Comparison of the lineage distribution of WBCs produced by receptor and ...
Structure, expression and function of a retroviral vector encoding a c-fms-flt3.
EFFECT OF C-KIT AND
FLT-3 OVEREXPRESSION ON
PRIMITIVE HEMATOPOIETIC CELLS
by Pak-Yan Pat Chu B.Sc.(Honours Biological Science), University of Windsor, 1996
A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Medical Genetics, Faculty of Medicine
We accept this thesis as conforming to the required standard
T H E UNIVERSITY OF BRITISH C O L U M B I A July 2003 © Pak-Yan Pat Chu, 2003
In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head, of my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d without my w r i t t e n p e r m i s s i o n .
Department of
AW/crt,/
GieneflcS
The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada
ABSTRACT Recent studies have demonstrated that hematopoietic stem cell (HSC) self-renewal divisions in vitro are favoured by exposure of the cells to elevated levels of Steel factor (SF) or flt-3 ligand (FL). This suggested that enhanced HSC self-renewal by these cytokines might be limited by the level of expression of the corresponding c-kit or flt-3 receptors on HSCs. In this thesis, I investigated this hypothesis by examining how retroviral-mediated overexpression of either c-kit or flt-3 affects the responsiveness of hematopoietic cells to SF and FL, respectively, first in a cell line model and then in primary mouse bone marrow cells,
c-kit and flt3-
transduced BaF3 cells were able to proliferate in lower concentrations of SF and F L , respectively, than control-transduced cells, although evidence of high dose inhibition was also noted. In primitive mouse bone marrow cells transduced with the same c-kit ory7f-3-encoding vectors, a similar effect on short term total cell expansion in vitro was seen. This included a cytokine-specific dose sensitization of progenitor and HSC expansion without detectable effects on their subsequent commitment or differentiation. Depressed responsiveness to very high cytokine concentrations was also seen in primary transduced cells such that no significant further net amplification of the transduced HSCs could be achieved in vitro.
In vivo
competitive reconstitution assays were performed to determine whether the enhanced cytokine sensitivity of receptor-overexpressing HSCs would give them a growth advantage in vivo. Although continued expression of functional receptors on the in vivo generated progeny of the transduced HSCs could be clearly documented and their self-renewal shown to be intact, their behaviour in vivo could not be distinguished from control-transduced cells, even when the corresponding ligand was administered exogenously or induced endogenously. It thus appears that neither c-kit nor flt-3 receptor levels on HSCs are limiting to their capacity to self-amplify in vivo. These results suggest that other molecular interactions of HSCs with their environment may be more potent regulators of HSC self-renewal responses in vivo and need to be identified to improve HSC amplification in vitro.
ii
TABLE OF CONTENTS ABSTRACT
ii
LIST OF TABLES
vi
LIST OF FIGURES
;
.........vii
LIST OF ABBREVIATIONS
ix
ACKNOWLEDGMENTS Chapter 1: 1.1.
xii
Introduction
1
Proj ect overview
1
1.2. Hematopoiesis 1.2.1. Hierarchical model of hematopoiesis 1.2.1.1. Pluripotent HSCs 1.2.1.2. Progenitor cells 1.2.1.3. Terminal maturation of blood cells 1.3. Characterization of HSCs 1.3.1. Definition of HSCs 1.3.2. Quantitation of HSCs 1.3.3. Phenotyping and purification of HSCs 1.4. Properties of HSCs 1.4.1. Self-renewal potential of HSCs 1.4.2. Commitment of HSCs 1.4.3. Cycling status of HSCs in vivo 1.4.4. Homing and mobilization of HSCs 1.5. Regulation of HSCs 1.5.1. Intrinsic factors / transcription factors 1.5.2. Environmental factors / cytokines 1.6. Effect of environmental factors on HSC self-renewal 1.6.1. Effect of growth-promoting cytokines 1.6.1.1. Instructive vs selective actions of cytokines 1.6.1.2. Kinetics of HSC stimulation 1.6.2. Effect of growth-inhibiting cytokines 1.6.3. Effect of morphogens 1.7. Sub-class III tyrosine kinase receptors and their corresponding ligands 1.7.1. c-kitandSF 1.7.1.1. Molecular structure 1.7.1.2. Expression of c-kit in primitive hematopoietic cells 1.7.2. Flt-3 and F L 1.7.2.1. Molecular structure 1.7.2.2. Expression of flt-3 in primitive hematopoietic cells 1.7.3. Roles of c-kit and flt-3 in primitive hematopoietic cell 1.7.3.1. In vitro studies 1.7.3.2. In vivo studies 1.7.4. Involvement of c-kit and flt-3 in hematopoietic diseases 1.8. Genetic manipulation of hematopoietic cells using recombinant retroviruses iii
1 2 5 6 6 7 7 7 9 11 11 12 12 13 14 14 16 18 18 19 21 21 22 23 25 25 26 27 27 28 29 29 33 34 34
1.8.1. Retrovirus biology 1.8.2. Production of recombinant retroviruses 1.8.3. Recombinant retroviral vectors 1.8.4. Murine HSCs as target for retroviral-mediated gene transfer 1.9. Present studies and thesis objective.
Chapter 2:
Materials and methods
40
2.1. Reagents 2.1.1. Cell lines 2.1.2. Cytokines 2.1.3. Mice 2.1.4. Isolation of mouse B M cells 2.2. Retoviral vectors construction and molecular analysis 2.2.1. Vector construction 2.2.2. Vector sequence validation 2.3. Transduction protocols 2.3.1. Production of retroviral supernatant 2.3.2. Transduction protocols for cell lines 2.3.3. Transduction protocol for murine B M cells 2.3.4. Assessment of gene transfer efficiency in bulk cell populations 2.3.5. Viral titer and helper virus assays 2.4. In vitro assays 2.4.1. BaF3 cell proliferation assays 2.4.2. Primary murine B M hematopoietic cell culture 2.5. Progenitor and stem cell assays 2.5.1. C F C assays 2.5.2. C R U assay for HSCs 2.5.2.1. Transplantation procedure 2.5.2.2. Assessment of HSCs in engrafted mice 2.5.2.3. Secondary B M transplantations 2.6. Flow cytometry and cell sorting 2.7. Statistical methods
Chapter 3:
3.1. Introduction 3.2. Results 3.2.1. Construction and validation of c-kit and flt-3 retroviral vectors 3.2.2. Generation of polyclonal and derivative monoclonal BaF3cell populations expressing different levels of c-kit and flt-3 3.2.3. Altered mitogenic responses of M-KIT and M-FLT-transduced BaF3 cells 3.3. Discussion
49 49 50 50 52 57 70
Effect of overexpressing c-kit or flt-3 on the in vitro self-renewal and
differentiation responses of primitive hematopoietic cells 4.1. 4.2.
40 40 40 41 41 41 41 42 43 43 43 44 44 45 45 45 45 46 46 46 46 46 47 47 48
The effect of variable levels of c-kit and flt-3 receptor expression on
responsiveness to the cognate ligand in a cell line model
Chapter 4:
35 36 36 37 38
Introduction Results
iv
73
4.2.1. Optimization of a protocol for retroviral transduction of primitive murine B M cells 74 4.2.2. Effect of forced overexpression of c-kit or flt-3 in primary B M cells on total cell expansion in vitro in response to SF and F L stimulation 81 4.2.3. Effect of forced overexpression of c-kit or flt-3 in primary B M cells on progenitor cell expansion in vitro in response to SF and F L stimulation 87 4.2.4. Effect of forced overexpression of c-kit or flt-3 in primary B M cells on the expansion of HSCs in vitro in response to SF or F L 89 4.3. Discussion 100
Chapter 5:
The role of c-kit and flt-3 in HSC expansion in vivo
5.1. Introduction 5.2. Results 5.2.1. In vivo competitive reconstitution experiments 5.2.2. Effect of in vivo injection of stimulatory factors 5.3. Discussion
Chapter 6:
General discussion
103 103 105 105 115 119
121
REFERENCES
125
v
LIST OF TABLES Table 4.1.
Titers of different c-kit retroviruses and the relative level of expression of c-kit in transduced BaF3 cells 76
Table 4.2.
Improved retrovirus production and transduction of primitive murine B M cells...... 79
Table 4.3.
Calculated repopulating activity of HSCs (CRUs) present in retrovirally transduced populations 90
Table 4.4.
Data used to calculate the expansion in vitro of M-KIT-transduced HSCs cultured with different concentrations of SF 97
Table 4.5.
Data used to calculate the expansion in vitro of M-FLT-IG-transduced HSCs cultured with different concentrations of F L
98
Table 5.1.
Calculated net expansion of transduced HSCs in primary mice
112
Table 5.2.
Level of engraftment in recipients of mixtures of receptor and MIG-transduced cells before and after injection of SF or LPS
116
vi
LIST OF FIGURES Figure 1.1.
The current model of the hematopoietic hierarchy
4
Figure 1.2.
The general molecular structure of the subclass III receptor T K family and their corresponding ligands 24
Figure 1.3.
Clone formation and progenitor expansion from single cell cultures of CD34 CD38" adult human B M cells
32
Figure 3.1.
Retroviral constructs
51
Figure 3.2.
FACS analysis and sorting of transduced BaF3 cells
53
Figure 3.3.
Fluorescence profiles of various polyclonal populations of transduced BaF3 cells
54
Mean and range of fluorescence intensity of individual clones of transduced BaF3 cells
56
+
Figure 3.4.
Figure 3.5.
Mitogenie responses of M-KIT and M-FLT-transduced BaF3 populations to IL3 58
Figure 3.6.
Mitogenic responses of M-KIT and M-FLT-transduced BaF3 populations to SF andFL 61
Figure 3.7.
Variation in the mitogenic responses of BaF3 cells to SF and F L according to their relative levels of expression of c-kit or flt-3
65
Figure 4.1.
Structures of the retroviral constructs
75
Figure 4.2.
Protocol for producing high titer retroviral supernatants
78
Figure 4.3.
Detection of transduced receptors on murine B M cells
80
Figure 4.4.
Effect of overexpression of c-kit and flt3 on total cell expansion in vitro
82
Figure 4.5.
Ligand-specific cell-autonomous expansion of receptor-transduced cells
86
Figure 4.6.
Effect of overexpression of c-kit and flt3 on C F C production in vitro
88
Figure 4.7.
Gene transfer efficiencies to HSCs
92
Figure 4.8.
Comparison of the lineage distribution of WBCs produced by receptor and control-transduced HSCs after their transplantation into mice
94
Figure 4.9.
Method used to calculate the extent of HSC expansion achieved in vitro
96
Figure 4.10.
Effect of overexpression of c-kit and flt3 on the maintenance of HSCs in vitro ..99
vii
Figure 5.1.
In vivo competitive reconstitution experimental design
Figure 5.2.
FACS analysis of WBCs from recipients of mixed populations of control and receptor-transduced B M cells 107
Figure 5.3.
Time course study of the competitive reconstituting activity of receptortransduced HSCs in primary recipients
109
Competitive HSC-reconstituting activity of receptor-transduced HSCs in primary recipients assessed by transplantation into secondary recipients
Ill
Figure 5.4.
106
Figure 5.5.
Cytokine sensitivities of B M cells from mice engrafted with receptor-transduced HSCs 114
Figure 5.6.
