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Growth properties and genetic manipulation of murine hemopoietic stem cells Pawliuk, Robert James 1996

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GROWTH  PROPERTIES AND MURINE  GENETIC  HEMOPOIETIC  MANIPULATION  STEM  OF  CELLS  by R O B E R T J A M E S PAWLIUK B . S c . H . , T h e University of A l b e r t a ,  1991  A T H E S I S S U B M I T T E D IN 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 FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES Medical Genetics Programme  W e a c c e p t t h i ^ h e s i s a s conforming to the required s t a n d a r d  T H E UNIVERSITY O F BRITISH C O L U M B I A S e p t e m b e r , 1996 © Robert J a m e s P a w l i u k ,  1996  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  scholarly purposes may be her  representatives.  permission.  Department The University of British Columbia Vancouver, Canada  for  an advanced  Library shall make it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  DE-6 (2/88)  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  ABSTRACT The  development  of  recombinant  retroviral  vectors  a b l e to  transfer  e x o g e n o u s genetic material into h e m o p o i e t i c target c e l l s h a s p l a y e d a pivotal role in our current u n d e r s t a n d i n g of h e m o p o i e s i s a n d h a s p l a y e d a p i o n e e r i n g role in the field of g e n e therapy. H o w e v e r , with the efficiency of g e n e transfer to m u r i n e s t e m c e l l s only 1 5 % the p o w e r of recombinant retroviral g e n e transfer is currently s e v e r e l y c o m p r o m i s e d by the efficiency of retroviral infection. T o optimize the utility of r e c o m b i n a n t retroviruses, the h u m a n C D 2 4 cell s u r f a c e antigen w a s d e v e l o p e d a s a d o m i n a n t s e l e c t a b l e m a r k e r in a retroviral v e c t o r to e n a b l e the identification a n d s e l e c t i o n of retrovirally t r a n s d u c e d murine b o n e m a r r o w c e l l s , including t h o s e with long term in vivo repopulating ability. Following infection of d a y 4 5 - F U treated m u r i n e b o n e m a r r o w c e l l s a n d s e l e c t i o n of retrovirally t r a n s d u c e d c e l l s u s i n g a n a n t i - C D 2 4 a n t i b o d y a n d F l u o r e s c e n c e A c t i v a t e d C e l l S o r t i n g ( F A C S ) , functional a n a l y s i s of s e l e c t e d C D 2 4 + c e l l s d e m o n s t r a t e d the p r e s e n c e of h e m o p o i e t i c c e l l s at v a r i o u s s t a g e s of d e v e l o p m e n t , including in vitro c l o n o g e n i c progenitors, d a y 12 CFU-S,  a n d c e l l s with totipotent  long-term  repopulating  potential.  Further  e x p e r i m e n t s d e m o n s t r a t e d the ability to r e g e n e r a t e the h e m o p o i e t i c s y s t e m s of m y e l o a b l a t e d recipient m i c e with c e l l s d e r i v e d e x c l u s i v e l y from provirally m a r k e d s t e m c e l l s a n d that the t r a n s f e r r e d  C D 2 4 g e n e w a s e x p r e s s e d in v a r i o u s  p h e n o t y p i c a l l y d e f i n e d p o p u l a t i o n s of c e l l s in v i v o i n c l u d i n g m a r r o w s t e m c e l l c a n d i d a t e s d e f i n e d by the S c a + L i n " cell s u r f a c e p h e n o t y p e . T h u s , C D 2 4 c a n b e utilized not only a s a s e l e c t a b l e marker but a l s o a s a m e a n s to track a n d p h e n o t y p e t r a n s d u c e d c e l l s a n d their p r o g e n y in vitro a n d in vivo. T o provide information o n the recovery of h e m o p o i e t i c s t e m cells following b o n e m a r r o w transplant, irradiated recipient m i c e w e r e injected with v a r i o u s n u m b e r s of d a y 14.5 fetal liver or d a y 4 5FU  adult  bone marrow  e s t i m a t e d to c o n t a i n  10, 1 0 0 or 1 0 0 0  Competitive  R e p o p u l a t i n g Units ( C R U ) . A n a l y s i s of the f e m o r a l m a r r o w of primary r e c i p i e n t s s h o w e d c o m p l e t e recovery of b o n e marrow  ii  cellularity a n d c l o n o g e n i c progenitor content a n d a n e a r full r e c o v e r y of d a y 12 CFU-S  numbers  i r r e s p e c t i v e of the  number  or o r i g i n  of t h e  cells  initially  t r a n s p l a n t e d . W h i l e the r e c o v e r y of d o n o r - c e l l - d e r i v e d C R U w a s i n c o m p l e t e in all c a s e s , fetal liver w a s m a r k e d l y s u p e r i o r to t h o s e f r o m adult b o n e  marrow.  M o r e o v e r , proviral integration a n a l y s i s of m i c e r e c e i v i n g retrovirally t r a n s d u c e d C D 2 4 + s e l e c t e d b o n e m a r r o w c e l l s p r o v i d e d e v i d e n c e for a > 3 0 0 - f o l d c l o n a l a m p l i f i c a t i o n of a s i n g l e t r a n s d u c e d s t e m c e l l . T h e s e s t u d i e s h a v e  provided  p r o c e d u r e s for the s e l e c t i o n , tracking a n d p h e n o t y p i n g of m u r i n e b o n e m a r r o w c e l l s , i n c l u d i n g t h o s e with c o m p e t i t i v e long t e r m l y m p h o - m y e l o i d  repopulating  ability. T h e availability of s u c h p r o c e d u r e s s h o u l d i n c r e a s e the p o w e r of retroviral marking  studies, and  be  advantageous  in  studies  aimed  at  the  genetic  m a n i p u l a t i o n of h e m o p o i e t i c s t e m c e l l s a n d their p r o g e n y , a s w e l l a s in the d e v e l o p m e n t of v e c t o r s a b l e to o p t i m i z e the e x p r e s s i o n of t r a n s f e r r e d g e n e s in s p e c i f i c target c e l l s of interest for u s e in h u m a n g e n e t h e r a p y trials. M o r e o v e r , t h e s e f i n d i n g s s e t the s t a g e for a t t e m p t s to e n h a n c e h e m o p o i e t i c s t e m c e l l r e g e n e r a t i o n p o s t - t r a n s p l a n t by the a d m i n i s t r a t i o n of e x o g e n o u s a g e n t s or the e x p r e s s i o n of intracellular factors that m a y e n h a n c e the r e g e n e r a t i v e potential of stem cells.  iii  TABLE  OF  CONTENTS Pag i ii iv vii viii ix xi  TITLE ABSTRACT T A B L E OF CONTENTS LIST O F FIGURES LIST O F T A B L E S ABBREVIATIONS ACKNOWLEDGMENT C H A P T E R 1. Introduction 1.1. T h e hemopoietic system 1.1.1. Overview of hemopoiesis 1.2. A s s a y s for early hemopoietic cells 1.2.1. In vitro clonogenic progenitors 1.2.2. Colony-forming units-spleen ( C F U - S ) 1.2.3. T h e hemopoietic stem cell ( H S C ) 1.2.4. Phenotyping a n d purification of H S C s 1.3. O n t o g e n y of the murine hemopoietic s y s t e m 1.3.1. Development of the hemopoietic s y s t e m 1.3.2. C o m p a r i s o n of s t e m cells from fetal liver a n d adult tissues 1.4. Properties of hemopoietic stem cells 1.4.1. Cycling status 1.4.2. D e v e l o p m e n t a l potential a n d d y n a m i c s of H S C s 1.4.3. Self-renewal a n d aging of H S C s 1.5. Regulation of hemopoiesis 1.5.1. Regulation by extracellular factors 1.5.1.1. Hemopoietic growth factors 1.5.1.2. T h e extracellular matrix 1.5.2. Regulation by intracellular factors 1.6. G e n e t i c manipulation of hemopoietic cells using r e c o m b i n a n t retroviruses 1.6.1. T h e lifecycle of retroviruses 1.6.2. R e c o m b i n a n t retroviruses a s vectors for g e n e transduction.. 1.6.2.1. Production of helper-free replication-defective retroviruses 1.6.3. Optimization of retroviral g e n e transfer 1.6.3.1. Retroviral infection strategies 1.6.3.2. Targeting of virions to host cells 1.6.3.3. U s e of s e l e c t a b l e markers to i n c r e a s e the utility of recombinant retroviral vectors 1.6.4. Retroviral vector design 1.7. T h e s i s objectives a n d general strategy C H A P T E R 2. Materials and M e t h o d s 2 . 1 . C o n s t r u c t i o n of retroviral vectors, virus production a n d viral a s s a y s . . . 2.1.1. Recombinant retroviral vectors 2.1.2. Viral packaging a n d other cell lines 2.1.3. Generation of viral producer cell lines 2.1.4. Viral titering a n d helper virus a s s a y iv  1 1 3 3 5 6 9 13 13 14 16 16 16 19 23 23 23 25 27 28 29 33 34 36 36 38 40 40 44  46 46 47 47 48  2.2. Hemopoietic cell culture and a s s a y s 2.2.1. M i c e 2.2.2. Viral infection of bone marrow cell a n d cell lines 2.2.3. In vitro clonogenic progenitor a s s a y 2.2.4. C F U - S a s s a y 2.2.5. B o n e marrow transplantation a n d quantitation of competitive repopulating units ( C R U ) 2.3. Molecular analysis 2.3.1. Southern blot analysis 2.3.2. Antibody staining procedures 2.3.3. F l u o r e s c e n c e activated cell sorting ( F A C S ) C H A P T E R 3. S e l e c t i o n of retrovirally t r a n s d u c e d h e m o p o i e t i c cells u s i n g CD24 as a marker of gene transfer 3.1. Introduction 3.2. Results 3.2.1. T h e C D 2 4 viral vector 3.2.2. F A C S selection of C D 2 4 - t r a n s d u c e d in vitro c l o n o g e n i c progenitors and C F U - s p l e e n ( C F U - S ) 3.2.3. Selection by F A C S of CD24-virus-infected C R U 3.3. Discussion  48 49 49 50 50 50 52 52 53 55  57 58 58 60 61 69  C H A P T E R 4. High level reconstitution with p r e s e l e c t e d h e m o p o i e t i c cells e x p r e s s i n g a t r a n s d u c e d gene e n c o d i n g a cell s u r f a c e antigen 4.1. Introduction 76 4.2. Results 77 4 . 2 . 1 . Viral vectors and experimental design 77 4 . 2 . 2 . T h e majority of donor-derived cells in recipients contain intact provirus 78 4 . 2 . 3 . Efficient g e n e transfer to a n d e x p r e s s i o n of C D 2 4 a m o n g S c a + L i n " b o n e marrow s t e m cell candidates.... 85 4.3. Discussion 89 C H A P T E R 5. E v i d e n c e of both o n t o g e n y and transplant d o s e e x p a n s i o n of hemopoietic stem cell in vivo 5.1. Introduction 5.2. Results 5.2.1. Overall experimental design 5.2.2. Kinetics of reconstitution of the terminal c o m p a r t m e n t s 5.2.3. Reconstitution of the marrow 5.2.4. Reconstitution of the marrow C R U compartment 5.2.5. R e g e n e r a t i v e ability of a single C R U a s s e s s e d using retroviral marking 5.3. Discussion CHAPTER Discussion  6.  CHAPTER References  7  regulated 93 94 94 94 95 97 99 101  107 \ 114  v  LIST O F CHAPTER F i g u r e 1.1 F i g u r e 1.2. F i g u r e 1.3. F i g u r e 1.4. F i g u r e 1.5. F i g u r e 1.6. F i g u r e 1.7. CHAPTER Figure 3.1. F i g u r e 3.2. F i g u r e 3.3. F i g u r e 3.4. F i g u r e 3.5.  Figure 3.6.  F i g u r e 3.7.  CHAPTER Figure 4 . 1 . Figure 4 . 2 . Figure 4.3.  Figure 4.4.  Figure 4 . 5 .  CHAPTER Figure 5.1. F i g u r e 5.2.  FIGURES  1 S c h e m a t i c representation of the hemopoietic hierarchy S c h e m a t i c representation of the competitive repopulating unit ( C R U ) assay. V a r i o u s o r g a n s in w h i c h h e m o p o i e s i s t a k e s p l a c e during ontological development S c h e m a t i c representation of a m o d e l to e x p l a i n H S C behavior following bone marrow transplant S c h e m a t i c representation of a typical retrovirus Critical features of the retroviral lifecycle S t e p s involved in the production of recombinant retrovirus.. 3. S c h e m a t i c of the J Z e n C D 2 4 t k n e o provirus F l o w cytometric a n a l y s i s of C D 2 4 e x p r e s s i o n of B a / F 3 cells infected with the J Z e n C D 2 4 t k n e o retrovirus F A C S selection of C D 2 4 virus-infected in vitro c l o n o g e n i c progenitors a n d day 12 C F U - S S e l e c t i o n of C D 2 4 virus-infected C R U by F A C S H e m o p o i e t i c reconstitution from C D 2 4 retrovirus-infected competitive repopulating c e l l s a s a s s e s s e d by S o u t h e r n blot Proportion of recipient m i c e e x p r e s s i n g the transferred C D 2 4 g e n e at early a n d late time points posttransplant a s a s s e s s e d by F A C S F l o w cytometric a n a l y s i s of C D 2 4 e x p r e s s i o n in the h e m o p o i e t i c t i s s u e s of o n e repopulated m o u s e 4 m o n t h s post transplantation  2 10 15 19 32 33 35  59 60 62 65  68  71  72  4. Viral vectors u s e d a n d F A C S selection of retrovirally transduced bone marrow cells 79 Detection of high levels of intact provirus in recipient m i c e by Southern blot analysis 83 A s s e s s m e n t of proviral integration in primary or s e c o n d a r y recipients a n d d a y 12 s p l e e n c o l o n i e s by S o u t h e r n blot analysis 86 E x p r e s s i o n of the transferred C D 2 4 g e n e in primary m a r r o w s t e m cell c a n d i d a t e s a s defined by the S c a + L i n " cell s u r f a c e phenotype 87 Proportion of s t e m cell c a n d i d a t e s defined by the S c a + L i n " cell s u r f a c e phenotype positive for C D 2 4 e x p r e s s i o n at 2 4 w e e k s post transplant 88 5. R e g e n e r a t i o n of Ly5.1 d o n o r - d e r i v e d c e l l s following the transplantation into s e c o n d a r y recipients 98 Demonstration of C D 2 4 provirus in b o n e marrow (B), s p l e e n (S), a n d thymus (T) D N A of primary a n d s e c o n d a r y transplant recipients 101 vi  LIST O F T A B L E S CHAPTER T a b l e 1.1.  CHAPTER Table 3.1.  T a b l e 3.2.  CHAPTER Table 4.1.  1 O p t i o n s in selectable/reporter m a r k e r s for retroviral vectors  3 Proviral integration a n d C D 2 4 e x p r e s s i o n on c e l l s from individual s p l e e n c o l o n i e s derived from sorted a n d u n s o r t e d b o n e marrow cells following C D 2 4 virus infection 63 Proviral integration a n d C D 2 4 e x p r e s s i o n on c e l l s from competitively repopulated mice a s s e s s e d 5 w e e k s post transplant 67 4 F l o w cytometric a n a l y s i s of C D 2 4 e x p r e s s i o n in v a r i o u s h e m o p o i e t i c t i s s u e s in recipient m i c e 2 4 w e e k s posttransplant  CHAPTER  5  Table 5.1.  Proportion of Ly5.1 + peripheral blood c e l l s from primary recipients R e g e n e r a t i o n of cellularity, C F C a n d d a y 12 C F U - S populations in the femurs of primary recipient m i c e 8 months post transplant E x p a n s i o n of donor-derived C R U in primary recipients of fetal liver or adult bone marrow cells  T a b l e 5.2.  T a b l e 5.3.  41  vii  81  95  96 99  LIST O F  7AAD ADA AGM AIDS ATTC BFU-E BFU-Mk BM bp BSA CAFC cDNA CD CFU CFU-E CFU-G CFU-GEMM CFU-GM CFU-M CFU-S cGy CRU Cs CS dCTP DMEM DNA EC EDTA Epo ES FACS FCS FITC FL 5-FU G418 G-CSF GFP GM-CSF G6PD Gpi HBS HF HPP-CFC HSA HSC  ABBREVIATIONS  7-amino actinomycin D adenosine deaminase aortic g o n a d a l m e s e n e p h r o s region acquired immune deficiency syndrome A m e r i c a n T y p e Culture C o l l e c t i o n burst forming unit-erythroid burst forming unit-megakaryocyte b o n e marrow b a s e pair bovine s e r u m albumin c o b b l e stone a r e a forming cell complementary deoxyribonucleic acid cluster d e s i g n a t i o n c o l o n y forming unit c o l o n y forming unit-erythroid c o l o n y forming unit-granulocyte c o l o n y forming unit-granulocyte-erythroid-monocytemegakaryocyte c o l o n y forming 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 o l o n y forming u n i t - m a c r o p h a g e c o l o n y forming unit-spleen centiGray competitive repopulating unit cesium calf s e r u m d e o x y c y t i d i n e triphosphate D u l b e c c o ' s Modified E a g l e s M e d i u m deoxyribonucleic acid e m b r y o n i c c a r c i n o m a cell ethylenediaminetetraacetic acid erythropoietin e m b r y o n i c s t e m cell f l u o r e s c e n c e activated cell sorter fetal calf s e r u m fluorescein-isothiocyanate flk2/flt3 ligand 5-fluorouracil geneticin granulocyte c o l o n y stimulating factor g r e e n f l u o r e s c e n c e protein 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 stimulating factor glucose 6 phosphate dehydrogenase glucose phosphate isomerase h e p e s buffered solution H a n k ' s b a l a n c e d salt solution/2%fetal calf s e r u m high proliferative potential c o l o n y forming cell heat stable antigen h e m o p o i e t i c s t e m cell viii  HXM ig IL IRES LFA LIF LTC LTC-IC LTR M-CSF MDR-1  MESV MHC  hypoxanthine-xanthine-mycophenolic acid immunoglobulin interleukin internal r i b o s o m a l entry site l e u k o c y t e function a s s o c i a t i o n l e u k e m i a inhibitory factor long term culture long term culture-initiating cell long terminal repeat m a c r o p h a g e c o l o n y stimulating factor multi-drug resistance-1 protein murine e m b r y o n i c s a r c o m a virus major histocompatibility c o m p l e x  m a c r o p h a g e inhibitory p r o t e i n - 1 a MoAb m o n o c l o n a l antibody MoMuLV M o l o n e y murine l e u k e m i a virus MPSV myeloproliferative s a r c o m a virus M R A - C F U - •S m a r r o w repopulating ability-colony forming unit-spleen MSCV murine s t e m cell virus n e w b o r n calf s e r u m NCS NH4CI a m m o n i u m chloride ORF o p e n reading f r a m e P phosphate PB peripheral blood PCMV P C C 4 e m b r y o n a l c a r c i n o m a c e l l - p a s s a g e d myeloproliferative s a r c o m a virus PCR p o l y m e r a s e c h a i n reaction PGK phosphoglycerate kinase PI p r o p i d i u m iodide RBC red b l o o d cell Rh rhodamine RNA ribonucleic acid R-PE R-phycoerythrin RPMI R o s w e l l P a r k M e m o r i a l Institute RT r e v e r s e transcription repopulating unit RU SCCM s p l e e n cell c o n d i t i o n e d m e d i u m SD s t a n d a r d deviation s o d i u m d o d e c y l sulfate SDS SEM s t a n d a r d error of the m e a n s o d i u m chloride s o d i u m citrate SSC TE tris-EDTA  MIP-1a  TGF-p U VCAM VLA VSV-G W WBC WGA  transforming growth factor-p units v a s c u l a r cellular a d h e s i o n m o l e c u l e v e r y late antigen v e s i c u l a r stomatitis virus G glycoprotein W h i t e spotting mutation white b l o o d cell w h e a t g e r m agglutinin  ix  ACKNOWLEDGMENTS  I w o u l d like to thank a n d e x p r e s s my gratitude: to my s u p e r v i s o r Dr. R. Keith H u m p h r i e s for the opportunity to d o graduate training at the Terry F o x Laboratory a n d for his enthusiastic support a n d tireless g u i d a n c e throughout this project. to D r s . C o n n i e J . E a v e s a n d P e t e r L a n s d o r p for their collaborative efforts a n d m a n y stimulating d i s c u s s i o n s . I would a l s o like to thank Dr. E a v e s for her invaluable contributions to writing the m a n u s c r i p t s d e s c r i b e d in this t h e s i s . to the m a n y m e m b e r s of the H u m p h r i e s laboratory for providing a n invigorating scientific environment in w h i c h to study. to Patty R o s t e n , G a y l e Thornbury, V i s i a D r a g o w s k a , M a y a S t - C l a i r a n d F r e d J e n s o n for expert technical a s s i s t a n c e . to D r s . Dixie M a g e r , Muriel Harris a n d R o s s M a c G i l l i v r a y for s e r v i n g on my graduate committee. to the M e d i c a l R e s e a r c h C o u n c i l of C a n a d a for financial support. to my parents for their y e a r s of support a n d understanding, to H e a t h e r M u r r a y for believing in m e a n d to the D r a g o n H a g s for m a k i n g life fun.  x  CHAPTER  1  INTRODUCTION  1.1  The  1.1.1  hemopoietic s y s t e m  O v e r v i e w of  hemopoiesis  H e m o p o i e s i s is the e s s e n t i a l , lifelong p r o c e s s w h e r e b y multiple t y p e s of highly  specialized blood  cells are  generated. T h e s e cells  include  those  r e s p o n s i b l e for carrying out specific functions s u c h a s c a r b o n d i o x i d e a n d o x y g e n transport (erythrocytes), b l o o d clotting (platelets), h u m o r a l (B l y m p h o c y t e s ) a n d cellular (T l y m p h o c y t e s ) immunity a s w e l l a s m o u n t i n g p h a g o c y t i c r e s p o n s e s to foreign o r g a n i s m s a n d their p r o d u c t s ( g r a n u l o c y t e s / m o n o c y t e s / m a c r o p h a g e s ) . In the n o r m a l h u m a n adult it is e s t i m a t e d that a p p r o x i m a t e l y 2 0 0 billion e r y t h r o c y t e s (1) a n d 6 0 billion n e u t r o p h i l i c  l e u k o c y t e s (2)  are produced everyday. This  o b s e r v a t i o n h a s stimulated a great d e a l of interest in the c e l l s that are ultimately r e s p o n s i b l e for a c c o m m o d a t i n g this e n o r m o u s daily output of c e l l s , a n d in the m e c h a n i s m s that regulate this p r o c e s s . T h e c e l l t y p e s m e n t i o n e d a b o v e c a n b e functionally d i v i d e d into two distinct g r o u p s t e r m e d m y e l o i d a n d l y m p h o i d (Figure 1.1). D u r i n g n o r m a l adult life m y e l o i d c e l l s are p r o d u c e d e x c l u s i v e l y within the b o n e m a r r o w (3) while c e l l s of the l y m p h o i d l i n e a g e s a r e p r o d u c e d to v a r y i n g d e g r e e s in the b o n e marrow, s p l e e n , t h y m u s a n d l y m p h n o d e s . M a t u r e functional e n d c e l l s a n d their i m m e d i a t e p r e c u r s o r s h a v e a limited l i f e s p a n a n d a limited proliferative c a p a c i t y a n d h e n c e are not self-maintaining. T h u s , t h e s e c e l l s must b e c o n t i n u o u s l y r e p l a c e d from a pool of m o r e primitive proliferating c e l l s . T h e s e c e l l s constitute a h i e r a r c h y of c e l l s with i n c r e a s i n g proliferative potential a n d w i d e r differentiative c a p a c i t i e s .  1  F i g u r e 1.1. S c h e m a t i c representation of the organization of the h e m o p o i e t i c s y s t e m a n d s o m e of the a s s a y s u s e d to e v a l u a t e H S C s a n d v a r i o u s p o o l s of progenitor c e l l s ; H S C , h e m o p o i e t i c s t e m cell; C R U , competitive repopulating unit a s s a y ; R U , r e p o p u l a t i n g unit a s s a y ; L T C - I C , c e l l s with the ability to initiate in vitro long-term cultures; C A F C , in vitro a s s a y for c o b b l e s t o n e forming a r e a c e l l s ; d a y 12 or d a y 8 C F U - S , colony-forming-unit s p l e e n c e l l s a b l e to p r o d u c e s p l e n i c n o d u l e s 12 or 8 d a y s p o s t i n o c u l a t i o n r e s p e c t i v e l y ; B l a s t c o l . , in vitro blast c o l o n y a s s a y . T h e relative potential for proliferation a n d s e l f - r e n e w a l of v a r i o u s g r o u p s of c e l l s is s h o w n to the right.  Ultimately, all c e l l s of both the m y e l o i d a n d l y m p h o i d l i n e a g e s are d e r i v e d from c e l l s referred to a s totipotent s t e m cells w h i c h are e s t i m a t e d to c o m p r i s e only 0.01%  of the total marrow c o m p a r t m e n t . T h e s e c e l l s are operationally d e f i n e d by  their c a p a c i t y to regenerate a n d sustain both the myeloid a n d lymphoid a r m s of the h e m o p o i e t i c s y s t e m for long p e r i o d s of time following t r a n s p l a n t a n d by their e x t e n s i v e c a p a c i t y for s e l f - r e n e w a l , the p r o c e s s of cellular division resulting in the p r o d u c t i o n of d a u g h t e r c e l l s w h i c h a r e functionally i n d i s t i n g u i s h a b l e f r o m p a r e n t c e l l s in t e r m s of their proliferative a n d differentiative  potential.  the  Many  important q u e s t i o n s r e m a i n u n a n s w e r e d r e g a r d i n g the nature a n d regulation of  2  totipotent h e m o p o i e t i c s t e m c e l l s . T h e s e include a b a s i c u n d e r s t a n d i n g of their n u m b e r s , biological potential, u s a g e o v e r time a n d the g e n e s e n c o d i n g extrinsic a n d / o r intrinsic factors w h i c h are r e s p o n s i b l e for the regulation of t h e s e biological c h a r a c t e r i s t i c s . M o r e o v e r , with a n i n c r e a s i n g e m p h a s i s o n the d e v e l o p m e n t of c l i n i c a l s t r a t e g i e s that d e p e n d u p o n the r e g e n e r a t i o n of H S C n u m b e r s  (eg.  autograft purging a n d the genetic therapy of inherited h e m a t o l o g i c a l d i s o r d e r s ) the a s s e s s m e n t of the self-renewal potential of H S C s is of particular interest. T h e ability to manipulate H S C s genetically h a s p r o v i d e d a powerful tool to b e g i n to a d d r e s s the a b o v e i s s u e s . T h e overall g o a l of the w o r k p r e s e n t e d in this t h e s i s w a s to d e v e l o p m e t h o d o l o g i e s to i n c r e a s e the utility of g e n e transfer for the efficient genetic manipulation a n d tracking of H S C s a n d to utilize t h e s e p r o c e d u r e s to define m o r e clearly the s e l f - r e n e w a l potential of H S C s following b o n e m a r r o w transplant. T h e following introduction e x a m i n e s the current state of k n o w l e d g e of the p h e n o t y p i c a n d functional properties of H S C s a s well a s the m e t h o d s a v a i l a b l e for their genetic manipulation. 1.2.  A s s a y s for early hemopoietic cells.  1.2.1. In vitro c l o n o g e n i c  progenitors  T h e ability to culture h e m o p o i e t i c cells in vitro h a s p r o v i d e d a large a m o u n t of i n f o r m a t i o n o n the c e l l u l a r o r g a n i z a t i o n , the proliferative a n d  differentiative  potential, a n d growth factor requirements of cells at v a r i o u s s t a g e s of h e m o p o i e t i c d e v e l o p m e n t . A s s a y s first d e s c r i b e d by B r a d l e y a n d Metcalf (4), a n d P l u z n i k a n d S a c h s (5) involved the growth of h e m o p o i e t i c c e l l s in a s e m i - s o l i d matrix of a g a r that a l l o w e d c o l o n i e s of h e m o p o i e t i c cells derived from s i n g l e c e l l s to be identified a n d c h a r a c t e r i z e d . S u b s e q u e n t a s s a y s for s u c h " c l o n o g e n i c p r o g e n i t o r s " utilized p l a s m a c l o t s or, n o w m o s t often, m e t h y l c e l l u l o s e to p r o v i d e s e m i - s o l i d m e d i u m . S u c h growth m e d i u m is typically s u p p l e m e n t e d with nutrients a n d growth factors required by the dividing cells a n d w h i c h h a v e b e e n p r o v i d e d historically in part by poorly d e f i n e d " c o n d i t i o n e d " cell m e d i u m . T o d a y a large n u m b e r of h e m o p o i e t i c  3  growth  f a c t o r s h a v e b e e n purified a n d their g e n e s c l o n e d r e s u l t i n g  in  availability of pure r e c o m b i n a n t f a c t o r s ( r e v i e w e d in (6)). T h e v a s t majority  the of  c l o n o g e n i c progenitors detectable in a s s a y s of normal b o n e m a r r o w are of uni- or bipotent potential that are a b l e to give rise to c o l o n i e s c o n s i s t i n g of g r a n u l o c y t e s and m o n o c y t e s / m a c r o p h a g e s (colony-forming  units g r a n u l o c y t e - m a c r o p h a g e ;  C F U - G M ) (7), pure granulocytes or m o n o c y t e s / m a c r o p h a g e s ( C F U - G or C F U - M ) (4, 5), erythrocytes ( B F U - E a n d C F U - E ) (8), a n d m e g a k a r y o c y t e s ( B F U - M k ) (9). M o r e recently a s s a y s for cells with B, but not T, lymphoid potential h a v e b e e n d e s c r i b e d (10). S u c h c e l l s , while s o m e t i m e s p o s s e s s i n g c o n s i d e r a b l e proliferative  potential  yielding c o l o n i e s of s e v e r a l t h o u s a n d s of cells, h a v e limited or no c a p a c i t y for selfr e n e w a l a s d e t e r m i n e d by their inability to generate equivalent s e c o n d a r y c o l o n i e s in s u b s e q u e n t replatings (11). T h e s e cells a l s o a p p e a r to b e actively c y c l i n g u n d e r n o r m a l s t e a d y - s t a t e conditions in vivo s i n c e they are highly s u s c e p t i b l e to killing by c y c l e - s p e c i f i c cytotoxic d r u g s s u c h a s 5-fluorouracil ( 5 - F U ) (12). S u c h c e l l s , t h e n , are b e l i e v e d to be relatively late in the hemopoietic hierarchy. S u c h in vitro a s s a y s a l s o e n a b l e the identification of e a r l i e r p r o g e n i t o r s c h a r a c t e r i z e d by their ability to p r o d u c e c o l o n i e s c o n s i s t i n g of multiple l i n e a g e s (ie. g r a n u l o c y t e / erythrocyte / m a c r o p h a g e / m e g a k a r y o c y t e from C F U - G E M M ) (13, 14), of great s i z e (eg. high proliferative potential c o l o n y - f o r m i n g c e l l s , H P P - C F C ) (15), a n d / o r c e l l s with a primitive undifferentiated c e l l u l a r m o r p h o l o g y (ie. blast c o l o n y forming cells) (16-18). C F U - G E M M a n d blast c o l o n y forming c e l l s a r e a l s o c h a r a c t e r i z e d by a capacity for self-renewal a s d e m o n s t r a t e d by the ability of s o m e of their c l o n a l p r o g e n y to form s e c o n d a r y a n d l e s s frequently tertiary multi-lineage a n d blast c o l o n i e s in replate a s s a y s (16, 19). 1.2.2. C o l o n y - f o r m i n g  units-spleen  (CFU-S)  T h e first a s s a y a v a i l a b l e to study h e m o p o i e t i c c e l l s s h o w n to p o s s e s s "stem c e l l - l i k e " p r o p e r t i e s w a s the in vivo s p l e e n c o l o n y a s s a y d e s c r i b e d by Till a n d M c C u l l o c h in 1961 (20). T h i s a s s a y is b a s e d on the ability of certain c e l l s ( C F U - S ,  4  for c o l o n y f o r m i n g  unit-spleen)  to  h o m e to the  spleen and grow  to  form  m a c r o s c o p i c h e m o p o i e t i c n o d u l e s d e t e c t a b l e o n the s u r f a c e of the s p l e e n 8 - 1 2 d a y s post-transplant. T h e origin of individual n o d u l e s from a s i n g l e cell (ie. c l o n a l in origin) w a s first d e t e r m i n e d by injecting b o n e m a r r o w c e l l s from m i c e harboring u n i q u e radiation-induced c h r o m o s o m a l abnormalities (21, 22) , a n d this w a s later c o n f i r m e d u s i n g unique retroviral integration e v e n t s a s m a r k e r s (23, 24). Individual s p l e e n c o l o n i e s d e t e c t e d on d a y 8 post-transplant u s u a l l y a r e restricted in the t y p e s of c e l l s they c o n t a i n (either erythroid or granulocytic but not both (25, 26)) and  rarely p r o d u c e d a u g h t e r c o l o n i e s u p o n s u b s e q u e n t r e t r a n s p l a n t a t i o n  (11).  T h u s , d a y 8 C F U - S r e s e m b l e uni-potential in vitro c l o n o g e n i c p r o g e n i t o r s .  In  a d d i t i o n , a large proportion of d a y 8 C F U - S a r e a l s o s e n s i t i v e to killing by the c y c l e - a c t i v e drug 5 - F U (27). H o w e v e r , t h o s e C F U - S w h i c h result in large c o l o n i e s d e t e c t e d o n d a y 12 are often c o m p o s e d of c e l l s of m u l t i - l i n e a g e s (20), a r e m o r e resistant to killing by 5 - F U a n d are often able to g e n e r a t e n u m e r o u s d a u g h t e r C F U S (28). W h e t h e r t h e s e cells p o s s e s s the potential to p r o d u c e c e l l s of the l y m p h o i d l i n e a g e s r e m a i n s controversial (29-31). A l t h o u g h s p l e e n c o l o n y - f o r m i n g c e l l s w e r e o n c e thought to constitute the m o s t primitive h e m o p o i e t i c c o m p a r t m e n t s i n c e they p o s s e s s a large c a p a c i t y for proliferation, a r e multi-potential a n d h a v e significant s e l f - r e n e w a l ability, recently the u s e of counterflow centrifugal elutriation h a s b e e n u s e d to s h o w that d a y 12 C F U - S c a n b e p h y s i c a l l y s e p a r a t e d from c e l l s with long term r e p o p u l a t i n g ability (32). 1.2.3. T h e  hemopoietic stem cell  It h a s b e e n p r o p o s e d that the  most  useful, rigorous definition  of  a  h e m o p o i e t i c s t e m cell s h o u l d be b a s e d on s u c h a cell having a n in vivo c a p a c i t y for the long term production of all blood cell l i n e a g e s (33). E v i d e n c e for the e x i s t e n c e of c e l l s with s u c h c h a r a c t e r i s t i c s h a s primarily c o m e from transplantation m o d e l s . F o r e x a m p l e , W u et. a l . (22) detected c o m m o n c h r o m o s o m a l a b n o r m a l i t i e s in both  5  the m y e l o i d a n d lymphoid c o m p a r t m e n t s of m y e l o a b l a t e d recipient m i c e that h a d r e c e i v e d a transplant of marrow c e l l s from d o n o r m i c e harboring u n i q u e radiation induced  chromosomal  abnormalities.  Subsequent evidence  suggesting  the  e x i s t e n c e of totipotent H S C s w a s provided by N a k a n o et. a l . (34) w h o p e r f o r m e d marrow  transplants  between  congenic  strains  of  mice  which  possessed  d i s t i n g u i s h a b l e h e m o g l o b i n a n d i s o e n z y m e m a r k e r s . M o r e o v e r , the transplant of retrovirally m a r k e d adult d a y 4 5 - F U b o n e marrow (35) or fetal liver c e l l s (36) into irradiated (35) or genetically a n e m i c W / W 1  V  (23) m i c e h a s a l s o d e m o n s t r a t e d the  e x i s t e n c e of l y m p h o - m y e l o i d repopulating s t e m c e l l s b a s e d o n the d e t e c t i o n of common  proviral  integrants  in  cells  c o m p a r t m e n t s in the recipient m i c e .  of  both  the  lymphoid  and  myeloid  A s d e s c r i b e d in the s u b s e q u e n t s e c t i o n ,  h e m o p o i e t i c s t e m c e l l s c a n n o t yet be positively identified o n the b a s i s of a n y u n i q u e m o r p h o l o g i c a l , p h y s i c a l or c e l l s u r f a c e c h a r a c t e r i s t i c s . T h u s ,  rigorous  identification of totipotent hemopoietic s t e m c e l l s relies o n functional a s s a y s b a s e d o n the ability of s u c h c e l l s to regenerate a n d s u s t a i n the h e m o p o i e t i c s y s t e m s of myeloablated  or  genetically  anemic  (ie.  W/W ) v  recipients.  A  variety  of  transplantation strategies h a v e b e e n d e s i g n e d in a n effort to quantify h e m o p o i e t i c s t e m c e l l s d e r i v e d from v a r i o u s c e l l p o p u l a t i o n s . O n e s u c h a p p r o a c h i n v o l v e s assessing  the  survival  of  irradiated  recipient  mice  for  30  days  following  transplantation with limiting n u m b e r s of marrow c e l l s (39-41). H o w e v e r , s u c h a n a s s a y is c o m p l i c a t e d by the potential contribution from residual recipient c e l l s , a n d the p o s s i b i l i t i e s that s u c h short term radioprotective c a p a c i t y m a y in part d e r i v e f r o m m o r e m a t u r e c e l l t y p e s (42, 4 3 ) . M o r e o v e r , the t r a n s p l a n t of  insufficient  n u m b e r s of c e l l s with short-term radioprotective c a p a c i t y c a n result in the d e a t h of the recipient before cells with long term repopulating c a p a c i t y c a n be r e a d out. The W locus, located on chromosome 5, encodes the receptor for Steel factor which is a hemopoietic growth factor shown to support the proliferation of both immature and lineage restricted lymphoid and myeloid progenitor cells in combination with other hemopoietic growth factors (37, 38). Mice harboring mutations at the W locus are characterized by an intrinsic defect in primitive hemopoietic stem cells and thus, the hemopoietic systems of these mice can be replaced with normal wild type hemopoietic cells without the use of conditioning regimens such as irradiation. 