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A study of the erythropoietin requirements of erythroid progenitors in polycythemia vera Cashman, Johanne Dianne 1982

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A STUDY OF THE ERYTHROPOIETIN REQUIREMENTS OF ERYTHROID PROGENITORS IN POLYCYTHEMIA VERA by JOHANNE DIANNE CASHMAN B.Sc, McGi.ll University, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1982 ©JOHANNE DIANNE CASHMAN, 1982 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of Pathology The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 15, 1982 DE-6 (3/81) - i i -ABSTRACT Erythroid progenitor c e l l s of the abnormal clone i n polycythemia vera (PV) are capable of colony formation in vitro without the addition of erythropoietin, the regulatory hormone required f o r normal in vivo and-in vitro erythropoiesis. This property of "erythropoietin-indepen-dent" colony formation has been considered a marker for the abnormal clone i n PV, although recent studies i n d i c a t e that not a l l erythropoietic members of the clone may be capable of e x h i b i t i n g t h i s abnormal phenotype. The present studies were undertaken to investigate the l e v e l of mat-uration at which establishment of an erythropoietin-independent phenotype might be determined. : A series of experiments was'performed;, on the replated progeny of single p r i m i t i v e hemopoietic c e l l s already committed to erythropoiesis (primitive BFU-E). F i r s t , conditions were established to maximize the number of erythroid colonies obtainable i n secondary assays of replated primary colonies of p r i m i t i v e BFU-E o r i g i n . Time course studies and experiments with i r r a d i a t e d peripheral blood "feeder" c e l l s treated i n d i f f e r e n t ways established that r e s u l t s were best when primary colonies were allowed to grow for 9 days p r i o r to r e p l a t i n g and when 9 day o l d feeders stored at 4°C were included i n the secondary assay medium. Second, a technique was developed f or d i v i d i n g such colonies between 2 secondary assay cultures. Experiments with normal primary colonies t r a n s f e r r e d to 2 secondary assays, both containing erythropoietin, showed that the v a r i a t i o n between true r e p l i c a t e s was random, i n d i c a t i n g that the procedure used divided each primary colony - i i i -equally. Third, i t was shown that secondary assays to which no erythropoietin was added f a i l e d to support erythroid colony formation by progenitors present i n normal 9 day old primary colonies. F i n a l l y , the d i s t r i b u t i o n of erythropoietin-dependent and eryt h r o p o i e t i n -independent phenotypes i n i n d i v i d u a l colonies derived from p r i m i t i v e BFU-E from 5 patients with. PV was assessed by r e p l a t i n g experiments. Most of the replated colonies from PV cultures that y i e l d e d e r y t h r o i d colonies i n secondary assays containing 3 units of ery t h r o p o i e t i n per ml also produced some erythroid colonies i n the paired r e p l i c a t e that contained < 0.01 units of erythropoietin per ml. However, fewer colonies were c o n s i s t e n t l y obtained i n the low eryth r o p o i e t i n cultures. These r e s u l t s i n d i c a t e that i n PV, most of the p r i m i t i v e erythroid bursts that generate phenotypically abnormal progeny capable of er y t h r o i d colony formation under conditions which are non-permissive f o r normal c e l l s also produce s i g n i f i c a n t numbers of progeny that are phenotypically normal i n t h i s respect. I t i s concluded that the capacity for erythropoietin-independent growth and maturation exhibited i n vitro by terminally d i f f e r e n t i a t i n g members of the abnormal clone i n PV i s not commonly f i x e d at or p r i o r to the p r i m i t i v e BFU-E stage of erythropoietic c e l l development. - i v -TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES . v i LIST OF FIGURES . : v i i ACKNOWLEDGEMENTS ix INTRODUCTION I. Clonal Assay Systems 1 A. The Spleen Colony Assay 2 B. In vitro Colony Assays 4 1. Granulocyte/macrophage colony formation 5 2. Erythroid colony formation 6 3. Megakaryocytic colony formation 10 4. Lymphoid colony formation 11 5. Mixed colony formation 12 C. Summary - 13 II . Regulation of Erythropoiesis 15 A. Regulation of Stem C e l l P r o l i f e r a t i o n 16 1. C e l l cycle status 16 2. Decision between self-renewal versus d i f f e r e n t i a t i o n 20 3. Commitment 22 B. Molecular Regulators of Erythropoietic C e l l 23 D i f f e r e n t i a t i o n 1. Erythropoietin 24 2. The ro l e of non-erythropoietin factors i n regulating e r y t h r o p o i e t i c d i f f e r e n t i a t i o n 26 C. Summary 28 -v-TABLE OF CONTENTS Page I I I . Polycythemia Vera 29 A. General Features of the Myel o p r o l i f e r a t i v e Diseases 29 B. S p e c i f i c Features of Polycythemia Vera 30 1. Cytogenetics 31 2. Erythropoietin-independence and c l o n a l dominance 32 C. Experimental Rationale 35 MATERIALS AND METHODS 1. Patients 38 2. C e l l Preparation 41 3. Primary Colony Assays 41 4. Secondary Colony Assays 42 a. Phytohemagglutinin-stimulated leukocyte conditioned media 42 b. Peripheral blood feeder c e l l s 43 5. Replating Technique 44 6. S t a t i s t i c a l Analysis 48 RESULTS 1. Conditions f o r Maximizing Erythroid Colony Counts i n Secondary 49 Assays of Replated Primary Colonies 2. Normal Er y t h r o i d Colony Formation i n Secondary Assay 51 Replicates With and Without Added Erythropoietin 3. Replating Experiments with Primary Colonies from PV 56 Patients DISCUSSION.AND CONCLUSIONS 58 REFERENCES 63 - v i -LIST OF TABLES Page Table 1. C l i n i c a l Data on Patients with a Diagnosis of Polycythemia Vera. 39 Table 2. C r i t e r i a of the Polycythemia Vera Study Group. 40 Table 3. Dependence of Secondary E r y t h r o i d Colony Formation by Replated Primary Colony C e l l s on the Use and P r i o r Treatment of Normal Peripheral Blood Feeder C e l l s . 52 Table 4. Number of Secondary Erythroid Colonies Obtained i n Duplicate Replaces of Primary Colonies from 3 Normal Individuals. 54 Table 5. Number of Replated Primary Colonies from 3 Normal Individuals Y i e l d i n g Erythroid Colonies i n Secondary Assays With and Without Added Erythropoietin. 55. Table 6. Number of Erythroid Colonies i n Secondary Assays With and Without Added Erythropoietin from Replated Primary Colonies from 5 PV Patients. 57 - V i i -LI.ST OF FIGURES Figure 1. Photographs of human erythroid colonies grown i n methylcellulose cultures. A. A large e r y t h r o i d burst, containing approximately 50 c l u s t e r s , photographed a f t e r 19 days of incubation. B. A small e r y t h r o i d burst, composed of 4 erythro-b l a s t c l u s t e r s , photographed a f t e r 12 days of incubation. C. An e r y t h r o i d colony composed of a s i n g l e c l u s t e r of about 50 erythroblasts, photographed a f t e r 9 days of incubation. Page 8/10 Figure 2. Diagrammatic representation, of the hierarchy of hemopoietic progenitor compartments c u r r e n t l y i d e n t i f i e d by colony assay procedures. 14 Figure 3. Three types of stem c e l l . t r a n s i t i o n s where regulatory 17/18 mechanisms may act to influence stem c e l l behaviour. A. Changes i n c e l l cycle status. B. Self-renewal versus d i f f e r e n t i a t i o n . C. Commitment to a s p e c i f i c d i f f e r e n t i a t i o n pathway. Figure 4. The experimental design developed to study the erythropoietin requirements of replated progeny of i n d i v i d u a l e r y t h r o i d progenitors (primitive BFU-E) from patients with PV. 37 Figure 5. A 9 day old colony of the type selected f o r r e p l a t i n g . 45 Figure 6. Examples of 9 day o l d secondary colonies obtained i n dishes containing about 3 u/ml of erythropoietin. Both colonies were derived from c e l l s from normal i n d i v i d u a l s . A. A t y p i c a l secondary colony, estimated to contain about 30 erythroblasts. B. A larger secondary colony estimated to contain 150 erythroblasts 47 - v i i i -LI.ST OF FIGURES Page Figure 7. Time course study of erythroid colony y i e l d s from replated primary colonies allowed to grow for varying periods p r i o r to assay i n secondary cultures with and without feeders 49 - i x -ACKNOWLEDGEMENT S I would l i k e to express my gratitude: To Dr. A l l e n Eaves .(Department of Pathology), my research supervisor, fo r h i s expert guidance and support, f o r obtaining the patient material used i n t h i s study, and for submitting without complaint to my many requests f o r "feeder"' cell-s; To Dr. Connie Eaves (Department of Medical,. Genetics)_;,rfor:'her.icritical evaluation of t h i s manuscript, and for her immense cont r i b u t i o n to my academic and t e c h n i c a l t r a i n i n g ; To Dr. Don Brunette (Faculty of D e n t i s t r y ) , and Dr. Don Brooks (Department of Pathology), members of my graduate committee, f o r t h e i r i n t e r e s t and h e l p f u l suggestions, and to Dr. Richard Pearce (Department of Pathology) f o r h i s assistance i n coping with u n i v e r s i t y buracracy; To Don Henkelman, for h i s help with the s t a t i s t i c a l a n alysis presented i n t h i s t h e s i s ; To Dr. Gerry K r y s t a l , Dr. Fumio Takei, Dr. Laure Coulombel, Ian Dube and the other s t a f f and students of the Terry Fox Laboratory f o r providing a stimulating environment f or research and learning; To Dianne Reid and Anne-Marie MacDougall f or t h e i r expert t e c h n i c a l assistance; To Sharon B e l l and Cathy Walker f o r typing t h i s manuscript; And f i n a l l y , to my husband Fred, f o r h i s lov i n g support and remarkable patience. INTRODUCTION I. CLONAL ASSAY SYSTEMS The mature, c i r c u l a t i n g c e l l s of the perip h e r a l blood have a f i n i t e l i f e span and are themselves incapable of c e l l d i v i s i o n . Accordingly they must be constantly produced from l e s s d i f f e r e n t i a t e d c e l l s . This occurs i n the marrow where for each lineage there e x i s t s a c l a s s of c e l l s that r e t a i n a considerable p r o l i f e r a t i v e capacity, but are r e s t r i c t e d i n t h e i r d i f f e r e n t i a t i o n p o t e n t i a l . These "committed progenitors" i n turn, a r i s e from a common plu r i p o t e n t stem c e l l compartment whose members are capable of maintaining t h e i r own population size by a process of s e l f -renewal, while also g i v i n g r i s e to c e l l s capable of progression down any one of the myeloid c e l l pathways. Associated with the progressive l o s s of p r o l i f e r a t i v e capacity and d i f f e r e n t i a t i o n p o t e n t i a l with maturation, i s the appearance of the s p e c i f i c morphological and biochemical markers of mature red c e l l s , p l a t e l e t s , and granulocytes (1). Much of our present knowledge regarding the precusor-progeny r e l a t -ionships which r e f l e c t ' t h i s balance of 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 has been obtained from observations . of ^ colonyrformation^by .'.hemppoietic progenitors. During the l a s t twenty years, both in vivo and in vitro colony.:assaysj have.Lbe.en developed forimest .classes of .'.hemopoietiooprogeriitor c e l l s ( 2 ). In these c l o n a l assay systems, single hemopoietic progenitor c e l l s 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 to form i n d i v i d u a l d i s c r e t e colonies of varying s i z e . The c e l l u l a r composition of each colony i s determined by the appearance of the morphological and functional markers character-i s t i c of the mature c e l l s . Such observations, coupled with appropriate -1--2 -manipulations of the culture conditions, have permitted inv e s t i g a t o r s to theorize about the functioning and regulation of various stages of hemo-p o i e s i s . Central to the concept of hemopoietic lineages i s the assump-t i o n , based on genetic and k i n e t i c studies, that i n d i v i d u a l colonies a r i s e from s i n g l e c e l l s . Only by determining the v a l i d i t y of t h i s assumption can investigators r e l a t e observations of the terminal c e l l s i n the pathway to regulatory e f f e c t s at the l e v e l of a stem c e l l or a committed progenitor. In t h i s section, I s h a l l review the various colony assay systems used to i d e n t i f y d i f f e r e n t classes of hemopoietic c e l l s , and how the sin g l e c e l l o r i g i n of colonies was reaffirmed as new culture methods were developed. A. The Spleen Colony Assay The spleen colony assay developed by McCulloch and T i l l i n 1961 (3) involves the i n j e c t i o n of appropriate numbers of hemopoietic c e l l s from donor mice into heavily i r r a d i a t e d r e c i p i e n t mice. When the spleens of the r e c i p i e n t mice, are examined 8 to 10 days l a t e r , macroscopic nodules are v i s i b l e on the surface. M i c r o s c o p i c a l l y these nodules are seen to consist of recognizable c e l l s of the various myeloid s e r i e s . Although a single lineage i s usually predominant i n each colony, "mixed" colonies are also found. In these e r y t h r o c y t i c , granulocytic, and mega-karyocytic c e l l s may a l l be present (4,5,6). Early observations of a. .linear r e l a t i o n s h i p between the number of nucleated marrow c e l l s i n j e c t e d and the number of spleen colonies formed l e d to the hypothesis that