Structure, expression and function of a retroviral vector encoding a c-fms-flt3 chimeric receptor 117
viii
LIST OF ABBREVIATIONS 5-FU AGM
5-fluorouracil Aorta-gonad-mesonephros
ALL
Acute lymphoblastic leukemia
AML
Acute myelogenous leukemia
APC
Allophycocyanin
BFU-E
Burst-forming unit erythroid
BIT
Bovine serum albumin, insulin, transferrin,
BM
Bone marrow
BMP
Bone morphogenetic proteins
BrdU
Bromodeoxyuridine
CD
Cluster differentiation
CFC
Colony-forming cell
CFC-E
Colony-forming cell - erythroid
CFC-Eo
Colony-forming cell - eosinophil
CFC-G
Colony-forming cell - granulocyte
CFU-GM
Colony-forming cell - granulocyte macrophage
CFC-M
Colony-forming cell - macrophage
CFC-Ma
Colony-forming cell - mast cell
CFC-Mk
Colony-forming cell - megakaryocyte
CFU-S
Colony-forming unit-spleen
CML
Chronic myelogenous leukemia
CRU
Competitive repopulation unit
CSF
Colony stimulating factors
CXCR4
Chemokine CXC receptor 4
Cy-5
Cyanine 5-succinimidylester
DMEM
Dulbecco's modified Eagle medium
ECM
Extracellular matrix
EGFR
Epidermal growth factor receptor
EPO
Erythropoietin ix
E C cells
Embryonic carcinoma cells
ES cells
Embryonic stem cells
EST
Expressed sequence tag
FACS
Fluorescent activated cell sorting
FCS
Fetal calf serum
FITC
Fluorescein isothiocyanate
FL
Flt-3 ligand
G-CSF
Granulocyte - colony stimulating factors
GFP
Green fluorescent protein
GM-CSF
Granulocyte/macrophage - colony stimulating factors
HF
Hank's Balanced Salt Solution plus 2% fetal calf seru
HSC
Hematopoietic stem cell
ICAM-1
Intercellular adhesion molecule-1
Ig
Immunoglobulin
IMDM
Iscove's modified Dulbecco's medium
IRES
Internal ribosomal entry site
IL
Interleukins
JAK
Janus Kinase
LIF
Leukemia inhibitory factor
Lin
Lineage
LIST
Ligand-receptor signaling-threshold
LPS
Lipopolysaccharide (bacterial)
LTC
Long-term culture
LTC-IC
Long-term culture-initiating cell
LTR
Long-term reconstituting or long terminal repeat
2-ME
2-mercaptoefhanol
MCP-1
Monocyte chemoattractant protein-1
M-CSF
Macrophage - colony stimulating factors
MFI
Mean fluorescence intensities
MlP-la
Macrophage inflammatory protein-1 alpha
MMuLV
Moloney murine leukemia virus
MSCV
Murine Stem Cell Virus X
NOD/SCID
Non-obese diabetic-severe combined immunodeficiency
PAS
Para-aortic splanchnopleure
PE
Phycoerythrin
PI
Propidium iodide
Rh-123
Rhodamine-123
RTK
Receptor tyrosine kinase
SI
Steel
SDF-1
Stroma-derived factor-1
SEM
Standard error of mean
SF
Steel factor
Shh
Sonic hedgehog
STAT
Signal transducer and activation of transcription
STR
Short-term reconstituting
TGF-P
Transforming growth factor-beta
TK
Tyrosine kinase
TNF
Tumor necrosis factor
TPO
Thrombopoietin
VLA-4
Very late antigen-4
W
White-spotting
WGA
Wheat germ agglutinin
Wnt
Wingless
xi
ACKNOWLEDGMENTS This thesis project is the most challenging task I have ever encountered. Throught out this project, I have gained, besides knowledge, a chance to explore the complexity of nature. It is truly one of the most valuable experiences.
I want to thank all the people in Terry Fox
Laboratory from whom I have gained a lot of help andfriendshipsover these years. There are several people whom I would like to especially thank here, because I think they have given me the most significant help: Dr. Connie Eaves, my supervisor, for giving me great help and support throughout this project. Her incredible energy, knowledge, enthusiasm and professionalism have a profound impact not just on this project but also on my personal development. My parents and grandparents, for their unconditional support.
Their support has
provided me with a rare opportunity of complete freedom and a safety net to pursue the path I have chosen. Gigi, for all the happy moments she has spent with me. Because of them, I feel very fortunate. Finally, I would also like to thank Roman M . Babicki for his generous financial support in the form of studentships for 2 years of my graduate program.
xii
CHAPTER 1: INTRODUCTION 1.1.
Project overview A stem cell is a cell that has the ability both to self-replicate and to give rise to
differentiated mature cells.
Hematopoietic stem cells (HSCs) are the cells ultimately
responsible for the life long production of all blood cell types. HSCs have been postulated to exist for more than 50 years, and their transplantation is routinely used to treat patients with cancers and other inherited disorders of the blood and immune systems. However, in spite of the intensive research in this field for the last several decades, many aspects of H S C biology remain unresolved. In particular, we still know very little about the molecular mechanisms that determine how HSCs maintain their long-term multilineage differentiation and extensive proliferative potential through many cell cycles. In this thesis, I have used a genetic approach to investigate whether the maintenance of these properties may be altered by changes in the levels of receptors that allow HSCs to respond to growth factors that can alter H S C selfrenewal in a concentration-dependent fashion. I believe that the theoretical model being tested in this thesis is important because further understanding of the mechanisms of H S C maintenance in vitro and in vivo is relevant to the future successful manipulation of HSCs for a variety of clinical applications.
1.2.
Hematopoiesis The hematopoietic system contains at least 10 different mature blood cell types, each
with a set of specialized properties crucial for life-supporting functions. These include oxygen transport, killing and removal of microorganisms, resolution of inflammatory responses and blood clotting. The mature blood cells are distributed throughout the body by a network of arteries, veins and lymphatics whereby the cellular and plasma components can be brought into contact with all of the other tissues. Most mature blood cells have a relatively short normal lifespan (ranging in humans from approximately 48 hours for neutrophils to 120 days for erythrocytes and in mice, from approximately 18 hours to 40 days for the same cell types). Most are also incapable of further division (Beulter et al. 1995). As a result, large numbers of blood cells are lost from the body each day (estimated as ~ 10 Abkowitz et al. 1995).
1
/day in humans) (Ogawa 1993;
The fact that the number of mature blood cells is maintained at relatively constant levels throughout adult life indicates the existence of a process whereby newly differentiated blood cells must be continuously produced from more primitive precursors.
This process,
which is referred to as hematopoiesis, is complex and dynamic, both in terms of the different types and numbers of blood cells produced and in the mechanisms that regulate their outputs in response to changing needs. Studies in mice have shown that the para-aortic splanchnopleure (PAS) and aortagonad-mesonephros (AGM) region are the earliest intra-embryonic sites of hematopoiesis (Godin et al. 1993; Medvinsky et al. 1993; Dzierzak et al. 1998; Nishikawa et al. 2001). It is believed that many HSCs first arise in the P A S / A G M region, from which they enter the embryonic circulation and then colonize the developing liver. However, some HSCs may also arise independently in the yolk sac blood islands where the first mature blood cells are formed (Dzierzak and Medvinsky 1995).
The liver then becomes the major site of hematopoiesis
throughout the rest of fetal life. After birth, hematopoiesis shifts to the bone marrow (BM).
1.2.1. Hierarchical model of hematopoiesis A major advance of understanding of hematopoiesis was prompted by early studies that demonstrated the rescue of lethally irradiated mice by the intravenous injection of histocompatible B M cells and the ability of a small subset of the injected B M cells to generate macroscopic colonies in the spleen of the irradiated recipients (Till and McCulloch 1961). Further analysis indicated that by 12 days post-transplant, these spleen colonies contain different lineages of hematopoietic cells, all of which are the progeny of a single rare cell that was called a colony-forming unit-spleen (CFU-S).
These studies also showed that spleen
colonies generated in this way contain daughter CFU-S, indicating that the original CFU-S were capable of self-generation (Till et al. 1964). These observations gave rise to the concept that CFU-S are HSCs.
Subsequent studies of the effect of 5-fluorouracil (5-FU) provided
evidence of pre-CFU-S cells (Hodgson and Bradley 1979).
Later, improved methods for
fractionating B M cells on the basis of a variety of different parameters showed that >90% of the CFU-S in normal adult mouse B M do not have long term repopulating ability (Hodgson and Bradley 1979; Spooncer et al. 1985; Ploemacher and Brons 1989; Ploemacher et al. 1993; Szilvassy and Cory 1993). Thus, while the CFU-S assay is useful for quantitating a primitive
2
multi-potent myeloid progenitor, it does not reliably or specifically measure the most primitive HSCs with long term repopulating activity (Jones et al. 1990). The use of semi-solid media for B M cell cultures allowed another major advance in the understanding of hematopoiesis (Bradley and Metcalf 1966; Ichikawa et al. 1966; Metcalf and Nicola 1984).
Such media allow the formation of individual colonies of specific or mixed
lineages of hematopoietic cells in vitro to be visualized.
These colonies were shown to
originate from single cells, and the initiating cells were termed (in vitro) colony-forming cells (CFCs).
CFCs are more numerous than CFU-S and their presence in spleen colonies
demonstrated that they were produced by CFU-S (Wu et al. 1967). Most of the colonies that develop from unseparated suspensions of hematopoietic cells consist of cells of only a single lineage, regardless of the mixture of cytokines/growth factors present in the culture medium. This suggests that the progenitors of these colonies are "committed" to a particular lineage differentiation pathway before being placed in the culture. Most CFCs also appear to lack selfrenewal capacity. They are therefore thought to represent a type of intermediate progenitor. More recently, strategies to separate progenitors with particular differentiation potentialities, based on differences in their physical and surface marker characteristics have also been developed (Ploemacher and Brons 1989; Jones et al. 1990; Wolf et al. 1993; Lemieux et al. 1995). Taken together, these observations support a model of hematopoiesis in which the hematopoietic differentiation process spans multiple steps leading to the generation of a hierarchy of progenitor subsets of generally increasing frequency. For simplicity, these are conventionally categorized as: stem cells, progenitor cells and terminally differentiating cells - the cells in each compartment representing the amplified progeny of cells from the preceding compartment (Zon 2001) (Figure 1.1a).
3
a)
b)
Dividing and terminally differentiating cells . . •
....
Figure 1.1. The current model of the hematopoietic hierarchy (a) A simplified model of the compartments in the hematopoietic hierarchy, illustrating the increasing size of the compartments, unidirectional progress of differentiation (vertical white arrows) and the accompanying decrease of cellular proliferative potential (inverted triangle). Commitment occurs in the stem cells (black) with no transdifferentiation between lineages (compartments separated by vertical dashed lines), (b) Additional subpopulations in the hierarchy defined by different in vivo or in vitro functional assays. In the stem cell (black) compartment, competitive repopulation units (CRUs) define the stem cells, whereas the colony-forming unitspleen (CFU-S) and long-term culture-initiating cells (LTC-ICs) include/comprise mainly shortterm repopulating cells. The extent of overlap between cell populations is not well defined. CFC, colony-forming cell (Figure adapted from Zon 2 0 0 1 ) .
According to this model, the production of mature blood cells from HSCs is unidirectional and achieved through the processes of cell amplification and differentiation along the myeloid and lymphoid lineages. This model also assumes that the blood cells in the later (more mature) levels of the hierarchy have less proliferative and differentiation potential, and that no dedifferentiation or transdifferentiation occurs between lineages. More recently, many additional experimental studies have suggested further sub-populations within each compartment (see below) leading to a more complex hierarchical model (Figure 1.1b). However, the basic pattern and assumptions remain the same.
1.2.1.1. Pluripotent HSCs The continued development of increasingly sophisticated cell separation technologies has allowed pluripotent HSC populations to be isolated in highly enriched form based on their unique constellations of physical and cell-surface properties (Spangrude et al. 1988; Morrison and Weissman 1994) (see also section 1.3.3).
Several experiments have shown that some
single HSCs can be sufficient to produce most of the blood cells generated in a mouse for many months (Jordan and Lemischka 1990; Lemischka 1992; Osawa et al. 1996a). However, other studies have also documented the functional heterogeneity of HSCs (Dick et al. 1985; Lemischka et al. 1986; Jordan and Lemischka 1990; Morrison and Weissman 1995; Goodell et al. 1996). Heterogeneity is revealed by the different repopulation abilities of phenotypically indistinguishable HSC-containing cell populations after their transplantation into irradiated mice
or by
the
demonstration
of
sustained
multi-lineage
repopulating abilities
by
phenotypically distinct cells (see also section 1.3.2). In general, adult mouse B M cells with multi-lineage repopulating ability have been found to comprise 2 separable populations: 1) long-term reconstituting (LTR) cells that can sustain hematopoiesis for more than 4 months in irradiated recipients, and 2) short-term reconstituting (STR) cells that can initially regenerate all of the blood elements but only for a few weeks (Jones et al. 1989; Jordan and Lemischka 1990; Keller and Snodgrass 1990; Harrison and Zhong 1992; Harrison et al. 1993; Zhong et al. 1996) (Figure 1.1b). Differences in the surface antigen profile of these 2 populations suggest that they may represent different stages of differentiation (Uchida et al. 1996; Zhao et al. 2000).
While the L T R cells are
assumed to be the more primitive, they may be unable to protect animals from lethal irradiation when injected in low numbers (Zhao et al. 2000). STR cells are thought to have less self5
renewal capacity, but may play an important role in the early recovery of mature blood cells in the immediate post-transplant period (Jordan and Lemischka 1990; Morrison and Weissman 1994; Uchida et al. 1996; Zhao et al. 2000). Recently, subsets of STR cells that are either myeloid or lymphoid-restricted have also been identified (Kondo et al. 1997; Akashi et al. 2000; Glimm et al. 2001).
