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Quantitation of human megakaryocyte progenitor cells in semi-solid cultures Fanning, Stephen Francis 1994

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Q U A N T I T A T I O N O F H U M A N M E G A K A R Y O C Y T E P R O G E N I T O R C E L L S I N S E M I - S O L I D C U L T U R E S . b y S T E P H E N F R A N C I S F A N N I N G M . B . B . S . , Univers i ty of Queensland, 1980. A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Pathology and Labora tory M e d i c i n e ) W e accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A December 1994 © S T E P H E N F R A N C I S F A N N I N G , 1994 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date 3 o D**./??^. DE-6 (2/88) A B S T R A C T In v i t ro assays for the study of human megakaryocy te ( M k ) progeni tor cel ls have been compromised by the exquis i te sens i t iv i ty o f these ce l l s to t ransforming growth factor-P ( T G F - P ) (which is found in : inh ib i to ry concentrat ions in serum and plasma) and a need to use i m m u n o c y t o c h e m i c a l methodology to speci f ica l ly identify c lona l populations of M k lineage ce l l s . The present studies were undertaken to ident i fy condi t ions that w o u l d overcome both of these problems and a l low human M k progenitors to be detected at o p t i m a l e f f i c i ency . The f i n a l procedure deve loped i n v o l v e d p l a t i n g n o r m a l adult human bone marrow ce l l s i n chamber -s l ides i n c e l l cul ture grade agarose d i s so lved in I scove ' s med ium supplemented w i t h 1% a lbumin , 200 M-g/ml iron-saturated transferr in, 10 ( i g / m l i n s u l i n , 40 m m o l / L l o w dens i ty l i pop ro t e in s , 5 x l 0 " ^ M 2-mercaptoethanol and a g rowth factor cockta i l consisting of I L - 3 , I L - 6 , G M - C S F and Steel factor. After 2 to 3 weeks at 3 7 ° C , cultures were f ixed in methanol: acetone and colonies conta in ing cel ls e x p r e s s i n g g l y c o p r o t e i n I l b / I I I a i d e n t i f i e d by i m m u n o p e r o x i d a s e s t a i n i n g . C o l o n i e s con ta in ing increas ing numbers of p o s i t i v e l y stained ce l l s appeared after i nc reas ing periods o f t ime consis tent w i t h the presence i n normal human bone marrow of a h ie ra rchy o f M k progeni tor ce l l s o f d i f f e r i n g p r o l i f e r a t i v e p o t e n t i a l . These condit ions were found to be superior to serum- or plasma-con ta in ing medium in their ab i l i ty to support the pro l i fe ra t ion of human M k progeni tor ce l l s . Dose response studies were undertaken to con f i rm that the concentrat ions of a lbumin and 2-mercaptoethanol were op t ima l . F o r 2 of 3 bone marrows, the number of M k colonies seen. wi th up to 80 m m o l / L low density l ipoproteins was the same as when these were not added to the medium. The growth factor combinat ion of insu l in , I L - 3 , I L - 6 , G M - C S F and Steel factor was superior to other combinat ions tested ( inc lud ing some con ta in ing G - C S F and /o r I L - 1 1 ) and p r o v i d e d m a x i m a l s t i m u l a t i o n o f the l a rge r (>50 ce l l s /colony) M k colonies. Under these conditions, the M k colony y ie ld after 18 to 20 days was l inear ly related to the number of cells plated over a wide range of c e l l concentrations. These findings validate the use of this assay for future studies of the factors regulat ing both the development and subsequent d i f ferent ia t ion of human M k progeni tor ce l l s . T A B L E O F C O N T E N T S A B S T R A C T T A B L E O F C O N T E N T S L I S T O F F I G U R E S A N D T A B L E S L I S T O F A B B R E V I A T I O N S A C K N O W L E D G E M E N T S C H A P T E R I I N T R O D U C T I O N 1 Out l ine o f hematopoiesis 1 1.1 Ontogeny and organizat ion of the hematopoiet ic system 1 1.2 H ie r a r chy of hematopoiet ic progeni tor ce l l s 4 1.2.1 Plur ipotent / h igh prol i fera t ive potent ial p rogen i to r ce l l s 5 1.2.2 Lineage-res t r ic ted / l ow prol i fera t ive potent ia l progeni tor ce l l s 9 1.3 O v e r v i e w of the molecular regulat ion o f hematopoiesis 11 2 M e g a k a r y o c y t o p o i e s i s 13 2.1 M e g a k a r y o c y t i c p rogeni to r ce l l s 14 2.2 L i g h t d e n s i t y - C F U - M k and transi t ional immature m e g a k a r y o c y t e s 16 2.3 T e r m i n a l maturat ion of megakaryocytes 19 2.4 Gene express ion dur ing M k different ia t ion 20 2.5 A n t i g e n express ion dur ing M k di f ferent ia t ion 21 2.6 P o s i t i v e regula t ion o f megakaryocy topo ies i s 23 2.6.1 I n t e r l e u k i n - 3 and g r a n u l o c y t e - m a c r o p h a g e co lony s t imula t ing factor 25 i i i v v i i i x x i V 2.6.2 I n t e r . l e u k i n - 6 27 2.6.3 I n t e r l e u k i n - 1 1 28 2.6.4 L e u k e m i a i n h i b i t o r y factor 30 2.6.5 Steel factor 30 2.6.6 G r a n u l o c y t e c o l o n y - s t i m u l a t i n g factor 32 2.6.7 I n t e r l e u k i n - 1 32 2.6.8 E r y t h r o p o i e t i n 33 2.6.9 B a s i c f ibroblast growth factor 35 2.6.10 T h r o m b o p o i e t i n 36 2.7 Nega t ive regula t ion of megakaryocy topo ies i s 37 2.7.1 T r a n s f o r m i n g growth factor-p 3 8 2.7.2 Platelet factor 4 39 2.7.3 I n t e r l e u k i n - 4 4 0 2.7.4 I n t e r f e r o n s 41 3 Thes is object ives 42 C H A P T E R II M A T E R I A L S A N D M E T H O D S 1 Prepara t ion of ce l l s 44 2 M e g a k a r y o c y t e progeni tor c e l l assay 44 2.1 A g a r o s e cul tures 45 2.2 Immunocy tochemica l ident i f ica t ion of co lon ies conta in ing M k cel ls 47 3 C l o n o g e n i c progeni tor c e l l assays 48 v i C H A P T E R III T H E D E V E L O P M E N T O F A Q U A N T I T A T I V E IN V I T R O A S S A Y F O R M E G A K A R Y O C Y T E P R O G E N I T O R C E L L S 1 I n t r o d u c t i o n 53 2 R e s u l t s 54 2.1 A b i l i t y of agarose cultures to support hematopoietic p rogeni to r c e l l - d e r i v e d c o l o n y fo rmat ion 55 2.2 Inh ib i to ry effect of serum and p lasma-conta in ing med ium 57 2.3 O p t i m a l concentrations of 2-mercaptoethanol , a lbumin and low density l ipoproteins 57 2.4 O p t i m a l growth factor ' combinat ions 62 2.5 P l a t i ng density studies 62 2.6 T ime course studies 69 3 D i s c u s s i o n and conc lus ions 71 R E F E R E N C E S 77 A P P E N D I X i P repara t ion o f agarose 106 i i Preparat ion of media for agarose cultures 106 i i i Es tab l i shment of agarose cultures 109 i v F i x a t i o n of agarose cultures 111 v Immunocy tochemica l ident i f ica t ion of M k progeni tor c e l l - d e r i v e d co lon i e s 112 v i S c o r i n g c r i te r ia for M k progenitor ce l l -de r ived co lonies 115 L I S T O F F I G U R E S A N D T A B L E S v i i F i g , 1.1. Summary o f the various described megakaryocyt ic p rogen i to r c e l l s . . 1 8 F i g . 1.2. Schematic summary of the ' two factor hypothesis ' of the regulat ion of M k development. 24 F i g . 2 . 1 . . Schematic diagram depict ing the M k progenitor c e l l assay culture system. 46 F i g . 2 . 2 . S m a l l M k colony in an agarose culture containing serum-substitute medium and I L - 3 , I L - 6 , G M - C S F and S F . 50 F i g . 2 . 3 . Large , mult iclustered M k colony in an agarose culture containing serum-substitute medium, I L - 3 , I L - 6 , G M - C S F and SF . . 51 F i g . 2 . 4 . B ipheno typ ic erythroid M k colony i n . an agarose culture conta ining serum-substitute medium, I L - 3 , I L - 6 , G M - C S F , S F and Ep . 52 Tab .3 .1 . Compar ison of the abi l i ty of agarose cultures in petri dishes or chamber slides to support hematopoietic progenitor c e l l der ived co lony formation wi th that ob ta ined w i t h m e t h y l c e l l u l o s e - c o n t a i n i n g . cu l tu res . 56 F i g . 3 . 1 . The effect of fetal ca l f serum (30%), normal plasma (30%), aplastic plasma (30%) and a serum-substitute on M k colony formation in cultures containing I L - 3 , I L - 6 , G M - C S F and SF. , 58 F i g . 3 . 2 . Effec t o f increas ing concentrations of 2-mercaptoethanol . on M k - c o l o n y fo rma t ion . . 59 F i g . 3 . 3 . Effect of increasing concentrations of a lbumin on M k -c o l o n y fo rma t ion . 60 F i g . 3 . 4 . Effect of increasing concentrations of low density l ipopro te ins on M k - c o l o n y format ion . 61 F i g . 3 . 5 a . Effect of different growth factor combinat ions on M k - c o l o n y product ion i n agarose cultures conta in ing a serum substitute. 63 F i g . 3 . 5 b . * Results from Fig .3 .5a . subcategorised according to co lony size (3-20 M k / c o l o n y ) . 64 F i g . 3 . 5 . c . Results from Fig .3 .5a . subcategorised according to co lony size (21-49 M k / c o l o n y ) . 65 V 111 F i g . 3 . 5 . d . Results from Fig .3 .5a . subcategorised according to colony size (>50 M k / c o l o n y ) . 66 F i g . 3 . 6 . Effect of the addition of erythropoietin on M k - c o l o n y product ion in agarose cultures conta ining a serum substitute, I L - 3 , I L - 6 , G M - C S F and S F . 67 F i g . 3 . 7 . P la t ing density study of mononuclear cells in agarose cultures containing a serum substitute and . I L - 3 , I L - 6 , G M - C S F and SF. 68 F i g . 3 . 8 . T ime course study of the development of M k colonies in agarose cultures containing I L - 3 , I L - 6 , G M - C S F and S F . 70 L I S T O F A B B R E V I A T I O N S A c h E a c e t y l c h o l i n e s t e r a s e a g a r - L C M agar-st imulated leukocyte condi t ioned med ium b F G F basic f ibroblast growth factor B F U - E b u r s t - f o r m i n g u n i t - e r y t h r o i d B F U - M k b u r s t - f o r m i n g u n i t - m e g a k a r y o c y t e B l - C F C blast c o l o n y - f o r m i n g c e l l B M bone mar row B S A bov ine serum a l b u m i n C F U - E c o l o n y - f o r m i n g u n i t - e r y t h r o i d C F U - G c o l o n y - f o r m i n g u n i t - g r a n u l o c y t e C F U - G E M M c o l o n y - f o r m i n g u n i t - g r a n u l o c y t e , e r y t h r o c y t e , mac rophage and m e g a k a r y o c y t e C F U - G M c o l o n y - f o r m i n g u n i t - g r a n u l o c y t e m a c r o p h a g e C F U - M c o l o n y - f o r m i n g u n i t - m a c r o p h a g e C F U - M k c o l o n y - f o r m i n g u n i t - m e g a k a r y o c y t e C F U - S c o l o n y - f o r m i n g u n i t - s p l e e n C M L ch ron ic m y e l o i d l e u k e m i a C R U c o m p e t i t i v e r epopu la t ing unit CSF c o l o n y - s t i m u l a t i n g fac tor E G F ep ide rmal growth factor E p e r y t h r o p o i e t i n FCS fetal ca l f serum 5 - F U 5 - f l u o r o u r a c i l G A T A consensus sequence of the 0 - g l o b i n gene enhancer reg ion G - C S F g r a n u l o c y t e - c o l o n y - s t i m u l a t i n g f ac to r G M - C S F g r a n u l o c y t e - m a c r o p h a g e - c o l o n y - s t i m u l a t i n g f a c t o r G P g l y c o p r o t e i n H P P - C F C h igh p ro l i fe ra t ive potent ia l c o l o n y - f o r m i n g c e l l H S horse serum I F N . i n t e r f e r o n I L i n t e r l e u k i n L D l ight density L D L s l ow density l ipoprote ins L I F l e u k e m i a i n h i b i t o r y factor L T C long term culture L T C - I C long term cu l tu re - in i t i a t ing c e l l M - C S F macrophage c o l o n y - s t i m u l a t i n g factor 2 - M E 2 - m e r c a p t o e t h a n o l M k m e g a k a r y o c y t e M k - C F U - S sp len ic megakaryocy te c o l o n y - f o r m i n g uni t M k - C S A m e g a k a r y o c y t i c c o l o n y - s t i m u l a t i n g a c t i v i t y M e g - C S F m e g a k a r y o c y t e c o l o n y - s t i m u l a t i n g fac tor M o A b m o n o c l o n a l an t ibody m S C F membrane-bound steel factor P F 4 platelet factor-4 P H A - L C M phy tohemag lu t in in s t imula ted l eukocy te cond i t i oned m e d i u m P 1 A 1 . p la te le t a n t i g e n - A l SF Steel factor (synonyms: c-ki t l igand, mast ce l l growth factor, X stem ce l l factor) S l / S l d S t e e l / S t e e l d i c k i e genotype TBS tris buffered saline TGF-(3 t r ans fo rming growth factor-p TSF . t h r o m b o p o i e s i s - s t i m u l a t i n g fac tor T S P t h r o m b o s p o n d i n v W F von W i l l e b r a n d factor A C K N O W L E D G E M E N T There are numerous people who have assisted in the development of this project and I wou ld l ike to gratefully acknowledge their assistance and support. I am par t icular ly grateful to D r . Conn ie Eaves , my supervisor, who has offered guidance and support throughout the course of this project. I wish to thank Dr . A l l e n Eaves for a l lowing me the pr iv i lege of s tudying at the Terry F o x Laboratory and for his administrat ive support that was essential for the undertaking of this project. I w o u l d also l ike to thank D r . D a v i d W a l k e r , Cha i rman of my Supervisory Committee and Dr . D . Hogge, Dr . J . Emerman and Dr . D . Brunette, the other members of this committee. Discussions and advice from D r . Graeme Dougher ty and Dr . Peter Lansdorp were very helpful . I wish to acknowledge Dianne R e i d , Ka ren L a m b i e , Jessica M a l t m a n , Git te Berghard and members of the Stem C e l l Assay Service for expert technical assistance and their patience i n teaching me various molecular and c e l l b io logy techniques. I w o u l d l ike to acknowledge my fe l low students from the "Eaves ' L a b " - E i b h l i n C o n n e a l l y , Saghi Ghaf fa r i , Made l e ine L e m i e u x , L u i s a P o n c h i o , and C i n d y M i l l e r - who have offered their u n s o l i c i t e d adv i ce , taunts, encouragement and whose friendships have enr iched my ongo ing lea rn ing experience wi th in the Terry F o x Laboratory . I am very grateful to the Nat iona l Cancer Institute of Canada and The L e u k e m i a Foundat ion o f Queensland for f inanc ia l support. W i t h o u t the f i n a n c i a l support of these two groups it w o u l d have been i m p o s s i b l e to undertake these studies. F i n a l l y I wish to thank Karen , Jonathon and E l y s e , who have demonstra ted amaz ing patience, tolerance and support w h i l s t these studies were b e i n g under taken. i C H A P T E R I I N T R O D U C T I O N 1 O U T L I N E O F H E M A T O P O I E S I S It has been estimated that 2.5 b i l l i o n red cel ls , 2.5 b i l l i o n platelets and 1.0 b i l l i o n neutrophils per k i l og ram body weight are produced da i ly from precursor cells present in the bone marrow of normal adults. A l l of these cells are b e l i e v e d to or iginate u l t imate ly f rom a r e l a t ive ly s m a l l popu l a t i on o f t o t i p o t e n t i a l h e m a t o p o i e t i c c e l l s w i t h o b v i o u s e n o r m o u s p r o l i f e r a t i v e p o t e n t i a l . T h i s rate o f b l o o d c e l l p r o d u c t i o n is n o r m a l l y ma in t a ined und imin i shed throughout the human l i fespan to replace senescent b lood cel ls w h i c h are removed at a corresponding rate from the c i r cu la t ion . H o w e v e r , the rate o f b l o o d c e l l p roduc t ion can also be t rans ient ly increased upon p h y s i o l o g i c demand (eg, i n response to hemorrhage or infect ion) (Ers lev and L i c h t m a n , 1990; Metca l f , 1989; R e n c r i c c a et a l , 1970). The hematopoiet ic system is thus a c e l l renewal system in wh ich many specia l ized end cel ls are p roduced from more p r i m i t i v e precursors. Th i s process is regulated by a c o m p l e x series o f mechanisms that balance c e l l s u r v i v a l , p ro l i f e r a t ion and d i f f e r e n t i a t i o n . 1.1 O N T O G E N Y A N D O R G A N I Z A T I O N O F T H E H E M A T O P O I E T I C S Y S T E M , E v i d e n c e o f hematopo ies i s can be detec ted very ea r ly i n embryogenesis. The first erythroid cells are found in the human embryo at 19 days o f ges t a t i on . G r a n u l o p o i e t i c p rogen i to r s ( c o l o n y - f o r m i n g un i t s -2 granulocyte-macrophage ( C F U - G M ) ) have been demonstrated i n the fetal b lood as early as 5 weeks of gestation, even though mature neutrophils are, not found at this stage (Johnson and Jones, 1973). B o t h e ry th ro id and g r a n u l o c y t i c precursors are be l i eved to arise f irst in the y o l k sac f rom undifferent iated progenitors of mesodermal o r ig in ( M o o r e and M e t c a l f , 1970; Hasse ldah l and Larsen , 1971). Some of these hematopoiet ic p r im i t i ve cel ls then migrate from the y o l k sac to the fetal l i ve r w h i c h then becomes the major source of red b lood cells . unt i l the bone marrow takes over this function at 24 weeks of gestation. The spleen also has a minor hematopoietic role in the midd le per iod of fetal development. Ev idence of megakaryocytopoies is and g r a n u l o c y t o p o i e s i s is seen t ransient ly i n the fetal l i v e r but these occur predominant ly in the bone marrow from the 12th week of gestation onward . M a t u r e neut rophi ls or megakaryocytes have not been found in the human y o l k sac pr ior to the onset of hematopoiesis in the fetal l ive r (Hesseldahl and Larsen , 1971). Ce l l s wi th a l ympho id morphology do appear, however , before the splenic or hepatic stages of hematopoiesis begin (Guest and B r o w n , 1957; W e i n b e r g and S tohlman, 1976). Megakaryocytes can be demonstrated i n the c i rcu la t ing b lood at 12 weeks of gestation, and mature neutrophils are seen in human fetuses after 16 weeks o f deve lopmen t ( M r s e v i c et a l , 1970) . G r a n u l o c y t o p o i e s i s lags b e h i n d e r y t h r o p o i e s i s t h r o u g h o u t i n t r a u t e r i n e deve lopment . M a t u r i n g B lymphocytes are detectable in the fetal l i v e r beg inn ing at 8-9 weeks of gestation and subsequently in the bone marrow (Tavassol i , 1979; R o i t et a l , 1985). Mature B cells migrate to the peripheral l y m p h o i d t issues ( l y m p h nodes, spleen and m u c o s a L a s s o c i a t e d l y m p h o i d tissues) where they are stimulated to differentiate into plasma cells (Roi t et a l , 1985). The first T lymphocyte precursors are be l ieved to migrate to the thymus where the thymic ep i the l i a l environment appears to be essential for them to complete their differentiation into mature T cel ls . 3 Hematopoies is in the marrow takes place in the spaces between the 'vascular sinuses (Wintrobe et a l , 1981). The sinus w a l l forms a barrier between the hematopoiet ic compartment and the c i r cu la t ion . Th i s barr ier is composed of a l u m i n a l layer of endothel ia l cel ls that form a complete inner l i n i n g , and an ab lumina l coat of cel ls described as advent i t ia l re t icular cel ls that form an incomple te . outer coat. The endothelial cel ls overlap extens ively , but because they lack tight junctions are thought to be able to sl ide over one another. Bone marrow endothelial cells are act ively endocytot ic and form the system that controls the t r a f f i ck ing o f part icles and molecules between the c i r c u l a t i o n and the hematopoie t ic spaces. The adven t i t i a l r e t i cu la r ce l l s syn thes i ze r e t i cu l a r f ibers , that a long w i t h the i r c y t o p l a s m i c processes , provide phys ica l support for adjacent hematopoietic cel ls (Tavasso l i , 1979). A reduc t ion i n the advent i t ia l c e l l cove r ing on the a b l u m i n a l surface , o f the sinus may facilitate penetration of the endothelial cel ls by mature b lood ce l l s . The marrow st romal microenenvi ronment consists of a ne twork of f ibroblasts , macrophages and adipocytes (L ich tman , 1981; A l l e n and Dexter , 1984; G i m b l e , 1990). These cells are believed to affect the development of blood c e l l s t h r o u g h d i r e c t su r face - to - su r face i n t e r a c t i o n s w i t h h e m a t o p o i e t i c precursor ce l l s . N o n r a n d o m associat ions observed between granulocytes and ad ipocy te s and f i b rob la s t s , between e ry throblas t s . and macrophages , and between lymphocytes and fibroblasts in the bone marrow support this concept (L ich tman , 1984). Macrophages are thought to ingest extruded red ce l l nuclei and senescent or defective ce l ls , and along with fibroblasts, act as a l oca l source of growth factors. B l o o d cel ls enter the c i r cu la t ion by exer t ing pressure on the e n d o t h e l i a l membrane and f o r m i n g a m i g r a t i o n pore th rough the cytoplasm of the endothelial cells (de Bruyn et a l , 1971). The mechanisms that regulate the associat ion of mature cel ls wi th the endothel ium have not been 4 establ ished, but it has been proposed that neutrophils ac t ive ly migrate toward the s i n u s o i d under the i n f l u e n c e o f a va r i e ty o f f ac to r s , i n c l u d i n g g l u c o c o r t o c o i d s , a n d r o g e n i c s t e r o i d s , e n d o t o x i n , and c o m p o n e n t s o f complement (Tavassol i ,1989; Deinard and Page, 1974; Ghebrehiwet and M u l l e r -Eberhard, 1979; M i t c h e l l et a l , 1967; V o g e l et a l , 1967). Megakaryocytes appear to fenestrate the endothe l ia l c e l l w i th mul t ip l e c y t o p l a s m i c processes and release cy top lasmic fragments (platelet f ields) d i rec t ly into the c i r cu l a t i on (de B r u y n et a l , 1971). Erythropoiet in has been shown to have ret iculocyte c e l l -re leas ing ac t i v i t y , probably by reducing the advent i t i a l c e l l cover o f the sinus w a l l and faci l i ta t ing the access of the ret iculocyte to the endothel ia l c e l l (L ich tman et a l , 1989). It has been suggested that the release of mature b lood ce l l s f rom the marrow may also depend upon their de fo rmabi l i ty because mature e ry th rocy te s and g ranu locy te s are more d e f o r m a b l e than the i r precursors (Roths te in , 1993). Hematopoies i s is no rma l ly c o n f i n e d , to the bone marrow after bir th . Gradua l ly during ch i ldhood the long bones are f i l l ed wi th fat cel ls and hematopoiesis becomes l imi t ed to the vertebrae, r ibs, sternum, s k u l l , pe lv is and p rox ima l regions of the femur and humeri , dur ing adult l i fe . 