s t i c s o f Transformed c e l l  virus replication,  f o l l o w i n g i n f e c t i o n of  including representatives  Marrow-derived l i n e s were a l s o c o n s i s t e n t l y n e g a t i v e However, SV-40 t r a n s f o r m a t i o n  c e l l s as shown by the  cells  even i n q u i e s c e n t  l a s t i n g e f f e c t o f SV-40 on these same c e l l s  T h i s can be seen as a decreased p o p u l a t i o n  cells  has a l s o now been  doubling  time  103  10-  1  10°  10  1  10  2  10  3  Fluorescence Intensity (arbitrary units)  FIGURE 15.  FACS P r o f i l e of CFUST-CL16 C e l l s S t a i n e d 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 t e s t sample i n each case. The d o t t e d l i n e shows the p r o f i l e of the c o r r e s p o n d i n g n e g a t i v e c o n t r o l sample.  104  FIGURE 16.  FACS P r o f i l e of a Suspension of Spontaneously Immortalized Lymphoblastoid Cells (Panel A ) , and HUVE-EC-E C e l l s 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  cells.  Capacity for anchorage-independent growth was tested by plating transformed and uninfected c e l l s in semi-solid medium.  SV-40 transformed lines  routinely formed d i s t i n c t colonies containing more than 100 c e l l s within 20 days in culture (Figure 18 A ) .  In contrast,  c e l l s from non-infected control  cultures yielded only a few small clusters of 4 to 8 c e l l s and did not form any colonies containing more than 50 c e l l s (Figure 18 B). plating efficiency  the  of the 3 lines tested remained high (>5%) and was  independent of c e l l concentration (Figure 19), colony-forming efficiency of different  (D)  After cloning,  although some v a r i a b i l i t y in the  lines was seen (data not shown).  Induction of Growth Factor Production  Recent reports have shown that fibroblasts and endothelial c e l l s of various tissue origins c o n s t i t u t i v e l y produce low or undetectable levels of hemopoietic growth factors,  but upon stimulation with various secretory  products of macrophages, including I L - 1 , hemopoietic growth factor production is rapidly and markedly enhanced (2,3,21,22).  This response  thus appears to be  a t i g h t l y regulated part of the functional program of a variety of mesenchymal c e l l populations. also stimulates  Recent data from the Terry Fox Lab have shown that  IL-lg  the production of hemopoietic growth factor production by c e l l s  in the adherent layer of long-term human marrow cultures (23). therefore of interest  to evaluate  It was  the a c t i v i t y of media conditioned by the  various transformed c e l l lines before and after exposure to IL-1(3. of two representative experiments are shown in Table IX.  The results  It can be seen that  addition of 12 units/ml IL-16 greatly enhanced the production and release of  106  FIGURE 17.  T r i t i a t e d - T h y m i d i n e Uptake (Panel A) o f SV-40 I n f e c t e d ( S o l i d L i n e s ) and U n i n f e c t e d (Broken L i n e s ) MH C e l l s .  107  FIGURE 17. . Growth Rate (Panel B) o f SV-40 I n f e c t e d ( S o l i d and U n i n f e c t e d (Broken L i n e s ) MH C e l l s .  Lines)  108  FIGURE 18.  A Colony o f 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 C u l t u r e 14 Days A f t e r Seeding the C u l t u r e s w i t h MH C e l l s I n f e c t e d With SV-40 V i r u s ( P a n e l A ) .  109  FIGURE 18.  C o n t r o l ( U n i n f e c t e d ) MH C e l l s F a i l e d Y i e l d Colonies (Panel B).  To  110  .  A n a l y s i s of the Clonogenic C a p a c i t y of MH2SV-CL1 C e l l s P l a t e d in Methylcellulose. Each data p o i n t r e p r e s e n t s the mean + 1 SEM of v a l u e s obtained i n each of two d i f f e r e n t experiments ~ (shown s e p a r a t e l y as c i r c l e s and t r i a n g l e s ) .  Ill T a b l e IX.  Evidence  f o r IL-16 Induced P r o d u c t i o n o f Hemopoietic  A c t i v i t y by R e p r e s e n t a t i v e  SV-40 Transformed Human C e l l  Erythroid Colonies  Addition  Exp 1  to M e t h y l c e l l u l o s e Assay  Colony-Stimulating  Exp 2  Granulocyte-Macrophage Colonies 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  EC22  0  2  9  2  EC22 + IL-16  9  4  97  26  Skin F i b r o b l a s t s  1  -  6  -  S k i n F i b r o b l a s t s + IL-16  2  -  56  -  No a d d i t i o n  0  0  0  0  IL-16  3  4  47  23  (1.2 u n i t s / m l )  99  M2-10B4 (mouse marrow f i b r o b l a s t  line)  -  3  -  12  M2-10B4 (mouse marrow f i b r o b l a s t + IL-16  line)  -  3  -  25  18  9  126  115  5  3  168  86  Human LCM (10%) GM-CSF (8 ng/ml)  Lines  * C e l l s were incubated with or without 12 u n i t s / m l of IL-16 f o r 24 hours as d e s c r i b e d i n the M a t e r i a l s and Methods. C o n d i t i o n e d media were added to m e t h y l c e l l u l o s e assays at a f i n a l c o n c e n t r a t i o n of 10% ( v / v ) . E r y t h r o i d c o l o n i e s i n c l u d e a l l c a t e g o r i e s (CFU-E p l u s BFU-E d e r i v e d ) . M2-10B4 c e l l s a r e a spontaneously immortalized cloned l i n e o f c e l l s p h e n o t y p i c a l l y s i m i l a r to the MH and CFUST l i n e s d e s c r i b e d here but d e r i v e d i n t h i s l a b o r a t o r y from a c u l 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 d i f f e r e n t i a t i o n lineages. tested in this way.  