1.2.1.2. Progenitor cells Within the intermediate class of progenitor cells identified as CFCs, a clear substratification is evident, based on the developmental potentialities these cells display.
In
general, 3 types of colonies can be seen in short term cultures. These are thought to reflect 3 hierarchically ordered sub-classes of CFCs as follows: 1) immature "blast CFCs" that form colonies in which a high proportion of the cells at the end of 2 weeks are, themselves, CFCs and thus produce secondary colonies of multiple lineages when replated into secondary cultures (Keller and Phillips 1982; Nakahata and Ogawa 1982); 2) multi-lineage CFCs, such as CFC-GEMM
(CFC-granulocyte, erythrocyte, megakaryocyte and macrophage) that form
colonies containing mature cells of all of these lineages (Johnson and Metcalf 1977; Fauser and Messner 1979; Humphries et al. 1981) as well as some CFCs; and 3) more lineage-restricted CFCs that form colonies containing mature cells of one or 2 lineages. The latter include C F C E (CFU-E) (erythroid), CFC-Eo (eosinophil), C F C - G (granulocyte), C F C - M (macrophage), CFC-Mast (mast cell), CFC-Mk (megakaryocyte), etc. In addition, single lineage CFCs can be stratified according to their proliferative potential, the more primitive CFCs giving rise to larger colonies in which mature elements appear later.
1.2.1.3. Terminal maturation of blood cells Morphologically recognizable blood cell precursors can be observed and followed for the last 3 to 5 consecutive cell cycles of their development. The changes that allow these cells to be distinguished and ordered in a sequence can be readily visualized by light microscopy of stained cell preparations. In summary, the features of the hematopoietic hierarchy are: 1) an initial loss of self-renewal potential; 2) a progressive distribution of restricted differentiation potentialities among increasingly larger populations of progenitor cells, and 3) an assumed irreversibility in the establishment of progenitor subpopulations (Zon 2001).
1.3.
Characterization of HSCs
1.3.1. Definition of HSCs HSCs are defined in this thesis as cells that are able to reconstitute all of the lymphoid and myeloid lineages for the lifetime of the individual (Lemischka et al. 1986). The existence of such cells was first demonstrated by studies that utilized radiation-induced chromosomal markers in primitive murine B M cells to track the common origin of lymphoid and myeloid cells regenerated in engrafted recipients (Wu et al. 1968; Abramson et al. 1977).
These
observations were subsequently confirmed using the unique integration site of retrovirallytransduced mouse B M or fetal liver cells to identify multi-lineage clones regenerated in vivo (Mintz et al. 1984; Dick et al. 1985; Keller et al. 1985; Lemischka et al. 1986; Capel and Mintz 1989; Jordan and Lemischka 1990; Keller and Snodgrass 1990).
Such experiments clearly
demonstrated that a small number of HSCs can be sufficient for the long-term restoration of normal multi-lineage hematopoiesis in a myeloablated mouse. In humans, the first evidence for HSCs came from the recognized involvement of multiple lineages in patients with myeloproliferative diseases (Dameshek 1951) that were subsequently shown to represent clonal disorders (Fialkow 1979). The existence of normal lympho-myeloid repopulating human HSCs was first formally demonstrated in 1989 by D N A analysis of recipients of allogeneic B M transplants (Turhan et al. 1989) and later demonstrated experimentally by limiting dilution xenografting experiments (Bhatia et al. 1997a; Conneally et al. 1997).
1.3.2. Quantitation of HSCs The detection and quantitation of HSCs relies on the use of functional assays that allow their rigorous discrimination from closely related primitive hematopoietic cells with limited reconstituting activity (e.g., short term repopulating cells) and/or more restricted differentiation potentialities (see also section 1.2.1.1). The reason for this is that a unique set of molecular correlates of the functional attributes of HSCs are not yet known. Accordingly, HSCs cannot be identified or measured directly (see below). assays are used to quantify HSCs.
In the murine system, 2 types of functional
Both rely on the transplantation of the test cells into
recipients whose endogenous hematopoiesis has been greatly suppressed, either by irradiation and/or because the recipients carry a mutation that results in deficient HSC activity. This is done to optimize the sensitivity of the assay by maximally stimulating hematological recovery
and H S C activation. Both assays use a prolonged (>4 months) post-transplant end-point of engraftment by the test cells.
Demonstration of such prolonged engraftment is essential to
ensure specificity of the read-out for long term repopulating cell activity since the progeny of other types of more restricted transplantable cells can also be detected for periods of up to 4 months (Jordan and Lemischka 1990; Morrison and Weissman 1994; Zhong et al. 1996; Miller etal. 1997). One functional assay for HSCs is based solely on the principle of competitive repopulation. This method compares the long-term repopulating ability of 2 sources of cotransplanted hematopoietic
cells whose progeny can be distinguished by
genetically
determined D N A , phenotypic or biochemical markers. The first of these populations is termed the competitor population, and usually consists of a pre-determined number of normal adult mouse B M cells that, by themselves, will readily repopulate and radioprotect the recipients. Various numbers of B M cells from a second population, termed the test population, are simultaneously injected with high numbers of the competitor population (generally 10 normal 6
adult B M cells).
The relative contributions to hematopoiesis by both cell populations are
measured 4 months post-transplantation (or later).
The frequency of HSCs in the test
population is then estimated based on the assumed existence of an inverse relationship between the variance and the input HSC number as predicted by the binomial equation, assuming a similar output potential of the HSCs in the test and competitor populations (Harrison et al. 1993). The other functional assay measures the frequency of HSCs directly using a limiting dilution analysis procedure (Szilvassy et al. 1990). This assay also incorporates the principle of competitive repopulation in that all recipients must contain (or receive) a minimal number of HSCs sufficient to ensure their survival. Survival of all recipients is necessary to avoid data bias, since the frequency of HSCs in the test suspension is determined from a statistical analysis of the proportion of mice transplanted with various doses of test cells that fail to show long-term (>4 months) lympho-myeloid engraftment by the test cells (above a defined detection threshold). Survival of the recipients is achieved either by co-transplanting a small number of HSCs of the same genotype as the recipient, or by using a recipient with a mutation that results in deficient endogenous HSC activity, such as the C57BL/6-W /W ' 4!
4
(W41) mouse.
In the latter instance, a lower (sublethal) dose of irradiation can then be used (Miller and Eaves
8
1997). However, the number of competitor or endogenous HSCs present in the recipient needs to be kept to a minimum in order to maximize the sensitivity of the assay (Szilvassy et al. 1990; Trevisan and Iscove 1995). Genetic markers, such as the Y chromosome for tracking the progeny of grafts of male cells into female recipients (Szilvassy et al. 1990), or a cell surface allo-antigen expressed on both lymphoid and myeloid cells (e.g., CD45/Ly5.1 or Ly5.2) (Spangrude and Scollay 1990; Szilvassy and Cory 1993; Rebel et al. 1994) can be used to identify the test cell origin of the blood cells produced post-transplant. Only recipients that are significantly (i.e., >1%) repopulated by both myeloid and lymphoid elements derived from the test cells for at least 4 months are considered positive (Miller and Eaves 1997). The input cell quantified by this assay is termed a competitive repopulation unit (CRU). Murine CRUs are present in the B M of normal adult mice at a frequency of approximately one per 10 cells 4
(Szilvassy et al. 1990; Miller and Eaves 1997). Because the quantitation of HSCs from the in vivo C R U assay is dependent on their reaching the B M microenvironment of the recipient and being stimulated to proliferate there, the type of treatment used to condition the recipients can influence C R U detection. Recent evidence also suggests that the cycling status of HSCs may affect their ability to be detected in a transplant assay (see section 1.4.4).
1.3.3. Phenotyping and purification of HSCs Functional assays for HSCs require them to proliferate and to differentiate for extended periods of time. Therefore, more immediate methods for the direct identification of HSCs are highly desirable. In the murine system, numerous strategies to obtain enriched populations of HSCs have been developed. These include bulk immunomagnetic and rosetting procedures, as well as multi-parameter fluorescent activated cell sorting (FACS) to remove or positively select for subpopulations according to their different physical characteristics (such as buoyant density and/or size), immunophenotypes (detected by antibody staining of variably expressed cell-surface markers) and abilities to efflux certain fluorescent dyes. Comparative studies of these properties exhibited by different types of B M cells have allowed many characteristics of HSCs in adult B M to be delineated, as follows: i) HSCs are relatively small cells with a lowdensity and a blast morphology (Orlic et al. 1993); ii) they express high levels of the Ly-6A/E (Sca-1) antigen (Spangrude et al. 1988; Spangrude and Brooks 1992), class I major histocompatibility antigens (H-2K) (Szilvassy et al. 1989; Spangrude and Scollay 1990), the CD38 antigen (Randall et al. 1996; Zhao et al. 2000) and c-kit (Ogawa et al. 1991; Ikuta and
9
Weissman 1992; Orlic et al. 1993) (see also section 1.7.1.3); iii) HSCs in normal adult mouse B M also express no or low levels of the sialomucin known as CD34 (a ligand for a cell adhesion molecule, L-selectin) (Osawa et al. 1996a; Sato et al. 1999; Zhao et al. 2000) nor many antigens present on mature blood cells (Muller-Sieburg et al. 1988; Spangrude et al. 1988; Spangrude and Brooks 1992; Orlic et al. 1993).
Such lineage-restricted cell surface
markers include CD3, CD4, CD8 CD45R (B220), CD90 (Thy-1), IgM (lymphoid), Ly-6G (Gr1), (myeloid) and Terll9 (erythroid); iv) HSCs are also largely separable from most other hematopoietic cells by their ability to efflux rhodamine-123 (Rh 123) (Mulder and Visser 1987; Ploemacher and Brons 1989; Orlic et al. 1993; Schinkel et al. 1997) and Hoechst 33342 (Baines and Visser 1983; Goodell et al. 1996; Zhou et al. 2001). Recent studies have demonstrated that HSCs can be enriched > 1000-fold by isolating subpopulations of adult mouse B M using various combinations of the above strategies to achieve HSC frequencies of 1 in 5 test cells (Wolf et al. 1993; Morrison and Weissman 1994; Spangrude et al. 1995; Osawa et al. 1996a; Wagers et al. 2002). However, these values may be underestimates because not every HSC thus isolated would be expected to home to the hematopoietic tissue and be activated following injection in vivo although this concept has recently been challenged (see section 1.4.4).
Moreover, it is important to note that
heterogeneity in repopulating behaviour has been observed even among the most enriched HSC populations. It is also important to note that as yet no known phenotypes can be used for the direct and definitive identification of HSCs. Numerous examples of dissociation in the regulation of surface and physiological marker expression and maintenance of HSC functions have been described (Rebel et al. 1994; Spangrude et al. 1995; Randall and Weissman 1997; Sato et al. 1999). In some cases, these changes appear to be related to HSC activation (Srour et al. 1992; Sato et al. 1999) that, in turn, may be variably related to cell cycle progression (Oh et al. 2000). Based on studies of human cells, it was believed that most of the primitive hematopoietic cells in the mouse would be likewise found to express high levels of the CD34 surface antigen (Krause et al. 1996; Bhatia et al. 1997b; Conneally et al. 1997). However, recent studies have shown that CD34 expression on murine HSCs appears only when they are activated (Osawa et al. 1996a; Goodell et al. 1997; Sato et al. 1999; Tajima et al. 2001; Ogawa 2002). In adult
10
humans, some primitive progenitors have now been found to be CD34" and capable of generating CD34 progeny (Bhatia et al. 1998; Zanjani et al. 1998; Nakamura et al. 1999). +
1.4. Properties of HSCs 1.4.1. Self-renewal potential of HSCs At the single cell level, a HSC self-renewal division can be operationally defined as proliferation without differentiation (i.e., maintenance of the undifferentiated state with all lineage options open).
Experimentally, evidence of such HSC self-renewal divisions can be
demonstrated and even quantitated by the analysis of the HSC progeny of primary HSCs regenerated in primary recipients.
This requires transplantating the cells from the primary
recipients into secondary recipients to demonstrate that progeny HSCs with longterm multilineage repopulating activity had been produced in the primary recipients. Several studies have clearly demonstrated the ability of HSCs to execute self-renewal divisions both in vivo (Lemischka et al. 1986; Jordan and Lemischka 1990; Keller and Snodgrass 1990) and in vitro (Fraser et al. 1990; Miller and Eaves 1997; Ema et al. 2000; Oostendorp et al. 2000). In some of these, it could be shown either directly (Ema et al. 2000; Oostendorp et al. 2000) or by integration site analysis of retrovirally marked clones (Lemischka et al. 1986; Fraser et al. 1990; Jordan and Lemischka 1990; Keller and Snodgrass 1990) that numerous recipients could be reconstituted by the progeny of a single original HSC. This provided a rigorous proof of self-renewal at the level of single HSCs. However, the numbers of self-renewal divisions that individual HSCs can execute and the extent to which this function can be altered by external factors (such as cytokines) have remained controversial issues (see section 1.6.1). Following B M transplantation, the donor HSC population is able to expand in vivo up to 100-fold in the following 8 to 12 month period (Osawa et al. 1996b; Pawliuk et al. 1996). Nevertheless, normally HSCs regenerate to levels that are 85% of highly enriched murine B M HSCs to proliferate in single-cell serum-free cultures (Nakauchi et al. 2001).