1.2 H I E R A R C H Y O F H E M A T O P O I E T I C P R O G E N I T O R C E L L S Over the past 30 years, k inet ic studies of bone marrow cel ls in c u l t u r e and after t r ansp lan t a t i on have c o n f i r m e d the e x i s t e n c e o f a progeni tor c e l l hierarchy wi th in the hematopoiet ic system. Sustained ce l lu l a r p roduc t ion depends on the presence o f a poo l of p r i m i t i v e totipotent cel ls capable of both se l f renewal and d i f ferent ia t ion . D u r i n g a series of success ive d i v i s i o n s ce l l s w i t h more res t r ic ted d i f fe ren t i a t ion po ten t ia l i t i e s 5 are generated eventual ly g i v i n g rise to uni -potent ia l progeni tor ce l l s w h i c h then r a p i d l y undergo a series o f te rminal d i v i s i o n s . D u r i n g the later c e l l cyc les , the f ina l different iat ion 'program of the l ineage is expressed and the cel ls acquire the morpho log ica l ly recognizable features of mature b lood cel ls . M o s t of these have a finite and l imi ted l ife span and are incapable of further d i v i s i o n (Ers lev and L i c h t m a n , 1990). 1.2.1 P L U R I P O T E N T / H I G H P R O L I F E R A T I V E P O T E N T I A L P R O G E N I T O R C E L L S Gene (or chromosomal) mark ing strategies in combina t ion wi th bone mar row t ransplanta t ion have es tab l i shed the ex is tence o f to t ipotent lympho-mye lopo ie t i c cells in the bone marrow of adult mice ( W u et a l , 1968; Abramson et a l , 1977; D i c k et al . 1985; Ke l l e r et al , 1985; Lemischka et al , 1986). The presence o f ce l l s wi th s imi l a r properties i n adult human mar row was i n i t i a l l y inferred f rom studies o f the different l ineages represented in the neoplas t ic c lone . in patients wi th various hematopoiet ic mal ignanc ies (Whang et al , 1963; Prchal et a l , 1978; Raskind and F ia lkow, 1987). For example, studies i n c h r o n i c m y e l o i d l e u k e m i a ( C M L ) have demons t ra ted the cons i s t en t presence o f the P h i l a d e l p h i a c h r o m o s o m e i n g ranu locy te s , macrophages , e r y t h r o i d , megaka ryocy t e s and somet imes i n B - and T - l y m p h o i d c e l l s suggest ing that this disease t y p i c a l l y arises in a c e l l wi th l y m p h o - m y e l o i d dif ferent ia t ion potential (Rask ind and F i a k l o w , 1987). M o r e recently, Turhan et al have provided evidence that l ympho-mye lo id repopulat ing cel ls exist in normal adult human marrow (Turhan et a l , 1989). A m o n g s t p l u r i p o t e n t i a l hematopo ie t i c c e l l s , some h i e r a r c h i c a l organizat ion appears to exist. Fo r example, studies of both murine and human 6 ce l l s have demonstrated that subpopulat ions of plur ipotent cel ls may vary in the r ap id i ty w i th w h i c h they g ive rise to detectable . numbers o f mature progeny and, converse ly , as to . whether the p roduc t ion o f such progeny is sustained or self l imi ted . However , some of this var iab i l i ty may reflect the s tochast ic nature o f p r i m i t i v e hematopoiet ic c e l l response to g rowth factor s t imulated choices between differentiat ion and self renewal (Eaves and Eaves , 1992) . T i l l and M c C u l l o c h were the first to develop a quantitative in v ivo assay for murine pluripotent hematopoietic cel ls ( T i l l and M c C u l l o c h , 1961). The ce l l s they desc r ibed fo rm m a c r o s c o p i c , m u l t i l i n e a g e c o l o n i e s i n the spleens o f t ransplanted mye loab la t ed or gene t i ca l ly c o m p r o m i s e d ( W 7 W V ) recipients ; hence the term co lony - fo rming units-spleen ( T i l l and M c C u l l o c h , 1961; Boggs et al , 1982; M c C u l l o c h et a l , 1964). Further studies showed that such mul t i l ineage spleen colonies may contain va ry ing numbers of daughter C F U - S (ie, cel ls capable of generating new macroscopic mul t i l ineage spleen co lon i e s upon, in jec t ion into secondary i r radia ted hosts) ( M c C u l l o c h et a l , 1964) . H o w e v e r , heterogenei ty amongst ce l l s def inable as C F U - S has subsequently been described. M a g l i et al (1982) have shown that most spleen co lon ies iden t i f ied as macroscop ica l ly v i s ib l e surface nodules after 7-8 days are ne i t he r m u l t i p o t e n t i a l nor s e l f - m a i n t a i n i n g and most w i l l have disappeared from the spleen wi th in 72 hours. In contrast, the colonies present after 14 days in mice injected with normal mouse bone marrow contain more than one l ineage o f hematopoiet ic different ia t ion as w e l l as precursor ce l l s c a p a b l e o f g e n e r a t i n g s i m i l a r m u l t i l i n e a g e s p l e e n c o l o n i e s on retransplantation. Several lines of evidence suggest that most C F U - S do not have long- te rm l y m p h o - m y e l o i d repopula t ing a b i l i t y . C F U - S and ce l l s capable of long- term repopula t ion differ in their sens i t iv i ty to 5 - f luo rourac i l and can be separated by sedimentation veloci ty (Ploemacher and B r o n s , 1989; Jones et al , 1990; Iscove, 1990). 7 Sz i lvassy et al (1990) have described a procedure for quantitating the long- t e rm repopula t ing c e l l content o f a g iven c e l l suspens ion us ing l i m i t i n g d i lu t ion analysis techniques. Decreas ing numbers of test cel ls wh ich must be der ived from genet ica l ly d is t inguishable (male) donors are injected into i r radia ted (female) recipients together w i th a mye lopro tec t ive graft o f (female) bone marrow cel l s that have been compromised in their long- term recons t i tu t ing potent ia l . The p ropor t ion of recipients s h o w i n g detectable (>5%) reconst i tut ion of their m y e l o i d and l y m p h o i d tissues wi th test (male) ce l l s is used to calculate the frequency of repopula t ing precursors (termed ' compet i t ive repopulat ing units ' or C R U ) in the o r ig ina l male c e l l suspension assayed using Poisson statistics (Porter and Ber ry , 1963; T a s w e l l et a l , 1981). A n a l y s i s of thymic and marrow reconsti tution by re t rovira l ly marked C R U has p rov ided evidence that , C R U are capable of reconst i tut ing both l y m p h o and m y e l o i d tissues long-term (Fraser et a l , 1992). O g a w a has described a type of co lony that can be produced in vi t ro wh ich contains a high frequency of daughter cel ls that are detectable as c lonogen ic progenitors when replated into secondary assays. In the murine system, these may include B l y m p h o i d as w e l l as m y e l o i d progenitors . The colonies consist exc lus ive ly of blasts at the time they are first recognized and the c e l l from which they are derived has been termed the C F U - B l a s t (Nakahata and Ogawa, 1982; Suda et a l , 19.83). These progenitors can remain dormant in vitro in G 0 for at least two weeks before proliferat ion is init iated, and in man are found amongst the CD34+ , H L A - D R - , C D 3 8 - , C D 3 3 - cells in the bone marrow (Terstappen et a l , 1991). Other types of in vi tro colonies that appear to be der ived from v e r y p r i m i t i v e p r o g e n i t o r c e l l s are those c h a r a c t e r i z e d by a h i g h 8 pro l i fe ra t ive potent ia l f o l l o w i n g s t imula t ion wi th m u l t i p l e , g rowth factors, or by growth factors in conjunction with factors produced by s tromal f ibroblasts . In many cases, secondary rep la t ing o f these c o l o n i e s has r evea led the presence in them of daughter progeni tors o f m u l t i p l e m y e l o i d l ineages (Bradley and Hodgson, 1979; Dowding and Gordon, 1992). Long- te rm bone marrow cultures ( L T C ) first descr ibed by Dexter et al (Dexter et al , 1977; Gartner arid Kaplan , 1980) have also proved to a l low the deve lopment of an ext remely useful ' assay ' for the quant i ta t ion o f very pr imi t ive progenitor cells (Sutherland et a l , 1990). The L T C system enables the p r o d u c t i o n i n v i t ro o f C F U - S , in v i t ro c l o n o g e n i c c e l l s and mature g ranu locy tes and macrophages f rom an i n i t i a l i n o c u l u m of mouse bone marrow for several months. In L T C , cel ls are d is t r ibuted between two fractions: an adherent fract ion, wh ich contains several types o f s t romal cel ls as w e l l as the majority of the more p r imi t ive hematopoiet ic ce l l s , and a nonadhe ren t f r a c t i o n , w h i c h cons i s t s m o s t l y o f mature g r a n u l o c y t e s , macrophages and their immediate precursors (Dexter et a l , 1984). S t romal cel ls have been shown to be an important component of the L T C by several f ind ings : (a) genet ica l ly defective hematopoiesis can be dup l ica ted or cured dependent on the stromal ce l l source; (b) preferential l oca l i za t ion of p r imi t ive hematopo ie t i c ce l l s i n the adherent f rac t ion p rov ides c lose contact w i t h stromal ce l l s ; (c) the c y c l i n g status of these pr imi t ive progenitors is dependent on pos i t ive and negative signals p rov ided from wi th in the adherent f ract ion; and (d) in the absence of an exogenous supply of appropriate growth factors there is an absolute dependence of ce l l s capable o f i n i t i a t i n g long- t e rm hematopoiesis on the presence of stromal cells (Dexter and M o o r e , 1977; M a u c h et al, 1980; Toksoz et al, 1980; Coulombel et al, 1983; Cashman et al , 1990; Eaves.et al , 1990). The number of progenitors detected in standard in v i t ro co lony 9 assays (ie, C F U - G M , B F U - E and C F U - G E M M ) in 5 week-old human L T C can be used to provide a relative measure of the number of ' L T C - i n i t i a t i n g ce l l s ' ( L T C -IC) present in the input suspension (Sutherland et a l , 1989). Th i s , however , recognizes the use of standardized condi t ions that inc lude seeding the input ce l l s onto a pre-established irradiated normal marrow L T C adherent layer, (or a competent f ibroblast ce l l layer as a substitute). Under these condi t ions , it has been shown that the c lonogenic c e l l output is l inear ly related to the input number of cel ls down to l i m i t i n g numbers of ce l l s , so that at l i m i t i n g d i l u t i on Po i s son statistics can be used to derive absolute L T C - I C frequencies (Sutherland et al , 1990). The frequency of L T C - I C in human marrow is ~ 1 per 3 x l 0 4 cells which is s imilar to the frequency of C F U - G E M M , C F U - B l a s t , B l - C F C and H P P - C F C (Sutherland et al , 1990). The L T C - I C has been shown by l imi t ing d i lu t ion analysis to have an average clonogenic ce l l output at the 5-week time point of 4 clonogenic cells ( C F U - G M plus B F U - E and occasional ly C F U - G E M M ) per L T C - I C , and at least a quarter of L T C - I C can be shown to exh ib i t p l u r i p o t e n t i a l capac i ty under the cond i t i ons p r e v a i l i n g i n these cul tures (Sutherland et a l , 1990). 1.2.2 L I N E A G E - R E S T R I C T E D / L O W P R O L I F E R A T I V E P O T E N T I A L P R O G E N I T O R C E L L S Hematopoie t ic progenitor cel ls that are commit ted to differentiate in a l ineage- res t r ic ted manner can be quanti tated by in v i t ro c l o n o g e n i c assays. In these, co lony formation depends on the p rov i s ion of appropriate nutrients and growth factor support for expression o f the pro l i fe ra t ive and d i f f e r en t i a t i ve po ten t i a l o f the progeni tors to be detected. C o n d i t i o n s appropriate for different ^types o f progenitor ce l l s i n c l u d i n g most types of 10 human m y e l o i d progenitors and mature human B and T cel ls (but less w e l l defined for early l y m p h o i d cel ls) have now been recognized (Metca l f , 1977; Golde and Takaku, 1985; M c C u l l o c h , 1984; Klu in-Nelemans and W i l l e m a z a , 1987; G o u b e de Lafores t and Rozensza jan , 1984). Quan t i t a t ion o f i n d i v i d u a l c lonogenic cells is possible when cells are plated at sufficiently low density to a l l ow . discrete colonies to be i n d i v i d u a l l y evaluated and a l inear re la t ionship between the number of ce l l s plated and the number o f co lon ies p roduced e x i s t s . Ery thropoie t i c progenitors generate colonies that are made up of hemoglob in ized erythroblasts at the end of their growth phase. The presence of hemoglob in gives these cells (and hence the . colonies that contain them) a d i s t i n c t i v e red c o l o r thereby a l l o w i n g the i r s p e c i f i c i d e n t i f i c a t i o n i n uns ta ined cul tures . The detect ion o f normal human e ry th ro id c l o n o g e n i c progenitors is dependent on the presence of erythropoiet in in the culture. A h i e r a r chy o f e r y t h r o i d progeni tors can be de l inea ted a c c o r d i n g to the i r p ro l i f e ra t ive capaci t ies . In humans, c o l o n y - f o r m i n g un i t -e ry thro id ( C F U - E ) ce l l s produce 1-2 clusters of erythroblasts con ta in ing 8-100 h e m o g l o b i n i z e d cel ls wi th maturation of such colonies being complete by 10-12 days. Burs t -f o r m i n g un i t - e ry th ro id ( B F U - E ) ce l l s may be s u b d i v i d e d in to those that p roduce s m a l l (3-8 c lus ters ) , in termediate (9-16 c lus te rs ) , or large (>16 clusters). Mature B F U - E (ie, those producing 3-8 clusters) and C F U - E represent a more r ap id ly tu rn ing over popu la t ion i n normal marrow than p r i m i t i v e B F U - E and are physical ly larger cells (Eaves and Eaves, 1985; Eaves et a l , 1979). C l o n o g e n i c progeni tors o f granulocytes ( C F U - G ) , macrophages ( C F U - M ) or both ( C F U - G M ) can be ident i f ied by their ab i l i t y to produce co lon ies con ta in ing a m i n i m u m of 20-50 mature ce l l s . A h ie rarchy of g r anu lopo ie t i c progeni tors w i th inc reas ing p ro l i f e ra t ive potent ia l and hence requ i r ing longer times to generate mature progeny has been obtained ( B o l 11 and W i l l i a m s , 1980; Ferrero et a l , 1983). S i m i l a r to the p r imi t ive B F U - E , c l o n o g e n i c g ranu lopo ie t i c progeni tors that produce co lon i e s o f very large numbers of cel ls (>500 cel ls) and which are quiescent i n normal human bone marrow can be identified (Eaves and Eaves, 1987). C lonogen ic M k progenitors are the least w e l l characterized of the . human c l o n o g e n i c p rogeni to r ce l l s . M k co lon ies are best i d e n t i f i e d by pos i t ive s ta ining for l ineage-specif ic markers because of the resemblance of some o f these ce l l s to macrophages in unstained preparat ions. Because t e r m i n a l d i f fe ren t ia t ion o f megakaryocytes usua l ly i n v o l v e s p o l y p l o i d i z a t i o n , a c lonogenic c e l l that produces two megakaryocytes may be considered to be ana logous to a c l o n o g e n i c e r y t h r o i d or g r a n u l o p o i e t i c c e l l capab le o f undergoing 3-5 d iv i s ions . M k colonies are therefore usual ly defined on the basis of their content of at least 2 or 3 megakaryocytes. Co lon i e s conta in ing mul t ip le lineages of mature b lood ce l l s can also be seen in c lonogenic assays and are der ived from a common progenitor referred to as a C F U - G E M M (Fauser and Messner, 1979). 1.3 O V E R V I E W O F T H E M O L E C U L A R R E G U L A T I O N O F H E M A T O P O I E S I S The extr insic regulation of hematopoiesis is achieved by mul t ip le in te rac t ing factors. Some o f these appear to be produced by s p e c i a l i z e d s t romal ce l l s in the marrow whose cont ro l of stem c e l l numbers and the product ion by stem cells of progenitor cells committed to the formation of cells in a par t icular l ineage appears to i nvo lve interactions wi th membrane-bound or loca l i zed growth factors (Hogge et al , 1994). Other regulatory, molecules that s t imulate the prol i fera t ion of progenitor ce l l s and their progeny and ini t ia te 1 2 the maturation events necessary to produce ful ly mature cells appear to act as released soluble or extracel lular matr ix-bound molecules (Metca l f , 19,91); The r egu l a t i on o f hematopo ie t i c p rogen i to r c e l l p r o d u c t i o n and d i f f e ren t i a t ion i m p l i e s the exis tence of feedback con t ro l mechanisms that result in ei ther s t imulat ion or inhib i t ion of progenitor cells (Eaves et al , 1991). Hematopoiesis appears to be regulated by a balance between the hematopoie t i c g rowth factors and a number of inhibi tory molecules. The hematopoiet ic growth factors exert their b i o l o g i c a l ac t iv i ty upon b i n d i n g to spe.cific receptors on target c e l l s . These receptors are expressed at low numbers on responsive cells (in general between 10-1000 per pos i t ive bone marrow cel l ) ( N i c o l a , 1987 and 1989). Sequence analysis of receptor c D N A s have shown these to be transmembrane proteins c o m p r i s i n g an e x t r a c e l l u l a r and i n t r a c e l l u l a r c o m p o n e n t l i n k e d by a s i n g l e in t ramembranous segment w h i c h remains constant in size between receptors (Ihle et al , 1994). The extracellular regions of the M - C S F receptor, . the human I L - 6 recep tor , and the I L - 1 receptor have sequence c h a r a c t e r i s t i c s o f the immunog lobu l in supergene family ( W i l l i a m s , 1987). Receptors for G M - C S F , G -C S F , e r y t h r o p o i e t i n , I L - 3 , I L - 4 and I L - 6 a l l have s i g n i f i c a n t sequence homologies wi th each other and also with the I L - 2 receptor beta chain, and I L -7, pro lac t in and growth hormone receptors. This group has been referred to as the hemopoiet in receptor superfamily (Bazan, 1989). , These receptors generally exist in both a h igh and low aff ini ty form wi th variable numbers of each form on different ce l l types ( N i c o l a and Metca l f , 1991). A l t h o u g h the specif ic i ty of b ind ing of a growth factor receptor wi th its l igand is extremely high, the b ind ing of one type of growth factor to its receptor can affect the b i n d i n g o f other receptors to their respect ive l igands , ( W a l k e r et a l , 1985). The b i o l o g i c a l effects observed 1 3 f o l l o w i n g b ind ing of a l igand to its receptor is the result of a b i o c h e m i c a l sequence o f events induced by the l igand-receptor in te rac t ion . T h i s is referred to as s ignal transduction (reviewed in D e v a l i a and L i n c h , 1991). The M - C S F receptor has been shown to have tyros ine kinase a c t i v i t y and cons ide rab le h o m o l o g y to the p l a t e l e t -de r ived g rowth factor ( P D G F ) receptor ( U l r i c h and Schlessinger, 1990; Roussel et a l , 1987; D o w n i n g et a l , 1988;. Sengupta et a l , 1988). S imi la r findings exist for related members of this receptor f ami ly wh ich include c-ki t (the receptor for Steel factor) and f l k -2 (the receptor for the f lk-2/ f l t -3 l igand (Hannum et a l , 1994). Even though other h e m o p o i e t i n receptor supe r f ami ly members l ack i n t r i n s i c ty ros ine k inase domains , they also q u i c k l y become tyros ine phosphory la ted themse lves and/or s t imula te r ap id and transient t y ros ine p h o s p h o r y l a t i o n s w i t h i n target ce l l s , due to the presence of a (permanently or t rans ient ly) associated tyrosine kinase J A K - 1 , 2 (Si lvennoinen et a l , 1993). A number of i n t r ace l lu l a r proteins ( P L C y , c-raf-1, PI-3 kinase and G A P ) have recently been shown to also become tyrosine phosphorylated by and associated wi th certain t y r o s i n e k ina se c o n t a i n i n g receptors upon l i g a n d b i n d i n g ( U l r i c h and Sch les s inge r , 1990). 