A l l showed this response.  A t o t a l of 15 lines were  Although there was some  v a r i a t i o n in the f i n a l a c t i v i t y of the conditioned media from different  lines  exposed to IL-10, an a c t i v i t y equivalent to or greater than 80 ng/ml of GM-CSF was often obtained.  In contrast, s i m i l a r l y prepared conditioned media obtained  from IL-16 stimulated marrow stromal lines of murine o r i g i n had no a c t i v i t y on human progenitors (Table IX).  This suggests that the weak a c t i v i t y sometimes  seen in controls given IL-16 alone was due to endogenous production of colonystimulating a c t i v i t y by co-existing c e l l s 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 a t t a i n maximal growth factor release (Figure 20).  Conditioned media were also assayed (Dr.  P.  Lansdorp, Terry Fox Laboratory) for IL-6 a c t i v i t y as measured by their a b i l i t y to stimulate ^H-thymidine incorporation into B13.29 c e l l s , c e l l l i n e that is s p e c i f i c a l l y responsive to IL-6 (24).  a murine hybridoma  Results from these  studies have indicated that the production of IL-6 b i o a c t i v i t y by the SV-40 transformed l i n e s , consistently  l i k e the production of colony-stimulating b i o a c t i v i t y was  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 c e l l s but also on target populations of widely diverse embryonic o r i g i n (25),  I also investigated  the p o s s i b i l i t y that medium  conditioned by SV-40 marrow immortalized lines might have b i o l o g i c a l a c t i v i t y on normal mesenchymal c e l l s .  As shown in Table X, conditioned media from one  113  FIGURE 20.  Hemopoietic C o l o n y - S t i m u l a t i n g A c t i v i t y o f Media C o n d i t i o n e d f o r 24 Hours by CFUST-CL16 o r MH2SV-CL1 C e l l s ( o r No C e l l s ) as a F u n c t i o n o f the C o n c e n t r a t i o n o f IL-16 Used as a S t i m u l a n t . The maximal number of c o l o n i e s o b t a i n a b l e from the p r o g e n i t o r s present i n the assay as i n d i c a t e d by s t i m u l a t i o n o f these w i t h an o p t i m a l c o n c e n t r a t i o n of recombinant human GM-CSF (8 ng/ml) i s a l s o shown f o r comparison. C e l l l i n e c o n d i t i o n e d media were present i n the m e t h y l c e l l u l o s e assays at a c o n c e n t r a t i o n o f 10% ( v / v ) .  114  T a b l e X. E f f e c t s o f C o n d i t i o n e d Medium o f an IL-1 S t i m u l a t e d , SV-40 Immortalized Marrow Stromal C e l l L i n e (CFUST-16) on CFU-F Formation  Addi t i o n ( f i n a l concentration i n CFU-F assay)  No. o f CFU-F/Dish Exp. (Day  1  Exp. 2  12)  1  Exp. 3  (Day 5) (Day 10)  13  10  58  Human PHA S t i m u l a t e d Leukocyte CM (10%)  29  24  62  CFUST-16 with IL-1 CM ( 1 0 % )  28  24  95  Medium  (a-10%)  2  CFUST-16 without IL-1 CM (10%) GM-CSF (8 ng/ml)  (Day 6)  8  14  7 29  iNo. o f days o f CFU-F colony growth p r i o r  22  to f i x a t i o n and  100  24  staining.  ^ C o n d i t i o n e d medium from c o n f l u e n t CFU-STC116 c e l l monolayers i n c u b a t e d f o r 24 hours w i t h or without 12 U/ml recombinant human IL-16 ( B i o g e n ) .  115  line  tested  (CFUST-16) shared w i t h GM-CSF the a b i l i t y  e f f i c i e n c y of f r e s h normal  (E)  Irradiation  bone marrow  that SV-40 c o u l d t r a n s f o r m human marrow s t r o m a l  their radiobiological properties, c e l l  were o b t a i n e d from normal irradiation.  fibroblasts.  studies  To examine the p o s s i b i l i t y c e l l s without a l t e r i n g  to s t i m u l a t e the p l a t i n g  and  survival  SV-40 transformed l i n e s a f t e r s i n g l e dose  In both cases, s u r v i v a l curves are e x p o n e n t i a l and  w i t h a DQ of 1.3  Gy  curves  shouldered  ( F i g u r e 21) which i s i n accord with p r e v i o u s l y r e p o r t e d  d a t a on c u l t u r e d bone marrow f i b r o b l a s t s (26).  3.  DISCUSSION  In proceeds  humans, hemopoiesis  i s normally r e s t r i c t e d  to the bone marrow where i t  i n c l o s e a s s o c i a t i o n w i t h as yet p o o r l y c h a r a c t e r i z e d  mesenchymal elements.  