1  6  A s e c o n d , very powerful a p p r o a c h to the quantification of h e m o p o i e t i c s t e m c e l l s h a s b e e n the u s e of the competitive repopulation a s s a y first d e v e l o p e d by H a r r i s o n (44, 45). T h i s a s s a y is b a s e d upon c o m p a r i n g the long-term repopulating abilities of two s e p a r a t e populations of h e m o p o i e t i c c e l l s w h i c h are d i s t i n g u i s h a b l e o n the b a s i s of allelic d i f f e r e n c e s in h e m o g l o b i n a n d Gpi-1 i s o e n z y m e m a r k e r s . O n e of the cell populations (the "competitor") c o n s i s t s of a fixed n u m b e r (usually 12 x 1fj6 cells) of fresh marrow that s e r v e s a s a s t a n d a r d for repopulating potential. V a r y i n g n u m b e r s of a "donor" or "test" s o u r c e of s t e m c e l l s a r e then injected a n d the m e a n relative contribution of the two p o p u l a t i o n s to h e m o p o i e s i s m e a s u r e d . R e p o p u l a t i n g units (RU) are c a l c u l a t e d a c c o r d i n g to the formula R U = % ( C ) / ( 1 0 0 - % ) , w h e r e % is the m e a s u r e d p e r c e n t a g e of peripheral blood c e l l s in the recipient with the d o n o r p h e n o t y p e , a n d C is the n u m b e r of f r e s h c o m p e t i t o r m a r r o w c e l l s u s e d / 1 0 . E a c h repopulating unit represents the repopulating ability s h o w n by 1 x 5  10  5  f r e s h m a r r o w c e l l s from the competitor p o o l . T h i s m e t h o d h a s a n u m b e r of  a d v a n t a g e s o v e r 3 0 d a y s u r v i v a l a s s a y s . R e c i p i e n t m i c e a r e a n a l y z e d at long periods post transplant  p r o v i d i n g a r i g o r o u s m e a s u r e of s t e m c e l l f u n c t i o n .  M o r e o v e r , the short term h e m a t o l o g i c a l r e s c u e of m y e l o a b l a t e d recipient m i c e is not d e p e n d e n t u p o n the cell population b e i n g t e s t e d s i n c e life s p a r i n g d o s e s of d o n o r c e l l s are p r o v i d e d in the competitor cell p o p u l a t i o n . H o w e v e r , this m e t h o d c a n n o t be u s e d to a s s e s s properties of individual H S C s s u c h a s their proliferative c a p a c i t y s i n c e o n e cannot distinguish b e t w e e n differing proliferative c a p a c i t i e s a n d differing n u m b e r s of H S C s . T h e C o m p e t i t i v e R e p o p u l a t i n g Unit a s s a y d e v e l o p e d by S z i l v a s s y et. a l . c o m b i n e s limiting dilution a n d c o m p e t i t i v e r e p o p u l a t i n g p r o c e d u r e s to  quantify  totipotent repopulating s t e m cells. In its original form, the C R U a s s a y i n v o l v e s the co-injection of limiting n u m b e r s of m a l e test c e l l s a l o n g with a fixed n u m b e r (2 x 1 0 ) of f e m a l e c o m p e t i t o r c e l l s into m y e l o a b l a t e d f e m a l e recipient m i c e . T h e 5  competitor cells are derived from mice which h a v e u n d e r g o n e two p r e v i o u s rounds  7  of h e m o p o i e t i c transplantation a n d thus, are relatively d e p l e t e d of c e l l s with l o n g t e r m r e p o p u l a t i n g c a p a b i l i t y . T h e function of the h e l p e r c e l l s is to e n s u r e the s u r v i v a l of the m y e l o a b l a t e d recipient e v e n w h e n limiting n u m b e r s of test c e l l s are t r a n s p l a n t e d . R e c i p i e n t s d e m o n s t r a t i n g repopulation by d o n o r H S C s a r e d e f i n e d a s t h o s e with a significant contribution (ie. > 5%) from m a l e c e l l s to both the l y m p h o i d a n d m y e l o i d c o m p a r t m e n t s a s identified by probing D N A e x t r a c t e d from h e m o p o i e t i c t i s s u e s with Y c h r o m o s o m e s p e c i f i c s e q u e n c e s . T h e f r e q u e n c y of h e m o p o i e t i c s t e m c e l l s , or c o m p e t i t i v e r e p o p u l a t i n g units ( C R U ) a s t h e y a r e referred to in this a s s a y , is d e t e r m i n e d by a s s e s s i n g the proportion of recipient m i c e w h i c h meet the repopulation criteria d e s c r i b e d a b o v e w h e n limiting dilution is r e a c h e d . T h e f r e q u e n c y of C R U in a test cell population is s u b s e q u e n t l y c a l c u l a t e d u s i n g P o i s s o n statistics a c c o r d i n g to: C R U frequency = 1 / n u m b e r of m a r r o w "test" c e l l s that result in 3 7 % of recipient m i c e being negative for repopulation. T h e C R U a s s a y h a s s i n c e b e e n m o d i f i e d by the u s e of n o r m a l m a r r o w c e l l s ( 1 0 ^ ) a s competitor c e l l s (46) a n d the u s e of m o u s e strains that e n a b l e the identification of d o n o r v e r s u s recipient cells on the b a s i s of allelic differences at a g e n e e n c o d i n g a peripheral blood leukocyte cell surface antigen (Ly5.1 or Ly5.2) (46-48) ( s e e F i g u r e 1.2). T h e ability of this a s s a y to detect totipotent ( l y m p h o - m y e l o i d )  repopulating  s t e m c e l l s h a s b e e n d e m o n s t r a t e d by the o b s e r v a t i o n of c o m m o n reconstitution of l y m p h o i d a n d m y e l o i d t i s s u e s by limiting n u m b e r s of c e l l s , a n d by  retroviral  marking p r o c e d u r e s (43, 49). O n e of the great a d v a n t a g e s of the C R U a s s a y is that it c a n be u s e d to quantify quickly a n d accurately the f r e q u e n c y of h e m o p o i e t i c s t e m c e l l s with long term l y m p h o - m y e l o i d reconstituting ability in a n y population of test c e l l s (43). S z i l v a s s y a n d his c o l l e a g u e s h a v e d e m o n s t r a t e d that C R U f r e q u e n c i e s d e t e r m i n e d at 10 w e e k s post transplant w e r e virtually identical to t h o s e o b t a i n e d at later t i m e p o i n t s s u g g e s t i n g that this a s s a y c a n be u s e d to o b t a i n estimations of totipotent H S C n u m b e r s in little over two months time. 1.2.4. P h e n o t y p i n g and  purification of  8  HSCs  accurate  The  frequency  of  repopulating  HSCs  among  m o n o n u c l e a r c e l l s h a s b e e n estimated to be 1 in 1 0  4  to 1 0  adult 5  bone  marrow  (43, 5 0 - 5 3 ) . T h i s low  f r e q u e n c y a n d the fact that the identification of H S C s involves functional a s s a y s (ie. are retrospective in nature b a s e d upon the long term repopulation of irradiated or g e n e t i c a l l y a n e m i c recipient mice) h a v e h a m p e r e d direct a n a l y s i s of s e l f - r e n e w a l a n d early e v e n t s a s s o c i a t e d with H S C commitment a n d differentiation. M a j o r effort h a s t h u s b e e n directed t o w a r d s purification of cells with s t e m cell c h a r a c t e r i s t i c s . P h y s i c a l , i m m u n o l o g i c a l a n d supravital stains h a v e all b e e n e m p l o y e d a l o n e or in c o m b i n a t i o n a s s t r a t e g i e s to e n r i c h for H S C s .  Physical techniques  (velocity  s e d i m e n t a t i o n (54), density gradient s e p a r a t i o n (55-57) a n d counterflow centrifugal elutriation (32, 58)) s e p a r a t e cells on the b a s i s of buoyant density a n d / o r cell s i z e . U s i n g t h e s e t e c h n i q u e s , H S C s h a v e b e e n g e n e r a l l y c h a r a c t e r i z e d a s relatively s m a l l , low density cells with a n undifferentiated blast cell-like m o r p h o l o g y . With the advent of flow cytometry, the goal of isolating purified populations of H S C s took a major leap forward. H S C s p o s s e s s m e d i u m forward light scatter a n d low to m e d i u m o r t h o g o n a l light scatter properties indicative of intermediate  size  a n d low granularity respectively (59). T h e d e v e l o p m e n t of m o n o c l o n a l a n t i b o d i e s ( M o A b ) (60) a n d the e x i s t e n c e of lectins w h i c h bind distinct s u g a r m o i e t i e s o n the cell s u r f a c e (ie. W h e a t G e r m A g g l u t i n i n ; W G A ) h a s e n a b l e d the e n r i c h m e n t of purified s t e m c e l l s c a n d i d a t e s on the b a s i s of two distinct strategies; positive a n d n e g a t i v e s e l e c t i o n p r o c e d u r e s . P o s i t i v e s e l e c t i o n p r o c e d u r e s are b a s e d u p o n the identification a n d s e l e c t i o n of c e l l s w h i c h e x p r e s s s p e c i f i c c e l l s u r f a c e a n t i g e n s a n d / o r lectins. M o A b s d i r e c t e d a g a i n s t L y - 6 A / E ( S c a - 1 ) , M H C - c l a s s I m o l e c u l e s (31, 4 8 , 5 3 , 5 9 , 61-64), W G A (53, 65-67), Thy-1 a n d the c-kit receptor h a v e b e e n widely u s e d for the enrichment of s t e m cells from adult b o n e m a r r o w . In addition,  9  neg. control  test sample  Q.  Figure 1.2. S c h e m a t i c r e p r e s e n t a t i o n of the limiting dilution a s s a y for C R U . Limiting n u m b e r s of Ly5.1 + test c e l l s d e r i v e d from P e p C 3 F 1 d o n o r m i c e a r e c o injected a l o n g with 1 0 L y 5 . 2 helper c e l l s into B 6 C 3 F 1 (Ly5.2) recipients following 9 5 0 c G y of w h o l e b o d y irradiation. T e s t c e l l s a r e p h e n o t y p i c a l l y d i s t i n g u i s h a b l e f r o m both h e l p e r a n d s u r v i v i n g e n d o g e n o u s host c e l l s o n t h e b a s i s of a l l e l i c differences at the L y 5 l o c u s . T h e proportion of m i c e s h o w i n g > 1% L y 5 . 1 c e l l s of both l y m p h o i d a n d m y e l o i d l i n e a g e s is d e t e r m i n e d by s t a i n i n g p e r i p h e r a l b l o o d c e l l s with a n antibody s p e c i f i c a l l y r e c o g n i z i n g the Ly5.1 a n t i g e n a n d a n a l y s i s by F A C S . T h e f r e q u e n c y of C R U is then c a l c u l a t e d u s i n g P o i s s o n statistics. N B M : n o r m a l b o n e m a r r o w c e l l s o b t a i n e d from a u n m a n i p u l a t e d control m o u s e . S S C : side scatter. 5  +  10  M o A b A A 4 . 1 h a s b e e n u s e d for the isolation of H S C s from fetal liver a n d the yolk s a c of the early m o u s e e m b r y o (63, 64). Alternatively, or in addition, negative s e l e c t i o n p r o c e d u r e s c a n b e u s e d to r e m o v e c e l l s w h i c h are not of interest. H S C s derived from adult b o n e m a r r o w h a v e b e e n s h o w n not to e x p r e s s a n u m b e r of m a r k e r s c h a r a c t e r i s t i c of later l i n e a g e restricted c e l l s (68). T h u s , m a r r o w c e l l s e x p r e s s i n g s o c a l l e d l i n e a g e or " L i n " m a r k e r s c a n be identified a n d r e m o v e d using M o A b s r e c o g n i z i n g B 2 2 0 ( B - c e l l s ) , C D 4 , C D 8 , C D 3 a n d C D 5 (T-cells), Mac-1 a n d 15-1.1 ( m o n o m y e l o c y t i c cells), Gr-1 (myeloid c e l l s ) , a n d T e r 1 1 9 a n d 10-2.2 (erythroid cells) (31, 5 3 , 6 4 , 6 7 - 7 4 ) . T h e e n r i c h m e n t of s t e m c e l l s a c h i e v e d v a r i e s to certain d e g r e e s d e p e n d i n g u p o n w h i c h s e t s of M o A b s are e m p l o y e d , but generally, the s e l e c t i o n of c e l l s with either the Sca-1+Lin-Thy1.1>o  ( 3 1 , 4 8 , 75) or S c a - 1 + L i n " W G A + (53, 67) c e l l s u r f a c e  p h e n o t y p e h a v e resulted in enrichment factors of a p p r o x i m a t e l y 5 0 0 to 1 0 0 0 - f o l d . T h e s e e n r i c h m e n t factors result in detectable H S C f r e q u e n c i e s of 1 in 10 (76) to 1 in 3 0 (46) s t e m cell c a n d i d a t e s . A l t h o u g h it w o u l d s e e m that H S C s h a v e not yet b e e n purified to h o m o g e n e i t y , o n e must c o n s i d e r the possibility that the long-term repopulating a s s a y h a s a lower limit to the n u m b e r of t r a n s p l a n t e d H S C s that will reproducibly result in d o n o r reconstitution. T h i s limit m a y result from the s e e d i n g efficiency of H S C s to the marrow, competition from H S C s in the c o m p e t i t o r c e l l p o p u l a t i o n or surviving e n d o g e n o u s H S C s , or ill d e f i n e d regulatory m e c h a n i s m s that direct test H S C s into q u i e s c e n c e or to differentiate rather than s e l f - r e n e w u p o n arrival in the m a r r o w c o m p a r t m e n t . T h u s , the enrichment factor s t a t e d a b o v e m a y be a n underestimation of H S C f r e q u e n c i e s in purified s u b p o p u l a t i o n s . Differential retention of certain f l u o r e s c e n t d y e s s u c h a s R h o d a m i n e 1 2 3 ( R h 1 2 3 ) a n d H o e c h s t 3 3 3 4 2 h a s a l s o b e e n e m p l o y e d to s e p a r a t e H S C s from m o r e mature progenitors. T h e retention of t h e s e d y e s by H S C s t e n d s to be poor (41, 7 7 80); in the c a s e of R h 1 2 3 this is d u e to the functioning of a P - g l y c o p r o t e i n p u m p w h i c h actively r e m o v e s R h 1 2 3 from the cell (81). T h e u s e of s u c h d y e s c a n e n a b l e  11  a n a p p r o x i m a t e 500-fold e n r i c h m e n t of s t e m cell c a n d i d a t e s . Interestingly, s o m e p h e n o t y p i c d i f f e r e n c e s b e t w e e n H S C s d e r i v e d from differing o n t o l o g i c a l s o u r c e s h a v e b e e n reported. F o r e x a m p l e , S c a + L i n " s u b p o p u l a t i o n s e n r i c h e d for  HSCs  d e r i v e d from d a y 14.5 fetal liver stain with the A A 4 . 1 , a n t i - M a c , a n d a n t i - C D 4 5 R B a n t i b o d i e s a n d retain R h o d a m i n e 123 ( R h 1 2 3 ' 9 ) w h i l e adult b o n e m a r r o w b r  n t  S c a + L i n " c e l l s similarly e n r i c h e d for H S C s are A A 4 . 1 " , M a c - 1 " , C D 4 5 R B " a n d Rh123dull. D e s p i t e the  fact  that  i m p r e s s i v e a d v a n c e s in the  identification  and  purification of H S C s h a s b e e n m a d e o v e r the past y e a r s , a s u r f a c e m a r k e r s p e c i f i c a l l y e x p r e s s e d o n H S C s h a s not b e e n reported. M o r e o v e r , a n u m b e r of g r o u p s h a v e s h o w n that c e l l s u r f a c e p h e n o t y p e is not a l w a y s a n a c c u r a t e prediction of biological capability. R e b e l et. a l . d e m o n s t r a t e d that a l t h o u g h the in vitro c u l t u r e of S c a + L i n " W G A + c e l l s for four w e e k s in s e r u m f r e e  medium  supplemented^/vith steel factor, IL-3, IL-6 a n d E p o resulted in >1000-fold i n c r e a s e in the n u m b e r of c e l l s with the starting s u r f a c e p h e n o t y p e , the n u m b e r  of  repopulating H S C s in t h e s e cultures w a s 1.3-fold lower than input v a l u e s (46). In addition, S p a n g r u d e et. a l . d e m o n s t r a t e d that following transplantation of recipient m i c e with 2 0 0 T h y - 1 . 1  l o w  Lin"Sca+Rh-123  l o w  m a r r o w c e l l s , c e l l s with this s u r f a c e  p h e n o t y p e w e r e e x p a n d e d to a p p r o x i m a t e l y 1000-fold o v e r input l e v e l s . D e s p i t e t h i s c o n s i d e r a b l e i n c r e a s e in the n u m b e r of c e l l s with the s t a r t i n g phenotype, these cells p o s s e s s e d poor reconstituting  ability;  o n l y 8 of  s e c o n d a r y transplant recipients (9.6%) receiving 5, 10, or 2 0 R h - 1 2 3 4 0 0 Thy-1 . l '  0 W  Lin  n e  surface  l o w  83  c e l l s or  9 L y - 6 A / E + c e l l s o b t a i n e d from primary r e c i p i e n t s e x h i b i t e d  d o n o r - d e r i v e d c e l l s in p e r i p h e r a l b l o o d 12 w e e k s p o s t t r a n s p l a n t (76). T h u s , functional a s s a y s remain the most reliable a n d rigorous m e t h o d to a s s e s s for H S C numbers.  1.3.  O n t o g e n y of the murine hemopoietic  12  system  1.3.1. D e v e l o p m e n t of the  hemopoietic s y s t e m  During vertebrate ontogeny, h e m o p o i e s i s is in a state of c o n t i n u o u s c h a n g e in t e r m s of the g e n e s w h i c h are e x p r e s s e d , the cellular constituents a n d the site(s) of production. F o r e x a m p l e , during ontogeny h e m o p o i e s i s t a k e s p l a c e s e q u e n t i a l l y in the e m b r y o n i c yolk s a c , paraaortic g o n a d a l m e s e n e p h r o s r e g i o n ( A G M ) , fetal liver, s p l e e n a n d finally the adult b o n e m a r r o w . W h i l e the m o l e c u l a r s t e p s that r e g u l a t e t h e s e c h a n g e s a r e poorly u n d e r s t o o d , recent s t u d i e s h a v e b e g u n to d e l i n e a t e e a r l y e v e n t s a n d p a t t e r n s of c e l l u l a r m i g r a t i o n that c o n s t i t u t e  this  d e v e l o p m e n t a l p r o g r a m . R e c e n t l y there h a s b e e n a great d e a l of interest in the u s e of h e m o p o i e t i c cells derived from d e v e l o p m e n t a l l y early s o u r c e s s u c h a s fetal liver or umbilical c o r d blood for u s e in h u m a n b o n e marrow transplantation. T h i s interest h a s b e e n stimulated by the findings of s e v e r a l studies ( d i s c u s s e d in m o r e detail in s e c t i o n 1.3.2) hinting  that H S C s d e r i v e d from o n t o l o g i c a l l y e a r l y s o u r c e s  may  p o s s e s s a s u p e r i o r r e g e n e r a t i v e c a p a c i t y a s c o m p a r e d to adult b o n e m a r r o w H S C s . In C h a p t e r 5 of this t h e s i s data are p r e s e n t e d demonstrating a n d quantifying the s u p e r i o r regenerative c a p a c i t y of H S C s derived from fetal liver a s c o m p a r e d to adult b o n e marrow following transplant. H e m o p o i e s i s b e g i n s in the e m b r y o n i c yolk s a c at a p p r o x i m a t e l y d a y 7 . 2 5 . B e t w e e n d a y s 7.5 a n d 8.5 the yolk s a c is a s o u r c e of in vitro c l o n o g e n i c progenitors (82), B l y m p h o i d p r e c u r s o r s (83) a n d d a y 12 C F U - S (82) with H S C s c a p a b l e of long term l y m p h o - m y e l o i d repopulating ability d e t e c t e d by d a y 11 (82, 84). M o r e r e c e n t l y , s i m i l a r h e m o p o i e t i c c e l l p o p u l a t i o n s h a v e b e e n d e m o n s t r a t e d to  be  p r e s e n t within the A G M region with C F U - S activity d e t e c t a b l e o n d a y 8 to d a y 11 (85, 86) a n d long term repopulating H S C s by d a y 10 (84). T h e production of blood c e l l s within the yolk s a c a n d A G M regions b e g i n s to d e c l i n e a s the fetal liver t a k e s o v e r a s the major site of e m b r y o n i c h e m o p o i e s i s . H e m o p o i e s i s is d e t e c t a b l e in murine fetal liver by d a y 10 of gestation a n d i n c r e a s e s b e t w e e n d a y s 1 0 - 1 3 (11). T h e fetal liver r e m a i n s the major site of h e m o p o i e s i s until birth w h e r e u p o n it  13  d e c r e a s e s rapidly. Erythropoiesis is detectable in the s p l e e n by d a y 15 although by day  17 g r a n u l o p o i e s i s p r e d o m i n a t e s in this o r g a n . H e m o p o i e s i s in the s p l e e n  r e a c h e s its p e a k at a p p r o x i m a t e l y 4 - 8 d a y s following birth w h e r e it d e c r e a s e s steadily a s the marrow b e g i n s to take over a s the predominant site of h e m o p o i e s i s for the r e m a i n d e r of the a n i m a l ' s life. F i g u r e 1.3 s h o w s the contribution of v a r i o u s o r g a n s to h e m o p o i e s i s during ontological d e v e l o p m e n t . 1.3.2. C o m p a r i s o n  of stem cells from fetal liver and adult t i s s u e s  T h e relationship b e t w e e n fetal liver a n d adult b o n e m a r r o w H S C s r e m a i n s u n c l e a r . A l t h o u g h H S C s from t h e s e two differing p o p u l a t i o n s a r e b e l i e v e d to b e ontologically related, that is d e r i v e d from the s a m e initial pool of primordial s t e m cells, fetal liver cells display functional characteristics that differ from t h o s e found in the adult m a r r o w . F e t a l liver d a y 8 C F U - S c e l l s for e x a m p l e p o s s e s s a s u p e r i o r s e l f - r e n e w a l c a p a c i t y a s c o m p a r e d to their adult b o n e marrow 100  n 1 —i 10D 12D 14D 16D 18D EMBRYONIC AGE BIRTH  1  1  2D  1  4D  7TI  1  1  r  6D 8D 2Wk 4Wk 6Wk POST NATAL AGE  Figure 1.3. C h a n g i n g sites of h e m o p o i e s i s reflected in the relative proportion of in vitro c l o n o g e n i c progenitors cells found within the e m b r y o n i c yolk s a c , fetal liver, s p l e e n a n d b o n e marrow during ontogeny a n d post natally. c o u n t e r p a r t s (87), a n d fetal liver c e l l s s h o w a g r e a t e r c o m p e t i t i v e  repopulating  ability a s c o m p a r e d to adult b o n e m a r r o w c e l l s in in vivo t r a n s p l a n t a t i o n a s s a y s (88). R e b e l et. a l . h a v e s h o w n that limiting n u m b e r s of fetal liver C R U are a b l e to 14  p r o d u c e a greater output of mature blood cells in vivo a s c o m p a r e d to adult b o n e m a r r o w (89). M o r e o v e r , it w a s a l s o d e m o n s t r a t e d that w h e n m a r r o w c e l l s from primary  r e c i p i e n t s of limiting  n u m b e r s of fetal liver C R U w e r e i n j e c t e d  into  s e c o n d a r y recipients, a significantly higher p e r c e n t a g e of t h e s e s e c o n d a r y m i c e showed  donor  cell-derived  reconstitution  of  their  lymphoid  and  myeloid  c o m p a r t m e n t s a s c o m p a r e d to m i c e that h a d r e c e i v e d m a r r o w c e l l s from primary recipients of similar n u m b e r s of adult b o n e marrow C R U . O n e p o s s i b l e e x p l a n a t i o n for the d i f f e r e n c e s in regenerative potential b e t w e e n t h e s e two cell p o p u l a t i o n s is that fetal liver C R U p o s s e s s a g r e a t e r intrinsically d e t e r m i n e d p r o b a b i l i t y  to  u n d e r g o s e l f - r e n e w a l v e r s u s differentiation d i v i s i o n s w h e n proliferating within the adult m a r r o w m i c r o e n v i r o n m e n t . V a s i r i et. a l . (90) h a v e recently p r o p o s e d the t h e o r y that the p r o g r e s s i v e l o s s of t e l o m e r i c D N A with e a c h r o u n d of c e l l u l a r division m a y act a s a mitotic c l o c k a n d therefore be the m o l e c u l a r b a s i s by w h i c h fetal liver c e l l s m a y be a b l e to u n d e r g o a greater n u m b e r of cell d i v i s i o n s prior to undergoing  senescence.  H o w e v e r , this  explanation  d o e s not  preclude  the  possibility that fetal liver H S C s m a y maintain differences in cell c y c l e t i m e s or the n u m b e r of c e l l s recruited or m a i n t a i n e d within the m i c r o e n v i r o n m e n t of the adult m a r r o w , or e x p r e s s additional intrinsically d e f i n e d m o l e c u l e s that m a y direct the c e l l into f a v o r i n g s e l f - r e n e w a l v e r s u s differentiation  d i v i s i o n s . Intriguing n e w  e v i d e n c e of p o s s i b l e intrinsic determinants of self-renewal w a s recently reported by S a u v a g e a u et. a l . in s t u d i e s of the H o m e o b o x family of transcription f a c t o r s (91). E n g i n e e r e d o v e r e x p r e s s i o n of H O X B 4 , a g e n e w h o s e e x p r e s s i o n is n o r m a l l y restricted to the most primitive adult bone marrow cells, w a s found to result in up to a  50-fold  i n c r e a s e in the  r e g e n e r a t i o n of retrovirally  c o m p a r e d to m a r k e r g e n e - t r a n s d u c e d control cells.  1.4.  Properties of hemopoietic stem  1.4.1. C y c l i n g  status  15  cells  transduced H S C s  as  In contrast to c o m m i t t e d c l o n o g e n i c progenitors a n d a large proportion of d a y 8 a n d 12 C F U - S , the vast majority of long term repopulating H S C s exist in a state of q u i e s c e n c e within the m a r r o w u n d e r n o r m a l h o m e o s t a t i c c o n d i t i o n s a s d e t e r m i n e d by their ability to survive a single injection of the c y c l e - s p e c i f i c cytotoxic drug 5 - F U (92). H o w e v e r , following treatment with 5 - F U the m o s t primitive  HSCs  a r e recruited into c y c l e s i n c e they b e c o m e highly sensitive to a s e c o n d d o s e of 5F U given 3-5 d a y s later (93). 1.4.2. D e v e l o p m e n t a l potential and  d y n a m i c s of  A l t h o u g h the e x i s t e n c e of totipotent H S C s  HSCs  is n o w a w e l l  documented  p h e n o m e n o n , the n u m b e r of H S C s a n d their u s a g e in s t e a d y state h e m o p o i e s i s r e m a i n s u n c l e a r . O n e theory that h a s b e e n put forth to e x p l a i n the d y n a m i c s of H S C utilization is the c l o n a l s u c c e s s i o n m o d e l p r o p o s e d by K a y in 1 9 6 5 (94). T h i s m o d e l p r o p o s e s that h e m o p o i e s i s is m a i n t a i n e d by the s e q u e n t i a l activation of s t e m cell c l o n e s that proliferate, differentiate a n d eventually b e c o m e e x h a u s t e d . In this m o d e l H S C s are akin to fuel for h e m o p o i e s i s , continually b e i n g a c t i v a t e d a n d " c o n s u m e d " from a r e s e r v e pool of c e l l s . A significant b o d y of d a t a from b o n e m a r r o w transplantation s t u d i e s h a s provided support for this theory. S t u d i e s b a s e d u p o n the t r a n s p l a n t of v a r i o u s m i x t u r e s of retrovirally m a r k e d or e n z y m a t i c a l l y distinguishable adult b o n e marrow or fetal liver s t e m cells into either irradiated (24, 95) or g e n e t i c a l l y a n e m i c W / W  v  m i c e (36, 96) r e v e a l e d major fluctuations in the  contribution of individual H S C s to h e m o p o i e s i s within the first six m o n t h s following transplant. During this period n u m e r o u s H S C c l o n e s w e r e o b s e r v e d to contribute significantly but transiently to h e m o p o i e s i s , the s p a n of their contribution lasting on a v e r a g e for only a few months. Further e v i d e n c e supporting the c l o n a l s u c c e s s i o n theory w a s a l s o o b t a i n e d from large a n i m a l m o d e l s . A b k o w i t z et. a l . p r o v i d e d d a t a demonstrating  large  chromosome-linked  clonal enzyme  fluctuations  in  cats  heterozygous  for  glucose-6-phosphate dehydrogenase  the  X  (G6PD)  following t r a n s p l a n t s of a u t o l o g o u s m a r r o w (97). H o w e v e r , the a n i m a l s u s e d in  16  t h e s e e x p e r i m e n t s w e r e only a n a l y z e d for relatively short p e r i o d s of time following transplant  (ie.  respectively).  1-5  months  or 1-1.5  Subsequent studies  y e a r s for m u r i n e using  retrovirally  and feline marked  recipients  marrow  cells  s e q u e n t i a l l y a n a l y z e d the c l o n a l contribution to the p e r i p h e r a l b l o o d of m u r i n e recipients for longer p e r i o d s of time (ie. 7-12 months) post transplant (64, 9 8 , 99). F o l l o w i n g a p e r i o d of c l o n a l instability for up to 6 m o n t h s p o s t  transplant,  h e m o p o i e s i s b e c a m e d o m i n a t e d by a s m a l l n u m b e r of totipotent s t e m cell c l o n e s that c o n t i n u e d to function for long p e r i o d s of time, often for the r e m a i n d e r of the a n i m a l ' s life. Interestingly, the c l o n a l fluctuations o b s e r v e d in felines t r a n s p l a n t e d with a l l o g e n e i c m a r r o w d e s c r i b e d by A b k o w i t z et. a l . in 1 9 9 0 (97) w e r e f o u n d to stabilize w h e n t h e s e s a m e recipients w e r e a n a l y z e d at longer t i m e s post transplant (3.5-6 y e a r s ) (100). O n e e x p l a n a t i o n for the c l o n a l fluctuation initially o b s e r v e d following transplant is that it is d u e to the utilization of m o r e "mature" s t e m c e l l s w h i c h p o s s e s s a limited c a p a c i t y for proliferation a n d s e l f - r e n e w a l . H S C s  are  b e l i e v e d to be o r g a n i z e d a s a hierarchy of c e l l s with d e c r e a s i n g s e l f - r e n e w a l a n d proliferative potential (refer to F i g u r e 1.1) a n d heterogeneity with r e g a r d s to both p h y s i c a l a n d functional properties of H S C s h a v e b e e n d o c u m e n t e d . T h u s , m o r e m a t u r e H S C s w o u l d quickly b e c o m e e x h a u s t e d a n d e v e n t u a l l y be r e p l a c e d by m o r e primitive H S C s w h i c h p o s s e s s a m o r e long-lived c a p a c i t y for and  proliferation  s e l f - r e n e w a l . A n alternative e x p l a n a t i o n of H S C b e h a v i o r f o l l o w i n g  bone  m a r r o w transplant h a s b e e n provided by J o r d a n a n d L e m i s c h k a (98). T h e a u t h o r s p r o p o s e d that the initial period of c l o n a l fluctuation o b s e r v e d w a s the result of a n e x p a n d i n g pool of totipotent H S C s undergoing s t o c h a s t i c c o m m i t m e n t v e r s u s selfr e n e w a l e v e n t s . T h e s e e v e n t s are portrayed in Figure 1.4. A c c o r d i n g to this m o d e l , the c l o n a l fluctuation o b s e r v e d within the first six months post transplant (depicted in F i g u r e 1.4A) a r e the c o n s e q u e n c e of either s t o c h a s t i c m e c h a n i s m s or the d e m a n d s of the radiation-ablated h e m o p o i e t i c s y s t e m resulting in the c o m m i t m e n t of the H S C s without significant s e l f - r e n e w a l . T h u s , a l t h o u g h this c l o n e initially  17  c o n t r i b u t e s to h e m o p o i e s i s , without s e l f - r e n e w a l it is u n a b l e to m a i n t a i n  its  n u m b e r s a n d is e x h a u s t e d . S t e m cell c l o n e s w h i c h are o b s e r v e d to contribute to h e m o p o i e s i s at both short a n d long p e r i o d s post t r a n s p l a n t a r e t h e result of c o m m i t m e n t d e c i s i o n s w h i c h o c c u r in parallel with a significant d e g r e e of selfr e n e w a l ( F i g u r e 1.4B). A l t e r n a t i v e l y , s o m e c l o n e s m a y u n d e r g o a s u b s t a n t i a l d e g r e e of s e l f - r e n e w a l shortly after transplant. S u c h c l o n e s , a l t h o u g h they w o u l d not  b e o b s e r v e d to c o n t r i b u t e  to h e m o p o i e s i s d u r i n g t h e  initial  period  of  r e g e n e r a t i o n w o u l d ultimately c o m e to d o m i n a t e h e m o p o i e s i s for lengthy p e r i o d s of time, p o s s i b l y for the remainder of the animal's life (Figure 1.4C).  A.  B.  Commitment  Self-Renewal + Commitment  o  o  • • M Extinct  L  Self-Renewal  O / \  /\ /\  > M  C.  o  o  /\ •  L /  \  ^  O  o  O •  / \i  M  o  nonO  / \ / \  o  contributing  o o  Continued Expansion  L  o  o  Months PostTransplant  4-6  Continued Expansion  • •OO M  • •OO  L  M  Stable  L Stable  12"16  F i g u r e 1.4. M o d e l p r o p o s e d by J o r d a n a n d L e m i s c h k a (64) to e x p l a i n t h e b e h a v i o r of H S C s at v a r i o u s time points following b o n e m a r r o w transplant. O p e n c i r c l e s d e s i g n a t e totipotent H S C s while filled c i r c l e s r e p r e s e n t c e l l s c o m m i t t e d to either m y e l o i d or lymphoid l i n e a g e s .  18  In s u m m a r y , it r e m a i n s u n c l e a r w h e t h e r the c l o n a l f l u c t u a t i o n  initially  o b s e r v e d following b o n e m a r r o w transplant is r e p r e s e n t a t i v e of the utilization of m o r e mature H S C s with limited proliferative a n d s e l f - r e n e w a l c a p a c i t i e s , or is the result of s t o c h a s t i c m e c h a n i s m s acting upon an e x p a n d i n g pool of totipotent H S C s . 1.4.3. Self-renewal and  a g i n g of  HSCs  S t u d i e s u s i n g retroviral m a r k i n g of H S C s h a v e d e m o n s t r a t e d c o n v i n c i n g l y the ability of H S C s to u n d e r g o self-renewal both in vitro (101) a n d in vivo (24, 9 8 , 99) through the detection of identical proviral b a n d i n g patterns in the b o n e m a r r o w a n d / o r t h y m u s of m o r e t h a n o n e primary or s e c o n d a r y recipient. A l t h o u g h the s t u d i e s m e n t i o n e d a b o v e directly demonstrate that s e l f - r e n e w a l of H S C s c a n o c c u r it remains  unknown  to what extent this process  can occur s i n c e the m a g n i t u d e of  s e l f - r e n e w a l e v e n t s h a v e not b e e n d o c u m e n t e d . T h i s h a s b e e n in part d u e to the fact that quantitative a s s a y s for m e a s u r i n g s t e m c e l l s with l o n g t e r m in v i v o reconstituting  ability w e r e not a v a i l a b l e until relatively r e c e n t l y ( 4 3 , 4 5 ) . A s  p r e v i o u s l y s t a t e d , the requirement for a functional a s s a y w h e n a n a l y z i n g long term repopulating s t e m cell function is e s s e n t i a l s i n c e a n u m b e r of g r o u p s h a v e s h o w n that cell s u r f a c e p h e n o t y p e is not a n a c c u r a t e determination of biological capability (46, 76). M o r e o v e r , the inefficiency of retroviral m a r k i n g p r o c e d u r e s h a s m a d e the t r a c k i n g of individual H S C s in vivo laborious. T h u s , m e t h o d s w h i c h i n c r e a s e the utility of r e c o m b i n a n t retroviral m a r k i n g w o u l d be a d v a n t a g e o u s t o w a r d s s t u d i e s a i m e d at m o r e clearly defining the regenerative potential of H S C s . C h a p t e r 3 of this t h e s i s d e s c r i b e s the d e v e l o p m e n t of m e t h o d s to e n h a n c e the p o w e r of retroviral marking procedures. T h a t H S C s h a v e a n unlimited ability to replenish their n u m b e r s through the p r o c e s s of s e l f - r e n e w a l h a s b e e n brought into q u e s t i o n by a n u m b e r of s t u d i e s d e m o n s t r a t i n g that e v e n a s i n g l e t r a n s p l a n t a t i o n  p r o c e d u r e c a n r e d u c e the  repopulating ability of b o n e marrow dramatically (102-109). F o r e x a m p l e , u s i n g the T 6 c h r o m o s o m e m a r k e r s y s t e m , R o s s et. a l . d e m o n s t r a t e d that the proportion of  19  d o n o r cell m i t o s e s in recipients of b o n e marrow w h i c h h a d b e e n s u b j e c t e d to o n e round of transplantation w a s only 1 0 % of that d e t e c t e d w h e n n o r m a l b o n e m a r r o w w a s u s e d (102). T h e possibility that the l o s s in repopulating ability of b o n e m a r r o w following transplant is d u e to d a m a g e to the recipients m i c r o e n v i r o n m e n t by the irradiation r e g i m e n w a s e x c l u d e d s i n c e s i m i l a r r e s u l t s w e r e o b s e r v e d u s i n g genetically anemic W / W  v  recipient m i c e . M a u c h a n d H e l l m a n (110) e v a l u a t e d the  long-term c o n s e q u e n c e s of transplanting limiting n u m b e r s of h e m o p o i e t i c c e l l s a n d d e m o n s t r a t e d a direct relationship b e t w e e n d o n o r cell d o s e a n d d a y 8 C F U - S r e c o v e r y a n d s e l f - r e n e w a l c a p a c i t y that w a s not r e f l e c t e d in p e r i p h e r a l b l o o d c o u n t s or b o n e m a r r o w cellularity. Further, the d e c r e a s e d r e c o v e r y of p a r a m e t e r s d i d not c h a n g e with t i m e after t r a n s p l a n t a t i o n  CFU-S  s u g g e s t i v e of  a  p e r m a n e n t l o s s of m a r r o w r e g e n e r a t i v e c a p a c i t y . H a r r i s o n et. a l . h a v e p r o v i d e d d a t a s u g g e s t i n g that t h e  r e d u c t i o n in H S C n u m b e r a n d f u n c t i o n  following  transplant is the result of two m e c h a n i s m s (111). T h e y c a l c u l a t e d that t r a n s p l a n t e d m a r r o w s h o w e d a two-fold reduction in s t e m cell n u m b e r s a s c o m p a r e d to f r e s h marrow. M o r e o v e r , they found that the a v e r a g e proportion of d o n o r d e r i v e d c e l l s in r e c i p i e n t s of p r e v i o u s l y t r a n s p l a n t e d m a r r o w w a s a p p r o x i m a t e l y 1 5 % of that of f r e s h m a r r o w . T h u s , t r a n s p l a n t e d m a r r o w d e m o n s t r a t e d a s e v e n - to eight-fold r e d u c t i o n in r e p o p u l a t i n g ability a s c o m p a r e d to f r e s h m a r r o w . T h e s e r e s u l t s s u g g e s t that not only are transplant recipients c o m p r o m i s e d in t e r m s of the quantity of s t e m c e l l s w h i c h they p o s s e s s but they are a l s o deficient in t e r m s of the quality of s t e m c e l l s . O n e e x p l a n a t i o n for the l o s s in the r e g e n e r a t i v e c a p a c i t y of b o n e m a r r o w following transplant is the inability of transplanted s t e m c e l l s to reconstitute fully t h e H S C c o m p a r t m e n t to n o r m a l (non-transplant) l e v e l s d u e to a limited c a p a c i t y for s e l f - r e n e w a l . A n alternative h y p o t h e s i s is that transplantation p l a c e s a high d e g r e e of s t r e s s o n the H S C s to u n d e r g o differentiative rather t h a n selfr e n e w a l e v e n t s in order to provide sufficient n u m b e r s of functionally mature c e l l s to reconstitute h e m o p o i e s i s in the m y e l o a b l a t e d recipient. A n o t h e r possibility is that  20  H S C regeneration following b o n e marrow transplant is limited d u e to the action of negative regulatory f e e d b a c k m e c h a n i s m s in vivo that c a n limit s t e m cell e x p a n s i o n prematurely e v e n though the H S C compartment is far from being r e g e n e r a t e d b a c k to n o r m a l levels. T h i s s c e n a r i o might o c c u r through the production of s u c h factors a s M I P - 1 a or T G F - p that h a v e b e e n s h o w n to d e c r e a s e the proportion of primitive h e m o p o i e t i c c e l l s in c y c l e (112-115). T h e o b s e r v e d d e c l i n e in r e p o p u l a t i n g c a p a c i t y f o l l o w i n g transplant  bone  marrow  r a i s e d the q u e s t i o n a s to w h e t h e r H S C s within the m a r r o w of o l d  u n m a n i p u l a t e d m i c e might a l s o s h a r e s u c h a defect. T h e h y p o t h e s i s , c a l l e d the g e n e r a t i o n - a g e h y p o t h e s i s (116), s u g g e s t e d that a n entire lifetime d e m a n d o n the HSCs  might  result  in a p o o l  of  HSCs  which  were  compromised  in  their  regenerative c a p a c i t y s i n c e they w e r e forced to h a v e u n d e r g o n e a greater n u m b e r of cell division e v e n t s . H o w e v e r , using the competitive repopulation a s s a y H a r r i s o n a n d his c o l l e a g u e s f o u n d no d i f f e r e n c e s in the r e p o p u l a t i n g ability b e t w e e n the m a r r o w of y o u n g a n d old d o n o r s (117). B o n e m a r r o w d e r i v e d from m i c e 2 to 2.5 y e a r s in a g e c o m p e t e d e q u a l l y well a g a i n s t the c o m p e t i t o r c e l l fraction a s d i d m a r r o w c e l l s d e r i v e d from y o u n g m i c e 3 to 6 months of a g e . M o r e o v e r , it w a s a l s o s h o w n that b o n e m a r r o w d e r i v e d from old m i c e c o m p e t e d e q u a l l y well in s e r i a l transplantation e x p e r i m e n t s a s t h o s e d e r i v e d from y o u n g m i c e . F o r e x a m p l e , the proportion of d o n o r d e r i v e d c e l l s in recipients of p r e v i o u s l y t r a n s p l a n t e d  marrow  d e r i v e d from either y o u n g or old donor m i c e w a s approximately 1 0 % a s c o m p a r e d to  fresh,  non-transplant  marrow.  