2 M E G A K A R Y O C Y T O P O I E S I S M e g a k a r y o c y t o p o i e s i s is cu r ren t ly unders tood as a c o m p l e x regulated process in w h i c h commi t t ed precursors at var ious stages o f M k d i f f e r e n t i a t i o n i n t e r a c t w i t h h e m a t o p o i e t i c g r o w t h f a c t o r s a n d / o r e x t r a c e l l u l a r mat r ix molecu les to result in the coo rd ina t ed r egu la t ion o f platelet product ion ( L o n g , 1993). In 1969, Harker and F i n c h first proposed that separate processes must regulate M k mass (ie, the p ro l i fe ra t ion of M k . 1 4 . progeni tor ce l l s ) versus M k size (ie, M k differentiat ion) (Harker and F i n c h , 1969). However , the recent c lon ing of a growth factor, mp l - l i gand , wi th potent M k p rogen i t o r - s t imu la t i ng ac t i v i t y i n v i t ro and p l a t e l e t - s t i m u l a t i n g ac t iv i ty i n v i v o has now provided a single candidate molecula r regulator of megakaryocytopoiesis (Lok et al , 1994; Kaushansky et a l , 1994; W e n d l i n g et a l , 1994; de Sauvage et al, 1994). . S i m i l a r to other l ineages, early megakaryocyte progeni tors can b e ' character ized in terms of ce l l s of va ry ing prol i fera t ive potential ( f ig . 1.1). E a r l y precursors ( M k progenitor cel ls) consist of cel ls capable of undergoing v a r y i n g degrees o f p r o l i f e r a t i o n . I m m a t u r e m e g a k a r y o c y t e s ( i e , p romegaka ryob la s t s ) are t r ans i t i ona l between the mi to t i c and e.ndomitotic state and have l i m i t e d p ro l i fe ra t ive potent ia l . Matu ra t ion - re l a t ed ac t iv i t i e s consis t of endomitot ic and cy toplasmic changes resul t ing in increased p l o i d y , increased c e l l s ize, increased antigenic content and cy top lasmic maturat ion of organelles. Mature M k cells do not proliferate and are the source of platelet product ion . (Rev iewed in L o n g , 1993). 2.1 M E G A K A R Y O C Y T E P R O G E N I T O R C E L L S Severa l observations by different groups have suggested that a hierarchy o f megakaryocyte progenitors exists. (Thean et a l , 1983; L e v i n et a l , 1981; L o n g et a l , 1985). This hierarchy has been defined by the t ime of appearance and c e l l u l a r c o m p o s i t i o n o f c o l o n i e s d e r i v e d f r o m such progenitors ( L e v i n et a l , 1981; L o n g et a l , 1985; Paulus et a l , 1982). B y an analysis of the cumulat ive dis t r ibut ion of the number of doubl ings undergone by M k progenitor cel ls dur ing the process of co lony format ion in v i t ro and subsequent quant i ta t ion o f the D N A content o f the ce l l s c o m p r i s i n g these 15 colonies , Paulus et al (1982) identif ied three discreet classes of M k progenitor cells . L e v i n et al (1981) and L o n g et al (1985) have also reported the existence of at least two subpopulations of M k progenitor cel ls . L e v i n et al noted that the existence of two types of M k colonies wi th ce l lu la r components hav ing dis t inct ly different D N A contents. Several groups have used this approach to study M k di f ferent ia t ion ( W i l l i a m s and Jackson , 1982; Sega l et a l , 1988; C h a t e l a i n and B u r s t e i n , 1984). It is genera l ly accepted that a l though a hierarchy of M k progenitor cells does exist, classes of progenitor cells are not r i g i d l y def ined, and that M k progeni tor cel ls can best be thought of as ex i s t ing i n a cont inuum. Despi te this l imi t a t ion , the c lass i f i ca t ion of M k progeni tor ce l l s into subpopulat ions provides informat ion that may be useful i n d e f i n i n g c e l l u l a r stages at w h i c h important b i o l o g i c a l processes occur dur ing M k progenitor development (Hoffman, 1989). Three classes of M k progenitor cells have been identif ied to date: the bu r s t - fo rming un i t -megakaryocy te ( B F U - M k ) , the c o l o n y f o r m i n g unit-megakaryocyte ( C F U - M k ) , and the l ight density M k progenitor ce l l ( L D - C F U -M k ) (Long et a l , 1985; Nakeef and Dan ie l s -McQueen , 1976; M e t c a l f et a l , 1975; M c C l e o d et al , 1976, Vainchenker et al , 1974; M a z u r et a l , 1981a; Messner et al , 1982; Solberg et al , 1985; K i m u r a et al, 1984; Geissler et al , 1983; Chatelain et al , 1988; B r i d d e l l et al , 1988a). The B F U - M k and C F U - M k have been identif ied in both human and rodent systems whi ls t the L D - C F U - M k has been defined only in the mouse. The B F U - M k is the most p r imi t ive progenitor c e l l commit ted to the M k l ineage. B F U - M k - d e r i v e d colonies require longer incubat ion times to generate mature progeny in vitro (21 days for B F U - M k and 12 days for C F U - M k in humans; 12 days for B F U - M k and 5 days for C F U - M k in mice). B F U - M k -der ived colonies contain larger numbers of cells and are composed of mult iple clusters compared wi th the p r imar i ly uni foca l C F U - M k - d e r i v e d colonies ( L o n g 16 et al , 1985; Br idde l l et al, 1988a). B F U - M k and C F U - M k also, differ with respect to their sensit ivity to pretreatment wi th 5-f luorouraci l ( 5 - F U ) . The c lon ing eff ic iency of C F U - M k is markedly reduced by exposure to 5 - F U whils t the c lon ing eff iciency of B F U - M k is unaltered by such treatment (Br idde l l et a l , 1988b). The abi l i ty of the B F U -M k to survive 5 - F U treatment has been shown to be characteristic of p r imi t ive hematopoiet ic progenitor cel ls (Hodgson and Brad ley , 1979; V a n Zant , 1989). Thean et a l (1983) have descr ibed a 5-FU-res is tant murine progeni tor c e l l capable o f f o r m i n g splenic M k colonies ( M k C F U - S ) in le tha l ly i r radia ted recipients wh ich appears to be closely related to the B F U - M k . B F U - M k and C F U - M k also have different phys i ca l characterist ics and can be separated by v e l o c i t y sedimenta t ion gradients and cen t r i fuga l elutriat ion (Long et a l , 1985; B r i d d e l l et a l , 1988a and 1988b) Hoffman et a l , 1987) . 2.2 L I G H T D E N S I T Y - C F U - M K A N D T R A N S I T I O N A L I M M A T U R E M E G A K A R Y O C Y T E S The L D - C F U - M k described by Chate la in et al (1988) . is a more mature progenitor than the C F U - M k . L D - C F U - M k can be separated from the other classes of M k progenitor cel ls by density gradient sedimentat ion, they have a lower prol i ferat ive capacity and generate progeny of a higher p lo idy class than those found in C F U - M k der ived co lonies . These characteris t ics suggest that the L D - C F U - M k appears to reside at a stage of development close to where the megakaryocy te p rogeni to r c e l l ceases to undergo mi tos i s and acquires the capacity for endomitosis. This ce l l has only been described in the mur ine sys tem. 1 7 A ce l l located developmentally at a transit ional stage between M k p rogen i to r ce l l s and mature M k ce l l s has also been suggested by the o b s e r v a t i o n o f s m a l l m o n o n u c l e a r c e l l s that express p l a t e l e t - s p e c i f i c p h e n o t y p i c m a r k e r s , but are not m o r p h o l o g i c a l l y i d e n t i f i a b l e as megakaryocytes. Such cel ls have been described in both rodents and humans ( M a z u r et a l , 1981a; Jackson, 1973; L o n g and Henry, 1979; L o n g and W i l l i a m s , 1981; Rabel l ino et al , 1981; L o n g et a l , 1982a; Y o u n g and Weiss , 1987). These c e l l s represent a p p r o x i m a t e l y 5% of m a r r o w M k e lements and are d i s t inguishab le from the L D - C F U - M k by their i nab i l i t y to fo rm co lon ies in vi tro (Rabel l ino et a l , 1981; Chatelain et al , 1988). A large number of mature M k ce l l s appear when these t rans i t iona l ce l l s are exposed to appropr ia te growth factors ( Y o u n g and Weiss , 1987; L o n g et a l , 1982b). These findings suggest that t rans i t ional ce l l s represent a p ivo ta l stage in M k development dur ing which the ce l l leaves the proliferative pool to enter a pool of cel ls that undergo endomi tos i s . A c u t e a r t i f i c i a l changes in the platelet count appear to have marked effects on the number o f t rans i t iona l ce l l s in the . bone marrow sugges t ing that impor tan t regula tory factors may exert the i r effect on megakaryocy topo ies i s at this l eve l of M k c e l l development ( Jackson, 1973; L o n g and Henry , 1979; Straneva et a l , 1986; K a l m a z et a l , - 1981; Lepore et a l , 1984) . Uncommit ted totipotent hematopoietic stem cell Burst forming unit - Megakaryocyte - M o s t pr imi t ive progenitor commit ted to M k l i n e a g e - B F U - M k colonies take a long time to appear (21 d a y s ) - B F U - M k colonies large, mult ic lustered - B F U - M k cel l is 5 - F U resistant - demonstrated in human and murine systems Colony forming unit - Megakaryocyte - C F U - M k colonies appear early (12 days) - C F U - M k colonies are small , unifocal (ie smaller pro l i fera t ive capaci ty than B F U - M k ) . - C F U - M k cel l is 5 - F U sensitive - demonstrated in human and murine systems. L i g h t density megakaryocyte progeni tor cel l - separated by l ight density - lower prol iferat ive capacity than C F U - M k - higher p lo idy than C F U - M k - demonstrated in murine system only to date T r a n s i t i o n a l i m m a t u r e m e g a k a r y o c y t e - smal l mononuclear cells expressing M k lineage antigens - unable to form colonies - act ively synthesizing D N A - mature into, terminal ly mature M k - demonstrated in human and murine systems T e r m i n a l l y m a t u r e m e g a k a r y o c y t e 1.1. Summary of the various described M k progenitor cells . 1 9 2.3 T E R M I N A L M A T U R A T I O N O F M E G A K A R Y O C Y T E S M e g a k a r y o c y t i c ma tu ra t ion i n v o l v e s m u l t i p l e processes that result in ' endomitos is and cy top la smic maturat ion. A r b i t r a r y stages of M k different ia t ion and maturation (stages I through I V ) from the megakaryoblas t to the p l a t e l e t - e l a b o r a t i n g m e g a k a r y o c y t e have been d e f i n e d ( Z u c k e r -F r a n k l i n , 1990). Endomi tos i s is the synchronous d i v i s i o n of the nucleus without d i v i s i o n o f the cytoplasm. This process results in a p o l y p l o i d nucleus wh ich may be 4, 8, 16, 32, 64 or 128n. The degree of lobulation is unrelated to ploidy and may continue after D N A synthesis has ceased. The size of normal M k cells correlates wi th their p lo idy , wi th cells of low plo idy being smaller than those of h igh p l o i d y (Ade le et a l , 1970; Penington and Streat f ie ld , 1975). In pa tho log ica l states this relat ion may not exist (eg, acute thrombocytopenia is usually associated with large M k cells in a l l p loidy classes, Queisser et a l , 1971; Nomura et a l , 1983; Harker and F inch , 1969; Ebbe et al , 1988). C y t o p l a s m i c maturat ion denotes the a c q u i s i t i o n o f c y t o p l a s m i c granules, ves ic les , and demarcat ion membranes wi th a concomitant reduct ion i n free and membrane-associated r ibosomes to a f i n a l stage commensura te , wi th the elaboration of platelets (reviewed in Zucke r -F rank l in , 1990). Platelets are bel ieved to be formed from large fragments o f M k cy top lasm. The actual mechanism of platelet format ion, however , is unclear. E l e c t r o n m i c r o s c o p i c s tud ies have s h o w n m e g a k a r y o c y t e s e x t e n d i n g pseudopod-l ike processes into the bone marrow sinusoids. These processes are thought to consis t of a s t r ing of platelet f ields w h i c h fragment into the c i rcula t ion (Thiery and Bessis , 1956; Becker and de B r u y n , 1976; L ich tman et a l , 1978). There is also increasing evidence that mature intact megakaryocytes migra te into s inuso ids , enter the b l o o d and fragment i n the p u l m o n a r y 2 0 vascular bed (Melamed et a l , 1966; Kaufman et al , 1965; Tavassol i and A o k i , 1981; Slater et al , 1983; Shoff et al , 1987). Platelets have important functions i n hemostasis , e labora t ion of c y t o k i n e s ( P D G F , T G F - p ) , and a therogenesis . P a t h o l o g i c a l states o f th rombocy topen ia ( low platelet count) may be caused either by a decreased p r o d u c t i o n or an increased des t ruct ion of platelets (or both) and are c h a r a c t e r i z e d c l i n i c a l l y by b l e e d i n g ep i sodes . States a s soc i a t ed w i t h , t h r o m b o c y t h e m i a (ie, h igh platelet count) are charac te r ized by increased t h r o m b o t i c d i so rde r s . 2.4 G E N E E X P R E S S I O N D U R I N G M E G A K A R Y O C Y T E D I F F E R E N T I A T I O N The molecular control of l ineage commitment . and different ia t ion i n v o l v e s the es tab l i shment o f spec i f i c patterns o f gene e x p r e s s i o n by i n t e r a c t i n g ne tworks o f l i neage - res t r i c t ed and non- re s t r i c t ed . t r a n s c r i p t i o n factors. ' Some of the G A T A z inc finger t ranscript ion factors are thought to have important roles in the control of hematopoiesis ( O r k i n , 1992). The ( T / A ) G A T A ( A / G ) consensus element bound by these factors, f i rs t iden t i f i ed w i t h i n the promoters and enhancers of several genes expressed in e ry thro id ce l l s , has also been found in the promoters of genes expressed unique ly in cel ls of the M k lineage such as GPI Ib (CD41b) and P F 4 ( R a v i d et a l , 1991; Lemarchande l et a l , 1993). V i s v a d e r and A d a m s (1993) have shown that enforced overexpression of G A T A - 1 , G A T A - 2 or G A T A T 3 in a p r imi t ive murine m y e l o i d ce l l l ine (416B) induced differentiation restricted to the M k lineage. Other findings in this study suggested that G A T A - 2 and G A T A - 3 l ie upstream of G A T A - 1 in a regulatory hierarchy and that, in 416B cel ls , G A T A - 1 may mediate the phenotypic changes induced by G A T A - 2 and G A T A - 3 . The c - m p l gene has recently been imp l i ca t ed as an important gene con t ro l l i ng megakaryocytopoies is . Th is gene encodes a member of the hema topo ie t i c receptor super fami ly whose l i g a n d has on ly recent ly been ident i f ied ( L o k et a l , 1994; W e n d l i n g et a l , 1994; Kaushansky et a l , 1994; de Sauvage et a l , 1994). Wend l ing et al (1992) have shown high levels of c -mpl expression in human leukemic and normal cells of the M k lineage. Studies on the express ion of c - m p l have shown that the c -mp l - encoded receptor is expressed in ear ly hematopoie t ic ce l l s , but its express ion appears to be downregula ted dur ing the various pathways of d i f ferent ia t ion except for the M k lineage. Anti-sense oligonucleotides complementary to c -mpl m R N A led to a marked inh ib i t ion of in vitro M k colony formation without effecting C F U -G M or e ry thro id colonies ( M e r t h i a et a l , 1993). Recen t ly c -mp l has been iden t i f i ed as the receptor for a new cy tok ine w h i c h s p e c i f i c a l l y regulates megakaryocy topo ies i s , s t rongly suggest ing that c - m p l has an important ro l e ' i n the processes of M k prol i fera t ion, maturation and platelet product ion ( L o k et al , 1994;. Wend l ing et al , 1994; Kaushansky et al , 1994; de Sauvage et al , 1994). Th is is further reviewed in section 2.6.10. 2.5 A N T I G E N E X P R E S S I O N D U R I N G M E G A K A R Y O C Y T E D I F F E R E N T I A T I O N B F U - M k and C F U - M k have both been shown to express the C D 3 4 ant igen, an antigen k n o w n to be present on a wide var ie ty o f human hematopoietic progenitor cells ( L u et a l , 1988; Watt et a l , 1987). C F U - M k were found to express the major h i s t o c o m p a t a b i l i t y type-I I an t igen ( H L A - D R 22 . an t igen i n humans or l a ant igen in mice) whereas B F U - M k expressed undetectable quanti t ies of this ant igen ( B r i d d e l l et a l , 1988b) . T h i s is consistent wi th the general observation that the C D 3 4 + D R - fract ion of adult bone marrow contains progeni tor ce l l s that are more p r i m i t i v e than those present in the C D 3 4 + D R + fraction. However , controversy exists regarding the consis tency and t i m i n g of expression of the platelet membrane g lycopro te ins (GPIb , GPI Ib , GPIIIa) on C F U - M k . Several groups have presented evidence to indicate that the expression of these antigens may occur on some C F U - M k but probably not on a l l (Berridge et a l , 1985; Fraser et al , 1986; Levene et a l , 1985; K a n z et a l , 1988; Vainchenker et a l , 1986). M o r e recently, D e b i l i et al (1992) us ing F A C S - s o r t e d bone • marrow cells found that 2%. of the C D 3 4 + cel ls co-expressed the ' G P I I I a antigen and the C D 3 4 + G P I I I a + and C D 3 4 - G P I I I a + p o p u l a t i o n s o f c e l l s were a lmos t a l l immatu re m e g a k a r y o b l a s t i c c e l l s . U l t r a s t ruc tu ra l evidence of maturat ion (the development of a - g r a n u l e s and demarca t ion membranes) was present in the C D 3 4 - G P I I I a + ce l l s and absent from the CD34+GPII Ia+ cel ls . The expression of GPIIb appeared to mimic that o f G P I I I a . G l y c o p r o t e i n l b , h o w e v e r , was e x p r e s s e d la ter d u r i n g d i f f e r e n t i a t i o n and its appearance c o i n c i d e d w i t h the b e g i n n i n g o f p o l y p l o i d i z a t i o n . The synthesis of von W i l l e b r a n d Fac to r was present in CD34+GPI I I a+ cel ls indicat ing that synthesis of this marker begins at an early stage of d i f ferent ia t ion . C e l l s ident i f ied m o r p h o l o g i c a l l y as mature M k have also been reported to exhibi t heterogeneity in their expression of c e l l surface antigens. A n t i g e n density is mentioned by many investigators as va ry ing from c e l l to c e l l (Va inchenker et a l , 1982a; Rabe l l i no et a l , 1979 and 1981; Wor th ing ton , 1984; Levene et al , 1985; L o n g , 1993), and, L o n g et al (1993) for example, have reported variations in expression of GPIIb/IIIa, v W F , T S P , P F 4 and P 1 A 1 . Rare M k ce l l s were observed that were negative for a par t icu la r ant igen even 2 3 though other M k in the v i c in i ty were strongly posi t ive . U s i n g a panel of antibodies with specif ic i ty for M k and platelets, • R a b e l l i n o et a l (1979), have described three groups of ce l l u l a r epitopes on deve lop ing M k . Group A antigens were observed on ly on C F U - M k and immature M k . Group B antigens were expressed on developing M k and then lost d u r i n g their t e rmina l d i f ferent ia t ion . G r o u p C antigens cons i s ted of p la te le t epi topes ( p r i m a r i l y platelet GPI Ib / I I I a ) w h i c h w e r e , found to be expressed throughout the M k di f ferent ia t ion process. In add i t ion , Z u c k e r -F r a n k l i n et al (1986) have shown that not a l l antibodies to platelet antigens react wi th M k , perhaps due to lack of exposure of the target antigen (Stahl et a l , 1986). 