fixed  R e c e n t l y , an v i t r o model has been developed  long-term maintenance and  r e g u l a t e d turnover of p r i m i t i v e  p r o g e n i t o r s o c c u r s i n the absence  of exogenously  However, a key component of t h i s c u l t u r e system l a y e r c o n t a i n i n g a v a r i e t y of mesenchymal c e l l  i n which  hemopoietic  p r o v i d e d growth  factors.  i s a complex adherent  cell  types of marrow o r i g i n .  To  f a c i l i t a t e a n a l y s i s of the i n d i v i d u a l r o l e s of these v a r i o u s mesenchymal types, I used SV-40 v i r u s as an i m m o r t a l i z i n g agent. was  prepared and  i t s biological activity  mesenchymal c e l l s . c u l t u r e adherent  the  A high t i t e r  cell  virus stock  t e s t e d on both murine and human  L i n e s c o u l d be r e a d i l y o b t a i n e d from human long-term marrow  l a y e r s , and marrow f i b r o b l a s t  c e l l s with properties s i m i l a r  cultures.  to those e x h i b i t e d  Both of these c o n t a i n  by a l l marrow-derived  cell  116  FIGURE 21.  C e l l S u r v i v a l Curve f o r 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 e n d o t h e l i a l c e l l s EC CL22 (open diamond), F a c t o r V I I I p o s i t i v e c e l l h y b r i d 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 l i n e s 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 c e l l s have been published.  In one, phenotypic  characterization suggested a f i b r o b l a s t i c o r i g i n of the l i n e (27). other,  In the  the presence of "round" c e l l s 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  c e 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) HUV-EC-C endothelial c e l l s with SV-40 suggesting  transformation of  that SV-40 may cause some  phenotypic a l t e r a t i o n of mesenchymal c e l l antigen expression when used as a transforming agent.  This was not surprising since preliminary data have  indicated that SV-40 transformed marrow c e l l s were also grossly abnormal cytogenetically.  On the other hand, I have found in a collaborative study that  SV-40 transformed c e l l s are stimulated by GM-CSF (29) as was also noted by Singer et a l (28).  As mentioned e a r l i e r such responsiveness  has further been  demonstrated to be a feature of a variety of developmentally unrelated c e l l s of non-hemopoietic o r i g i n (25).  GM-CSF responsiveness  to indicate a close relationship to hemopoietic  can therefore not be used  cells.  SV-40 transformed c e l l s grew at a faster rate than non-infected c e l l s and showed other properties of transformed c e l l s such as loss of contact i n h i b i t i o n and  the a c q u i s i t i o n of anchorage-independence.  Interestingly,  this could not  be correlated with tumorigenicity since at least one l i n e when injected  into  4 nude mice f a i l e d to generate any tumors (data not shown), a finding also reported by others for SV-40 transformed c e l l s (30).  This could not be  118 correlated either with increased r a d i o s e n s i t i v i t y since both SV-infected and non-infected c e l l s had similar response to ionizing r a d i a t i o n . The a b i l i t y of transformants to form colonies  in semi-solid medium,  p a r t i c u l a r l y at very low c e l l concentrations was consistent with the l i k e l y single c e l l o r i g i n of the colonies obtained under these conditions.  This was  confirmed by assessment of a methylation sensitive r e s t r i c t i o n fragment polymorphism in the X-linked HPRT gene (31) a female heterozygote Laboratory).  length  in the DNA of a clone isolated from  (data not shown, obtained by Dr. A l i Turhan, Terry Fox  Thus, these studies have demonstrated the f e a s i b i l i t y of  generating a large number of clonal lines and subclones by exploiting a b i l i t y to form colonies  their  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 c e l l populations to show a marked induction of hemopoietic colony-stimulating factor production, including the.production of at least GM-CSF and I L - 6 .  This has been confirmed at the molecular l e v e l by Northern  B l o t t i n g 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  p r o l i f e r a t i o n of very primitive hemopoietic c e l l s in methylcellulose has been reported (33).  L a s t l y , the finding that medium conditioned by IL-16 stimulated  SV-40 transformed lines stimulates  the clonal growth of fresh normal  f i b r o b l a s t i c c e l l s is i n t r i g u i n g .  It raises  inflammatory c e l l s ,  monocytes for instance,  the tantalizing p o s s i b i l i t y  that  may recruit mesenchymal c e l l s  l o c a l l y to produce hemopoietic growth factors which could in turn participate  119 in various  physiopathologic  myelofibrosis  and  p o s s i b l y d e s m o p l a s t i c r e a c t i o n of tumors.  