Thus, although  data  from  murine  serial  transplantation s t u d i e s h a v e s u g g e s t e d that H S C s m a y h a v e a finite c a p a c i t y for s e l f - r e n e w a l it s e e m s , u s i n g the a s s a y s a v a i l a b l e , that this limit is not r e a c h e d during the normal life s p a n of the a n i m a l . A l t h o u g h the findings of n u m e r o u s studies s u g g e s t that the c a p a c i t y of H S C s to u n d e r g o s e l f - r e n e w a l is not unlimited, few d a t a are a v a i l a b l e o n the extent  to  w h i c h s e l f - r e n e w a l of H S C s from various ontological s o u r c e s c a n o c c u r . S u c h d a t a  21  w o u l d b e v a l u a b l e in light of the crucial role that H S C s play in a n u m b e r of clinical p r o c e d u r e s including autograft p u r g i n g , the g e n e t i c t h e r a p y of v a r i o u s heritable h e m a t o l o g i c a l d i s o r d e r s , attempts at e x vivo e x p a n s i o n of H S C s a s well a s the u s e of alternative s o u r c e s of t r a n s p l a n t a b l e s t e m cell p o p u l a t i o n s (ie. fetal liver a n d u m b i l i c a l c o r d b l o o d ) . C h a p t e r 5 of this t h e s i s d e s c r i b e s d a t a c o l l e c t e d o n t h e regenerative c a p a c i t y of H S C s derived from adult b o n e marrow or fetal liver.  1.5. R e g u l a t i o n of  hemopoiesis  T h e p r o c e s s by w h i c h a s m a l l n u m b e r of H S C s c o n t i n u o u s l y g e n e r a t e t h e appropriate  n u m b e r of mature blood c e l l s constituting the eight major h e m o p o i e t i c  l i n e a g e s is a c o m p l e x , regulated p r o c e s s . M o r e o v e r , the entry of mature blood c e l l s into t h e c i r c u l a t i o n , their l o c a l i z a t i o n to the a p p r o p r i a t e t i s s u e s a s w e l l a s their functional activation is a l s o under strict regulation. T h e k n o w n m o l e c u l e s w h i c h a r e r e s p o n s i b l e for regulating t h e s e v a r i o u s a s p e c t s of h e m o p o i e s i s c a n g e n e r a l l y b e d i v i d e d into two g r o u p s : extracellular factors ( c o m p o s e d of both h u m o r a l factors a n d cell o r m a t r i x - a s s o c i a t e d factors), a n d intracellular factors ( e g . growth factor r e c e p t o r s a n d transcription factors). A l t h o u g h there is a n a b u n d a n c e of e v i d e n c e supporting the role of m o l e c u l e s belonging to both of t h e s e g r o u p s in the regulation of h e m o p o i e s i s their relative i m p o r t a n c e a n d relationship to o n e a n o t h e r i s , at present, unclear. 1.5.1. R e g u l a t i o n by extracellular factors 1.5.1.1. H e m o p o i e t i c growth f a c t o r s The  m o s t e x t e n s i v e l y c h a r a c t e r i z e d g r o u p of e x t r i n s i c f a c t o r s a r e t h e  h e m o p o i e t i c growth factors. T h i s group is c o m p o s e d of the h e m o p o i e t i c c o l o n y s t i m u l a t i n g f a c t o r s ( G - C S F , M - C S F , G M - C S F ) , t h e interleukins (IL-1 to IL-17), h e m o p o i e t i c inhibitors (TGF-|3 a n d MIP-1oc) a n d "stem cell" factors (Steel factor a n d flk2/flt3 ligand). T h e s e factors a r e glycoproteins of 10-70 kilodaltons, a r e a b l e to act at e x t r e m e l y low c o n c e n t r a t i o n s (ie. 10"6 M ) , a n d a r e p r o d u c e d by a variety of cell  22  t y p e s throughout the b o d y (reviewed in (118) a n d (38)). T o d a t e m o r e t h a n 2 5 distinct h e m o p o i e t i c growth factors h a v e b e e n identified a n d the g e n e s for m o s t h a v e b e e n c l o n e d . T h e s e factors h a v e b e e n s h o w n to play a role in the s u r v i v a l , proliferation  and  differentiation  of  hemopoietic  c e l l s at v a r i o u s  stages  of  d e v e l o p m e n t (119). B y far, the majority of d a t a that h a v e b e e n c o l l e c t e d o n the f u n c t i o n of t h e s e growth f a c t o r s h a s b e e n their a c t i o n o n the m o r e  mature  h e m o p o i e t i c c e l l t y p e s . M u c h l e s s d a t a r e g a r d i n g their e f f e c t s u p o n H S C s  is  available. H o w e v e r , s e v e r a l growth factors h a v e b e e n identified that are involved in the activation a n d regulation of the proliferation of primitive h e m o p o i e t i c c e l l s a n d their p r o g e n y . E n t r y of q u i e s c e n t blast c o l o n y f o r m i n g c e l l s into the c e l l c y c l e is regulated by multiple synergistic factors, including IL-6 (120), 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 ( G - C S F ) (121), IL-11 (122, 123), IL-3 (124), a n d S t e e l factor (125). In contrast, factors s u c h a s M I P - 1 a a n d T G F - p h a v e b e e n s h o w n to inhibit the entry of primitive murine a n d h u m a n progenitors into the cell c y c l e (113, 126, 127). In addition to bringing q u i e s c e n t primitive h e m o p o i e t i c c e l l s into c y c l e , S t e e l factor, the ligand for the c-kit receptor, a l s o plays a role in the proliferation of t h e s e cells. A l t h o u g h S t e e l factor by itself h a s little effect upon the proliferation of primitive h e m o p o i e t i c c e l l s , w h e n c o m b i n e d with o t h e r s f a c t o r s s u c h a s IL-3, G M - C S F , g r a n u l o c y t e colony-stimulating factor ( G - C S F ) , IL-4,  IL-1oc,  IL-11 a n d IL-12, S t e e l  factor h a s a potent effect u p o n the proliferation of both i m m a t u r e a n d l i n e a g e restricted l y m p h o i d a n d m y e l o i d c e l l s ( 1 2 8 - 1 3 5 ) . M o r e recently a growth  factor  w h o s e r e c e p t o r is e x p r e s s e d o n a m o r e primitive s u b g r o u p of h e m o p o i e t i c c e l l s h a s b e e n d e s c r i b e d . U s i n g Northern blot a n d r e v e r s e t r a n s c r i p t a s e - p o l y m e r a s e c h a i n reaction ( R T - P C R ) a n a l y s i s , e x p r e s s i o n of the flt3/flk2 r e c e p t o r w a s largely c o n f i n e d to primitive h e m o p o i e t i c cell p o p u l a t i o n s w h i c h i n c l u d e c e l l s with l o n g t e r m l y m p h o - m y e l o i d r e p o p u l a t i n g ability (136, 137). S i m i l a r l y , in h u m a n s , the  23  flt3/flk2 r e c e p t o r is e x p r e s s e d on t h y m i c , s p l e n i c a n d b o n e m a r r o w  progenitors  p o s i t i v e for e x p r e s s i o n of the C D 3 4 c e l l s u r f a c e a n t i g e n , a m a r k e r f o u n d o n primitive h e m o p o i e t i c cells a n d endothelial cells (138, 139). T h e flt3/flk2 ligand (FL) ( 1 4 0 , 141) is a b l e to stimulate the proliferation of primitive m u r i n e a n d h u m a n h e m o p o i e t i c p r o g e n i t o r c e l l s w h e n c o m b i n e d with o t h e r h e m o p o i e t i c  growth  factors, reminiscent of the effects of S t e e l factor. C o m b i n e d with S t e e l factor a l o n e , or S t e e l f a c t o r / I L - 3 / G M - C S F , F L i n c r e a s e s the proportion of A A 4 . 1 + S c a - 1 + L i n g h i  m u r i n e fetal liver c e l l s in c y c l e a s d e t e r m i n e d by [3|H]thymidine  n  incorporation  a s s a y ( 1 4 0 ) a n d the n u m b e r of in vitro c o l o n i e s in c l o n o g e n i c a s s a y s following plating of T h y ' ° S c a - 1 L i n " s t e m cell c a n d i d a t e s purified from murine b o n e m a r r o w +  (142). Furthermore, w h e n c o m b i n e d with IL-7, F L i n d u c e s proliferation of immature d a y 14 m u r i n e fetal T - c e l l p r e c u r s o r s s u g g e s t i n g a role for F L in  lymphoid  d e v e l o p m e n t (142). A l t h o u g h both S t e e l factor a n d F L a p p e a r to be important early in h e m o p o i e s i s t h e s e factors a r e not e s s e n t i a l s i n c e m i c e l a c k i n g e x p r e s s i o n of S t e e l factor, F L or their receptors, d u e to natural mutations or g e n e k n o c k o u t , are still v i a b l e ( 1 4 3 , 144). T h e s e o b s e r v a t i o n s s u g g e s t the e x i s t e n c e of a d d i t i o n a l u n k n o w n s t e m cell factors a n d / o r known growth factors a b l e to c o m p e n s a t e for the l o s s of e x p r e s s i o n of S t e e l factor/FL or their receptors. W h i l e s o m e growth f a c t o r s play a role in the d e v e l o p m e n t of c e l l s of n u m e r o u s h e m o p o i e t i c l i n e a g e s (ie. IL-3), others a p p e a r to b e m o r e restricted. F o r e x a m p l e , erythropoietin, t h r o m b o p o i e t i n , G - C S F a n d M - C S F p r e d o m i n a n t l y act in the  development  of  erythrocytes,  platelets,  neutrophilic  granulocytes  and  m a c r o p h a g e s / m o n o c y t e s respectively. M o r e o v e r , s e v e r a l of the interleukins (ie. IL2 a n d IL-7) function predominantly in lymphoid d e v e l o p m e n t . 1.5.1.2. T h e  extracellular  matrix  The hemopoietic microenvironment  is c o m p o s e d of a v a r i e t y of n o n -  hemopoietic cells which include fibroblasts, adipocytes, endothelial cells a n d reticular c e l l s w h i c h t o g e t h e r p r o d u c e the e x t r a c e l l u l a r matrix.  24  Morphological  studies have demonstrated a close physical association between stromal and b l o o d c e l l s within the marrow cavity (145-147). In addition to p r o d u c i n g a variety of growth factors (147) ,the a b o v e cell types a l s o p r o d u c e a n u m b e r of other proteins w h i c h h a v e b e e n h y p o t h e s i z e d to play a role in regulating the s u r v i v a l , proliferation a n d differentiation of h e m o p o i e t i c c e l l s by facilitating c o m m u n i c a t i o n b e t w e e n c e l l s v i a cell s u r f a c e receptors a n d through the l o c a l i z e d concentration a n d presentation of s e q u e s t e r e d growth factors to hemopoietic cells. E x a m p l e s of the different t y p e s of p r o t e i n s c o m p o s i n g the e x t r a c e l l u l a r matrix i n c l u d e a n u m b e r of c o l l a g e n s ( T y p e s I, III, IV a n d V ) , g l y c o p r o t e i n s ( t h r o m b o s p o n d i n , fibronectin, h e m o n e c t i n , laminins a n d t e n a s c i n ) , a n d g l y c o s a m i n o g l y c a n s (hyaluronic a c i d , h e p a r a n sulfate, d e r m a t a n sulfate a n d chondroitin sulfate). G l y c o p r o t e i n s s u c h a s fibronectin a n d t h r o m b o s p o n d i n a s well a s g l y c o s a m i n o g l y c a n s s u c h a s hyaluronic acid a n d h e p a r i n sulfate are thought to function in h e m o p o i e s i s by acting u p o n on primitive hemopoietic  progenitors.  For e x a m p l e , fibronectin  and thrombospondin  are  a d h e s i v e ligands for a variety of h u m a n progenitors including C F U - G E M M , B F U - E , C F U - G M a n d L T C - I C . ( 1 4 8 , 149). M o r e o v e r , hyaluronic a c i d , h e p a r i n sulfate a n d chondroitin sulfate are involved in the a d h e s i o n of primitive h u m a n progenitors to s t r o m a in long term cultures in vitro a n d are thought to e n h a n c e h e m o p o i e s i s either by binding both primitive progenitors a n d growth factors that c a n stimulate t h e m , by c o n c e n t r a t i n g t h e s e growth factors at the site of their c e l l u l a r r e c e p t o r s , or by e n h a n c i n g the a t t a c h m e n t of primitive c e l l s to the s t r o m a l f e e d e r l a y e r (150). Interestingly, a n i n c r e a s e in the p r o d u c t i o n of c h o n d r o i t i n s u l f a t e in l o n g t e r m cultures h a s b e e n correlated with a n e n h a n c e m e n t of d a y 10 C F U - S a n d C F U - G M p r o d u c t i o n (151). In addition, integrins s u c h a s L F A - 1 a n d V L A - 4 a n d the C D 4 4 g l y c o p r o t e i n w h i c h a r e e x p r e s s e d o n C D 3 4 + h u m a n b o n e m a r r o w c e l l s (152) a p p e a r to b e important in m e d i a t i n g interactions b e t w e e n primitive h e m o p o i e t i c progenitors a n d s t r o m a s i n c e the addition of a n t i b o d i e s directed a g a i n s t V L A - 4 or C D 4 4 retards l y m p h o - a n d m y e l o p o i e s i s in long term cultures (153, 154). Further,  25  V L A - 4 interactions with fibronectin, V C A M - 1 a n d L-selectin h a v e b e e n implicated in mediating the in vivo h o m i n g of hemopoietic progenitors to the m a r r o w a n d s p l e e n a n d the s a m e interactions m a y be involved in the mobilization of progenitors from the marrow to the blood (149, 155). T h e a b o v e d a t a strongly s u g g e s t that a d h e s i o n m o l e c u l e s a n d m o l e c u l e s of the e x t r a c e l l u l a r matrix p l a y a role in the regulation of primitive h e m o p o i e t i c progenitor c e l l s (and p o s s i b l y totipotent H S C s ) . T h e s e m o l e c u l e s m a y function by i n c r e a s i n g the r e s p o n s e of progenitors cells to v a r i o u s h e m o p o i e t i c growth factors s i n c e t h r o m b o s p o n d i n h a s b e e n o b s e r v e d to i n c r e a s e the r e s p o n s e of the c e l l s to IL-3 a n d G M - C S F (156). 1.5.2. R e g u l a t i o n by intracellular factors S i g n a l t r a n s d u c t i o n v i a growth factors i n v o l v e s the b i n d i n g of the g r o w t h f a c t o r to its a p p r o p r i a t e r e c e p t o r f o l l o w e d by d i m e r i z a t i o n of the r e c e p t o r a n d initiation of d o w n s t r e a m s i g n a l i n g p a t h w a y s involving a variety of c y t o p l a s m i c i n t e r m e d i a t e s s u c h a s protein k i n a s e s a n d p h o s p h a t a s e s a n d their s u b s t r a t e s . G r o w t h factors are b e l i e v e d to influence a variety of cellular functions including cell s u r v i v a l , c y c l i n g status, proliferation a n d p o s s i b l y differentiation. A l t h o u g h m a n y of the d o w n s t r e a m  m o l e c u l e s i n v o l v e d in t h e s e p a t h w a y s  h a v e not yet  identified, s e v e r a l transcription factors are n o w r e c o g n i z e d a s k e y  been  regulators in  h e m o p o i e s i s . T r a n s c r i p t i o n factors that a p p e a r to be i n v o l v e d in the regulation of early h e m o p o i e t i c cell d e v e l o p m e n t a n d self-renewal h a v e recently b e e n identified through o v e r e x p r e s s i o n a n d g e n e disruption e x p e r i m e n t s (157). Disruption of the G A T A - 2 g e n e in m i c e results in a m a r k e d reduction in all h e m o p o i e t i c progenitors (158). M o r e o v e r , the o v e r e x p r e s s i o n of H O X B 4 h a s b e e n s h o w n to b e a s s o c i a t e d with a n i n c r e a s e in the r e g e n e r a t i v e ability of H S C s f o l l o w i n g b o n e  marrow  transplant (91). S o m e factors are m o r e restricted in their patterns of e x p r e s s i o n . T h e S C L / T a l - 1 a n d Ikaros transcription factors, for e x a m p l e , are e x p r e s s e d in c e l l s of the myeloid/erythroid a n d lymphoid l i n e a g e s respectively (159, 160). A n u m b e r  26  of transcription factors that a p p e a r to be involved in the d e v e l o p m e n t a l regulation of s p e c i f i c h e m o p o i e t i c l i n e a g e s h a v e recently b e e n identified. T h e transcription factors M Z F 1 , N F A T a n d O c t - 2 / P a x 5 are specifically e x p r e s s e d in neutrophils, T c e l l s a n d B c e l l s respectively, while G A T A - 1 is n e c e s s a r y for the d e v e l o p m e n t of c e l l s of the erythroid, mast a n d m e g a k a r y o c y t e l i n e a g e s (157, 1 6 1 , 162). T h u s , a s u b s t a n t i a l a m o u n t of d a t a e x i s t s s u g g e s t i n g that  transcription  f a c t o r s play a c r u c i a l role in early h e m o p o i e t i c d e v e l o p m e n t . A s a result i n t e n s e r e s e a r c h efforts are now being f o c u s e d upon identifying t h o s e g e n e s w h o s e activity is r e g u l a t e d by t h e s e transcription factors a n d w h i c h m a y e n c o d e the m o l e c u l a r f a c t o r s w h i c h a r e r e s p o n s i b l e for g o v e r n i n g the s e l f - r e n e w a l , proliferation  and  c o m m i t m e n t of H S C s .  1.6.  Genetic  manipulation  of  hemopoietic  cells  using  recombinant  retroviruses B a s e d u p o n their c a p a c i t y for highly efficient infection a n d n o n - t o x i c a n d s t a b l e integration into the g e n o m e of a w i d e r a n g e of c e l l t y p e s , r e c o m b i n a n t retroviruses represent the most attractive v e h i c l e for e x o g e n o u s g e n e transfer into m a m m a l i a n target cells . The  ability to t r a n s f e r e x o g e n o u s g e n e s into H S C s u s i n g  recombinant  r e t r o v i r u s e s (ie. g e n e t i c a l l y mark them) h a s p r o v i d e d significant insight into the proliferative a n d differentiative potential of totipotent H S C s a n d h a s p r o v i d e d direct e v i d e n c e of the ability of t h e s e c e l l s to s e l f - r e n e w . In a d d i t i o n , g e n e t r a n s f e r m e t h o d o l o g i e s h a v e p r o v i d e d the m e a n s to test critically v a r i o u s g e n e s e n c o d i n g putative  regulatory  molecules which  may  play  a  role  in c o n t r o l l i n g  HSC  proliferation, differentiation a n d / o r self-renewal at the m o l e c u l a r level. F o r e x a m p l e , the transfer a n d o v e r e x p r e s s i o n of a variety of g e n e s e n c o d i n g h e m o p o i e t i c growth factors in murine recipients resulted in myeloproliferative d i s o r d e r s s i m i l a r to t h o s e o b s e r v e d in m a n y h u m a n l e u k e m i a patients s u g g e s t i v e that the d y s r e g u l a t e d  27  e x p r e s s i o n of h e m o p o i e t i c growth factors m a y play a role in h u m a n d i s e a s e v i a autostimulatory  mechanisms  (163-167).  In  addition,  these  experiments  d e m o n s t r a t e d c o n c l u s i v e l y the role of growth factors in h e m o p o i e t i c d e v e l o p m e n t and regulation  in v i v o , s u p p o r t i n g  results obtained from  in vitro c l o n o g e n i c  progenitor a s s a y s . M o r e o v e r , g e n e transfer is central to the newly d e v e l o p e d field of h u m a n g e n e therapy. G e n e therapy b e c a m e reality in 1 9 9 0 w h e n a y o u n g girl, born with a defective v e r s i o n of the g e n e e n c o d i n g a d e n o s i n e d e a m i n a s e ( A D A ) , r e c e i v e d injections of her o w n T c e l l s t r a n s d u c e d with a r e c o m b i n a n t  retrovirus  e x p r e s s i n g a n o r m a l functioning c o p y of the A D A g e n e . S i n c e then m o r e t h a n 100 clinical trials a i m e d at treating d i s e a s e s ranging from inherited d i s o r d e r s s u c h a s c y s t i c fibrosis a n d G a u c h e r s d i s e a s e to c a n c e r a n d A c q u i r e d I m m u n e D e f i c i e n c y S y n d r o m e ( A I D S ) h a v e b e e n a p p r o v e d in the U . S . a l o n e . Unfortunately, d e s p i t e this flurry of activity, the results that h a v e b e e n a c h i e v e d with the current g e n e t r a n s f e r m e t h o d o l o g i e s h a v e b e e n rather d i s a p p o i n t i n g . T h i s is d u e to p r o b l e m s a s s o c i a t e d with the efficiency of g e n e transfer protocols, e x p r e s s i o n of transferred g e n e s a s well a s a lack of u n d e r s t a n d i n g of the growth a n d survival r e q u i r e m e n t s of h u m a n H S C s . M a r k i n g s t u d i e s , the investigation of putative regulatory g e n e s in h e m o p o i e t i c d e v e l o p m e n t a n d g e n e t h e r a p y s h a r e at l e a s t o n e of two  major  r e q u i r e m e n t s : 1) the efficient a n d stable transduction of the h e m o p o i e t i c s t e m cell a n d 2) appropriate e x p r e s s i o n of the t r a n s d u c e d g e n e s in the d e s i r e d c e l l t y p e of interest. T h e r e m a i n d e r of this introduction d e a l s with the a d v a n c e s that h a v e b e e n m a d e using recombinant retroviruses a s a vector for g e n e transfer. 1.6.1. T h e  lifecycle of  retroviruses  R e t r o v i r u s e s do not exist a s distinct genetic e l e m e n t s within their host c e l l s but p e r m a n e n t l y integrate into the g e n o m e in the form of a D N A provirus a n d thus are stably transmitted from parent cell to progeny c e l l . H o w e v e r , during the c o u r s e of their lifecycle, the virus alternates from a n R N A to a D N A i n t e r m e d i a t e . T h e critical features of the retroviral lifecycle involve the s y n t h e s i s a n d p a c k a g i n g of a  28  g e n o m i c R N A c o p y of the provirus into structural viral proteins; s h e d d i n g of the intact virion particle from the s u r f a c e of the host cell; e n t r a n c e of the virion into the target cell v i a s p e c i f i c cell s u r f a c e receptors; reverse transcription of the viral R N A into a d o u b l e - s t r a n d e d c D N A c o p y ; integration of the proviral c D N A into the g e n o m i c D N A of its host cell; a n d e x p r e s s i o n of the viral g e n e s . In g e n e r a l , all retroviruses p o s s e s s three distinct o p e n reading f r a m e s ( O R F ) c a l l e d g a g , pol a n d e n v . T h e s e g e n e s e n c o d e polyproteins c o m p o s e d of e s s e n t i a l structural proteins s u c h a s the matrix ( M A ) , c a p s i d ( C A ) a n d n u c l e o c a p s i d ( N C ) (gag O R F ) , the e n z y m e s required for the reverse transcription (RT) of viral R N A into c D N A a n d integration (IN) of the c D N A into the g e n o m e of the host cell (pol O R F ) , a n d the s u r f a c e ( S U ) a n d t r a n s m e m b r a n e (TM) proteins r e s p o n s i b l e for e n t r a n c e into its target c e l l v i a interaction with s p e c i f i c cell s u r f a c e r e c e p t o r s (env O R F ) ( r e v i e w e d in (168) a n d (169)). In the proviral form, the g a g , pol a n d e n v g e n e s are b o u n d e d o n e i t h e r s i d e by l o n g t e r m i n a l r e p e a t s ( L T R s ) w h i c h c o n t a i n  the  e n h a n c e r a n d promoter regulatory e l e m e n t s a s well a s the transcriptional initiation a n d p o l y a d e n y l a t i o n s i g n a l s . T h e s e e l e m e n t s a r e r e s p o n s i b l e for d i r e c t i n g the s y n t h e s i s of both the cellular R N A transcripts for the production of the viral proteins a s well a s the full length g e n o m i c R N A c o p y of the provirus utilizing the c e l l u l a r R N A p o l y m e r a s e II e n z y m e . A s c h e m a t i c d i a g r a m of a typical murine retrovirus is s h o w n in F i g u r e 1.5. P a c k a g i n g of the g e n o m i c R N A into virion p a r t i c l e s is d e p e n d e n t upon interaction of the viral structural proteins with a s p e c i f i c s e q u e n c e c a l l e d \j/ w h i c h  l i e s d o w n s t r e a m of t h e  5' L T R ( 1 7 0 ) .  However,  additional  s e q u e n c e s p r e s e n t within the d o w n s t r e a m g a g g e n e a r e n o w a l s o k n o w n to b e e s s e n t i a l for efficient R N A p a c k a g i n g (171, 172). O n c e s u r r o u n d e d in a c o r e of viral p r o t e i n , the virion particle b u d s from the cell obtaining a s u r r o u n d i n g e n v e l o p e c o m p o s e d of c e l l u l a r m e m b r a n e s t u d d e d with viral e n v e l o p e g l y c o p r o t e i n s . T h e infectious virion particle s u b s e q u e n t l y g a i n s e n t r a n c e into a potential target c e l l t h r o u g h the interaction of the viral e n v e l o p e proteins with s p e c i f i c c e l l s u r f a c e  29  receptors. S e v e r a l of the cell surface receptors which retroviruses h a v e exploited to g a i n entry into target c e l l s h a v e recently b e e n identified. T h e s e i n c l u d e r e c e p t o r s for ecotropic murine retroviruses ( M C A T ) (173, 174), feline i m m u n o d e f i c i e n c y virus ( C D 9 ) (175, 176), h u m a n a n d s i m i a n i m m u n o d e f i c i e n c y v i r u s e s ( C D 4 ) ( 1 7 7 - 1 7 9 ) , a v i a n l e u k o s i s v i r u s t y p e A (Tval) (180), b o v i n e l e u k e m i a v i r u s (Blvr)  (181)  a m p h o t r o p i c murine retroviruses ( R a m - 1 ) (182, 183) a n d the c o m m o n r e c e p t o r for g i b b o n a p e l e u k e m i a virus (184), feline l e u k e m i a virus group B (185) a n d s i m i a n s a r c o m a - a s s o c i a t e d virus (186) (Glvr-1). Interestingly, the M C A T , Glvr-1 a n d R a m - 1 r e c e p t o r s are transport proteins w h i c h perform e s s e n t i a l h o u s e k e e p i n g f u n c t i o n s ; M C A T s e r v e s a s a cationic a m i n o a c i d transporter of arginine, lysine a n d ornithine ( 1 7 3 , 187) w h i l e  R a m - 1 a n d Glvr-1 a r e both s o d i u m - d e p e n d e n t  phosphate  s y m p o r t e r s (188). Entry into the target c e l l results in the t r a n s f o r m a t i o n of the q u i e s c e n t e n v e l o p e d virion into a n e n z y m a t i c a l l y active nucleoprotein c o m p l e x that b e g i n s to r e v e r s e t r a n s c r i b e the g e n o m i c R N A t e m p l a t e into a d o u b l e s t r a n d e d c D N A c o p y (189, 190). S u c c e s s f u l integration of the proviral c D N A into the g e n o m e of the host cell h a s b e e n s h o w n to require that the cell b e actively c y c l i n g ( 1 9 1 , 192), with the m a i n barrier to s u c c e s s f u l retroviral integration b e i n g the p r e s e n c e of the n u c l e a r m e m b r a n e (193). U p o n d i s s o l u t i o n of the n u c l e a r m e m b r a n e  at  p r o p h a s e of mitosis the viral D N A enters the n u c l e u s a n d inserts itself stably into the g e n o m e of its host through the action of the viral e n c o d e d i n t e g r a s e w h i c h " r a n d o m l y " n i c k s the g e n o m i c D N A c r e a t i n g a 4-6 b a s e pair s t a g g e r e d cut a n d ligating it to the s t a g g e r e d cut viral D N A . A s u m m a r y of the critical s t e p s in the retroviral lifecycle are s h o w n in Figure 1.6. 1.6.2. R e c o m b i n a n t retroviruses as v e c t o r s for gene t r a n s d u c t i o n At p r e s e n t , s e v e r a l h u r d l e s remain to be o v e r c o m e for the s a f e , s t a b l e a n d efficient u s e of recombinant retroviruses a s vectors for g e n e transfer. T h e s e include the ability to p r o d u c e retroviral s t o c k s free of replication c o m p e t e n t wild t y p e virus, the ability to introduce the recombinant retrovirus into the target cell through  30  Figure 1.5. S c h e m a t i c representation of a typical retrovirus s h o w i n g the g a g , pol a n d e n v g e n e s , s p l i c e a c c e p t o r ( S A ) a n d s p l i c e d o n o r ( S D ) s i t e s , a n d the \\i p a c k a g i n g s i g n a l . T h e L T R is c o m p o s e d of three r e g i o n s ; U 3 , R a n d U 5 . U 3 c o n t a i n s two direct repeat 7 2 bp e n h a n c e r s a n d the transcriptional start s i g n a l s . T h e R N A c a p site a n d polyadenlyation site is located at the b e g i n n i n g a n d e n d of the R region respectively.  31  Figure 1.6. C r i t i c a l f e a t u r e s of the retroviral l i f e c y c l e . Infectious v i r i o n s e n t e r a target cell through interaction with specific receptors on the cell s u r f a c e w h e r e u p o n the viral R N A is r e v e r s e t r a n s c r i b e d into a c D N A c o p y . T h e viral c D N A u n d e r g o e s integration into the g e n o m e of the host cell a n d a g e n o m i c length R N A m o l e c u l e is p r o d u c e d a n d p a c k a g e d into viral p a r t i c l e s c o m p o s e d of viral s t r u c t u r a l a n d e n z y m a t i c proteins. T h e virion b u d s from the s u r f a c e of the host cell a n d the c y c l e repeats itself. interaction with specific cellular receptors, a n d the ability to e x p r e s s the transferred g e n e at high l e v e l s in the a p p r o p r i a t e c e l l t y p e f o l l o w i n g integration into the g e n o m e of the host cell. 1.6.2.1. P r o d u c t i o n of  helper-free replication-defective  retroviruses  T h e ability to p r o d u c e replication incompetent recombinant retroviruses free of a n y c o n t a m i n a t i n g helper, or replication competent retrovirus is e s s e n t i a l to both g e n e t h e r a p y a n d g e n e m a r k i n g s t u d i e s . T h e p r e s e n c e of replication c o m p e t e n t retrovirus c a n result in the c o n t i n u o u s production of infectious virus particles from target c e l l s p r o d u c i n g i n c r e a s i n g l y n u m e r o u s a n d c o m p l e x m a r k i n g p a t t e r n s in  32  c l o n a l a n a l y s i s , a n d h a s b e e n s h o w n to be a s s o c i a t e d with l y m p h o c y t i c l e u k e m i a in a n o n - h u m a n primate g e n e therapy trial (194). T h e production of replication-defective retroviruses c a n be a c h i e v e d through the r e p l a c e m e n t of the viral g a g , pol a n d e n v g e n e s with a p a r t i c u l a r g e n e of interest u s i n g s t a n d a r d g e n e t i c e n g i n e e r i n g p r o c e d u r e s a n d p l a s m i d c o n s t r u c t s . T h e recombinant retroviral vector is then introduced into a p a c k a g i n g cell line u s i n g c a l c i u m p h o s p h a t e precipitation. A p a c k a g i n g cell line is a fibroblast d e r i v e d line into w h i c h h a s b e e n stably introduced the g a g , pol a n d e n v g e n e s of a wild t y p e retrovirus a n d thus, is the s o u r c e of the e s s e n t i a l retroviral proteins. H o w e v e r , t h e s e viral s e q u e n c e s are e n g i n e e r e d s o that although they c o n t i n u o u s l y p r o d u c e all of the r e q u i r e d viral proteins, their R N A is u n a b l e to be p a c k a g e d . T h u s , g e n o m i c length R N A m o l e c u l e s d e r i v e d from the recombinant  retroviral v e c t o r c o m b i n e s  with the viral proteins a n d b u d s from the p a c k a g i n g cells resulting in the r e l e a s e of infectious r e c o m b i n a n t retroviral particles c a p a b l e of o n e round of infection. U p o n entry into a target c e l l a n d integration into the host g e n o m e , the  recombinant  retrovirus is u n a b l e to undergo further rounds of replication d u e to the a b s e n c e of the required viral g e n e s . T h e provirus is stably p r o p a g a t e d in the target cell a n d the p r o g e n y of the target c e l l a n d e x p r e s s e s the t r a n s f e r r e d g e n e of interest ( s e e Figure 1.7). T h e first p a c k a g i n g cells lines (\|/2 (195), \ | / A M (196) a n d P A 3 1 7 (197)) w e r e c o n s t r u c t e d by d e l e t i n g a p p r o x i m a t e l y 3 5 0 n u c l e o t i d e s of the 5' r e g i o n of the retrovirus w h i c h c o n t a i n e d the \|/ p a c k a g i n g signal (\j/2, \|/AM) a s well a s the 3' L T R a n d a portion of the 5' L T R ( P A 3 1 7 ) . H o w e v e r , replication c o m p e t e n t v i r u s e s c o u l d b e g e n e r a t e d t h r o u g h rare r e c o m b i n a t i o n e v e n t s during r e v e r s e t r a n s c r i p t i o n in w h i c h the regions deleted c o u l d be corrected for by intact s e q u e n c e s o b t a i n e d from the recombinant retroviral vector. T o d a y , third generation p a c k a g i n g lines ( G P + E 8 6 (198, 199) a n d \ | / C R E (200)) are a v a i l a b l e in w h i c h the viral g a g , pol a n d e n v  33  Retroviral Packaging  Sequences  Recombinant retroviral vector TARGETTED GENE  Figure 1.7. P a c k a g i n g c e l l s l i n e s c o n t i n u a l l y p r o d u c e the g a g , p o l a n d e n v proteins but viral R N A is u n a b l e to be p a c k a g e d d u e to deletion of the \|/ s i g n a l s e q u e n c e a n d s e p a r a t i o n of the g a g / p o l a n d e n v g e n e s onto different p l a s m i d s . Introduction of a functional r e c o m b i n a n t retroviral v e c t o r d e l e t e d of viral g e n e s results in the production of g e n o m i c length recombinant R N A w h i c h are p a c k a g e d into virion p a r t i c l e s a n d b u d from the p a c k a g i n g c e l l . T h e r e l e a s e of i n f e c t i o u s r e c o m b i n a n t retroviral p a r t i c l e s are s u b s e q u e n t l y a b l e to u n d e r g o 1 r o u n d of infection a n d integration into a host target cell.  34  g e n e s a r e s e p a r a t e d onto two s e p a r a t e p l a s m i d c o n s t r u c t s with g a g a n d pol o n o n e a n d e n v o n the other. T h e s e lines require three recombinational e v e n t s for the production of helper virus to o c c u r thus greatly d e c r e a s i n g the likelihood of this event. O n e d r a w b a c k of using the lines m e n t i o n e d a b o v e is that the production of s t a b l e , h i g h titre viral p r o d u c e r c e l l s c a n t a k e s e v e r a l w e e k s . M o r e r e c e n t l y , ecotropic (able to infect m o u s e a n d rat cells only) ( B O S C ) a n d a m p h o t r o p i c (able to infect m o u s e , h u m a n a s w e l l a s other c e l l t y p e s ) ( B I N G ) p a c k a g i n g c e l l l i n e s d e r i v e d from a highly transfectable 2 9 3 T cell line h a v e b e e n p r o d u c e d e n a b l i n g t h e rapid, transient production of high titre viral s u p e r n a t a n t s free of h e l p e r virus within 7 2 h o u r s (201) . 