each colony developed from a si n g l e c e l l (3,7). -3-More d i r e c t evidence was obtained by cytogentic studies using recogniz-able, p e r s i s t e n t chromosomal markers, randomly generated i n bone marrow c e l l s by r a d i a t i o n (8,9). The presence of a given marker i n 95-100% of metaphases from one colony and i t s absence from a l l metaphases of adjacent spleen colonies was strong evidence for the single c e l l o r i g i n of spleen colonies. Once t h i s had been demonstrated, c e r t a i n properties of the c e l l of o r i g i n , the colony forming unit-spleen (CFU-S) could be i n f e r r e d from analysis of the c e l l u l a r composition of i n d i v i d u a l spleen colonies. For CFU-S i n normal marrow these properties included: 1) p l u r i p o t e n t i a l i t y , as shown by the presence of more than one c e l l type i n "mixed" spleen colonies; 2) extensive p r o l i f e r a t i v e capacity, evidenced by the large size of colonies, which may contain i n excess of l O ^ c e l l s ; 3) the a b i l i t y to self-renew, since a c e l l suspension made from a sing l e colony i s capable of producing comparable colonies containing a l l three myeloid lineages i n a secondary r e c i p i e n t (10,11,12). These properties of the CFU-S f u l f i l l the c r i t e r i a f o r a stem c e l l , which must have the capacity for maintaining i t s own population, while giving r i s e to large numbers of f u l l y d i f f e r e n t i a t e d progeny. The spleen colony assay i s considered a quantitative method for detecting these c e l l s . At the present time, the CFU-S i s thought: to be the .myeloid.stem . c e l l . Although chromosomal and G-6PD isoenzyme studies have shown that a common ancestral c e l l e x i s t s for both the lymphoid and myeloid lineages (9,13,14,15) no d i r e c t c y t o l o g i c a l evidence for the presence of lymph-ocytes i n spleen colonies has been reported. -4-Despite the extensive a p p l i c a t i o n of the spleen colony assay, several problems associated with i t s use remain unresolved.. F i r s t , not a l l of the c e l l s capable of forming colonies reach the spleen (10) and.many which do may not be retained there (16). Though retrans-plantation.studies have been used to estimate the f r a c t i o n " f " that s e t t l e i n the spleen, values obtained for " f " range from .03 to .24 (10,17). Moreover, since no method i s a v a i l a b l e to c o r r e c t for stem c e l l s which may be damaged during the transplantation process a l l estimates of " f " must be considered as minimum values. Furthermore, perturbations induced i n the mouse to determine q u a n t i t a t i v e e f f e c t s on the CFU-S population, may also a f f e c t - t h e i r spleen seeding e f f i c i e n c y , i . e . " f " i t s e l f can vary (18). Second, cytogenetic studies have shown that more than one colony may a r i s e from a single c e l l (19). Third, i n d i v i d u a l estimates of the CFU-S content of a given c e l l suspension show wide f l u c t u a t i o n s and accurate values require the use of large numbers of r e c i p i e n t s (3). F i n a l l y , e t h i c a l considerations obviously preclude the a p p l i c a t i o n of t h i s assay i n man. Accordingly much e f f o r t has been expended i n attempts to develop appropriate in vitro culture systems f o r the study of hematological disorders. B. In Vitro Colony Assays Knowledge about the second compartment of hemopoietic c e l l s , the committed progenitor compartment, has been obtained l a r g e l y from studies using i n vitro colony assay systems. The existence of t h i s type of population was f i r s t suggested by the r e s u l t s of studies of erythro-p o i e t i n stimulated red c e l l production i n polycythemic mice (20). -5-These showed that the erythropoietin-responsive c e l l s (ERC) detected by t h i s assay were more p r i m i t i v e than the f i r s t morphologically recogniz-able c e l l , the proerythroblast, but d i f f e r e n t from CFU-S. From these studies the concept of an intermediate progenitor c e l l type was put forward. Such progenitors were envisaged as lineage r e s t r i c t e d but morphologically u n d i f f e r e n t i a t e d . 1. Granulocyte/macrophage colony formation The f i r s t r e a l evidence for a p r i m i t i v e committed progenitor c e l l followed the development, i n 1965, by two independent groups (21,22) of the f i r s t in vitro hemopoietic colony assay system. Dilute agar was employed to form a semisolid matrix, i n which marrow c e l l s were held immobilized, permitting the formation of d i s c r e t e colonies containing 50 to several hundred c e l l s of the granulocyte-macrophage (GM) s e r i e s . The c e l l of o r i g i n was named the colony-forming u n i t - c u l t u r e (CFU-C). The presence of colony stimulatory factors (CSF) was found to be e s s e n t i a l for GM colony formation. This requirement can be met by the use of a v a r i e t y of c e l l feeder systems (21,22,23) or by a d d i t i o n of "conditioned" medium i n which such c e l l s have been incubated (24,25,26). Although CSF's appear unique i n t h e i r a b i l i t y to stimulate the p r o l i f e r a t i o n of granulocyte-macrophage progenitors in vitro (27) controversy s t i l l e x i s t s concerning a possible p h y s i o l o g i c a l r o l e in vivo (28,29). Direct microscopic observations of the formation of a GM colony from a si n g l e c e l l have been reported by several i n v e s t i g a t o r s (30) using a v a r i e t y of techniques, including: a) experiments in v o l v i n g r e p l a t i n g of c e l l s from primary colonies (31); b) t r a n s f e r of single c e l l s to i n d i -v i d u a l culture dishes by micromanipulation (32); and c) p h y s i c a l i s o -l a t i o n of i n d i v i d u a l c e l l s by p l a s t i c rings (22). Human bone marrow -6-(33,34) and peripheral blood c e l l s (35) have been cultured s u c c e s s f u l l y by t h i s method, and a common progenitor f o r human granulocytes and macrophages established by isoenzyme studies of s i n g l e colonies (36). CFU-C and CFU-S i n the mouse have been distinguished by differences i n a number of properties such as c e l l s i z e and density (37) , c y c l i n g k i n e t i c s (38) and r a d i a t i o n s e n s i t i v i t y (39). Analysis of the content of CFU-S and CFU-C i n individual spleen colonies (40,41) have indi c a t e d that a close r e l a t i o n s h i p e x i s t s between ;these .two progenitors. 2. Erythroid colony formation The f i r s t successful i-n vitro colony assay system for erythroid progenitors was reported i n 1971 (42) using murine f e t a l l i v e r c e l l s plated i n a plasma c l o t system supplemented with erythropoietin. A f t e r 2 days incubation eryt h r o i d colonies c o n s i s t i n g of small c l u s t e r s of 8 to 64 hemoglobin containing c e l l s are seen. The c e l l of o r i g i n , termed the colony forming u n i t - e r y t h r o i d , (CFU-E), was subsequently shown to i d e n t i f y a r e l a t i v e l y l a t e progenitor on the erythroid pathway (43). Improvements i n the culture system permitted the detection of another erythroid progenitor i n murine bone marrow, the burst forming u n i t erythroid (BFU-E), capable of forming large, multi-clustered colonies 3 4 containing up to 10 to 10 c e l l s , a f t e r 7 days of incubation (44). Subsequently, an intermediate population of e r y t h r o p o i e t i c colony-forming was discovered. These yielded small bursts a f t e r 3 - 4 days of culture and were d i s t i n c t from both CFU-E and the progenitors of large bursts seen a f t e r 7 days of culture (45,46). Accordingly, the terms "mature" and " p r i m i t i v e " BFU-E were proposed .(47). The concept of a hierarchy of e r y t h r o p o i e t i c progenitor c e l l classes - 7 -detectable by the d i f f e r e n t sized colonies they generated was validated i n a number of ways. Co r r e l a t i o n analysis of the progenitor content of i n d i v i d u a l spleen colonies showed that CFU-S, p r i m i t i v e BFU-E,mature BFU-E and' CFU-E were sequentially r e l a t e d but had ;discrete; compartments, . (48 ) . Differences between CFU-E and BFU-E, i n terms of t h e i r responsiveness to erythropoietin stimulation or withdrawal, c e l l volume, 3 p r o l i f e r a t i v e capacity, s e n s i t i v i t y to H-thymidine, and time course of colony formation were consistent with such a sequence ( 4 4 - 4 7 ) . The m u l t i -c l u s t e r e d appearance of large e r y t h r o i d colonies i s due to a period of m o b i l i t y during the f i r s t stages of colony formation ( 4 4 ). As the c e l l s d i f f e r e n t i a t e to the CFU-E stage, mobility i s l o s t , so that with further d i v i s i o n , the c e l l s remain together and form a t i g h t c l u s t e r , comparable i n s i z e and appearance to a CFU-E colony. The number of c l u s t e r s present i n a burst thus provides a convenient measure of colony s i z e which i n turn appears to be predetermined by the stage of d i f f e r e n t i a t i o n of the progenitor c e l l that gave r i s e to i t (see Figure 1 ) . This r e l a t i o n s h i p has been d i r e c t l y demonstrated i n the mouse by r e p l a t i n g immature large bursts, and observing, a f t e r a furt h e r period of incubation, the growth of CFU-E and small bursts ( 4 8 ) . Erythroid colony-forming c e l l s , capable of y i e l d i n g d i f f e r e n t s i z e d erythroid colonies can also be detected i n human bone marrow ( 4 9 - 5 2 ) and periph e r a l blood ( 5 3 - 5 4 ) using both plasma c l o t and methylcellulose culture systems. Comparisons of murine and human erythroid progenitor properties suggest colony s i z e ( i . e . c l u s t e r content) can be used to i d e n t i f y a comparable hierarchy of c e l l types i n the human system ( 4 5 , 5 0 ) . Formal evidence f o r the single c e l l o r i g i n of murine CFU-E colonies -8-F i g . 1. Photographs of human erythroid colonies grown i n methyl-c e l l u l o s e cultures. A. A. large e r y t h r o i d burst, containing approximately 50 c l u s t e r s , photographed a f t e r 19 days of incubation. Each c l u s t e r contains, about 50 hemoglobin-synthesizing erythroblasts. Erythroid colonies composed of 16 or more such c l u s t e r s develop from progenitor c e l l s termed p r i m i t i v e burst forming u n i t - e r y t h r o i d ...(BFU-E) .", This process takes on average 18-20 days (51). P r i m i t i v e BFU-E are considered to represent the e a r l i e s t c e l l s committed to the erythroid lineage (47). B. A small e r y t h r o i d burst, composed of 4 erythroblast c l u s t e r s , photographed a f t e r 12 days of incubation. Progenitor c e l l s which give r i s e to colonies containing 3-8 c l u s t e r s are c a l l e d "mature" BFU-E. These c e l l s occupy an intermediate p o s i t i o n i n the erythroid c e l l pathway (47). Small bursts reach maturity f a s t e r than large bursts and the period required i s 10-12 days (51). C. An erythroid colony composed of a sin g l e c l u s t e r of about 50 erythroblasts, photographed a f t e r 9 days of incubation. The c e l l of o r i g i n of t h i s colony i s termed a colony-forming unit-erythroidi'.(.CFU-E).. and..is_thbught..to ^ be ..the. .most j,dif f eren-t i a t e d erythroid precursor (47). A progenitor c e l l i s considered to be a CFU-E i f i t gives r i s e to a colony containing two small c l u s t e r s , or to a colony composed of a sing l e c l u s t e r of 8 or more erythroid c e l l s . Such colonies are the f i r s t to appear and can be recognized a f t e r 6-9 days of incubation (51) . Magnification 200X... -10-was obtained by Cormack (55), who photographically recorded the devel-opment of colonies from single c e l l s . In the mouse, d i r e c t evidence for the single c e l l o r i g i n of e r y t h r o i d bursts was obtained by seeding male and female hemopoietic c e l l s i n culture, and determining, by demonstra-t i o n of the presence or absence of the Y chromosome, the homogeneity of male and female colonies (56). The c l o n a l nature of human bursts was demonstrated i n cultures i n i t i a t e d with c e l l s from females heterozygous at the G-6-PD isoenzyme locus. Isoenzyme analysis of i n d i v i d u a l bursts showed them to contain only one isoenzyme type, although colonies with ei t h e r type were present (.57) . 