of permanent cloned regulated  ability  the r o l e that turnover.  processes i n c l u d i n g wound h e a l i n g , a t h e r o s c l e r o s i s ,  l i n e s of c e l l s with a stromal  to produce such f a c t o r s should  these c e l l s may  The  availability  phenotype that e x h i b i t  the  f a c i l i t a t e future studies  p l a y i n the c o n t r o l of hemopoietic stem  cell  of  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 c u l t u r e s 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.  2.  Z u c a l i 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 s t i m u l a t e s f i b r o b l a s t s to produce granulocyte-macrophage c o l o n y - s t i m u l a t i n g a c t i v i t y and p r o s t a g l a n d i n 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 marrowd e r i v e d f i b r o b l a s t s to produce m u l t i l i n e a g e h e m a t o p o i e t i c growth f a c t o r s . Exp Hematol 15: 983, 1987.  4.  Moore MAS. 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U n r e g u l a t e d p r o l i f e r a t i o n o f p r i m i t i v e c h r o n i c myeloid leukemia p r o g e n i t o r s i n the presence o f normal marrow adherent c e l l s . Proc N a t l Acad S c i USA 83: 5306, 1986.  15.  F r a n t z CN, Duerst RE, Ryan DH, C o n s t i n e LS, Gelsomino N, Rust L, Gregory P. A monoclonal a n t i - n e u r o b l a s t o m a a n t i b o d y that d i s c r i m i n a t e s between human nonhematopoietic and h e m a t o p o i e t i c c e l l types. Hybridoma 5: 297, 1986.  16.  Abboud CN, Duerst RE, F r a n t z CN, Ryan DH, L i e s v e l d J L , Brennan JK. of human f i b r o b l a s t c o l o n y - f o r m i n g c e l l s and e n d o t h e l i a l c e l l s by monoclonal a n t i b o d y (6-19) and complement. Blood 68: 1196, 1986.  17.  H a y f l i c k L, Morehead PS. The s e r i a l c u l t i v a t i o n o f human d i p l o i d strains. Exp C e l l Res 25: 585, 1961.  18.  Todaro JG, Green H. An assay f o r c e l l u l a r V i r o l o g y 23: 117, 1964  19.  Eddy BE, Borman GS, Grabbs GE, Young RD: I d e n t i f i c a t i o n o f the oncogenic substance i n Rhesus monkey kidney c e l l c u l t u r e s as s i m i a n v i r u s 40. V i r o l o g y 17:65, 1962  20.  Rigby PWJ, Lane DP: S t r u c t u r e and f u n c t i o n of s i m i a n v i r u s 40 l a r g e T a n t i g e n , i n K l e i n G ( e d ) : Advances i n V i r a l Oncology, V o l 3, New York, Raven P r e s s , p 31, 1983  21.  Munker R, Gasson J , Ogawa M, K o e f f l e r HP: Recombinant human TNF induces p r o d u c t i o n o f granulocyte-monocyte c o l o n y - s t i m u l a t i n g f a c t o r s . Nature 323:79, 1986  22.  Lee M, Segal GM, Bagby GC: I n t e r l e u k i n - 1 induces human bone marrowd e r i v e d f i b r o b l a s t s to produce m u l t i l i n e a g e h e m a t o p o i e t i c growth f a c t o r s . Exp Hematol 15:983, 1987  23.  Eaves AC, Eaves C J : Maintenance and p r o l i f e r a t i o n c o n t r o l o f p r i m i t i v e hemopoietic p r o g e n i t o r s i n long-term c u l t u r e s o f human marrow c e l l s . Blood C e l l s ( i n p r e s s )  24.  Aarden LA, Groot ER, Schapp 0L, Lansdorp PM. P r o d u c t i o n of hybridoma growth f a c t o r by human monocytes. Eur J Immunol 17: 1411, 1987.  25.  Baldwin GC, D i p e r s i o , J , Kaufman SE, Duan SG, Gasson JC. C h a r a c t e r i z a t i o n of human GM-CSF r e c e p t o r s on non-hematopoietic c e l l s . Blood 70 ( s u p p l 1) : 166a, 1987  26.  Laver J , E b e l l W, C a s t r o - M a l a s p i n a H. R a d i o b i o l o g i c a l p r o p e r t i e s of the human h e m a t o p o i e t i c microenviroment: C o n t r a s t i n g s e n s i t i v i t i e s of p r o l i f e r a t i v e c a p a c i t y and h e m a t o p o i e t i c f u n c t i o n to i n v i t r o i r r a d i a t i o n  Lysis  cell  t r a n s f o r m a t i o n by SV-40.  122  27.  H a r i g a y a K, Handa H: G e n e r a t i o n of f u n c t i o n a l c l o n a l c e l l l i n e s human bone marrow stroma. Proc N a t l Acad S c i USA 82:3477, 1985  from  28.  S i n g e r JW, Charbord P, K e a t i n g A, Nemunaitis J , Raugi G, Wignt TN, Lopez JA, Roth GJ, Dow LW, Fialkow PJ: Simian v i r u s 40-transformed adherent c e l l s from human long-term marrow c u l t u r e s : Cloned c e l l l i n e s produce c e l l s with s t r o m a l and h e m a t o p o i e t i c 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 f a c t o r a c t i v e on a v a r i e t y of c e l l types of non-hemopoietic o r i g i n . S c i e n c e (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 s i m i a n v i r u s 40 to form tumors i n athymic mice. Proc N a t l Acad S c i USA 72:4971, 1975  31.  V o g e l s t e i n B, Fearon ER, Hamilton SR, P r e i s e n g e r AC, W i l l a r d FW, M i c h e l s o n AM, Riggs AD, O r k i n SH: C l o n a l a n a l y s i s u s i n g recombinant DNA probes from the X-chromosome. Cancer Res 47:4806, 1987  32.  