However, optimal
retention of HSC activity requires additional exposure to a ligand that will adequately activate gpl30 (Audet et al. 2001). Investigation of the effects of both relative and absolute differences in SF and F L cytokine concentrations on HSC responses has indicated that their viability, 20
proliferation, and self-renewal behave differently.
Promotion of HSC self-renewal requires
exposure to the highest cytokine concentrations (Zandstra et al. 1997b) (see section 1.7.3).
1.6.1.2. Kinetics of HSC stimulation The kinetics and/or intensity of stimulation (Marshall 1995) (see section 1.7.3.1), and other factors to which a cell is exposed (Sachs 1996; Callard et al. 1999; Schlessinger 2000) may have a considerable influence on how it responds. Variant forms of cytokines, such as membrane-bound, matrix-associated or membrane-anchored forms, may also elicit different biological outcomes (Gordon et al. 1987; Anderson et al. 1990; Otsuka et al. 1991; Lisovsky et al. 1996) (see section 1.7.3.1). Many stromal cell types can support some HSC maintenance in vitro (Toksoz et al. 1992; Koller et al. 1997; Brandt et al. 1998; Bennaceur-Griscelli et al. 1999), although the concentration of cytokines found in stromal cultures is well below what is commonly added to stroma-free cultures (Burroughs et al. 1994; Punzel et al. 1999a). Moreover, membrane forms of SF have been found to be indispensable for normal hematopoietic development, with soluble forms being unable to substitute for them (Witte 1990). These observations suggest that the membrane-bound form of at least some cytokines is likely to be important to HSC regulation in vivo. In addition to potentially altering signaling events when the ligand cannot be internalized, the effective local concentration of a cytokine may be increased when it is bound on the cell membrane or to E C M components, such as glycosaminoglycans (Gordon et al. 1987; Long et al. 1992), thus providing a more potent stimulus for enhancing HSC self-renewal.
1.6.2. Effect of growth-inhibiting cytokines In addition to growth-promoting cytokines, a number of cytokines may selectively inhibit the self-renewal of HSCs, or may even induce cell death. Examples are the TGF-P proteins (Eaves et al. 1991) and members of the chemokine family, such as macrophage inhibitory M l P - l a (Graham et al. 1990; Dunlop et al. 1992; Broxmeyer et al. 1993; Eaves et al. 1993), monocyte chemoattractant protein-1 (MCP-1) (Cashman et al. 1998) and stromaderived factor 1 (SDF-1) (Cashman et al. 2002).
It has been shown that in unperturbed
stromal-cell containing long-term cultures (LTCs) of human cells, the primitive cells contained within the adherent cell layer become quiescent after a week, but can be transiently activated by media changes. In contrast, the more mature CFCs proliferate continuously (Cashman et al. 1985). Later it was discovered that the molecular mechanism responsible for the endogenously 21
induced quiescence involved the co-operative activity of TGF-p (Cashman et al. 1990) and chemokines such as MCP-1 (Cashman et al. 1998) and SDF-1 (Cashman et al. 2002). In immunodeficient non-obese diabetic-scid (NOD/SCID) mice engrafted with human hematopoietic cells, the function of these inhibitory cytokines in vivo was also examined. TGF-P, MCP-1, M l P - l a or SDF-1 when injected into engrafted mice were able to arrest the cycling of different types of human progenitor cells. However, only the injection of TGF-P and SDF-1 could inhibit the proliferation of human HSCs in this in vivo model (Cashman et al. 1999; Cashman et al. 2002).
1.6.3. Effect of morphogens Recent evidence has suggested that Notch, BMP, Shh and Wnt signaling pathways also play a role in controlling the self-renewal activities of HSCs (Bhatia et al. 1999; VarnumFinney et al. 2000; Bhardwaj et al. 2001; Reya et al. 2003). Characterization of the roles of these factors in non-hematopoietic systems have indicated that considerable cross talk can exist between these signaling pathways (Polakis 2000) and the Wnt signaling pathway has been shown to synergize with Notch, BMP, and Shh functions (Kuhl et al. 2000). The Wnt proteins belong to a major family of developmentally important signaling molecules, first identified for their instructive roles during embryonic development in Drosophila
melanogaster (Sharma 1973).
The Wnt gene family encodes
secreted
glycoproteins that act as paracrine or autocrine factors and are highly conserved in vertebrates. Members of the Wnt family confer distinct cellular functions depending on the nature of the target cell and expression of the corresponding Frizzled (Frz) receptors (Huelsken and Birchmeier 2001). Evidence for a role of Wnt signaling in regulating mesodermal cell fate, from which hematopoiesis is initiated (Mead and Zon 1998), combined with the expression of Wnt in primitive hematopoietic cells (Van Den Berg et al. 1998), has suggested a potential role for Wnt in the regulation of primitive hematopoietic cells.
Moreover, hematopoietic
progenitors from mouse and humans have shown increased self-renewal activities in response to conditioned media from Wnt-expressing cells or purified Wnt protein (Austin et al. 1997; Van Den Berg et al. 1998; Murdoch et al. 2003; Willert et al. 2003) A very recent study has further shown that increased HSC self-renewal could be achieved by modulating p-catenin, a major component of the Wnt signaling pathway (Reya et 22
al. 2003).
In this study, the investigators found that over-expression of P-catenin in HSCs
resulted in increased proliferation of purified HSCs candidates while significantly inhibiting their differentiation in vitro for a period of several weeks. They also obtained evidence that Wnt signaling may be required for the growth response of normal HSCs to other cytokines in vitro and may be important to HSC expansion in transplanted mice. Finally, they have shown that both HoxB4 and Notchl are upregulated in response to Wnt signaling in HSCs.
This
raises the possibility that the effects of Wnt signaling on HSCs are mediated through HoxB4 and/or Notchl.
1.7.
Sub-class III tyrosine kinase receptors and their corresponding ligands The sub-class III receptor tyrosine kinase (RTK) family includes c-fms, c-kit,flt-3and
the PDGF A and B receptors (Gronwald et al. 1988; Matsui et al. 1989).
Homologies in
receptor chain composition, structure, and sub-molecular motif characteristic of this R T K family have been recognized (Figure 1.2).
Moreover, the genes that encode this family
demonstrate overall conservation in exon size, number, sequence, and intro/exon boundaries, suggesting that they arose from duplications of an ancestral gene (Agnes et al. 1994). Although most of these family members and their corresponding ligands are known to have important roles in hematopoiesis, as mentioned above, the c-kit and flt-3 ligand-receptor pairs appear to have unique activities in more primitive hematopoietic cells.
23
Receptor tyrosine kinase Ig domians TM JM TK1 IK
SP
i-
i
/
Size (amino acid number)
-540
-20
-100
TK2
-170
Ligand of RTK SP
Size (amino acid number)
H1-4
-190
TM CT
-27
Figure 1.2. The general molecular structure of the subclass III receptor T K family and their corresponding ligands The approximate size of Ig, transmembrane (TM) and TK1 and 2 domains of a typical subclass III RTK family member are indicated (upper panel). The approximate size of the extracellular and T M domains of the type 1 transmembrane isoforms of the RTK ligand are also shown (lower panel). The cleavage site for the formation of soluble ligand isoforms is in the tether region, represented by the arrow. SP, signal peptide (removed in the post-translational process); JM, juxtamembrane domain; IK, interkinase domain; CT, carboxy-terminal domain; H 1-4, helix region 1 to 4 (adapted from Lyman and Jacobsen 1998).
24
1.7.1. c-kit and SF 1.7.1.1. Molecular structure Initial knowledge of the physiological function of c-kit was developed from analyses of mice with naturally occurring mutations at the white-spotting (W) locus.
Such mice exhibit
defects in the development of multiple tissues including hematopoietic cells as well as melanocytes (Paulson and Bernstein 1995).
Reciprocal B M transplantation experiments
demonstrated that wild-type B M cells rescue the hematopoietic defect in W mutants, even without irradiation of the recipient. However, B M cells from mice with more pronounced W mutations do not generate macroscopic spleen colonies or rescue irradiated recipients (Bernstein 1959; McCulloch 1964). These findings suggested that a gene at the W locus was essential for HSC function. Later it was shown that the relevant gene at the W locus is c-kit (Chabot et al. 1988; Geissler et al. 1988), a gene first identified as a component of an oncogenic retrovirus (Chabot et al. 1988) and subsequently identified structurally as belonging to the R T K family of growth factor receptors. Mice with mutations at the Steel (SJ) locus have a phenotype virtually identical to W mutant mice (severe anemia, pigmentation defect, and sterility) (Russell 1979). However, in the case of SI mutant mice, these defects were found to be due to alterations in the environment in which the affected cells develop, rather than in the affected cells, themselves. Thus, B M cells from SI mutant mice can repopulate lethally irradiated wild-type or W animals, but the macrocytic anemia in SI mutant mice is not cured by the transplantation of wild-type B M cells (McCulloch et al. 1965). Later it was shown that the SI gene encodes a growth factor (SF) that is the ligand for c-kit (Copeland et al. 1990; Nocka et al. 1990; Williams et al. 1990; Zsebo et al. 1990). The murine and human c-kit receptors are both 976 amino acids in length, have 9 potential sites for N-linked glycosylation in their extracellular domains (Yarden et al. 1987; Qiu et al. 1988), and are glycosylated at one or more of these sites (Majumder et al. 1988). The c-kit receptor contains 5 immunoglobulin (Ig)-like domains in the extracellular region that are involved in ligand binding and receptor dimerization. The cytoplasmic region contains a split T K domain containing an ATP-binding region and a phosphotransferase domain (Figure 1.2).
The predicted size of the protein backbone alone is approximately 108 kD. However,
25
immunoprecipitation shows 2 proteins of approximately 140 kD and 155 kD, (Yarden et al. 1987), presumably due to glycosylation differences. The murine and human SF proteins are each 273 amino acids in length, with a 25 amino acid leader, a 185 amino acid extracellular domain, a 27 amino acid transmembrane domain, and a 36 amino acid cytoplasmic tail (Figure 1.2). The primary translation product of the SF gene is a type 1 transmembrane protein, i.e., the N-terminus of the protein is located outside of the cell. There are 4 cysteine residues that are conserved between SF and F L (see also section 1.7.2.1). These cysteine residues form 2 intramolecular disulfide bonds that establish the 3 dimensional structure of the protein (Lu et al. 1991).
Although SF forms homodimers in
solution, they are not covalently linked (Arakawa et al. 1991). The mature mouse and human SF proteins undergo proteolytic cleavage to generate a soluble, biologically active, 164-165 amino acid protein (Anderson et al. 1990; Huang et al. 1990; Martin et al. 1990; Zsebo et al. 1990) with a primary cleavage site encoded within exon 6 (Martin et al. 1990) and a secondary cleavage site within exon 7 that could be targeted if the primary site were absent due to alternative splicing of the transcript (Anderson et al. 1990; Flanagan et al. 1991; Toksoz et al. 1992). Both high (kd, 16 to 310 pmol/L) and low (kd, 11 to 65 nmol/L) affinity binding of SF to its receptor have been reported (Broudy et al. 1992; Turner et al. 1992; Broudy et al. 1994). Some primary cells and cell lines have only high affinity sites, whereas others have both (Broudy et al. 1992; Turner et al. 1992). However, another study suggested that neither the number of c-kit receptors per cell nor the types of receptors present correlated with the ability of cells to proliferate in response to SF (Broudy et al. 1994).
1.7.1.2. Expression of c-kit in primitive hematopoietic cells c-kit is expressed on cells of a number of lineages and from a number of tissues. During embryogenesis, c-kit is first expressed in the ectoderm (Orr-Urtreger et al. 1990; Motro et al. 1991). By Embryonic day 8.5 (E8.5), c-kit is expressed in the yolk-sac blood islands. Later in development, it is expressed by the hematopoietic cells that appear in the A G M , and then c-kit cells parallel the subsequent progression of hematopoiesis at different sites - i.e., +
first in the fetal liver, then in the spleen, and finally the B M (Orr-Urtreger et al. 1990; Motro et al. 1991).