3. Megakaryocytic colony formation Megakaryocytic colony forming progenitors were f i r s t detected by Nakeff and associates (58) using bone marrow c e l l s from v i n b l a s t i n e treated mice, cultured i n agar over feeder layers of mouse embryo f i b r o b l a s t s . The addition of conditioned media prepared by incubating spleen c e l l s i n the presence of pokeweed mitogen res u l t e d i n the formation of l arger colonies containing up to 80 c e l l s (59). I d e n t i f i c a t i o n of the mature c e l l s was obtained by observations of the morphology, DNA content, polyploidy, and the presence of high l e v e l s of a c e t y l c h o l i n -esterase i n the cytoplasm (60). P l a t e l e t production from megakaryocytes i n colonies formed i n plasma c l o t cultures containing ery t h r o p o i e t i n has also been demonstrated (61) . A .linear r e l a t i o n s h i p has been shown between the.number .of c e i l s plated .and the, number of,, colonies formed,, consistent with the idea that each colony arose from a single c e l l , termed the colony-forming unit-megakaryocyte (CFU-M) (59,60). -11-4. Lymphoid colony formation The observation that c e r t a i n plant l e c t i n s , such as phytohemagglutinin (PHA) and pokeweed mitogen (PWM) could cause transformation of small lymphocytes i n a l i q u i d culture system (62,63), prompted investigators to apply these substances to agar cultures of human peripheral lymphocytes (64). To obtain colony formation these authors f i r s t p r e s e n s i t i z e d the lymphocytes with PHA i n a l i q u i d suspension culture and then plated the c e l l s i n agar medium to which fresh PHA was again added. Two types of colonies were generated by t h i s method, large colonies of 200 to 500 c e l l s , and smaller colonies of 50 to 150 c e l l s . I n t e r e s t i n g l y , the smaller colonies appeared l a t e r i n the culture dishes (6 to 7 days of incubation) than the larger colonies (3 to 4 days). This l e d the authors to postulate an inductive e f f e c t of the e a r l i e r , larger colonies. The addit i o n of sheep red blood c e l l s to the agar has eliminated the need for preincubation of the colony forming c e l l s with the mitogen (65). The c e l l s comprising the colonies were i d e n t i f i e d as T lymphocytes i n human assays by t h e i r rosette forming a b i l i t y , and i n mouse assays by the presence of the theta antigen. Single c e l l o r i g i n was v e r i f i e d by enclosing single c e l l s with pyrex rings and observing colony growth by microscopic examination (66). Bilymphocyte colony formation i n agar by murine c e l l s has been obtained by the addition of 2-mercaptoethanol and sheep red blood c e l l s to the agar ] (67). The mature c e l l s were i d e n t i f i e d as B lymphocytes by the presence of Fc and receptors, and c e l l membrane immunoglobulins on the c e l l surface. These i n v e s t i g a t o r s provided evidence f o r the sing l e c e l l o r i g i n of these colonies by enriching spleen suspensions for NIP -12-(4-Hydroxy-3-ido-5 nitrophenyl acetic acid) binding c e l l s and demon-s t r a t i n g that a l l the c e l l s i n a single colony were eit h e r hapten binding or non-binding. Later i n v e s t i g a t o r s were able to produce B lymphocyte colonies by preincubation of the c e l l suspension with mitogens, i n a manner analogous to the production of T c e l l colonies (68). 5. Mixed colony formation The f i r s t in vitro culture techniques detected committed precursors, i . e . c e l l s that appeared to be l i m i t e d i n t h e i r capacity for d i f f e r e n t i a -t i o n to the production of a single mature blood c e l l type. Recently, optimization of culture conditions has resulted i n the production of "mixed" colonies containing more than one lineage. The f i r s t example of t h i s was the erythroid-megakaryocytic colonies described by McLeod and h i s associates for murine bone marrow cultures containing erythropoietin (.61). Evidence f o r the c l o n a l nature of these colonies was presented i n l a t e r studies u t i l i z i n g chromosomal markers. (69). Larger colonies, con-t a i n i n g c e l l s from a l l three myeloid c e l l l i n e s were subsequently discovered i n cultures of murine f e t a l l i v e r c e l l s plated i n agar to which had been added mitogen-stimulated, spleen c e l l conditioned medium (70). I n i t i a l l y , these authors hypothesized that the m u l t i p o t e n t i a l stem c e l l of o r i g i n was a f e t a l stem c e l l type, but subsequent i n v e s t i g a t i o n s demonstrated that comparable colonies could also be obtained i n assays of mouse adult bone marrow c e l l s , although at a considerably lower frequency (.71,72,73). Such colonies were shown to contain demonstrable CFU-S (.74) and following optimization of secondary assay conditions s e l f -renewal of the p l u r i p o t e n t c e l l of o r i g i n was r e a d i l y demonstrable (75). The s i n g l e c e l l o r i g i n of mixed colonies was determined by d i r e c t obser-vation of colony formation from si n g l e c e l l s (70,76). -13-Th e f i r s t evidence of analogous mixed colonies of human o r i g i n was obtained by Fauser and Messner (77). They obtained colonies i n which both granulocytic and e r y t h r o i d c e l l s were present i n methylcellulose cultures of human bone marrow, perip h e r a l blood, and cord blood. The c e l l of o r i g i n was named CFU-G/E. Subsequently, these authors were able to obtain mixed colonies i n human bone marrow cultures i n which mega-karyocytes and macrophages were present i n addition to granulocytes and erythroid c e l l s and a new term, CFU-GEMM, was coined (78). Recently i t was reported that such colonies may also contain c e l l s . t h a t . c a n d i f f e r e n t i a t e into T-lymphocytes (79). The single c e l l o r i g i n of human mixed colonies was established by co-culturing male and female bone marrow c e l l s and demonstrating by analysis of the d i s t r i b u t i o n of fluorescent Y bodies i n i n d i v i d u a l colonies that each colony was derived from a s i n g l e donor and hence presumably a single c e l l (77). C. Summary An o u t l i n e of hemopoiesis, as defined by in vivo and in vitro c l o n a l assays i s presented i n Figure 2.. (80) . In such a scheme, 3 stages of hemopoietic c e l l development i s emphasized. The smallest compartment consists of stem c e l l s , defined by the dual properties of p l u r i -p o t e n t i a l i t y and self-renewal capacity. These c e l l s can be detected by t h e i r a b i l i t y to form large mixed colonies in vitro or spleen colonies i n mice. The second compartment consists of committed progenitor c e l l s which s t i l l possess extensive p r o l i f e r a t i v e capacity, but are r e s t r i c t e d to one pathway of d i f f e r e n t i a t i o n and appear to possess l i t t l e , i f any, a b i l i t y for self-renewal. In vitro, subpopulations of committed progenitors -14-ERYTHROIO PROGENITORS MEGAKARYOCYTE PROGENITORS GRANULOCYTE PROGENITORS F i g . 2. Diagrammatic representation of the hierarchy of hemopoietic progenitor compartments c u r r e n t l y i d e n t i f i e d by colony assay procedures. Colonies are grown i n semisolid media, such as methylcellulose or agar, with the addition of serum, e s s e n t i a l nutrients and appropriate stimulatory growth f a c t o r s . According to t h i s model the state of d i f f e r e n t i a t i o n of the colony forming c e l l determines the s i z e of the colony i t produces in vitro. From reference (80); used with permission. - 1 5 -may be detected by v a r i a t i o n s i n the size and time of appearance of the. colonies they produce. The t h i r d , and the l a r g e s t compartment i s composed of morpho-l o g i c a l l y recognizable, maturing c e l l s . These c e l l s are very l i m i t e d i n t h e i r p r o l i f e r a t i v e p o t e n t i a l and have no capacity for self-renewal. As they mature, fu n c t i o n a l and morphological markers which characterize the terminal c e l l become in c r e a s i n g l y apparent and the a b i l i t y to d i v i d e . i s l o s t . These c e l l s are then released into the c i r c u l a t i o n where they f u l f i l l t h e i r f u n c t i o n a l r o l e before they die and are replaced by new c e l l s . Considerable evidence e x i s t s for the presence of a common lymphoid-myeloid stem c e l l , and very recently, human colonies containing both myeloid and lymphoid elements have been reported. The grouping of hemopoietic c e l l s into three compartments i s a convenient method of describing the h i e r a r c h i c a l arrangement of the system. However, i t must be kept i n mind that d i f f e r e n t i a t i o n from the stem c e l l to the terminal c e l l i s a continuous process, and many intermediate.populations of c e l l s may e x i s t . For example, bipotent progenitors such as human CFU-G/E or murine progenitors of e r y t h r o i d -megakaryocyte colonies may represent intermediate stages between the p l u r i p o t e n t stem c e l l and various committed progenitor compartments. II . REGULATION OF ERYTHROPOIESIS D i f f e r e n t i a t i o n i n the e r y throid pathway i s a multistage process,. during which progression through successive stages of maturation i s co r r e l a t e d with decreasing p r o l i f e r a t i v e capacity and changes i n -16-s e n s i t i v i t y to s p e c i f i c regulatory f a c t o r s . Control mechanisms may operate at a number of l e v e l s s t a r t i n g with the p l u r i p o t e n t stem c e l l and-continuing a l l the way down to the mature red c e l l . In t h i s section, I s h a l l f i r s t review what i s known about the c o n t r o l of stem c e l l , p r o l i f e r a t i o n and committment to form unipotent erythropoietic progenitors. I s h a l l then discuss the r o l e of various factors believed to influence c e l l flow as c e l l s pass from the p r i m i t i v e BFU-E stage to the CFU-E stage and u l t i m a t e l y undergo terminal maturation into red blood c e l l s . A. Regulation of Stem C e l l P r o l i f e r a t i o n Since most information on the functioning of the hemopoietic system has been obtained from observations on terminal c e l l s , our understanding of e a r l i e r regulatory mechanisms at the stem c e l l l e v e l are l i m i t e d . 3 However, experiments u t i l i z i n g H-thymidine to determine the proportion of CFU-S i n a c t i v e cycle, and analysis of spleen colonies to determine the spectrum of e a r l y progeny obtained from i n d i v i d u a l CFU-S suggest three types of stem c e l l t r a n s i t i o n s that may or may not be independently regulated. These three t r a n s i t i o n s are: 1) changes i n c e l l cycle status; 2) self-renewal versus d i f f e r e n t i a t i o n ; and 3) commitment to a s p e c i f i c d i f f e r e n t i a t i o n pathway,(see Figure 3). 1. C e l l cycle status Lajtha and h i s associates (81) were the f i r s t to postulate that changes i n the more d i f f e r e n t i a t e d c e l l compartment would a f f e c t the p r o l i f e r a t i v e status of the stem c e l l . In t h e i r model the stem c e l l could e x i s t i n two states; i n a n o n - p r o l i f e r a t i v e , r e s t i n g state termed -17-Fig.3. Three types of stem c e l l t r a n s i t i o n s where regulatory mechanisms may act to influence stem c e l l behaviour: A, changes i n c e l l cycle status; B, self-renewal versus d i f f e r e n t i a t i o n ; C, commitment to a s p e c i f i c d i f f e r e n t i a t i o n pathway. -18-/ -19-G (82), and i n the p r o l i f e r a t i v e cycle (G n, S, G_, or M phase). O L A Subsequent experiments by other i n v e s t i g a t o r s provided evidence for 3 t h i s .model. Experiments u t i l i z i n g -,H-thymidine or other cycle a c t i v e drugs indicated that although the CFU-S i n a normal, i n t a c t mouse are i n a r e s t i n g state, the majority of the stem c e l l population can be triggered i n t o a c t i v e cycle by hemopoietic perturbation such as by s u b l e t h a l . i r r a d i a t i o n (83,84), the administration of cycle a c t i v e cytotoxic - drugs (84-88), endotoxin (89) or bleeding (90). Such studies indicated that the stem c e l l population i s capable of responding to a demand for new c e l l s by increasing i t s p r o l i f e r a t i v e a c t i v i t y . Both l o c a l regulatory mechanisms and long range humoral f a c t o r s have been implicated i n causing t h i s c e l l c ycle t r a n s i t i o n . Some experiments indi c a t e that depletion of stem c e l l s i n one part of the body r e s u l t s i n an increased p r o l i f e r a t i v e a c t i v i t y i n an unaffected s i t e , suggesting that long range mechanisms may e x i s t (91). In studies with mice undergoing p a r t i a l body i r r a d i a t i o n , i n v e s t i g a t o r s observed that the CFU-S i n the shielded areas were also induced i n t o active cycle. However, the p r o l i f e r a t i v e a c t i v i t y of the CFU-S i n the shielded marrow quickly reverted to the pretreatment state, even though the CFU-S i n the i r r a d i a t e d area were s t i l l present i n reduced numbers and were c y c l i n g r a p i d l y (92). The e a r l y t r a n s i e n t increase i n p r o l i f e r a t i v e a c t i v i t y has been postulated to r e s u l t from a decrease i n CFU-S i n the shielded area, due to accelerated d i f f e r e n t i a t i o n (92) or by migration (93-95) which activates a l o c a l mechanism capable of responding to a decrease i n the - surrounding stem c e l l population (96-98). -20-More recently Wright and h i s associates have separated two a c t i v i t i e s from bone marrow.conditioned media which appear to be capable.of reversibly..altering the p r o l i f e r a t i v e a c t i v i t y of the stem c e l l i n e i t h e r a p o s i t i v e or negative fashion (99-100). Although the s i z e of the l o c a l CFU-S compartment appears to be the primary factor i n determining stem c e l l p r o l i f e r a t i o n , the mechanism by which t h i s t r i g g e r operates i s not known. Extensive work i n Byron's laboratory has demonstrated that a number of substances can increase the f r a c t i o n of stem c e l l s i n DNA synthesis in vitro. B-adrenergic agents (101-102), c h o l i n e r g i c agents (102), and histamine (103) are capable of t r i g g e r i n g the stem c e l l in vitro, and t h e i r effects.appear to be mediated through the cyclase system. The addition of c y c l i c nucleotides to a bone marrow suspension w i l l also increase CFU-S c y c l i n g (104-104). Such findings l e d t h i s i n v e s t i g a t o r to postulate that high l e v e l s of phosphodiesterase i n G q c e l l s may prevent an increase i n the concentration of c y c l i c nucleotides s u f f i c i e n t to permit the i n i t i a t i o n of DNA synthesis. Other substances which may increase the number of stem c e l l s in active cycle are androgens (104,106,107) and prosta-glandin (108). These.substances do not appear to exert t h e i r e f f e c t s through the cyclase system. 