Eaves CJ, Cashman JD, Ross R, Raines E, Eaves AC: F a c t o r s that a c t i v a t e q u i e s c e n t hemopoietic p r o g e n i t o r s i n normal long-term human marrow cultures. Blood 68 (Suppl 1 ) : 141a, 1986 "  33.  I k e b u c h i K, Wong GG, C l a r k SC, I h l e JN, H i r a i Y, Ogawa M. Interleukin-6 enhancement of I n t e r l e u k i n - 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 p r o g e n i t o r s . Blood 70 (Suppl 1):173a, 1987  123  CHAPTER  V  SUMMARY AND FUTURE DIRECTIONS  In through cells  humans, hemopoiesis  i s maintained  f o r the l i f e t i m e of the  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  (1).  These c e l l s o r i g i n a t e i n the y o l k sac mesoderm and  the e x t r a v a s c u l a r spaces of the bone marrow of the a d u l t .  themselves  There  Very l i t t l e  i s known about  s e v e r a l y e a r s evidence has accumulated  i n the  However, over the past  to support the i d e a that  p r o l i f e r a t i v e s t a t e of hemopoietic stem c e l l s  which  (2).  the m o l e c u l a r events i n v o l v e d  commitment of p l u r i p o t e n t hemopoietic stem c e l l s .  in  they are  i n c l o s e p r o x i m i t y to a number of f i x e d bone marrow elements  c o l l e c t i v e l y c o n s t i t u t e a hemopoietic "microenvironment"  stem  then undergo a  s e r i e s o f m i g r a t i o n s e a r l y i n ontogeny to e v e n t u a l l y e s t a b l i s h  found  individual  the  i s e x t r i n s i c a l l y regulated  through s h o r t - r a n g e i n t e r a c t i o n s with n o n - c i r c u l a t i n g bone marrow c e l l s ( 3 ) . While full  the exact nature of the s i g n a l s i n v o l v e d  i n these i n t e r a c t i o n s  awaits  b i o c h e m i c a l c h a r a c t e r i z a t i o n , an a t t r a c t i v e h y p o t h e s i s i s that marrow  stromal c e l l s results  respond  to p e r t u r b a t i o n s a f f e c t i n g mature blood c e l l s which  i n the l o c a l s e c r e t i o n of hormone-like  hemopoietic growth f a c t o r s or  the e x p r e s s i o n by s t r o m a l c e l l s of s u r f a c e bound forms of such growth P r i m i t i v e hemopoietic c e l l s cannot S t u d i e s on the e a r l i e s t  factors.  be r e c o g n i z e d m o r p h o l o g i c a l l y .  p r o l i f e r a t i v e events o c c u r r i n g i n hemopoietic  d i f f e r e n t i a t i o n have t h e r e f o r e come to r e l y mainly on i n v i t r o techniques that d e t e c t these c e l l s by measuring under d e f i n e d c o n d i t i o n s .  culture  t h e i r developmental  potential  In v i t r o colony assays have been i n v a l u a b l e i n  p r o v i d i n g a c o n c e p t u a l framework about  the h i e r a r c h i c a l o r g a n i z a t i o n o f the  124  hemopoietic system.  They have a l s o l e d to the i n i t i a l d i s c o v e r y and  i d e n t i f i c a t i o n o f a number o f d i s t i n c t  hemopoietic growth f a c t o r s ( 4 ) .  However, colony assays have c e 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 . support p r o g e n i t o r c e l l they minimize c e l l u l a r play a c r i t i c a l  limited self-renewal.  First,  they o n l y  Second, by t h e i r v e r y n a t u r e ,  i n t e r a c t i o n s w i t h s t r o m a l c e l l s which a r e s u s p e c t e d to  r o l e i n the r e g u l a t i o n of stem c e l l s  i n vivo ( 5 ) .  d e s c r i p t i o n by Dexter et a l . ( 6 ) o f an a l t e r n a t i v e i n v i t r o system  Thus, the which  a l l o w s the long-term maintenance o f high p r o l i f e r a t i v e p o t e n t i a l p r o g e n i t o r c e l l s was n o t h i n g l e s s  than r e v o l u t i o n a r y .  For the f i r s t  time, i t became  p o s s i b l e to study the turnover o f hemopoietic stem c e l l s under i n v i t r o c o n d i t i o n s which a l l o w , and l i k e l y r e q u i r e , c l o s e i n t e r a c t i o n s between stem c e l l s and marrow s t r o m a l c e l l c u l t u r e system e v i d e n c e that unanswered.  their  U t i l i z a t i o n of the long-term marrow  by many d i f f e r e n t groups has s i n c e p r o v i d e d c o n s i d e r a b l e t h i s i s the case.  However, many important q u e s t i o n s a r e s t i l l  For example, the nature of the c e l l u l a r r e g u l a t o r y s i g n a l s  r e g u l a t e stem c e l l has  types.  that  turnover i n t h i s system has not y e t been e s t a b l i s h e d nor  origin.  These i s s u e s cannot  be r e s o l v e d 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 c u l t u r e system and then d e v e l o p i n g e x p e r i m e n t a l approaches adherent  layer.  