1.6.3. Optimization of retroviral gene transfer 1.6.3.1. Retroviral infection  strategies  T h e efficiency of infection of H S C s is d e p e n d e n t u p o n a large n u m b e r of factors including their c y c l i n g status, the addition of e x o g e n o u s h e m o p o i e t i c growth factors a n d the method of infection. A s p r e v i o u s l y m e n t i o n e d , s u c c e s s f u l retroviral infection r e q u i r e s that t h e target cell b e in a state of active c y c l e (191, 192). Unfortunately, the v a s t majority of H S C s a r e in a state of q u i e s c e n c e u n d e r n o r m a l p h y s i o l o g i c c o n d i t i o n s (93). Following the injection of the c y c l e specific cytotoxic drug 5 - F U , h o w e v e r , a larger proportion of H S C s a r e actively c y c l i n g (93) a n d a r e m o r e s u s c e p t i b l e to retroviral infection (202, 2 0 3 ) . A l t h o u g h H S C s c a n s u r v i v e for a p e r i o d of time a n d b e s u s c e p t i b l e to retroviral t r a n s d u c t i o n in t h e a b s e n c e of e x o g e n o u s l y a d d e d h e m o p o i e t i c growth factors (204) , the infectability of d a y 12 C F U - S w a s found to b e greatly i n c r e a s e d in t h e p r e s e n c e of IL-3 (24, 2 0 5 ) . M o r e o v e r , v a r i o u s c o m b i n a t i o n s of I L - 3 , IL-6, S t e e l factor, IL-1 a n d l e u k e m i a inhibitory factor (LIF) w e r e f o u n d to e n h a n c e retrovirus m e d i a t e d g e n e transfer into murine a s well a s h u m a n a n d n o n - h u m a n  35  primate H S C s (124, 2 0 6 - 2 1 2 ) . In addition to bringing c e l l s into a state of active cell c y c l e growth factors m a y a l s o play a role in increasing the n u m b e r a n d / o r affinity of r e c e p t o r m o l e c u l e s o n target c e l l s (213). A l t h o u g h growth f a c t o r s i n c r e a s e the efficiency of g e n e transduction, caution must be u s e d s i n c e the culture of m a r r o w c e l l s in the p r e s e n c e of growth factors for e x t e n d e d p e r i o d s of time in vitro m a y impair the engrafting ability of the H S C s upon transplantation into irradiated h o s t s (214). T h e m e t h o d of e x p o s u r e of the retrovirus to the target m a r r o w c e l l s a l s o h a s a large effect u p o n the efficiency of viral transfection. T h e most s u c c e s s f u l results u s i n g murine b o n e marrow c e l l s a s targets h a v e b e e n a c h i e v e d by co-culturing the viral p r o d u c e r a n d m a r r o w c e l l s together ( 2 1 5 - 2 1 7 ) . W h i l e early results u s i n g a n o n - h u m a n primate m o d e l f a v o r e d the u s e of filtered viral p r e p a r a t i o n s f r e e of p r o d u c e r c e l l s (218) (219-221), m o r e recent s t u d i e s h a v e s h o w n that the efficiency of s u p e r n a t a n t infection of primitive h u m a n h e m o p o i e t i c c e l l s c a n b e i n c r e a s e d t h r o u g h the u s e of a n a u t o l o g o u s s t r o m a l s u p p o r t l a y e r d u r i n g the  infection  p r o c e d u r e (222-224). T h e stromal layer most likely i n c r e a s e s the efficiency of viral infection v i a the production of v a r i o u s defined a n d / o r undefined growth factors a n d v i a cell-cell m e d i a t e d interaction. Interestingly, Moritz et. a l . h a v e reported that the p r e s e n c e of the extracellular matrix m o l e c u l e fibronectin c a n i n c r e a s e the efficiency of s u p e r n a t a n t infection of c o m m i t t e d progenitor c e l l s a n d primitive c e l l s with long t e r m culture initiating ability ( L T C - I C ) (225). It h a s b e e n h y p o t h e s i z e d that this m o l e c u l e is s o m e h o w a b l e to bring both the target cell a n d retrovirus into c l o s e proximity thus i n c r e a s i n g the probability of retroviral infection. R e c e n t l y , a m e t h o d by w h i c h retroviral s u p e r n a t a n t is a l l o w e d to flow t h r o u g h a p o r o u s m e m b r a n e u p o n w h i c h the target c e l l s lie h a s b e e n d e s c r i b e d (226). W h i l e normal s u p e r n a t a n t infection relies upon B r o w n i a n motion for virions to c o m e into contact with a target cell (a v e r y inefficient p r o c e s s ) , the flow-through t r a n s d u c t i o n s y s t e m o v e r c o m e s this limitation by "flowing" all virus particles past the target c e l l s resulting in a m u c h  36  higher probability of contact b e t w e e n virus a n d cell. T h i s s y s t e m c a n l e a d to high transfection efficiencies e v e n at low viral titre a n d c a n be d o n e in the a b s e n c e of p o l y c a t i o n s s u c h a s protamine sulfate or p o l y b r e n e w h i c h h a v e b e e n historically utilized a s m e t h o d s to i n c r e a s e g e n e transfer efficiency. 1.6.3.2. Targeting of virions to host cells T h e transduction of murine H S C s using ecotropic retroviral v e c t o r s c a r r y i n g a variety of g e n e s (ie. n e o ^ (23, 3 5 , 2 0 4 , 205), a d e n o s i n e d e a m i n a s e (215-217), pglobin (227) a n d g l u c o c e r e b r o s i d a s e (228-230)) h a s , in g e n e r a l , b e e n f o u n d to be m u c h m o r e efficient t h a n g e n e t r a n s f e r into H S C s from larger a n i m a l s u s i n g a m p h o t r o p i c v i r u s e s . O n e current limitation  to retroviral m e d i a t e d g e n e transfer  into the b o n e m a r r o w of m i c e or larger a n i m a l s is that the d e s i r e d target c e l l , the totipotent H S C , is both rare a n d largely n o n - c y c l i n g (93). U s i n g s t a n d a r d infection p r o t o c o l s L u s k e y et. a l . h a s e s t i m a t e d the efficiency of g e n e transfer to m u r i n e HSCs  to  b e 2 0 % u s i n g S o u t h e r n blot  a n a l y s i s of  D N A obtained  from  the  h e m o p o i e t i c t i s s u e s of recipients t r a n s p l a n t e d with retrovirally t r a n s d u c e d b o n e m a r r o w at 4 m o n t h s post t r a n s p l a n t a s m e a s u r e d by proviral integration  and  g e n o m e c o p y n u m b e r a n a l y s i s (206). M o r e o v e r , E i n e r h a n d et. a l . h a v e e s t i m a t e d the efficiency of g e n e transfer to marrow repopulating a b i l i t y - C F U - S ( M R A - C F U - S ) w h i c h is a cell that h o m e s to the marrow of the recipients following transplant a n d proliferates  to  produce  numerous  day  12-CFU-S  cells. Thirteen  days  post  transplant the recipient m o u s e is sacrificed a n d its b o n e marrow u s e d to a s s a y for d a y 12 C F U - S by injection into s e c o n d a r y recipients. T h u s , through the a n a l y s i s of individual d a y 12 s p l e e n c o l o n i e s , E i n e r h a n d a n d his c o l l e a g u e s e s t i m a t e d the efficiency of g e n e transfer to t h e s e primitive cells to be 1 5 % (207). T h e efficiency of g e n e transfer to H S C s d e r i v e d from larger a n i m a l s s u c h a s n o n - h u m a n p r i m a t e s (219, 2 2 1 , 2 3 1 ) , felines (232), c a n i n e s (233-235) or h u m a n s (236, 237) a p p e a r s to be c o n s i d e r a b l y lower. Quantitative  P C R performed  on D N A obtained  from  peripheral blood a n d b o n e marrow cells at a m i n i m u m of o n e y e a r post transplant  37  s u g g e s t e d that the efficiency of g e n e transfer to H S C s from larger a n i m a l s is only 0 . 1 % to 5 % . T h i s low g e n e transfer efficiency m a y in part be d u e to the e x p r e s s i o n of low l e v e l s of the a m p h o t r o p i c receptor R a m - 1 (188). S e v e r a l investigators h a v e a t t e m p t e d to b y p a s s this p r o b l e m through the u s e of viral p s e u d o t y p i n g w h i c h i n v o l v e s r e p l a c i n g the a m p h o t r o p i c e n v e l o p e g e n e with a n e n v g e n e f r o m  a  different virus w h o s e receptor is e x p r e s s e d at a m u c h higher density on the d e s i r e d target c e l l . C o m b i n i n g the M o l o n e y murine l e u k e m i a virus g a g a n d pol g e n e s with the e n v g e n e d e r i v e d from the g i b b o n a p e l e u k e m i a v i r u s ( 2 3 8 - 2 4 0 ) or the v e s i c u l a r stomatitis virus G g l y c o p r o t e i n ( V S V - G ) ( 2 4 1 , 242) h a s r e s u l t e d in a n i n c r e a s e in g e n e transfer to h u m a n cell lines, c o m m i t t e d h u m a n progenitor c e l l s and LTC-IC. S o m e investigators h a v e attempted to target retroviral infection to s p e c i f i c cell t y p e s by creating a hybrid env g e n e e n c o d i n g a portion of the viral e n v protein a n d a ligand r e c o g n i z e d by a s p e c i f i c receptor o n the s u r f a c e of the target c e l l . U s i n g this a p p r o a c h , r e t r o v i r u s e s h a v e b e e n t a r g e t e d to s p e c i f i c c e l l s v i a the erythropoietin receptor (243), the low density lipoprotein receptor (244), e p i d e r m a l growth factor receptor (245) a s well a s major histocompatibility c l a s s I a n d c l a s s II antigen receptors (246). A l t h o u g h this method d o e s s h o w p r o m i s e , c a u t i o n must be u s e d s i n c e the fusion of the viral env with a foreign m o l e c u l e m a y result in a n o n functional e n v e l o p e protein a n d thus a non-infectious virus particle. 1.6.3.3.  Use  of  selectable  markers  to  increase  the  utility  of  r e c o m b i n a n t retroviral v e c t o r s T o aid in the identification, e n r i c h m e n t a n d t r a c k i n g of t r a n s d u c e d target c e l l s a variety of s e l e c t a b l e m a r k e r s h a v e b e e n incorporated into retroviral v e c t o r s . T h e m o s t widely u s e d of t h e s e h a v e b e e n g e n e s w h i c h c o n f e r r e s i s t a n c e to toxic c o m p o u n d s s u c h a s n e o m y c i n a n d h y g r o m y c i n . A variety of o t h e r s s u c h a s pg a l a c t o s i d a s e a n d a n u m b e r of cell surface antigens h a v e a l s o b e e n utilized. T a b l e 1.1 s h o w s the v a r i o u s g e n e s that have b e e n utilized a s s e l e c t a b l e m a r k e r s to date.  38  T h e ability to identify a n d select for retrovirally t r a n s d u c e d H S C s rapidly a n d n o n - t o x i c a l l y c o u l d e n h a n c e the p o w e r of m a r k i n g s t u d i e s , efforts to a s s e s s the effects of o v e r e x p r e s s i n g putative H S C regulatory m o l e c u l e s a n d current efforts at h u m a n g e n e therapy. H o w e v e r , at the time that the work d e s c r i b e d in this t h e s i s w a s initiated, the u s e of g e n e s e n c o d i n g c e l l s u r f a c e a n t i g e n s a s s e l e c t a b l e m a r k e r s for the s e l e c t i o n of retrovirally t r a n s d u c e d in vivo repopulating H S C s h a d not b e e n reported. A s p r e v i o u s l y m e n t i o n e d , the work d e s c r i b e d in C h a p t e r 3 of this t h e s i s d e s c r i b e s the n o v e l u s e of the h u m a n cell s u r f a c e a n t i g e n C D 2 4 a s a dominant s e l e c t a b l e marker in a retroviral vector to e n a b l e the efficient s e l e c t i o n of t r a n s d u c e d murine B M c e l l s including t h o s e with totipotent long term repopulating ability. 1.6.3.4. Retroviral vector  design  S e v e r a l factors c a n influence the p e r f o r m a n c e of a n y particular  retroviral  c o n s t r u c t including the regulatory e l e m e n t s u s e d to drive e x p r e s s i o n , the n u m b e r a n d s i z e of t r a n s c r i p t i o n a l  units, the viral b a c k b o n e u s e d , the d i r e c t i o n  of  transcription a n d the p r e s e n c e or a b s e n c e of s e l e c t a b l e m a r k e r s . A l t h o u g h lineage restricted promoters m a y be ideal if the transferred g e n e is to b e e x p r e s s e d e x c l u s i v e l y in particular cell t y p e s (247, 2 4 8 ) , viral L T R s h a v e c o n s i s t e n t l y b e e n s h o w n to result in higher levels of g e n e e x p r e s s i o n a s c o m p a r e d to a variety of internal promoters of viral or cellular origin (249, 2 5 0 ) . Unfortunately, viral L T R s s u c h a s the M o l o n e y M u r i n e L e u k e m i a V i r u s ( M o M u L V ) h a v e b e e n f o u n d to be subject to transcriptional s h u t d o w n following long p e r i o d s of time in vivo (215, 251) a n d in primitive cell types s u c h a s e m b r y o n i c c a r c i n o m a ( E C ) c e l l s ( 2 5 2 - 2 5 4 ) , e m b r y o n a l s t e m ( E S ) c e l l s (255) a n d primitive h e m o p o i e t i c c e l l l i n e s (256) p o s s i b l y d u e to methylation of the regulatory e l e m e n t s (257). S e v e r a l viral m u t a n t s h a v e b e e n i s o l a t e d that are a b l e to e x p r e s s t r a n s f e r r e d g e n e s at high l e v e l s in E C a n d E S cells. T h e P C M V ( P C C 4 e m b r y o n a l c a r c i n o m a c e l l - p a s s a g e d m y e l o p r o l i f e r a t i v e s a r c o m a virus) (258) a n d the d l 5 8 7 r e v v i r u s (259)  39  possess  Table 1.1. O p t i o n s in S e l e c t a b l e / R e p o r t e r M a r k e r s for Retroviral V e c t o r s Marker G e n e S i z e in bp* M e t h o d s of Quantification of Selection G e n e Expression at S i n g l e C e l l Resolution n e o m y c i n (260, 261) h y g r o m y c i n (262) puromycin methotrexateR(263,264) cytosine deaminase (265) M D R - 1 (266) thymidine k i n a s e (267) 0  R  p - g a l a c t o s i d a s e (268, 269) alkaline phosphatase (270) Leu-1 (271) transferrin receptor (271) M D R - 1 (272) truncated nerve growth factor receptor (273) IL-2 receptor (274) Heat S t a b l e A n t i g e n ( H S A ) (275) mutated murine prion protein (276) Green Fluorescence Protein ( G F P ) (277) C D 2 4 (278) Thy-1 (279)  800 1200 700 700 1600  drug r e s i s t a n c e  no  FACS  yes  immuno-based ( F A C S , panning)  yes  3800 1400 3°°0 4000  2300 2800 3800 1500 1400 228 1200 720 240 488  * A p p r o x i m a t e s i z e of c D N A e n c o m p a s s i n g c o d i n g region  40  1  v a r i o u s d e l e t i o n s a n d b a s e pair mutations w h i c h w e r e f o u n d to r e m o v e s e v e r a l transcriptional b l o c k s located within the L T R a n d primer binding sites of the original v i r u s e s (280-282). It is the r e m o v a l of t h e s e transcriptional b l o c k s that e n a b l e s the e x p r e s s i o n of transferred g e n e s under the control of t h e s e regulatory e l e m e n t s in both E C a n d E S c e l l s . R e c e n t l y , H a w l e y et. a l . (283) h a v e p r o d u c e d a s e r i e s of vectors b a s e d upon a Murine Stem Cell Virus ( M S C V ) backbone which c o m b i n e s the L T R from P C M V , the 5' untranslated region from the d l 5 8 7 r e v v i r u s , a n d a n u m b e r of c o n v e n i e n t c l o n i n g sites. D a t a will be p r e s e n t e d in this C h a p t e r 4 of this t h e s i s to s u g g e s t that the M S C V vectors are a b l e to t r a n s c r i b e t r a n s d u c e d g e n e s efficiently in primitive primary h e m o p o i e t i c s t e m cell c a n d i d a t e s a n d their p r o g e n y for e x t e n d e d periods of time in vivo. P l a c i n g a g e n e of interest u n d e r the regulatory c o n t r o l of a n promoter  d o w n s t r e a m of the viral L T R r e g u l a t o r y  internal  s e q u e n c e s m a y result  in  e x p r e s s i o n p r o b l e m s d u e to promoter interference b e t w e e n the viral a n d internal regulatory e l e m e n t s . I n d e e d , the e x p r e s s i o n of internally driven g e n e s h a s b e e n f o u n d to be u n p r e d i c t a b l e . In s o m e c a s e s efficient e x p r e s s i o n from the internal p r o m o t e r w a s d e t e c t e d (172, 284) while in other c a s e s little or no e x p r e s s i o n w a s o b s e r v e d ( 2 1 6 , 2 8 5 - 2 8 7 ) . A s a m e a n s to b y p a s s this potential p r o b l e m selfinactivating or "crippled" v e c t o r s c a n be u s e d . T h e s e v e c t o r s p o s s e s s a deletion of a portion of the 3' L T R a n d are d e s i g n e d s u c h that following infection of the target cell the viral e n h a n c e r a n d promoter regulatory e l e m e n t s are non-functional (288290). T h e n u m b e r of transcriptional units present within the retroviral v e c t o r c a n a l s o influence the efficiency of transferred g e n e e x p r e s s i o n . A l t h o u g h s o m e s t u d i e s h a v e s u g g e s t e d that s u p e r i o r results are obtained u s i n g a simplified v e c t o r w h i c h c o n t a i n s o n l y o n e t r a n s c r i p t i o n a l unit (215, 2 9 1 ) , o t h e r s h a v e f o u n d that the i n c l u s i o n of a n a d d i t i o n a l g e n e e n c o d i n g a d o m i n a n t s e l e c t a b l e m a r k e r in the v e c t o r c a n be beneficial (249, 2 9 2 , 293). H o w e v e r , s o m e studies h a v e reported that  41  c e l l s s e l e c t e d for o n the b a s i s of e x p r e s s i o n of the s e l e c t a b l e m a r k e r c a n result in the s u p p r e s s i o n of the a d j a c e n t g e n e , m o s t likely d u e to p r o m o t e r i n t e r f e r e n c e b e t w e e n internal a n d viral regulatory e l e m e n t s in multi-gene v e c t o r s (274, 2 9 4 ) . A solution to this p r o b l e m h a s b e e n to e x p r e s s both g e n e s from a s i n g l e regulatory e l e m e n t by linking t h e m together u s i n g a n internal r i b o s o m a l entry site ( I R E S ) e l e m e n t . I R E S e l e m e n t s are discrete fragments of D N A (approximately 5 0 0 - 6 0 0 - b p in  size)  derived  from  the  5'  untranslated  regions  of  the  polio  or  e n c e p h a l o m y o c a r d i t i s virus (295, 2 9 6 ) . I R E S e l e m e n t s a l l o w r i b o s o m e s to bind internally on the m R N A rather than at the 5' c a p site a s normally o c c u r s a n d t h u s , e n a b l e s m o r e than o n e transcriptional unit to be t r a n s l a t e d from a s i n g l e m R N A m o l e c u l e . T h e s e e l e m e n t s h a v e b e e n s h o w n to link up to three s e p a r a t e g e n e s thus allowing the regulation of multiple transcriptional units from a s i n g l e regulatory e l e m e n t (297, 298).  1.7.  T h e s i s objectives and general strategy A l t h o u g h recombinant retroviruses currently  remain the most  efficient  m e t h o d for i n t r o d u c i n g e x o g e n o u s g e n e t i c m a t e r i a l into target c e l l s , t h e infection e f f i c i e n c y of H S C s r e p r e s e n t s a s e r i o u s hurdle to effective marking  studies  and  gene  therapy.  Moreover,  although  gene  low  retroviral transfer  m e t h o d o l o g i e s h a v e p r o v i d e d the critical m e a n s to test g e n e s e n c o d i n g putative r e g u l a t o r y m o l e c u l e s w h i c h m a y play a role in c o n t r o l l i n g H S C p r o l i f e r a t i o n , differentiation a n d / o r self-renewal v i a o v e r e x p r e s s i o n of the transferred g e n e , at the time that the work d e s c r i b e d in this t h e s i s w a s initiated, the direct d e m o n s t r a t i o n that a n y r e g u l a t o r y e l e m e n t w a s a b l e to drive high a n d s u s t a i n e d l e v e l s of transferred g e n e e x p r e s s i o n in H S C s h a d not b e e n a c h i e v e d . T h e first objective of this t h e s i s work w a s to d e v e l o p p r o c e d u r e s that w o u l d i n c r e a s e the utility of retroviral g e n e transfer p r o c e d u r e s . T h e s e s t u d i e s f o c u s e d o n d e t e r m i n i n g the feasibility of utilizing a c D N A e n c o d i n g the h u m a n C D 2 4 c e l l s u r f a c e antigen a s a dominant s e l e c t a b l e marker in a retroviral vector to e n a b l e the 42  rapid, efficient a n d non-toxic identification a n d s e l e c t i o n of retrovirally t r a n s d u c e d m u r i n e b o n e m a r r o w c e l l s , i n c l u d i n g t h o s e with totipotent l o n g t e r m  in v i v o  repopulating ability ( C h a p t e r 3). M y s e c o n d objective w a s to exploit the s e l e c t i o n p r o t o c o l d e v e l o p e d in C h a p t e r 3 to d e m o n s t r a t e the  r e g e n e r a t i o n of the h e m o p o i e t i c s y s t e m s of  m y e l o a b l a t e d recipient m i c e with c e l l s d e r i v e d e x c l u s i v e l y from provirally m a r k e d H S C s a n d to quantify the levels of e x p r e s s i o n of the transferred C D 2 4 m a r k e r g e n e in v a r i o u s p h e n o t y p i c a l l y d e f i n e d p o p u l a t i o n s of c e l l s in vivo i n c l u d i n g c a n d i d a t e H S C s d e f i n e d by the S c a + L i n " cell s u r f a c e phenotype. T h e results of t h e s e s t u d i e s ( p r e s e n t e d in C h a p t e r 4) d e m o n s t r a t e the u s e f u l n e s s of the C D 2 4 s e l e c t i o n p r o c e d u r e to e n a b l e effective tracking of the contribution of individual s t e m c e l l s to the regeneration of the H S C a n d more mature cell c o m p a r t m e n t s in m y e l o a b l a t e d recipient m i c e following b o n e marrow transplant, a n d to quantify transferred g e n e e x p r e s s i o n both in vitro a n d in vivo. A l t h o u g h retroviral m a r k i n g s t u d i e s h a v e p r o v i d e d direct e v i d e n c e of the e x i s t e n c e of totipotent H S C s , a n d h a v e provided significant insight into the ability of t h e s e c e l l s to r e g e n e r a t e the h e m o p o i e t i c s y s t e m s of m y e l o a b l a t e d t r a n s p l a n t recipients (ie. their proliferative a n d differentiative potential), to date quantitative d a t a a s s e s s i n g the extent to w h i c h t h e s e c e l l s c a n r e g e n e r a t e their  numbers  following transplant (ie. self-renew) is limited. T h e final objective of this work w a s to d e f i n e m o r e c l e a r l y the r e g e n e r a t i v e (self-renewal) c a p a c i t y of H S C s  following  b o n e m a r r o w transplant. T h e recovery of totipotent long term repopulating s t e m cell n u m b e r s in lethally irradiated recipient m i c e w a s a s s e s s e d a s a f u n c t i o n of the n u m b e r a n d s o u r c e (adult b o n e m a r r o w v s . fetal liver) of c e l l s t r a n s p l a n t e d . In a d d i t i o n , u s i n g the C D 2 4 s e l e c t i o n p r o c e d u r e ( C h a p t e r 3) the c o n t r i b u t i o n  of  individual H S C c l o n e s to the regeneration of the H S C c o m p a r t m e n t w a s a s s e s s e d in s o m e e x p e r i m e n t s . T h e results of this work are p r e s e n t e d in C h a p t e r s 4 a n d 5.  43  CHAPTER MATERIAL  2.1.  Construction  of  retroviral  AND  2 METHODS  vectors,  virus  production  and  viral  assays 2.1.1. R e c o m b i n a n t retroviral  vectors  E x p e r i m e n t s d i s c u s s e d in C h a p t e r s 3, 4 a n d 5 utilized a retrovirus d e r i v e d from the J Z e n l retroviral b a c k b o n e kindly provided by Dr. S . C o r y (Walter a n d E l i z a H a l l Institute, M e l b o u r n e , A u s t r a l i a ) . T h e 3' L T R of J Z e n l is d e r i v e d f r o m  the  myeloproliferative s a r c o m a virus ( M P S V ) ( 1 6 7 ) . T o construct J Z e n C D 2 4 t k n e o , a 3 1 0 bp S a l I fragment c o n t a i n i n g bp 1 to 3 0 3 of the p u b l i s h e d C D 2 4 c D N A s e q u e n c e (299) a n d e n c o m p a s s i n g the entire 2 4 0 bp c o d i n g r e g i o n w a s r e m o v e d  from  P A X 1 1 4 (300) a n d i n s e r t e d into the X h o I site of J Z e n t k n e o u s i n g s t a n d a r d p r o c e d u r e s . J Z e n t k n e o w a s constructed by inserting into the H p a I - H i n d III sites of J Z e n l a 1 0 9 2 b a s e pair S m a I- H i n d III fragment from p T Z 1 9 R t k n e o that c o n t a i n s the n e o R g e n e linked to a mutant p o l y o m a virus e n h a n c e r t a n d e m r e p e a t a n d H e r p e s S i m p l e x virus t h y m i d i n e k i n a s e g e n e p r o m o t e r i s o l a t e d f r o m p M C I n e o (301). M S C V n e o l R E S C D 2 4 , u s e d in e x p e r i m e n t s p r e s e n t e d in C h a p t e r 4 , w a s c o n s t r u c t e d u s i n g the M S C V n e o E B v e c t o r (283) (kindly p r o v i d e d by Dr. R o b e r t H a w l e y , S u n n y b r o o k H e a l t h S c i e n c e C e n t e r , T o r o n t o , O N ) originally d e r i v e d from the M E S C retroviral vector of G r e z et. al. (302) a n d the L N retroviral v e c t o r s of Miller et. a l . (303). P G K n e o w a s r e m o v e d from M S C V n e o E B by digesting with B g l l l / B a m H I a n d religating to c r e a t e M S C V ( - ) . A 9 4 8 - b p E c o R I / X h o l fragment from a p r e v i o u s l y d e s c r i b e d c o n s t r u c t (304) e n c o m p a s s i n g the e n c e p h a l o m y o c a r d i t i s v i r u s internal r i b o s o m a l entry site ( I R E S ) s e q u e n c e a n d the 2 4 0 - b p c o d i n g region of the h u m a n C D 2 4 cell s u r f a c e a n t i g e n c D N A (299) w a s ligated into a n E c o R I / X h o l d i g e s t e d M S C V ( - ) . Lastly, a 8 4 7 - b p blunted M l u l / S a l l fragment d e r i v e d from p M C I n e o (301)  44  c o n t a i n i n g a n e o m y c i n r e s i s t a n c e g e n e w a s inserted by blunt e n d ligation into the E c o R I site of M S C V I R E S C D 2 4 to create M S C V n e o l R E S C D 2 4 . 2.1.2. Viral p a c k a g i n g and other cell lines T h e e c o t r o p i c p a c k a g i n g cell line, G P + E - 8 6 (199), a n d the a m p h o t r o p i c cell line, G P + A M 1 2 (198), w e r e u s e d to g e n e r a t e helper-free r e c o m b i n a n t retrovirus. T h e c e l l lines w e r e m a i n t a i n e d in H X M m e d i u m c o m p o s e d of D u l b e c c o ' s M o d i f i e d E a g l e s M e d i u m ( D M E 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 , British C o l u m b i a ) , 1 0 % h e a t - i n a c t i v a t e d ( 5 5 ° C for 3 0 minutes) n e w b o r n calf s e r u m ( G i b c o / B R L C a n a d a ; B u r l i n g t o n , Ontario), h y p o x a n t h i n e (15 m g / m l ; S i g m a C h e m i c a l C o . , S t . L o u i s , M O ) , x a n t h i n e (250 m g / m l ; S i g m a ) , a n d m y c o p h e n o l i c a c i d (25 m g / m l ; Sigma). The maintained  IL-3-dependent  in R P M I with  murine  h e m o p o i e t i c c e l l line B a / F 3  (305)  was  1 0 % fetal calf s e r u m a n d 5 % m o u s e s p l e e n c e l l  c o n d i t i o n e d m e d i u m ( S C C M ) ( S t e m C e l l T e c h n o l o g i e s ) . A l l c e l l s w e r e cultured at 3 7 ° C in a humidified a t m o s p h e r e of 5 % C O 2 in air. 2.1.3. G e n e r a t i o n of viral p r o d u c e r cell lines P u r i f i e d proviral p l a s m i d D N A w a s introduced into the G P + A M - 1 2 p a c k a g i n g c e l l line u s i n g the c a l c i u m p h o s p h a t e ( C a P 0 4 ) t r a n s f e c t i o n  technique.  DNA  precipitate w a s f o r m e d by c o m b i n i n g 18 u,g of purified D N A , 5 0 uJ of 2.5 M C a C l 2 a n d d H 2 0 up to 0.5 ml, w h i c h w a s slowly a d d e d to 0.5 m L of 2 X H B S ( 5 0 m M H e p e s , 3 M N a C I , 1.5 m M N a 2 H P 0 4 , p H 7.12) while gently bubbling with air to mix. T h e solution w a s a l l o w e d to s t a n d at room t e m p e r a t u r e for 3 0 m i n u t e s a n d t h e n a d d e d d r o p w i s e to 9 ml of m e d i u m on 2 x 1 0  5  G P + A M - 1 2 p a c k a g i n g c e l l s plated in  a 100 m m t i s s u e culture d i s h ( B e c k t o n D i c k i n s o n , L i n c o l n P a r k N J ) 2 4 h o u r s previously. After 2 4 hours the m e d i u m w a s r e p l a c e d with fresh m e d i u m containing 1 m g / m l (approximately 0.6 m g / m l active c o m p o u n d ) of the n e o m y c i n a n a l o g G 4 1 8 ( G i b c o / B R L ) . M e d i u m w a s r e p l a c e d e v e r y three d a y s . M e d i u m w a s p l a c e d o n d i s h e s of confluent G 4 1 8  R  G P + A M - 1 2 cells a n d 2 4 hours later the supernatant w a s  r e m o v e d , filtered (0.22u.m filter, Millipore, B e d f o r d , M A ) a n d o v e r l a y e d onto 1 x 1 0  45  5  G P + E - 8 6 e c o t r o p i c viral p a c k a g i n g c e l l s in the p r e s e n c e of 7 u,g/ml p o l y b r e n e ( S i g m a ) . T w e n t y four hours later the m e d i u m w a s r e m o v e d a n d r e p l a c e d with fresh m e d i u m containing 1 mg/ml G 4 1 8 . 2.1.4. Viral titering and helper virus  assay  V i r a l titres w e r e d e t e r m i n e d by a s s a y i n g m e d i u m c o n d i t i o n e d by  viral  p r o d u c e r cell lines for transfer of n e o m y c i n r e s i s t a n c e to N I H - 3 T 3 c e l l s ( A m e r i c a n Type  Culture Collection [ATCC], Rockville, MD). Medium was  subconfluent  placed  atop  1 0 0 m m d i s h e s of viral p r o d u c e r c e l l s a n d 2 4 h o u r s later  the  s u p e r n a t a n t w a s r e m o v e d a n d filtered. V a r i o u s dilutions of the viral s u p e r n a t a n t w e r e p l a c e d in a final v o l u m e of 2 mis a n d p l a c e d on top of 2 x 1 0  3 T 3 c e l l s plated  5  in 6 0 m m t i s s u e culture d i s h e s ( B e c t o n D i c k i n s o n ) with 7 |ig/ml p o l y b r e n e a d d e d . T w e n t y four h o u r s later the supernatant w a s r e p l a c e d with fresh m e d i u m c o n t a i n i n g 1 m g / m l G 4 1 8 . M e d i u m w a s r e p l a c e d e v e r y three d a y s . G 4 1 8  R  colonies were  s c o r e d after staining with m e t h y l e n e blue to derive the n u m b e r of infectious virus particles carrying n e o  g e n e r a t e d by the viral p r o d u c e r s (colony forming units per  R  ml). A s s a y for the p r e s e n c e of h e l p e r virus w a s p e r f o r m e d by a t t e m p t i n g serially transfer n e o  R  to 3 T 3 cells (195). Confluent d i s h e s of G 4 1 8  obtained following  infection with 5 m i s of filtered u n d i l u t e d viral  R  to  3 T 3 cells were supernatant  f o l l o w e d by G 4 1 8 s e l e c t i o n a s d e s c r i b e d a b o v e . M e d i a w a s c h a n g e d o n confluent d i s h e s of G 4 1 8  R  3 T 3 cells a n d 2 4 hours later the m e d i u m w a s r e m o v e d , filtered a n d  p l a c e d o n top of 2 x 1 0  5  3 T 3 cells plated out 2 4 hours previously. T w e n t y four h o u r s  later the m e d i u m w a s r e m o v e d a n d r e p l a c e d with 5 mis of fresh m e d i u m c o n t a i n i n g 1 m g / m l G 4 1 8 . C o l o n i e s w e r e s u b s e q u e n t l y s c o r e d 2 - 3 w e e k s later a s d e s c r i b e d above.  2.2.  H e m o p o i e t i c cell culture and  2.2.1.  assays  Mice  46  M i c e u s e d in t h e s e e x p e r i m e n t s w e r e 8 to  12 w e e k o l d ( C 5 7 B I / 6 J x  C 3 H / H e J ) F 1 ( B 6 C 3 F 1 ) m a l e a n d f e m a l e m i c e bred a n d m a i n t a i n e d in the a n i m a l facility of the British C o l u m b i a C a n c e r R e s e a r c h C e n t r e f r o m p a r e n t a l  strain  b r e e d e r s originally obtained from the J a c k s o n L a b o r a t o r i e s ( B a r H a r b o r , M A ) . M i c e u s e d a s b o n e m a r r o w d o n o r s in competitive repopulation e x p e r i m e n t s w e r e 10 to 14 w e e k old m a l e or f e m a l e ( C 5 7 B I / 6 L y - P e p 3 b x C 3 H / H e J ) F 1 ( P e p C 3 F 1 ) m i c e . B 6 C 3 F 1 a n d P e p C 3 F 1 m i c e are p h e n o t y p i c a l l y d i s t i n g u i s h a b l e o n the b a s i s of a l l e l i c d i f f e r e n c e s at the L y 5 c e l l s u r f a c e a n t i g e n l o c u s ;  B 6 C 3 F 1 mice are  h o m o z y g o u s L y 5 . 2 a n d P e p C 3 F 1 m i c e are L y 5 . 1 / L y 5 . 2 h e t e r o z y g o t e s . All a n i m a l s w e r e h o u s e d in micro isolator c a g e s a n d p r o v i d e d with sterile f o o d a n d a c i d i f i e d sterile water. 2.2.2. Viral infection of b o n e marrow cells and cell lines B o n e m a r r o w c e l l s from adult m a l e or f e m a l e B 6 C 3 F 1 or P e p C 3 F 1 m i c e injected 4 d a y s previously with 5-fluorouracil in sterile p h o s p h a t e - b u f f e r e d s a l i n e (5F U , 150 m g / k g b o d y weight; H o f f m a n - L a R o c h e Ltd., M i s s i s s a u g a , Ontario) w e r e flushed  from  femoral  shafts  with  alpha  medium  and  5%  F C S (StemCell  T e c h n o l o g i e s ) . F o r c o - c u l t u r e infections 3 x 1 0 ^ m a r r o w c e l l s w e r e i n c u b a t e d o n a confluent m o n o l a y e r of irradiated (1500 c G y X - r a y s ) C D 2 4 viral p r o d u c e r c e l l s for 2 4 to 4 8 h o u r s in m e d i u m c o m p o s e d of D M E M ,  1 5 % F C S , 10 n g / m l  human  interleukin-6 (IL-6), 6 ng/ml murine IL-3, 100 ng/ml murine S t e e l factor, a n d 7 m g / m l p o l y b r e n e ( S i g m a ) . A l l growth f a c t o r s w e r e u s e d a s diluted s u p e r n a t a n t s f r o m t r a n s f e c t e d C O S c e l l s p r e p a r e d in the T e r r y F o x L a b o r a t o r y . C e l l s u s e d for c o m p e t i t i v e r e p o p u l a t i o n e x p e r i m e n t s w e r e i n c u b a t e d for 4 8 h o u r s in the a b o v e m e d i u m in the a b s e n c e of p o l y b r e n e prior to co-culture with viral p r o d u c e r c e l l s in a n effort to e n h a n c e g e n e transfer to the most primitive c e l l s (124, 2 1 7 ) . L o o s e l y a d h e r e n t a n d n o n - a d h e r e n t c e l l s w e r e r e c o v e r e d by gentle agitation a n d w a s h i n g of d i s h e s with D M E M a n d 1 5 % F C S . C e l l s w e r e p e l l e t e d , r e s u s p e n d e d in f r e s h  47  culture m e d i u m a n d incubated for a further 4 8 hours at 3 7 ° C to allow for e x p r e s s i o n of the transferred C D 2 4 g e n e . T h e m u r i n e c e l l line B a / F 3 w a s i n f e c t e d by e x p o s u r e to f i l t e r e d viral s u p e r n a t a n t f r o m the J Z e n C D 2 4 t k n e o e c o t r o p i c viral p r o d u c e r c e l l line. V i r a l s u p e r n a t a n t w a s s u p p l e m e n t e d with 7 u.g/ml p o l y b r e n e . C e l l s w e r e t h e n plated in m e t h y l c e l l u l o s e ( d e s c r i b e d below) in the p r e s e n c e of G 4 1 8 (1 mg/ml). Independent G418  R  c o l o n i e s w e r e p i c k e d a n d e x p a n d e d in the p r e s e n c e of G 4 1 8 to be u s e d a s  positive controls for F A C S a n d S o u t h e r n blot a n a l y s i s . 2.2.3. In vitro c l o n o g e n i c  progenitor  assay  S o r t e d a n d u n s o r t e d b o n e marrow c e l l s w e r e plated in 3 5 m m petri d i s h e s ( 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 , British C o l u m b i a ) in 1.1 ml culture m i x t u r e s c o n t a i n i n g 0 . 8 % m e t h y l c e l l u l o s e in a l p h a m e d i u m s u p p l e m e n t e d with 3 0 % F C S , 1% bovine s e r u m albumin ( B S A ) , 1 0 " M (3-mercaptoethanol, 3 U/ml h u m a n urinary 4  erythropoietin leukocyte  ( 1 0 0 , 0 0 0 units/mg), 2 % S C C M a n d 1 0 % a g a r - s t i m u l a t e d  conditioned  m e d i u m , all of w h i c h  were  obtained  human  from S t e m C e l l  T e c h n o l o g i e s Inc.. C e l l s w e r e plated in the p r e s e n c e or a b s e n c e of 1.5  mg/ml  (approximately 0.9 mg/ml active c o m p o u n d ) of G 4 1 8 ( G i b c o / B R L ) a n d i n c u b a t e d at 3 7 ° C in 5 % C O 2 . L a r g e single a n d multi-lineage c o l o n i e s w e r e s c o r e d after 8-14 d a y s incubation a c c o r d i n g to standard criteria (19). 2.2.4.  CFU-S  assay  Lethally irradiated B 6 C 3 F 1 mice (910-950 c G y , 110 c G y / m i n , w e r e i n j e c t e d i n t r a v e n o u s l y with 1 x 1 0 ^ to 1 x 1 0  4  1  3  7  C s y-rays)  cells from indicated cell  f r a c t i o n s . T w e l v e d a y s later, a n i m a l s w e r e s a c r i f i c e d v i a c e r v i c a l d i s l o c a t i o n a n d well isolated m a c r o s c o p i c s p l e e n c o l o n i e s individually d i s s e c t e d a n d s u s p e n d e d for flow cytometric a n d D N A a n a l y s i s . 