2. Decision between self-renewal versus d i f f e r e n t i a t i o n The nature of the mechanism which determines the choice between self-renewal and r e s t r i c t i o n of d i f f e r e n t i a t i o n p o t e n t i a l i s not well defined. Indeed, i t i s not c l e a r that these are i n f a c t mutually exclusive processes, although for s i m p l i c i t y they are usually modelled as such. A major problem i s the lack of information about the -21-immediate changes that characterize the c e l l s produced when " d i f f e r e n t i a t i o n " rather than self-renewal occurs. This i s perhaps not s u r p r i s i n g since stem c e l l s can s t i l l only be detected by inference from looking at t h e i r progeny. Most studies of self-renewal have therefore simply looked at the preservation or l o s s of spleen colony forming a b i l i t y under a v a r i e t y of circumstances. For example stem c e l l s from W/WV mice do not form spleen colonies when i n j e c t e d i n t o normal +/+ l i t t e r m a t e s , yet CFU-S from normal syngeneic mice do form such colonies i n W/W mice (109). In contrast stem c e l l s from S l / S l mice are capable of forming spleen colonies when i n j e c t e d i n t o +/+ normal syngenic mice, but transplanted stem c e l l s from +/+ l i t t e r m a t e s d. do not form spleen colonies i n S l / S l hosts (110), suggesting that .the : microenvironment i n Sl/Sl* 3 , mice may be responsible for the p r o l i f e r a t i v e f a i l u r e of the transplanted normal stem c e l l s . Retransplantation studies have shown that although most spleen colonies contain CFU-S, t h e i r d i s t r i b u t i o n v a r i e s widely from colony to colony (10). I t has been suggested that t h i s heterogeneity r e s u l t s from -a.lax c o n t r o l of the . self-renewal process at the stem c e l l l e v e l where the actual choice between self-renewal and f a i l u r e to self-renew at the single c e l l l e v e l i s made. This choice i s made at random, but with a given p r o b a b i l i t y which can. be determined by examination of the behaviour of the population as a whole (111). The r e p l a t i n g experiments of Humphries and h i s colleagues (74,112,75) demonstrated that CFU-S production i n i n d i v i d u a l macroscopic mixed colonies showed the same heterogeneity observed for spleen colonies in vivo. These findings rein f o r c e d the model proposed by T i l l et al. ( I l l ) and suggested that -22-self-renewal i s not normally determined by e x t r i n s i c influences. However, some heterogeneity i n self-renewal may be due to non-stochastic processes. Examination of the self-renewal capacity of CFU-S which survive treatment with cytotoxic drugs, such as bulsulphan and isopropyl methane sulfonate, suggest that such drugs may act s e l e c t i v e l y on c e r t a i n CFU-S subpopulations (113-114). S e r i a l t ransplantation experiments ind i c a t e that a progressive loss of self-renewal capacity occurs when a r e p e t i t i v e p r o l i f e r a t i o n stimulus i s applied to the stem c e l l (115). These experiments i n d i c a t e that the h i s t o r y of the CFU-S, p a r t i c u l a r l y i t s organ of o r i g i n and the previous p r o l i f e r a t i v e demands made upon i t may influence the choice between self-renewal and d i f f e r e n t i a t i o n (116). This has led some authors to postulate an age structure for stem c e l l s , such that the CFU-S compartment i s not formed of a homogenous population, but of a "continuum" of c e l l s of decreasing self-renewal capacity (117). In t h i s model, as the stem c e l l s progress i r r e v e r s i b l y through the continuum, they experience a decline i n s e l f -renewal capacity, an increased p o t e n t i a l f o r d i f f e r e n t i a t i o n , and an increase i n c e l l c y c l e a c t i v i t y before eventually leaving the compartment as committed progenitors (117-118). 3. Commitment Various models have been formulated i n an attempt to elucidate the mechanism by which the c e l l "determines" the pathway i t w i l l follow. An e a r l y model postulated that pathway s p e c i f i c inducers, such as erythropoietin, may act upon a common plu r i p o t e n t stem c e l l to " d i r e c t " d i f f e r e n t i a t i o n i n t o a p a r t i c u l a r c e l l l i n e (119). This model pre d i c t s that competing demands made upon the stem c e l l by these -23-regulatory molecules would r e s u l t i n an increase i n one c e l l l i n e occurring at the expense of the other. Other investigators have suggested that the choice of pathways, l i k e the choice between self-renewal and d i f f e r e n t i a t i o n , i s made at random, with f i x e d p r o b a b i l i t i e s for each c e l l l i n e . In t h i s model, control i s exerted through the operation of pathway s p e c i f i c f a c t o rs which amplify t h i s random choice at l a t e r maturation stages, when appropriate, to meet the demand f o r mature c e l l s (111). Although r e s u l t s consistent with a competition model have been claimed, these cannot be interpreted rigorously since choice of pathway was i n f e r r e d from the numbers of terminal c e l l s produced and the manipulations used would have been expected to a f f e c t population flow of already committed progenitors. This c r i t i c i s m can i n part be overcome by measurements of p r i m i t i v e committed progenitor c e l l numbers. In such studies as have been under-taken i t has been found that increased output of one p r i m i t i v e c e l l type i s on average accompanied by an increased output of other p r i m i t i v e c e l l types (48). This i s the r e s u l t predicted by the f i x e d p r o b a b i l i t y model. B. Molecular Regulators of E r y t h r o p o i e t i c C e l l D i f f e r e n t i a t i o n Evidence f o r a lineage s p e c i f i c regulator of erythropoiesis goes back several decades. The hormone i d e n t i f i e d as a r e s u l t of these e a r l y studies has recently been p u r i f i e d to homogeneity (120). More re c e n t l y the a v a i l a b i l i t y of colony assays for erythropoietic progenitors at d i f f e r e n t stages of development has led to the discovery of a second type of f a c t o r . I t i s r e f e r r e d to here as burst enhancing factor or BEF, although other terms such as BPA (burst promoting a c t i v i t y ) and BFA (burst feeder a c t i v i t y ) have also been used by some in v e s t i g a t o r s . -24-In t h i s section I w i l l review what i s known about the r e l a t i v e importance of erythropoietin and BEF i n re g u l a t i n g c e l l s at d i f f e r e n t p o s i t i o n s on the.red c e l l pathway. 1. Erythropoietin The concept that red blood c e l l production was regulated by a c i r c u l a t i n g factor was developed i n 1906 by Carnot and Deflandre (121). D i r e c t evidence f o r the existence of t h i s humoral factor was not obtained u n t i l 1950, when Reissman (122) demonstrated that both members of a p a r a b i o t i c p a i r became polycythemic when one r a t was exposed to hypoxia. Subsequently, several i n v e s t i g a t o r s documented the presence of a substance i n the plasma of anemic animals which could stimulate erythropoiesis when in j e c t e d into normal r e c i p i e n t s (123-126). Experiments with nephrectomized rats established the kidney as the s i t e of e r y t h r o p o i e t i n production i n adult animals (127). Such f i n d i n g s l e d to the concept of a feedback mechanism for erythropoietic regulation (128-129) where the kidney, acting as the main sensory organ i n the system,secretes ery t h r o p o i e t i n i n response to a decrease i n t i s s u e oxygen tension. The increased l e v e l of erythropoietin stimulates erythropoiesis i n the bone marrow and t h i s a l l e v i a t e s the tissue hypoxia. Studies on the biochemistry of e r y t h r o p o i e t i n have established that i t i s a glycoprotein with a molecular weight of about 40,000 daltons of which about 29% i s carbohydrate (120). Approximately 7.5% of the molecule i s s i a l i c acid, which i s necessary for in vivo function. A s i a l o erythropoietin, although f u l l y f u n c t i o n a l in vitro, i s r a p i d l y cleared from the c i r c u l a t i o n by the l i v e r (130). -25-Direct evidence f o r the requirement of erythropoietin f o r red blood c e l l formation was f i r s t given by Jacobson and h i s colleagues (127). They showed that a l l recognizable erythroid precursors disappeared from the marrow and spleen of mice whose hematocrits were a r t i f i c i a l l y r a i s e d by hypertransfusion. I n j e c t i o n of a single dose of eryth r o p o i e t i n r e s u l t e d i n the reappearance within 48 hours of a wave of maturing erythroid c e l l s . More re c e n t l y the same type of protocol has been used to evaluate the r o l e of erythropoietin on CFU-E and BFU-E production. I t was found that polycythemia s u b s t a n t i a l l y reduces the number of CFU-E i n both spleen and marrow and t h i s e f f e c t i s r e v e r s i b l e by eryth r o p o i e t i n administration (43,44,131,132). Although regulation of normal CFU-E numbers by er y t h r o p o i e t i n i s well established, there i s also evidence i n d i c a t i n g that some CFU-E can be generated even when eryth r o p o i e t i n l e v e l s are gr e a t l y reduced (43, 133-135). However, production of the splenic and marrow CFU-E that 59 occurs i n polycythemic mice i s not followed by an increase i n Fe incorporation, or by an increase i n r e t i c u l o c y t e number. A requirement for erythropoietin can thus be assumed to be v i r t u a l l y absolute at these l a t e r stages. In vitro, evidence for an absolute ery t h r o p o i e t i n requirement f o r CFU-E 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 has been shown by delayed add i t i o n experiments (136). In contrast to CFU-E polycythemia has l i t t l e e f f e c t i f any on mature or p r i m i t i v e BFU-E numbers even i n mice undergoing repopulation of t h e i r CFU-E compartments following i r r a d i a t i o n and marrow trans-p l a n t a t i o n (44,46,137). Although some BFU-E are i n a r e s t i n g state i n normal mice (46), neither hypertransfusion nor anemia changes the -26-proportion i n active c e l l cycle (.133) . These findings l e d to the concept that erythropoietin l e v e l s have l i t t l e i f any regulatory e f f e c t on c o n t r o l l i n g events that take place i n p r i m i t i v e e r y t h r o p o i e t i c c e l l s , 1. e. at or p r i o r to the mature BFU-E stage. However, there i s some evidence that mature BFU-E, can respond to eryt h r o p o i e t i c s t i m u l i by increasing the proportion of c e l l s i n S-phase (137). Furthermore, erythropoietin administration a f t e r bulsulphan treatment r e s u l t s i n a prompt recovery of mature BFU-E numbers without a detectable increase i n the CFU-S or p r i m i t i v e BFU-E populations (138). The pre c i s e stage at which c e l l s f i r s t become responsive to ery t h r o p o i e t i n has not yet been established. 2. The r o l e of non-erythropoietin factors i n regulating erythropoietic  d i f f e r e n t i a t i o n The f a c t that hypertransfusion ( i . e . reduction of erythropoietin levels) has almost no e f f e c t on the rate of p r i m i t i v e BFU-E recovery i n i r r a d i a t e d mice suggested that t h i s compartment might be subject to co n t r o l by fa c t o r s other than erythropoietin. The f i r s t demonstration of such factors operating on BFU-E was reported by Aye (139). He observed that factors released by incubated human leukocytes or human embryo kidney c e l l s increased red c e l l colony formation i n cultures i n i t i a t e d with human marrow that had been f i r s t passed through a nylon wool column to remove adherent c e l l s . Such f a c t o r s were therefore c a l l e d burst enhancing factors or BEF. Subsequent studies showed that colony formation by mouse BFU-E also was enhanced by factors derived from mitogen stimulated mouse spleen c e l l s (136,140) or i r r a d i a t e d mouse marrow (141). Moreover t h i s could be more r e a d i l y demonstrated when -27-endogenous marrow sources were reduced simply by p l a t i n g the marrow at 4 concentrations below 8x10 c e l l s per ml (141). In both mouse and man a requirement for BEF was c l e a r l y demonstrable for p r i m i t i v e BFU-E, l e s s so for mature BFU-E and barely demonstrable for CFU-E (136,141,142). Early p u r i f i c a t i o n studies showed that-theLBEF.' s..