The close correlation between c-kit expression, de novo development of
hematopoietic cells, and their presumed migration to new sites of hematopoiesis support the 26
belief that c-kit activation is involved in the survival and/or migration of hematopoietic cells during early fetal development. In the adult B M , c-kit is expressed by approximately 8% of hematopoietic cells (Ogawa et al. 1991).
Half of these c-kit murine B M cells co-express lineage-specific cell surface +
antigens such as Gr-1 and Mac-1 (lin ) that are characteristic of maturing myeloid cells. The +
other half of c-kit B M cells are lin". These cells express higher levels of c-kit suggesting that +
terminal myeloid differentiation is accompanied by a down-regulation of c-kit expression (Ogawa et al. 1991). Many studies have suggested that most, if not all, HSCs (purified by various methods from B M , fetal liver, or the AGM) are in this c-kit lin~ population (Okada et +
al. 1991; fkuta and Weissman 1992; Orlic et al. 1993; Okuda et al. 1996; Osawa et al. 1996a; Sanchez etal. 1996).
1.7.2. Flt-3 and FL 1.7.2.1. Molecular structure The flt-3 gene was cloned independently by 2 different groups. One group exploited the sequence homology offlt-3 to other members of the R T K gene family (Rosnet et al. 1991). A second group used degenerate oligonucleotides (based on conserved regions within the kinase domain of RTKs) in a PCR-based strategy to isolate a novel receptor fragment from highly purified murine fetal liver stem cells (Matthews et al. 1991). The gene encoding the ligand for flt-3 (FL) was also cloned by different groups using different strategies.
One
screened a T-cell derived cDNA library to isolate a cell-surface protein that would bind to the soluble form of flt-3 (Lyman et al. 1993a).
The other used the purified protein to design
degenerate oligonucleotide primers to amplify a portion of the F L gene by PCR, which was then used to isolate a bacterial clone containing a full-length murine cDNA (Hannum et al. 1994). Once the murine F L cDNA had been isolated, it was used to isolate a human F L cDNA (Lyman etal. 1994). Murine and human flt-3 receptors contain 1000 and 993 amino acids, respectively, and have 9 and 10 potential extracellular N-linked glycosylation sites, respectively (Lyman et al. 1993b). Immunoprecipitation has shown 2 proteins of 130-143 kD and 155-160 kD (Lyman et al. 1993b; Rosnet et al. 1996).
The predicted size of the protein backbone alone is
approximately 110 kD. Pulse-chase analysis demonstrated that the larger protein arises from the smaller protein (likely as a result of glycosylation changes). 27
Only the larger protein is
found on the cell surface (Lyman et al. 1993b). One isoform of the murine flt-3 receptor is missing the fifth of the 5 Ig-like regions in the extracellular domain as a result of the skipping of 2 exons during transcription (Lavagna et al. 1995). The physiologic significance of this flt-3 receptor isoform is unknown. The primary translation product of the F L gene is a type 1 transmembrane protein (Figure 1.2).
The mouse and human proteins contain 231 and 235 amino acids, respectively.
The mouse and human F L proteins are 72% identical at the amino acid level, and homology is greater in the extracellular region (73%) than in the cytoplasmic domain (57%).
Like SF,
preliminary data suggest that F L also exists as a noncovalently linked homodimer, which contains 3 intramolecular disulfide bonds (Lyman and Jacobsen 1998). Multiple isoforms of both mouse and human F L have been identified. The predominant isoform of human F L is the transmembrane protein that is biologically active on the cell surface (Lyman et al. 1993a; Hannum et al. 1994; Lyman et al. 1994). This isoform is also found in the mouse, although it is not the most abundant isoform (Lyman and Jacobsen 1998). These transmembrane F L proteins can be proteolytically cleaved to generate a soluble form of the protein that is also biologically active (Lyman et al. 1993a). The most abundant isoform of murine F L is a 220 amino acid membrane-bound (Lyman and Jacobsen 1998). Soluble F L can also be synthesized from a relatively rare isoform in both mouse and human cells by alternative splicing of exon 6, which introduces a stop codon at the end of the extracellular domain (Lyman and Jacobsen 1998). The binding affinity of human F L for the flt-3 receptor on human myeloid leukemia cells has been estimated to be 200 to 500 pmol/L (Turner et al. 1996) and only high-affinity binding is seen. There is no evidence that SF or F L bind to any other protein other than the ckit and flt-3 receptors, respectively.
Similarly, no other ligands that bind the flt-3 and c-kit
receptors are known (Lyman and Jacobsen 1998). 1.7.2.2. Expression of flt-3 in primitive hematopoietic cells The flt-3 gene is expressed in the placenta beginning at El3.5, and increases until birth (Rosnet et al. 1991). The flt-3 gene is also expressed in sites of hematopoiesis in the fetal liver. In the thymus, flt-3 begins to be expressed in the fetal thymus on El6.5. F L is also expressed in the yolk sac, fetal liver, and placenta (Hannum et al. 1994; Lyman et al. 1994). In the adult,
28
flt-3 and F L are co-expressed in spleen, B M , thymus, ovary, testis, liver, kidney, and intestine (Lyman and Jacobsen 1998). Flt-3 expression on hematopoietic cells appears restricted predominantly to primitive cells. Consistent with the cloning of flt-3 from fetal liver-enriched progenitors, 96% of those fetal liver cells and 88% of lin"Sca kit B M cells express flt-3 (Rasko et al. 1995). These cells +
+
contain distinct flt-3 and flt-3" subpopulations and the long-term repopulating activity appears +
to be predominantly but not exclusively found in the flt-3" fraction. A greater proportion of flt3
+
HSCs are in active cell cycle as compared to flt-3" HSCs and growth factor treatment
upregulates flt-3 expression in HSC-enriched populations (Zeigler et al. 1994). Therefore it is possible that originally flt-3" HSCs upregulate flt-3 in response to other growth factors, and acquire F L responsiveness in this fashion before their functions as HSCs are altered.
1.7.3. Roles of c-kit and flt-3 in primitive hematopoietic cell 1.7.3.1. In vitro studies Investigation of the effects of incubating primitive hematopoietic cells in vitro to different relative and absolute concentrations of SF and F L has helped to delineate the different roles of c-kit and flt-3 in normal hematopoiesis. Several studies involving single-cell cloning and delayed addition of cytokines also demonstrated that SF and F L act directly on primitive hematopoietic cells, and not through secondary effects mediated by other cells (Lyman and Jacobsen 1998). In the absence of other cytokines, both SF (Bodine et al. 1992; Katayama et al. 1993; L i and Johnson 1994; Keller et al. 1995) and F L (Muench et al. 1995; Rasko et al. 1995; Takahira et al. 1996; Veiby et al. 1996) appear able to selectively promote the viability rather than the proliferation of primitive hematopoietic cells. Soluble SF can also promote the adhesion of hematopoietic cells to extracellular matrix proteins in vitro, such as fibronectin, (Levesque et al. 1996) or to adhesion molecules expressed by cells in the microenvironment, such as V C A M - 1 (Kovach et al. 1995), which may then have a later effect on H S C selfrenewal (see also section 1.6.1.2). SF appears to be more efficient than F L at recruiting murine HSCs into the cell cycle, independent of which other cytokine is used as the synergistic factor (Lyman et al. 1993a; Hannum et al. 1994; Zeigler et al. 1994; Broxmeyer et al. 1995; Hudak et al. 1995; Jacobsen et al. 1995; Rasko et al. 1995; Ramsfjell et al. 1996; Ramsfjell et al. 1997; Audet et al. 2002). In contrast, several studies indicate that F L is more efficient than SF (or at least as efficient as SF) 29
in stimulating primitive human cells to proliferate (Gabbianelli et al. 1995; Petzer et al. 1996b; Rusten et al. 1996; Shah et al. 1996; Dao et al. 1997; Zandstra et al. 1997b). Studies of relative cytokine dose-responses and interaction parameters have indicated a possible quantitative way of how c-kit and flt-3 regulate different populations in the hematopoietic hierarchy (HSCs, CFCs and total cells) (Zandstra et al. 1997b; Audet et al. 2002).
These studies have
demonstrated that it is possible to enhance HSC amplification by increasing the extracellular concentration of cytokines.
Notably this required at least 10-fold higher cytokine
concentrations than were necessary to stimulate maximal expansion of CFCs or total cells from a common primitive starting population in the same cultures (Zandstra et al. 1997b; Audet et al. 2002). Moreover, from the study of these effects at the single cell level, it was possible to demonstrate that the effects on HSC expansion could not be explained by changes in H S C survival. (Figure 1.3).
This dose-dependent change in primitive cell responses suggests that
different response outcomes can be regulated by different intensities of activated intracellular signaling mechanisms (Zandstra et al. 2000).
An example of a similar threshold model is
found in T and B cell signaling, where a sufficient number of receptors must be triggered to yield a full cellular response (Valitutti et al. 1995; Chidgey and Boyd 1997; Valitutti and Lanzavecchia 1997; Bachmann et al. 1999; Benschop et al. 1999). According to such a model, when a relevant ligand-receptor interaction is kept above a required threshold level in HSCs, differentiation would continues to be suppressed, thus permitting a self-renewal division to occur. a A more detailed, large scale statistical analysis of the effects on murine HSCs of IL11, SF, and F L concentrations and their interactions showed that IL-11 has a maximal stimulatory effect on HSC expansion at 20 ng/mL with higher concentrations being inhibitory (Audet et al. 2002). In contrast, no saturation or decrease in HSC expansion was observed as the SF and F L concentrations were increased slightly beyond 300 ng/mL, suggesting that HSCs might be further expanded with even higher SF or F L concentrations. However, an unexpected negative interaction between SF and F L on HSCs was also observed that caused a significant decrease in H S C expansion when SF and F L were combined at high concentrations. reason for this negative interaction on HSC expansion is unclear.
The
However, it has been
speculated that, as the concentration of F L and SF increases, heterodimers of SF and F L form in solution and these in turn cause the formation of receptor heterodimers (Audet et al. 2002). It has been reported that formation of heterodimers between different members of the 30
epidermal growth factor receptor (EGFR) family is possible (Hackel et al. 1999) and crossactivations of c-kit and flt-3 have also been reported (Otto et al. 2001).
31
High cytokine Self-renewal
/\
/ \ / \ /\
Low cytokine Differentiation
/\
/ \ / \ /\
Figure 1. 3. Clone formation and progenitor expansion from single cell cultures of CD34 CD38 adult human BM cells +
In the study by Zandstra et. al., human B M HSC candidates (CD34 CD38") cells were isolated and cultured as single cells in serum-free medium containing either high (300 ng/mL SF, F L , and 60 ng/mL IL-3) or low (30 ng/mL SF, F L , and 6 ng/mL IL-3 ) cytokine concentrations for 10 days. Clone formation and C F C and LTC-IC expansion were determined at the end of a 10-day culture period. The frequency of input cells that proliferated was the same for both cytokine cocktails but the expansion of primitive cells (gray circles) was higher in the cocktail containing the higher concentrations of cytokines. These results suggest that self-renewal versus differentiation decisions can be modulated by the cytokine signaling intensity induced by the activated cytokine/receptor complex (Figure adapted from Zandstra et. al. 1997). +
32
1.7.3.2. In vivo studies As noted above, W and SI mutant mice exhibit defects in hematopoiesis.
Despite the
severe macrocytic anemia and the resulting embryonic lethality associated with some alleles of W and SI, some mutants are viable. In these, disruption of hematopoiesis appears restricted to erythropoiesis and mast cell generation. Specifically, in the B M of W41 mutant mice that have a partial c-kit signaling deficiency, the numbers of myeloid, pre-B, erythroid, multipotent progenitor cells, lin"Sca-l candidate stem cells and LTC-ICs are at near-normal levels (Miller +
et al. 1996). However, long-term repopulating HSC numbers are reduced about 20-fold (Miller et al. 1996). W41 fetal liver cells are qualitatively and quantitatively similar to normal mice in their short-term reconstituting ability but have less competitive long-term reconstituting ability than normal fetal liver HSCs (Miller et al. 1997). Sl/Sl mice lack functional SF and die at day 15 or 16 of gestation (Lyman and Jacobsen 1998). However, the number of cells in the HSC candidate populations (lin Sca-l Thy-l ) +
+
l0
and also CFU-S numbers in these mice increase normally in Sl/Sl mice from day 13 to 15 (Ikuta and Weissman 1992). Nevertheless, the enhanced production of SF seen in adults given myeloablative treatments (Hunt et al. 1992; Yan et al. 1994) and the ability of endogenously produced or exogenously administered SF to promote the survival of and recovery of hematopoiesis in myeloablated mice (Zsebo et al. 1992; Neta et al. 1993; Patchen et al. 1994; Yan et al. 1994) is consistent with the view that SF plays a role in promoting H S C reconstitution. Taken together, these observations suggest that SF might not be essential for HSC development in the mouse embryo but that, under certain circumstances, adult murine HSCs self-renewal might be SF-dependent. Whether flt-3 or F L is required for normal hematopoiesis has also been addressed by creating mice that carry a homozygous deletion of most of the gene encoding flt-3 (Mackarehtschian et al. 1995) or F L (McKenna et al. 2000). flt-3 null mice have normal levels of peripheral blood cells and are generally healthy and fertile, in contrast to the lethality observed in mice homozygous for the deletion of the genes encoding the c-kit receptor or its ligand (Bernstein et al. 1991). However, the loss of functional flt-3 receptors does result in a reduced number of early B-cell precursors and a defect in HSCs, as measured in a long-term competitive repopulation assay. This defect was demonstrable as a reduced ability of primitive cells from flt-3 null mice to compete with their wild-type counterparts in irradiated recipient 33
reconstitution assays (Mackarehtschian et al. 1995). flt-3 null mice crossed with mice carrying mutations in the c-kit receptor generated W/W-flt-3 null mice that died between 20 and 50 days after birth with more severely reduced numbers of hematopoietic cells than either of the parental strains (Mackarehtschian et al. 1995). These findings demonstrated that both flt-3 and c-kit receptors have distinct roles in ensuring the generation of a full complement of HSCs in the adult. F L null mice, like flt-3 receptor null mice, have a normal, healthy appearance, but also have reduced numbers in myeloid and B-lymphoid progenitor cells, dendritic cells, and natural killer cells (McKenna et al. 2000).