2.6 P O S I T I V E R E G U L A T I O N O F M E G A K A R Y O C Y T O P O I E S I S Studies i n a var ie ty of a n i m a l models suggest that platelet p r o d u c t i o n i n v i v o can be a l tered by h u m o r a l factors . I n d u c t i o n o f th rombocytopen ia in rodents is accompanied by an increase in bone marrow M k numbers, s ize, and D N A content whi ls t thrombocytosis created by platelet transfusions results in rec iproca l changes (Ode l l et a l , 1976; Burs t e in et a l , 1981; Ebbe et a l , 1966). Further evidence for the existence of a regulatory mechan i sm can be seen in the constancy o f the normal per iphera l b l o o d platelet count and the qualitative alterations observed in platelets and M k that a c c o m p a n y c l i n i c a l t h r o m b o c y t o p e n i a s and t h r o m b o c y t o s e s o f v a r y i n g etiologies (Penington and Olsen, 1970; Adams, et al , 1978; Sul l ivan et a l , 1979). The regulation of megakaryocytopoiesis is a complex process and remains incomple te ly understood. Schemat ica l ly M k development has been d iv ided into two phases: characterized first by a phase of prol iferat ion and •2 4 M E G A K A R Y O C Y T O P O I E S I S : T W O F A C T O R H Y P O T H E S I S Different growth factors are required for: 1. P r o l i f e r a t i o n ® St imula t ion of M k - p r o g e n i t o r cel ls to proliferate. @ \ 2. D i f f e r e n t i a t i o n ( a ) Endomitosis - cont inued mitosis wi thout cytoplasmic d iv i s ion resulting in 4 N , 8 N , 1.6N, 3 2 N , 6 4 N or 124N. ( b ) Cytoplasmic maturation - development o f organel les (eg alpha-granules , demarcat ion membranes) found in platelets. ( c ) Antigenic maturation - express ion o f antigens found on platelets (GPIIb/I I Ia , G P I b ) . 16N 32N F i g . 1.2. Schematic summary of the ' T w o Factor ' hypothesis of the regulation of M k development. 25 later by a maturation phase. Factors act in i t i a l ly to stimulate prol i ferat ion of M k progenitor cel ls committed to the M k lineage and these are ident i f ied as M k c o l o n y s t i m u l a t i n g factors ( M k - C S F s ) . L a t e - a c t i n g factors p romote endoredupl ica t ion (po lyp lo id i za t i on ) , cy top lasmic dif ferent ia t ion and enlargement , and the accumula t ion of l ineage-spec i f i c markers r e su l t ing i n the acquisi t ion of the characteristics of mature M k (fig. 1.2). This ' two-factor ' hypothesis is based on a number of f indings. L e v i n et al (1980) demonstrated that there were no changes in marrow C F U - M k up to 14 days after the induct ion of thrombocytopenia in rodents whi l s t at the same time profound changes in M k number and size occurred. Burs te in et a l (1981) noted that the total body content of C F U - M k . in mice did not rise unti l 90 hours after the induc t ion o f thrombocytopenia , despite an ear l ier two- fo ld increase in M k number and size at 65 hours. W i l l i a m s et a l (1982) were able to fractionate W E H I - 3 c e l l condi t ioned medium previously shown to stimulate M k co lony formation and demonstrated that i nd iv idua l fractions d id not support M k c o l o n y g rowth , a l though M k co lony growth c o u l d be obta ined when combinat ions of fractions were tested. 2.6.1 I N T E R L E U K I N - 3 A N D G R A N U L O C Y T E - M A C R O P H A G E C O L O N Y S T I M U L A T I N G F A C T O R B o t h in te r l euk in -3 ( I L T 3 ) and g ranu locy te -macrophage c o l o n y s t imula t ing factor ( G M - C S F ) have been shown to be M k - C S F s in addi t ion to the i r a b i l i t y to affect the g rowth of progeni tors of a number o f other hematopoietic lineages (Bruno et a l , 1988; Quesenberry et a l , 1985; L o p e z et a l , 1987; M i z o g u c h i et a l , 1986; Teramura et a l , 1988). IL -3 appears to have the abi l i ty to promote the formation of greater numbers of M k colonies than does 26 G M - C S F (Quesenberry et al,1985; Messner et al,1987) and the effects of IL -3 and G M - C S F are addi t ive (Robinson et a l ,1987; M c N i e c e et a l ,1988; Donahue et a l ,1988). A n I L - 3 / G M - C S F fusion protein has also been shown to. stimulate B F U - M k (but not C F U - M K ) - d e r i y e d colonies to the same extent as the addition of op t ima l concentrat ions of both I L - 3 and G M - C S F added together (Bruno et a l ,1992) . I L - 3 has been shown to enhance B F U - M k - d e r i v e d co lony format ion. M o r e o v e r , I L - 3 not only increased co lony format ion, but also increased the number of cells in ind iv idua l C F U - M K - d e r i v e d colonies . In addi t ion, Teramura et al (1988) found that I L - 3 stimulated the growth of M k progenitor cel ls in non-adherent, T - c e l l depleted mononuclear cel ls plated in serum-free med ium suggesting that IL -3 stimulates the growth of M k progenitor cel ls without any requirement for addi t ional factors present in serum or plasma. Inter leukin-3 has been shown to have a direct effect on s ingle m u r i n e M k r e s u l t i n g i n an increase in c e l l d i ame te r , p l o i d y and a c e t y l c h o l i n e s t e r a s e a c t i v i t y , i . e . , changes a s s o c i a t e d w i t h m a t u r a t i o n (Ishibashi and Burs te in , 1986). Con t inuous infus ions o f human I L - 3 i n pr imates resu l ted i n profound increases in platelet counts ( K r u m w i e h and Se i le r , 1988 and 1989) and A g l i e t t a et al (1991) found that patients with normal hematopoiesis who were treated with G M - C S F also showed an increase in the prol iferat ive act ivi ty o f M k progeni tor ce l l s and increased maturation of M k . Pr imates first in jected wi th I L - 3 and then subsequently wi th G M - C S F showed a dose-dependent increase in platelet numbers even though I L - 3 and G M - C S F , at the doses used, had no significant effect on platelet, numbers. Stahl et a l (1992) have presented evidence to suggest that the administration of I L - 3 fo l lowed by G M - C S F treatment to primates increases thrombopoies is by inc reas ing M k numbers and matura t ion and that these effects are d i m i n i s h e d by the simultaneous administrat ion of these two cytokines . 27 These data indicate that I L - 3 and G M - C S F can have addi t ive effects on megakaryocytopoies is both in vi t ro and in v i v o . T h i s may be expla ined by the observations that G M - C S F and I L - 3 may cross compete for receptor b ind ing (Park et a l , 1989), and, that the G M - C S F and I L - 3 receptors share a common p-chain that can associate with and convert either receptor to a h igh affinity form (Nico la ,1991) . 2.6.2 I N T E R L E U K I N - 6 In ter leukin-6 ( IL-6 ) is a 26,000 k D g lycopro te in wi th mul t ip le b i o l o g i c a l ac t iv i t i e s i n c l u d i n g the a b i l i t y to enhance Ig secre t ion by B lymphocytes , to induce an ant ivi ra l state in fibroblasts, to serve as a second s i g n a l i n m u r i n e T c e l l a c t i v a t i o n and t o . s t imula te or enhance the prol i fera t ion of different types of hematopoietic progenitor cel ls (Hirano et a l , 1986; V a n Damme et al , 1987; Zilberstein et al, . 1986; Garmen et al , 1987; W o n g et a l , 1988). I L - 6 has also been found to have the abi l i ty to activate pr imi t ive quiescent progenitor cel ls into cyc l e , a l l o w i n g them to become responsive to I L - 3 and G M - C S F (Ikebuchi et al , 1987). Interleukin-6 m R N A has been found in murine bone marrow stromal ce l l s , activated T-ce l l s and macrophage c e l l l ines ( C h i u et a l , 1988). Th is expression is i nduc ib l e , e.g. by I L - 1 and l ipopolysacchar ide (Bagby, 1989;. J i r i k et a l , 1989). Severa l groups have demonstrated that I L - 6 can have a direct effect on murine M k to promote their maturation. I L - 6 can also potentiate the action of I L - 3 to stimulate M k progenitor cells direct ly (Ishibashi et a l , 1989a; W i l l i a m s et a l , 1990; Warren et a l , 1989; Lo tem et a l , 1989). Ishibashi et al (1989a) and W i l l i a m s et al (1990) found that, al though human I L - 6 had no in f luence on murine M k co lony format ion , it d i d synerg ize wi th I L - 3 to 28 increase the number of M k colonies in serum-free agar cultures. In l i qu id m a r r o w cu l tu res , I L - 6 alone p romoted marked increments in M k s i ze , acetylchol inesterase ac t iv i ty , and higher p lo idy classes (ie, changes associated wi th te rmina l maturation). W h e n single M k cel ls were isolated these same f indings were present ind ica t ing a direct matura t ion-promot ing effect o f I L - 6 on M k and ind ica t ing that I L - 6 receptors must be present on these ce l l s (Ishibashi et a l , 1989a). Lo tem et al (1989) have found that the addition of a M o A b specific for murine I L - 6 to serum-containing agar cultures of unfractionated murine bone marrow cells stimulated by either IL -3 alone or I L - l a alone resulted in a marked reduct ion in C F U - M k - d e r i v e d co lony format ion. Th is suggests that the st imulatory effect of IL -3 and I L - l a is mediated by endogenous product ion _ of I L - 6 i n these cultures. S tud ies on mi ce and pr imates g i v e n I L - 6 i n v i v o have demonstrated a dose-dependent increase in platelet counts wi th an associated ; m a r k e d increase in M k s i ze , aga in i n d i c a t i n g I L - 6 p r o m o t i o n o f M k maturation. In these studies, no changes in M k or C F U - M k numbers were observed (Ishibashi et al , 1989b; Asano et al , 1990). 2.6.3 I N T E R L E U K I N - 1 1 Interleukin-11 ( IL-11) is a recently described cy tokine that also appears to be a mul t i - l ineage regulator of murine and human bone marrow cells (Paul et al , 1990). Al though IL-11 has no inherent M k - C S F act ivi ty it appears able to synergize wi th I L - 3 to promote both human and murine M k co lony format ion in v i t ro . Y o n e m u r a et al (1992) demonstrated that IL -11 synergized wi th opt imal and suboptimal concentrations of I L - 3 to enhance the 29 growth of murine M k colonies and to increase the size and p lo idy of the cells p r o d u c e d . I L - 1 1 a lone was . a lso found to inc rease the s i ze and ace ty lchol ines te rase ( A c h E ) content o f M k ce l l s in l i q u i d cul ture wi thou t increasing the number of M k cells (Yonemura et al , 1992). Bruno et a l ( 1 9 9 0 ) s h o w e d that I L - 1 1 s y n e r g i z e d w i t h s u b o p t i m a l (but not- o p t i m a l ) concentrations of I L - 3 to promote human M k colony formation in vi t ro. . In v i v o studies have demonstrated that I L - 1 1 can cause an increase in platelet numbers, M k p lo idy and numbers of M k progenitor cel ls i n normal mice and non-human primates. Post-transplant mice exposed to I L -11 (ei ther by exogenous admin i s t r a t ion or by endogenous p r o d u c t i o n by infec t ion of the donor bone marrow with an IL-11 c D N A conta in ing re t rovira l vector) showed accelerated recovery of platelets (Neben et a l , 1993; Bree et a l , 1991; Goldman et al , 1991; D u et al, 1993; Paul et al, 1991; Hangoc et al, 1993). I L - 1 1 has a l so been s h o w n to s t imula t e m e g a k a r y o b l a s t i c l eukemia c e l l l ines i n a dose-dependent manner. G r o w t h - o f these cel ls was inh ib i t ed by ant i - IL-11 antibody and I L - 1 1 antisense o l igonuc leo t ides . I L - 1 1 transcripts were also found in these cel ls . These f indings suggest that IL -11 might be an autocrine growth factor for megakaryoblast ic cel ls ( K o b a y a s h i et al, 1993). . . These studies indicate that I L - 1 1 is an impor tant synerg i s t i c factor i n megakaryocytopoies is wi th effects on both M k . progeni tor ce l l s and terminal ly differentiating M k . P rec l in i ca l studies suggest that IL-11 may be of use to accelerate platejet r e c o v e r y after bone marrow transplantation. A role for IL-11 in the development of megakaryoblast ic leukemia has also been s u g g e s t e d . 2.6.4 L E U K E M I A I N H I B I T O R Y F A C T O R 30 L e u k e m i a inh ib i to ry factor ( L I F ) was pur i f i ed and c loned based on its ab i l i ty to induce differentiation i n and suppress the prol i ferat ion of M l murine mye lo id leukemic cells (Tomida et al , 1984; H i l t on et a l , 1988; Gear ing et a l , 1987). Receptors for murine L I F have been demonstrated on immature and mature murine M k cells (Metca l f et al , 1991). L I F alone was not found to have any effect in vi tro on normal murine M k progenitor ce l ls . L I F d id , however, synergize with IL-3 but not with G M - C S F , G - C S F or M - C S F to stimulate murine M k colony formation (Metcalf et al , 1991; D e b i l i et al , 1993). In v ivo studies have demonstrated that LIF- in jec ted mice show an increase in platelet counts with a rise in M k up to 2-fold in the bone marrow and 5-fold in the spleen, wi th an associated 10-fold increase in M k progenitor cel ls in the spleen (Metca l f et a l , 1990). L I F - i n d u c e d these increases in M k p rogen i to r ce l l s and M k before increases i n p la te le t l eve l s were seen, suggesting that the latter was based on the formation of increased numbers of M k and d id not merely result from an induced release of platelets f rom pre-exis t ing M k . The magnitude of the M k and platelet increases induced by L I F was equal to, or greater than, those reported after the injection of I L - 3 , I L - 6 or th rombopoie t in , suggest ing a possible c l i n i c a l role of L I F therapy for the treatment of thrombocytopenias (Metca l f et a l , 1986; M c D o n a l d , 1988; Ishibashi et a l , 1989b). 2.6.5 S T E E L F A C T O R S t e e l F a c t o r ( S F ) e x h i b i t s potent s y n e r g i s t i c a c t i o n s i n conjunct ion wi th other C S F s result ing in the prol iferat ion of both mye lo id and 3 1 l y m p h o i d hematopoietic progenitor cells ' and is bel ieved to play a major role in stem ce l l development (Wi l l i ams et al , 1990a; Zsebo et a l , 1990; Broxmeyer et a l , 1991; M c N i e c e et al , 1991; Bernstein et a l , 1991). S F has also been variously referred to as c-ki t l igand, mast ce l l growth factor ( M G F ) , or stem cel l factor ( S C F ) by various investigators (Dexter and M o o r e , 1977b; Copeland et a l , 1990; Flanagan and Leder, 1990; Mar t in et al , 1990; Zsebo et al , 1990; Huang et al , 1990; Anderson et a l , 1990; W i l l i a m s et al , 1990b; Witte, 1990). Different forms o f S F produced by d i f ferent ia l R N A process ing have been described. This results in an obl igatory membrane-bound species (mSF) as we l l as soluble molecules. M k have been shown to express receptors for S F (c-kit) and Avraham et al (1992) have shown that the adhesion of M k to bone marrow fibroblasts in vi t ro can be mediated in part by an interact ion between m S F and c-ki t on the M k . This interaction results in the s t imulat ion of M k pro l i fe ra t ion and provides a model whereby M k and their progeni tor ce l l s may be . anchored to s t romal c e l l elements w i t h i n the bone marrow microenv i ronment and posi t ioned to respond to cytokines . S l / S l d mice have defective hematopoiesis as a consequence of their i nab i l i t y to produce a membrane-bound form of S F and are k n o w n to have abnormal thrombopoiesis as w e l l as being anemic. The i r bone marrow has subs tan t i a l ly fewer M k than no rma l (+/+) mice and the i r M k are macrocy t ic . Platelet mass and platelet turnover in S l / S l ^ mice has, however , been reported as, normal . Interestingly adminis t ra t ion of S F was found to cause a thrombocytosis (Ebbe et al , 1973, 1978 and 1986; Adrados et a l , 1984). Such f indings suggested that S F may play an important role in the normal r egu la t ion o f megaka ryocy topo ie s i s . ' Tanaka et al (1992) found that S F alone d id not support the growth of M k colonies from normal murine bone marrow, but d id synergize wi th I L - 3 to result in a s igni f icant increase not only in the number of M k colonies 32 obtained but also in the number of M k per colony. However , the size and D N A content o f i n d i v i d u a l M k in the colonies was not increased. A different ia l effect o f S F ac t ing on different subsets o f M k progeni tor ce l l s has been described by Br idde l l et al (1991). SF was found to, synergize with I L - 3 , but not G M - C S F to increase the number of B F U - M K - d e r i v e d co lon ies f rom normal human bone marrow. S F synergized with both IL -3 and G M - C S F to increase the number and size of C F U - M K - d e r i v e d colonies. D e b i l i et al (1993) found that human M k grown in the presence of IL -3 and S F were of larger size, had increased p lo idy and contained both a granules and demarca t ion membranes wi th platelet shedding compared with I L - 3 alone wh ich d id not demonstrate this degree of maturation. Taken together, these studies indicate a definite effect of S F on M k progenitor proliferat ion in vi t ro, however, effects on M k maturation are less clear and may vary among species. 2.6.6 G R A N U L O C Y T E C O L O N Y - S T I M U L A T I N G F A C T O R Granulocy te co lony-s t imula t ing factor ( G - C S F ) by i t se l f has been shown to have no M k - C S F act ivi ty. G - C S F is, however, capable of increasing the number and the size of M k colonies formed in the presence of I L - 3 as compared to assays containing IL -3 alone ( M c N i e c e et a l , 1988; B r u n o et a l , 1988). O n the other hand, G - C S F did not synergize with G M - C S F to increase either the number or size of M k colonies. 2.6.7 I N T E R L E U K I N - 1 Severa l studies have demonstrated that in ter leukin- loc ( I L - l a ) by 3 3 i t se l f is also unable to st imulate M k co lony format ion (Bruno et a l , 1988; Takahash i et a l , 1991) but may synergize wi th I L - 3 to increase M k co lony format ion. However , Warren et al (1989) found that I L - l a was able to increase M k colony formation only in the presence of both IL -3 and I L - 6 . The effects of I L - l a on hematopoiesis have been, i n part, attributed to their ab i l i ty to cause either endothelial cel ls , fibroblasts, and T lymphocytes to elaborate a number of cytokines inc luding G M - C S F , I L - 3 , G - C S F , or I L - 6 , which might then di rect ly influence progenitor ce l l prol i fera t ion (Bagby et a l , 1986; Sief f et a l , 1988; Y a n g et al , 1988). Us ing C D 3 4 + D R - bone marrow cells in a serum substitute assay, B r i d d e l l and Hoffman (1990) found that the synergist ic r e l a t ionsh ip of I L - l a and IL -3 could be, abrogated by addi t ion of an I L - l a neut ra l iz ing antibody but not by a G M - C S F neutral iz ing antiserum, suggesting that I L - l a acts direct ly on M k progenitor cells and not by s t imulat ing marrow accessory ce l l s . 2.6.8 E R Y T H R O P O I E T I N The effect of erythropoie t in (Ep) on M k co lony format ion has been ex tens ive ly studied wi th c o n f l i c t i n g results. The express ion of E p receptors on M k cel ls has been inferred from the b ind ing of r a d i o l a b e l e d Ep to rat and mouse marrow M k cells (but not to platelets) (Fraser et al , 1989). S o m e i n v i v o s tudies have sugges ted that E p p romotes megakaryocytopoies is . Chron ic administration of E p to rats resulted in early, but unsustained increases i n platelet counts and an increase in t h y m i d i n e labe l ing of M k (Berr idge et a l , 1988). However transgenic mice expressing the human E p gene d id not show increases in their platelet counts, al though mice administered, single high doses of E p d id develop an increase in platelet 3 4 levels (Semenza et al , 1989; M c D o n a l d et a l , 1987). In some, but not a l l human E p trials, there was a slight thrombocytosis (Eschbach et a l , 1989; Stone et a l , 1988). Whether these were direct or indirect effects could not be determined. Recen t ly , L o n g m o r e et a l (1993) have demonstrated that mice infec ted wi th a recombinant spleen focus - fo rming re t rovi rus express ing an oncogenic E p receptor had an increase in splenic M k and platelet leve ls . M u l t i p o t e n t i a l , mixed erythroid and M k and pure M k progenitor cel ls were a l l increased in these animals. The abnormal Ep receptor was expressed in the M k progeni tors suggest ing that a direct effect on megakaryocytopoies i s in these mice had occurred (Longmore et al , 1993). In vitro studies have also y ie lded conf l i c t ing results for an effect o f E p on megakaryocy topo ies i s . E p in serum-free or p l a sma-con ta in ing cultures fa i led to stimulate M k progenitor Cells above baseline under a variety of culture condit ions ( M a z u r et a l , 1981b; Yakahash i et a l , 1991; Z a u l i et a l , 1992). H o w e v e r , other studies have demonstrated an effect of E p on megaka ryocy topo ie s i s w h i c h appeared to be dependent on the presence of serum or subopt imal (but not optimal) concentrations of P H A - L C M (Tsukada et a l , 1992; Dessypris et a l , 1987). U s i n g neutral izing antibodies it was shown that E p could synergize with a. serum factor that was distinct from I L - l a , I L - 3 , IL-4 , IL-6, . G - C S F or G M - C S F (Tsukada. et al, 1992). One explanat ion for the inconsis tency of these in v i t ro results might be due to contaminat ion of part ial ly purif ied E p preparations wi th other cytokines (Hoffman, 1989). Other variables such as the species o r ig in of the E p preparations, the culture and M k progenitor ce l l detection systems, and the c o n c e n t r a t i o n o f E p used may also be impor tan t i n e x p l a i n i n g these differences. The data of Ross i et al (1989) suggest that E p has l i t t le influence on megakaryocytopoies is at the doses known to e l ic i t a max ima l erythropoiet ic 3 5 response. They have suggested that al though subsets of murine splenic M k progeni tors may be in f luenced by ex t raord ina r i ly h igh doses o f E p , it is u n l i k e l y that this phenomenon is a s igni f icant factor in the normal ove ra l l regula t ion of megakaryocytopoies is in v i v o . de Sauvage et a l (1994) have recen t ly demons t ra ted . 