these l i n e s , are  important  myself  to r e c o n s t r u c t  the f u n c t i o n p r o v i d e d by the  The purpose o f my r e s e a r c h was to i n i t i a t e s t u d i e s a l o n g  based on the assumption  that  the  i n the r e g u l a t i o n o f hemopoiesis.  mesenchymal c e l l s of the marrow To a c h i e v e t h i s g o a l , I s e t  the task o f i s o l a t i n g and then a n a l y z i n g these elements  term marrow c u l t u r e The  from the l o n g -  system.  f i r s t g o a l was to develop an assay that would a l l o w q u a n t i t a t i o n o f  the hemopoietic s u p p o r t i v e f u n c t i o n of long-term c u l t u r e adherent  l a y e r s which  125 might then l a t e r be applied to assess the essential  c e l l u l a r 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 c e l l s (e.g. peripheral blood).  regular marrow aspirates or  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 c e l l s would markedly reduce the s e n s i t i v i t y selection  of the assay,  of a suitable target population was the major concern.  Results of cocultivation experiments i n i t i a t e d with T-depleted l i g h t density peripheral blood c e l l s 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 p r o l i f e r a t i v e 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 s e l f establish an adherent layer (7).  did not contain c e l l s able to  These results validated the f e a s i b i l i t y and  usefulness of reconstitution experiments to study the regulation of hemopoiesis  by non-hemopoietic stromal c e 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 source of hemopoietic progenitor c e l l s .  to bone marrow as an alternative However, because bone marrow  aspirates contain mesenchymal progenitor c e l l s i t was necessary strategy  to develop a  to adequately eliminate or suppress these c e l l s to reduce the  background in control dishes to an acceptable l e v e l . investigators  Although many  have described potential methods for this (8,9,10,11), most  126 proved u n s a t i s f a c t o r y .  One  which d i d work however was  e v i d e n c e i n the murine system which suggested (CHP)  c o u l d be used  to s e l e c t i v e l y  based on e x p e r i m e n t a l  that C i s - 4 - H y d r o x y - L - P r o l i n e  i n h i b i t stromal c e l l  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 c o l l a g e n s y n t h e s i s . effects  proliferation.  These s t u d i e s showed that CHP mesenchymal c e l l  on these two c e l l  c o u l d be used  p r o l i f e r a t i o n at doses  is  When I t e s t e d i t s  on the s t r o m a l and hemopoietic components of normal  a significantly d i f f e r e n t i a l effect  CHP  human bone marrow  types was r e v e a l e d .  to completely b l o c k marrow  that had no e f f e c t  on the stem  cell  r e g u l a t o r y f u n c t i o n s of such c e l l s once they had a l r e a d y been e s t a b l i s h e d as a confluent  layer in v i t r o .  These f i n d i n g s p r o v i d e d f u r t h e r evidence  that  c o l l a g e n - p r o d u c i n g mesenchymal c e l l s of marrow o r i g i n p l a y an important in  promoting  hemopoiesis  i n long-term marrow c u l t u r e s .  Moreover they showed  that supplementation of long-term marrow c u l t u r e medium w i t h CHP possible  makes i t  to use unseparated marrow c e l l suspensions to i n v e s t i g a t e and d e f i n e  mesenchymal c e l l  types that may  To f u r t h e r c h a r a c t e r i z e  have stem c e l l  regulatory  properties.  the c e l l p o p u l a t i o n s present i n long-term marrow  c u l t u r e s and  t h e i r r e s p e c t i v e f u n c t i o n s as part of the hemopoietic  environment,  a major e f f o r t was  individual vitro,  then d i r e c t e d at o b t a i n i n g c l o n e d l i n e s of  marrow s t r o m a l c e l l s .  Since human mesenchymal c e l l s senesce i n  some i m m o r t a l i z a t i o n procedure was  have r e p o r t e d that SV-40 v i r u s can be used type without a l t e r a t i o n I it  role  needed.  Recently, several  groups  to transform v a r i o u s human c e l l s  of the o r i g i n a l d i f f e r e n t i a t e d  t h e r e f o r e s e t out to generate a high t i t e r  phenotype of the  p r e p a r a t i o n of SV-40 and  cells.  then  use  to i s o l a t e a l a r g e 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 e n d o t h e l i a l c e l l  origin.  L i n e s were d e r i v e d from long-term c u l t u r e adherent fibroblasts.  These a l l expressed  l a y e r s and marrow  the same phenotype as c e l l s present i n the  127  non-infected  population.  laminin,  negative  but  A l l were p o s i t i v e f o r c o l l a g e n I, c o l l a g e n IV,  for factor VIII.  