\which..,were" a c t i v e on mouse and human progenitors were glycoproteins of approximately the same molecular weight (35-40,000 daltons) (136,143). A d i r e c t e f f e c t of BEF on the c e l l c ycle a c t i v i t y of BFU-E has been 3 demonstrated in vitro by H-thymidine studies (144) . In the presence of optimal concentrations of t h i s f a c t o r , colony formation by mouse and human BFU-E can proceed f o r as many as 7 days of c u l t u r e i n the absence of er y t h r o p o i e t i n (136,145). This p r i m i t i v e BFU-E may undergo 5 to 7 d i v i s i o n s before reaching a stage where contact with erythropoietin becomes c r u c i a l . Since an e f f e c t of p a r t i a l l y p u r i f i e d BEF on DNA synthesis and s u r v i v a l of CFU-S has also been demonstrated (144), BEF may also regulate i n part, the d i f f e r e n t i a t i o n of committed erythroid progenitors from the stem c e l l . BEF may be obtained from a v a r i e t y of c e l l sources including pokeweed mitogen stimulated murine spleen c e l l s (146) and human embryo kidney c e l l s , but i t s p h y s i o l o g i c a l importance in vivo l i k e that of CSF remains c o n t r o v e r s i a l . The f a c t that BEF replaces an a c t i v i t y that can be provided by other marrow c e l l s (139,142) suggests the existence of a l o c a l regulatory mechanism i n bone marrow that could have fu n c t i o n a l s i g n i f i c a n c e in vivo. The production of BEF a c t i v i t y by peripheral blood c e l l s , and i t s presence i n serum and urine also suggest a possible systemic r o l e (141,143). -28-C• Summary Although our knowledge about how erythropoiesis i s regulated;, i s s t i l l very l i m i t e d c e r t a i n postulations canibe made. Most stem c e l l s and some p r i m i t i v e BFU-E appear to be quiescent i n the normal adult, but these can be triggered into cycle by a number of f a c t o r s , i n c l u d i n g BEF. Control of BFU-E production from CFU-S appears to be secondary to mechanisms that c o n t r o l CFU-S p r o l i f e r a t i o n and self-renewal and strong evidence for mechanisms that d i r e c t the d i f f e r e n t i a t i o n of CFU-S towards a s p e c i f i c pathway has not been obtainable. The e a r l i e s t d i f f e r e n t i a t i o n steps between CFU-S and p r i m i t i v e BFU-E do not appear to require erythropoietin and p r i m i t i v e BFU-E numbers are not changed by manipulations that a l t e r erythropoietin l e v e l s in vivo. In vitro, a major regulatory influence of BEF on p r i m i t i v e BFU-E can be demonstrated. Exposure to BEF stimulates p r i m i t i v e BFU-E in t o cycle and can support t h e i r progeny through several d i v i s i o n s , p o s s i b l y to the mature BFU-E stage. Thereafter, d i f f e r e n t i a t i n g e r y t h r o p o i e t i c c e l l s develop a demonstrable and increasing s e n s i t i v i t y to erythropoietin. Concomitantly, a response to BEF becomes in c r e a s i n g l y d i f f i c u l t to demonstrate. A requirement f o r erythropoietin becomes pronounced when c e l l s begin to proceed out of the mature BFU-E compartment. Mature BFU-E appear to correspond to the ERC detected by in vivo assays. For d i f f e r e n t i a t i o n beyond the CFU-E stage, erythropoietin appears to be an absolute requirement. The mechanism of ac t i o n of BEF and erythropoietin on t h e i r target c e l l s i s s t i l l a subject of conjecture. One p o s s i b i l i t y i s that -29 d i f f e r e n t i a t i o n proceeds according to a predetermined program, subject to regulation only by factors that control p r o l i f e r a t i o n and/or s u r v i v a l (47). I I I . ' POLYCYTHEMIA VERA. A. General Features of the 'Myeloproliferative Disorders In 1951, Dameshek (147) introduced the term "myeloproliferative disorders" (MPD) to describe a group of c l o s e l y r e l a t e d syndromes -polycythemia vera (PV), e s s e n t i a l thrombocytosis (ET), chronic myeloid leukemia (CML), and myeloid metaplasia with myelofibrosis. These were grouped together because they were a l l believed to o r i g i n a t e from d i s -ordered growth of a p l u r i p o t e n t stem c e l l . Several overlapping c l i n i c a l and pathological c h a r a c t e r i s t i c s are seen i n t h i s group, most outstanding of which i s the tendency f o r a l l myeloid c e l l types to be elevated, although overproduction of mature blood c e l l s i s u s u a l l y p a r t i c u l a r l y pronounced f o r a given pathway according to the s p e c i f i c disease. The demonstration of the Philadelphia chromosome as a unique and consistent marker i n erythrocytes, granulocytes, macrophages, and megakaryocytes from patients with CML provided the f i r s t evidence for the c l o n a l o r i g i n of diseases included i n the MDP group (148-150). Another approach to the study of c l o n a l i t y i n the MPD has been the use of a d i f f e r e n t genetic marker system, the isoenzymes of glucose-6-phosphate dehydrogenase (G6pD), The gene for G6PD i s on the X-chromosome, and i n women one X-chromosome i n each c e l l undergoes i n a c t i v a t i o n early i n embryogenesis (151). The process i s a random one, and the choice i s f i x e d so that a l l the progeny of t h i s e a r l y c e l l have the same active -30-sin g l e X-chxomosome. An adult female heterozygous at the G6PD locus for A B the usual gene Gd , and a variant such as Gd , i s therefore a mosaic of two c e l l types, one synthesizing only type A G6PD, and the other only type B. The two isoenzymes may be separated by electrophoresis. Since X - i n a c t i v a t i o n occurs before t i s s u e determination most ti s s u e s i n G6PD female heterozygotes are also composed of equal numbers of c e l l s synthesizing one or the other isoenzyme type. Using t h i s marker system, female G6PD heterozygotes with CML (.152) , PV (57) , and myeloid metaplasia with myelofibrosis (153) have shown only one isoenzyme type i n the c i r c u l a t i n g red c e l l s , granulocytes and p l a t e l e t s , even though mosaicism was c l e a r l y demonstrable i n t h e i r skin f i b r o b l a s t s . Though ample evidence e x i s t s that these diseases o r i g i n a t e from a l e s i o n i n a plur i p o t e n t stem c e l l that permit i t s progeny to completely dominate the mature c e l l compartment, the nature of the mechanism that confers such a s e l e c t i v e advantage on these c e l l s i s unknown. Of great i n t e r e s t as well, i s the basis f o r the predominant elevation of one p a r t i c u l a r c e l l l i n e of the abnormal clone. B. S p e c i f i c Features of Polycythemia Vera The d i s t i n g u i s h i n g feature of PV i s an absolute increase i n the red c e l l mass. The disease i s a progressive one, of long duration, involving constant cytopathological p r o l i f e r a t i v e changes i n the marrow r e s u l t i n g i n d i f f e r i n g mature c e l l population r a t i o s . I f untreated, the great increase i n c i r c u l a t i n g c e l l mass may lead to stroke or myocardial i n f a r c t i o n . When treated, the l i f e s p a n of the patient i s lengthened, and the disease may progress to a spent phase, characterized by extramedullary hemopoiesis and f i b r o s i s and s c l e r o s i s of the bone -31-marrow (154). In 1-15% of patients, the disease terminates i n leukemic changes t y p i c a l of acute myeloblastic leukemia with progressive anemia and thrombocytopenia and the appearance of b l a s t c e l l s (155-158). 1. Cytogenetics Though no s p e c i f i c chromosomal marker comparable to the P h i l a d e l p h i a chromosome has been found i n PV, a number of patients (10 to 25%) do possess genetic abnormalities i n the d i v i d i n g marrow c e l l s (159). About 10-15% have a chromosomally marked c e l l l i n e or clone (160). Some chromosomes e s p e c i a l l y chromosome numbers 1, 8, 9, 20, seem to be affected more often than others, and a f a i r l y common cytogenetic f i n d i n g , i n about 20% of patients, i s the 20q deletion, which i s not found i n other hematological disorders (161-164). Other chromosomal abnormalities include trisomies, s t r u c t u r a l rearrangements such as deletions and translocations, aneuploidy, polyploidy, and hyper- and hypodiploidy (165-166). Karyotypic aberrations are more extensive i n patients with a longstanding h i s t o r y of the disease, and i n those who 32 have been treated with P (160). The importance of chromosomal changes i n the e t i o l o g y of PV i s unknown. Although a higher incidence of karyotypic abnormalities i s seen i n treated PV patients when the disease transforms into acute leukemia (160), no d i r e c t c o r r e l a t i o n was seen between the presence of chromosomal abnormalities and progression to acute leukemia (159). Conversely, the presence of a normal karyotype i n PV does not eliminate the p o s s i b i l i t y that the disease w i l l terminate i n a leukemic trans-formation. The prognostic value of cytogenetic findings i n PV at t h i s time therefore appears to be very l i m i t e d . -32-2. Erythropoietin-independence...and clonal- dominance PV patients have an inappropriate increase i n red c e l l mass without a concomitant .increase i n the l e v e l of erythropoietin. This led to the idea e a r l y on that erythropoiesis i n these subjects might be "autonomous", i . e . outside the regulatory influence of erythropoietin (167). Some inv e s t i g a t o r s have postulated that another humoral factor might s t i l l exert some control of red blood c e l l production i n PV (168) when normal counts have been exceeded, and erythropoietin l e v e l s are no longer detectable (169). However, a number of experiments have shown that PV patients may respond normally to erythropoietic s t i m u l i . For example, both phlebotomy and hypoxia lead to an increased production of erythropoietin (170) and concomitant with the appearance of erythro-p o i e t i n i n the urine, an increase i n i r o n turnover and r e t i c u l o c y t e production occurs, i n d i c a t i v e of augmented erythropoiesis (167). In vitvo studies, u t i l i z i n g the plasma c l o t (52) agar (171), and methylcellulose (139,17.2,173 ,142) culture .systems -have demonstrated the presence of erythroid progenitors i n PV capable of colony formation i n culture without the a d d i t i o n of erythropoietin. Such "erythropoietin-independent" erythroid colony formation i s not seen i n normal subjects, or i n patients with secondary polycythemia i f care i s taken to ensure that l e v e l s of erythropoietin i n other components of the culture (e.g. in the f e t a l c a l f serum) are i n s i g n i f i c a n t (174,175). Controversy e x i s t s however, as to whether erythropoietin-independent progenitors present i n PV patients are t r u l y autonomous of the normal requirement for continuing contact with erythropoietin. An a l t e r n a t i v e p o s s i b i l i t y i s that these c e l l s are e x q u i s i t i v e l y s e n s i t i v e to erythropoietin and -33-can s a t i s f y t h e i r requirement i n the presence of the trace amounts of the hormone contributed by the f e t a l c a l f serum i n the culture medium. Zanjani and h i s associates (.172) were able to i n h i b i t endogenous colony formation by using an antiserum r a i s e d against a preparation containing erythropoietin but the s p e c i f i c i t y of t h e i r antiserum was not r i g o r o u s l y evaluated. Other in vitvo experiments have provided evidence that at l e a s t some erythroid precursors i n PV r e t a i n a normal ery t h r o p o i e t i n response mechanism. A stimulatory e f f e c t of added erythropoietin on the number of colonies obtained i n PV culture was f i r s t demonstrated by Prchal and Axelrad (.52) and has subsequently been confirmed by a number of i n v e s t i -gators (139,142,172,173). This suggests that such erythroid progenitors may be subject to normal regulation by v a r i a b l e erythropoietin l e v e l s in vivo. Further studies have confirmed that two populations of erythroid progenitors e x i s t i n PV: 1) a phenotypically abnormal popula-t i o n capable of colony formation i n cultures containing l e s s than .001 u/ml of erythropoietin; and 2) a phenotypically normal population with a normal erythropoietin responsiveness (142). The use of G6PD isoenzyme markers i n female heterozygotes with PV have shown that a l l erythroid precursors capable of erythropoietin-independent colony formation are members of the abnormal clone. Colonies containing the second isoenzyme varia n t could be demonstrated only when eryth r o p o i e t i n was added at a concentration that supports colony formation by normal progenitors (176). In addition these authors showed that the r a t i o .of: type A and type B colonies obtained i n the presence of erythropoietin was i n d i c a t i v e of a subpopulation of c e l l s belonging to the abnormal clone but not capable -34-of erythropoietin-independent growth. Erythropoietin independent growth can thus be considered as a unique but not.consistent marker of a l l members of the abnormal clone. I t s presence i n some c e l l s has, however, suggested the basis f o r a p o s s i b l e pathogenic mechanism i n PV (172,142). I t would be expected that low c i r -c u l a t i n g l e v e l s of erythropoietin i n the PV patient would provide a con^ siderable growth advantage f o r abnormal c e l l s that could complete the eryth r o p o i e t i c program under such conditions. This could explain how they come to dominate the mature c e l l compartment. However, .the demon-s t r a t i o n that only one isoenzyme type