50% h o m o l o g y ( tak ing into account conserva t ive subst i tu t ions) between the N -t e r m i n a l 153 res idues o f . E p and m p l - l i g a n d ( t h r o m b o p o i e t i n ) . The s ign i f icance of this observation is uncertain. In summary, E p receptors have been demonstrated on M k and although effects of Ep have been observed on megakaryocytopoiesis in vi t ro, it is s t i l l uncertain as to whether these are direct or indirect , require synergism wi th other factors and/or are p h y s i o l o g i c a l l y s igni f icant . 2.6 .9 B A S I C F I B R O B L A S T G R O W T H F A C T O R Bas ic fibroblast, growth factor ( b F G F ) is a mul t i funct ional growth factor produced by bone marrow stromal cel ls and is known to be a potent modulator of hematopoiesis (Gabbianel l i et a l , 1990; G a l l i c c h i o et a l , 1991; O l i v e r et al , 1990; W i l s o n et a l , 1991; Han et a l , 1992). Both b F G F and the. b F G F -receptor have been i d e n t i f i e d , in human platelets and - M k suggest ing a poss ib le role i n megakaryocytopoies is (Brunner et a l , 1993; B i k f a l y i et a l , 1992). However , Bruno et al (1993) have cited evidence to suggest that b F G F exerts its effects on megakaryocytopoiesis v i a two dist inct mechanisms. O n the one hand, b F G F may act directly at the level of the M k progenitor ce l l as shown by its abi l i ty to increase the size of M k colonies derived from pure populations of sorted C D 3 4 + cells . On the other hand the data of this group and others also indicate that b F G F acts indirect ly v i a B M accessory cells to promote the release 36 and/or synthesis of cy tokines that may also st imulate M k co lony format ion (Han et al , 1992). 2.6 .10 T H R O M B O P O I E T I N ( M P L - L I G A N D ) The concept of a th rombocytopoies i s - s t imula t ing factor ( T S F ) or t h rombopo ie t i n ( T P O ) was first proposed 30 years ago and refers to the exis tence o f a l ineage-speci f ic humora l factor that regulates the number o f c i r cu l a t i ng platelets by causing an increase in the number and size of M k (Yamamoto, 1957; Kelemen et al , 1958). More recent studies indicated that such a factor is most l ike ly released in response to a decrease in the number, mass or funct ion of c i rcu la t ing platelets ( L e v i n and Evatt , 1979; M c D o n a l d , 1981; O d e l l , 1974; Ebbe, 1976). The plasma, serum and urine from thrombocytopenic animals and patients have been used to study T S F and in 1975 a T S F was shown to be produced by H E K cells in culture (Ode l l et a l , 1961; Evat t et a l , 1974; M c D o n a l d , 1975; Nakeff and Roozendaal , 1975; M c D o n a l d et a l , 1975). The work of M c D o n a l d et al demonstrated that the medium from kidney cel ls in culture contained a factor that stimulated thrombopoieisis in mice as measured by the 3^s i n c o r p o r a t i o n . in to p la te le t s . A dose response r e l a t i o n s h i p was demonstra ted between the vo lume of t h r o m b o c y t o p o i e t i c a l l y ac t ive m e d i u m injected and the l eve l of isotope that subsequently appeared in the c i rcu la t ing platelets. Severa l studies have shown that such T S F - c o n t a i n i n g preparations potentiate the action of incompletely defined cytokines with M k - C S F act ivi ty to increase the number of M k colonies in vitro ( M c D o n a l d et a l , 1985; W i l l i a m s et a l , 1979 and 1984; Hoffman et al , 1985). They may also directly stimulate small A c h E + precursor cel ls to become large mature M k ( L o n g et a l , 1982a and 1982b) . • 3 7 Recent ly , the c D N A s for both human and murine T P O have been cloned (de Sauvage et al , 1994; L o k et al , 1994; Wendl ing et a l , 1994). These three groups u t i l i z e d the observa t ion that the m y e l o p r o l i f e r a t i v e l e u k e m i a virus (mpl) isolated by Souyr i et al (1990) had transduced in its envelope gene a ce l lu la r gene (c-mpl) that was subsequently shown to have special relevance as a receptor for cells of the M k lineage (Mer th ia et a l , 1993). U s i n g different approaches the l i g a n d for c - m p l was i so la ted and shown to encode a g lycopro te in w h i c h has selective actions on M k prol i fera t ion in vi t ro and an abi l i ty to increase platelet numbers in v ivo ( L o k et a l , 1994; W e n d l i n g et a l , 1994; Kaushansky et al , .1994; de Sauvage et al , 1.994). 2.7 N E G A T I V E R E G U L A T O R S O F M E G A K A R Y O C Y T O P O I E S I S S e v e r a l observat ions have suggested that m e g a k a r y o c y t o p o i e s i s is also under the control of a variety of negative regulators. The f ind ing that p l a sma is superior to serum in support ing M k co lony format ion in v i t ro produced early evidence that platelets might elaborate inhibi tors of M k co lony format ion (Messner et a l , 1982; So lberg et a l , 1985; K i m u r a et a l , 1984; V a i n c h e n k e r et a l , 1982b). Hypertransfusion of platelets into exper imenta l animals has been shown to result in a decrease in the size, p lo idy and number of M k , an effect attributable to the suppression of a thrombopoiet ic hormone or re lease o f i n h i b i t o r s o f m e g a k a r y o c y t o p o i e s i s ( P e n i n g t o n , 1981) . A d m i n i s t r a t i o n of platelet extracts also inh ib i t ed megakaryocytopoies is ( K r i z s a et a l , 1977). Several groups have reported that transforming growth factor-(3 ( T G F - ( 3 ) w h i c h is found in re la t ive ly h igh concentrat ions in platelets and serum, might be responsible for this i nh ib i to ry ac t iv i ty , at least in part (Solberg et al , 1987; Ishibashi et al , 1987; M i t j a v i l a et a l , 1988). The inhibitory • 3 8 effects o f TGF-(3 are not l imi ted to the M k lineage. Other cytokines wi th apparent M k inh ib i to ry ac t iv i ty inc lude platelet factor-4 (PF4) , some o f the interferons ( IFN) and inter leukin-4 ( IL-4 ) . 2.7.1 T R A N S F O R M I N G G R O W T H F A C T O R - p T r a n s f o r m i n g growth factor-p (TGF - P ) is a family of polypeptide factors known to have mult iple activit ies on many different ce l l types! ' T G F - p exists in at least five isoforms coded for by different genes (Roberts et al , 1990; Sporn et a l , 1990). Differential expression of T G F - P isoforms in different ce l l types has been observed and suggests that the b i o l o g i c a l effects o f the isoforms may differ ( M i l l e r et al , 1989; Jacobsen et al , 1991). Platelets contain the highest k n o w n concentrat ion of T G F - P and are major storage sites of T G F - P in the body (Assoian et al , 1983). T G F - P may be stored in platelets as a 400,000-dalton complex (Pircher et a l , 1986) and then secreted into the serum dur ing c lo t t ing and dur ing th rombin- induced platelet secretion (Childs et a l , 1982; Assoian and Sporn, 1986). T G F - P exists in the serum in a nonactivated form and may be activated in a loca l i zed manner to inhibi t (or stimulate) ce l l population growth (Sporn et a l , 1987). It is a potent inh ib i to r o f co lony formation by p r imi t i ve hematopoiet ic progenitors and has a reduced inhibi tory act ivi ty on more committed progenitors (S ing et a l , 1988; Ottmann and Pelus, 1988; H i n o et a l , 1988; Hampson et a l , 1989), some of which appear to be stimulated by T G F - P (Jacobsen et al , 1991). T G F - P has been ident i f ied as a potent inh ib i to r of M k - c o l o n y growth where it is active at p icomolar concentrations. Ishibashi et a l (1987) have demons t r a t ed that a s i g n i f i c a n t i n h i b i t i o n o f a c e t y l c h o l i n e s t e r a s e a c t i v i t y (a marker o f mur ine M k c y t o p l a s m i c maturat ion) occur red when 3 9 murine bone marrow cel ls were, cul tured in the presence of TGF - .p and that when isolated M k cel ls were cultured in serum-free media in the presence of T G F - P a reduction in the IL -3 induced increase in M k size was noted. Co lony assays demonstrated a , reduction in the number of M k colonies obtained in the presence o f T G F - P (Ishibashi et a l , 1987). Signif icant inh ib i t ion of A c h E act ivi ty was first observed at 2 p m o l / L (50 pg/ml) wh ich is the concentration equivalent to the amount of T G F - p in 5 x l 0 6 platelets (based on data suggesting that 1 ng of the factor can be released from 10^ platelets) ( A s s o i a n and Sporn , 1986). Kutter et al (1992) using a rat bone marrow culture system found that human T G F - p i n h i b i t e d the number of M k produced and i n h i b i t e d endomitos is after 3 days. An t ibod ie s to T G F - P were found to comple te ly abrogate the inhibi tory effect of serum in these cultures. Several groups have demonstrated an inhib i tory effect of T G F - p on human M k progenitor cel ls (Zau l i et a l , 1992; Bruno et a l , 1989; H a n et al ,1989; Solberg et a l , 1987). Berthier et al (1993) also found that a nearly complete i nh ib i t i on of M k colonies occurred when 500 pg /ml ; of T G F - p j was added to serum-free l i qu id cultures containing I L - 3 and I L - 6 and that the poor growth o f M k colonies in p lasma-conta in ing cultures compared to serum-free cultures could be abrogated by the addition of an antibody to T G F - p . The mechanism of action of T G F - p remains poor ly understood. 2.7.2 P L A T E L E T F A C T O R - 4 Platelet factor-4 (PF4) is a Mk/pla te le t - spec i f ic oc-granule pro te in that exists as a tetramer composed of identical 7.8 k D monomers (Deuel et a l , 1977; Hermodson et a l , 1977). W h e n platelets are activated, the protein is 4 0 ex t ruded f rom the oc -g ranu le c o m p l e x e d w i t h a h igh m o l e c u l a r we igh t p ro teog lycan (Barber et a l , 1972). Subsequent ly , the complex is r ap id ly c leared f rom the c i r cu la t ion , perhaps by b ind ing to endothel ia l surfaces and hepatocytes ( R u c i n s k i et a l , 1986). A number of ac t iv i t ies have been attributed to P F 4 . G e w i r t z et al (1989) have demonstrated that P F 4 can i n h i b i t m e g a k a r y o c y t o p o i e s i s w i t h i ts effect b e i n g p r e d o m i n a n t l y to i n h i b i t matura t ion rather than M k progeni tor c e l l p ro l i f e r a t i on . U s i n g synthet ic C O O H - t e r m i n a l P F 4 peptides, this group was able to loca l ize the M k inhib i tory act ivi ty to this domain of the molecule. P F 4 was shown to induce expression of c-myc and c-myb in H E L cel ls , a f inding consistent wi th the hypothesis that P F 4 inhibi ts maturation. (As cells differentiate the expression of c-myc and c-myb usually declines: West in et al , 1982; Lachman and Skoul tch i , 1984; G o w d a et al, 1986). M i t j a v a l a et a l (1988) have also shown that P F 4 i n h i b i t s megakaryocytopoies i s . 2.7.3 I N T E R L E U K I N - 4 Inter leukin-4 ( IL-4) is a 20,000 dalton T c e l l product that has mul t ip l e effects w i th in the i m m u n o l o g i c a l and hematopoiet ic system ( L o n d o n and M c K e a r n , 1990). Sonoda et al (1993) found that recombinant human I L - 4 s t rongly inh ib i t ed pure and mixed M k co lony format ion in a dose-dependent manner. De layed addi t ion experiments suggested that I L - 4 acts on an early stage of prol i ferat ion of M k . progenitors. The inhib i tory effect was also seen i n serum-free cond i t ions us ing C D 3 4 + D R + ce l l s as the target popu l a t i on i nd i ca t i ng that the inh ib i to ry effect on megakaryocytopoies is was l i k e l y due 4 1 to a direct effect of I L - 4 on the progenitor and not mediated by secondary release of cytokines by accessory cel ls . However , a lack of effect of I L - 4 on human megakaryocytopoies i s has also been reported. B r u n o et a l (1989) reported that va ry ing concentrations of I L - 4 d id not affect I L - 3 - or G M - C S F -p r o m o t e d M k c o l o n y fo rma t ion . T h i s group used w h o l e bone mar row mononuclear cel ls as the target populat ion at a re la t ively high density. It is poss ib le that marrow accessory ce l l s elaborate cy tokines that modula te the effects of I L - 4 on M k colony formation. B r i d d e l l and Hoffman (1990) reported that I L - 4 d i d not affect I L - 3 - or G M - C S F - p r o m o t e d B F U - M k - d e r i v e d co lony format ion us ing C D 3 4 + D R - cel ls as the target populat ion. These investigators used a much lower concentration of I L - 4 than Sonoda 's group wh ich may not have been sufficient to observe . an effect. Pesche l et al (1987) have reported that I L - 4 c o u l d s t imulate mur ine M k c o l o n y fo rma t ion , ei ther alone or i n c o m b i n a t i o n w i t h other cy tok ines . Th i s observat ion is different from that observed i n the human system and raises the possibil i ty, that I L - 4 may function differently i n the two s p e c i e s . , 2.7.4 I N T E R F E R O N S Interferons ( I F N s ) are na tu ra l ly o c c u r r i n g , i n d u c i b l e pro te ins that exert several b i o l o g i c a l functions, i n c l u d i n g modula t ion of hematopoiesis (Borden and B a l l , 1981; T r i n c h i e r i and Perussia , 1985; Zoumbos et a l , 1985; P iac ibe l lo et a l , 1985). IFNs have been shown to have inhibi tory effects on the growth of hematopoietic progenitor cel ls f rom normal bone marrow as w e l l as C M L patients (Neumann and Fauser, 1982; Broxmeyer et a l , 1983; Ganser et a l , .1987; W i l l i a m s et al , 1981). Patients treated with I F N - a frequently develop a • 4 2 reduction i n platelet counts suggesting a possible inhib i tory effect of I F N - a on the M k lineage in v i v o . I F N - a has been used as an effective treatment to l ower the platelet count in patients w i t h essent ial th rombocytos i s (Ta lpaz , 1989) . Several groups have demonstrated an inhib i tory effect o f I F N s on m e g a k a r y o c y t o p o i e s i s i n v i t r o . Ganse r et a l (1987) found that human megakaryocytopoies i s was markedly inh ib i t ed by I F N - a and I F N - y and that the i n h i b i t o r y effect o f I F N - a was due to a di rect ac t ion on hematopoie t ic progeni tor ce l l s , whereas the effect of I F N - y was mediated to a s igni f icant degree through accessory ce l l populations. Bo th Ca r lo -S te l l a et a l (1987) and G r i f f i n and Grant (1990) have demonstrated that M k progeni tor ce l l s f rom norma l and mye lopro l i fe ra t ive human bone marrows are equa l ly sensi t ive to I F N s . H i g h e r concentrat ions of I F N - y were required to achieve the same degree of inhib i t ion of M k colonies by comparison to I F N - a . I F N - a and I F N - y were also found to synergize wi th each other to produce inh ib i to ry effects greater than or equivalent to those of 10-100 fo ld higher concentrat ions o f e i ther a lone. 3 T H E S I S O B J E C T I V E S The study of M k progenitor cells in vi t ro has been hindered by the exquisi te sensit ivity of these cells to T G F - p (which is present at inhibi tory concen t ra t ions i n both serum and p lasma) . Because uns ta ined c o l o n i e s conta in ing M k cel ls cannot be re l iably ident i f ied by l ight microscopy alone an alternative means to speci f ica l ly identify these cells is required. A n assay for M k progenitor cells wou ld therefore consist of 3 main components- (a) the use o f cul ture condi t ions op t imized for the prol i fera t ion of M k progeni tor ce l l s , (b) the use of a method to fix the entire culture in situ, and (c) the use of a 43 p r o c e d u r e for the i m m u n o c y t o c h e m i c a l i d e n t i f i c a t i o n o f M k - c o n t a i n i n g c o l o n i e s . A t the time I began my thesis research, previously described M k co lony assay systems had opt imized one or two of these requirements but not a l l three. The objective of my thesis was therefore to see i f it were possible to develop a reproducible assay for human M k progenitors that met a l l three of these r e q u i r e m e n t s and w o u l d the re fore make p o s s i b l e the fu r ther cha rac te r i za t ion o f these ce l l s i n normal i n d i v i d u a l s and in patients w i th per turbed hematopoies i s as w e l l as for studies o f thei r p r o d u c t i o n and d i f fe ren t i a t ion under different cul ture cond i t ions . C H A P T E R II 44 M A T E R I A L S A N D M E T H O D S 1 P R E P A R A T I O N O F C E L L S Fresh human bone marrow aspirate ce l l s were obtained as left over mater ia l f rom healthy ind iv idua l s donat ing bone marrow for a l logene ic bone mar row t ransp lan ta t ion w i t h i n f o r m e d consent . These c e l l s were co l lec ted in preservative-free sterile heparin at a f ina l concentrat ion of 100 -200 U / m l . In some experiments cryopreserved cadaveric bone marrow was used f o l l o w i n g thawing in the presence o f 20% fetal c a l f serum ( F C S ) and DNase (50 |o.g/ml). In both cases, the l ight density ce l l fraction was separated by centrifugation of the cel ls on 1.077 gm/ml F i co l l -Paque (Pharmacia) at 400 g for 30 minutes. Interface cells were then collected, washed twice in Iscove's D M E M (without F C S ) , resuspended in Iscove ' s D M E M and counted us ing a h e m a t o c y t o m e t e r . 2 M E G A K A R Y O C Y T E P R O G E N I T O R C E L L A S S A Y S Megakaryocy te progenitor c e l l assays differ f rom other types of progeni tor c e l l assays because of the culture condi t ions required to promote M k co lony format ion and the necessity for cultures to be f ixed and undergo i m m u n o c y t o c h e m i c a l i d e n t i f i c a t i o n o f c o l o n i e s c o n t a i n i n g M k - l i n e a g e restr icted ce l l s . The method developed dur ing the course of this thesis is descr ibed be low. The experiments to validate the various parameters chosen are presented in Chapter III. 2.1 A G A R O S E C U L T U R E S Test ce l l s were cu l tu red i n an agarose -medium c o n t a i n i n g a serum substitute mod i f i ed s l igh t ly from that o r i g i n a l l y descr ibed for murine erythroid progenitor cells (Iscove et a l , 1980; Lansdorp et a l , 1993). The f ina l p la t ing mixture consisted of 0.3% cel l -cul ture grade agarose (S igma St. L o u i s , M o . ) w i t h 1% d e i o n i z e d bov ine serum a l b u m i n (Stem C e l l T e c h n o l o g i e s , V a n c o u v e r , B C ) , 10 u.g/ml i n s u l i n ( C o l l a b o r a t i v e Resea rch Inc . , W a l t h a m , Mass . ) , 200 u.g/ml iron-saturated transferrin ( I . C . N . B i o m e d i c a l s , O h i o ) , 6.7 u . l /m l l o w densi ty l ipopro te ins ( S i g m a St. L o u i s , M o . ) and 5 x l 0 " 5 M 2-mercap toe thano l ( S i g m a St . L o u i s , M o ) i n I s c o v e ' s m e d i u m . V a r i o u s combinat ions of recombinant human growth factors were added to cultures at the f o l l o w i n g f inal concentrations: 20 ng/ml I L - 3 (Sandoz), 20 ng /ml I L - 6 (Terry Fox Laboratory, Vancouver , B C ) , 14 ng/ml G M - C S F (Sandoz), 50 ng/ml Steel factor (Amgen) , 20 ng/ml G - C S F (Amgen) and 20 ng/ml IL-11 (Genetics Institute). Unless otherwise stated, a l l cultures contained I L - 3 , I L - 6 , G M - C S F and S F at these concentrations. N o r m a l l y 2.7 mis per tube of serum' subst i tute m e d i a was prepared, a l iquoted and then kept frozen pr ior to use. B o n e marrow at an appropriate concentration (to y i e ld <100 M k colonies) in a 0.3 ml volume and 22 u.1 of low density lipoproteins were added to the tube. To this was added 0.3 mis of 3.3% agarose in water wh ich had been l iquef ied by heating and then a l lowed to equilibrate to 3 7 ° C . This mixture was then thoroughly vortexed and the contents plated in 0.75 ml volumes per w e l l of a chamber s l ide (Nunc) . These chamber slides consisted of a sterile 76x26 mm plastic microscope slide onto w h i c h two detachable rubber seals and two plast ic chamber wa l l s were positioned. A l l assays were set up in duplicate or quadruplicate 0.75 m l 46 Megakaryocyte progenitor-cell assay Cells Serum-substitute media Agarose IL-3 IL-6 GM-CSF SF Chamber slide 18 days 37°C 5% C O 2 I F i g . 