2.2.5. B o n e marrow transplantation and quantification of  CRU  In the e x p e r i m e n t s d e s c r i b e d in C h a p t e r s 3 a n d 4 , 1 x 1 0  4  to 4 x 1 0 ^  retrovirally t r a n s d u c e d C D 2 4 + s e l e c t e d or u n s e l e c t e d b o n e m a r r o w c e l l s d e r i v e d 48  f r o m P e p C 3 F 1 (Ly5.1/I_y5.2) d o n o r s w e r e i n t r a v e n o u s l y injected into irradiated ( 1 3 7 C s y-rays, 9 5 0 c G y , 110 c G y / m i n ) B 6 C 3 F 1 (Ly5.2) recipients with or without a lifesparing d o s e of L y 5 . 2 competitor b o n e marrow cells; either 1 x 1 0 from a normal m o u s e or 2 x 1 0  5  5  marrow cells  marrow cells from a c o m p r o m i s e d a n i m a l (59). T h e  function of t h e s e latter c e l l s is to e n s u r e the short term s u r v i v a l of the recipient following the irradiation p r o c e d u r e . T h e level of reconstitution of r e c i p i e n t s with d o n o r (Ly5.1) cells a n d e x p r e s s i o n of the transferred C D 2 4 g e n e w a s a s s e s s e d at 5 to 3 2 w e e k s post-transplantation by flow cytometric a n a l y s i s of p e r i p h e r a l b l o o d s a m p l e s (50-100 JLLI) obtained by tail vein puncture. F o r the e x p e r i m e n t s d e s c r i b e d in C h a p t e r 5 fetal livers w e r e r e m o v e d from d a y 14.5 e m b r y o s o b t a i n e d from timed m a t i n g s of C 5 7 B I / 6 L y - P e p 3 b ( L y 5 . 1 ) m a l e and C3H/HeJ(Ly5.2) female  m i c e . C e l l s w e r e s u s p e n d e d in a l p h a  medium  c o n t a i n i n g 5 % F C S ( S t e m C e l l T e c h n o l o g i e s ) by r e p e a t e d gentle aspiration through 5 ml, 2 ml a n d 1 ml pipettes followed by 18- a n d 2 1 - g a u g e n e e d l e s . B o n e m a r r o w cells  from m a l e or f e m a l e P e p C 3 F 1 m i c e injected 4 d a y s p r e v i o u s l y with 5 -  fluorouracil ( 5 - F U ; 150 m g / k g body weight) w e r e f l u s h e d from f e m o r a l s h a f t s with a l p h a m e d i u m a n d 5 % F C S a n d a single cell s u s p e n s i o n similarly o b t a i n e d . C e l l s were counted using a standard hemocytometer.  Cell survival w a s >98%  as  d e t e r m i n e d by trypan blue e x c l u s i o n . F e t a l liver c e l l s or post 5 - F U b o n e m a r r o w c e l l s from Ly5.1+ d o n o r s w e r e injected in combination with a lifesparing d o s e of 1 0  5  b o n e m a r r o w c e l l s from normal 8-12 w e e k old B 6 C 3 F 1 (Ly5.2+) m i c e (59) into the tail v e i n of recipient B 6 C 3 F 1 (Ly5.2+) m i c e p r e v i o u s l y i r r a d i a t e d with 9 5 0 c G y (110cGy/min,  1  3 C s y-rays). C R U w e r e m e a s u r e d by injecting g r o u p s of 7 lethally 7  irradiated B 6 C 3 F 1 (Ly5.2+) recipients in c o m b i n a t i o n with 1 0  5  s y n g e n i c (Ly5.2+)  n o r m a l b o n e m a r r o w c e l l s a n d a s s e s s i n g the r e c i p i e n t s 16 w e e k s later for the p r e s e n c e of Ly5.1+ lymphoid a n d m y e l o i d c e l l s in their peripheral b l o o d . R e c i p i e n t m i c e w e r e c o n s i d e r e d positive if > 1 % of e a c h of m y e l o i d a n d l y m p h o i d p e r i p h e r a l b l o o d c e l l p o p u l a t i o n s (identified by their unique forward a n d s i d e s c a t t e r profiles,  49  respectively) d e m o n s t r a t e d the d o n o r Ly5.1 cell s u r f a c e p h e n o t y p e . C R U f r e q u e n c y w a s c a l c u l a t e d by determining the n u m b e r of negative recipients a s a function of the n u m b e r of test c e l l s injected a n d a p p l y i n g P o i s s o n statistics u s i n g the m e t h o d of least likelihood (306, 307).  2.3.  Molecular  analysis  2.3.1. S o u t h e r n Blot DNA  was  Analysis  purified  from  NaDodS04/proteinase K-digested  cells  by  p h e n o l / c h l o r o f o r m extraction (308) or u s i n g the D N A z o l r e a g e n t ( C a n a d i a n Life T e c h n o l o g i e s , Burlington, Ontario). D N A w a s d i a l y z e d for 16 h o u r s a g a i n s t 1 x T E (10 m M Tris p H 7 . 5 , 1 m M E D T A p H 8.0) buffer a n d 10-20 u g d i g e s t e d with X b a l , S s t l or E c o R I ( G i b c o / B R L ) at 3 7 ° C for 12-16 hours. Following ethanol precipitation, D N A w a s d i s s o l v e d in 2 0 uJ of 1 x T E buffer a n d s e p a r a t e d on a 0 . 8 % a g a r o s e g e l . G e l s w e r e then treated for 3 5 minutes with Solution I (0.5M N a O H , 1.5 NaCI) a n d for 3 5 minutes with Solution 2 (1M Tris p H 7.0, 2 M NaCI). D N A w a s then transferred to a nylon m e m b r a n e ( Z e t a - P r o b e ; B i o - R a d Laboratories, R i c h m o n d C A ) in 1 0 X S S C by s t a n d a r d blotting m e t h o d s . B l o t s w e r e p r e h y b r i d i z e d at 6 0 ° C for 2 h o u r s in 4 . 4 X S S C , 7 . 5 % f o r m a m i d e , 0 . 7 5 % S D S , 1.5mM E D T A , 0 . 7 5 % s k i m milk, 3 7 0 m g / m l of s a l m o n s p e r m D N A . B l o t s w e r e then h y b r i d i z e d for 2 0 h o u r s at 6 0 ° C u n d e r the s a m e c o n d i t i o n s a s a b o v e with the i n c l u s i o n of 7 . 5 % d e x t r a n s u l f a t e ( S i g m a ) . M e m b r a n e s w e r e s e p a r a t e l y p r o b e d u s i n g a X h o l / S a l l fragment of p M C I n e o (301) containing n e o  R  s p e c i f i c s e q u e n c e . P r o b e s w e r e l a b e l e d with 3 2 p - d C T P ( 3 0 0 0  C i / m m o l ; A m e r s h a m ) by r a n d o m priming a n d purified on a S e p h a d e x - G 5 0 c o l u m n before hybridization. M e m b r a n e s w e r e s u b s e q u e n t l y w a s h e d twice at 6 0 ° C for 3 0 m i n u t e s e a c h in 0 . 3 X S S C , 0 . 1 % S D S a n d 1 m g / m l of s o d i u m p y r o p h o s p h a t e . A u t o r a d i o g r a p h y w a s performed with K o d a k X A R - 5 film a n d a n intensifying s c r e e n at - 7 0 ° C for 1-10 d a y s . M e m b r a n e s w e r e stripped for re-probing by boiling in 1% S D S , a n d w a s h i n g for 4 0 m i n u t e s . B l o t s w e r e r e - p r o b e d with the K p n l / M s e l  50  f r a g m e n t of p X M ( E R ) - 1 9 0 w h i c h r e l e a s e s the full length erythropoietin  receptor  c D N A (kindly p r o v i d e d by A . D ' A n d r e a , D a n a - F a r b e r C a n c e r Institute, B o s t o n , M A ) for a n internal control for D N A loading. Densitometric a n a l y s i s w a s p e r f o r m e d u s i n g a p h o s p h o - i m a g e r with I m a g e Q u a ^ M software ( M o l e c u l a r D y n a m i c s , S u n n y v a l e , CA) 2.3.2. A n t i b o d y staining  procedures  T o a n a l y z e C D 2 4 cell surface antigen expression following  retroviral  infection cells w e r e w a s h e d o n c e in a l p h a m e d i u m with 5 % F C S , r e s u s p e n d e d (1 to 7 x 1fj6 cells/ml) in 0.2 to 0.4 ml of m e d i u m conditioned by h y b r i d o m a 2 . 4 G 2 w h i c h s e c r e t e s a n a n t i - m o u s e IgG F c receptor antibody (309), a n d i n c u b a t e d on ice for 3 0 m i n u t e s in a n effort to r e d u c e n o n - s p e c i f i c staining. C e l l s w e r e then w a s h e d o n c e with H a n k ' s b a l a n c e d salt s o l u t i o n c o n t a i n i n g 2 % F C S ( H F ) . T e t r a m o l e c u l a r c o m p l e x e s of m o n o c l o n a l antibodies w e r e u s e d for the staining p r o c e d u r e (310) by m i x i n g a n t i - C D 2 4 antibody 3 2 D 1 2 ( a g e n e r o u s gift from Dr. S F u n d e r u d , O s l o , N o r w a y ) , anti-R phycoerythrin antibody ID3 a n d F(ab')2 f r a g m e n t s of the anti-IgG antibody P 9 (311) at a 1:2:3 molar ratio. S u c h tetrameric c o m p l e x e s provide a rapid a n d flexible m e a n s of g e n e r a t i n g l a b e l e d a n t i b o d y from e v e n s m a l l q u a n t i t i e s of starting material a n d are equivalent to directly l a b e l e d antibody. T h e s e tetrameric a n t i b o d y c o m p l e x e s w e r e u s e d for staining at a final c o n c e n t r a t i o n of 0.8 u.g/ml. C e l l s w e r e incubated on ice for 4 0 minutes, w a s h e d twice with H F , a n d then s t a i n e d with R-phycoerythrin ( R - P E ) at 2 u.g/ml. After a further 4 0 minutes o n ice, c e l l s w e r e w a s h e d t w i c e with H F a n d r e s u s p e n d e d in H F c o n t a i n i n g 1 | i g / m l of  7-amino  a c t i n o m y c i n D ( 7 A A D , S i g m a ) to distinguish d e a d c e l l s prior to a n a l y s i s by flow c y t o m e t r y u s i n g a F A C S c a n cell a n a l y z e r ( B e c t o n D i c k i n s o n a n d C o . , S a n J o s e , C A ) . C e l l s u s e d in sorting e x p e r i m e n t s w e r e s t a i n e d with p r o p i d i u m i o d i d e (PI, S i g m a ) to distinguish d e a d c e l l s . B o n e marrow c e l l s cultured a l o n e or o n G P + E - 8 6 p a c k a g i n g c e l l s w e r e u s e d a s negative controls.  51  R e p o p u l a t i o n of recipient m i c e with d o n o r - d e r i v e d c e l l s w a s a s s e s s e d b y staining peripheral blood c e l l s , b o n e marrow s p l e e n a n d t h y m u s c e l l s with a n F I T C c o n j u g a t e d anti-Ly5.1 m A b kindly provided by Dr. G . S p a n g r u d e ( R o c k y M o u n t a i n Laboratory,  H a m i l t o n , M T ) a n d a n a l y s i s by flow c y t o m e t r y .  Peripheral  blood  s a m p l e s w e r e o b t a i n e d v i a tail v e i n p u n c t u r e a n d d e p l e t e d of e r y t h r o c y t e s b y incubating t h e m for 10 minutes o n i c e in the p r e s e n c e of 4 v o l u m e s of sterile 1 M NH4CI  solution. Thymic a n d splenic cells were obtained by teasing these organs  apart a n d p a s s a g i n g the c e l l s through a n 21 g a u g e n e e d l e in o r d e r to obtain a s i n g l e cell s u s p e n s i o n . B o n e marrow cells w e r e f l u s h e d from f e m u r s a n d tibia u s i n g a n 21 g a u g e n e e d l e a n d a l p h a m e d i u m a n d 5 % F C S ( S t e m C e l l T e c h n o l o g i e s ) . In s o m e e x p e r i m e n t s p h e n o t y p i c a n a l y s i s of Ly5.1 d o n o r d e r i v e d p e r i p h e r a l b l o o d l e u k o c y t e s w a s a c h i e v e d t h r o u g h d o u b l e a n t i b o d y l a b e l i n g with L y 5 . 1 - F I T C in c o m b i n a t i o n with o n e of G r - 1 - P E (from h y b r i d o m a R B 6 - 8 C 5 p r o v i d e d b y D r . G . S p a n g r u d e ) to identify g r a n u l o c y t e s , M a c - 1 - P E (from h y b r i d o m a M 1 / 7 0 , A T C C , R o c k v i l l e , M D ) to identify m a c r o p h a g e s , B 2 2 0 - P E (from h y b r i d o m a R A 3 - 6 B 2 , Dr. G . S p a n g r u d e ) to identify B lymphocytes or L y 1 - P E (from h y b r i d o m a T I B 1 0 4 , A T C C ) to identify T l y m p h o c y t e s a s d e s c r i b e d below. L e v e l s of e x p r e s s i o n of t h e transferred C D 2 4 g e n e in c e l l s from repopulated m i c e w e r e a s s e s s e d b y s t a i n i n g c e l l s from h e m o p o i e t i c t i s s u e s with a n t i - C D 2 4 tetrameric antibody c o m p l e x e s a n d R - P E a s described above. C D 2 4 expression among peripheral  blood leukocytes w a s  a n a l y z e d b y staining peripheral blood s a m p l e s with a n t i - C D 2 4 / R - P E t e t r a m e r s in combination  with  FITC  labeled  G r - 1 to  identify  granulocytes,  M a c - 1 for  m a c r o p h a g e s , Ly-1 for T cells a n d B 2 2 0 for B c e l l s . C D 2 4 e x p r e s s i o n o n peripheral b l o o d e r y t h r o c y t e s w a s a s s e s s e d by staining p e r i p h e r a l b l o o d s a m p l e s prior to e x p o s u r e to  NH4CI.  T h e e x p r e s s i o n of the transferred C D 2 4 g e n e o n m a r r o w s t e m  c e l l c a n d i d a t e s d e f i n e d b y the S c a + L i n " c e l l s u r f a c e p h e n o t y p e w a s a c h i e v e d through  multiple a n t i b o d y  l a b e l i n g of b o n e m a r r o w c e l l s with a n t i - C D 2 4 / R - P E  52  tetramers in c o m b i n a t i o n with G r - 1 - F I T C , M a c - 1 - F I T C , B 2 2 0 - F I T C , L y - 1 - F I T C a n d S c a - 1 - C y - 5 ( E 1 3 - 1 6 1 . 7 , Dr. G . S p a n g r u d e ) . 2.3.3. F A C S  sorting  C e l l s w e r e sorted on a F A C S t a r + (Beckton D i c k i n s o n a n d C o . , S a n J o s e , C A ) e q u i p p e d with a 5 W argon a n d a 3 0 m W helium n e o n laser. C e l l s w e r e c o l l e c t e d in sterile eppendorf vials in a l p h a m e d i u m with 5 0 % F C S .  53  CHAPTER  SELECTION CELLS  OF  RETROVIRALLY  3  TRANSDUCED  USING CD24 A S A M A R K E R  OF GENE  HEMOPOIETIC TRANSFER  T h e results p r e s e n t e d in this C h a p t e r h a v e b e e n d e s c r i b e d in: Pawliuk, R., R. K a y , P. M . L a n s d o r p , a n d R. K. H u m p h r i e s . 1994. S e l e c t i o n of retrovirally t r a n s d u c e d hematopoietic cells using C D 2 4 a s a m a r k e r of g e n e transfer. B l o o d 8 4 : 2 8 6 8 - 2 8 7 7 . Pawliuk, R., R. K a y , P . M. L a n s d o r p a n d R. K. H u m p h r i e s . 1995. C D 2 4 a s a m a r k e r g e n e for the selection a n d tracking of retrovirally t r a n s d u c e d s t e m c e l l s . In: M o l e c u l a r B i o l o g y of H e m o g l o b i n Switching. E d s . G . S t a m a t o y a n n o p o u l o s a n d A . N i e n h u i s . V o l . 2 of the P r o c e e d i n g s of the 9th C o n f e r e n c e on H e m o g l o b i n S w i t c h i n g , pp. 2 3 1 - 2 4 7 .  54  3.1.  Introduction T h e d e v e l o p m e n t of recombinant retroviruses a s v e c t o r s for g e n e transfer  has  provided  a powerful  t o o l to a d d r e s s c u r r e n t  questions  regarding  n u m b e r s , biological potential, kinetics and regulation. M o r e o v e r ,  HSC  recombinant  retroviruses h a v e p l a y e d a pioneering role in the field of g e n e t h e r a p y . H o w e v e r , the p o w e r of retroviral g e n e t r a n s f e r is currently limited by the p o o r  infection  efficiency of H S C s d u e to their rarity, cycling status a n d paucity of viral receptors. T o aid in the identification, e n r i c h m e n t a n d tracking of t r a n s d u c e d target cells, a variety of s e l e c t a b l e m a r k e r s h a v e b e e n incorporated into retroviral v e c t o r s . T h e most widely u s e d of t h e s e h a v e b e e n intracellular c o m p o n e n t s w h i c h c o n f e r r e s i s t a n c e to toxic c o m p o u n d s s u c h a s n e o m y c i n (23, 3 5 , 2 0 5 ) , h y g r o m y c i n (262, 312,  313),  chloramphenicol  (314),  methotrexate  (216,  234,  263,  315),  m y c o p h e n o l i c a c i d (316), or v a r i o u s c h e m o t h e r a p e u t i c a g e n t s ( 2 6 6 , 3 1 7 - 3 1 9 ) . H o w e v e r , u s e of t h e s e m a r k e r s in s e l e c t i o n p r o t o c o l s c a r r y d i s a d v a n t a g e s that i n c l u d e n o n - s p e c i f i c drug toxicity a n d difficulties in quantifying e x p r e s s i o n l e v e l s . T h e b a c t e r i a l ( 3 - g a l a c t o s i d a s e g e n e (lacZ) a n d the h u m a n p l a c e n t a l  alkaline  p h o s p h a t a s e g e n e h a v e a l s o b e e n e m p l o y e d to s e l e c t t r a n s d u c e d c e l l s in vitro (268, 269) a n d a s reporter m o l e c u l e s both in vitro (268, 2 7 0 , 3 2 0 , 321) a n d in vivo ( 3 2 2 - 3 2 5 ) . H o w e v e r , the p r e s e n c e of a n e n d o g e n o u s m a m m a l i a n l y s o s o m a l (3g a l a c t o s i d a s e a n d p r o b l e m s in a c h i e v i n g a d e q u a t e l e v e l s of e x p r e s s i o n of the e x o g e n o u s p-gal g e n e h a v e limited its effective u s e . G e n e s e n c o d i n g cell s u r f a c e antigens h a v e a l s o b e e n utilized a s s e l e c t a b l e m a r k e r s of g e n e transfer to fibroblasts (271, 2 7 4 , 319) a n d m o r e recently to h u m a n peripheral  blood lymphocytes  (326). T h e u s e of s u c h g e n e s offers  several  potentially significant a d v a n t a g e s including: the rapid a n d quantitative detection of transferred g e n e e x p r e s s i o n in the d e s i r e d target cell population by flow cytometry; the efficient a n d non-toxic s e l e c t i o n of t r a n s d u c e d target c e l l s by F A C S or other i m m u n o - b a s e d selection t e c h n i q u e s ; a n d the tracking of t r a n s d u c e d c e l l s a n d their  55  p r o g e n y both in vitro a n d in vivo. H o w e v e r , at the time that the w o r k d e s c r i b e d in this t h e s i s w a s initiated the applicability of this a p p r o a c h to primitive h e m o p o i e t i c c e l l s h a d not yet b e e n d e m o n s t r a t e d . M o r e o v e r , the g e n e s w h i c h h a d b e e n u s e d a s s e l e c t a b l e m a r k e r s are relatively large, leaving limited s p a c e in the retroviral v e c t o r for other g e n e s of interest. R e c e n t l y , c D N A s e n c o d i n g the h u m a n h e m o p o i e t i c c e l l s u r f a c e a n t i g e n C D 2 4 , a n d its murine h o m o l o g u e the heat stable antigen ( H S A ) h a v e b e e n c l o n e d (299, 3 2 7 ) . T h e function of t h e s e m o l e c u l e s is not yet r e s o l v e d although C D 2 4 h a s b e e n a s s o c i a t e d with activation a n d differentiation e v e n t s in B c e l l s a s well a s the o x i d a t i v e burst r e s p o n s e in g r a n u l o c y t e s (299, 3 2 7 ) , a n d roles for H S A in T cell d e v e l o p m e n t a n d activation h a v e b e e n s u g g e s t e d (328, 3 2 9 ) . B o t h a n t i g e n s a r e g l y c o p r o t e i n s a t t a c h e d to the outer s u r f a c e of the p l a s m a m e m b r a n e by a g l y c o s y l p h o s p h a t i d y l i n o s i t o l lipid a n c h o r a n d are e x p r e s s e d o n multiple  l i n e a g e s of  h e m o p o i e t i c cells. T h e mature peptides are only 3 0 - 3 5 a m i n o a c i d s in s i z e with the entire c o d i n g r e g i o n b e i n g e n c o m p a s s e d within a n a p p r o x i m a t e 2 4 0 bp D N A fragment.  In a d d i t i o n , the m a t u r e C D 2 4 a n d H S A p r o t e i n s s h a r e o n l y  limited  s e q u e n c e h o m o l o g y (57%) with o n e another a n d a n t i b o d i e s to C D 2 4 a n d H S A a r e not c r o s s reactive (300, 327). T h e s e features of s m a l l c o d i n g s i z e , potential for cell s u r f a c e e x p r e s s i o n o n multiple  h e m o p o i e t i c l i n e a g e s a n d limited  homology,  s u g g e s t e d to m e that H S A a n d C D 2 4 w o u l d be p o s s i b l e c a n d i d a t e s for s e l e c t a b l e m a r k e r s of g e n e transfer to h e t e r o l o g o u s h e m o p o i e t i c cells. In the w o r k p r e s e n t e d in this C h a p t e r I d e m o n s t r a t e the feasibility of utilizing C D 2 4 for the identification a n d s e l e c t i o n of retrovirally t r a n s d u c e d primary c e l l s of the m u r i n e h e m o p o i e t i c s y s t e m including t h o s e with long-term l y m p h o - m y e l o i d repopulating ability.  3.2.  RESULTS  3.2.1. T h e  C D 2 4 viral vector  56  T o explore the p o s s i b l e u s e of C D 2 4 a s a s e l e c t a b l e cell s u r f a c e marker, the retroviral v e c t o r d e p i c t e d in F i g u r e 3.1 w a s c o n s t r u c t e d . T h i s v e c t o r c o n t a i n s t h e m i n i m a l 2 4 0 bp C D 2 4 c D N A s e q u e n c e e n c o m p a s s i n g the c o m p l e t e c o d i n g region u n d e r t h e c o n t r o l of t h e M P S V long t e r m i n a l repeat e n h a n c e r a n d p r o m o t e r regulatory e l e m e n t s . F o r initial feasibility s t u d i e s the n e o m y c i n r e s i s t a n c e g e n e u n d e r the control of the thymidine k i n a s e g e n e promoter w a s a l s o i n c l u d e d in the v e c t o r to a i d in viral titering a n d to provide a n i n d e p e n d e n t m e a n s to a s s e s s g e n e t r a n s f e r . A C D 2 4 viral p r o d u c e r w a s g e n e r a t e d u s i n g t h e e c o t r o p i c G P + E - 8 6 p a c k a g i n g line a n d h a d a titre of ~ 5 x 1 0 C F U / m l a s a s s e s s e d b y n e o 5  R  gene  transfer to N I H - 3 T 3 cells.  tk  CD24 I  LTR  neo  1 200 bp  Figure 3.1. S c h e m a t i c of the J Z e n C D 2 4 t k n e o provirus. It i n c o r p o r a t e s a 2 4 0 - b p portion of the C D 2 4 c D N A e n c o m p a s s i n g the c o m p l e t e c o d i n g region; a thymidine k i n a s e - n e o m y c i n r e s i s t a n c e c a s s e t t e (tkneo) from p M C I n e o ; a n d L T R s e q u e n c e s from the M P S V a s d e s c r i b e d in Material a n d M e t h o d s in C h a p t e r 2 .  T r a n s f e r of the C D 2 4 g e n e to hemopoietic c e l l s w a s initially e v a l u a t e d in IL3 - d e p e n d e n t murine B a / F 3 c e l l s . A p p r o x i m a t e l y 8 0 % of B a / F 3 c e l l s w e r e f o u n d to e x p r e s s high l e v e l s of s u r f a c e C D 2 4 antigen after 2 d a y s of co-cultivation with C D 2 4 viral p r o d u c e r s a n d a further 5 d a y s of growth in the a b s e n c e of G 4 1 8 s e l e c t i o n (Figure 3.2). S i m i l a r levels of e x p r e s s i o n w e r e d e t e c t e d a s e a r l y a s 12 h o u r s post infection (data not s h o w n ) . 3.2.2.  FACS  progenitors  selection  of  and CFU-spleen  CD24-transduced  in  vitro  clonogenic  (CFU-S)  D a y 4 5 - F U b o n e marrow cells w e r e co-cultivated with C D 2 4 viral p r o d u c e r s for 2 4 hours a n d r e c o v e r e d non-adherent cells w e r e cultured for a further 4 8 h o u r s 57  to allow e x p r e s s i o n of the transferred C D 2 4 g e n e prior to flow cytometric a n a l y s i s a n d cell sorting. A s s h o w n in a representative F A C S profile for o n e experiment,  Uninfected Ba/F3  CD24 virus infected Ba/F3  Log fluorescence  Figure 3.2. F l o w c y t o m e t r i c a n a l y s i s of C D 2 4 e x p r e s s i o n by B a / F 3 c e l l s previously cocultivated with C D 2 4 viral p r o d u c e r c e l l s a n d then cultured for 5 d a y s in the a b s e n c e of G 4 1 8 . Infected a n d control (uninfected) c e l l s w e r e s t a i n e d with a n a n t i - C D 2 4 - b a s e d tetramolecular antibody c o m p l e x c o u p l e d to R - P E a s d e s c r i b e d in Materials a n d M e t h o d s (Chapter 2).  approximately 1/3 of the b o n e marrow cells r e c o v e r e d after co-cultivation infection without prior growth factor prestimulation w e r e positive for the C D 2 4 c e l l s u r f a c e antigen (see Figure 3.3 top panel). In 3 e x p e r i m e n t s 9 6 ± 3 . 6 % of in vitro c l o n o g e n i c progenitors r e c o v e r e d in the C D 2 4 + fraction w e r e G 4 1 8 - r e s i s t a n t c o m p a r e d to 3 5 6 9 % in the u n s o r t e d m a r r o w population ( s e e F i g u r e 3.3 bottom p a n e l ) . S o m e G 4 1 8 - r e s i s t a n t progenitors (12-27%) w e r e a l s o d e t e c t e d in the C D 2 4 " f r a c t i o n , likely a s a result of overlap b e t w e e n the C D 2 4 + a n d C D 2 4 " c e l l p o p u l a t i o n s a n d the relatively low sorting threshold c h o s e n . CD24'and  C D 2 4 + fractions w e r e a l s o a s s a y e d for d a y 12 C F U - S .  A  s u m m a r y of findings from 3 e x p e r i m e n t s are p r e s e n t e d in T a b l e 3 . 1 . A l l s p l e e n c o l o n i e s derived from the C D 2 4 + fraction (37 of 3 7 a n a l y z e d ) w e r e positive both for 58  proviral  D N A s e q u e n c e s and significant  l e v e l s of C D 2 4 e x p r e s s i o n a b o v e  b a c k g r o u n d staining (range 5 - 7 9 % of c e l l s a n a l y z e d ) . T h e o b s e r v a t i o n that not all c e l l s within a c o l o n y e x p r e s s e d detectable levels of C D 2 4 is likely a result of s o m e admixture of contaminating inter-colony c e l l s a s well a s differences in the a b s o l u t e level  of  C D 2 4 e x p r e s s i o n on  c e l l s within e a c h c o l o n y .  In  contrast,  only  a p p r o x i m a t e l y 5 0 % of s p l e e n c o l o n i e s derived from the unsorted marrow (13 of 30) s h o w e d e v i d e n c e of g e n e transfer by S o u t h e r n blot a n a l y s i s . F u r t h e r m o r e , only 4 of 13 m a r k e d c o l o n i e s w e r e positive for e x p r e s s i o n of the t r a n s f e r r e d C D 2 4 g e n e . S u r p r i s i n g l y , a significant proportion of s p l e e n c o l o n i e s d e r i v e d f r o m the C D 2 4 " fraction (15 of 26) w e r e a l s o f o u n d to c o n t a i n intact p r o v i r u s a l t h o u g h o n l y 3 e x p r e s s e d d e t e c t a b l e l e v e l s of C D 2 4 . T h u s retrovirally-transduced d a y 12 C F U - S c a n be s u c c e s s f u l l y e n r i c h e d b a s e d on their immediate e x p r e s s i o n of a t r a n s d u c e d C D 2 4 g e n e . In a d d i t i o n , the differentiating  d a y 12 p r o g e n y of the C F U - S  thus  s e l e c t e d a l s o s h o w maintained e x p r e s s i o n of the t r a n s d u c e d C D 2 4 g e n e in vivo. 3.2.3. S e l e c t i o n by  F A C S of  CD24-virus-infected  CRU  S u b s e q u e n t e x p e r i m e n t s w e r e c o n d u c t e d to d e t e r m i n e the feasibility of s e l e c t i n g C D 2 4 t r a n s d u c e d C R U by F A C S . In a n effort to facilitate g e n e transfer to repopulating c e l l s , d a y 4 5 - F U b o n e marrow c e l l s w e r e prestimulated with growth factors for 4 8 h o u r s prior to co-culture with C D 2 4 viral p r o d u c e r c e l l s . T h e C D 2 4 e x p r e s s i o n profiles of n o n - a d h e r e n t c e l l s r e c o v e r e d 4 8 h o u r s after the c o - c u l t u r e period for 2 e x p e r i m e n t s are s h o w n in F i g u r e 3.4. G r e a t e r than 5 0 % of c e l l s w e r e C D 2 4 + u s i n g this infection protocol c o m p a r e d to 3 3 % with no pre-stimulation ( s e e F i g u r e 3.3 top panel). U n s o r t e d a n d sorted C D 2 4 or C D 2 4 + c e l l s (Figure 3.4 expt -  1) f r o m Ly5.1 d o n o r m i c e w e r e injected into lethally irradiated L y 5 . 2 r e c i p i e n t s under competitive  r e p o p u l a t i n g c o n d i t i o n s . In p r e l i m i n a r y s t u d i e s injection  of  limiting n u m b e r s of b o n e m a r r o w c e l l s r e c o v e r e d following the sorting p r o c e d u r e r e v e a l e d a c o m p e t i t i v e repopulating cell f r e q u e n c y of a p p r o x i m a t e l y 1/3 x 1 0 . 4  T h e r e f o r e , recipients w e r e transplanted with 1 0 or 4 x 1 0 4  59  4  unsorted or sorted c e l l s  Uninfected BM  CD24 virus infected BM  1(  r  io  u  10  10  io  J  10  L o g fluorescence  120  expt 1  expt2  • •  unsorted Fracl(CD24-)  •  F r a c II ( C D 2 4 + )  expt 3  Figure 3.3. F A C S s e l e c t i o n of C D 2 4 virus-infected in vitro c l o n o g e n i c progenitors a n d d a y 12 C F U - S . ( U p p e r panel) E x p r e s s i o n of C D 2 4 o n d a y 4 5 - F U B M c e l l s 4 8 hours after coculture with C D 2 4 viral p r o d u c e r s c o m p a r e d to uninfected d a y 4 5 - F U B M c e l l s . C e l l s w e r e s t a i n e d with a n t i - C D 2 4 / R - P E tetrameric a n t i b o d y c o m p l e x e s a n d a n a l y z e d by flow cytometry. Infected c e l l s w e r e s o r t e d into C D 2 4 " (I) a n d C D 2 4 + (II) fractions by F A C S a n d a s s a y e d for in vitro c l o n o g e n i c progenitors a n d day 12 C F U - S . ( L o w e r panel) P r o p o r t i o n of G 4 1 8 - r e s i s t a n t in vitro c l o n o g e n i c p r o g e n i t o r s in t h e C D 2 4 " (I), C D 2 4 + (II) a n d presort B M p o p u l a t i o n s for t h r e e i n d e p e n d e n t e x p e r i m e n t s . P r e s o r t B M c e l l s w e r e not o b t a i n e d in e x p e r i m e n t 3 b e c a u s e of lack of c e l l s . N o G 4 1 8 resistant c l o n o g e n i c progenitors w e r e o b s e r v e d in uninfected control B M cells. R e s u l t s of a n a l y s i s of d a y 12 C F U - S - d e r i v e d s p l e e n c o l o n i e s a r e p r e s e n t e d in T a b l e 3.1.  60  Table 3.1. P r o v i r a l Integration a n d C D 2 4 E x p r e s s i o n o n C e l l s f r o m Individual S p l e e n C o l o n i e s D e r i v e d from S o r t e d a n d U n s o r t e d B o n e M a r r o w C e l l s F o l l o w i n g C D 2 4 V i r u s Infection. Cells Transplanted  Total N o . Colonies Analyzed  No. Colonies Positive for C D 2 4 Expression  No. Colonies Positive for Proviral Integration  Unsorted  30  4  13  F r a c II (CD24+)  37  37  37  Frac I (CD24-)  26  3  15  R e c i p i e n t m i c e w e r e transplanted with 1 x 1 0 to 1 x 1 0 b o n e marrow c e l l s , w h i c h h a d b e e n c o - c u l t u r e d with C D 2 4 viral p r o d u c e r c e l l s , from o n e of e a c h of the presort, C D 2 4 " or C D 2 4 + marrow fractions a s d e s c r i b e d in the l e g e n d to F i g 3.3. W e l l i s o l a t e d s p l e e n c o l o n i e s w e r e d i s s e c t e d a n d a n a l y z e d for C D 2 4 e x p r e s s i o n u s i n g flow cytometry a n d for proviral integration using S o u t h e r n blot a n a l y s i s . T h e table r e p r e s e n t s d a t a a c c u m u l a t e d over 3 s e p a r a t e e x p e r i m e n t s . * A s p l e e n c o l o n y w a s c o n c l u d e d to be positive for C D 2 4 e x p r e s s i o n if >5% of the c e l l s b o u n d significant levels of the a n t i - C D 2 4 tetramolecular antibody c o m p l e x . 3  4  in a n effort to m i n i m i z e the likelihood of C D 2 4 " C R U contributing to the transplant d u e to c o n t a m i n a t i o n  of the C D 2 4 + f r a c t i o n . B a s e d u p o n the p r o p o r t i o n  of  uninfected control c e l l s found within the C D 2 4 + sorting w i n d o w (0.7%, F i g u r e 3.4 expt. 1) our c a l c u l a t i o n s predict that for every 2 x 104 c e l l s sorted l e s s than 0 . 0 0 5 C D 2 4 " (i.e. contaminating) C R U w o u l d be f o u n d within the positive sort w i n d o w d u e to o c c u r r e n c e s s u c h a s n o n - s p e c i f i c binding of the C D 2 4 / R P E  tetrameric  antibody c o m p l e x . F o r recipients transplanted with cells from the C D 2 4 + fraction, 10 of 11 w e r e found  to  be  transplantation  reconstituted  with  ( s e e T a b l e 3.2).  provirus-containing E x p r e s s i o n of  CRU 5  C D 2 4 on  weeks  post-  peripheral  blood  l e u k o c y t e s of t h e s e m i c e r a n g e d from 7 - 2 4 % . In 3 m i c e , e v i d e n c e of the s a m e p r o v i r a l integration f r a g m e n t s in c e l l s of both m y e l o i d a n d l y m p h o i d t i s s u e s s u g g e s t e d g e n e transfer to a totipotent repopulating cell. Retrovirally-infected C R U w e r e a l s o f o u n d in the C D 2 4 fraction with 8 of 11 m i c e s h o w i n g e v i d e n c e of -  61  proviral m a r k i n g in either b o n e m a r r o w a n d / o r t h y m u s D N A . H o w e v e r , n o n e of t h e s e 8 r e c i p i e n t s s h o w e d d e t e c t a b l e l e v e l s of C D 2 4 e x p r e s s i o n o n p e r i p h e r a l b l o o d l e u k o c y t e s . Of 5 recipients injected with u n s o r t e d  b o n e m a r r o w c e l l s , all 5  w e r e repopulated with retrovirally m a r k e d cells but only 2 s h o w e d detectable levels of C D 2 4 e x p r e s s i o n in peripheral blood c e l l s . T h e s e results d e m o n s t r a t e that, a s for d a y 12 C F U - S C D 2 4 e x p r e s s i o n in combination with F A C S c a n be u s e d for the s e l e c t i o n of retrovirally t r a n s d u c e d C R U w h o s e progeny maintain e x p r e s s i o n of the t r a n s f e r r e d C D 2 4 g e n e for at least 5 w e e k s post transplantation. T h e s e f i n d i n g s w e r e c o n f i r m e d a n d e x t e n d e d in a s e c o n d experiment in w h i c h retrovirally infected Ly5.1  bone  CD24  +  m  e  d  marrow  cells were  , and C D 2 4  + n i  g  h  sorted  into C D 2 4 " a s w e l l a s  CD24 ' +  0 W  ,  fractions 4 8 hours post infection (see F i g u r e 3.4 expt  2). Limiting n u m b e r s (1 x 1 0 ) of c e l l s from e a c h of the 4 f r a c t i o n s w e r e t h e n 4  i n j e c t e d into lethally irradiated L y 5 . 2 r e c i p i e n t s u n d e r c o m p e t i t i v e  repopulating  c o n d i t i o n s . B a s e d u p o n the sorting w i n d o w s c h o s e n ( F i g u r e 3.4 e x p t 2) o u r c a l c u l a t i o n s s u g g e s t that for every 2 x 1 0  4  cells s o r t e d , l e s s t h a n 0 . 0 3 , 0.01 a n d  0.0001 C D 2 4 - C R U w e r e s o r t e d , respectively in the C D 2 4 +  l o w  , CD24  +  m  e  d  ,  and  C D 2 4 h i g h w i n d o w s , d u e to o c c u r r e n c e s s u c h a s n o n - s p e c i f i c antibody staining. In +  t h e s e e x p e r i m e n t s , r e c i p i e n t s w e r e a n a l y z e d for e v i d e n c e of Ly5.1 d o n o r c e l l derived  repopulation  a n d C D 2 4 g e n e e x p r e s s i o n at 5 a n d  transplant, a s well a s for e x p r e s s i o n of the n e o  R  16 w e e k s  post  g e n e a n d e v i d e n c e of proviral  m a r k i n g at 16 w e e k s post transplant. M i c e t r a n s p l a n t e d with C D 2 4  + l o w  ,  CD24  +  m  e  d  ,  or C D 2 4  + h i  g  h  c e l l s all  s h o w e d significant levels of multi-lineage (i.e., lymphoid a n d myeloid) Ly5.1 donorc e l l - d e r i v e d repopulation a n d all recipients of C D 2 4 + s e l e c t e d m a r r o w c e l l s a g a i n s h o w e d e v i d e n c e of proviral marking in b o n e marrow a n d / o r t h y m u s D N A (Figure 3.5). In 6 of t h e s e m i c e ( m o u s e m l , C D 2 4 C D 2 4  +  m  e  d  fraction; and mice m l  + , 0 W  fraction; m o u s e m l , m 3 a n d m 4 ,  and m2, C D 2 4  + h  ig  n  f r a c t i o n ; F i g u r e 3.5)  retroviral marking patterns o b s e r v e d by S o u t h e r n blot a n a l y s i s of b o n e marrow a n d  62  Expt 1  Expt 2  Uninfected BM  Uninfected BM  CD24 virus infected BM  CD24 virus infected BM  21% 30%  Log fluorescence  Log fluorescence  Figure 3 . 4 . S e l e c t i o n of C D 2 4 v i r u s - i n f e c t e d C R U by F A C S . T h e C D 2 4 e x p r e s s i o n profiles of d a y 4 5 - F U uninfected control m a r r o w c e l l s (top p a n e l s ) a n d d a y 4 5 - F U B M cocultured with C D 2 4 viral p r o d u c e r c e l l s (bottom p a n e l s ) 4 8 h o u r s postinfection are s h o w n for two i n d e p e n d e n t e x p e r i m e n t s . C e l l s w e r e s t a i n e d with a n t i - C D 2 4 / R - P E tetrameric antibody c o m p l e x e s a n d a n a l y z e d by flow cytometry. In experiment 1, infected c e l l s w e r e sorted into C D 2 4 " (I) a n d C D 2 4 + (II) fractions a n d i n j e c t e d into lethally irradiated r e c i p i e n t m i c e u n d e r c o m p e t i t i v e r e p o p u l a t i n g conditions at 1 x 1 0 to 4 x 1 0 c e l l s per m o u s e . M i c e w e r e a n a l y z e d 5 w e e k s post t r a n s p l a n t a t i o n for both C D 2 4 c e l l s u r f a c e e x p r e s s i o n o n p e r i p h e r a l b l o o d l e u k o c y t e s a n d proviral integration in B M a n d t h y m u s (results a r e s h o w n in T a b l e 3.2). F o r e x p e r i m e n t 2, B M c e l l s in presort, C D 2 4 " , C D 2 4 , CD24 and C D 2 4 + ' 9 e x p r e s s i n g fractions w e r e injected into lethally irradiated recipient m i c e under competitively repopulating conditions at 1 x 1 0 c e l l s per m o u s e . M i c e w e r e a n a l y z e d 16 w e e k s posttransplantation for C D 2 4 cell s u r f a c e e x p r e s s i o n o n their peripheral blood c e l l s a n d proviral integration in B M a n d t h y m u s (results are s h o w n in T a b l e 3.3 a n d Figure 3.5). P e r c e n t a g e s of cells in the sort w i n d o w s are indicated. 4  4  + l o w  n  n  4  63  +  m  e  d  thymus  were  clearly  indicative  of  gene transfer  to  a totipotent  long-term  repopulating c e l l . T h e d e g r e e of Ly5.1 d o n o r cell repopulation a m o n g t h e s e m i c e strongly  c o r r e l a t e d with the  intensity  of  proviral  marking  (Figure  3.5),  an  o b s e r v a t i o n c o n s i s t e n t with g e n e transfer to all r e p o p u l a t i n g c e l l s in the C D 2 4 + fractions. A l t h o u g h C D 2 4 e x p r e s s i o n w a s d e t e c t e d in the majority of recipients of C D 2 4 + s e l e c t e d marrow at 5 w e e k s post transplant, e x p r e s s i o n l e v e l s w e r e poorly m a i n t a i n e d at the later 16 w e e k time point a n a l y z e d in this experiment (Figure 3.6). In this c a s e , in only 2 of 8 m i c e repopulated with c e l l s from the C D 2 4 + f r a c t i o n s (mice m2 and m3, C D 2 4  +  m  e  d  cells) w e r e C D 2 4 + p e r i p h e r a l b l o o d ( 4 . 