i s found i n the c i r c u l a t i o n even when red c e l l l e v e l s (and hence presumably erythropoietin levels) are returned to normal i n the treated patient, i s at variance with erythro-poietin-independence being the sole pathological mechanism (176). Furthermore such a phenotype does not account f o r the increase i n granulocyte and megakaryocyte c e l l l i n e s usually seen i n these patients. Other inv e s t i g a t o r s have postulated that the defect i n an abnormal stem c e l l may lead to an expansion of the committed stem c e l l pool, occurring p r e f e r e n t i a l l y i n the er y t h r o i d c e l l l i n e (177). Recent experimental data on large numbers of patients and controls i n d i c a t e that populations of committed progenitors are not elevated i n PV (80)-: I t has also been found that the r a t i o of p r i m i t i v e e r y t h r o p o i e t i c to granulopoietic progenitor numbers do not d i f f e r from that found i n normal i n d i v i d u a l s (142) even when members within the abnormal clone are s p e c i f i c a l l y looked at using the G6PD marker system (178). These experimental findings suggest that the l e s i o n . i n the abnormal stem c e l l does not a f f e c t e a r l y commitment events, but, by some unknown mechanism, -35-p r e f e r e n t i a l a m p l i f i c a t i o n of the progeny of the abnormal clone occurs at l a t e r stages of maturation. I t has been suggested that the pre-dominance of abnormal c e l l s i n l a t e r compartments may r e s u l t i n part from suppression of normal erythropoiesis at a d i f f e r e n t i a t i o n step between BFU-E and the more mature CFU-E (.178,179). Though no mechanism has been defined to account for suppression of erythroid progenitors, some evidence has been presented suggesting that the decrease i n the normal granulocyte population may be due to fewer CFU-C i n a c t i v e cycle (180). In summary, PV i s a disease of c l o n a l o r i g i n , that may be described as a neoplastic disorder (179), i n which the progeny of a si n g l e stem c e l l , i n s e n s i t i v e to normal growth regulatory mechanisms, completely f i l l s the mature c e l l compartments in vivo. Unlike CML r e s i d u a l normal stem c e l l s have been r e a d i l y demonstrable in vitro. However, as the disease progresses these decrease i n number and may become e x t i n c t . Although chromosomal abnormalities are found i n a minority of patients with PV, there i s no evidence as yet that such genetic rearrangements play a primary r o l e i n the o r i g i n and evolution of the neoplastic c h a r a c t e r i s t i c s of the abnormal clone. C. Experimental Rationale The present studies were undertaken to obtain further information about the mechanism that leads to an a l t e r e d e r y t h r o p o i e t i n responsive-ness. S p e c i f i c a l l y , we sought to determine whether the generation of phenotypically normal and abnormal erythroid c e l l s r e f l e c t s differences established very e a r l y on.(for example, at the l e v e l of the p l u r i p o t e n t stem c e l l compartment or during the process of r e s t r i c t i o n of -36-d i f f e r e n t i a t i o n potential) or, whether the p o t e n t i a l to express either phenotype p e r s i s t s considerably beyond t h i s point. I f the f i r s t model were co r r e c t then c e l l s derived from i n d i v i d u a l BFU-E would be expected to be of one phenotype or the other but not both. If the second model were correct, some BFU-E would be expected to produce both phenotypes. Preliminary studies suggested that i t should be p o s s i b l e to d i s t i n g u i s h between these two a l t e r n a t i v e s by r e p l a t i n g primary colonies of p r i m i t i v e BFU-E o r i g i n . Accordingly, experiments were undertaken (1) to define conditions that y i e l d e d the highest number of erythroid colonies i n secondary assays, (2) to e s t a b l i s h whether primary colonies could be equally s p l i t between 2 secondary cultures and (.3) to compare the number of secondary erythroid colonies obtained i n assays containing high and low e r y t h r o p o i e t i n concentrations when i n d i v i d u a l primary colonies from cultures of PV c e l l s were replated. The r e s u l t s obtained are not consistent with segregation of phenotypes p r i o r to the p r i m i t i v e BFU-E stage i n PV and suggest a new approach to i d e n t i f y i n g a subpopulation of p r i m i t i v e BFU-E that appear r e s t r i c t e d to the production of pheno-t y p i c a l l y normal progeny. -3.7-THE EXPERIMENTAL DESIGN Progenitor cell o ^ 9 days Primary colony <5b Secondary colonies with epo without epo The experimental design developed to study the erythropoietin requirements of replated progeny of individual erythroid progenitors .(primitive BFU-E) from patients with PV. Primary colonies are incubated in the presence of erythropoietin at 37°C for 9 days, individually removed and resuspended i n a s l i g h t l y modified secondary culture medium (see Materials and Methods) and divided equally between two secondary assays, one with a high and one with a low erythropoietin concentration. After a further 9 to 10 days of incubation, secondary cultures were examined for the presence of erythroid colonies -38-MATERIALS AND METHODS 1. Patients Five patients with PV were used i n t h i s study (Table 1). Three of these were cultured within a few months of diagnosis and met the diagnostic c r i t e r i a of the Polycythemia Vera Study Group (PVSG) (181). The c r i t e r i a established by the PVSG were made p a r t i c u l a r l y stringent i n order to obtain a homogenous group of patients f o r the purpose of evaluating various courses of treatment. I f a pat i e n t has a l l three of the major c r i t e r i a , i . e . an elevated red blood c e l l mass, a normal a r t e r i a l oxygen saturation and splenomegaly, he i s considered to have PV. I f splenomegaly i s not present, then the patient must have at l e a s t two of the minor c r i t e r i a (see Table 2);. The other two PV pat i e n t s had a longstanding h i s t o r y of the disease. The diagnosis of Patient 4 had been established 9 years p r i o r to the present study. This patient f u l f i l l e d a l l the c r i t e r i a of the PVSG except that two attempts to obtain a r t e r i a l blood gases had been unsuc-c e s s f u l and the patient subsequently refused further t e s t s . Patient 5 had been diagnosed 17 years p r i o r to t h i s study, and a r t e r i a l blood gases and a red c e l l mass had not been measured. However, t h i s p a t i e n t had other convincing c r i t e r i a (Table 1) and had required regular phlebotomies since diagnosis. Control blood samples were obtained from three healthy adult volunteers. Informed consent was obtained i n a l l cases. TABLE 1. C l i n i c a l Data on Patients with a ..Diagnosis of Polycythemia Vera patient Number 1* 2* 3* 4 5 Sex M M F F F Age at diagnosis 47 74 72 32 55 Age at culture 47 74 72 41 72 Duration of known disease (yr) <1 <1 <1 9 17 Red c e l l mass (ml/kg) 54 46 32 41 NA A r t e r i a l oxygen saturation (%) 97 92 93 NA NA Splenomegaly Yes No No Yes Yes -3 P l a t e l e t s (per yH x 10 ) 280 642 725 763 584 _3 White c e l l count (per u£ x 10 ) 8.0 8.8 7.1 14.5 20.0 LAP score 218 197 110 77 NA Serum B 1 2 (pg/ml) NA NA NA 1,600 NA Marrow panmyelosis Yes NA Yes Yes Yes Marrow ir o n s t a i n Neg. NA Trace Neg. Neg. Marrow f i b r o s i s No NA No No No Basophils (per u&) 520 176 198 296 198 Giant p l a t e l e t forms Yes Yes No Yes Yes Post-bath p r u r i t u s No Yes No Yes Yes * - F u l f i l l the c r i t e r i a of the Polycythemia Vera Study Group NA- Not a v a i l a b l e . -40-TABLE 2. C r i t e r i a of the Polycythemia Vera Study Group Major' C r i t e r i a : 1) increased red c e l l mass male >. 36 ml/kg female >. 32 ml/kg 2) normal a r t e r i a l oxygen saturation ( i . e . >. 92%) 3) splenomegaly In the absence of splenomegaly, two of the following may be substituted: Minor C r i t e r i a : 1) leukocytosis white blood c e l l count > 12,000/uA 2) thrombocytosis p l a t e l e t count > 400,000/u& 3) elevated leukocyte a l k a l i n e phosphatase (LAP) score (> 100) 4) an elevated serum vitamin B (> 900 pg/ml) or unbound B.,0 binding capacity (> 2,200 pg/ml) From reference (181). -41-2. C e l l Preparation Peripheral blood specimens were c o l l e c t e d i n preservative-free s t e r i l e heparin at a f i n a l concentration of 50 units/ml. Ten ml aliquotes of blood were layered over 15 ml of Ficoll-Hypaque (LSM-Bionetics, Kensington Maryland) and spun f o r 30 minutes at 1,700 rpm at room temperature. The l i g h t density mononuclear c e l l f r a c t i o n f l o a t i n g at the p l a s m a / F i c o l l -Hypaque i n t e r f a c e was then removed and washed twice i n 10 ml of a medium (Gibco, Calgary, Alberta) containing 2% f e t a l c a l f serum (FCS, Flow Labor-a t o r i e s , Inglewood, C a l i f o r n i a ) and resuspended i n 2% FCS f o r p l a t i n g . This c e l l suspension was then added to the methylcellulose assay mixture to e s t a b l i s h primary colonies or i r r a d i a t e d and then added as a source of "feeders" (see below). The f i n a l concentration of c e l l s used to generate primary colonies 4 5 ranged from 5 x 10 c e l l s / m l to 4 x 10 c e l l s / m l , depending on the i n d i v i d ual growth c h a r a c t e r i s t i c s of each patient. Since a l l patients had been previously evaluated f o r erythroid colony growth t h i s could be determined by looking at the r e s u l t s of previous assays. Usually several plates at two d i f f e r e n t concentrations were established to ensure that adequate num-bers of large, w e l l - i s o l a t e d erythroid colonies would be obtained. Under the above conditions from 5 to 50 colonies were obtained i n each dish. With 50 randomly d i s t r i b u t e d colonies, each having a diameter of 1 mm i n a volume of 1.1 ml the p r o b a b i l i t y of colony overlay i n a d i s h i s only 0.014 3. Primary Colony Assays The basic methylcellulose assay mixture used contained 0.8% methylcellulose, (Dow Chemicals, Vancouver, B.C.), 30% FCS, 1% deionized -4 bovine.;serum albumin (BSA: Sigma, St. Louis, Missouri), 10 M 2-mercapto-ethanol (Sigma), c e l l s and a media. Sheep plasma Step 3 erythropoietin -42-(Connaught Laboratories, Toronto) or p a r t i a l l y p u r i f i e d .erythropoietin from the urine of a patient with, a p l a s t i c anemia (.182) was added to the culture medium to give a f i n a l concentration of 2.5 u/ml. This mixture was prepared by adding appropriate volumes of each component and then vortexing vigorously f o r 1-2 seconds. In the culture system used to establish, primary colonies, 9% (v/v) leukocyte conditioned media (.LCM) was added. LCM was prepared by the standard agar-medium overlay procedure (26), but with an increase i n the c e l l concentration i n the agar to 4 x 10^ c e l l s / m l and the addi t i o n of -4 10 . M 2-mercaptoethanol to the medium. A l l components of the cultures had been pretested f o r optimal growth supporting capacity against previous standards. In t h i s way, v a r i a t i o n due to unknown factors i n d i f f e r e n t batches of FCS, BSA, LCM and erythropoietin was minimized. The complete methylcellulose culture mixture containing the c e l l s as plated i n r e p l i c a t e 35 mm Lux p e t r i dishes, using a syringe with a 15 gauge needle. A volume of 1.1 ml was delivered into each dish. These were then incubated at 37° i n an atmosphere of 5% CO^ and high humidity f o r nine days. 4. Secondary Colony Assays Secondary cultures were prepared using the methylcellulose and a medium mixture described above with 2 modifications found to increase p l a t i n g e f f i c i e n c y i n r e p l a t i n g assays.(see Results). a. Phytohemagglutinin-stimulated leukocyte conditioned media F i r s t , phytohemagglutinin-stimulated leukocyte conditioned media (PHA-LCM) was used i n place of the leukocyte conditioned media added to the primary cultures. PHA-LCM was prepared by incubating normal human -4 3-periph e r a l blood buffy coat c e l l s for 7 days at 37°C i n a medium -4 containing 10 M 2-mercaptoethanol, 10% FCS, 1% BSA, 1% PHA (PHA M 6 Form, Gibco) with the c e l l s at a f i n a l concentration of 4 x 10 per ml. The conditioned medium was then centrifuged at 2,000 rpm for 30 minutes to remove c e l l s and debris. Individual batches of PHA-LCM were tested for t h e i r a b i l i t y to enhance large erythroid burst formation i n assays of normal non-adherent human marrow c e l l s (.51) , and combined, aliquoted, and kept frozen at -20°C p r i o r to use. PHA-LCM produced i n t h i s way was ro u t i n e l y found to be a potent source of burst enhancing and granulocyte colony stimulating a c t i v i t y when added to assays of non-adherent human marrow c e l l s at a f i n a l concentration of 5% (v/v) (175). b. Peripheral blood feeder c e l l s Previous r e p l a t i n g experiments performed with mouse c e l l s i n our laboratory (75), had shown that the addi t i o n of fresh, i r r a d i a t e d marrow feeder c e l l s to the secondary c u l t u r e dishes gave a 5-10 f o l d increase i n the number of large mixed colonies formed. Because of obvious d i f f i c u l t i e s i n obtaining human marrow on demand, a se r i e s of experiments were undertaken to determine i f peri p h e r a l blood c e l l s might be found to increase the y i e l d of secondary erythroid colonies obtainable i n the present studies. To prepare feeder c e l l s , blood was separated on Ficoll-Hypaque as described above and the l i g h t density mononuclear f r a c t i o n obtained. These c e l l s were then given 2,500 rads using a 280 KV Picker X-ray machine (HVL, 1.7 mm Cu, dose rate 155 rads/min) to prevent further c e l l d i v i s i o n , and then added to the secondary assay 5 mixture at a f i n a l concentration of 4 x 10 c e l l s / m l . A l l feeder -44-c e l l s were obtained from the same donor (A.E.)