2 . 1 . S c h e m a t i c d i a g r a m d e p i c t i n g the M k p r o g e n i t o r c e l l assay cul ture sys tem. Hema topo ie t i c ce l l s are cu l tu red i n s e m i - s o l i d agarose cultures conta in ing a serum substitute and other defined components. 47 volumes and then incubated at 3 7 ° C in a humidif ied atmosphere of 5% C 0 2 i n air. 2.2 I M M U N O C Y T O C H E M I C A L I D E N T I F I C A T I O N O F C O L O N I E S C O N T A I N I N G M K C E L L S Cultures were f ixed after 17-20 days incubat ion unless otherwise stated. 1 The wal l s and rubber seal of the chamber were removed wi thout disrupt ing the culture and strips of fi l ter paper placed over the slide . The s l ide and cultures wi th the o v e r l y i n g f i l te r paper were then subjected to centr ifugation at 1500 rpm for 10 minutes in a cytocentrifuge (modif ied from B a i n e s , 1989). F o l l o w i n g this , the s l ide , cul ture and f i l t e r paper was immedia te ly placed in methanol'.acetone (1:3 v/v) for 20 minutes. The f i l ter paper was then removed without disrupt ing the culture and the f ixed culture a l l owed to air dry overnight. Immedia te ly pr ior to s ta in ing, the cultures were rehydrated for 10 minutes in tris buffered saline ( T B S ) at p H 7.6. Nonspec i f i c b ind ing of ant ibody was b locked by further incubat ing the cultures i n 8% human serum i n Hanks so lu t ion wi th 0.0:1% sodium azide for 15 minutes. The p r imary ant ibody ( ' 1 0 E 5 ' mouse monoc lona l I g G 2 a specific for the human glycoprote in I lb / I I Ia complex k ind ly provided by Dr . B . Co l l e r , Stonybrook, N e w Y o r k ) was applied and left for 1 hour at room temperature (Col le r et al," 1986). D u r i n g this t ime add i t iona l ant ibody was intermit tent ly added and the s l ides frequently rocked to facil i tate even appl ica t ion to a l l areas of the. culture. . Endogenous peroxidase ac t iv i ty was b locked by the appl ica t ion of pure methanol for 10 minutes, a solut ion of 1.5% methanol:periodic ac id i n methanol for 5 minutes and then pure methanol again for a f ina l 10 minutes ( C l a r k and Dessypr i s , , 4 8 1 9 8 5 ; D e s s y p r i s , p e r s o n a l c o m m u n i c a t i o n ) . A r a b b i t a n t i - m o u s e IgG:perox idase conjugated antibody was then appl ied for 1 hour, again wi th attention to frequent appl ica t ion of extra antibody and rock ing o f the s l ides . The perox idase substrate ( d i a m i n o - b e n z i d i n e , hydrogen pe rox ide ) was then appl ied for 10 minutes fo l lowed by G i l l ' s H e m a t o x y l i n for 45 seconds. Ten minute T B S washes were performed between each of these steps. S ta ined cultures were air dr ied and scored for the presence o f co lon ies con ta in ing s p e c i f i c a l l y stained orange-brown cel ls express ing GPIIb / I I Ia . W i t h each batch o f s ta in ing , fresh cultures cons i s t ing of outdated platelets (obta ined f rom the Canadian R e d Cross) suspended at a h igh concentrat ion in 0 .3% agarose were stained wi th 1 0 E 5 to serve as a posi t ive cont ro l , and cultures c o n t a i n i n g numerous large C F U - G M - d e r i v e d co lon ies were stained w i t h an i r re levan t i so type spec i f ic con t ro l ( a n t i - T N P , p rov ided by D r . P L a n s d o r p , Ter ry F o x Laboratory, Vancouver) to serve as negative controls. M k colonies were defined as any discrete co l lec t ion of. 3 or more p o s i t i v e l y stained ce l l s . These colonies were further c lass i f i ed accord ing to whether they contained 3-20, 21-49, or >50 posi t ive cel ls (fig.2.2 and f ig .2.3) . W h e n E p was added to the cultures, colonies consis t ing of both posi t ive cells and erythroid cel ls were noted and these scored as biphenotypic E r y t h r o i d - M k colonies (fig.2.4). M o s t of these did not appear to contain either granulocytic or mac rophage c e l l s . L a r g e c o l o n i e s w i t h p r e d o m i n a n t l y nega t ive (nonerythroid) cel ls wi th occasional posi t ive cel ls were scored as being C F U -G E M M and were not included amongst the counts of (pure) M k colonies. 3 . C L O N O G E N I C P R O G E N I T O R C E L L A S S A Y S P r i m i t i v e ery thropoie t ic ( B F U - E ) , g ranulopoie t ic ( C F U - G M ) , and m u l t i - l i n e a g e ( C F U - G E M M ) progen i to r ce l l s were quant i ta ted i n s tandard m e t h y l c e l l u l o s e assays as p r e v i o u s l y desc r ibed ( C o u l o m b e l et a l , 1983) . B r i e f l y , cells were cultured in a 0.8-0.9% methylcel lulose (4000cps) di luted in I scove ' s med ium supplemented wi th 30% F C S , 1 m g / m l of de ion ized bovine se rum a l b u m i n , 10% v /v aga r - s t imu la t ed human l e u k o c y t e c o n d i t i o n e d m e d i u m ( S t e m C e l l T e c h n o l o g i e s , V a n c o u v e r , B C ) , 3 U / m l e r y t h r o p o i e t i n ( S t e m C e l l Technologies ) , and 1 0 " 4 M 2-mercaptoethanol (S igma, St. L o u i s ; M o ) . Cu l tu res were mainta ined at 3 7 ° C i n an atmosphere of 5% carbon d iox ide atmosphere wi th 95% humidi ty . Co lon ies . were scored 18-21 days, after the in i t ia t ion of the cultures. A l l assays . were set up i n duplicate or quadruplicate 1.1 m l volumes. 50 F i g . 2 . 2 . S m a l l M k - c o l o n y i n an agarose cu l tu re c o n t a i n i n g s e rum-substitute medium and I L - 3 , I L - 6 , G M - C S F and S F . The culture was f ixed and M k - c o l o n i e s i d e n t i f i e d f o l l o w i n g i m m u n o c y t o c h e m i s t r y . P o s i t i v e c o l o n i e s conta in ce l l s w h i c h are stained brown ind ica t ing the presence of GPIIb / I I Ia . These c o l o n i e s were u n i f o c a l , appeared ear ly , and were s i m i l a r to those p rev ious ly described as of C F U - M k o r ig in ( L o n g et a l , 1985; B r i d d e l l et a l , 1988a) . F i g . 2 . 3 . Large , mult ic lustered M k - c o l o n y in an agarose culture containing serum-substitute medium, I L - 3 , I L - 6 , G M - C S F and S F . C u l t u r e s were f i x e d and M k - c o l o n i e s iden t i f i ed f o l l o w i n g i m m u n o c y t o c h e m i s t r y . Large M k - c o l o n i e s were mul t i foca l , contained a large number of posi t ive cel ls and appeared late, and were s imi lar to those previously described as of B F U - M k or ig in (Long et a l , 1985; Br idde l l et al , 1988a). 52 F i g . 2 . 4 . B ipheno typ i c e r y t h r o i d - M k co lony in an agarose culture conta in ing serum-substitute medium, I L - 3 , I L - 6 , G M - C S F , S F and E p . Co lon ie s were ident i f ied f o l l o w i n g immunocy tochemis t ry . E r y t h r o i d - M k c o l o n i e s con t a ined m u l t i p l e dense clusters o f e ry th ro id ce l l s w i t h scat tered ce l l s pos i t ive for the presence o f GPIIb/ I I Ia . C H A P T E R III T H E D E V E L O P M E N T O F A N IN V I T R O Q U A N T I T A T I V E A S S A Y F O R H U M A N M E G A K A R Y O C Y T E P R O G E N I T O R C E L L S 1 I N T R O D U C T I O N Progress in the study of human megakaryocyte progeni tor ce l l s has lagged behind that of other hematopoietic lineages by at least ten years because o f unique d i f f i cu l t i e s in d e v e l o p i n g sens i t ive and spec i f i c assays suitable for the routine quantitation of human M k progeni tor ce l l s . These relate to the exquisite sensit ivity of these cells to TGF-fJ wh ich is present in normal serum and plasma at concentrations that are inhib i tory to M k cel ls and to the need for spec i f i c i m m u n o - h i s t o c h e m i c a l procedures to ensure the accurate and sensitive detection of a l l M k colonies (which may contain as v few as 3 M k ) . This latter requirement necessitates the use of a procedure to a l low the entire culture to be f ixed in situ. T o date several approaches have been used for the assay of M k progenitor cel ls , each of wh ich has both advantages and disadvantages (Zau l i et al , 1992; Asso ian et a l , 1983;. Sporn et a l , 1986; Levene et a l , 1987; Vainchenker et a l , 1979; M a z u r et a l , 1981; Bruno et al , 1988; Han et al , 1989; Clark and Dessypris, 1985). M e t h y l c e l l u l o s e , agar and p lasma/ f ibr in c lo t have a l l been used as the suppor t ing matr ix for M k progeni tor c e l l assays. M e t h y l c e l l u l o s e cul tures have the disadvantage that they cannot be f i xed in s i tu . The recogni t ion of M k colonies in such cultures has therefore had to rely on the detect ion of very large mature M k cel l s in each c o l o n y , or requi red the 54 p l u c k i n g o f e v e r y c o l o n y and t r ans fe r to s l i d e s fo r subsequen t i m m u n o c y t o c h e m i c a l . s taining to identify cel ls of the M k lineage (Messner et a l , 1982; K i m u r a et a l , 1984). Such an approach would clearly not be feasible except for very smal l scale experiments. . Assays using agar require that the culture be transferred from the culture dish to a glass sl ide pr ior to f ixa t ion . P l a sma / f ib r in clot cultures have the disadvantage that they w i l l often undergo f i b r i n o l y s i s w i t h i n 14 days mak ing assessment o f co lon ies after 7-10 days di f f icul t on a routine basis. Cul tu re media used to stimulate M k progeni tor c e l l p ro l i fe ra t ion have inc luded normal human plasma, aplastic plasma, a g a r - L C M , P H A - L C M and de f ined serum components w i t h var ious combina t i ons o f spec i f i c g rowth factors. r M e d i a containing either F C S or horse serum have been found to be unsui tab le because o f the i nh ib i t o r y effect o f T G F - P i n such sera on M k progeni tor c e l l g rowth . F o r this reason, p lasma c lo t cu l tures also have potent ia l disadvantages over other semi-so l id matrix systems. Cul tures have been f ixed by dehydrat ion us ing f i l ter paper, or the b l o w i n g of air currents across the surface of the cul ture . A l t h o u g h m u r i n e M k can be i d e n t i f i e d by a s i m p l e c y t o c h e m i c a l r eac t i on for acetylcholinesterase, this enzyme is not expressed in human M k cel ls and the i den t i f i c a t i on o f co lon ies con ta in ing human M k l ineage ce l l s has therefore r equ i red the use o f i m m u n o c y t o c h e m i c a l methods i n v o l v i n g s t a in ing w i t h ant ibodies spec i f ic for the human M k l ineage-spec i f ic g l y c o p r o t e i n antigens l i b , I l i a or lb (Mazur et al, 1981b; Col ler et al, 1986). 2 R E S U L T S The method for assaying human M k progeni tor ce l l s developed 5'5 during the course of this thesis is described in Chapter II. In the development o f this method, opt imal culture condit ions and an opt imal period o f . incubat ion p r i o r to f i x a t i o n and s ta in ing were de termined . P l a t i n g densi ty studies demons t ra ted a l inea r r e l a t i onsh ip between M k c o l o n y y i e l d and the concentration of cells plated. The experimental data are presented be low. 2.1 A B I L I T Y O F A G A R O S E C U L T U R E S T O S U P P O R T H E M A T O P O I E T I C P R O G E N I T O R C E L L D E R I V E D C O L O N Y F O R M A T I O N B e c a u s e m e t h y l c e l l u l o s e cul tures cannot be f i x e d , a d i rec t compar ison of the abi l i ty of agarose and methylcel lulose to support M k colony format ion is not poss ible . A s an alternative approach, the ab i l i ty of 0 .3% agarose to support hematopoie t ic p rogeni to r c e l l - d e r i v e d c o l o n y fo rma t ion was assessed by compar ing granulopoiet ic and erythroid co lony format ion in 0.3% agarose and 0.8% methylce l lu lose cultures conta in ing otherwise iden t ica l m e d i u m components . M e t h y l c e l l u l o s e cultures were establ ished i n 35 mm petri dishes (Greiner) . Agarose cultures were established in both 35 mm petri dishes and in chamber slides (Nunc) ; C F U - G M - d e r i v e d colony numbers (both 20-500 and >500 cel ls /colony) were similar in a l l cultures (Table 3.1). The total number o f e ry thro id colonies was also s imi l a r in a l l cul tures; however , the agarose cul tures fa i l ed to support the p ro l i f e r a t ion o f large B F U - E (that produce ery thro id colonies conta ining > 9 clusters o f erythroblasts) regardless of the culture vessel . This has been noted previously for erythroid colonies cul tured in agar (C . Eaves, personal communicat ion) . 56 i i 60 o c 3 03 a 2 2 3 3 O E « o3 3 -3 m .2 c l i O 03 43 1 - 1 S o S l l l l 03 o 60 Hi 03 <~ 3 03 0) (U Ui 4 1 T3 3 ^ ' ° O 03 3 3 M g ° O ii C3 O o 41 03 B fa w w pa -—-fa • v© 03 -—• fa ^ pa •—-fa ^ H 43 in (N. CN O oo o O o r-4 (N O (N VO <n oo VO 00 00 in oo i—i 00 ON m i 1 T-t c/3 8 "2 43 —| o 5 43 8 "2 43 - i O u (i <u " c o a o "e * js 3 i_ 3 ^ c: 4 1 03 03 a « <D m tH)in 6043 o T3 <u - <o u £ S 2 6 2 E 6 .2 c3 T 3 60 3 .5 O T 3 EX U Oi * 2 ° 43 4£ O hi 3 ^ i O M 3 O 3 u O (U 3 4 1 <u «, O 3 ' O l l t « 43 i <U •a in O 03 E 3 -4 1 "3 3 CJ 43 S - E ' 3 3 in CO u <D 4 = 4 1 60 3 3 "* -a •a S =3-S O o £ a, 4 1 O 1-5 -rj ° 3 O CI, O 1-1 o | §•8 o3 6 u <^  n O a) ^ ^ t/3 >, t-3 S 1 1 «1 ill 03 0) S3 >i ^5 3 £ | -T3 O 3 r l 03 IT) -03 C3 O T3 u n 3 E 3 ° c « 4—* 2 2 ^ (D <l> S 03 o3 O o u. i i 03 E o I l l 03 60 03 S 2 2 © 43 -3 o j3 43 O 1-t3 S i i o o <u O 43 03 *-' 3 S O u DH (U 60 3 •a .S 3 - '3 43 4 1 60 3 j 2 u * 3 o 4 3 T 3 9j ' IX, 3 03 > 2.2 I N H I B I T O R Y E F F E C T O F S E R U M A N D P L A S M A -C O N T A I N I N G M E D I U M 57 The effects of different types of plasma (30%), serum (30%) and a serum subst i tute on M k c o l o n y fo rma t ion i n o therwise s i m i l a r m e d i u m (containing I L - 3 , 20 ng/ml ; I L - 6 , 20 ng/ml; G M . C S F , 14 ng /ml ; S F , 50 ng/ml ; 1% B S A and 5 x l 0 " 5 M 2-mercaptoethanol in Iscove's D M E M ) were studied ( F i g 3.1). C u l t u r e s c o n t a i n i n g 30% F C S had reduced numbers o f M k c o l o n i e s in c o m p a r i s o n to cultures con ta in ing either normal or aplast ic p l a sma or the serum substitute. M a x i m a l numbers of M k colonies were a lways obtained w i t h the serum substitute. Cul tures con ta in ing p lasma f rom patients w i th aplast ic anemia were unique in that they contained numerous pure e ry thro id , m i x e d e r y t h r o i d - M k and m i x e d e r y t h r o i d - M k - g r a n u l o p o i e t i c co lon ies i n spite of the fact that no exogenous erythropoietin was added. This is consistent wi th the expec ta t ion that c i r c u l a t i n g e ry th ropo ie t in leve ls are e levated in such patients (Cotes, 1982). 2.3 O P T I M A L C O N C E N T R A T I O N S O F 2 - M E R C A P T O E T H A N O L , A L B U M I N A N D L O W D E N S I T Y L I P O P R O T E I N S The opt imal concentration of each of several o f the components o f the serum substitute was determined by independent ly manipu la t ing each and then assessing the effect on M k colony yields (Figs. 3.2-4). Fo r each dose response study, a l l components of the serum substitute cock ta i l except for the componen t be ing s tudied were ma in ta ined constant at the concen t ra t ions given in Chapter II. O p t i m a l concentrations of 2-mercaptoethanol and a lbumin f e l l 58 120 100 H 80 H 60 H 40 20 A p PI Ser Sub FCS N PI F i g . 3.1. The effect o f fetal c a l f serum (30%), no rma l p l a sma (30%), aplastic p lasma (30%) and a serum substitute (see Mater ia l s and Methods) on M k colony formation in cultures containing I L - 3 , I L - 6 , G M - C S F and S F . E a c h bar indicates the mean plus 1 S E M of the average count from assays of each of 3 different bone marrows assessed in replicate. Cultures contained 1 0 5 l i gh t densi ty mononuclear bone marrow cel l s and were f i x e d , s ta ined and scored after 18-20 days. The results for each set of cultures are expressed as a percent of the max imum number of colonies obtained wi th each bone marrow. W i t h a l l of the bone marrows the maximum number of M k colonies occurred in the serum substitute medium. Cul tures conta in ing aplast ic p lasma contained numerous e ry th ro id and G M - c o l o n i e s . 59 1 0 " 7 1 0 " 6 1 0 " 5 1 0 " 4 1 0 ~ 3 Concen t ra t ion of 2-mercaptoethanol ( M ) -F ig .3 .2 . Ef fec t o f increas ing concentrat ions o f 2-mercaptoethanol on M k c o l o n y f o r m a t i o n . The number of M k colonies is expressed as a percent of the max imum number o f co lon ies observed wi th increas ing concentrat ions o f 2-mercaptoethanol i n each experiment . E a c h point represents the mean +/- 1 S E M of the average count from assays of each of 3 different bone marrows assessed in replicate. 60 100 0 0 .5 1.0 1.5 2 . 0 3 .0 A l b u m i n concen t r a t ion ( g m / l O O m l ) F i g . 3 . 3 Effec t of increas ing concentrat ions of a l b u m i n on M k c o l o n y f o r m a t i o n . The number of M k colonies is expressed as a percentage of the m a x i m u m number o f co lon ies observed wi th inc reas ing concentra t ions o f a l b u m i n in each experiment . E a c h point represents the mean +/- 1 S E M of the average count from assays of each of 3 different bone marrows assessed in replicate. C u l t u r e s con ta ined 1 0 5 l ight density mononuclear bone marrow ce l l s . A l l cultures contained I L - 3 , I L - 6 , G M - C S F and S F and were f ixed and stained after 18-20 days of culture. 61 120 20 H—1—i—1—i—1—i—1—i—'—i—1—i—•—i—i—i—•—i—•—i—i—I 0 20 40 60 80 100 120 140 160 180 200 Concentrat ion of L D L ( m m o l / L ) F i g . 3 . 4 Ef fec t o f increas ing concentrat ions o f l o w densi ty l ipopro te ins ( L D L ) on M k colony formation. The number of M k colonies is expressed as a percentage o f the m a x i m u m number o f co lon ies observed wi th increas ing concentrat ions of l ow densi ty l i popro te ins i n each exper iment . E a c h point represents the mean o f the counts obtained f rom 2 replicates for each of 3 different bone marrows (shown i n d i v i d u a l l y by different symbols ) . 62 be tween 5 x l O ~ 5 M and 1 0 " 5 M and 0.5-2% respect ive ly . H i g h e r and l ower concentra t ions were less support ive . S i m i l a r l y M k co lony y ie lds remained m a x i m a l in cultures containing between 20 and 80 m M L D L with an inh ib i tory effect seen with higher concentrations of L D L . For 2 of 3 B M samples tested m a x i m a l M k colony numbers were also obtained in the absence o f any added L D L . 