3 % a n d  1 7 . 1 % , r e s p e c t i v e l y ) or b o n e m a r r o w c e l l s ( 3 . 4 % a n d 4 2 % , r e s p e c t i v e l y ) still present. In addition, G 4 1 8  R  c l o n o g e n i c progenitors w e r e d e t e c t e d in the m a r r o w of  t h e s e two m i c e ( 3 % a n d 6 8 % respectively). In the other 6 m i c e , neither C D 2 4 + nor G418  R  c e l l s w e r e d e t e c t e d although provirally m a r k e d c e l l s w e r e p r e s e n t in the  b o n e m a r r o w and/or t h y m u s of all of t h e s e a n i m a l s (data not s h o w n ) . T h e proportion of recipient mice s h o w i n g e x p r e s s i o n of the transferred C D 2 4 g e n e o n peripheral blood l e u k o c y t e s a l s o d e c r e a s e d with time post transplant in a third e x p e r i m e n t in w h i c h recipients w e r e transplanted with 1 x 1 0  5  bone marrow  c e l l s from unsorted or C D 2 4 " or C D 2 4 + sorted fractions. R e c i p i e n t s w e r e a s s e s s e d for C D 2 4 e x p r e s s i o n at 8 a n d 3 2 w e e k s post transplant. W h i l e the v a s t majority of r e c i p i e n t s t r a n s p l a n t e d with C D 2 4 + s o r t e d m a r r o w s h o w e d e x p r e s s i o n of the t r a n s f e r r e d C D 2 4 g e n e at 8 w e e k s (14 of 15 mice) only a fraction w e r e f o u n d to c o n t i n u e to e x p r e s s C D 2 4 w h e n a n a l y z e d at 3 2 w e e k s (3 of 13) d e s p i t e the d e t e c t i o n of intact p r o v i r u s in all m i c e at this t i m e point. R e p r e s e n t a t i v e flow cytometric profiles for 1 of t h e s e mice are s h o w n in Figure 3.7. O n e of 8 m i c e transplanted with u n s e l e c t e d marrow s h o w e d the p r e s e n c e of C D 2 4 o n peripheral blood l e u k o c y t e s at 8 w e e k s post transplant although this m o u s e w a s f o u n d to b e n e g a t i v e for C D 2 4 e x p r e s s i o n at  the later t i m e point. N o m i c e  t r a n s p l a n t e d with C D 2 4 " sorted c e l l s d e m o n s t r a t e d C D 2 4 e x p r e s s i o n at either of  64  Table 3.2. Proviral Integration a n d C D 2 4 E x p r e s s i o n o n C e l l s from C o m p e t i t i v e l y R e p o p u l a t e d M i c e A s s e s s e d 5 W e e k s P o s t -Transplantation (Expt. 1). Cells Transplanted Unsorted  Sort Fraction I (CD24-)  Sort Fraction II (CD24+)  Reconstitution with Retrovirally Marked Cells*  5/5  8/11  10/11  C D 2 4 Cell Surface E x p r e s s i o n in P . B . C e l l s (>5%)+.  2/5  0/11  10/11  S h o w n a r e the n u m b e r of m i c e f o u n d to be p o s i t i v e for r e c o n s t i t u t i o n with retrovirally m a r k e d c e l l s or C D 2 4 cell s u r f a c e e x p r e s s i o n o n p e r i p h e r a l b l o o d (P.B.) l e u k o c y t e s over the total a n a l y z e d at 5 w e e k s post transplantation. * Retroviral marking w a s a s s e s s e d by S o u t h e r n blot a n a l y s i s of b o n e m a r r o w a n d t h y m u s from e a c h transplanted recipient. Blots w e r e s e p a r a t e l y p r o b e d with P l a b e l e d fragments of the n e o g e n e a n d C D 2 4 c D N A with identical results. * E x p r e s s i o n of the C D 2 4 antigen on peripheral blood l e u k o c y t e s w a s a s s e s s e d by s t a i n i n g p e r i p h e r a l b l o o d s a m p l e s d e p l e t e d of erythrocytes with a n t i - C D 2 4 / R - P E tetrameric antibody c o m p l e x e s a n d a n a l y s i s by flow cytometry. 3  R  65  2  unsorted  M U b  contrl  ml B T B  m2  CD24 ml  T B T  m2  CD24' m3  ml  l o w  ml  CD24* '» m  m2  CD24' 9  J  m3  h|  m4  ml  m2  h  m3  B T  Figure 3 . 5 . H e m o p o i e t i c reconstitution from C D 2 4 retrovirus-infected competitive repopulating c e l l s a s a s s e s s e d by S o u t h e r n blot a n a l y s i s of proviral integration in B M (B) a n d t h y m u s (T) 16 w e e k s posttransplantation. R e c i p i e n t s r e c e i v e d 1 x 1 0 c e l l s of C D 2 4 virus-infected marrow from presort, C D 2 4 " , C D 2 4 , CD24 , or C D 2 4 ' g h fractions a s s h o w n for Expt 2 F i g u r e 3.4. Individual recipient m i c e a r e l a b e l e d a s m 1 - m 4 . D N A (20 |ig) from e a c h t i s s u e s a m p l e w a s d i g e s t e d with E c o R l , a n e n z y m e that cuts o n c e within the C D 2 4 provirus s e q u e n c e . S h o w n are results of a blot p r o b e d with a 3 P - l a b e l e d fragment of the n e o g e n e ; identical results w e r e o b s e r v e d u s i n g the C D 2 4 c D N A a s a p r o b e . T h e positive control r e p r e s e n t s D N A o b t a i n e d from a retrovirally infected B a / F 3 c l o n e h a r b o r i n g two c o p i e s of provirus. 4  + l o w  + m e d  + n  2  R  the time points a n a l y z e d in this experiment (see Figure 3.6). Additional a n a l y s i s of b o n e marrow a n d t h y m u s D N A from t h e s e mice revealed no g r o s s r e a r r a n g e m e n t s in proviral structure to account for the lack of e x p r e s s i o n in t h e s e recipients. L a c k of C D 2 4 e x p r e s s i o n a l s o did not a p p e a r to be a result of promoter interference s i n c e G418  R  c l o n o g e n i c progenitors w e r e d e t e c t e d only in t h o s e m i c e s h o w i n g C D 2 4  e x p r e s s i o n on peripheral blood l e u k o c y t e s . T h e proportion of G 4 1 8  R  clonogenic  p r o g e n i t o r s t e n d e d to c o r r e l a t e with the proportion of C D 2 4 + p e r i p h e r a l b l o o d l e u k o c y t e s in recipient mice, ranging from 3 % to 6 8 % . 66  3.3.  Discussion In the work p r e s e n t e d in this C h a p t e r , I h a v e t e s t e d the utility of a c D N A  e n c o d i n g C D 2 4 , a h u m a n cell s u r f a c e a n t i g e n , for the post-infection s e l e c t i o n of h e m o p o i e t i c c e l l s t r a n s d u c e d with a retrovirus  c o n t a i n i n g this c D N A .  FACS  a n a l y s i s in c o m b i n a t i o n with functional s t u d i e s r e v e a l e d that u n d e r the c o n d i t i o n s u s e d , B a / F 3 c e l l s , a f a c t o r - d e p e n d e n t h e m o p o i e t i c cell line, a s w e l l a s p r i m a r y m a r r o w in vitro c l o n o g e n i c progenitors, d a y 12 C F U - S a n d , most significantly, the e a r l i e s t c e l l s c a p a b l e of c o m p e t i t i v e  long-term  e x p r e s s e d C D 2 4 within 4 8 h o u r s of t e r m i n a t i o n  hemopoietic  repopulation  of the i n f e c t i o n  all  procedure.  S o u t h e r n blot a n a l y s i s of b o n e marrow a n d t h y m u s c e l l s from m i c e competitively r e p o p u l a t e d with limiting n u m b e r s of s e l e c t e d C D 2 4 + c e l l s d e m o n s t r a t e d proviral integration in virtually all a n i m a l s in w h i c h d o n o r c e l l s w e r e d e t e c t e d . B e c a u s e long-term repopulating cells are s u c h rare cells a n d h a v e b e e n difficult to purify to h o m o g e n e i t y , it h a s b e e n difficult to a n a l y z e the d i v e r s i t y in p r o p e r t i e s  and  b e h a v i o r of individual cells of this type, particularly following their transplantation in v i v o . T h e ability to obtain, prior to transplant, a population of c e l l s that are 1 0 0 % provirally m a r k e d s h o u l d n o w greatly e n h a n c e the p o w e r of s t u d i e s a i m e d at a d d r e s s i n g s u c h q u e s t i o n s . In addition this type of strategy s h o u l d be u s e f u l a s a preclinical m o d e l for the d e v e l o p m e n t of m o r e effective g e n e transfer s t r a t e g i e s to long-term h u m a n repopulating s t e m cells for u s e in g e n e therapy. T h e r e c o v e r y of c o m p e t i t i v e r e p o p u l a t i n g c e l l s in the top 8 % of C D 2 4 e x p r e s s i n g c e l l s p r o v i d e s indirect e v i d e n c e that the M P S V L T R e n h a n c e r a n d p r o m o t e r a r e a b l e to drive high level g e n e e x p r e s s i o n in the m o s t  primitive  h e m o p o i e t i c c e l l s p r e s e n t in adult m a r r o w t i s s u e . T h e u s e of s u c h a s e l e c t a b l e m a r k e r s h o u l d be useful for optimizing vectors to a c h i e v e high a n d s u s t a i n e d l e v e l s of transferred g e n e e x p r e s s i o n in very primitive h e m o p o i e t i c c e l l s a n d ultimately for s t u d i e s a i m e d at the g e n e t i c m a n i p u l a t i o n of s t e m cell b e h a v i o r . A n interesting finding of this study w a s that C D 2 4 e x p r e s s i o n o b s e r v e d in primitive  67  retrovirally  infected cells at the time of selection w a s a l s o maintained in their p r o g e n y although this did d e c r e a s e with time after transplantation. T h e m o s t d r a m a t i c e v i d e n c e of c o n t i n u e d C D 2 4 e x p r e s s i o n w a s s e e n at the l e v e l of C F U - S ;  1 0 0 % of  the  retrovirally-infected d a y 12 C F U - S in the sorted C D 2 4 + fraction g a v e rise to s p l e e n c o l o n i e s w h i c h w e r e a l s o positive for C D 2 4 e x p r e s s i o n . In contrast, d e s p i t e the d e t e c t i o n of intact p r o v i r u s in a p p r o x i m a t e l y half of the C F U - S (unsorted)  in the  initial  or C D 2 4 " fraction, only a minority of t h e s e y i e l d e d c o l o n i e s of c e l l s  e x p r e s s i n g detectable levels of C D 2 4 . O n e explanation for this o b s e r v a t i o n is that t h e e x p r e s s i o n of the t r a n s f e r r e d C D 2 4 g e n e is integration  site  dependent.  Similarly, most mice transplanted with limiting n u m b e r s of sorted C D 2 4 + C R U w e r e f o u n d to h a v e C D 2 4 e x p r e s s i n g p e r i p h e r a l b l o o d l e u k o c y t e s 5-6 w e e k s p o s t transplantation  (expt. 1), w h e r e a s s u c h c e l l s w e r e not o b s e r v e d in a n i m a l s  r e p o p u l a t e d with C R U from the C D 2 4 " fraction despite the p r e s e n c e of provirus in the m a r r o w a n d / o r t h y m u s of a n u m b e r of t h e s e . S u s t a i n e d e x p r e s s i o n of C D 2 4 in r e c i p i e n t s of initially s e l e c t e d C D 2 4 + C R U w a s a l s o o b s e r v e d 4 m o n t h s p o s t transplantation although in a s m a l l proportion of m i c e d e s p i t e the p e r s i s t e n c e of retrovirally m a r k e d c e l l s in m y e l o i d a n d / o r l y m p h o i d t i s s u e s in all. N e i t h e r g r o s s r e a r r a n g e m e n t in proviral structure (as a s s e s s e d by S o u t h e r n blot a n a l y s i s ) or p r o m o t e r interference b e t w e e n the C D 2 4 a n d n e o  R  g e n e s c o u l d a c c o u n t for the  l a c k of C D 2 4 e x p r e s s i o n o b s e r v e d in t h e s e m i c e . T h e l o s s of C D 2 4 e x p r e s s i o n in s o m e l o n g t e r m r e p o p u l a t e d m i c e t r a n s p l a n t e d with C D 2 4 + s e l e c t e d c e l l s is s u g g e s t i v e of a s h u t d o w n of e x o g e n o u s promoter activity in vivo, a p h e n o m e n o n w h i c h h a s b e e n previously reported (215, 251). S u c h s h u t d o w n of promoter activity m a y b e related to the methylation status of the regulatory e l e m e n t s (257, 3 3 0 , 331). H o w e v e r , others h a v e reported the continued e x p r e s s i o n of transferred g e n e s s u c h a s h u m a n C D 8 (291) a n d the h u m a n g l u c o c e r e b r o s i d a s e g e n e (332) for l o n g p e r i o d s post transplantation. It is important to note, h o w e v e r , that in t h e s e s t u d i e s recipients w e r e p u r p o s e l y transplanted with only o n e or few s t e m c e l l s to e n a b l e  68  Figure 3.6. P r o p o r t i o n of recipient m i c e f o u n d to be e x p r e s s i n g the t r a n s f e r r e d C D 2 4 g e n e at early a n d late time points post transplant. L y 5 . 2 recipient m i c e w e r e transplanted with 1 x 1 0 to 1 x 1fj5 retrovirally infected Ly5.1 B M c e l l s from the sort fractions i n d i c a t e d . E r y t h r o c y t e - d e p l e t e d peripheral b l o o d s a m p l e s w e r e t e s t e d 53 2 w e e k s post transplantation with a n t i - C D 2 4 tetrameric antibody c o m p l e x e s / R - P E a n d a n a l y s i s by flow cytometry. A n i m a l s w e r e s c o r e d positive if > 2 % of c e l l s w e r e C D 2 4 + . T h e total n u m b e r of a n i m a l s t e s t e d is s h o w n in p a r e n t h e s e s a n d the proportion positive for C D 2 4 e x p r e s s i o n i n d i c a t e d in p e r c e n t . R e p o p u l a t i o n of r e c i p i e n t s with Ly5.1 d o n o r - d e r i v e d c e l l s w a s a s s e s s e d by s t a i n i n g with F I T C conjugated Ly5.1 antibody a n d a n a l y s i s by flow cytometry. 4  69  Control mouse  Mouse repopulated with CD24 virus infected marrow  Peripheral blood mononuclear cells  Peripheral red blood ceils  Bone marrow  Spleen  Thymus  L o g fluorescence  Figure 3.7. F l o w c y t o m e t r i c a n a l y s i s of C D 2 4 e x p r e s s i o n in t h e h e m o p o i e t i c t i s s u e s of a m o u s e repopulated with C D 2 4 retrovirus-infected B M 4 m o n t h s post t r a n s p l a n t a t i o n . C e l l s w e r e s t a i n e d with a n t i - C D 2 4 / R - P E t e t r a m e r i c a n t i b o d y c o m p l e x e s . T o a s s e s s C D 2 4 e x p r e s s i o n o n red b l o o d c e l l s s a m p l e s w e r e not d e p l e t e d of erythrocytes prior to the staining p r o c e d u r e .  70  the a n a l y s i s of g e n e e x p r e s s i o n at the c l o n a l l e v e l w h e r e a s in the  studies  m e n t i o n e d a b o v e recipients w e r e not t r a n s p l a n t e d at limiting dilution. M y results w o u l d s u g g e s t that at least for L T R controlled g e n e e x p r e s s i o n the p h e n o m e n o n of promoter  shutdown  may  be m o r e w i d e s p r e a d t h a n  previously appreciated.  M o r e o v e r , it m a y be that m i c e repopulated with limiting n u m b e r s of C D 2 4 + s e l e c t e d c e l l s continue to e x p r e s s the transferred C D 2 4 g e n e but at levels b e l o w that w h i c h c a n b e d e t e c t e d with F A C S . B e c a u s e of the e a s e a n d sensitivity of m e t h o d s for m o n i t o r i n g t r a n s d u c e d C D 2 4 e x p r e s s i o n in p e r i p h e r a l b l o o d c e l l s , this v e c t o r construct s h o u l d be well suited for further s t u d i e s of vector modifications that m a y a b r o g a t e p r o m o t e r s h u t d o w n in primitive h e m o p o i e t i c c e l l s a n d their  long-term  progeny. N o g r o s s a b n o r m a l i t i e s in h e m o p o i e s i s w e r e o b s e r v e d in m i c e e x p r e s s i n g high l e v e l s of C D 2 4 following transplantation with C D 2 4 v i r u s - i n f e c t e d  marrow  c o m p a r e d to n o r m a l control a n i m a l s . Further, s u s t a i n e d e x p r e s s i o n in recipient m i c e at least 4 m o n t h s post transplantation d e m o n s t r a t e s that the u s e of s u c h a foreign a n t i g e n a s a retroviral m a r k e r is c o m p a t i b l e with long t e r m e x p r e s s i o n in h e m o p o i e t i c a l l y reconstituted lethally irradiated recipients. T h e w o r k p r e s e n t e d in this C h a p t e r h a s d e m o n s t r a t e d the feasibility of utilizing the C D 2 4 c e l l s u r f a c e a n t i g e n a s a s e l e c t a b l e m a r k e r a n d a reporter m o l e c u l e in primary h e m o p o i e t i c c e l l s including the most primitive e l e m e n t s of this s y s t e m . T h i s t e c h n i q u e s h o u l d prove useful a s a m e t h o d for i n c r e a s i n g the p o w e r of retroviral m a r k i n g s t u d i e s , rapidly testing v a r i o u s retroviral infection p r o t o c o l s a n d the identification of regulatory e l e m e n t s w h i c h o p t i m i z e g e n e e x p r e s s i o n in primitive hemopoietic s t e m cells or other target cells of interest.  71  CHAPTER  HIGH  LEVEL  CELLS  RECONSTITUTION  EXPRESSING  WITH  A TRANSDUCED SURFACE  4  PRESELECTED GENE  HEMOPOIETIC  ENCODING  A  CELL  ANTIGEN.  T h e results p r e s e n t e d in this C h a p t e r h a v e b e e n d e s c r i b e d in: P a w l i u k , R., a n d R. K. H u m p h r i e s . High level reconstitution with p r e s e l e c t e d h e m o p o i e t i c c e l l s e x p r e s s i n g a t r a n s d u c e d g e n e e n c o d i n g a cell s u r f a c e a n t i g e n . M a n u s c r i p t in preparation.  72  4.1.  Introduction G e n e therapy r e p r e s e n t s a n attractive strategy for the treatment of v a r i o u s  h u m a n heritable d i s o r d e r s , c a n c e r a n d A c q u i r e d I m m u n e D e f i c i e n c y S y n d r o m e ( A I D S ) . S u c c e s s f u l g e n e t h e r a p y of h e m a t o l o g i c a l d i s o r d e r s r e q u i r e s that two major g o a l s be met: (1) efficient a n d stable transduction of the h e m o p o i e t i c s t e m cell ( H S C ) a n d (2) appropriate s u s t a i n e d e x p r e s s i o n of t r a n s d u c e d g e n e ( s ) in the c e l l s of interest. A l t h o u g h significant p r o g r e s s h a s b e e n m a d e t o w a r d s a c h i e v i n g t h e s e g o a l s , efficient high level retroviral g e n e transfer to long t e r m repopulating s t e m c e l l s r e m a i n s a c h a l l e n g e , likely d u e to a multiplicity of factors including low levels of viral receptors a n d the largely quiescent nature of H S C s . T o o v e r c o m e t h e s e difficulties, incorporation  a number  of s t r a t e g i e s  of s e l e c t a b l e m a r k e r g e n e s into retroviral  involving  vectors have  the been  d e v e l o p e d to e n a b l e the e n r i c h m e n t of t r a n s d u c e d target c e l l s . T h e m o s t w i d e l y u s e d of t h e s e h a v e b e e n t h o s e w h i c h confer r e s i s t a n c e to toxic c o m p o u n d s s u c h a s n e o m y c i n (23, 3 5 , 205) or h y g r o m y c i n (262, 3 1 2 ) . T h e a n a l y s i s of  murine  r e c i p i e n t s e n g r a f t e d with b o n e m a r r o w c e l l s s u b j e c t e d to d r u g s e l e c t i o n either before (215, 2 2 7 , 260) or after (266) transplant h a s s h o w n that the proportion of provirally m a r k e d c e l l s in the recipient c a n be i n c r e a s e d u s i n g this strategy. M o r e recently, a n u m b e r of c D N A s e n c o d i n g cell s u r f a c e a n t i g e n s , including the h u m a n low affinity nerve growth factor receptor (273, 326), Thy-1 (279), a n d M D R - 1 (multid r u g r e s i s t a n c e - 1 ) (272), h a v e b e e n utilized a s d o m i n a n t s e l e c t a b l e m a r k e r s . R i c h a r d s o n et. a l . u s e d a c D N A e n c o d i n g M D R - 1 a s a s e l e c t a b l e m a r k e r to d e m o n s t r a t e that p r e s e l e c t i o n of retrovirally t r a n s d u c e d m i d g e s t a t i o n a l fetal liver c e l l s l e a d s to a n i n c r e a s e in the p r o p o r t i o n  of c i r c u l a t i n g  peripheral  blood  l e u k o c y t e s e x p r e s s i n g M D R - 1 a s c o m p a r e d to m i c e receiving u n s e l e c t e d m a r r o w (333).  H o w e v e r , d e s p i t e the potential  that t h e s e p r o c e d u r e s p o s s e s s ,  their  e f f e c t i v e n e s s for a c h i e v i n g high level h e m o p o i e t i c reconstitution with e x c l u s i v e l y provirally  m a r k e d c e l l s h a s not yet b e e n fully e x p l o r e d . M o r e o v e r ,  73  despite  i n c r e a s i n g interest in g e n e t i c a l l y m a n i p u l a t i n g  H S C b e h a v i o r , little is  known  regarding regulatory e l e m e n t s which m a x i m i z e the e x p r e s s i o n of transferred g e n e s in t h e s e c e l l s . T h u s , m e t h o d s e n a b l i n g the efficient s e l e c t i o n , a s w e l l a s the tracking a n d quantification of transferred g e n e e x p r e s s i o n in primitive h e m o p o i e t i c c e l l s a n d their progeny for e x t e n d e d periods of time in vivo are required. I h a v e e x a m i n e d t h e s e i s s u e s in a s y s t e m utilizing the h u m a n c e l l s u r f a c e a n t i g e n C D 2 4 a s a d o m i n a n t s e l e c t a b l e m a r k e r in c o m b i n a t i o n with F A C S .  In  C h a p t e r 3 I d e m o n s t r a t e d that retrovirally t r a n s d u c e d totipotent in vivo repopulating s t e m c e l l s c o u l d be s e l e c t e d for on the b a s i s of C D 2 4 e x p r e s s i o n within 4 8 h o u r s of termination of the infection protocol (278). In the work d e s c r i b e d in this C h a p t e r , this  C D 2 4 selection  approach  is s h o w n  to  enable the  almost  exclusive  regeneration of h e m o p o i e s i s in m y e l o a b l a t e d recipient m i c e with provirally m a r k e d c e l l s . T h e contribution of individual H S C s to h e m o p o i e s i s w a s a n a l y z e d by proviral integration a n a l y s i s . M o r e o v e r , persistent e x p r e s s i o n of the transferred C D 2 4 g e n e w a s o b s e r v e d in a s i g n i f i c a n t proportion  of S c a + L i n " b o n e m a r r o w c e l l s (a  s u b p o p u l a t i o n k n o w n to be e n r i c h e d for c e l l s with long term in vivo r e p o p u l a t i n g ability), p e r i p h e r a l b l o o d l e u k o c y t e s , red b l o o d c e l l s , s p l e e n , t h y m u s a n d w h o l e b o n e m a r r o w c e l l s for a m i n i m u m of 6 m o n t h s post transplant. Finally, this s t u d y r e v e a l s intriguing e v i d e n c e of v e c t o r - b a s e d d i f f e r e n c e s in the ability to  drive  persistent e x p r e s s i o n of transferred g e n e s in vivo. 4.2.  Results  4.2.1. Viral v e c t o r s and experimental  design  In the c o u r s e of t h e s e e x p e r i m e n t s two v e c t o r s b a s e d o n different  viral  b a c k b o n e s (Figure 4.1 u p p e r panel) w e r e e m p l o y e d . In both v e c t o r s , the m i n i m a l 2 4 0 b p c o d i n g region of the C D 2 4 c D N A w a s p l a c e d u n d e r the control of the viral L T R regulatory e l e m e n t s . Both vectors a l s o c o n t a i n e d a c D N A e n c o d i n g r e s i s t a n c e to the n e o m y c i n a n a l o g G 4 1 8 . In the J Z e n C D 2 4 t k n e o vector (hereafter referred to a s J Z e n C D 2 4 ) the n e o m y c i n r e s i s t a n c e c a s s e t t e is d r i v e n from the  74  thymidine  k i n a s e g e n e p r o m o t e r , w h i l e in M S C V n e o l R E S C D 2 4 (hereafter r e f e r r e d to a s M S C V C D 2 4 ) both the n e o m y c i n r e s i s t a n c e a n d C D 2 4 c D N A s a r e driven from the viral L T R d u e to the inclusion of a n internal ribosomal entry site d e r i v e d from the 5' u n t r a n s l a t e d region of the e n c e p h a l o m y o c a r d i t i s v i r u s . B o t h the J Z e n C D 2 4 a n d M S C V C D 2 4 viral p r o d u c e r s y i e l d e d titres of a p p r o x i m a t e l y 5 x 1 0 a s s e s s e d by transfer of n e o  R  5  CFU/ml as  to N I H - 3 T 3 c e l l s with no h e l p e r virus d e t e c t e d in  either line. In a n effort to facilitate g e n e transfer to repopulating s t e m cells, d a y 4 5F U B M c e l l s w e r e prestimulated with growth factors for 4 8 hours prior to 4 8 hours of co-culture with irradiated viral p r o d u c e r cells. C e l l s r e c o v e r e d from co-culture w e r e s u b s e q u e n t l y s t a i n e d for C D 2 4  expression as detailed  in C h a p t e r 2 .  The  e x p r e s s i o n profiles a n d sort t h r e s h o l d s for 1 e x p e r i m e n t are s h o w n in F i g u r e 4.1 (lower panel). F o l l o w i n g F A C S s e l e c t i o n , 4 x 1 0  5  C D 2 4 + c e l l s per recipient w e r e  t r a n s p l a n t e d into a total of 14 m y e l o a b l a t e d m i c e (7 recipients per viral construct). T h i s t r a n s p l a n t d o s e w a s e s t i m a t e d to c o n t a i n 12 ± 4 c o m p e t i t i v e  repopulating  units ( C R U ) a s d e t e r m i n e d from p r e v i o u s e x p e r i m e n t s c a r r i e d out to d e t e r m i n e C R U f r e q u e n c i e s in the population of c e l l s r e c o v e r e d following the infection a n d s e l e c t i o n p r o c e d u r e (data not s h o w n ) . 4.2.2. T h e  majority of d o n o r - d e r i v e d cells  in recipients c o n t a i n  intact  provirus S i x m o n t h s post transplant, the a v e r a g e proportion of t r a n s p l a n t d e r i v e d (Ly5.1+) cells in recipients w a s 5 8 % for bone marrow (range 2 2 % to 6 8 % ) , 8 5 % for p e r i p h e r a l b l o o d m o n o n u c l e a r c e l l s (range 4 7 % to 9 5 % ) , a n d 9 3 % for t h y m u s ( r a n g e 4 7 % to 1 0 0 % ) ( s e e T a b l e 4.1). T h u s , the v a s t majority of m i c e s h o w e d virtually c o m p l e t e reconstitution of b o n e m a r r o w a n d t h y m u s with Ly5.1 d o n o r d e r i v e d c e l l s (taking  into a c c o u n t that a p p r o x i m a t e l y 3 0 % of the m a r r o w  is  c o m p o s e d of differentiating erythroid progenitors a n d their p r o g e n y w h i c h d o not e x p r e s s the Ly5.1 antigen). E x p r e s s i o n of the t r a n s d u c e d C D 2 4 g e n e w a s d e t e c t e d  75  VIRAL V E C T O R S A N D F A C S S E L E C T I O N JZenCD24tkneo LTR  TK  CD24  NEO  =1  3.0 Kb Sstl  MSCVneolRESCD24 LTR  IRES  NEO  CD24  -  LTR  2.5 Kb Sstl  4  CD24-PE  Figure 4.1. V i r a l v e c t o r s u s e d a n d F A C S s e l e c t i o n of retrovirally t r a n s d u c e d b o n e m a r r o w c e l l s . ( U p p e r p a n e l ) S c h e m a t i c of t h e J Z e n C D 2 4 t k n e o a n d M S G V n e o l R E S C D 2 4 provirus. J Z e n G D 2 4 t k n e o i n c o r p o r a t e s a 2 4 0 - b p portion of the c D N A e n c o m p a s s i n g the c o m p l e t e c o d i n g r e g i o n ; a t h y m i d i n e k i n a s e n e o m y c i n r e s i s t a n c e c a s s e t t e (tkneo) from p N C I n e o ; a n d L T R s e q u e n c e s from M P S V . M S C V n e o l R E S C D 2 4 c o n t a i n s the n e p g e n e from p M C I n e o ; the c o d i n g r e g i o n of the C D 2 4 c D N A ; a n internal r i b o s o m a l entry s e q u e n c e f r o m t h e encephalomyocarditis virus; and L T R s e q u e n c e s from M S C V . (Lower panel) E x p r e s s i o n of C D 2 4 o n d a y 4 5 - F U B M c e l l s 4 8 h o u r s after c o - c u l t u r e with J Z e n C D 2 4 or M S C V C D 2 4 viral p r o d u c e r s c o m p a r e d to uninfected d a y 4 5 - F U B M c e l l s . C e l l s w e r e s t a i n e d with a n t i - C D 2 4 / R - P E tetrameric antibody c o m p l e x e s a n d a n a l y z e d by flow c y t o m e t r y . C D 2 4 + c e l l s w e r e s o r t e d a n d t r a n s p l a n t e d into m y e l o a b l a t e d recipient mice at 4 x 105 C D 2 4 + c e l l s per recipient.  76  o n peripheral b l o o d l e u k o c y t e s , red blood c e l l s a n d w h o l e b o n e m a r r o w c e l l s for all r e c i p i e n t s a n d on t h y m o c y t e s for all but 2 m i c e . T h e proportion of transplant d e r i v e d c e l l s e x p r e s s i n g C D 2 4 (ie. L y 5 . 1 C D 2 4 + cells) v a r i e d with the l i n e a g e of +  the c e l l type t e s t e d , a n d a s a function of the virus u s e d (Table 4.1). T h e greatest proportion of C D 2 4 + c e l l s w a s o b s e r v e d a m o n g B M , peripheral b l o o d l e u k o c y t e s a n d red b l o o d cells, with the lowest proportion found in t h y m u s . F o r e x a m p l e , 7 4 % of w h o l e B M , 9 0 % of red blood c e l l s a n d 5 8 % of peripheral b l o o d l e u k o c y t e s in recipient m 5 , transplanted with M S C V C D 2 4 t r a n s d u c e d marrow, w e r e positive for C D 2 4 e x p r e s s i o n c o m p a r e d to only 1 8 % of t h y m o c y t e s (Table 4.1). M i c e receiving M S C V C D 2 4 t r a n s d u c e d B M s h o w e d s o m e 6-fold higher proportion of C D 2 4 + c e l l s for all cell t y p e s tested a s c o m p a r e d to m i c e transplanted with J Z e n C D 2 4 infected marrow  (Table 4.1). A s an independent  a s s e s s m e n t of g e n e t r a n s f e r ,  the  p r o p o r t i o n of G 4 1 8 r e s i s t a n c e c l o n o g e n i c p r o g e n i t o r s in the b o n e m a r r o w  of  r e c i p i e n t s w a s d e t e r m i n e d a n d found to correlate with the proportion of C D 2 4 + c e l l s in total b o n e marrow (Table 4.1) Stringent  s o r t i n g t h r e s h o l d s d e p i c t e d in F i g u r e 4.1  (< 0 . 2 % of  non-  t r a n s d u c e d c o n t r o l b o n e m a r r o w c e l l s positive) w e r e c h o s e n in a n effort to m a x i m i z e the probability that only retrovirally t r a n s d u c e d s t e m c e l l s e x p r e s s i n g significant l e v e l s of C D 2 4 w o u l d b e i s o l a t e d . D e s p i t e this, o n a v e r a g e o n l y a proportion of transplant d e r i v e d c e l l s w e r e found to e x p r e s s C D 2 4 , s u g g e s t i v e of p r o m o t e r s h u t d o w n or the inefficient  s e l e c t i o n of t r a n s d u c e d s t e m c e l l s . T o  d i s c r i m i n a t e b e t w e e n t h e s e possibilities S o u t h e r n blot a n a l y s i s w a s c a r r i e d out to a s s e s s the p r e s e n c e of intact provirus in the B M , s p l e e n a n d t h y m u s of recipient m i c e . A s s h o w n in F i g u r e 4 . 2 , high levels of intact provirus w e r e d e t e c t a b l e in the h e m o p o i e t i c t i s s u e s of five r e p r e s e n t a t i v e r e c i p i e n t s of J Z e n C D 2 4 t r a n s d u c e d marrow.  C o m p a r a b l e results were  o b s e r v e d for  recipients  of  MSCVCD24  t r a n s d u c e d B M (data not s h o w n ) . Densitometric a n a l y s i s of the s i g n a l intensities o b t a i n e d w h e n m e m b r a n e s w e r e s e q u e n t i a l l y p r o b e d with a f r a g m e n t of the  77  Table 4.1. Flow Cytometric Analaysis of CD24 Expression in Various Hematopoietic Tissues in Recipients of JZenCD24 or MSCVCD24 Virus-Infected Marrow Assessed 24 Weeks Post-Transplant.  %G418 BM Prog. R  Whole BM Cells Transplanted  Mouse  % CD24+/ % Ly5.1 + Ly5.1+*  Peripheral RBCs % CD24+1  Thymus % CD24+ %Ly5.1 + Ly5.1 +  Peripheral Blood Leukocytes % CD24 %Ly5.1 + Ly5.1 +  +/  JZenCD24  ml m2 m3 m4 m5 m6 m7  64 66 58 57 66 67 67 62 + 2  8 18 16 5 12 21 25 15 + 3  7 3 6 4 2 15 13 7±3  98 98 98 96 99 99 99 98 + 0.5  0 1 3 0 3 1 7 2+1  93 94 93 82 91 90 92 91 +2  4 2 4 4 2 8 10 5+ 1  6 13 11 3 5 24 34 14 ± 6  MSCVCD24  ml m2 m3 m4 m5 m6  29 68 22 64 61 66 52+ 10  52 85 41 30 74 88 62 ± 1 1  25 44 23 29 90 41 42 ± 13  99 98 31 93 100  8 21 10 15 18 20 15 ± 4  70 81 47 92 95 90 79 ± 9  14 33 28 5 58 29 28 ± 9  56 90 40 64 87 90 71+ 10  99  87 ± 1 4  Repopulation of recipients with Ly5.1 donor-derived cells was assessed by staining erylhrocyte-depleted peripheral blood samples with FITC-conjugated Ly5.1 antibody and analysis by flow cytometry. Samples were also tested for expression of the transferred CD24 gene by staining cells with anti-CD24 tetrameric antibody complexes/R-PE and analysis by flow cytometry. CD24 expression on bone marrow stem cell candidates was achieved by combining anti-CD24 tetrameric antibody complexes with a cocktail of antibodies recognizing lineage specific antigens as explained in Chapter 2. The proportion of G418R clonogenic progenitors in BM was determined as described in Material and Methods. * The expression of CD24 in various hemopoietic tissues is given as the proportion of cells with the Ly5.1+ cell surface phenotype expressing CD24. +Ly5.1 is not expressed on red blood cells (RBCs). Therefore, the % CD24+ values given for RBC represents the % of total RBCs.  n e o m y c i n r e s i s t a n c e g e n e to detect r e c o m b i n a n t provirus a n d the  erythropoietin  r e c e p t o r g e n e a s a n e n d o g e n o u s control s h o w e d that the a v e r a g e proviral c o p y n u m b e r per haploid g e n o m e w a s 2.6 (range 1.2 to 4.2) a n d 2.5 (range 0.6 to 6.5) for r e c i p i e n t s of J Z e n C D 2 4 a n d M S C V C D 2 4 infected B M r e s p e c t i v e l y . T h e s e f i n d i n g s a r e c o n s i s t e n t with high level r e g e n e r a t i o n with provirally m a r k e d c e l l s a n d s u g g e s t that C D 2 4 e x p r e s s i o n in only a fraction of transplant d e r i v e d ( L y 5 . 1 ) +  c e l l s is m o s t likely the result of d o w n regulation of the t r a n s f e r r e d C D 2 4 g e n e rather than the a b s e n c e or g r o s s alteration of recombinant provirus. T o further a s s e s s the d e g r e e of reconstitution with provirally m a r k e d c e l l s , f e m o r a l B M from primary recipients w a s transplanted into s e c o n d a r y recipients to g e n e r a t e d a y 12 C F U - S . Of 19 a n d 2 0 individual s p l e e n c o l o n i e s g e n e r a t e d from the B M of recipients of J Z e n C D 2 4 a n d M S C V C D 2 4 infected marrow, respectively, all w e r e found to contain intact provirus. T h i s finding strongly s u g g e s t s that at the time  of  s a c r i f i c e e s s e n t i a l l y all h e m o p o i e s i s w a s d e r i v e d f r o m  retrovirally  t r a n s d u c e d s t e m c e l l s . Integration a n a l y s i s of h e m o p o i e t i c t i s s u e s of  primary  transplant recipients a n d of d a y 12 s p l e e n c o l o n i e s g e n e r a t e d from t h e s e m i c e are p r e s e n t e d in F i g u r e 4 . 3 . T w o of the s e l e c t e d m i c e m a n i f e s t e d different patterns of c l o n a l reconstitution. A n a l y s i s of s p l e n i c a n d thymic D N A o b t a i n e d from a primary recipient of J Z e n C D 2 4 t r a n s d u c e d marrow (top panel) s h o w s a c o m p l e x pattern of p r o v i r a l m a r k i n g indicative of p o l y c l o n a l reconstitution. T h e a n a l y s i s of d a y 12 s p l e e n c o l o n i e s g e n e r a t e d from the B M of this a n i m a l is c o n s i s t e n t with at least 4 c l o n a l integration patterns b e i n g d e t e c t e d a n d h e n c e , is s u g g e s t i v e of multiple H S C s actively contributing to h e m o p o i e s i s at the time of s a c r i f i c e . In contrast, the a n a l y s i s of B M a n d t h y m i c transduced  marrow  D N A obtained from  s h o w e d a pattern  of  a r e c i p i e n t of  proviral  marking  MSCVCD24  consistent  with  m o n o c l o n a l reconstitution ( F i g u r e 4 . 3 , bottom p a n e l ) . T h e i d e n t i c a l pattern  of  proviral m a r k i n g a m o n g all d a y 12 s p l e e n c o l o n i e s g e n e r a t e d from the B M of this m o u s e further s u p p o r t s this c o n c l u s i o n .  79  neg. Ctrl  ml B  S  m2 T  B  S  m3 T  B  S  m4 T  B  10  -  S  T  m5 B  S  ^ » f # ^  pos. ^  T  —  3.0 K b  Figure 4 . 2 . D e t e c t i o n of high l e v e l s of intact provirus in the b o n e m a r r o w (B), s p l e e n (S), a n d t h y m u s (T) of recipient m i c e t r a n s p l a n t e d with 4 x 1 0 ^ C D 2 4 + s e l e c t e d , J Z e n C D 2 4 virus infected b o n e marrow cells at 2 4 w e e k s post transplant by S o u t h e r n blot a n a l y s i s . Individual recipient m i c e are l a b e l e d a s m 1 - m 5 . D N A (10 jig) from e a c h t i s s u e s a m p l e w a s d i g e s t e d with S s t I, a n e n z y m e w h i c h cuts o n c e within e a c h proviral L T R . S h o w n are the results of a blot p r o b e d with a 3 2 p . l a b e l e d f r a g m e n t of the n e o g e n e (top) a n d a fragment of the erythropoietin r e c e p t o r g e n e a s a n e n d o g e n o u s control for D N A loading (bottom). T h e positive control r e p r e s e n t s D N A obtained from a retrovirally infected B a / F 3 c l o n e harboring two c o p i e s of provirus. R  80  T h u s , d e s p i t e the transplant of multiple t r a n s d u c e d H S C s , long term h e m o p o i e s i s w a s a s s o c i a t e d with m o n o c l o n a l s t e m cell activity. T h e s e differences in the pattern of h e m o p o i e t i c reconstitution, however, w e r e not related to the viral vector u s e d . B M from primary recipients of C D 2 4 + s e l e c t e d c e l l s w a s a l s o t r a n s p l a n t e d into s e c o n d a r y m y e l o a b l a t e d recipients in a n effort to a s s e s s the r e g e n e r a t i o n of transduced  long term  repopulating  stem  cells.  