1.7.4. Involvement of c-kit and flt-3 in hematopoietic diseases c-kit is commonly expressed on human acute myelogenous leukemia (AML) blasts and SF is co-expressed with c-kit in 30% of such cases (Wang et al. 1989; Ikeda et al. 1991; Broudy et al. 1992).
c-kit receptor levels on human A M L blast cells are variable but, in
general, are similar to, or less than, c-kit levels on normal HSCs and progenitor cells (Cole et al. 1996). The c-kit receptor is also expressed on the blasts in a majority of samples from chronic myelogenous leukemia (CML) patients in blast crisis (Buhring et al. 1991). Flt-3 receptors are seen even more frequently on human A M L blasts than c-kit. Flt-3 is expressed in 93% of patients with A M L and approximately 20% of patients with A M L have an internal tandem duplication of the juxtamembrane domain of Flt-3 (Nakao et al. 1996; Kiyoi et al. 1999) that correlates with a poor prognosis.
Flt-3 receptors are present on the blasts of
100% of patients with B cell acute lymphoblastic leukemia (ALL) (McKenna et al. 1996) and 75% of patients with T cell A L L (Birg et al. 1992; DaSilva et al. 1994). Flt-3 cells were +
detected in 29% of C M L patients with accelerated phase disease and in 75% of C M L patients in blast crisis (Birg et al. 1992).
1.8.
Genetic manipulation of hematopoietic cells using recombinant retroviruses Until recently the most commonly used vehicles for exogenous gene transfer to primary
hematopoietic cells have been replication-incompetent recombinant retroviral vectors (Kohn 1997). The advantage of these vectors is their ability to transduce a variety of cell types, as well as to integrate stably into the genome of the transduced cells in the form of a D N A provirus. The use of such vectors has provided a powerful approach to track the proliferation and differentiation of individual HSC clones (Jordan and Lemischka 1990; Keller and Snodgrass 1990; Pawliuk et al. 1996) and to genetically manipulate HSCs and their progeny. 34
1.8.1. Retrovirus biology All retroviruses are basically similar in virion structure, genomic organization and mode of replication (Coffin 1996).
One of the most intensively studied retroviruses is the
Moloney murine leukemia virus (MMuLV) (Varmus 1988). The M M u L V virion is about 100 nm in diameter, and is enveloped by a glycoprotein-containing lipid bilayer.
The virion
contains a R N A genome that is reverse-transcribed into D N A upon entry of the virus into the host cell. The viral cDNA is then integrated into the host cell genome. The structure of an integrated wild type M M u L V viral (or proviral) D N A includes a transcriptional control sequence, a polyadenylation signal, and sequences required for integration in the long terminal repeat (LTR) regions at the 5' and 3' ends of the provirus.
The sequence required for
packaging the viral genome into a virus particle is designated psi (cp), and is located downstream of the 5' LTR. The proteins required for replication and packaging the retrovirus are encoded in 3 distinct open reading frames (ORFs) of the provirus; namely, the gag (group specific antigens), pol (polymerase/reverse transcriptase/integrase), and env (envelope), with gag-pol-env as the 5' to 3' order of these genes. The gag gene encodes a polypeptide that is cleaved into at least 3 proteins designated as the matrix, capsid and nucleocapsid protein. The pol gene encodes 2 proteins, reverse transcriptase, an RNA-dependent D N A polymerase, and the integrase protein necessary for integration of the viral cDNA into the D N A of the host cell. The env gene encodes the glycoprotein that surrounds the virion, and is a heterodimeric complex of both transmembrane and surface domains.
The larger surface domain is
responsible for binding to the cell surface receptor. The transmembrane domain anchors the complex to the virion envelope and contains domains responsible for the fusion of viral and cellular membranes (Varmus 1988). The retroviral replication cycle can be divided into 2 phases. The first phase includes binding of the virus particle to a specific receptor on the cell surface, entry of the virion core into the host cell and integration of the viral genetic material into the host genome. The second phase consists of synthesis and processing the viral mRNAs and protein from the provirus, assembly of the virion and finally release (non-lytic budding) of replication-competent mature virions (Varmus 1988).
35
1.8.2. Production of recombinant retroviruses To generate a recombinant replication-incompetent retrovirus, the viral structural genes (gag, pol, env) are replaced with a marker gene or the gene of interest and the viral structural genes required for virus production are provided in trans by retroviral packaging cell lines. Many of these cell lines have been derived from fibroblasts that have been engineered to produce retroviral structural proteins through the introduction of gag, pol and env genes by stable transfection, but are unable to package viral R N A because the cp sequence is missing. Thus when a retroviral vector plasmid is introduced into the packaging cells, the retroviral R N A subsequently produced can combine with the retroviral proteins produced by the packaging cell line to produce infectious recombinant viral particles.
These are capable of
transducing competent target cells but are incapable of directing further virus production (Varmus 1988). Recently, highly transferable packaging cell lines have been produced (Pear et al. 1993) and co-transfection of these cells with a gag/pol plasmid and env plasmid as well as the vector plasmid allows the generation of higher titers of recombinant retrovirus than were previously possible.
Recombinant virus production using these cells is transient, with virus
production peaking 48 to 72 hours after co-transfection of the various plasmids (Pear et al. 1993). These transient virus production methods are attractive because conventional stable retrovirus-producingfibroblastsroutinely require >4 weeks to generate and even longer periods to allow the isolation and expansion of selected stable, high titer clones. The titers obtained by transient transfection can also be similar to those produced by selected stable producer cell lines. In practice, replication competent-free virus titers rarely exceed 10 particles/mL.
1.8.3. Recombinant retroviral vectors Several factors can influence the performance of any particular retroviral construct including the regulatory elements used to drive expression, the number and size of transcriptional units, the viral backbone used, the direction of transcription, and the presence or absence of selectable markers.
Viral LTRs have consistently been shown to be strong
promoters, resulting in higher levels of gene expression as compared to a variety of internal promoters of viral or cellular origin (Correll et al. 1994). However, viral LTRs in the M M u L V are also highly susceptible to transcriptional silencing in a variety of primitive cell types including embryonic carcinoma (EC) cells (Kempler et al. 1993), embryonic stem (ES) cells 36
(Seliger et al. 1986) and various "primitive" hematopoietic cell lines (Baum et al. 1995). Several viral mutants that are able to express transferred genes at high levels in E C and ES cells have been isolated (Colicelli and Goff 1987; Hilberg et al. 1987). By combining the LTR, the 5' untranslated regions, and a number of convenient cloning sites, Hawley et al (Hawley et al. 1994) have produced a series of Murine Stem Cell Virus (MSCV) vectors that are relatively resistant to silencing. Further data have suggested that the provirus from the M S C V vector is able to transcribe transduced genes efficiently in HSCs and their progeny for extended periods of time in vivo (Sorrentino et al. 1995; Pawliuk et al. 1997).
1.8.4. Murine HSCs as target for retro viral-mediated gene transfer Murine B M marking and transplantation studies have shown that HSCs can be transduced without compromising their ability to repopulate the lymphoid and myeloid lineages (Dick et al. 1985; Lemischka et al. 1986; Jordan and Lemischka 1990).
The
efficiency of transduction of HSCs depends on a number of parameters including whether or not they are cycling and whether or not they have upregulated expression of the receptor for the retrovirus at the time of exposure. In addition, studies of more mature progenitors suggest that growth factors activate other, as yet uncharacterized mechanisms that enhance retroviral transduction (Hogge and Humphries 1987). Because the vast majority of HSCs in the normal adult are quiescent (see section 1.4.3.), a number of strategies are used to stimulate them to start to proliferate prior to exposure to retroviruses. One is to pretreat mice by injecting them with 5-FU to selectively kill more mature hematopoietic progenitor cells, that then causes a large proportion of HSCs to be induced into cycle (Harrison and Lerner 1991).
Additional stimulation of the cells with
cytokine cocktails including various combinations of IL-3, IL-6, SF, FL, IL-1 and LIF have been found to further enhance retrovirus-mediated gene transfer into murine, human and nonhuman primate HSCs (Bodine et al. 1989; Luskey et al. 1992; Einerhand et al. 1993; Peters et al. 1996; Conneally et al. 1997).
Therefore, most protocols include such a 48-hour
"prestimulation" of the target cells prior to exposure to retrovirus (Bodine et al. 1989). During transduction, another important parameter is the number of virus particles to which each target cell is exposed.
Since standard retroviruses are not stable, they cannot be
concentrated and additional strategies to increase their titers or effective titers have proven helpful. One of these is the use of fibronectin to concentrate virus particles on a molecular 37
surface that co-localizes to primitive hematopoietic cells (Moritz et al. 1994). Both of these activities of fibronectin were subsequently found to be attributable to a single 30/35 kD fragment of the fibronectin molecule (Hanenberg et al. 1996). Another consideration is the duration of the period of viral exposure. It has been shown that the absolute number of HSCs decreases dramatically during the transduction period (particularly between day 2 and day 4 of the transduction procedure) (Antonchuk et al. 2002). Therefore, although longer exposure of the target cells to virus may increase HSC transduction efficiency, the absolute number of transduced H S C that can ultimately be recovered may be less than the number present after a shorter 2-day transduction procedure. The retroviral receptor is the primary determinant of the type of cells that can be transduced by a given virus. Many of these receptors have multiple transmembrane domains and
function as transporter molecules.
These include the receptor for ectropic murine
retrovirus (MCAT) (Kim et al. 1991; Wang et al. 1991), amphotropic murine retrovirus (Ram1) (Miller et al. 1994) and the common receptor for gibbon ape leukemia virus (Kavanaugh et al.
1994).
M C A T and Ram-1 receptors are transport proteins that perform essential
housekeeping functions. M C A T serves as a cationic amino acid transporter (Wang et al. 1991) and Ram-1 is a sodium-dependent phosphate symporter (Kavanaugh et al. 1994). Transduction of murine HSCs using ecotropic retrovirus has been found to be more efficient than using amphotropic virus, presumably due to the lower levels of expression of Ram-1 on primitive murine hematopoietic cells as compared to human hematopoietic cells (Kavanaugh et al. 1994).
1.9.
Present studies and thesis objective B M transplantation studies have demonstrated that HSCs numbers can expand by
>8000 fold during the course of several serial B M transplants(Iscove and Nawa 1997). However, to date, no studies have been able to demonstrate >5-fold reproducible H S C expansion in vitro (Miller and Eaves 1997). Recent in vitro studies have now shown that HSC expansion is critically influenced by both the types and concentrations of the cytokines used in the culture (Zandstra et al. 1997b; Audet et al. 2002). In particular, these studies demonstrated that an elevated level of F L or SF in the cultures would preferentially increase the expansion of the more primitive cell populations, but without significant increase in the growth of the more mature progenitor populations.