2.4 O P T I M A L G R O W T H F A C T O R C O M B I N A T I O N S M u l t i p l e c o m b i n a t i o n s o f r ecombinan t human g r o w t h factors were studied for their abi l i ty to support opt imal M k colony formation (F ig 3.5a-3.5d): I L - 3 (20 ng/ml) , I L - 6 (20 ng/ml) , IL-11 (20 ng/ml) , G M - C S F (14 ng/ml) , G - C S F (20 ng/ml) , and S F (50 ng/ml) . M a x i m a l s t imulat ion o f M k colony formation was obtained with the combination of I L - 3 , I L - 6 , G M - C S F and S F . The addit ion of E p (3 U / m l ) to agarose cultures conta ining I L - 3 , I L - 6 , G M - C S F and S F resulted in a reduct ion of the number of large (>50 M k / c o l o n y ) and intermediate s ized (21-49 M k / c o l o n y ) M k co lon ies and the appearance instead of b ipheno typ ic e r y t h r o i d - M k co lon ies . The number o f e r y t h r o i d - M k colonies that formed in the presence of E p was equivalent to the reduct ion in the number of large and intermediate pure M k colonies seen in the absence of E p ( F i g 3.6), suggesting that these originate from the same p r e c u r s o r s . 2.6 P L A T I N G D E N S I T Y S T U D I E S The effect of varying the concentration of light density B M cells 63 IL-3 , IL-6 , G M - C S F , SF IL-3 , IL-6 , IL-11* , G M - C S F , S F IL-3 , IL-11, G M - C S F , SF IL-3 , IL-6, SF IL-3 , SF IL-3 , IL-6 , IL-11, SF IL-3 , G M - C S F , S F IL-6, IL-11, G - C S F , G M - C S F , SF IL-3 , IL-6 , IL-11, G M - C S F EL-3, IL-6 , IL-11, G M - C S F , SF IL-3 , IL-6, G M - C S F IL-3 , IL-6, IL-11 IL-3 IL-3 , IL-6 IL-6, SF IL-3 , G M - C S F IL-3 , IL-11, G M - C S F IL-3 , IL-11 SF G M - C S F IL-6 " N i l 120 % max imum M k colonies F i g . 3.5a. Ef fec t o f different growth factor combina t ions on M k c o l o n y p roduc t ion i n agarose cultures conta in ing a serum substitute. E a c h culture c o n t a i n e d 10^ l igh t density mononuclear ce l l s and was f i x e d and scored f o l l o w i n g immunocy tochemis t ry after 18-20 days. E a c h bar represents the mean of the average count (obtained from replicate cul tures) f rom 2 different bone marrows. The results are expressed as a percent of the max imum number of M k colonies wh ich was obtained for both bone marrows in cultures containing I L - 3 , I L - 6 , G M - C S F and S F . G r o w t h factors were used at the fo l l owing concentrations: I L - 3 , 20 ng /ml ; I L -6, 20 ng /ml ; I L - 1 1 , 20 ng/ml (except I L - 1 1 * which was 100 ng/ml); G M - C S F , 14 ng/ml ; G - C S F , 20 ng/ml; and SF , 50 ng/ml. 64 IL-3 , IL-6, G M - C S F , SF IL-3 , IL-6 , IL-11*, G M - C S F , S F IL-3 , IL-11, G M - C S F , SF IL-3 , IL-6 , SF IL-3 , SF IL-3 , IL-6 , IL-11, SF IL-3 , G M - C S F , SF IL-6, IL-11, G - C S F , G M - C S F , SF IL-3 , IL-6 , IL-11, G M - C S F IL-3 , IL-6 , IL-11, G M - C S F , SF IL-3 , IL-6, G M - C S F IL-3 , IL-6, IL-11 IL-3 IL-3 , IL-6 IL-6, SF IL-3 , G M - C S F IL-3 , IL-11, G M - C S F IL-3 , IL-11 SF G M - C S F IL-6 N i l 0 10 —r~ 20 —i— 30 40 —i— 50 60 % maximum M k colonies F i g . 3.5b. Resul ts from F i g . 3 . 5 a . subcategorized accord ing to co lony s ize . E a c h bar represents the mean of the average count of M k colonies wh ich were 3-20 M k c e l l s / c o l o n y i n s ize (obta ined f rom rep l ica te cu l tures) f rom 2 different bone marrows. 65 EL-3, IL-6 , G M - C S F , SF IL-3 , IL-6 , IL-11*, G M - C S F , S F IL-3 , IL-11, G M - C S F , SF IL-3 , IL-6, SF IL-3 , SF IL-3 , IL-6 , IL-11, SF IL-3 , G M - C S F , SF IL-6 , IL-11 , G - C S F , G M - C S F , SF IL-3 , IL-6 , IL-11, G M - C S F IL-3 , IL-6 , IL-11, G M - C S F , SF IL-3 , IL-6, G M - C S F IL-3 , IL-6, IL-11 IL-3 IL-3 , IL-6 IL-6, SF IL-3 , G M - C S F IL-3 , IL-11, G M - C S F IL-3 , IL-11 SF G M - C S F IL-6 N i l - i — 10 —i— 20 30 % maximum M k colonies F i g . 3.5c. Resul ts f rom F i g . 3 . 5 a . subcategorized accord ing to c o l o n y s ize . E a c h bar represents the mean of the average count of M k colonies wh ich were 21-49 M k c e l l s / c o l o n y i n s ize (obtained f rom repl ica te cul tures) f rom 2 different bone marrows. 66 IL-3 , IL-6, G M - C S F , SF IL-3 , IL-6, IL-11*, G M - C S F , S F DL-3, IL-11, G M - C S F , SF IL-3 , IL-6 , S F IL-3 , S F IL-3 , IL-6 , IL-11, SF IL-3 , G M - C S F , SF IL-6 , IL-11, G - C S F , G M - C S F , SF IL-3 , IL-6 , IL-11, G M - C S F IL-3 , IL-6, IL-11, G M - C S F , SF IL-3 , IL-6, G M - C S F IL-3 , IL-6, IL-11 IL-3 IL-3 , IL-6 IL-6, SF IL-3 , G M - C S F IL-3 , IL-11, G M - C S F IL-3 , IL-11 S F G M - C S F IL-6 N i l mm* mmmm mm — i • 1 • — 10 20 % max imum M k colonies 30 F i g . 3.5d. Resul ts f rom F i g . 3 . 5 a . subcategorized accord ing to co lony s ize. Each bar represents the mean of the average count of M k colonies wh ich were > 50 M k ce l l s /colony in size (obtained from replicate cultures) from 2 different bone marrows. 67 120 3-20 Mk/colony • 21-49 Mk/colony 0 >50 Mk/colony Total pure M k colonies ^ M i x e d erythroid/Mk colonies Wi thout E p W i t h Ep F i g . 3.6. Effect of the addit ion of erythropoietin on M k colony product ion in agarose cultures containing a serum substitute, I L - 3 , I L - 6 , G M - C S F and S F (at 20, 20, 14 and 50 ng/ml respectively). Each culture contained 1 0 5 l ight density mononuclear ce l l s and was f ixed and scored f o l l o w i n g immunocy tochemis t ry after 18-20 days. Cultures were established both with and without E p 3 U / m l . E a c h bar represents the mean o f results f rom 2 different bone marrows assayed i n duplicate. The results are expressed as a percent o f the max imum number of M k colonies obtained from each bone marrow. M k colonies were subclassif ied as either being pure M k colonies containing either 3-20, 21-49 or >50 M k / c o l o n y or as being mixed with both erythroid clusters and M k present w i t h i n the co lony . 68 1000 q 1 - j 1 — i — i i 1 1 1 1 1 1 — i — i i 1 1 1 1 1 1 — i — i | 1 0 3 1 0 4 1 0 5 1 0 6 Number of l ight density bone marrow cells plated F i g . 3.7. P l a t i n g density study of mononuclear ce l l s i n agarose cultures containing a serum substitute and I L - 3 , I L - 6 , G M - C S F and S F (at 20, 20, 14 and 50 ng /ml respect ively) . Va r i ab l e numbers of low density mononuclear cel ls were p la ted i n 0.75 m l agarose cul tures and M k c o l o n i e s scored f o l l o w i n g i m m u n o c y t o c h e m i s t r y after 18-20 days incuba t ion . Data points and bars represent means +/- 1 S E M of from 3-16 replicate cultures. The two different bone marrows are indicated as B M #1 and B M #2. f o r m a t i o n . 69 present i n the assay cultures and the number of M k colonies formed was also studied. A t a l l ce l l plating densities, except for the highest density for B M #1, a l inear relat ionship was observed between these 2 parameters (F ig . 3.7). W i t h the h ighes t c e l l dens i t y for B M # 1 , numerous g r a n u l o c y t e - m a c r o p h a g e colonies were seen and these appeared to have had an inhib i tory effect on M k c o l o n y f o r m a t i o n . 2.5 T I M E C O U R S E S T U D I E S The time course of M k colony formation was assessed by f i x i n g and scor ing M k colonies after varying periods of incubation (F ig . 3. 8). S m a l l co lon ies cons i s t i ng mos t ly of 3-20 pos i t ive ce l l s in a loose c lus ter were observed as early as 6-9 days after in i t i a t ion o f the cultures. M a x i m u m numbers of M k colonies were noted between days 21 and 30. A t this t ime, large M k colonies consisting of >50 (sometimes up to 200) cells in mult iple foci were present at max imum numbers. The morphology of the M k wi thin colonies was also noted to vary marked ly over t ime, ranging from smal l round mononuclear ce l l s to very large p o l y p l o i d , mu l t i nuc l ea t ed ce l l s w i t h the appearance o f c y t o p l a s m i c shedding of platelet fields. The presence of the latter may be dependent on the i n c l u s i o n of C S F s i n the culture medium that promote M k maturation. (The growth factors that were used were chosen p r i m a r i l y for their p ro l i fe ra t ive effect on M k progenitor cel ls .) F r o m day 16 onwards , co lon ies con t a in ing non-nuc lea ted but pos i t ive ly stained cells were noted. Such colonies were not seen pr ior to this t ime , a l though pos i t ive ce l l s con ta in ing p y k n o t i c nuc l e i were seen ear l ie r pr imar i ly in smal l sized colonies. Once apparent, the size and frequency of 70 100 F i g . 3.8. T i m e course study of the development of M k colonies i n agarose cultures conta ining I L - 3 , I L - 6 , G M - C S F , and S F (at 20, 20, 14 and 50 ng/ml respec t ive ly) . Cul tures contained 1 0 5 l ight density mononuclear bone marrow cel ls and were performed in at least duplicate. Cul tures were f ixed and M k c o l o n i e s s co red f o l l o w i n g i m m u n o c y t o c h e m i s t r y at va r i ous t imes after in i t i a t ion o f the cultures. E a c h bar represents the mean f rom a representative bone marrow and is subcategorized according to M k colony size as shown. 7 1 c o l o n i e s c o n t a i n i n g n o n - n u c l e a t e d p o s i t i v e l y s t a i n e d c e l l s i n c r e a s e d continuously and by day 40 most colonies were of this type. 3 DISCUSSION A N D C O N C L U S I O N S Megakaryocy te progenitor cel ls in normal human bone marrow samples were detected us ing a tissue cul ture grade agarose-based m e d i u m con t a in ing a def ined serum substitute supplemented wi th a c o m b i n a t i o n o f g rowth factors and an immunope rox idase technique for the spec i f i c and sens i t ive i n situ iden t i f i ca t ion of a l l co lon ies con ta in ing GPI Ib / I I I a -pos i t i ve c e l l s . The method descr ibed offers several advantages over e x i s t i n g techniques. The use of defined culture conditions avoids the effects of T G F - P and other inhibi tory substances found in plasma and serum. A l s o , the assay is not rel iant on specif ic batches of plasma, serum or condi t ioned media and a l l o w s the effects o f speci f ic cy tok ines to be s tudied i n the absence of undefined st imulatory or inh ib i tory substances found in such reagents. The components o f the serum substitute used were taken f rom the fo rmula t ion d e r i v e d o r i g i n a l l y by Iscove and co l leagues (1980) for mur ine e r y t h r o i d progenitors as modif ied by Dain iak et al (1985). They, are easily prepared and readily stored at - 2 0 ° C for long periods without loss of activity . The use of an agarose preparat ion that gels at a low temperature enables the add i t ion of l iquef ied agarose to the medium conta ining the cel ls without heat damage to the latter. The establ ishment of cultures i n chamber sl ides was found to great ly fac i l i t a t e the process of f i x i n g the cul tures s ince this made i t unnecessary to transfer them to slides as required for cultures set up in petri 72 dishes. The use of the centrifugation method of Baines (1989) also s impl i f i ed the f ixa t ion process. The immunoperoxidase staining technique resulted i n a permanent ly stained cul ture w h i c h c o u l d then be stored and re t r ieved for repeated r ev iew as desi red. S ince the c o m p l e t i o n o f this thesis further deve lopmen t o f the method has been undertaken and p r e l i m i n a r y studies suggest that the use o f an a lka l ine phosphatase an t i - a lka l ine phosphatase ( A P A A P ) detect ion system gives super ior results to the peroxidase method because it avoids the problems of endogenous background peroxidase i n the c e l l preparat ion. In addi t ion, it avoids the risks associated wi th the use of benzidene wh ich is a potential carcinogen. M k colony formation was found to be markedly reduced in serum-con t a in ing cultures as compared to cultures con ta in ing a serum substi tute. Th is effect was attributed to the inhibi tory effect of T G F - P present i n serum as described by others (Berthier et a l , 1993). The serum substitute also appeared to be more effective than normal p lasma or even plasma from patients wi th aplastic anemia suggesting that even these plasmas may contain some T G F - p . M o r e o v e r the use of aplastic plasma resulted in a marked overgrowth of non-M k colonies wh ich may have contributed to an inhibi tory effect on M k colony number. Dose response studies indicated that opt imal M k colony growth could be obtained wi th 0.5-2% a lbumin and 1 0 ' ^ M to 5 x l 0 " ^ M 2 - m e r c a p t o e t h a n o l . The addit ion of L D L up to concentrations of 80 m M did not enhance M k colony fo rma t ion over that obta ined i n the absence o f added L D L i n 2 o f 3 e x p e r i m e n t s . O f the growth factor combinations tested, the combinat ion of I L -3, I L - 6 , G M - C S F and S F (at 20, 20, 14 and 50 ng/ml , respectively) produced m a x i m a l s t imulat ion of M k progenitor cel ls . Th is f inding is consistent wi th previous reports ind ic t ing that these cytokines have st imulatory effects on M k progenitor ce l ls . B o t h IL -3 and G M - C S F have been shown to have st imulatory . 73 ac t iv i ty on M k progenitor cel ls wi th I L - 3 appearing to have a greater abi l i ty than G M - C S F to promote the formation of M k colonies (Bruno et a l , 1988; Quesenberry et a l , 1985; Lopez et a l , 1987; MizogUch i et al , 1986; Teramura et a l , 1988; Messner et a l , 1987). The effects of I L - 3 and G M - C S F have been reported to be additive (Robinson et al , 1987; M c N i e c e et a l , 1988; Donahue et a l , 1988) and an I L - 3 / G M - C S F fusion protein has been shown to stimulate B F U -M k - d e r i v e d colonies (but not C F U - M k - d e r i v e d colonies) to the same extent as the addit ion of opt imal concentrations of both IL -3 and G M - C S F added together (Bruno et a l , 1992). IL -3 has also been shown to have a direct s t imulatory effect on M k progenitor cel ls without any requirement for addi t iona l factors produced by adherent ce l ls , T ce l ls , or present in plasma or serum (Teramura et a l , 1988). IL -3 has also been shown to have a direct effect on single murine M k r e s u l t i n g i n changes assoc ia ted w i t h M k c y t o p l a s m i c and nuc lea r maturation (Ishibashi and Burs te in , 1986). Interleukin-6 is be l ieved to play a centra l role in hematopoiesis by ac t iva t ing quiescent stem ce l l s into c y c l e , a l l owing them to become responsive to IL-3 and G M - C S F (Ikebuchi et a l , 1987). I L - 6 has also been shown to potentiate the action of I L - 3 to st imulate M k progeni tor cel ls direct ly (Ishibashi et a l , 1989a; W i l l i a m s et a l , 1990). S F synergizes wi th IL -3 and G M - C S F to increase the number of M k progenitor ce l l -de r ived colonies and the number of M k cel ls per co lony (Tanaka et a l , 1992; Br idde l l et al , 1991). The addi t ion o f IL -11 and G - C S F d id not further increase the number of M k colonies in this study. IL -11 has p rev ious ly been shown to act on ly synerg i s t i ca l ly wi th subopt imal concentrat ions o f I L - 3 to s t imulate h u m a n B F U - M k (but not C F U - M k ) and i n the presence o f o p t i m a l concentrations of I L - 3 , no effect of added IL-11 was seen (Bruno et a l , 1991). A l t h o u g h G - C S F has previous ly been shown to augment M k co lony format ion by I L - 3 ( M c N i e c e et a l , 1988; Bruno et a l , 1988), such an effect may not have 74 been demonstrable in this study due to the presence of opt imal concentrations o f other growth factors. Previous studies have g iven conf l ic t ing results as to the effect of E p on megaka ryocy topo i e s i s in v i t r o . Some showed that E p p romoted m e g a k a r y o c y t o p o i e s i s whereas others found no ev idence o f a response (Berridge et al , 1988; Semenza et al , 1989; M c D o n a l d et a l , 1987; Eschbach et a l , 1989; Stone et a l , 1988; Longmore et al , 1993; M a z u r et a l , 1981b; Yakahashi et al , 1991; Z a u l i et a l , 1992). One explanation for the inconsistency of these studies might be due to contaminat ion of the par t ia l ly pur i f ied E p preparations used wi th other cytokines (Hoffman, 1989). Some of these studies also required the presence o f serum or subopt imal concentrations of P H A - L C M to demonstrate an effect of E p on megakaryocytopoies is suggesting ..synergism wi th another factor (Tsukada et a l , 1992; Dessypris et al , 1987). In the present study, E p did not increase the number of M k colonies obtained al though m i x e d e ry thro id-M k co lon ies .were demonstrated i n the presence o f E p suggest ing that a biphenotypic progenitor ce l l restricted to these two lineages may exist. F r o m this study it w o u l d appear that 20-30% of M k progeni tor ce l l s have this capab i l i t y and require the presence o f E p for express ion o f their . e ry th ro id dif ferent ia t ion, potential . The presence, of such a b iphenotypic progenitor c e l l was recently hypothesized by M c D o n a l d and S u l l i v a n (1993). P l a t ing density studies demonstrated l inear i ty consistent w i t h a single c e l l o r ig in of each colony. The optimal time of f ixation of cultures was day 21 . A t this t ime M k c o l o n y numbers were m a x i m a l i n c l u d i n g , i n par t icular , the largest M k colonies (>50 ce l l s / co lony ) . The m o r p h o l o g i c a l appearance of pure M k colonies showed marked heterogeneity w i th a range in appearance from very smal l un i foca l colonies con ta in ing on ly 3 pos i t ive ce l l s to large spreading mul t i foca l colonies conta in ing > 200. pos i t ive ce l l s . T i m e course studies of the appearance of M k colonies of various sizes showed 75 that sma l l colonies appeared first (by day 6) and achieved m a x i m a l numbers sooner (by day 16) than the largest M k colonies which appeared later (day 12) and reached m a x i m a l numbers later (day 21) cons is ten t w i t h p r ev ious descriptions of B F U - M k and C F U - M k (Long et al , 1985; B r i d d e l l et al , 1988a). B F U - M k and C F T J - M k have also been found to differ in other properties that a l l ow their i so la t ion from one another and are consistent wi th their postulated sequential developmental relat ionship (Hodgson and Brad l ey , 1979; V a n Zant, 1989; L o n g et a l , 1985; Br idde l l et al , 1988b; Hoffman et al , 1987). Nevertheless, d i s t inc t classes of progeni tor ce l l s have not been r i g i d l y def ined, and M k progeni tor ce l l s are l i k e l y to compr ise a con t inuum of ce l l s of decreas ing p ro l i f e ra t ive potent ia l . F o r this reason we have a rb i t ra r i ly c l a s s i f i e d M k colonies as smal l (3-20 posi t ive ce l l s ) , intermediate (21-49 pos i t ive ce l l s ) or large (> 50 pos i t ive ce l l s ) . The f ind ing of colonies con ta in ing anucleate pos i t ive cel ls that increased i n number and size from day 16 onwards w o u l d suggest that these represent colonies of M k that were undergoing apoptosis. The method descr ibed in this thesis for the detect ion o f M k progenitor cel ls should prove useful for the future routine assay of these cel ls . The l ack o f a requirement for undefined reagents (eg, spec i f ic batches o f serum or plasma) wou ld suggest that the method could be easi ly established i n any c e l l culture laboratory. Because the assay avoids the inhibi tory effects of serum and contains defined culture components, it should also be an opt imal s y s t e m for s t u d y i n g the effects o f c y t o k i n e s whose i n f l u e n c e on m e g a k a r y o c y t o p o i e s i s has not yet been cha rac t e r i s ed (eg, the r ecen t ly descr ibed T P O wh ich has been shown to stimulate megakaryocytopoies i s , but whose effects on human M k progeni to r ce l l s have not yet been f u l l y described). 