S e c o n d a r y recipients  were  s a c r i f i c e d 5 m o n t h s post transplant a n d D N A from B M a n d t h y m u s a n a l y z e d by S o u t h e r n blot. T h e p r e s e n c e of recombinant provirus w a s d e t e c t e d in both B M a n d t h y m u s of all s e c o n d a r y r e c i p i e n t s ( F i g u r e 4.3) d e m o n s t r a t i n g that the f e m o r a l m a r r o w of the p r i m a r y recipient h a d b e e n r e g e n e r a t e d with H S C s that w e r e retrovirally t r a n s d u c e d . M o r e o v e r , in s e v e r a l i n s t a n c e s the pattern of  proviral  m a r k i n g o b s e r v e d in the h e m o p o i e t i c t i s s u e s of s e c o n d a r y recipients w a s identical to that o b s e r v e d a m o n g d a y 12 s p l e e n c o l o n i e s . F o r e x a m p l e , a n identical pattern of proviral marking w a s o b s e r v e d b e t w e e n m o u s e D a n d s p l e e n c o l o n i e s 2 a n d 5 (top panel); m o u s e A B M with s p l e e n c o l o n i e s 6-8 (top panel); a n d m i c e F - J with c o l o n i e s 1-9 (bottom panel). T h i s o b s e r v a t i o n d e m o n s t r a t e s that h e m o p o i e s i s in the primary recipients w a s being s u s t a i n e d by totipotent c e l l s with the c a p a c i t y for long term  hemopoietic  reconstitution.  In a d d i t i o n  Figure 4.3 provides clear  e v i d e n c e of s e l f - r e n e w a l of t r a n s d u c e d H S C s within the f e m o r a l m a r r o w of the p r i m a r y recipient. T h e m o s t striking e v i d e n c e of s e l f - r e n e w a l is s e e n a m o n g s e c o n d a r y recipients s h o w n in the bottom p a n e l . All 5 m i c e ( M S C V C D 2 4 F-J) s h o w the s a m e pattern of proviral m a r k i n g in B M a n d thymic D N A s u g g e s t i n g that the H S C s r e s p o n s i b l e for regenerating t h e s e m i c e w e r e d e r i v e d from a s t e m cell that h a d u n d e r g o n e a m i n i m u m of 4 self-renewal division e v e n t s in the f e m o r a l m a r r o w of the primary recipient. T o g e t h e r , t h e s e r e s u l t s d e m o n s t r a t e the utility of the C D 2 4  selection  a p p r o a c h to e n a b l e the regeneration of the h e m o p o i e t i c s y s t e m s of m y e l o a b l a t e d recipient m i c e to very high levels with provirally m a r k e d c e l l s , a n d a s a m e a n s to  81  track the s e l f - r e n e w a l of individual H S C s a n d their contribution to h e m o p o i e s i s following transplant. 4.2.3. G e n e transfer to and e x p r e s s i o n  of CD24 a m o n g S c a + L i n "  bone  marrow stem cell c a n d i d a t e s T o obtain more direct e v i d e n c e of e x p r e s s i o n of the transferred C D 2 4 g e n e in primitive h e m o p o i e t i c c e l l s , C D 2 4 e x p r e s s i o n w a s a n a l y z e d in the S c a + L i n " s u b p o p u l a t i o n of B M , a population k n o w n to be e n r i c h e d for c e l l s with long term repopulating ability. A n a l y s i s w a s carried out 4 8 h o u r s following termination of the infection p r o c e d u r e , a n d in reconstituted m i c e 6 m o n t h s post transplant. A n t i b o d y s t a i n i n g p e r f o r m e d 4 8 h o u r s after  retroviral  infection s h o w e d that both  the  M S C V C D 2 4 a n d J Z e n C D 2 4 v e c t o r s w e r e a b l e to drive high l e v e l s of C D 2 4 e x p r e s s i o n in the majority of S c a + L i n " c e l l s ( 7 5 % v s . 6 6 % respectively)  (Figure  4.4). N o difference in the a b s o l u t e level of C D 2 4 e x p r e s s i o n a m o n g c e l l s infected with either virus w a s d e t e c t e d . At 6 m o n t h s post transplant, the proportion of r e g e n e r a t e d S c a + L i n " c e l l s e x p r e s s i n g the t r a n s f e r r e d C D 2 4 g e n e v a r i e d a s a function of the v e c t o r u s e d . W h i l e 8 1 % (range 5 3 % - 9 7 % ) of the r e g e n e r a t e d S c a + L i n " c e l l s in recipients of M S C V C D 2 4 t r a n s d u c e d m a r r o w w e r e positive for C D 2 4 e x p r e s s i o n , o n l y 2 2 % (range  12%-45%)  of  C D 2 4 + S c a + L i n " c e l l s w e r e d e t e c t e d in r e c i p i e n t s of  J Z e n C D 2 4 t r a n s d u c e d m a r r o w ( F i g u r e 4.5). T h e s e r e s u l t s c o n t r a s t with the e s s e n t i a l l y c o m p l e t e reconstitution with t r a n s d u c e d c e l l s o b s e r v e d in the B M for both v e c t o r s . T h e e x p r e s s i o n of C D 2 4 w a s a l s o d e t e c t e d in s e c o n d a r y transplant recipients, although a g a i n , vector related differences in the proportion of S c a + L i n " c e l l s e x p r e s s i n g C D 2 4 w e r e d e t e c t e d . T h e a v e r a g e proportion of S c a + L i n " c e l l s e x p r e s s i n g C D 2 4 w a s 5 9 % (range 3 9 % - 8 4 % ) , a n d 8 % (range 0%-27%) recipients of M S C V C D 2 4 a n d J Z e n C D 2 4 infected marrow respectively.  82  for  JZenCD24tkneo pos. Ctrl. -|0 S T  day 12 spleen col.  A  B  2°  C  D  ctrl neg.  1 2 3 4 5 6 7 8 B T B T B S B T — 15Kb  ¥  — 8.8Kb — 5.8Kb — 4.8Kb  — 3.0Kb  MSCVneolRESCD24 pos. Ctrl. 1 0 BT  2°  day 12 spleen col.  F  G  H  I  J  neg. ctrl  1 2 3 4 5 6 7 8 9 B T B T B T B T B T  — 7.2Kb  — 4.5Kb  Figure 4 . 3 . A s s e s s m e n t of proviral integration in b o n e m a r r o w (B), s p l e e n (S), a n d / o r t h y m u s (T) in primary or s e c o n d a r y recipients a n d d a y 12 s p l e e n c o l o n i e s g e n e r a t e d from the femoral marrow of s e l e c t e d primary recipients by S o u t h e r n blot a n a l y s i s . P r i m a r y r e c i p i e n t s r e c e i v e d 4 x 1 0 C D 2 4 + s e l e c t e d J Z e n C D 2 4 or M S C V C D 2 4 infected b o n e m a r r o w c e l l s a n d w e r e s a c r i f i c e d at 2 4 w e e k s post t r a n s p l a n t . F e m o r a l b o n e m a r r o w from s e l e c t e d primary m i c e w e r e u s e d a s a s o u r c e of d o n o r c e l l s for the g e n e r a t i o n of d a y 12 s p l e e n c o l o n i e s a n d for transplant into s e c o n d a r y recipients. F o r the a n a l y s i s of d a y 12 s p l e e n c o l o n i e s lethally irradiated recipient m i c e w e r e t r a n s p l a n t e d with 1 x 1 0 f e m o r a l b o n e m a r r o w c e l l s . T w e l v e d a y s later a n i m a l s w e r e s a c r i f i c e d v i a c e r v i c a l d i s l o c a t i o n a n d i n d i v i d u a l s p l e e n c o l o n i e s d i s s e c t e d a n d D N A purified for S o u t h e r n blot a n a l y s i s . L e t h a l l y irradiated s e c o n d a r y transplant r e c i p i e n t s r e c e i v e d 2 x 10® f e m o r a l b o n e marrow c e l l s . M i c e w e r e sacrificed 5 months later a n d D N A extracted from b o n e marrow, s p l e e n a n d t h y m u s . Individual recipient mice are l a b e l e d a s 1 ° or 2 ° (A-J) while individual d a y 12 s p l e e n c o l o n i e s are numerically l a b e l e d (1-9). D N A (10 u.g) w a s d i g e s t e d with E c o R I , a n e n z y m e w h i c h c u t s o n c e within the proviral s e q u e n c e . S h o w n are the results of two s e p a r a t e blots (top; J Z e n C D 2 4 ; bottom; M S C V C D 2 4 ) p r o b e d with a P l a b e l e d fragment of the n e o g e n e . T h e positive control is D N A obtained from a retrovirally infected B a / F 3 c l o n e harboring two c o p i e s of the J Z e n C D 2 4 t k n e o provirus. T h e negative control is D N A o b t a i n e d from the s p l e e n of a normal unmanipulated control m o u s e . 5  5  3  2  R  83  0  v.-f»" • h 50 100 150 200 250 FSC-H  Control bm 0.3  0.1  -b  ° 6 50 100 150 200 250 FSC-H  FSC-H  JZenCD24tkneo infected bm  MSCVneolRESCD24 infected bm  65.5  4.4  75.2  6.3  11.0/fvv  6.2  48 hrs post inf UJ  0.  24 wks post tx  l  DC •  °  Q  o  o  a s : * ! ! ! i|^; ;^13.8  23.5^P" ? : :  10  -  0.1  2.6  16.3  4.4  <b  Sr  : - v ; 7.7  10° 10 10 10 10 1  «b  2  3  32.7  11.5  •b  ^fPf*v2.4  10" 10 10 10 10 1  2  3  10° 10 1  10  10 10' 3  2  3.6-  :  ^ f c . 3  "10° 101 10* 10 10' 3  Lln-FITC  Figure 4 . 4 . E x p r e s s i o n of t h e transferred C D 2 4 g e n e in p r i m a r y m a r r o w s t e m c e l l s c a n d i d a t e s a s d e f i n e d by the S c a + L i n " cell s u r f a c e p h e n o t y p e . U n i n f e c t e d control B M a n d J Z e n C D 2 4 or M S C V C D 2 4 virus infected m a r r o w w e r e a n a l y z e d at 4 8 h o u r s post infection with viral p r o d u c e r s or at 2 4 w e e k s post transplant into m y e l o a b l a t e d r e c i p i e n t m i c e . C e l l s w e r e s t a i n e d with S c a - 1 - C y 5 , a n t i - C D 2 4 t e t r a m e r i c / R - P E a n d a cocktail of antibodies r e c o g n i z i n g l i n e a g e s p e c i f i c a n t i g e n s including G r - 1 - F I T C a n d M a c - 1 - F I T C for g r a n u l o c y t e s / m a c r o p h a g e s , L y - 1 - F I T C for T l y m p h o c y t e s a n d B 2 2 0 - F I T C for B l y m p h o c y t e s . C e l l s w e r e g a t e d o n the b a s i s of uptake of propidium iodide, forward a n d s i d e scatter profiles, a n d e x p r e s s i o n of S c a - 1 (top p a n e l s ) a n d e x p r e s s i o n of C D 2 4 o n t h e g a t e d p o p u l a t i o n a s s e s s e d (lower p a n e l s ) .  84  100  "55 o  Q £ 0 - J  o >  c.E +  (0 o  •  a o  w 0)  a. x  UJ  1°JZen CD24  1°  MSCV CD24  2 ° JZen 2 ° MSCV CD24 CD24  Figure 4 . 5 . S h o w s the p r o p o r t i o n of s t e m c e l l c a n d i d a t e s d e f i n e d by t h e S c a + L i n " cell s u r f a c e p h e n o t y p e positive for C D 2 4 e x p r e s s i o n at 2 4 w e e k s post t r a n s p l a n t in r e c i p i e n t s of J Z e n C D 2 4 or M S C V C D 2 4 C D 2 4 + s e l e c t e d b o n e m a r r o w . F e m o r a l b o n e m a r r o w c e l l s from individual recipient m i c e w e r e s t a i n e d with S c a - 1 - C y 5 , a n t i - C D 2 4 t e t r a m e r i c / R - P E a n t i b o d y c o m p l e x e s a n d a c o m b i n a t i o n of a n t i - G r - 1 - F I T C , a n t i - M a c - 1 - F I T C , a n t i - L y - 1 - F I T C a n d B 2 2 0 - F I T C r e c o g n i z i n g g r a n u l o c y t e s / m a c r o p h a g e s , T a n d B l y m p h o c y t e s a n d a n a l y z e d by flow cytometry. M i c e r e p r e s e n t e d by solid circles w e r e u s e d a s m a r r o w d o n o r s into s e c o n d a r y recipients. H o r i z o n t a l lines s h o w the a v e r a g e proportion of S c a + L i n " C D 2 4 + s t e m cell c a n d i d a t e s in primary a n d s e c o n d a r y recipients of J Z e n or M S C V t r a n s d u c e d b o n e marrow. 85  4.3.  Discussion A k e y finding of the work d e s c r i b e d in this C h a p t e r w a s that p r e s e l e c t i o n of  t r a n s d u c e d c e l l s b a s e d upon the transfer a n d e x p r e s s i o n of a c D N A e n c o d i n g the C D 2 4 c e l l s u r f a c e a n t i g e n e n a b l e d the reconstitution of recipient m i c e a l m o s t e x c l u s i v e l y with provirally m a r k e d c e l l s . D e s p i t e the relatively s m a l l t r a n s p l a n t dose,  reconstitution  of r e c i p i e n t s with t r a n s p l a n t  derived ( L y 5 . 1 ) cells was +  e s s e n t i a l l y c o m p l e t e (Table 4.1). S o u t h e r n blot a n a l y s i s of D N A from B M , s p l e e n a n d t h y m u s r e v e a l e d the p r e s e n c e of high levels of r e c o m b i n a n t provirus (Figure 4 . 2 ) , with proviral c o p y n u m b e r s per h a p l o i d g e n o m e >1  in a l m o s t all m i c e .  M o r e o v e r , the finding that all d a y 12 s p l e e n c o l o n i e s c o n t a i n e d intact ( F i g u r e 4.3)  further s u p p o r t s  the  c o n c l u s i o n that all H S C s  provirus  contributing  to  h e m o p o i e s i s in t h e s e a n i m a l s w e r e t r a n s d u c e d . W h i l e recipient m i c e w e r e t r a n s p l a n t e d with n u m e r o u s t r a n s d u c e d H S C s , the n u m b e r of s t e m cells found to contribute to long term h e m o p o i e s i s w a s found to v a r y . In s o m e , h e m o p o i e t i c reconstitution w a s e s s e n t i a l l y m o n o c l o n a l w h i l e in o t h e r s a n u m b e r of i n d e p e n d e n t  clones contributed  to h e m o p o i e s i s . T h e s e  d i f f e r e n c e s s u g g e s t a s t o c h a s t i c p h e n o m e n o n attributable  to e i t h e r  seeding  e f f i c i e n c i e s that a r e < 1 0 0 % a n d / o r d e c i s i o n s relating to s e l f - r e n e w a l v e r s u s differentiation of the s t e m cell upon arrival in the marrow. It s h o u l d b e p o s s i b l e to e x p l o r e t h e s e i s s u e s in m o r e detail u s i n g the C D 2 4 s e l e c t i o n a p p r o a c h to p r o v i d e a population of H S C s in w h i c h all are retrovirally m a r k e d . T h e recovery of long term repopulating s t e m cells within the C D 2 4 + s e l e c t e d fraction indicates that both the M P S V (JZen) a n d M S C V L T R regulatory e l e m e n t s c a n drive significant levels of g e n e e x p r e s s i o n immediately post infection. T h i s is further s u p p o r t e d by the detection of C D 2 4 e x p r e s s i o n o n the majority of S c a + L i n " s u b p o p u l a t i o n of c e l l s following the infection protocol (Figure 4.4). M o r e o v e r , C D 2 4 e x p r e s s i o n persisted on regenerated S c a + L i n " cells in both primary a n d s e c o n d a r y transplant recipients (Figure 4.5). W h i l e both J Z e n a n d M S C V b a s e d v e c t o r s g a v e  86  similar a b s o l u t e levels of e x p r e s s i o n a n d proportions of t r a n s d u c e d c e l l s following i n f e c t i o n , M S C V p r o v e d s u p e r i o r to J Z e n in t r a n s p l a n t  r e c i p i e n t s d e s p i t e the  detection of roughly similar levels of intact provirus. T h i s w a s a l s o evident in the l e v e l s of C D 2 4 e x p r e s s i o n o b s e r v e d a m o n g more mature c e l l s in the B M , t h y m u s a n d p e r i p h e r y ( T a b l e 4.1). T h e s e d a t a s u g g e s t that the J Z e n L T R  regulatory  e l e m e n t s a r e m o r e s u s c e p t i b l e to s u p p r e s s i o n / s h u t d o w n (215, 2 5 1 ) , p e r h a p s d u e to the m e t h y l a t i o n of the p r o m o t e r a n d / o r e n h a n c e r e l e m e n t s ( 2 5 7 , 3 3 1 ) . T h e M S C V v e c t o r (283) c o m b i n e s the L T R from P C M V ( P C C 4 e m b r y o n a l c a r c i n o m a c e l l - p a s s a g e d myeloproliferative  s a r c o m a virus) (258) a n d the 5'  untranslated  region of the dl587rev virus (259), both v i r u s e s isolated a s mutants a b l e to e x p r e s s t r a n s f e r r e d g e n e s in e m b r y o n i c c a r c i n o m a ( E C ) a n d e m b r y o n i c s t e m ( E S ) c e l l lines. T h e s e retroviral mutants p o s s e s s v a r i o u s d e l e t i o n s a n d b a s e pair c h a n g e s b e l i e v e d to r e m o v e potential b l o c k s to transcription l o c a t e d within the L T R a n d primer binding sites of the original v i r u s e s (252, 2 8 0 - 2 8 2 , 334). M y s t u d i e s provide s t r o n g e v i d e n c e that t h e s e c h a n g e s m a y similarly h a v e important c o n s e q u e n c e s for t h e e x p r e s s i o n of t r a n s d u c e d g e n e s in H S C s a n d their p r o g e n y in v i v o . D i f f e r e n c e s in the e x p r e s s i o n of the transferred C D 2 4 g e n e w a s a l s o d e t e c t e d a m o n g c e l l s of different l i n e a g e s . T h e proportion of C D 2 4 + t h y m o c y t e s significantly l o w e r t h a n that o b s e r v e d a m o n g c e l l s of either the m y e l o i d  was (bone  marrow) or erythroid lineage (Table 4.1). Further s t u d i e s u s i n g m a r k e r s s u c h a s C D 2 4 m a y facilitate the identification of regulatory e l e m e n t s to further i n c r e a s e the levels a n d p e r s i s t e n c e of transferred g e n e e x p r e s s i o n in h e m o p o i e t i c s t e m c e l l s or other target cells of interest. In c o n c l u s i o n , t h e s e s t u d i e s d e m o n s t r a t e the feasibility of u s i n g the C D 2 4 cell surface antigen  in c o m b i n a t i o n with F A C S to e n a b l e virtually  complete  r e g e n e r a t i o n of the h e m o p o i e t i c s y s t e m s of m y e l o a b l a t e d recipient m i c e with provirally m a r k e d c e l l s . T h i s a p p r o a c h p r o v i d e s a powerful strategy to track the b e h a v i o r of individual H S C s in vivo, a n d a s a m e t h o d to detect a n d quantify levels  87  a n d p e r s i s t e n c e of g e n e e x p r e s s i o n in v a r i o u s phenotypically d e f i n e d p o p u l a t i o n s of cells in vivo. Efforts to c o m p a r e the levels of e x p r e s s i o n of g e n e e x p r e s s i o n from alternative retroviral v e c t o r s s u c h a s M F G , a n d to e x t e n d this a p p r o a c h to the h u m a n setting are currently in p r o g r e s s .  88  CHAPTER  EVIDENCE REGULATED  OF  BOTH  ONTOGENY  EXPANSION OF  5  AND  TRANSPLANT  HEMOPOIETIC S T E M  DOSE  C E L L S IN  VIVO  T h e results p r e s e n t e d in this C h a p t e r h a v e b e e n d e s c r i b e d in: P a w l i u k , R., C . J . E a v e s a n d R. K. H u m p h r i e s . 1996. E v i d e n c e of both ontogeny a n d transplant d o s e regulated e x p a n s i o n of hematopoietic s t e m c e l l s in vivo. B l o o d in p r e s s .  89  5 . 1 . Introduction T h e r e is a n i n c r e a s i n g e m p h a s i s o n the d e v e l o p m e n t of c l i n i c a l s t r a t e g i e s that d e p e n d on the regenerative potential of t r a n s p l a n t a b l e H S C s . T h e s e i n c l u d e efforts d i r e c t e d at autograft p u r g i n g , g e n e transfer (including attempts to s e l e c t t r a n s d u c e d cell populations) e x vivo e x p a n s i o n a n d the u s e of alternative s o u r c e s of t r a n s p l a n t a b l e h e m o p o i e t i c cell p o p u l a t i o n s ( s u c h a s fetal liver a n d u m b i l i c a l c o r d b l o o d ) . At the s a m e t i m e , the m o l e c u l a r m e c h a n i s m s that d e f i n e  the  r e g e n e r a t i v e potential of H S C , or that regulate its e x p r e s s i o n , particularly in v i v o , r e m a i n poorly u n d e r s t o o d . S e r i a l transplantation s t u d i e s h a v e p r o v i d e d e v i d e n c e that the s e l f - r e n e w a l c a p a c i t y of H S C s  m a y b e finite or at l e a s t s u b j e c t  e x h a u s t i o n (105, 1 0 7 , 109). In addition, c o m p a r a t i v e s t u d i e s of the  to  proliferative  activities of fetal liver a n d adult b o n e m a r r o w c e l l s h a v e s h o w n that, u n d e r the s a m e conditions, t h e s e m a y display differences s u g g e s t i n g intrinsically d e t e r m i n e d differences in regenerative potential. (87, 8 8 , 335). H o w e v e r , n o n e of t h e s e s t u d i e s h a s relied on the u s e of direct m e a s u r e m e n t s of H S C n u m b e r s , at least in part b e c a u s e quantitative a s s a y s for c e l l s with long-term repopulating ability h a v e only recently b e c o m e available (43, 45). T h e critical importance of s u c h a s s a y s is further u n d e r s c o r e d by a g r o w i n g b o d y of e v i d e n c e i n d i c a t i n g that the c e l l s u r f a c e p h e n o t y p e is not n e c e s s a r i l y a reliable indicator of retained s t e m cell function, nor is the p r e s e n c e of t e r m i n a l c e l l s a reliable reflection of the s i z e of the H S C c o m p a r t m e n t (53, 7 6 , 111). T h u s , p a r a m e t e r s that m a y affect the k i n e t i c s a n d u l t i m a t e extent  of r e g e n e r a t i o n of the H S C c o m p a r t m e n t  o b t a i n e d after  the  transplantation of different n u m b e r s or s o u r c e s of h e m o p o i e t i c c e l l s h a v e not b e e n d e f i n e d . T o a d d r e s s t h e s e q u e s t i o n s , I u s e d a limiting dilution a s s a y for c e l l s with long-term  competitive  repopulating  ability to m e a s u r e H S C r e g e n e r a t i o n  in  m y e l o a b l a t e d recipients of different n u m b e r s of H S C s from adult b o n e m a r r o w or fetal liver.  90  5.2.  Results  5.2.1. O v e r a l l experimental  design  T h e p u r p o s e of t h e s e e x p e r i m e n t s w a s to e x a m i n e a n d c o m p a r e the kinetics of r e c o v e r y of e v e r y l e v e l of h e m o p o i e t i c c e l l d e v e l o p m e n t in  myeloablated  r e c i p i e n t s a s a f u n c t i o n of both the C R U c o n t e n t a n d the origin of the c e l l s t r a n s p l a n t e d . D o n o r c e l l s w e r e o b t a i n e d from either d a y 14.5 fetal livers or the b o n e m a r r o w of adult m i c e injected 4 d a y s previously with 150 m g / k g of 5 - F U a n d g r o u p s of m i c e t h e n injected with a r a n g e of cell n u m b e r s e s t i m a t e d to c o n t a i n 1 0 0 0 , 100 or 10 C R U , ie. 1 0 % , 1% a n d 0 . 1 % of the total marrow C R U content of a n a v e r a g e untreated adult m o u s e (43) or 9 0 % , 9 % or 0 . 9 % of the C R U content of a s i n g l e d a y 14.5 fetal liver (335). All mice a l s o r e c e i v e d 1 0  5  b o n e m a r r o w c e l l s from  a n u n m a n i p u l a t e d adult L y 5 . 2 B 6 C 3 F 1 d o n o r c o n t a i n i n g a n e s t i m a t e d c o m p e t i n g graft of 10 C R U (53). R e c i p i e n t s of t h e s e grafts w e r e s a c r i f i c e d 8 - 1 2 following  transplantation  contribution  for a s s e s s m e n t of the test  graft  months  (Ly5.1 )-derived +  both to their r e c o n s t i t u t e d p e r i p h e r a l b l o o d c e l l s a s w e l l a s their  marrow C F C , C F U - S a n d C R U populations. 5.2.2. Kinetics  of reconstitution of the terminal c o m p a r t m e n t s  F l o w cytometric a n a l y s i s of mature c e l l s in the p e r i p h e r a l b l o o d 8 m o n t h s p o s t t r a n s p l a n t r e v e a l e d e x t e n s i v e reconstitution of both l y m p h o i d a n d m y e l o i d c o m p a r t m e n t s with L y 5 . 1 proportion of L y 5 . 1  +  +  test cells for all transplant g r o u p s ( s e e T a b l e 5.1). T h e  c e l l s contributing to both of t h e s e mature c o m p a r t m e n t s w a s  highly c o n s i s t e n t b e t w e e n e x p e r i m e n t s for e a c h t r a n s p l a n t d o s e a n d m a d e up nearly all (> 80%) of the cells in recipients of fetal liver c e l l s a n d in recipients of all but the lowest transplant d o s e of adult b o n e marrow c e l l s (containing 10 C R U ) . In t h e s e , the proportion of peripheral blood c e l l s that w e r e Ly5.1 + w a s slightly lower, albeit still a p p r o x i m a t e l y 5 0 % of the total, reflecting their origin from a n e q u i v a l e n t proportion (also - 5 0 % ) of all the C R U transplanted.  91  Table 5.1. Proportion of L y 5 . 1 P e r i p h e r a l B l o o d C e l l s from P r i m a r y R e c i p i e n t s T r a n s p l a n t e d with V a r y i n g N u m b e r s of Ly5.1 + S y n g e n i c Adult B o n e M a r r o w - or Fetal Liver-Derived C R U +  Source and Transplant D o s e  BM BM BM FL FL FL  2x10 2 x 10 2x10  6  5  4  1.7 x 1 0 1.7x10® 1.7x105  7  Estimated No. C R U Transplanted*  % L y 5 . 1 + P B cells  Expt. 1  Expt. 2  1000 100 10  9 1 + 2 (5) 7 7 + 4 (5) 4 2 ± 7 (13)  91 ± 2 (3) 81 ± 3 (3) 59 + 8 (13)  1000 100 10  91 ± 1 (5) 94 ± 0 (3) 79 ± 4 (5)  9 2 + 0 (2) 89 ± 1 (2) 82 ± 4 (2)  V a l u e s s h o w n are the m e a n ± S D (number of m i c e a n a l y z e d ) of the proportion of L y 5 . 1 + c e l l s in the c i r c u l a t i n g W B C p o p u l a t i o n s p r e s e n t in p r i m a r y t r a n s p l a n t recipients a n a l y z e d 8 months post transplantation in two individual e x p e r i m e n t s . * T h e estimation of n u m b e r of C R U transplanted is d e r i v e d from a control v a l u e of 1/2000 ( 9 5 % c o n f i d e n c e interval: 1 in 1300 to 1 in 5700) for adult 5 - F U B M (43) a n d 1/17000 ( 9 5 % c o n f i d e n c e interval: 1 in 1 1 5 0 0 to 1 in 2 6 , 0 0 0 ) for fetal liver (335).  5.2.3. Reconstitution of the  marrow  T w o m i c e per transplant group w e r e c h o s e n for further a n a l y s i s of the level of test transplant-derived c e l l s in the marrow 8 months post-transplant. In a n effort to detect the m a x i m u m levels of reconstitution attained by this s o u r c e , recipients in w h i c h at least 8 0 % of the peripheral blood c e l l s w e r e Ly5.1+ w e r e s e l e c t e d . In addition, the b o n e marrow cells of o n e of the recipients w e r e a l s o s t a i n e d with the Ly5.1 a n t i b o d y a n d u p o n F A C S a n a l y s i s w e r e f o u n d , like the b l o o d , to c o n t a i n >70% Ly5.1+ (ie. test transplant-derived) cells (data not s h o w n ) . The  total m a r r o w cellularity a n d C F C n u m b e r s in all p a i r s of  primary  recipients, irrespective of the n u m b e r or origin of the cells initially t r a n s p l a n t e d , h a d r e g e n e r a t e d to l e v e l s e q u i v a l e n t to t h o s e found in u n m a n i p u l a t e d a g e - m a t c h e d control m i c e (Table 5.2). In contrast, for d a y 12 C F U - S , this w a s true only for the recipients of the highest transplant d o s e of fetal liver cells. F o r recipients of  92  T a b l e 5.2. Regeneration of Cellularity, C l o n o g e n i c Progenitor and Day 12 C F U - S Content in Femoral Marrow of Mice T r a n s p l a n t e d with Various N u m b e r s of Adult B M or Fetal Liver C R U (8 Months Post Transplant)  Transplant Source  Adult B M  Fetal Liver  Normal Control  Estimated No. C R U Transplanted  Total n u c l e a t e d cells/femur (±SD)  10 100 1000  2.1 ±0.7  10 100 1000  2.1 ± 0 . 5 x 1 0 2.3±0x 10 2.4±0.7x 1 0  7  2.3 + O . l x 1 0  7  x 10  7  2.3 + 0.1 x 1 0 2.5±0.5x 10  7  D a y 12 C F U S/femur x 1 0 (±SD)  C F U C/femur x10 (±SD) 2  2  10.1 + 2 . 4  382 ± 6 1 . 0 504 ± 1 0 1  16.1 ± 1 . 8 17 + 1.5  4 7 5 + 160  7  7  7  11.8 ± 2 . 4  330 + 98.7 3 9 3 ± 17.9 3 9 4 ± 37.4  2 1 . 6 ± 1.0  476±138  24.4 ± 2.6  14.3 ± 1.0  B o n e m a r r o w c e l l s from primary recipients (2 mice per group) 8 months post transplant were c o u n t e d using a s t a n d a r d h e m o c y t o m e t e r a n d plated in methylcellulose at 2.7 x 1 0 cells/ml a n d c o l o n i e s s c o r e d 12 d a y s later. R e g e n e r a t i o n of day 12 C F U - S w a s a s s e s s e d by transplanting 7.5 x 1 0 - 1 x 1 0 marrow cells from pooled m a r r o w from two primary recipients into 5 irradiated ( 9 5 0 c G y ) B 6 C 3 F 1 mice/group. A n i m a l s w e r e sacrificed 12 d a y s later a n d m a c r o s c o p i c s p l e e n c o l o n i e s c o u n t e d . N o r m a l control a n i m a l s w e r e 6-8 month old u n m a n i p u l a t e d B 6 C 3 F 1 m i c e . S e e T a b l e 5 . 1 . for c a l c u l a t e d C R U f r e q u e n c i e s in unmanipulated adult b o n e marrow a n d fetal liver. F o r e a c h experiment 2 m i c e s h o w i n g the highest proportion of L y 5 . 1 cells in the peripheral blood w e r e c h o s e n a s d o n o r s for s e c o n d a r y transplantation. T h e a v e r a g e proportion of L y 5 . 1 cells in the peripheral blood of c h o s e n m i c e for e a c h group w a s ; adult marrow 10 C R U : 8 1 % , 100 C R U : 8 5 % , 1000 C R U : 9 4 % , fetal liver 10 C R U : 8 2 % , 100 C R U : 9 4 % , 1000 C R U : 9 2 % . 4  4  5  +  +  all other s o u r c e s or n u m b e r s of test cells, recovery of d a y 12 C F U - S n u m b e r s w a s i n c o m p l e t e ranging from 4 5 % to 8 5 % of normal v a l u e s (Table 5.2). A l t h o u g h there w a s a trend t o w a r d s a g r e a t e r r e c o v e r y of this m o r e primitive c o m p a r t m e n t  in  r e c i p i e n t s of h i g h e r initial transplant d o s e s , the a c t u a l d i f f e r e n c e s b e t w e e n the g r o u p s w e r e not statistically significant (p < 0.05). 5.2.4. Reconstitution of the  marrow C R U compartment  T o c o m p a r e the a c c o m p a n y i n g level of r e g e n e r a t i o n of L y 5 . 1  +  donor-  d e r i v e d s t e m c e l l s in the marrow of t h e s e s a m e pairs of primary recipients, C R U f r e q u e n c i e s a n d h e n c e n u m b e r s w e r e a l s o d e t e r m i n e d . T h e p r e s e n c e of Ly5.1  +  m y e l o i d a n d l y m p h o i d c e l l s in the peripheral blood of the s e c o n d a r y C R U a s s a y r e c i p i e n t s w a s e v a l u a t e d after 16 w e e k s a n d u s e d to d e r i v e the C R U n u m b e r s s h o w n in F i g u r e 5.1. In most c a s e s , the Ly5.1+ C R U population h a d r e g e n e r a t e d to a level c o r r e s p o n d i n g to only a s m a l l proportion of the normal C R U population a n d the l e v e l s a c h i e v e d correlated positively with the original d o s e of L y 5 . 1  +  C R U used  to reconstitute the primary recipients. Interestingly, the level of C R U r e g e n e r a t i o n w a s . c o n s i s t e n t l y higher (p < 0.01 to < 0.1) for fetal liver transplants a s c o m p a r e d to m a r r o w t r a n s p l a n t s containing the s a m e original n u m b e r of C R U . T h u s , o n a per input C R U b a s i s , C R U amplification in the fetal liver t r a n s p l a n t s w a s 1 0 X m o r e effective than with primary b o n e marrow transplants a n d , only in primary recipients of 1 0 0 0 C R U of fetal liver origin, did the t r a n s p l a n t e d C R U r e c o v e r to a n o r m a l s i z e d population within 8 months post-transplant. E f f e c t s of the t r a n s p l a n t  d o s e a s w e l l a s the t i s s u e origin a r e  more  dramatically revealed by using the s a m e data to calculate the extent of L y 5 . 1 C R U +  amplification in e a c h experimental situation tested. T h e results of s u c h c a l c u l a t i o n s a r e s h o w n in T a b l e 5.3. It c a n be s e e n that for both fetal liver a n d adult b o n e m a r r o w t r a n s p l a n t s , C R U e x p a n s i o n w a s inversely related to the initial n u m b e r of C R U transplanted although the extent of C R U e x p a n s i o n for input fetal  94  Normal  100  T  o z o  Adult BM  10-  Fetal Liver  0 > O O 0  rr D C O  1-  1 10  100  1000  10000  N u m b e r of C R U T r a n s p l a n t e d F i g u r e 5 . 1 . R e g e n e r a t i o n of L y 5 . 1 donor-derived cells following the t r a n s p l a n t a t i o n into s e c o n d a r y r e c i p i e n t s of b o n e m a r r o w c e l l s f r o m p r i m a r y r e c i p i e n t s originally t r a n s p l a n t e d with 10, 100 or 1 0 0 0 fetal liver or adult b o n e m a r r o w C R U 8 m o n t h s previously. C R U n u m b e r s w e r e d e t e r m i n e d by m e a s u r i n g the f r e q u e n c y of C R U by limiting dilution a n a l y s i s in s e c o n d a r y r e c i p i e n t s a n d c a l c u l a t i n g the n u m b e r of C R U in the marrow of primary transplant recipients a s c o m p a r e d to a n u n m a n i p u l a t e d control m o u s e b a s e d upon the a s s u m p t i o n that a n a v e r a g e adult m o u s e h a s approximately 2 0 0 million marrow cells. F o r e a c h transplant d o s e C R U regeneration by fetal liver w a s significantly greater than adult b o n e marrow; 10 C R U : p < 0.01, 100 C R U : p < 0 . 1 , 1000 C R U : p < 0 . 0 1 . F o r adult b o n e marrow the 10 C R U transplant d o s e is significantly lower than both the 100 a n d 1 0 0 0 C R U d o s e at p < 0 . 0 1 . F o r fetal liver the 10 C R U d o s e is significantly lower than both the 100 a n d 1000 C R U d o s e ; 100 C R U : p < 0 . 1 , 1 0 0 0 C R U : p < 0 . 0 1 . T h e 100 C R U d o s e is significantly lower than the 1000 C R U d o s e at p<0.1. T h e estimation of the n u m b e r of C R U transplanted is derived from a control v a l u e of 1/2000 ( 9 5 % c o n f i d e n c e interval: 1 in 1 3 0 0 to 1 in 5 7 0 0 ) for adult 5 - F U b o n e m a r r o w (43) a n d 1/17,000 ( 9 5 % c o n f i d e n c e interval: 1 in 11,500 to 1 in 26,000) for fetal liver (335). +  95  Table 5.3. E x p a n s i o n of D o n o r - D e r i v e d C R U in P r i m a r y R e c i p i e n t s of F e t a l Liver or Adult B o n e M a r r o w C e l l s Transplant Source  Estimated No. CRU Transplanted *  Adult b m  10 100 1000  100 1500 1800  (75-150) (1000-2100) (1300-2500)  10 15 2  F e t a l Liver  10 100 1000  1500 3100 6200  (1000-2100) (2300-4200) (4500-8500)  148 31 6  Untransplanted Control  -  Estimated No. B o n e Marrow C R U Post Expansion (range + S E M ) f  Fold Expansion  10000 (6300-14800)  -  * S e e T a b l e 5.1 for c a l c u l a t e d C R U f r e q u e n c i e s in u n m a n i p u l a t e d adult b o n e m a r r o w a n d fetal liver. f R e s u l t s are e x p r e s s e d a s the n u m b e r of C R U per m o u s e b a s e d o n the e s t i m a t e that 2 x 1 0 f e m o r a l m a r r o w c e l l s c o n s t i t u t e s a p p r o x i m a t e l y 1 0 % of the total h e m o p o i e t i c population of the m o u s e . 7  liver C R U w a s g r e a t e r t h a n that of their c o u n t e r p a r t s in b o n e m a r r o w .  The  m a x i m u m C R U e x p a n s i o n o b s e r v e d w a s 150-fold for fetal liver C R U by c o m p a r i s o n to a m a x i m u m C R U e x p a n s i o n of only 15-fold for adult b o n e marrow transplants. 5.2.5. R e g e n e r a t i v e ability of  a single C R U  assessed  using  retroviral  marking T o g a i n further insight into C R U regeneration I u s e d retroviral m a r k i n g for c l o n a l a n a l y s i s of s e l f - r e n e w a l a n d l y m p h o - m y e l o i d reconstitution. 5 - F U - t r e a t e d Ly5.1 m a r r o w c e l l s w e r e c o - c u l t u r e d with c e l l s p r o d u c i n g a retrovirus c o n t a i n i n g the c o d i n g region of the h u m a n C D 2 4 cell s u r f a c e antigen a n d w e r e then s t a i n e d with a n a n t i - C D 2 4 a n t i b o d y a n d C D 2 4 + c e l l s s e l e c t e d by F A C S 4 8 h o u r s postinfection a s d e s c r i b e d in C h a p t e r 2; Material a n d M e t h o d s . 10$ of t h e s e Ly5.1 + c e l l s (estimated to contain 3.0 + 1.0 C R U ) w e r e transplanted into Ly5.2+ recipients.  96  A l l of 13 s u c h recipients s h o w e d detectable levels of Ly5.1+ (donor c e l l - d e r i v e d ) repopulation with v a l u e s ranging from 3 - 3 2 % Ly5.1+ p e r i p h e r a l b l o o d l e u k o c y t e s (10/13 m y e l o i d / l y m p h o i d repopulation; 3/13 lymphoid-restricted repopulation). T h e h e m o p o i e t i c t i s s u e s of all of t h e s e mice a l s o s h o w e d the p r e s e n c e of intact provirus at 11 m o n t h s post transplant but in only 3 w a s C D 2 4 e x p r e s s i o n d e t e c t a b l e in the peripheral blood l e u k o c y t e s (where v a l u e s of 2 - 2 0 % C D 2 4 + w e r e m e a s u r e d ) . O n e of t h e s e recipients s h o w i n g 3 2 % d o n o r - d e r i v e d Ly5.1+ c e l l s , of w h i c h 6 3 % w e r e C D 2 4 + , w a s c h o s e n for further study to quantify the c l o n a l regeneration of Ly5.1 + C R U . S o u t h e r n blot a n a l y s i s of the b o n e marrow, s p l e e n a n d t h y m u s of this m o u s e s h o w e d identical proviral b a n d i n g patterns a n d b a n d intensities in all of t h e s e t i s s u e s c o n s i s t e n t with the r e p o p u l a t i o n of this p r i m a r y recipient by a s i n g l e t r a n s d u c e d totipotent repopulating s t e m cell. W h e n the marrow c e l l s of this m o u s e w e r e then a s s a y e d for their content of Ly5.1+ C R U , 0.2 ± 0.05 Ly5.1+ C R U per 1 0  5  m a r r o w c e l l s or 50 Ly5.1+ C R U per femur w e r e d e t e c t e d . T h i s r e p r e s e n t s 3 . 