A model of HSC self-renewal control has been proposed, 38
suggesting that the probability o f a self-renewal response o f H S C s is increased when the relevant ligand-receptor interaction is maintained above a threshold level (Zandstra et al.
2000). According to this model, the control o f H S C self-renewal is dependent o n at least 2 variables- the ligand concentration and the accessibility o f the corresponding receptors. In this thesis, I have focused on testing the second variable.
M y experimental approach was to
overexpress flt-3 or c-kit receptors by retroviral transduction o f H S C s and to then evaluate the effect o f these manipulations on the sensitivity o f H S C self renewal responses to F L and SF. The prediction was that an increased number o f receptors would sensitize the H S C s to attain a greater self-renewal response i n the presence o f a lower concentration o f ligand. M y specific objectives were therefore as follows: 1)
T o construct and validate retroviral vectors encoding functional c-kit or flt-3
cDNAs. 2)
T o compare the ligand-stimulated responses o f cloned lines o f c-kit or flt-3
transduced factor-dependent cells expressing different levels o f flt-3 or c-kit receptors. 3)
T o study the effect o f flt-3 or c-kit overexpression on H S C self-renewal and
differentiation in vitro. 4)
T o study the effect o f flt-3 and c-kit overexpression on H S C function in vivo
39
CHAPTER 2: MATERIALS AND METHODS 2.1. Reagents 2.1.1. Cell lines All cell lines were grown from periodically checked stocks that had been shown to be mycoplasma-free.
They were maintained at 37°C with 5% CO2 in medium containing 100
U/mL penicillin and 100 U/mL streptomycin obtained from StemCell Technologies Inc. (Vancouver, BC), unless specified otherwise. Phoenix-Eco cells are modified 293T cells, originally derived from human embryonic kidney tissue. They contain the SV40T antigen gene and the retroviral ecotropic env and gagpol genes to allow packaging of mRNA containing the retroviral packaging (\|/) sequence into ecotropic retrovirus (see section 1.8.1). Phoenix-Eco cells were obtained from Dr. G.P. Nolan (Stanford University, Palo Alto, CA) and were maintained in a growth medium consisting of Dulbecco's modified Eagle medium (DMEM), 2 m M L-glutamine and 10% fetal calf serum (FCS) that had been heat-inactivated (by incubation at 56 °C for 30 minutes). Cells were kept in continuous log phase growth by maintenance between 20% and 80% confluence. . NIH-3T3 murine fibroblasts were purchased from the American Type Tissue Collection (ATCC, Rockville, MD) and maintained in D M E M plus 2 m M L-glutamine and 10% bovine calf serum BaF3 cells are a murine IL-3-dependent murine cell line with features of pro-B cells (Perkins et al. 1996).
They were obtained from Dr. G. Krystal (Terry Fox Laboratory,
Vancouver, BC) and maintained in suspension cultures in RPMI 1640 plus 10% heatinactivated FCS, 10 M 2-mercaptoethanol (2-ME) and 10 ng/mL murine IL-3. 4
2.1.2. Cytokines Most of the cytokines used were purified recombinant murine (rm) or human (rh) proteins prepared in the Terry Fox Laboratory by transient expression of the appropriate cDNA in COS cells (rmIL-3 and rmSF). Rh F L was obtained from Immunex (Seattle, WA), rh IL-11 from Genetics Institute (Cambridge, M A ) , rh IL-6 from Cangene (Mississauga, ON), rh thrombopoietin (TPO) from Genenetech (San Francisco, CA) and rh erythropoietin (EPO) from StemCell Technologies.
40
2.1.3. Mice The
mice used i n these experiments were C 5 7 B L / 6 J (B6) mice and various B 6 -
congenic strains, i.e. Bd-W /]^ 41
41
(W41), and B6-Pep3b (Pep3b). In most experiments, Pep3B
mice were used as marrow donors and either B 6 or W41 mice as recipients, all at 8 to 14 weeks of age. The hematopoietic cells from the Pep3b strain are phenotypically distinguishable from those from B 6 or W41 mice on the basis o f allelic differences at the L y 5 (CD45) locus. Pep3b are homozygous Ly-5.1, whereas B 6 and W41 mice are homozygous for the Ly-5.2 allotype. A l l mice were bred and maintained i n the A n i m a l Facility i n the British Columbia Cancer Research Centre
from
parental strain breeders
Laboratories (Bar Harbor, M E ) .
originally obtained
from
The Jackson
They were housed i n micro-isolator cages with sterile water,
food, and bedding. Irradiated mice also received acidified water (pH 3.0) for 4 months after irradiation.
2.1.4. Isolation of mouse BM cells BM
cells were obtained
from
Pep3b (test) or B 6 (competitor) mice injected
intravenously 4 days previously with 150 mg/kg body weight o f 5 - F U (Faulding, Vaudreuil, PQ) i n phosphate-buffed saline, b y flushing dissected femurs and tibias using 22 and 26 gauge needles, respectively. Harvested B M cells were filtered through a cell strainer (100 u m N y l o n , Falcon, Becton Dickinson Labware, Franklin Lakes, NJ.) to remove clumps and resuspended i n D M E M supplemented with 2 % F C S at 10 to 10 cells/mL. 6
7
This cell suspension was then
placed carefully on top o f a 225 u M Ficoll solution (Type 400, Sigma) containing 257 m M sodium diatrizoate (Sigma) and centrifuged at 500g for 30 minutes at room temperature. The layer o f cells at the interface between the medium and Ficoll were recovered and washed twice. The cells were then finally re-suspended i n Iscove's modified Dulbecco's medium ( I M D M ) with 2 % F C S or other supplement as required for subsequent use i n direct assays or virus transduction studies.
2.2. Retoviral vectors construction and molecular analysis 2.2.1. Vector construction A l l viral vectors used i n this study were constructed from one o f 2 backbone vectors: M S C V - I R E S - G F P ( M I G , Figure 3.1) and a derivative o f this, referred to here as M S C V ( M ) and created b y removal o f the I R E S - G F P cassette as a Xho-Clal fragment from the M I G 41
vector. MIG contains the L T R sequences of the murine stem cell virus (MSCV) (Hawley et al. 1992), an internal ribosomal entry site (IRES) element (derived from the encephalomyocarditis virus) and the cDNA of the enhanced green fluorescent protein (GFP) (Antonchuk et al. 2001). It was obtained from Dr. K. Humphries (Terry Fox Laboratory, Vancouver, BC). The murine c-kit cDNA encompassing the complete coding sequence was isolated as an EcoRl-Hindlll
fragment (total 3.6 Kbp, with 2.9 Kbp coding sequence) from a plasmid
obtained from Dr. P. Besmer (Memorial Sloan-Kettering Cancer Center, New York, NY). It was inserted by cohesive-end ligation into the multiple cloning site upstream of the 3'-LTR of the M vector to create the MSCV-c-kit (M-KIT) vector (Figure 3.1). The murine flt-3 cDNA (total 3.2 Kbp, with 3.0 Kbp coding sequences) was cut into EcoRl-EcoRl and EcoRI-Xhol fragments, respectively, from a plasmid obtained from Dr. D. Birnbaum (INSERM, Marseille, France). The 2 fragments were then re-ligated and inserted simultaneously into the multiple cloning site (cleaved EcoRl and Xhol sites) of the MIG vector to create the MSCV-Flt-3-IRES-GFP (M-FLT-IG) vector.
The IRES-GFP fragment was
subsequently removed to create a M S C V - F L T (M-FLT) vector (Figure 3.1). A chimeric receptor cDNA composed of the M-CSF receptor (c-fms) extracellular domain and the flt-3 receptor intracellular domain (called fms-flt-3 [FF3]) was obtained from Dr. Lemischka (Princeton University, New Jersey). This cDNA was cut into EcoRl-EcoRl and EcoRl-Xhol fragments, respectively, and was re-ligated and inserted simultaneously into the multiple cloning site (cleaved EcoRl and Xhol sites) of the MIG vetor to create the MSCV-ff3IRES-GFP (M-FF3-IG)) vector (Figure 5.6a).
2.2.2. Vector sequence validation Highly purified (by QIAquickTM column, Qiagen, Missisauga, ON) M-KIT, M - F L T IG and M-FF3-IG vectors were diluted to 100 ng/uL in 10 m M Tris (pH=8.5) solution and used as templates for sequencing.
The Sanger method of sequencing, using fluorescence-
labelled dideoxy-dNTP-mediated chain termination was performed by the NAPS Unit at the University of British Columbia (Vancouver, BC). Complete c-kit, flt-3 and FF3 cDNAs were sequenced.
The results obtained for c-kit
and flt-3 were found to match published data (Qiu et al. 1988; Rosnet et al. 1991) and the data in the expressed sequence tag (EST) database in GenBank® (Boguski et al. 1993). However, a 42
single nucleotide sequence difference (C instead of A) was found in the FF3 cDNA at cDNA sequence #1565 (start count from the first A T G site, located in the transmembrane domain), that would result in a change in the encoded amino acid from Gin to His.
2.3.Transduction protocols 2.3.1. Production of retroviral supernatant Log-phase Phoenix-Eco cells were plated at 3.5 x 10 per 100 mm tissue culture dish in 6
12 mL growth medium and 22 hours later, when the cells were about 80% confluent, 6 mL was removed. At the same time, a transfection mixture was prepared by adding 30 p.g of retroviral vector D N A in 0.1X Tris-EDTA solution (TE, pH=8.0) plus 5 p.g of an ecotropic viral env gene-expressing plasmid (Env-1) and 5 p.g of a gag-pol gene-expressing plasmid (GP3, both obtained from Dr. R. Kay, Terry Fox Laboratory) with 250mM CaCh to give a final volume of 500 (J.L. Addition of the latter 2 plasmids increased the viral titer by approximately 5-fold (see section 4.2.1). This solution was then added dropwise (10 drops of ~ 50 uLper drop) to 500 uL of pre-warmed 2x HBS (pH= 7.05, 50 mM HEPES, 10 mM KC1, 12mM dextrose, 280 m M NaCl and 1.5 m M Na2HP04) with slow shaking to produce a fine D N A precipitate. The resultant mixture was then added immediately to the Phoenix-Eco cells.
These were then
incubated at 37 °C for 12 hours at which time the medium was removed and replaced with 12 mL of fresh growth medium. After a further 16 hours of incubation, the medium was again replaced and finally harvested another 20 hours. This virus-containing supernatant was then filtered through a 45 urn low-protein binding filter (Millex, Millipore Co. Bedford, M A ) and then kept on ice for use within a few hours or frozen and stored at -70 °C until required.
2.3.2. Transduction protocols for cell lines NIH-3T3 and BaF3 target cells were harvested and inoculated at 0.7 x 10 and 10 cells 5
5
per 35 mm well, respectively. NIH-3T3 cells were inoculated 24 hours prior to being exposed to virus-containing medium to allow adherence of the cells to the bottom of the well. BaF3 cells were inoculated immediately before being transduced. The target cells were covered by (in the case of NIH-3T3) or suspended in (in the case of BaF3) at least 0.4 mL of different dilutions of virus-containing supernatant to which 5 u.g/mL protamine sulphate was added and the cells incubated with virus for 4 hours. 4 mL of fresh growth medium was then added and the cells incubated for another 44 hours. 43
2.3.3. Transduction protocol for murine BM cells Isolated day 4 5-FU treated mouse B M cells (see section 2.1.4) were first cultured (prestimulated) for 48 hours without virus in Iscove's modified Dulbecco's medium (IMDM) containing 10 mg/mL bovine serum albumin, 10 mg/mL rh insulin, 0.2 mg/mL iron-saturated human transferrin, (BIT, StemCell), 40 p.g/mL low density lipoproteins (LDL, Sigma Chemicals, St. Louis, MO), 10" M 2-ME (Sigma) and the following cytokines: 300 ng/mL rm 4
SF, 1 ng/mL rh F L , and 20 ng/mL rh IL-11 (mHSC cytokine cocktail) to maximize the stimulation of the HSCs with optimal maintenance of their multilineage differentiation potential (Audet et al. 2002). At the end of 48 hours, the cells were then harvested, centrifuged and re-suspended at 2 to 5 x 10 cells per mL in viral supernatant diluted 1:1 (v/v) in D M E M 6
plus 15% heat-inactivated FCS, 5 |j.g/mL protamine sulphate and same growth factors as used in the pre-stimulation medium the HSC cytokine cocktail. The re-suspended cells were then placed in a volume of at least 0.7 mL in a 60 mm petri dish, that had been pre-coated with 3 p.g/cm fibronectin (Sigma) (overnight at 4 °C) and pre-loaded with viral supernatant (for 2 hour at 4 °C just before the transduction procedure). After 4 hours, 4 mL of fresh D M E M plus 15% FCS containing the mHSC cytokine cocktail were added to the culture and incubation continued for another 12 hours. The non-adherent cells in the transduction dishes were then harvested, centrifuged and re-suspended in a freshly prepared mixture of 1:1 diluted viral supernatant in D M E M plus serum and the mHSC cytokine cocktail. The cells were returned to the same dishes again for an another 4 hours of incubation prior to adding a further 4 ml of D M E M plus 15% FCS and the mHSC cytokine cocktail and incubated another 24 hours (Figure 4.2.). At the end of this time all cells were harvested, washed once in Hank's Balanced Salt Solution plus 2% FCS (HF) and re-suspended as required for in vitro and in vivo studies.