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Y o u n g K M , W e i s s L : M e g a k a r y o c y t o p o i e s i s : I n c o r p o r a t i o n o f t r i t i a t ed t h y m i d i n e by s m a l l a c e t y l c h o l i n e s t e r a s e - p o s i t i v e c e l l s i n m u r i n e bone marrow during ant ibody-induced thrombocytopenia. B l o o d 69: 290, 1987. Z a u l i G , V i t a l e L , B r u n e l l i M A , Bagnara G P : Prevalence of the p r imi t ive megakaryocyt ic progenitors ( B F U - M K ) i n adult human per ipheral b lood . E x p Hematol 20: 850, 1992. Zi lbers te in A , Ruggier i R , K o r n J H , Reve l M : Structure and expression of c D N A and genes for human in ter feron-beta-2 , a d i s t inc t species i n d u c i b l e by growth-stimulatory cytokines. E M B O J 5: 2529, 1986. Zoumbos N C , Gascon P , Djeu J Y , Y o u n g N S : Interferon is a mediator of hematopoet ic suppression i n aplast ic anemia i n v i t ro and poss ib ly in v i v o . Proc Nat l A c a d Sc i U S A 82: 188, 1985. Zsebo K M , W y p c h J , M c N i e c e I K , L u H S , Smi th K A , Karkare S B , Sachdev R K , Yuschenkof f V N , Birket t N C , W i l l i a m s L R , S.atyagel V N , Tung W , Bosse lman R A , M e n d i a z E A , L a n g l e y K E : I d e n t i f i c a t i o n , p u r i f i c a t i o n and b i o l o g i c a l cha rac te r i za t ion o f hematopoie t ic stem c e l l factor f rom buffa lo rat l i v e r -conditioned medium. C e l l 63: 195, 1990. Zsebo K M , W i l l i a m s D A , Geissler E N , Broudy V C , Mar t in F H , Atk ins H L , Hsu R Y , Birket t N C , Okino K H , Murdock D C , Jacobsen F W , Langley K E , Smith K A , Takeishi T, Cattanach G B M , G a l l i SJ , Suggs S V : Stem ce l l factor is encoded at the SI locus of the mouse and is the l igand for the c-ki t tyrosine kinase receptor. C e l l 63: 213, 1990. Z a u l i G , V i t a l e L , B r u n e l l i M A , Bagnara G P : Prevalence of the p r imi t ive megakaryocyte progenitors ( B F U - m e g ) i n adult human per ipheral b lood . E x p Hematol 20: 850, 1992. ' . Z u c k e r - F r a n k l i n D , Stahl C , Hyde P : An t igen i c d i s s imi la r i ty between platelet and megakaryocyte surface membranes. In, L e v i n e R F , W i l l i a m s N , L e v i n J 1 0 5 (eds): Megakaryocyte development and function, pp259, 1985. Z u c k e r - F r a n k l i n D : M o r p h o l o g y of megakaryocytes and platelets. In, W i l l i a m s W J , Beutler E , Ers lev A J , L ich tman M A (eds): Hematology (edition 4). M c G r a w H i l l , N e w Y o r k , p p l l 6 1 , 1990. A P P E N D I X 106 M E G A K A R Y O C Y T E P R O G E N I T O R C E L L A S S A Y i P R E P A R A T I O N O F 3.3% A G A R O S E IN W A T E R M e t h o d 1. A d d 1.65 gms of Agarose powder (Type V i l a low gel l ing temperature, tissue culture grade, Sigma, Cat N o #A-9045) to a clean 100 ml glass b o t t l e . 2. Au toc l ave bottle containing agarose powder and leave in d ry ing cab ine t o v e r n i g h t . 3. A d d 50 mis of ,s ter i le tissue culture grade water to the bottle in a H E P A filtered hood and heat to dissolve the agarose (either on a hot plate or in a microwave oven), do not a l low bo i l ing to occur for longer than a few seconds. 4. A l i q u o t the hot agarose solution into 1.3-1.5 m l volumes and cap t ight ly. These may be stored indefinitely at room temperature although the agarose may start to dry out after a few weeks i f the tubes are not sealed tightly in which case the tubes should be d i s c a r d e d . i i P R E P A R A T I O N O F M E D I A F O R D I L U T I O N O F T H E A G A R O S E C U L T U R E S a M a t e r i a l s 1. Iscove's D M E M media, (Stem C e l l Technologies, Inc.) 2. 2 X Iscove's D M E M media, (Stem C e l l Technologies,Inc.) 3. 1 0 " 2 M 2-mercaptoethanol in water, (Sigma, Cat N o #M7522) 4. 10% bovine serum albumin ( B S A ) in Iscove's D M E M (Stem C e l l Technologies, Inc.) Pr ior to use add 5 mis of 7% sodium bicarbonate in water to 40 mis B S A 10%). 5. Insul in l m g / m l , (Bovine insul in , Cat N o #40205 from Col labora t ive Research Inc., Wal tham, Mass . , 20 mg/bottle). 107 6. Transferr in 20 mg/ml (human transferrin- i ron saturated, Ca t No#823431 from I . C . N . , lgm/bot t le ) 7. Human Steel Factor 8. H u m a n In ter leukin-3 9. H u m a n In ter leukin-6 10. H u m a n G r a n u l o c y t e - m a c r o p h a g e c o l o n y - s t i m u l a t i n g factor 11. L o w density l ipoproteins from human plasma, 5 mg/bottle, (from Sigma, Cat N o #L2139) 12. Agarose 3.3% in water, prepared as above. b M e t h o d F i n a l concentra t ions are: 2- M e r c a p t o e t h a n o l 5 x 1 0 " 5 M Iscove's D M E M I X B o v i n e serum a lbumin 10 m g / m l I n s u l i n 10. u .g /ml T r a n s f e r r i n 200 u .g /ml P e n i c i l l i n / s t r e p t o m y c i n I X Steel Factor 50 ng /ml I L - 3 20 ng /ml I L - 6 20 ng /ml G M - C S F (Sandoz) 14 ng /ml L o w density l ipoprote ins 40 u .g /ml A g a r o s e 0 .3% Cel ls as required For example - for a 3.3 ml final volume tube: R e a g e n t Stock solut ion V o l u m e 2- M e r c a p t o e t h a n o l 1 0 " 2 M 3.5 u l I n s u l i n 1 m g / m l 33 (xl T r a n s f e r r i n 20 m g / m l 33 \±l Steel Fac tor 4,000 ng /ml 41 u l I L - 3 ' 5 , 000 ng /ml 13 u l " I L - 6 11,000 ng /ml 6 p.1 G M - C S F 2,100 ng /ml 22 u.1 108 Iscove's D M E M IX 1.7 mis 2 X Iscove's D M E M 2X - 0.37 ml A l b u m i n 10% in Iscove's 10% 0.37 m l wi th 7% N a bicarbonate (40 mis B S A : 5 mis bicarb) L o w densi ty l ipoprote ins 5-6.5 m g / m l 22 u.1 C e l l suspension ( Iscove 's) 0.33 ml A g a r o s e 3 .3% 0.30 ml The first 11 components above can be prepared and combined without adding the low density l ipoproteins, cells or agarose and can be stored at - 2 0 ° C for many weeks. No te s : 1. The volume of 2 X Iscove's D M E M volume = the volume of agarose+bicarbonate+Pen/Strep solutions added (a l l of w h i c h were d i sso lved in water only) . 2. T o prepare Insu l in : a. A d d 4 mis of Iscove's media to a bottle containing 20 mg (This w i l l give a 5 mg/ml solution) b . F i l te r sterilize by passage through a 0.22 [i filter. c . A l iquo t 200 u.1 per Falcon 2058 tube. . d. A d d 800 u.1 Iscove's D M E M to each tube ( insu l in concentrat ion is now 1 mg/ml ) . e. Store at - 2 0 ° C . 5. T o prepare t ransferr in : a. A d d 25 mis of Iscove's D M E M to a bottle containing 1 gm of T r a n s f e r r i n . b . F i l te r sterilize by passage through a 0.22 u. filter. c . A l i q u o t 500 u.1 per Falcon 2058 tube. d. A d d 500 u.1 Iscove's D M E M to each tube (transferrin concentrat ion is now 20 mg/ml) . e. Store at - 2 0 ° C . 109 i i i E S T A B L I S H M E N T O F A G A R O S E C U L T U R E S a M a t e r i a l s 1. 2 m l pipettes. 2. Monojec t sterile disposable 1ml syringes, Sherwood M e d i c a l #501S-TB. 3. Monojec t sterile blunt needles, Stem C e l l Technologies . 4. Nunc L a b - T e k chamber slides, sterile, permanox slide, 2 wel l s / s l ide , 96 slides/case Cat N o #177429, Gibco . 5. Serum-substitute media wi th added growth factors. 6. L o w density l ipoproteins (Sigma) . 7. Agarose 3.3%. b M e t h o d 1. T o Falcon 2058 tubes each containing 2.65 ml of the media containing the first 11 components add 22 p i of low density lipoproteins (keep L D L s sterile), and add 0.33 mis of the ce l l suspension (in Iscove's D M E M at lOx the f inal ce l l concentration desired). 2. Place the Fa lcon tube containing media, L D L s and cells in a 3 7 ° C water bath. 3. L iquefy the aliquoted agarose by loosening the cap of the tubes and placing them in a microwave oven. A v o i d bo i l ing . 15-20 seconds at a 'med ium ' setting is usually sufficient. 4. Place the melted agarose in the water bath for 10 minutes to coo l it to 3 7 ° C . The level of water in the bath should be higher than the top of the agarose in the tube to prevent the surface of the agarose from c o o l i n g and s o l i d i f y i n g . 5. U s i n g a blunt ended needle and a 1 ml disposable syringe, transfer 0.3 ml of agarose to the tube containing 3.0 mis of culture medium and cells and vortex. 6. Us ing a 2 ml pipette remove 1.5 ml of the final cells, agarose and media mixture and dispense .75 ml into each of the 2 wells of a chamber slide. Repeat with a second 1.5 ml volume from the same tube and plate onto a second chamber slide. Q u i c k l y rotate each slide on an angle to enable even spreading of the f inal mixture in the chamber before the agarose so l id i f ies . 7. Place each slide in a 100 m l petri dish containing a 35 m l petri dish f i l l ed wi th water to maintain humidi ty during incubat ion of the '• •' - 11 c u l t u r e s . 8. Place the sl ides.at 4 ° C for 10-15 minutes to a l low the cultures to comple t e ly s o l i d i f y . 9. Place in a humidif ied 37°'C incubator in which an atmosphere of 5% C O 2 i n air is maintained. M a x i m u m M k colony numbers are typ ica l ly seen 2-3 weeks later. No te s : ' 1. Th is method results i n a f inal concentration of . 3% agarose. 2. The agarose has the advantage of a low. ge l l ing temperature and can be left at room temperature for 5-10 minutes before so l id i fy ing . W h e n adding agarose to the media the agarose should s t i l l be i n a l i q u i d state without clumps. It should not be hot to touch. 3. The f inal number of ce l l s /ml w i l l be 1/10 th that of the or ig inal ce l l c o n c e n t r a t i o n . 4. The f ina l number of ce l l s /we l l can be calculated by retaining the f o l l o w i n g p ropo r t i ons : 4 . 4 x 1 0 $ cells per 3.3 ml vo l = 1x10$ cel ls /wel l (if 0.75 mis are added to each w e l l ) . 5. • P la t ing 0.75 mis of agarose cul ture/well results i n a f inal c e l l number of 0.227 x or iginal total ce l l number per 3.3 mis ; 6. A .3.3 m l volume therefore .results i n 2 chamber slides, ie 4 cultures. 7. It is important to a l low the culture to solidify before placing at 3 7 ° C (otherwise the culture may never adequately so l id i fy and the cel ls w i l l not be immob i l i z ed during their, period of growth in the i n c u b a t o r ) . , 111 i v F I X A T I O N O F T H E C U L T U R E S a M a t e r i a l s 1. Methano l - Methanol A C S (Cat N o #ACS531) , from B D H Inc, Toronto, Ontario. 2. Acetone - Acetone optima (Cat N o #A929-4), f rom F i she r Sc i en t i f i c . 3. Whatman filter paper #1, 150 mm (Cat N o #1001-150) from Whatman Lab D i v i s i o n , Spr ingf ie ld M i l l , Maids tone , Kent . England . 4. Shandon fi l ter cards, thick, white (Cat N o #190005) from Shandon Inc., Pit tsburgh, P A 15275. 5. Shandon cytospin 2, from Shandon Southern Instruments Inc, Sewick l ey , P A . , 6. 100 m l Tupperware container b M e t h o d 1. Place chamber slides in a refrigerator for 10-15 minutes. 2. Ensure that each slide is correctly labeled with a diamond point pen. Labe l ing that had been made with ink w i l l ' run ' and disappear once exposed to the methanokacetone. 3. Careful ly remove the plastic walls and rubber seal of the chamber slides without damaging the culture. Use forceps to lift the seal off the slide starting at a corner near the ident i f icat ion label . 4. Place a piece of Whatman #1 filter paper (cut to the approximate size of the slide, ie 26 x 76 mm) onto the top of cultures on the chamber s l ide . 5. Place a Shandon fi l ter card over the filter paper. 6. P lace the chamber slide wi th over ly ing fi l ter paper and fi l ter card into a cytocentr ifuge bucket. 7. Centrifuge at 1500 rpm for 10 minutes. 8. Careful ly remove the filter card leaving the filter paper on top. of the cu l tu re . 9. Transfer the chamber slide wi th filter paper s t i l l ove r ly ing the cultures on it to a Tupperware container in wh ich approximately 50-112 75 mis of methanol:acetone 1:3 has been placed. Be careful to lower the slide gently down into the fixative solution (the plastic slides have a tendency to float and may then become separated from the fi l ter paper strip to which the cultures are stuck). 10. Leave i n the methanokacetone solution for 20 minutes. 11. Care fu l ly remove the chamber slide with fi l ter paper s t i l l o v e r l y i n g it from the methanol:acetone solution and then lift the fi l ter paper from the slide using a forceps leaving the cultures behind on the sl ide. This should be done immediately fo l lowing removal from the methanol:acetone before the f i l ter paper starts to dry. 12. A l l o w the f ixed cultures to air dry overnight. 13. Cultures can then be stored at 4 ° C unt i l immunocytochemis t ry cari be performed (Slides can be stored for at least 1 month before immunocy tochemis t ry and s t i l l g ive satisfactory s ta ining) . I M M U N O C Y T O C H E M I C A L I D E N T I F I C A T I O N O F M k C O L O N I E S a . M a t e r i a l s 1. H S N 8% Hank ' s solution containing 8% human serum and 0 .01% sodium azide. 2. T B S p H 7.6 Tr izma Base Saline (0.05 M Tr izma base, 0.15 M sodium c h l o r i d e ) . 3. P r i m a r y an t ibody : 1 0 E 5 (mouse anti-human GPIIb/IIIa) obtained from Dr . Bar ry Co l l e r , Stonybrook. Suppl ied at 1 mg/ml . Use at 4-6 u.g/ml diluted in H S N 8%. 4. A n t i b o d y for negative cont ro l : ocTNP (mouse an t i -TNP, isotype = same as 10E5, ie IgG2a) obtained from Dr . P . Lansdorp (Terry F o x Labora tory) . Use at the same concentration as the pr imary antibody. 5. M e t h a n o l Methanol A C S (Cat N o #ACS531) , from B D H Inc, Toronto. 6. Periodic acid, Sigma (Cat N o #P-7875), 10 gm bottle. 7. Secondary A n t i b o d y : Rabbi t anti-mouse Ig: peroxidase conjugate (Sigma Cat N o #A 9044) Use at a 1:50 dilution in H S N 8%. 1 1 3 8. D A B , (3 ,3 ' d iamino-benzidine tetrahydrochloride) Sigma (Cat N o #D-5905), 10 mg tablets. 9. . Hydrogen peroxide 30%, 'Perhydrol 30%' , B D H ( C a r N o #M07209-76). 10. G i l l ' s formulat ion #1 hematoxyl in , Fisher Scient i f ic (Cat N o CS400-1D) , 1 liter. b M e t h o d 1. Remove the slides wi th the f ixed cultures from the refrigerator and a l low to come to room temperature. 2. Rehydrate the cultures by placing in T B S for 10 minutes. 3. A p p l y 300 \x\ of H S N 8% and leave for 15 minutes. 4. Gent ly shake off excess H S N 8%. 5. A p p l y primary antibody by covering each culture with 300 u.1. Leave for at least 1 hour at room temperature with gentle rock ing every 10 - 15 minutes to ensure even staining over the entire culture surface. 6. Wash to remove unbound primary antibody with T B S for 10 minutes (ie 4 x T B S washes of approximately 1, 3, 3 and 3 minutes). 7. B l o c k endogenous peroxidase act ivi ty by p lac ing the slides in fresh methanol for 10 minutes ( 2 x 5 minute washes) and then once in fresh periodic acid (1.5% in methanol) for 5 minutes fo l lowed by 2 more 5 minute washes in methanol. 8. Rehydrate in T B S - 10 minutes (use 3-4 changes of T B S during this p e r i o d ) . 9. A p p l y the secondary antibody (as for the primary antibody) and leave for 1 hour at room temperature. 10. Remove unbound secondary antibody by 4 washes in T B S for a total of 10 minutes (as for the pr imary antibody). . 1 1 . Place slides in D A B - h y d r o g e n peroxide solution for 10. m i n u t e s . 12. Wash slides in water for 1-2 minutes. 13. Place slides in G i l l ' s Hematoxyl in for 30-45 seconds. 14. Wash slides in water unt i l a l l excess hematoxyl in has been washed a w a y . 1 14 15. A l l o w slides to air dry. No te s : 1. Batches of 20 slides (or multiples of 20) are convenient for processing us ing the currently avai lable slide carriers and large c o p l i n jars . 2. Transfer of slides during washing steps needs to be done gently to prevent the cultures from detaching from the- sl ides. 3: T B S p H 7.6 is prepared as follows: 5XTBS: . D i s t i l l ed water 900 mis v S o d i u m ch lo r ide 43.5 gms T r i z m a Base 32 .5 gms p H to 7.6 with Hydroch lor ic acid (usually requires approximately 17.5 mis of 12N HC1.) Make up volume to 1 liter. Sodium chloride (AnalaR Cat N o #B 10241) from B D H Inc, Toronto. T r i z m a base [Tr is(hydroxymethyl)aminomethane] from S i g m a , (Cat N o ##T1503). Hydroch lor ic acid (AnalaR, Cat N o #B10125) from B D H . 1XTBS: 200 mis of 5 X T B S 800 mis of dist i l led water T B S should be made up fresh on the day of use. 4. H S N 8%: Hanks solution 94.95 mis Human serum 8 mis • Sodium azide 20% 50 p i H S N 8% is used to block Fc receptors on n o n - M k cells and therefore avoid nonspecific b inding of the primary antibody. Because the pr imary antibodies are of mouse or ig in and the secondary antibody binds to mouse Ig then rabbit serum (but not mouse) can potential ly be used instead of human serum. 5. Use fresh methanol and periodic acid during the endogenous peroxidase b lock ing step. It is important to wash out the periodic acid with 2 changes of methanol and 10 minutes of T B S prior to the . addi t ion of the secondary antibody (otherwise residual per iodic ac id could potent ial ly inactivate the peroxidase enzyme conjugated to the secondary an t ibody) . 6. D A B takes a few minutes to dissolve and it is best to prepare this immediately prior to the T B S washes fo l lowing the secondary antibody. D o this as fol lows: A l l o w 14 D A B tablets (10 mg/tablet) to equilibrate to • - 1 1 5 room temperature before r emoving from the f o i l packaging . A d d the 14 D A B tablets to 200 mis T B S in a conical flask wi th a stir bar and a l low these to dissolve. Proceed with T B S washes of the slides for 10 minutes. A d d 168 u l of hydrogen peroxide immediately prior to exposing the slides to the D A B solution. (Act iv i ty of D A B / H 2 0 2 can be checked by adding a smal l volume of this solution to the diluted secondary antibody. A brown-black color reaction should occur wi th in a few seconds of mixing. ) . 7. The plastic slides tend to float when in the staining jars, to avoid this place a weight (eg: 4 glass slides taped together) across the top of the slides i n the slide carrier. 8. Pos i t ive controls, are prepared as fo l lows : Iscove's D M E M 2.7 mis Platelet concentrate 0-3 mis Agarose 3.3% 0.3 mis Plate 0.75 mis per we l l into chamber slides. Place cultures i n the refrigerator to a l low sol id i f ica t ion to occur. These can t h e n . b e left i n the refrigerator overnight . F i x i n methanol:acetone 1:3 for 20 minutes after centrifugation. 9. Sl ides for negative controls should contain reasonable numbers of granulopoiet ic colonies. These slides should contain no posi t ive ce l l s / co lon ies f o l l o w i n g immunocytochemis t ry . P o s i t i v i t y i f present is due to either nonspecific b inding of antibodies or residual endogenous peroxidase ac t i v i t y . v i S C O R I N G C R I T E R I A F O R M k C O L O N I E S 1. S c o r i n g c r i t e r i a varies amongst invest igators . 2. It is generally accepted that because the process of endomitosis wi th in a single M k can result in the same number of nuclear divis ions as occurs in a small C F U - E or C F U - G M then 2-3 D A B positive cells in close association are sufficient to score as a colony derived from a ' C F U - M k ' . 3. Hoffman 's group noted that some. M k colonies can be very large and have adopted the terms B F U - M k and C F U - M k to dist inguish between - progenitors of large and smal l M k colonies, respectively, by analogy to B F U - E and C F U - E . on the erythroid pathway. However a s imilar dis t inct ion in the growth factor responsiveness of B F U - M k and C F U - M k has not yet been established. In this document we refer to a l l colonies containing D A B posit ive colonies s imply as C F U - M k - d e r i v e d . 4. However we also note that the number of cells i n Mk-co lon i e s can also vary markedly. W e have arbitrarily considered a colony to consist of at 116 least 3 D A B positive cells in close association. Colonies are then classified as consisting of 3-20 cells, 21-49 cells, and >50 cells as a means of d i s t ingu i sh ing progenitors of d i f fe r ing pro l i fe ra t ive potent ia l . 5. The morphology of cells wi th in M k colonies can also vary markedly, ranging from smal l round mononuclear cells to very large p o l y p l o i d , mult inucleated cel ls wi th the appearance of cytoplasmic shedding of platelet fields. The presence of the latter may be dependent on the inc lus ion of C S F s i n the culture medium that promote M k maturation. (The growth factors we have used were chosen p r imar i ly for their prol i fera t ive effect on M k progenitor ce l l s ) . 6. Some colonies consist almost entirely of non-nucleated D A B posit ive cel ls . T ime course studies suggest that these represent cells of the M k lineage that are undergoing apoptosis. These are thus presumed to represent the progeny of M k progenitors and are routinely scored as posi t ive colonies and sized according to the total number of cells present in each. 7. Co lon ie s containing both posi t ive and negative staining cel ls present a problem. If the majority of cells are D A B positive with a minori ty (eg <5%) being negative then we have tended to score these as pure M k colonies. If there is pr imari ly only an isolated focus of posit ive cel ls , or posi t ive cells are scattered throughout a colony of negative staining cells then we have scored these as being 'm ixed ' . If E p is added to the cul ture then b iphenotyp ic e r y t h r o i d / M k . co lonies are seen. 

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