None expressed any  d i f f e r e n t i a t i o n markers of hemopoietic c e l l s immortalized  T200 e x p r e s s i o n  was  with the one  noted i n one  did exhibit altered properties transformed c e l l s . a higher  sometimes e x p l o i t e d  present  that  and  the  information  about  f u n c t i o n a l s t u d i e s performed w i t h  the a b i l i t y of normal parent mesenchymal  the probable i d e n t i t i e s of the  i n GM-CSF and  u s i n g Northern b l o t t i n g and i t s e l f was  dependent c e l l  Humphries and IL-6  Dr R.  of r e a d i l y d e t e c t a b l e  obtained  demonstrated induction The  u s i n g an  presence IL-6  (12)  IL-6,  l e v e l s of hemopoietic  both of which can a c t on p r i m i t i v e  is a tantalizing finding.  However, I have p r e s e n t e d  e v i d e n c e that r e g u l a t i o n of p r i m i t i v e hemopoietic c e l l s the l o c a l l e v e l r a t h e r  v i v o i s simply  who  mRNA l e v e l s f o l l o w i n g  cells  line.  hemopoietic c e l l s  one  Kay  these  Additional  f a c t o r s produced was  f u r t h e r confirmed by s p e c i f i c b i o a s s a y s  enhanced p r o d u c t i o n  Obviously,  was  of  S - l p r o t e c t i o n a n a l y s i s techniques.  growth f a c t o r s such as GM-CSF and  at  to grow to  This l a t t e r property  s t i m u l a t i n g f a c t o r s upon i n d u c t i o n with I L - 1 .  a s u b s t a n t i a l increase  lines  lines.  i n a c o l l a b o r a t i v e study with Dr R.K.  The  time, a b i l i t y  i n long-term marrow c u l t u r e s to show a marked p r o d u c t i o n  h e m o p o i e t i c colony  of IL-6  these  that are g e n e r a l l y c h a r a c t e r i s t i c of  from the f i r s t  they r e t a i n e d  the  that weak a c t i v a t i o n of  However, a l l of  anchorage independence.  to clone  A major c o n c l u s i o n l i n e s was  exception  l i n e examined.  Several  These too resembled  These i n c l u d e a reduced d o u b l i n g  c e l l density,  the  that were examined.  e n d o t h e l i a l l i n e s were a l s o o b t a i n e d .  parent c e l l s p h e n o t y p i c a l l y ,  of  and  possibility due  is likely  than v i a the c i r c u l a t i o n of r e l e a s e d i s that  to o p e r a t e  factors.  the l o c a l i z a t i o n of responses apparent i n  to the d i l u t i o n of r e l e a s e d growth f a c t o r s to i n e f f e c t u a l  128  l e v e l s a t more d i s t a n t  regions.  R e c e n t l y , evidence was presented to i n d i c a t e  that GM-CSF can a l s o bind to glycosaminoglycans of the marrow e x t r a c e l l u l a r matrix.  T h i s suggests an i n t r i g u i n g mechanism by which r e l e a s e d growth  f a c t o r s might  be l o c a l l y c o n c e n t r a t e d i n t i s s u e s .  Another  possibility  enhanced p r o d u c t i o n of s e c r e t e d forms o f growth f a c t o r s might  i s that  be accompanied  by a b i o l o g i c a l l y more important enhanced p r o d u c t i o n o f membrane-bound growth f a c t o r molecules. (13).  Such a p o s s i b i l i t y has a precedent  i n the case o f M-CSF  However, these f i n d i n g s do not address the q u e s t i o n o f how n e g a t i v e  r e g u l a t i o n of p r i m i t i v e hemopoietic c e l l s vivo.  One p o s s i b i l i t y  regulatory c e l l s  i s achieved e i t h e r  i s that n e g a t i v e r e g u l a t i o n o c c u r s a t the l e v e l o f the  i . e . i n the absence  o f a p p r o p r i a t e s t i m u l a t i o n of the s t r o m a l  c e l l s hemopoietic growth f a c t o r p r o d u c t i o n d e c l i n e s to l e v e l s insufficient GQ s t a t e . specific Recent  that a r e  to a c t i v a t e nearby hemopoietic p r o g e n i t o r s and these then e n t e r a  Alternatively,  i t i s possible  that s t r o m a l c e l l s may a l s o  i n h i b i t o r y substances i n a f a s h i o n  d a t a from  the T e r r y Fox L a b o r a t o r y have i n d i c a t e d  term marrow c u l t u r e s f o l l o w i n g the a d d i t i o n o f I L - 1 . by s t r o m a l c e l l s and i t s p o s s i b l e involvement by marrow c e l l s w i l l be very i n t e r e s t i n g  that TGF-61 can that o c c u r s i n l o n g -  Whether TGF-61 i s made  i n the r e g u l a t i o n o f hemopoiesis  to e s t a b l i s h .  In summary, my s t u d i e s have i d e n t i f i e d l a y e r as l i k e l y  release  that i s s u b j e c t to r e g u l a t i o n .  o v e r r i d e the s t i m u l a t i o n of hemopoietic p r o g e n i t o r c e l l s  adherent  i n v i t r o or i n  c o l l a g e n - p r o d u c i n g c e l l s o f the  to be an important p o p u l a t i o n f o r the maintenance and  t u r n o v e r o f p r i m i t i v e hemopoietic c e l l s .  