2.3.4. Assessment of gene transfer efficiency in bulk cell populations To assess the efficiency of NIH-3T3 and BaF3 cell transduction, these cells were harvested at the end of the transduction protocol and then analyzed by flow cytometry to determine the proportion of GFP or receptor-positive cells, according to the virus used. For primary B M cells, aliquots of cells were diluted to 5 x 10 cells/ml in D M E M plus 15% FCS 4
plus the mHSC cytokine cocktail and cultured for an additional 24 hours prior to being analyzed by flow cytometry for GFP or transduced receptor expression.
44
2.3.5. Viral titer and helper virus assays Viral titers were determined by measuring the highest dilution of viral supernatant that was still able to transfer expression of GFP (MIG-based virus) or c-kit (M-KIT based virus) to at least 5% of murine NIH-3T3 or BaF3 cells as measured by flow cytometry (see below). To test for the presence of helper virus, supernatant from a confluent virus-transduced NIH-3T3 cell culture (that had been incubated for at least 16 hours) was harvested, filtered and transferred to fresh NIH-3T3 cells for another 16 hours.
Flow cytometric analysis was
performed on the indicator NIH-3T3 cells 48 hours later.
Absence of helper virus was
indicated by failure to detect transfer of GFP fluorescence (or c-kit expression) from transduced target cells to the indicator cells (Cone and Mulligan 1984).
2.4 Jn vitro assays 2.4.1. BaF3 cell proliferation assays BaF3 cells were washed, resuspended in fresh growth medium, and aliquoted at 10
4
cells/well (in U-shaped 96-well plates). Various test growth factors were then added to each well such that the final volume was 0.1 mL/well. After 20 (in IL-3 containing medium) or 38 (in SF or F L containing medium) hours of incubation at 37 °C, the cells were pulsed with 1 uCi of H-thymidine (specific activity of 2 Ci/mmol, Mandel NEN) for 4 or 10 hours, 3
respectively. The D N A of the cells was then harvested onto filter mats using a cell harvester (LKB Wallac 1295-001, Turku, Finland), and the quantity of H-thymidine incorporated into 3
D N A measured in a liquid scintillation counter (LKB 1205 Betaplate).
2.4.2. Primary murine BM hematopoietic cell culture Primary B M expansion cultures were initiated with 5 to 10 x 10 transduced B M cells 3
per mL of culture medium (medium volume varying from 3 to 20 mL depending on the type of assay). The cells were cultured for 4 or 7 days at 37 °C in a humidified atmosphere of 5% C 0
2
in air. A l l expansion cultures were performed in D M E M supplemented with 15% FCS plus 20 ng/mL human IL-11 and test cytokines as indicated. At the end of the incubation period, the cells in suspension were removed and adherent cells were detached either by incubation in 0.5 mL typsin-EDTA for 4 minutes at 37 °C, or by using a cell scraper (Falcon), followed by 2 rinses with D M E M containing 15% FCS. The harvested cells were centrifuged at 350 g for 5
45
minutes, and cell counts performed followed by flow cytometry analysis.
C F C and H S C
assays were undertaken using appropriate aliquots.
2.5.Progenitor and stem cell assays 2.5.1. CFC assays Appropriate dilutions of B M cells were suspended in a solution of 1 % methylcellulose in Alpha medium supplemented with 15% FCS, 1% BSA, 10~ M 2-Me, 10 mg/mL rh insulin, 4
200mg/mL human transferrin (iron-saturated), 2mM L-glutamine (MethoCult, StemCell) supplemented with 50 ng/mL rm SF, 10 ng/mL rm IL-3, 10 ng/mL rh IL-6 and 3 units/mL rh EPO and l . l m L aliquots were then plated in 35 mm petri dishes (StemCell Technologies). Cultures were incubated at 37 °C for 10 to 14 days and the numbers and types of colonies present were then scored as granulocyte-macrophage (from CFU-GM), predominantly erythroid (from BFU-E) or obviously mixed (from C F U - G E M M ) according to standard criteria (Humphries etal. 1981).
2.5.2. CRU assay for HSCs 2.5.2.1. Transplantation procedure B6 mice were given a lethal dose of irradiation (900 cGy, 110 cGy/min,
137
C s y-rays)
and W41 mice a sub-lethal dose of 400 cGy prior to being injected intravenously with test cells. In experiments in which B6 mice were injected with limiting numbers of CRUs (HSCs) in the test cell suspension (10-fold range of receptor levels (MFI values) and a narrow distribution of fluorescence values within the clone (Figure 3.4). FACS analysis was again performed on these selected clones after a further 2-week period of culture in IL-3-containing medium and the results confirmed that the level of receptor expression in each of these clones was also stable (see Figure 3.4, open symbols).
55
10000 1000 100 10 1000 100 10
H
y
"7
T°
*
#
0*
H
H MIG
•7
y y
tf
o
>
o
o o v> > v>
Figure 3.4. Mean and range offluorescenceintensity of individual clones of transduced BaF3 cells Each ofthe 8 M-KIT (a) and 8 M-FLT (b)-transduced BaF3 clonal populations selected for study were stained with PE-conjugated anti-receptor Abs 1 week (solid symbols) and 3 weeks (open symbols) after isolation and then analyzed by FACS. Each point shown represents the mean ± 2SD of the MFI. MIG control value [cross in the (b)] is also shown. Squares - subclones from the A polyclonal population; diamonds -subclones from B; triangles - subclones from C; circles - subclones from D.
56
3.2.3. Altered mitogenic responses of M-KIT and M-FLT-transduced BaF3 cells To first test whether the normal IL-3 responsiveness of the M-KIT or M - F L T transduced BaF3 cells remained the same, both polyclonal and monoclonal populations of transduced BaF3 cells expressing different levels of receptors were stimulated with IL-3 and their proliferative responses compared to control (MIG)-transduced BaF3 cells. As shown in Figure 3.5a, the 4 polyclonal M-FLT-transduced populations responded to IL-3 in a manner similar to the MIG controls. The same was true for most of the M - F L T clones, although 2 of these showed an unexplained reduced ability to respond to IL-3. Interesting, this behaviour was even more common and more pronounced in the M-KIT transduced cells (Figure 3.5a & b).
57
75
a) Polyclonal cells
MIG
5(H
4"Jfc*
y
M-FLT
'A
25%J*
ijt^—
*J?i
0
I
•
s
i
S
*
-*1
75
M-KIT
•
.
b) M-KIT clones MIG
*.
50
1.4
Q_ U
25
T • T • T
4
J'i T
T
A
M-KIT
75 c) M-FLT clones
f
50 H
{ T
MIG
I' • /
25 H j
i - ' j / i
.
T
0 -I 0.001
0.01
0.1
1
IL-3 (ng/ml) 58
10
100
Figure 3.5.
Mitogenic responses of M-KIT and
BaF3 populations
to
M-FLT-transduced
IL-3
10 MIG, M-KIT and M-FLT-transduced polyclonal (a) and clonal (b and c) BaF3 populations were cultured in 0.1 mL of medium containing different concentrations of IL-3 for 24 hours. H -thymidine was added for the last 4 hours of incubation. For M KIT (black) and M - F L T (gray)-transduced cells, squares, diamonds, triangles, circles represent populations derived from populations A , B, C and D. Crosses represent the MIG control values. Populations within an individual transduction group were averaged and were fit with a sigmoidal model by the method of least squares shown by the solid line. Data for the polyclonal populations (a) are representative results from a single experiment (n=2); data from the clonal populations (b & c) represent the average results from 3 independent experiments ± SEM. 4
3
59
Next, these c-kit and flt-3 expressing BaF3 populations were assayed for their ability to respond to their corresponding ligands, SF and F L (Figure 3.6). For the polyclonal populations of both M-KIT and M-FLT-transduced cells, data from population A and B (the cells with the lowest levels of transduced receptor expression) were grouped as "low expresser" (Figure 3.6a, open symbols). Similarly, data from populations C and D (the cells with the highest level of receptor expression) were grouped as "high expresser" (Figure 3.6a, solid symbols). The data for the clonal populations of both M-KIT and M-FLT-transduced cells with different levels of receptor expression were similarly grouped into one that contained the 4 populations with the lowest levels of expression of the transduced receptor (Figure 3.6b, open symbols) and one that contained the 4 populations with the highest levels of expression of the transduced receptor (Figure 3.6b, solid symbols).
The populations expressing the higher levels of c-kit or flt-3
showed some ability to proliferate in response to SF or F L as a substitute for IL-3.
For those
expressing low levels of these receptors, responsiveness to SF or F L assessed in this way was reduced or absent. MIG-transduced control cells showed no response to either SF or F L over the full range of concentrations tested (to 300 ng/mL). Overall, the flt-3-expressing cells were more sensitive to stimulation with F L than the c-kit-expressing cells were to stimulation with SF. Thus, responsiveness to F L was seen when M-FLT-transduced cells were exposed to F L concentrations ranging from 1 to 100 ng/mL, whereas responses of M-KIT-transduced cells required their exposure to >10 ng/mL SF.
60
a) M-KIT polyclonal cells 20
H
c
10 H
D CO
B, A
oiO
b) M-KIT clones 20
B3 10 H
D1, C1 T
0.001
0.01
0.1
1
SF (ng/ml)
61
10
100
D2 B2, B1, A2, A1
Z
1000
c) M-FLT polyclonal 20 H
10
D B, A iX
X
X -X- X i-X
d) M-FLT clones 20
I
C1
A—-4
C2
A
A
10 H
/ D1, D2, B1, A3
0 0.001
0.01
#^fa|^44*a*^ A2, A1 0.1
1
FL (ng/ml)
62
10
100
1000
Figure 3.6.
Mitogenic responses of M-KIT and
BaF3 populations to SF and
M-FLT-transduced
FL
10 MIG, M-KIT and M-FLT-transduced polyclonal (a & c) and clonal (b & d) BaF3 populations were cultured in 0.1 mL of medium containing different concentrations of SF (a & b) or F L (c & d) for 48 hours. H-fhymidine was added for the last 10 hours of incubation. For both M-KIT (a & b) and M - F L T (c & d)- transduced cells different symbols represent populations derived from populations with different levels of c-kit or flt-3 receptor expression (squares = A, diamonds = B, triangles = C, circles = D). Crosses represent the MIG control values. Populations within an individual transduction group were separated into high (solid symbols) and low (open symbols) receptor-expressing sub-groups (see text for detail). Data from the polyclonal populations (a & c) are representative results from a single experiment (n=2); data from the clonal populations (b & d) represent the average results from 3 independent experiments ± SEM. 4
3
63
Figure 3.7 shows a more detailed analysis of the mitogenic responsiveness of individual M-KIT and M-FLT-transduced BaF3 cells to specific concentrations of SF and F L , respectively, as a function of their levels of expression of c-kit and flt-3. Although a positive correlation in both cases was anticipated, such a simple relationship was borne out only in the case of the flt-3-expressing cells exposed to low concentrations of F L (Figure 3.7e, 0.2 ng/mL FL).
For the c-kit expressing cells, responses to a relatively low SF concentration appeared
independent of the level of receptor expression on the target cells (Figure 3.7a, 0.2 ng/mL SF). At higher levels of either SF or FL, the responses of the corresponding receptor-transduced cells were best fitted by a negative polynomial regression (Figure 3.7 c, d, f, g and h) indicative of an initial positive correlation with a reduced responsiveness above a certain level of receptor expression.
64
M-KIT
0.3 R=-0.04, p=0.84
a) 0.2 ng/mL SF T
0.2 -
II
0.1 CO
0.0 Q_ O
?
1
1.5 R =0.19, p=0.09 2
b) 3 ng/mL SF
1.0 ii
0.5 1 1 J_
^Eil 2^
0.0
1
T •
400
400 MFI
65
1*
800
1200
M-KIT r R =0.29 p=0.02* 2
c) 48 ng/mL SF
T
12-
1 •
CO
lt_dlJlt_j Q. O
18
T •
R =0.24, p=0.03