7 % of the C R U population in the femur of a normal adult B 6 C 3 F 1 m o u s e . S e v e n t e e n of t h e 19 s e c o n d a r y t r a n s p l a n t r e c i p i e n t s w h o s h o w e d the p r e s e n c e of Ly5.1  +  peripheral b l o o d c e l l s w e r e a l s o positive for C D 2 4 e x p r e s s i o n , with v a l u e s ranging f r o m 1.5% to 1 4 . 2 % C D 2 4 + p e r i p h e r a l b l o o d l e u k o c y t e s . Strikingly, the s a m e proviral b a n d i n g pattern s e e n in the primary a n i m a l w a s a l s o o b s e r v e d e x c l u s i v e l y in the h e m o p o i e t i c t i s s u e s of all of the s e c o n d a r y recipients w h o w e r e reconstituted with Ly5.1+ cells (Figure 5.2). T h i s observation indicates a 370-fold amplification of the  o r i g i n a l t r a n s d u c e d C R U d u r i n g the  p e r i o d of  11  months  after  it  was  transplanted into the primary recipient. T h i s result provides formal e v i d e n c e of C R U s e l f - r e n e w a l in vivo a n d e x t e n d s the p r e v i o u s f i n d i n g s indicating the extent to w h i c h this c a n occur. 5.3.  Discussion P r e v i o u s s t u d i e s h a v e i n d i c a t e d that the  c a p a c i t y of  most  primitive  h e m o p o i e t i c c e l l s c a p a b l e of regenerating the entire s y s t e m c a n n o t be m a i n t a i n e d 97  o n s e r i a l transfer. In this C h a p t e r I s h o w e d that this m a y be at least partially attributable to a c o m m o n failure of the C R U compartment to be fully regenerated  Primary Recipient 1  2A  Secondary Recipients 2B  b s b s b s t  2C  2D  b s t b s t  pos neg. ctrl. ctrl.  2E  2F  b s t b s t  2G b s  2H  21  t b s t  b s — 6.6 Kb — 4.0 Kb — 2.2 Kb  F i g u r e 5.2. D e m o n s t r a t i o n of C D 2 4 provirus in b o n e marrow (B), s p l e e n (S), a n d t h y m u s (T) D N A from primary a n d s e c o n d a r y transplant r e c i p i e n t s . R e c i p i e n t s r e c e i v e d 10® C D 2 4 + s e l e c t e d c e l l s in c o m b i n a t i o n with 1 0 n o r m a l m a r r o w c o m p e t i t o r c e l l s . Individual m i c e a r e l a b e l e d a s 1 for the p r i m a r y r e c i p i e n t ( s a c r i f i c e d at 11 m o n t h s p o s t - t r a n s p l a n t ) , or 2A-I for s e c o n d a r y r e c i p i e n t s (sacrificed at 13 w e e k s post-transplant). D N A (15 u.g) from e a c h t i s s u e s a m p l e w a s d i g e s t e d with E c o R l , a n e n z y m e that cuts o n c e within the C D 2 4 proviral s e q u e n c e . S h o w n are results of a blot probed with a 3 2 p - | | | fragment of the n e o g e n e . T h e positive control represents D N A obtained from C D 2 4 viral producer c e l l s w h i c h contain > 10 proviral c o p i e s . 5  R  a D e  e c  e v e n 8 m o n t h s post-transplant in spite of, i n d e e d p e r h a p s b e c a u s e of, a c o m p l e t e r e c o v e r y of later cell t y p e s including d a y 12 C F U - S a s well a s c e l l s d e t e c t a b l e a s C F C . Interestingly, there w a s , n e v e r t h e l e s s , a quantitative relationship b e t w e e n the extent of amplification s e e n in d o n o r C R U n u m b e r s a n d both the s i z e of the initial transplant a n d its ontological s o u r c e .  98  It h a s b e e n s u g g e s t e d by s e v e r a l authors that transplantable s t e m c e l l s m a y fail to r e g e n e r a t e the s t e m c e l l c o m p a r t m e n t to n o r m a l (non-transplant) b e c a u s e of a n i n h e r e n t l y  limited c a p a c i t y for s e l f - r e n e w a l ( 1 0 3 , 1 0 5 ,  levels 109).  A c c o r d i n g to s u c h a m o d e l , the a b s o l u t e extent of C R U a m p l i f i c a t i o n w o u l d b e a n t i c i p a t e d to d e c r e a s e a s the n u m b e r of s t e m c e l l s t r a n s p l a n t e d w a s d e c r e a s e d s i n c e the n u m b e r of s t e m c e l l s with the highest s e l f - r e n e w a l potential w o u l d a l s o d e c r e a s e proportionately. O n the other h a n d , it is p o s s i b l e that the transplantation of s m a l l e r n u m b e r s of m a r r o w c e l l s might p l a c e a higher " s t r e s s " o n the s y s t e m resulting in the production of stimuli that c o u l d favor differentiation rather than selfrenewal  r e s p o n s e s . E v i d e n c e of  decreased stem  cell  regeneration  under  c o n d i t i o n s that support their proliferation both in vitro (336), in utero (11) a n d after bone marrow transplantation  ( 1 0 2 , 1 0 3 , 1 0 5 , 1 0 9 , 117) h a v e b e e n r e p o r t e d .  H o w e v e r , s u c h s t u d i e s d o not n e c e s s a r i l y m e a s u r e the c a p a c i t i e s of the c e l l s t e s t e d but rather their r e s p o n s e under a given set of m o l e c u l a r l y u n d e f i n e d a n d poorly u n d e r s t o o d environmental conditions. T h e present s t u d i e s , w h i c h h a v e u s e d a quantitative a s s a y to provide a direct m e a s u r e m e n t of the s i z e of the r e g e n e r a t e d totipotent, t r a n s p l a n t a b l e s t e m cell c o m p a r t m e n t indicate that a h i g h e r d e g r e e of s t e m c e l l amplification is o b t a i n e d following the transplant of s m a l l e r n u m b e r s of s t e m c e l l s a s c o m p a r e d to the transplant of larger n u m b e r s of s t e m c e l l s , e v e n t h o u g h this is insufficient to a c h i e v e a c o m p a r a b l e level of regeneration of the s t e m cell p o p u l a t i o n by c o m p a r i s o n to its s i z e in u n p e r t u r b e d a n i m a l s ( T a b l e 5.3 a n d F i g u r e 5.1). T h u s , the extent to w h i c h either fetal liver or adult b o n e m a r r o w s t e m c e l l s e x p r e s s their full regenerative potential v a r i e s a c c o r d i n g to h o w m a n y of t h e m ( a n d / o r a c c o m p a n y i n g m a r r o w cells) are t r a n s p l a n t e d a n d this d e c r e a s e s with innoculum size.  A p o s s i b l e e x p l a n a t i o n for this finding w o u l d b e the activation of  negative f e e d b a c k regulatory m e c h a n i s m s in vivo that c a n limit s t e m cell e x p a n s i o n p r e m a t u r e l y , p e r h a p s v i a the p r o d u c t i o n by m a t u r e h e m o p o i e t i c c e l l s of s u c h f a c t o r s a s M I P - 1 a a n d T G F - 3 that m a y s e l e c t i v e l y d e c r e a s e the proportion of  99  primitive  h e m o p o i e t i c c e l l s in c y c l e ( 1 1 2 - 1 1 5 , 3 3 7 ) s u c h f a c t o r s might  thus  attenuate or e v e n terminate C R U e x p a n s i o n e v e n t h o u g h the n u m b e r s of t h e s e c e l l s might still be significantly below the "normal" level. It is interesting to s p e c u l a t e that this effect might be p r o m o t e d by the co-transplantation of large n u m b e r s of m a t u r e h e m o p o i e t i c c e l l s or their i m m e d i a t e p r e c u r s o r s . S u c h a p o s s i b i l i t y is c o n s i s t e n t with the o b s e r v a t i o n that the m a x i m u m d e g r e e of C R U e x p a n s i o n w a s o b s e r v e d with the s m a l l e s t transplant d o s e . A c c o r d i n g l y , it w o u l d be a n t i c i p a t e d that s t e m c e l l e x p a n s i o n might  be e n h a n c e d w h e n  purified s t e m c e l l s a r e  t r a n s p l a n t e d to r e d u c e the n u m b e r of mature cells present during the initial s t a g e s of engraftment. It is important to note that in t h e s e e x p e r i m e n t s only the r e g e n e r a t i o n of donor L y 5 . 1  +  C R U w e r e quantified. M y results clearly s h o w that the t r a n s p l a n t e d  L y 5 . 1 + C R U w e r e u n a b l e to reconstitute the C R U c o m p a r t m e n t  of  primary  transplant recipients to l e v e l s found in normal adult m i c e . H o w e v e r , the extent to w h i c h 10 Ly5.2+ C R U present in the competitor cell population or t h o s e surviving in the host might h a v e contributed to regeneration of the total C R U c o m p a r t m e n t is not k n o w n . It s e e m s highly unlikely that a large r e s e r v e of inactive L y 5 . 2  +  C R U would  h a v e b e e n present in the primary recipients s i n c e the vast majority of all the c e l l s in the marrow a n d the peripheral blood of t h e s e m i c e w e r e L y 5 . 1 . +  R e s u l t s from m a n y p r e v i o u s s t u d i e s h a v e i n d i c a t e d that the r e g e n e r a t i v e b e h a v i o r of h e m o p o i e t i c cells in a transplant setting is a function of their ontological state. T h u s , o v e r 2 0 y e a r s a g o , it w a s found that a transplant of fetal liver c e l l s w o u l d o u t c o m p e t e adult b o n e marrow following their c o m b i n e d transplantation into recipient m i c e (88). S i m i l a r l y , 10 y e a r s later, it w a s s h o w n that d a y 8 C F U - S d e r i v e d from fetal liver p o s s e s s a greater c a p a c i t y for s e l f - r e n e w a l a s c o m p a r e d to their adult b o n e marrow counterparts (87). Recently, R e b e l et. a l . (335) h a v e s h o w n that limiting n u m b e r s of fetal liver C R U are a b l e to p r o d u c e a g r e a t e r output of m a t u r e b l o o d c e l l s in vivo a s c o m p a r e d to adult b o n e m a r r o w . M o r e o v e r , w h e n  100  m a r r o w c e l l s from primary recipients of limiting n u m b e r s of fetal liver C R U w e r e injected into s e c o n d a r y recipients, a significantly higher p e r c e n t a g e of t h e s e m i c e s h o w e d d o n o r - d e r i v e d reconstitution of their lymphoid a n d m y e l o i d c o m p a r t m e n t s a s c o m p a r e d to m i c e that h a d r e c e i v e d m a r r o w c e l l s from primary r e c i p i e n t s of s i m i l a r n u m b e r s of adult b o n e m a r r o w C R U . T h e s e results a r e t h u s a l s o highly s u g g e s t i v e of a greater regenerative capability of fetal liver H S C by c o m p a r i s o n to adult b o n e marrow. H o w e v e r , b e c a u s e of their d e s i g n , n o n e of t h e s e s t u d i e s c o u l d d i s c r i m i n a t e b e t w e e n quantitative differences in the s e l f - r e n e w a l of t r a n s p l a n t a b l e H S C a n d p o s s i b l e differences in the extent of proliferation a c h i e v e d by their m o r e differentiated p r o g e n y . B y a d d r e s s i n g this q u e s t i o n h e r e , it h a s b e e n p o s s i b l e to e s t a b l i s h that fetal liver C R U a r e i n d e e d s u p e r i o r to their adult b o n e  marrow  c o u n t e r p a r t s both in t e r m s of the relative a n d a b s o l u t e n u m b e r s of C R U they will r e g e n e r a t e under similar conditions. A p o s s i b l e explanation for t h e s e differences is that fetal liver C R U p o s s e s s a greater intrinsically regulated probability for self-renewal w h e n stimulated to divide in the m i c r o e n v i r o n m e n t of the post-transplant m y e l o a b l a t e d m o u s e . Intriguing e v i d e n c e of c a n d i d a t e g e n e s that m a y be involved in the intrinsic control of selfr e n e w a l w a s recently reported by S a u v a g e a u et. a l . from s t u d i e s of the effects of H o x g e n e s o n h e m o p o i e s i s in vivo (91). T h e s e s h o w e d that o v e r e x p r e s s i o n of H O X B 4 , w h o s e e x p r e s s i o n is normally restricted to the most primitive adult b o n e m a r r o w c e l l s in the adult (91) resulted in a 50-fold i n c r e a s e in the r e g e n e r a t i o n of t r a n s d u c e d C R U a s c o m p a r e d to n e o - t r a n s d u c e d control c e l l s . H o w e v e r , the possibility that the g e n e s e x p r e s s e d within fetal liver a n d adult b o n e m a r r o w H S C i n f l u e n c e this s e l f - r e n e w a l probability d o e s not p r e c l u d e other m e c h a n i s m s that might influence the o b s e r v e d differences in the rates of fetal liver a n d adult m a r r o w C R U amplification.  F o r e x a m p l e , t h e s e m a y a l s o exhibit d i f f e r e n c e s in the time  r e q u i r e d to transit o n e c o m p l e t e c e l l c y c l e (338) or in the p r o p o r t i o n of c e l l s  101  recruited or m a i n t a i n e d within the m i c r o e n v i r o n m e n t of the m a r r o w of the adult mouse. T h e u s e of r e c o m b i n a n t  retroviruses a s genetic tags h a s b e e n u s e d  e x t e n s i v e l y to track the proliferative a n d differentiative b e h a v i o r of individual s t e m cell c l o n e s both in vitro (49) a n d in vivo (98, 99). In the p r e s e n t s t u d y I utilized a r e c o m b i n a n t retrovirus c o n t a i n i n g the c o d i n g r e g i o n of the h u m a n C D 2 4 c e l l s u r f a c e antigen  to confirm the totipotentiality of the C R U d e t e c t e d u s i n g the C R U  a s s a y , a n d to aid in the verification a n d quantification of the d e g r e e of e x p a n s i o n e x h i b i t e d by i n d i v i d u a l C R U in v i v o . O n l y o n e proviral b a n d i n g pattern  was  d e t e c t e d in b o n e m a r r o w a n d t h y m i c D N A in the p r i m a r y a n d all s e c o n d a r y t r a n s p l a n t recipients (Figure 5.2), highly s u g g e s t i v e that the r e g e n e r a t i o n of the s t e m c e l l c o m p a r t m e n t in the primary recipient w a s m o n o c l o n a l in nature. T h i s c l o n e r e g e n e r a t e d the C R U c o m p a r t m e n t to 3 . 7 % of n o r m a l level, c o m p a r a b l e to that o b s e r v e d in recipients of 10 adult b o n e marrow C R U . T h i s r e p r e s e n t s a C R U e x p a n s i o n of 370-fold, which w a s higher than that o b s e r v e d for a n y transplant d o s e of fetal liver or adult b o n e m a r r o w (Table 5.3). D i f f e r e n c e s in e s t i m a t e s of C R U e x p a n s i o n at the level of the w h o l e population v e r s u s individual c l o n e s likely reflect heterogeneity  amongst  e q u i v a l e n t individual  the  regenerative  activity  displayed  by  biologically  H S C r e s p o n d i n g to proliferative stimuli (19, 8 9 , 9 9 , 1 0 1 ,  339). H o w e v e r , at the s a m e time the present results confirm the ability of totipotent repopulating s t e m c e l l s to u n d e r g o s e l f - r e n e w a l d i v i s i o n s during c l o n a l e x p a n s i o n in vivo a n d d e m o n s t r a t e the e n o r m o u s regenerative potential that s o m e s t e m c e l l s m a y therefore p o s s e s s . T h e r e s u l t s of this s t u d y h a v e important  i m p l i c a t i o n s for b o n e  marrow  transplantation efforts. A sufficiently quantitatively or qualitatively i m p a i r e d p o o l of h e m o p o i e t i c s t e m cells c o u l d influence the longevity of a patient's graft, a n d further, might r e d u c e t o l e r a n c e to cytotoxic a g e n t s or other c i r c u m s t a n c e s w h i c h w o u l d i m p o s e a proliferative d e m a n d on the s t e m cell pool. T h i s study thus highlights the  102  i m p o r t a n c e of o p t i m i z i n g s t e m c e l l n u m b e r s in b o n e m a r r o w t r a n s p l a n t s  and  s u g g e s t s potential c o n s e q u e n c e s of transplanting different n u m b e r s or s o u r c e s of s t e m c e l l s in protocols s e e k i n g to r e s c u e , or, alternatively, to genetically modify the marrow.  T h e s e s t u d i e s a l s o s e t the  s t a g e for a t t e m p t s  to  enhance C R U  r e g e n e r a t i o n post-transplant by the administration of e x o g e n o u s a g e n t s or the e x p r e s s i o n of t r a n s d u c e d intracellular f a c t o r s that m a y e n h a n c e the r e g e n e r a t i v e potential of s t e m c e l l s e x p r e s s e d u n d e r a d e f i n e d condition in vivo.  103  apparent  experimental  CHAPTER  6  DISCUSSION T h e u s e of recombinant retroviruses to transfer e x o g e n o u s g e n e s into H S C s has  provided significant  insight  into the o r g a n i z a t i o n a n d r e g u l a t i o n  of  the  h e m o p o i e t i c s y s t e m a n d h a s p l a y e d a central role in the e m e r g i n g field of g e n e t h e r a p y . H o w e v e r , m a n y important q u e s t i o n s remain u n a n s w e r e d r e g a r d i n g the nature a n d regulation of totipotent H S C s , including a b a s i c u n d e r s t a n d i n g of their u s a g e o v e r time, their potential for s e l f - r e n e w a l a n d proliferation, a n d the g e n e s e n c o d i n g e x t r a c e l l u l a r a n d / o r i n t r a c e l l u l a r f a c t o r s w h i c h a r e r e s p o n s i b l e for r e g u l a t i n g t h e s e b i o l o g i c a l c h a r a c t e r i s t i c s . A l t h o u g h the ability to  genetically  m a n i p u l a t e H S C s using recombinant retroviruses p r o v i d e s a powerful tool to begin to a d d r e s s t h e s e i s s u e s , the poor infection efficiency to H S C s r e m a i n s a n o b s t a c l e . T h e overall g o a l of the work p r e s e n t e d in this t h e s i s w a s to d e v e l o p m e t h o d o l o g i e s to e n h a n c e the utility of current g e n e transfer p r o t o c o l s for the efficient g e n e t i c manipulation a n d tracking of H S C s a n d to utilize t h e s e p r o c e d u r e s to m o r e clearly define the self-renewal potential of H S C s following b o n e marrow transplant. W o r k d e s c r i b e d in C h a p t e r 3 d e m o n s t r a t e s the u s e of the C D 2 4 cell s u r f a c e a n t i g e n a s a d o m i n a n t s e l e c t a b l e m a r k e r in a r e c o m b i n a n t retroviral v e c t o r in c o m b i n a t i o n with F A C S to e n a b l e the rapid a n d non-toxic s e l e c t i o n of retrovirally t r a n s d u c e d m u r i n e b o n e m a r r o w c e l l s including in vitro c l o n o g e n i c p r o g e n i t o r s , d a y 12 C F U - S a n d totipotent repopulating s t e m c e l l s . T h e p r e s e n c e of totipotent repopulating s t e m cells within the C D 2 4 + fraction ( C h a p t e r s 3 a n d 4) d e m o n s t r a t e s that the M P S V a n d M S C V L T R regulatory e l e m e n t s are a b l e to drive h i g h - l e v e l g e n e e x p r e s s i o n in the m o s t primitive h e m o p o i e t i c c e l l s p r e s e n t in adult m a r r o w t i s s u e . T h e ability of t h e s e e l e m e n t s to drive g e n e e x p r e s s i o n in  primitive  h e m o p o i e t i c c e l l s w a s a l s o directly c o n f i r m e d in s t u d i e s d e s c r i b e d in C h a p t e r 4 with the a n a l y s i s of C D 2 4 e x p r e s s i o n a m o n g a S c a + L i n " s u b p o p u l a t i o n of b o n e m a r r o w c e l l s . T h e s e o b s e r v a t i o n s h a v e a n u m b e r of important i m p l i c a t i o n s . T h e  104  ability to a c h i e v e high a n d s u s t a i n e d l e v e l s of t r a n s f e r r e d g e n e e x p r e s s i o n in primitive h e m o p o i e t i c c e l l s is e s s e n t i a l for s t u d i e s a i m e d at t e s t i n g ,  through  o v e r e x p r e s s i o n , g e n e s e n c o d i n g m o l e c u l e s w h i c h m a y be i n v o l v e d in regulating the s u r v i v a l , mobility, c y c l i n g , proliferation, differentiation, or s e l f - r e n e w a l of H S C s . M o r e o v e r , the ability to obtain a population of c e l l s in w h i c h 1 0 0 % a r e provirally m a r k e d s h o u l d e n h a n c e the detection of p h e n o t y p e s w h i c h m a y be a s s o c i a t e d with the o v e r e x p r e s s i o n of potential putative s t e m c e l l r e g u l a t o r y m o l e c u l e s . T h e o v e r e x p r e s s i o n of H o x B 4 h a s b e e n s h o w n to i n c r e a s e the r e g e n e r a t i v e ability of H S C s following b o n e marrow transplant (91). Additional m o l e c u l e s w h i c h w o u l d be of interest to test u s i n g this p r o c e d u r e include the ligand for the flt3/flk2 receptor, s i n c e the e x p r e s s i o n of this receptor is restricted to primitive h e m o p o i e t i c c e l l s (137), a n d V L A - 4 w h o s e interactions with V C A M - 1 , fibronectin a n d L-selectin h a v e been  implicated  in m e d i a t i n g  the  in v i v o  homing  of  primitive  hemopoietic  progenitors to the marrow a n d s p l e e n (155). O n e intriguing finding d e s c r i b e d in C h a p t e r 4 of this t h e s i s w a s the detection of v e c t o r related d i f f e r e n c e s in the p e r s i s t e n c e of e x p r e s s i o n of the t r a n s f e r r e d C D 2 4 g e n e following e x t e n d e d p e r i o d s of time in v i v o . A g r e a t e r proportion of C D 2 4 + l y m p h o c y t e s , g r a n u l o c y t e s / m a c r o p h a g e s , erythrocytes a n d S c a + L i n " s t e m cell c a n d i d a t e s w e r e o b s e r v e d in the recipients of M S C V C D 2 4 t r a n s d u c e d b o n e m a r r o w a s c o m p a r e d to J Z e n C D 2 4 t r a n s d u c e d m a r r o w d e s p i t e the d e t e c t i o n of roughly equivalent levels of intact recombinant provirus. O n e e x p l a n a t i o n for this is s h u t d o w n of the viral (JZen) L T R regulatory e l e m e n t s in vivo, a p h e n o m e n o n w h i c h h a s b e e n previously reported  (215, 251), and which may be the  result  of  m e t h y l a t i o n of the p r o m o t e r a n d / o r e n h a n c e r e l e m e n t s (257, 3 3 1 ) . In a d d i t i o n , d i f f e r e n c e s in the e x p r e s s i o n of the transferred C D 2 4 g e n e w e r e d e t e c t e d a m o n g c e l l s of different l i n e a g e s . T h e proportion of C D 2 4 + t h y m o c y t e s w a s significantly l o w e r t h a n that o b s e r v e d a m o n g c e l l s of either the m y e l o i d ( b o n e m a r r o w )  or  erythroid lineage ( C h a p t e r 4). Together, t h e s e results s u g g e s t that d e s p i t e efforts to  105  construct/identify  vectors  a b l e to d r i v e  high  and persistent  l e v e l s of  gene  e x p r e s s i o n in all w h i c h contain recombinant provirus (irrespective of cell lineage), this g o a l h a s not yet b e e n r e a l i z e d . N e v e r t h e l e s s , the e f f e c t i v e n e s s of C D 2 4 a s a m e a n s to rapidly quantify the levels of transferred g e n e e x p r e s s i o n attainable from different v e c t o r s s u g g e s t s that this m a r k e r w o u l d be useful in efforts to identify regulatory e l e m e n t s that m a x i m i z e the levels a n d p e r s i s t e n c e of transferred g e n e e x p r e s s i o n in H S C s or a n y other target cell of interest. R e c e n t l y , a n o v e l v e c t o r c a l l e d M F G ( R i c h a r d Mulligan, W h i t e h e a d Institute for B i o m e d i c a l R e s e a r c h , M.I.T., C a m b r i d g e , M A ) h a s b e e n s h o w n to p r o v i d e higher l e v e l s of t r a n s f e r r e d  gene  e x p r e s s i o n t h a n M o l o n e y murine l e u k e m i a virus b a s e d v e c t o r s (340). In addition, Dr. D o n a l d K o h n a n d his c o l l e a g u e s ( C h i l d r e n s H o s p i t a l , L o s A n g e l e s , C A ) a r e currently  a t t e m p t i n g to s o l v e the p r o b l e m of p r o m o t e r  shutdown  in v i v o  by  identifying a n d r e m o v i n g potential methylation sites present in v e c t o r s b a s e d o n the M o l o n e y murine l e u k e m i a virus. It would be interesting to u s e C D 2 4 to quantify a n d c o m p a r e the levels a n d p e r s i s t e n c e of g e n e e x p r e s s i o n obtainable from t h e s e v e c t o r s a s c o m p a r e d to J Z e n a n d M S C V . C D 2 4 w o u l d a l s o be of u s e in efforts to rapidly s c r e e n alternative infection p r o t o c o l s i n v o l v i n g the u s e of n e w l y d i s c o v e r e d h e m o p o i e t i c  retroviral growth  factors (eg. flt3/flk2 ligand), or newly d e v e l o p e d strategies s u c h a s t h o s e involving the u s e of fibronectin (described in detail on p a g e 37-38) or the "flow-through" viral t r a n s d u c t i o n s y s t e m ( d e s c r i b e d on p a g e 38). A s a n e x a m p l e of this, Dr. C r a i g J o r d a n ( S o m a t i x , A l a m e d a , C A ) is currently u s i n g C D 2 4 a s a m a r k e r of g e n e transfer in c o m b i n a t i o n with F A C S in a n effort to correlate the c y c l i n g s t a t u s of primitive h u m a n h e m o p o i e t i c c e l l s with the efficiency of g e n e transfer ( p e r s o n a l communication). T h e selection a p p r o a c h d e s c r i b e d a n d utilized in C h a p t e r s 3 a n d 4 h a s n o w b e e n e x t e n d e d to the h u m a n setting. U s i n g retroviral v e c t o r s c o n t a i n i n g H S A , the m u r i n e h o m o l o g u e of C D 2 4 , in c o m b i n a t i o n with F A C S , C o n n e a l l y et. a l . h a s  106  d e m o n s t r a t e d the ability to e n r i c h for retrovirally t r a n s d u c e d primitive  human  h e m o p o i e t i c c e l l s , including L T C - I C (275). Efforts to determine w h e t h e r t r a n s d u c e d c e l l s c a p a b l e of repopulating immunodeficient m i c e c a n be e n r i c h e d for u s i n g this t e c h n i q u e are currently underway. In collaborative efforts with Dr. S t e f a n K a r l s s o n s ' lab (National Institutes of Health, B e t h e s d a , M D ) the selection a p p r o a c h d e v e l o p e d in C h a p t e r 3 h a s b e e n applied to test the therapeutic potential of v e c t o r s a i m e d at the genetic therapy of G a u c h e r s d i s e a s e (a h u m a n a u t o s o m a l r e c e s s i v e l y s o s o m a l s t o r a g e d i s o r d e r c a u s e d by a d e f i c i e n c y of the e n z y m e g l u c o c e r e b r o s i d a s e ) . A retroviral v e c t o r c o n t a i n i n g the c D N A e n c o d i n g H S A a s a s e l e c t a b l e m a r k e r in c o m b i n a t i o n with a t h e r a p e u t i c g l u c o c e r e b r o s i d a s e ( G C ) c D N A w a s c o n s t r u c t e d a n d u s e d to infect transformed B cell lines from G a u c h e r patients. W h i l e retrovirally t r a n s d u c e d , u n s e l e c t e d B cells s h o w e d a slight i n c r e a s e in the level of G C e n z y m e a s c o m p a r e d to u n t r a n s d u c e d B c e l l s , F A C S s e l e c t i o n of H S A + B c e l l s e n h a n c e d G C e n z y m e levels to more than 5-fold over u n t r a n s d u c e d cell l e v e l s , a n d e v e n to l e v e l s m o r e t h a n 2-fold o v e r t h o s e in n o r m a l n o n - G a u c h e r c e l l s (341). S u c h strategies b a s e d on this a p p r o a c h m a y e n a b l e the implantation of t r a n s d u c e d c e l l s to a n i m a l s or patients, although the u s e of cell s u r f a c e a n t i g e n s a s m a r k e r s for h u m a n g e n e t h e r a p y r e m a i n s c o n t r o v e r s i a l . N e v e r t h e l e s s , this a p p r o a c h s h o u l d prove  useful  in  preclinical  studies  (eg.  using  non-human  primates  or  i m m u n o c o m p r o m i s e d mice) a i m e d at optimizing the therapeutic potential of v e c t o r s for u s e in h u m a n g e n e therapy trials. T h e o b s e r v a t i o n that the C D 2 4 cell s u r f a c e a n t i g e n c a n be e x p r e s s e d on red blood c e l l s s u g g e s t s potential for this m o l e c u l e a s a s e l e c t a b l e m a r k e r in v e c t o r s a i m e d at the g e n e t i c treatment of s i c k l e c e l l a n e m i a a n d t h a l a s s e m i a s . In c o l l a b o r a t i v e efforts with Dr. P h i l i p p e L e B o u l c h (M.I.T., B o s t o n , M A ) , Dr. C o n n i e E a v e s (Terry F o x Laboratory, V a n c o u v e r , B . C . ) a n d Dr. R o n a l d N a g e l (Albert E i n s t e i n C o l l e g e of M e d i c i n e , B r o n x , N Y )  C D 2 4 is  currently b e i n g e m p l o y e d in retroviral c o n s t r u c t s c o n t a i n i n g a h u m a n  8/(3-globin  107  fusion g e n e d e s i g n e d for anti-sickling properties with the g o a l of testing t h e s e in a (3 (sickle) t r a n s g e n i c m o u s e m o d e l . s  T h e low efficiency of g e n e transfer to H S C s h a s m a d e efforts to track the utilization,  proliferation  a n d s e l f - r e n e w a l of i n d i v i d u a l  HSCs  using  retroviral  m a r k i n g t e c h n i q u e s extremely laborious. T h e ability to obtain, prior to transplant, a population of c e l l s in w h i c h 1 0 0 % are provirally m a r k e d w o u l d i n c r e a s e the p o w e r a n d e a s e of retroviral marking s t u d i e s a i m e d at a d d r e s s i n g t h e s e i s s u e s . R e s u l t s p r e s e n t e d in C h a p t e r 4 d e m o n s t r a t e that the C D 2 4 s e l e c t i o n a p p r o a c h c a n e n a b l e the isolation of a population of hemopoietic target cells in w h i c h virtually 1 0 0 % are provirally  m a r k e d a n d t h u s , a l l o w s o n e to track the  b e h a v i o r of aM. H S C s  t r a n s p l a n t e d . T h e p o w e r of this a p p r o a c h to track individual H S C s is d e m o n s t r a t e d in F i g u r e 4 . 3 . T h e transplant of e x c l u s i v e l y provirally m a r k e d H S C s e n a b l e d the d e t e r m i n a t i o n of w h e t h e r h e m o p o i e s i s w a s p o l y c l o n a l (top panel) or m o n o c l o n a l (bottom p a n e l ) , a n d a l s o a l l o w e d the a c c u r a t e determination of the n u m b e r a n d relative contribution of e a c h H S C to h e m o p o i e s i s in the primary recipient at the t i m e of s a c r i f i c e . T h e d y n a m i c s of H S C utilization u n d e r h o m e o s t a t i c c o n d i t i o n s r e m a i n s u n r e s o l v e d . O n e a p p r o a c h to a d d r e s s i n g this q u e s t i o n w o u l d b e to s e q u e n t i a l l y s a m p l e the p e r i p h e r a l b l o o d of m i c e t r a n s p l a n t e d with  retrovirally  t r a n s d u c e d H S C s o b t a i n e d following the C D 2 4 selection p r o c e d u r e . S t u d i e s u s i n g sequential  a n a l y s i s of  transplanted  peripheral  with retrovirally  blood  transduced  to  track  individual  (non-selected)  HSCs  marrow  in  mice  have  been  d e s c r i b e d (98, 99). Alternatively, a p r o c e d u r e h a s b e e n d e s c r i b e d w h e r e b y f e m o r a l m a r r o w c a n be sequentially obtained from a m o u s e by inserting a n e e d l e into the k n e e joint of a n a n e s t h e t i z e d m o u s e (342). S u f f i c i e n t q u a n t i t i e s  of D N A for  S o u t h e r n blot a n a l y s i s is obtained by using the extracted m a r r o w to g e n e r a t e d a y 12 C F U - S in s e c o n d a r y recipient m i c e . T h e u s e of this p r o c e d u r e in c o m b i n a t i o n with the C D 2 4 s e l e c t i o n a p p r o a c h w o u l d e n a b l e the contribution  of  individual  H S C s to h e m o p o i e s i s to be t r a c k e d , theoretically, for the entire life of the recipient  108  m o u s e a n d s h o u l d p r o v i d e insight into the n u m b e r a n d p e r s i s t e n c e of  HSCs  contributing to h e m o p o i e s i s under homeostatic conditions. T h e results of transplant s t u d i e s p r e s e n t e d in C h a p t e r 5 r e v e a l the inability of H S C n u m b e r s to r e c o v e r to normal levels following the transplant of e v e n large n u m b e r s of adult b o n e m a r r o w or fetal liver s t e m c e l l s . T h e s e results m a y h a v e r e l e v a n c e to clinical b o n e m a r r o w transplantation s t u d i e s . A l t h o u g h c l i n i c a l b o n e m a r r o w t r a n s p l a n t a t i o n h a s b e e n p e r f o r m e d for only 2 0 to 2 5 y e a r s , t o d a y it c o n s t i t u t e s a n major e l e m e n t of therapy for the treatment of n u m e r o u s f o r m s of cancer  a s well  as  heritable  hematological  disorders.  Indeed,  over  6,000  a u t o l o g o u s b o n e m a r r o w t r a n s p l a n t s w e r e p e r f o r m e d in North A m e r i c a in 1 9 9 4 a l o n e ( p e r s o n a l c o m m u n i c a t i o n : International B o n e M a r r o w T r a n s p l a n t R e g i s t r y ) . Might the poor r e c o v e r of s t e m cell n u m b e r s following b o n e marrow transplant be a contributing factor to marrow failure? It will be important to monitor the recipients of b o n e marrow grafts for long periods to attempt to determine w h e t h e r p o o r s t e m cell r e c o v e r y p l a y s a role in b o n e marrow failure a n d s u b s e q u e n t d e a t h of the patient. M o r e o v e r , the poor r e c o v e r y of H S C n u m b e r s following b o n e m a r r o w transplant s h o u l d b e t a k e n into c o n s i d e r a t i o n w h e n attempting h u m a n g e n e t h e r a p y , w h i c h m a y require the selection of t r a n s d u c e d s t e m cells, a s well a s the transplant of fetal liver or umbilical c o r d blood H S C s s i n c e both m a y require the transplant of m o d e s t n u m b e r s of cells into the patient. T h e ability to transplant H S C s in which 1 0 0 % are provirally m a r k e d p r o v i d e d intriguing insight into the r e g e n e r a t i v e c a p a c i t y of H S C s following b o n e m a r r o w transplant ( C h a p t e r 5). P r o v i r a l integration a n a l y s i s of m i c e r e c e i v i n g retrovirally t r a n s d u c e d , C D 2 4 + s e l e c t e d , b o n e marrow cells provided e v i d e n c e for a >300-fold c l o n a l amplification of a s i n g l e t r a n s d u c e d s t e m c e l l . A n u m b e r of q u e s t i o n s are r a i s e d by this finding. D o all H S C s p o s s e s s s u c h a c a p a c i t y for r e g e n e r a t i o n ? If s o , what factors or c i r c u m s t a n c e s will e n a b l e this potential to be fully r e a l i z e d following t r a n s p l a n t ? T h e s e findings set the s t a g e for attempts to m a x i m i z e the r e c o v e r y of  109  H S C s n u m b e r s following b o n e m a r r o w transplant through the a d m i n i s t r a t i o n e x o g e n o u s a g e n t s s u c h a s h e m o p o i e t i c growth  of  f a c t o r s or t h e e x p r e s s i o n of  t r a n s d u c e d g e n e s e n c o d i n g intracellular f a c t o r s . It w o u l d b e i n t e r e s t i n g l y  to  d e t e r m i n e whether the administration of the "stem cell" factors s u c h a s S t e e l factor a n d / o r F L following b o n e marrow transplant m a y i n c r e a s e the level of regeneration of s t e m cell n u m b e r s . Alternatively, s u c h a n experimental strategy c o u l d be utilized to test the role that particular g e n e s , found to be e x p r e s s e d in primitive h e m o p o i e t i c c e l l s , m a y play in regulating the s e l f - r e n e w a l or c o m m i t m e n t of t h e s e c e l l s by o v e r e x p r e s s i o n u s i n g r e c o m b i n a n t retroviral v e c t o r s c o m b i n e d with the u s e of a dominant selectable marker gene such as C D 2 4 . In s u m m a r y t h e s e studies h a v e provided m e t h o d s w h i c h e n h a n c e the utility of current g e n e transfer protocols a n d h a v e for the first time p r o v i d e d  quantitative  d a t a r e g a r d i n g the r e g e n e r a t i o n of H S C n u m b e r s f o l l o w i n g the t r a n s p l a n t  of  v a r i o u s n u m b e r s of H S C s from v a r i o u s ontological s o u r c e s . T h e s e s t u d i e s set the s t a g e for future e x p e r i m e n t s d e s i g n e d to provide further insight into the b e h a v i o r a n d biological potential of H S C s a s well a s studies a i m e d at d e v e l o p i n g i m p r o v e d v e c t o r s for u s e in g e n e therapy.  110  CHAPTER  7  REFERENCES 1.  E r s l e v , A . J . 1983. Production of erythrocytes. In H e m a t o l o g y . M c G r a w - H i l l , New York.  2.  D a n c e y , J . T . , K.A. D e u b e l l e i s s , L.A. Harker, a n d C . A . F i n c h . 1976. Neutrolphil kinetics in m a n . J . 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