-A ser i e s of preliminary experiments (see Results) determined that o feeder c e l l s incubated at 4 C for 9 to 10 days gave the best improvement i n secondary colony formation, and t h i s was therefore adopted as the standard procedure. 5. Replating Technique The primary cultures.were removed from the incubator a f t e r nine days and examined at :40X magnification,;, using an. inverted microscope, for. the presence of well-isolated..colonies..containing 100 to 200 large r e f r a c t i l e c e l l s arranged i n c l u s t e r s (Figure 5). Such colonies i n v a r i a b l y proved to be er y t h r o p o i e t i c . Colonies c o n s i s t i n g of a dense core of c e l l s with or without a surrounding halo of c e l l s were avoided as these were considered to be granulocytic. Colonies i n which hemo-globin synthesis was already evident were also avoided. Colonies selected for r e p l a t i n g were removed from the d i s h by mouth a s p i r a t i o n using a long f i n e l y drawn out Pasteur pipet containing a small quantity of a media. Each colony was placed i n a small tube containing 1 ml of the secondary assay mixture made up previously. A f t e r vortexing each tube to resuspend the c e l l s , the contents were c a r e f u l l y drawn up in t o a 1 ml pipette. The media containing the resuspended sin g l e colony was then equally divided between two adjacent wells of a Libro t i s s u e c u l t u r e tray (Model 96LV-TC). The large 96 well trays had been previously cut into 4 well blocks and each placed i n s i d e a 60 mm p e t r i d i s h to f a c i l i t a t e handling. On average, 0.4 ml was put into each p a i r of wells and the remaining two wells f i l l e d , with" d i s t i l l e d water f o r humidification. F i n a l l y , a 0.05 ml a l i q u o t of a media containing 27.5 -45-• « « »P _ * | « J 5 |Qp C F i g . 5. A 9 day o l d colony of the type s e l e c t e d f o r r e p l a t i n g . The scattered d i s t r i b u t i o n of s i n g l e c e l l s i n small groups together with the lack of evidence of any hemoglobin synthesis are features that i d e n t i f y e a r l y stage c o l o n i e s that go on to develop i n t o very large bursts a f t e r a f u r t h e r 9-10 days incubation (see Figure 1) or on r e p l a t i n g y i e l d the highest number of secondary co l o n i e s . (Magnification 200X) -46-u/ml of erythropoietin was added to one of the wells, g i v i n g a f i n a l concentration of ~ 3 u/ml. An equal volume of a media containing no e r y t h r o p o i e t i n was placed i n the opposite well to y i e l d a c u l t u r e containing l e s s than 0.01 u/ml of erythropoietin. The wells were then scanned at 100X magnification for...the presence of .aggregates of 2 or more c e l l s . I f such aggregates were found the dishes were discarded. Secondary assays were incubated at 37°C i n an atmosphere of 5% CO^ i n a i r for a further 9 days and then scored d i r e c t l y f o r the number of c l u s t e r s containing 8 or more hemoglobinized (orange-red) erythroblasts. The vast majority of these were t y p i c a l CFU-E type colonies (Figure 6) although occasionally bursts were also seen. Both types of colonies were included i n the data. The amount of erythropoietin present i n the secondary cultures to which no erythropoietin was i n t e n t i o n a l l y added was estimated from previous assays of the maximum possible l e v e l s of erythropoietin i n the complete methylcellulose medium (.< 0.002 units per ml) (45) and from estimates of erythropoietin carry-over i n the volume of primary culture added to each secondary assay. This was determined by adding 59 a given amount of Fe to the primary cultures and counting the amount of l a b e l t r ansferred to secondary assays. Twenty-four colonies were transferred and the amount of l a b e l carry-over showed that on average each primary colony had been removed i n a volume of 0.001 ml, and the maximum volume for a s i n g l e colony was 0.002 ml. Since primary cultures contained 3 u n i t s of erythropoietin, on average the carry-over into the secondary cultures to which no e r y t h r o p o i e t i n was added would have given 0.003 units of erythropoietin per ml with a maximum of 0.006. F i g . 6. Examples of 9 day o l d secondary c o l o n i e s obtained i n dishes containing about 3 u/ml of e r y t h r o p o i e t i n . Both c o l o n i e s were derived from c e l l s from normal i n d i v i d u a l s . A. A t y p i c a l secondary colony, estimated to contain about 30 e r y t h r o b l a s t s . Colonies of t h i s s i z e and appearance comprised the majority of c o l o n i e s obtained i n secondary assays. B. A l a r g e r secondary colony estimated to contain 150 e r y t h r o b l a s t s . Such large secondary c o l o n i e s were not common and were obtained only i n secondary assays to which e r y t h r o p o i e t i n had been added. (Magnification 200X) -48-6. S t a t i s t i c a l Analysis Chi-square analysis C183) was used to t e s t f o r equivalent colony growth, i n the two secondary cultures derived from each, primary colony. A chi-square value was f i r s t c a l c u l a t e d f o r each primary colony. The values f o r a l l the primary colonies, of each, patient were then summed y i e l d i n g a t o t a l chi-square value f o r the patient. The use of some secondary cultures with low expected frequencies does not i n v a l i d a t e the analysis since the average expected frequencies were reasonably high,(184), and the sample t o t a l chi-square values g r e a t l y exceeded the c r i t i c a l values. Analysis of variance was not used because of the complexities introduced by having unequal numbers of secondary colonies produced by each primary colony. - 4 9 -RESULTS 1. Conditions for Maximizing Erythroid Colony Counts i n Secondary  Assays of Replated Primary Colonies To maximize the number of secondary erythroid colonies obtained from each primary colony, the time of r e p l a t i n g and the possible p o t e n t i a t i n g e f f e c t of i r r a d i a t e d feeder c e l l s were examined. Primary cultures f o r time course studies were set up using peripheral blood from normal i n d i v i d -uals and from patients with PV as described i n the Methods. At d a i l y i n t e r v a l s primary cultures were examined and attempts made to d i s t i n g u i s h early stage large bursts. In preliminary experiments i t was established that between 6 and 11 days such colonies appear as d i s c r e t e c o l l e c t i o n s of 50 to 200 large, i r r e g u l a r l y d i s t r i b u t e d , r e f r a c t i l e c e l l s , without an obvious c e n t r a l core. P r i o r to Day 6 of c u l t u r e , these c r i t e r i a were not u s e f u l and i t was d i f f i c u l t to d i s t i n g u i s h early stage large bursts from early stage small bursts or granulocyte-jcolonies with a high degree of success. A f t e r Day 11, the number of large bursts i n which hemoglobin synthesis had not yet begun diminished r a p i d l y . Therefore, r e p l a t i n g studies were done on Day 6 through to Day 11. At each time point secondary colony y i e l d was based on the assessment of 20 i n d i v i d u a l l y replated primary colonies. The r e s u l t s of a t y p i c a l experiment are shown i n Figure.-7 (open symbols). I t can be seen that on average 9 day old primary colo:-->j_ nies yielded the highest number of secondary erythroid colonies. Several experiments of t h i s type confirmed that t h i s was true not only f o r primary colonies from normal i n d i v i d u a l s but a l s o for primary colonies from patients with PV (data not shown). Also evident i n Figure 7 i s the ±h to 2- f o l d increase i n secondary -50-> c o o O > CO E C 30,. 0. 25 L O O. to CD O O O CO •o c o o <D CO o z 20L « 15 10L 19 - 2 0 R e p l a t e d Colonies per Point NORMAL • F e e d e r s . o No F e e d e r s """I T 1 S if * Age of Primary Colonies When Replated ( Days ) Figure 7. Time course study of erythroid colony y i e l d s from replated primary colonies allowed to grow for varying periods p r i o r to assay i n secondary cultures with (- • ) and without (o) feeders. Values shown represent the mean -± SEM f o r counts from 19-20 i n d i v i d u a l l y replated primary colonies. -51-colony formation obtained when feeders were used. In t h i s and subsequent experiments feeders consisted of i r r a d i a t e d p e r i p h e r a l blood mononuclear c e l l s added to the secondary c u l t u r e medium at a f i n a l concentration of 5 o 4 x 10 c e l l s per millimeter, and stored at 4 C f o r 9 days p r i o r to the add i t i o n of c e l l s from primary colonies. The adoption of t h i s p a r t i c u l a r protocol was based on preliminary experiments which were i n i t i a l l y designed to see i f feeders could be e f f e c t i v e l y prepared before the time of r e -p l a t i n g (for convenience) and, i f so, what were the optimal conditions f o r storage. Table 3 summarizes the r e s u l t s of several experiments. A number of points emerge from these studies. Addition of p e r i p h e r a l blood mononuclear c e l l s to primary r e p l a t e assays enhanced secondary e r y t h r o i d colony formation only i t the feeders had been stored at 4°C for 9-10 days previously. Freshly prepared feeder c e l l s had no consistent enhancing o e f f e c t and the use of feeders stored at 37 C s i g n i f i c a n t l y i n h i b i t e d secondary colony formation. 2. Normal Erythroid Colony Formation i n Secondary Assay Replicates'With •-.and Without Added Erythropoietin The purpose of the present study was to determine whether erythro-poietin-dependent c e l l s as well as erythropoietin-independent c e l l s were produced by i n d i v i d u a l p r i m i t i v e BFU-E i n patients with PV. To assess t h i s the number of secondary erythroid colonies which developed i n paired r e p l i c a t e ^ r e p l a t e s with and without added erythropoietin was compared. If the capacity f o r generating erythropoietin-independent c e l l s was.fixed . at the l e v e l of p r i m i t i v e BFU-E then a l l primary colonies y i e l d i n g ery-t h r o i d colonies i n secondary assays without added erythropoietin.would be expected to y i e l d on average an equivalent number of colonies i n the -52-TABLE 3. Dependence of Secondary Erythroid Colony Formation by Replated Primary Colony C e l l s on the Use and P r i o r Treatment of Normal Peripheral Blood Feeder C e l l s * Replating Conditions Experiment No. Difference i n Growth Relative 1 2 3 4 Mean to No Feeders** No feeders 17. 9 8. 6 10. 7 9. 8 11. 7 -Fresh feeders 10. 0 19. 0 7. 6 10. 0 11. 6 Not s i g n i f i c a n t Stored feeders (37°C) 3 days 30. 2 6. 6 9. 6 15. 4 Not s i g n i f i c a n t 6-7 days 7. 7 5. 4 6. 6 S i g n i f i c a n t decrease 9-10 days . 0. 4 4. 2 5. 8 3. 0 3. 3 S i g n i f i c a n t decrease Stored feeders (4°C) 3 days 26. 6 9. 0 11. 8 15. 8 Not s i g n i f i c a n t 6-7 days 10. 8 13. 6 12. 2 Not s i g n i f i c a n t 9-10 days 26. 2 22. 8 22. 4 17. 8 22. 3 S i g n i f i c a n t increase Data presented are the number of secondary colonies obtained per replated primary colony based on the i n d i v i d u a l assessment of 20 primary colonies f o r each condition tested. Four separate experiments were performed i n each of which the same i n d i v i d u a l was used both as a source of primary colonies and as a source of feeder c e l l s . S i g n i f i c a n c e of the variance component due to the addi t i o n of the s p e c i f i e d type of feeder as determined by two-way analyses of variance. -53-paired secondary assays. Some v a r i a t i o n between r e p l i c a t e s would be expected s o l e l y on the basis, of sampling error but these would also be expected to be random and hence average out. To j t e s t t h i s assumption a t o t a l of 60 primary colonies, 20 from each of 3 normal i n d i v i d u a l s , were replated into two i d e n t i c a l secondary assays, and s u f f i c i e n t e rythropoietin added to both to bring the f i n a l concentration to 3 u n i t s per m i l l i l i t e r . The numbers of secondary colonies obtained i n each p a i r are shown i n Table 4. As predicted, there were di f f e r e n c e s between r e p l i c a t e s for most p a i r s . However, these were not consistent as shown by Chi-square a n a l y s i s (183), i n d i c a t i n g that on average an equal number of secondary erythroid colony-forming c e l l s was delivered into each r e p l i c a t e by the r e p l a t i n g procedure used. In a second c o n t r o l experiment the design was modified so that one secondary culture contained a high concentration of erythropoietin while the other contained no_added ery t h r o p o i e t i n . A t o t a l of 90 primary colonies, 30 from each of 3 normal i n d i v i d u a l s , were assessed i n t h i s way and the r e s u l t s are shown i n Table 5. F i f t y - n i n e y i e l d e d secondary erythroid colonies i n the r e p l i c a t e s to which er y t h r o p o i e t i n was added. No secondary colony formation occurred i n any of the r e p l i c a t e s to which erythropoietin was not added. This shows that the l e v e l of erythropoietin present i n secondary assays to which no e r y t h r o p o i e t i n was added was i n s u f f i c i e n t to support erythroid colony formation by normal progenitors, and hence v a l i d a t e d the use of such culture conditions to d i s t i n g u i s h erythropoietin-dependent and erythropoietin-independent phenotypes. -54-Table 4. Number of Secondary Erythroid Colonies Obtained i n Duplicate Replates of Primary Colonies from 3.Normal Individuals.