In a d d i t i o n , phenotypic and  f u n c t i o n a l c h a r a c t e r i z a t i o n of human marrow mesenchymal l i n e s have suggested that p r o d u c t i o n o f hemopoietic growth f a c t o r s may w e l l be i n v o l v e d r e g u l a t i o n o f p r i m i t i v e hemopoietic c e l l molecules may a l s o be i n v o l v e d .  proliferation.  i n the  However, o t h e r  For example, f u r t h e r 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 s u r f a c e primitive cells  to "home" to s p e c i f i c t i s s u e s .  whether molecules contribute  that  facilitate  to c e l l - m e d i a t e d  inter-cell  that  allow  S i m i l a r l y i t i s not known  interactions  growth f a c t o r responses  The a v a i l a b i l i t y o f s e v e r a l exhibit  determinants e x i s t  and thereby  exist.  c l o n e d human marrow mesenchymal l i n e s  the phenotype and r e g u l a t e d  a b i l i t y of cultured  that  marrow c e l l s to  produce hemopoietic growth f a c t o r s and s y n e r g i s t i c a c t i v i t i e s s h o u l d facilitate future  further  of t h i s type.  Of p a r t i c u l a r importance  w i l l be  t e s t s of the a b i l i t y of these l i n e s alone and/or i n combination to  reconstitute culture  studies  the hemopoietic s u p p o r t i v e f u n c t i o n  adherent  layers.  of i n t a c t long-term marrow  130  REFERENCES 1.  Dexter TM, Simmons P, P u r n e l l RA, Spooncer E, S c h o f i e l d R. The r e g u l a t i o n o f hemopoietic c e l l development by the s t r o m a l c e l l environment and d i f f u s i b l e r e g u l a t o r y molecules. I n : A p l a s t i c anemia: Stem c e l l b i o l o g y and advances i n treatment. A l a n R L i s s , New York, pp 131, 1984.  2.  T r e n t i n J J . I n f l u e n c e of hematopoietic organ stroma ( h e m a t o p o i e t i c i n d u c t i v e microenvironment) on stem c e l l d i f f e r e n t i a t i o n . I n : " R e g u l a t i o n o f hematopoiesis", Appleton-Century C r o f t s , e d u c a t i o n a l d i v i s i o n , M e r i d i t h Corp, pp 61, 1970.  3.  Dexter TM, Spooncer E, Toksoz D, L a j t h a LG. The r o l e o f c e l l s and t h e i r products i n the r e g u l a t i o n o f i n v i t r o stem c e l l p r o l i f e r a t i o n and g r a n u l o c y t e development. J o u r n a l o f Supramolecular S t r u c t u r e 13: 513-524, 1980.  4.  M e t c a l f D. Hemopoietic c o l o n i e s i n v i t r o : C l o n i n g o f normal and leukaemic c e l l s , S p r i n g e r V e r l a g , B e r l i n , pp227, 1977.  5.  Eaves AC, Cashman JD, Gaboury LA, Eaves C J . C l i n i c a l s i g n i f i c a n c e o f long-term c u l t u r e s 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, L a j t h a LG. C o n d i t i o n s 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 P h y s i o l 91: 335, 1977.  7.  Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves C J . Unregulated p r o l i f e r a t i o n of p r i m i t i v e c h r o n i c myeloid leukemia p r o g e n i t o r s i n the presence o f normal marrow adherent c e l l s . Proc N a t l Acad S c i USA 83: 5306, 1986.  8.  G i l b e r t SF, Migeon BR. D - v a l i n e as a s e l e c t i v e agent f o r normal human and rodent e p i t h e l i a l c e l l s , i n c u l t u r e . C e l l 5: 11, 1975.  9.  E b e l l W, C a s t r o - M a l a s p i n a H, Moore MAS, O ' R e i l l y RJ. D e p l e t i o n o f s t r o m a l c e l l elements i n human marrow g r a f t s s e p a r a t e d by soybean agglutinin. Blood 65: 1105, 1985.  10.  Paraskeva C, Buckle B.G, Thorpe PE. S e l e c t i v e k i l l i n g of c o n t a m i n a t i n g human f i b r o b l a s t s i n e p i t h e l i a l c u l t u r e s d e r i v e d from c o l o r e c t a l tumours u s i n g an a n t i - T h y - 1 a n t i b o d y - r i c i n c o j u g a t e . Br J Cancer 51: 131, 1985.  11.  Abboud CM, Duerst RE, F r a n t z CN, Ryan DH, L i e s v e l d J L , Brennan JK. of human f i b r o b l a s t c o l o n y - f o r m i n g c e l l s and e n d o t h e l i a l c e l l s by monoclonal a n t i b o d y (6-19) and complement. Blood 68: 1169, 1986.  Lysis  131  12.  Ikebuchi K, Wong GG, C l a r k SC, I h l e JN, H i r a i Y, Ogawa M. I n t e r l e u k i n - 6 enhancement o f i n t e r l e u k i n - 3 dependent p r o l i f e r a t i o n o f m u l t i p o t e n t i a l hemopoietic p r o g e n i t o r s . Blood 70 ( S u p p l l ) : 1 7 3 a , 1987.  13.  Rettenmier CW, Roussel MF, Ashmun RA, Ralph P, P r i c e K, Sherr C J . S y n t h e s i s of membrane-bound c o l o n y - s t i m u l a t i n g F a c t o r - 1 (CSF-1) and downmodulation o f CSF-1 r e c e p t o r s i n NIH 3T3 c e l l s transformed by c o t r a n s f e c t i o n of the human CSF-1 and c-FMS (CSF-1 r e c e p t o r ) genes. M o l e c u l a r and C e l l u l a r B i o l o g y 7: 2378, 1987.  

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