* Colony Number #1 #2 #3 a b a Lb a b 1 5 9 27 24 8 14 2 19 13 6 6 31 37 3 27 27 18 21 4 7 4 16 22 15 13 2 2 5 8 5 12 15 11 9 6 7 13 7 9 2 2 7 6 7 14 20 17 27 8 7 6 13 10 26 29 9 24 39 6 7 5 3 10 3 4 9 13 7 11 11 6 5 74 83 47 49 12_ 25 21 8 9 44 33 13 12 14 13 16 41 41 14 6 4 7 9 5 6 15 5 3 8 7 5 3 16 9 7 27 20 14 10 17 5 7 20 17 5 7 18 4 3 40... 46 13 17 19 3 5 6 7 2 1 20 7 12 7 8 7 6 TOTALS 204 226 337 360 296 314 Total Chi-Square (JD.F.) 13.6(20) n.s. ..6..'4.(2P) n. s. 11. 2 ( 2 0 ) n ' S -D.F. degrees of freedom n.s. not s i g n i f i c a n t * A f t e r 9 days culture, 20 primary colonies from each i n d i v i d u a l were divided equally between 2 duplicate secondary cultures (a and b), each containing about 3 u/ml of erythropoietin. -55-Table 5. Number of Replated Primary Colonies From 3 Normal Individuals Y i e l d i n g Erythroid Colonies i n Secondary Assays With and Without Added Erythropoietin.* Experiment Number +Erythropoietin -Erythropoietin 1 20/30 0/30 2 22/30 0/30 3 17/30 0/30 Total 59/90 0/90 * A f t e r 9 days of cu l t u r e 30 primary colonies from each i n d i v i d u a l were divided equally between 2 secondary cultures, one containing 3 u/ml of erythropoietin, the other containing < 0.01 u/ml of erythropoietin. -56-3. Replating Experiments, with. Primary Colonies from EV Patients. Table 6 shows the r e s u l t s obtained when primary colonies were removed from assays of peripheral blood from PV patients and transferred to paired secondary cultures with and without added ery t h r o p o i e t i n . Five separate experiments were performed, each with, a d i f f e r e n t PV patient. Of the 109 primary colonies replated, 21 produced erythroid colonies i n the secondary assay containing 3 units of erythropoietin per ml, but not i n the paired r e p l i c a t e that contained < 0.01 units, per ml. The remaining 88 produced er y t h r o i d colonies under both conditions, but the colonies obtained i n the low e r y t h r o i p o i e t i n appeared c o n s i s t e n t l y smaller, and i t can be seen from Table 6 that the number of secondary colonies i n the low e r y t h r o p o i e t i n assays was also c o n s i s t e n t l y lower. This was true for a l l 64 primary colonies from the f i r s t 4 PV patients studied that y i e l d e d colonies i n the low erythropoietin r e p l i c a t e s , and f o r 20 of 24 such colonies obtained from PV patient 5. The d i f f e r e n c e i n counts between high and low erythro-p o i e t i n assay r e p l i c a t e s was s i g n i f i c a n t (p < 0.05) as determined by c h i -square analysis (Table 6). Since the experimental design does not permit i d e n t i f i c a t i o n of c e l l s not belonging to the abnormal clone, the primary colonies which yielded secondary colonies only i n the high e r y t h r o p o i e t i n r e p l i c a t e s were not included i n the s t a t i s t i c a l a n a l y s i s due to the p o s s i b i l i t y that they were members of a r e s i d u a l normal population. The r e s u l t of the analysis indicates that most primary colonies that contained c e l l s capable of secondary erythroid colony formation i n cultures with < 0.01 u n i t s of erythropoietin per m l i a l s o contained c e l l s that appeared phenotypically normal ( i . e . , incapable of secondary erythroid colony ... formation under these conditions). -57-TABLE 6. Number of Erythroid Colonies i n Secondary Assays with and with-out Added Eryth r o p o e i t i n (.Ep) from Replated Primary Colonies from 5 PV Patients* PV Patient Number 1 2 3 4 5 +Ep -Ep +Ep -Ep +Ep -Ep +Ep -Ep +Ep -Ep Primary colonies 17 0 32 0 22 0 14 0 that yielded 11 0 29 0 17 0 14 0 secondary colo- 20 0 17 0 14 0 nies only i n 17 0 15 0 5 0 the high Ep dish 10 0 9 0 4 0 5 0 9 0 3 0 Primary colonies 25 4 56 4 33 7 that yielded 21 4 35 8 35 4 secondary colo- 16 8 29 8 33 4 nies i n l b o t h 12 6 24 9 32 5 dishes 14 3 17 5 27 1 10 6 15 4 18 5 10 4 13 6 17 4 9 6 13 2 16 3 6 4 9 4 13 3 2 1 9 3 9 2 8 2 8 2 8 2 7 1 5 2 4 3 5 2 4 1 5 1 3 2 4 2 3 3 3 2 2 1 24 7 49 22 24 7 27 24 14 6 24 16 11 6 24 13 11 6 22 13 12 4 18 16 10 3 27 6 10 3 20 13 6 4 16 13 6 2 11 14 6 2 12 10 6 1 10 9 5 2 11 8 5 2 11 5 5 1 9 7 5 1 . 9 5 3 2 9 4 3 2 2 7 3 1 . 5 4 3 1 4 4 4 3 3 1 1 2 2 1. Totals 125 46 255 64 268 53 172 63 -331 220 T o t a l q S S S S Chi-Square (DF) 43.5C10) 122.3(16) 154.8(18) 54.6(20) 44.6(24) *After 9 days culture up to 30 primary colonies from each i n d i v i d u a l were divided equally between 2 secondary cultures and incubated a further 9 days with or without added erythropoietin as described i n the Methods. S - s i g n i f i c a n t -58-DISCUSSION AND CONCLUSIONS Altered erythropoietin responsiveness i s a well-established feature of erythropoietic c e l l s i n patients with PV, a c l o n a l disorder o r i g i n -ating i n the p l u r i p o t e n t stem c e l l compartment (185). Evidence of t h i s 59 phenomenon was f i r s t obtained from studies of Fe uptake i n short-term suspension cultures of PV marrow c e l l s (186). Subsequently, as assays for human erythroid colony-forming c e l l s were developed, i t was d i s -covered that cultures of PV c e l l s y ielded recognizable erythroid colonies at concentrations of e r y t h r o p o i e t i n i n s u f f i c i e n t to support the growth of normal c e l l s (52). This i n turn l e d to more d e t a i l e d studies of the frequency of expression of t h i s abnormal phenotype i n i n d i v i d u a l patients and amongst PV patients considered as a group. Such studies have shown the following: In most PV patients only a proportion of the t o t a l number of progenitors (CFU-E and BFU-E) y i e l d s u f f i c i e n t " erythropoietin-independent" progeny to be c l a s s i f i e d as phenotypically abnormal. The remainder appear i d e n t i c a l to normal progenitors i n terms of the concen-t r a t i o n of erythropoietin they require f o r colony formation (142,173). A l l progenitors that are capable of erythropoietin-independent erythroid colony formation appear to be members of the neoplastic ( i . e . , dominant) clone. However, not a l l erythropoietic progenitors belonging to t h i s clone have t h i s capacity (176). F i n a l l y , from the r e s u l t s of a recent survey of a large number of unselected patients with PV, i t would appear that a l l clones that lead to t h i s disease also produce r e a d i l y detectable numbers of phenotypically abnormal erythroid progenitors (.187) . This i s c l e a r l y d i f f e r e n t from the s i t u a t i o n i n chronic myelogenous leukemia, a r e l a t e d c l o n a l disorder, where progenitors capable of e r y t h r o p o i e t i n --59-independent erythroid colony formation have been found occasionally but not c o n s i s t e n t l y (188-190). The present experiments were undertaken to examine the d i s t r i b u t i o n of erythropoietin-independent and -dependent progenitors i n i n d i v i d u a l clones, each derived from a p r i m i t i v e e r y t h r o p o i e t i c c e l l . A technique was devised that makes possible the assessment of such c e l l s by r e p l a t i n g primary colonies into secondary cultures containing feeders. Using t h i s technique i t was possible to show that c e l l s i n normal 9 day old colonies obtained i n primary c u l t u r e s with 2.5 units of erythropoietin per ml c o n s i s t e n t l y yielded substantial numbers of CFU-E-like colonies i n secondary cultures when erythropoietin l e v e l s were maintained. If on the other hand, erythropoietin l e v e l s i n the secondary cultures were reduced to < 0.01 u n i t s per ml, no erythroid colonies formed. In a d d i t i o n i t was p o s s i b l e to show that the procedure used to s p l i t i n d i v i d u a l primary colonies d e l i v e r e d equal amounts to two secondary cultures. A p p l i c a t i o n of t h i s r e p l a t i n g procedure to i n d i v i d u a l colonies obtained i n primary assays of PV peripheral blood revealed two patterns of growth. Most primary colonies (88/109 or 81%) y i e l d e d secondary colonies i n both secondary replates, although the number and s i z e of colonies obtained with erythropoietin (~ 3 units per ml) was c o n s i s t e n t l y greater than that seen i n the secondary r e p l i c a t e to which no erythro^. p o i e t i n was added (concentration < 0.01 u n i t s per ml). In only 4 of the 88 were the numbers of erythroid colonies i n the secondary assays without added erythropoietin equal to or greater than the number i n the secondary assays with a high l e v e l of erythropoietin. These findings - 6 0 -c l e a r l y e s t a b l i s h that i n PV both erythropoietin-independent and -dependent phenotypes are commonly produced by s i n g l e p r i m i t i v e BFU-E p r o l i f e r a t i n g and d i f f e r e n t i a t i n g in vitro. This i n d i c a t e s that f i x a t i o n of phenotypes does not r e g u l a r l y occur p r i o r to the p r i m i t i v e BFU-E stage. These data also confirm and explain the findings of Prchal et al. (176) who showed that some CFU-E and BFU-E of the neoplastic clone were erythropoietin-dependent. Since they also demonstrated that a l l erythropoietin-independent c e l l s were members of the neoplastic clone (176), i t can be assumed that p r i m i t i v e BFU-E that give r i s e to erythropoietin-independent c e l l s are a l s o members of the neoplastic clone. The present study shows that when such p r i m i t i v e BFU-E 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 in vitro they give r i s e to s i g n i f i c a n t numbers of erythropoietin-dependent progeny as well. The large d i f f e r e n c e s i n secondary colony s i z e seen between assay r e p l i c a t e s with and without eryt h r o p o i e t i n suggest that the capacity f o r erythropoietin-independent growth may never be f i x e d during the process of erythroid c e l l d i f f e r e n t i a t i o n . Some heterogeneity i n secondary colony s i z e s was expected since c e l l s vary i n t h e i r c e l l cycle times and divide at d i f f e r e n t rates. A f t e r just a few d i v i s i o n s , t h i s asynchrony i n the primary colony could lead to large d i f f e r e n c e s i n secondary colony s i z e . However, i t was noted that i n secondary assays of primary colonies from PV patients only very small colonies of 8-16 erythroblasts were seen i n the dishes without added erythropoietin, even when bursts of 2-3 c l u s t e r s and large c l u s t e r s of approximately 50 erythroblasts were seen i n the r e p l i c a t e assay where a high concentration of erythropoietin -6SL-was present. If the c e l l s i n the 9 day old primary colony had already chosen t h e i r phenotype text her erythropoietin-independent or erythro-... . u . poietin-dependent), and i f t h i s choice were transmitted to a l l of t h e i r progeny, fewer secondary colonies, would have been expected i n the dishes with, erythropoietin but the range of colony s i z e s should have been the same i n both r e p l i c a t e s . Since t h i s was not the case, the f i n d i n g of smaller secondary colonies i n the dishes without e r y t h r o p o i e t i n strongly suggests that the mechanisms responsible for erythropoietin independence are not f i x e d but may come and go with r e s u l t a n t . d i f f e r e n t i a l e f f e c t s on the production, s u r v i v a l and eventual hemoglobinization of c e l l s that are normally dependent on continuous contact with erythropoietin. Progenitor c e l l s capable of erythropoietin-independent growth have been found i n neonatal lamb bone marrow (191) and human cord blood (192). This a b i l i t y may therefore be viewed more c o r r e c t l y not as a "new" a c q u i s i t i o n of the PV c e l l but as a r e a c t i v a t i o n of mechanisms normally operative i n f e t a l e r y t h r o p o i e t i c c e l l s but which are shut down a f t e r - b i r t h . The second growth pattern obtained when colonies from primary PV assays were replated was i d e n t i c a l to that seen with colonies from normal subjects - i . e . erythroid colonies i n the r e p l i c a t e to which eryth r o p o i e t i n was added but none i n the r e p l i c a t e to which erythropoietin was not added. In most instances the number of colonies scored i n the high erythropoietin r e p l i c a t e was s u f f i c i e n t l y high that f a i l u r e to detect an erythropoietin-independent subpopulation seemed u n l i k e l y . I t has been shown that i n PV r e s i d u a l normal stem c e l l s may p e r s i s t and give r i s e to a detectable proportion of the c e l l s i n the BFU-E JW. -62-compartme'nt (.178) , although, in vivo these do not go on to y i e l d mature red blood c e l l s (.185). 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