International Journal of Cell Cloning 8:11-25 Suppl 1 (1990)
Cell Interactions and Gene Expression in Early Hematopoiesis Myrtle Z Gordon, Anthony M . Ford, Melvyn E Greaves Leukaemia Research Fund Centre, Institute of Cancer Research, London, England
Key Words. Gene expression
Stem cells
Microenvironment
Cell adhesion
Abstract. As part of an investigation of the mechanisms controlling gene expression during lineage commitment, we have investigated the transcriptional status of hematopoietic lineage-specific genes and the interactions of early hematopoietic progenitor cells with stromal cells of the marrow microenvironment. The results indicate that a subset of otherwise lineage-restrictedgenes are transcriptionally active and/or DNAse I hypersensitive (i.e., “primed” for transcription) in multipotent, interleukin 3-dependent hematopoietic cells, and that they may become inaccessible and transcriptionallysilent when cells are induced to adopt a single lineage during commitment. The external influences regulating gene expression in hematopoietic cells include binding interactionswith stromal cells and exposure to locally presented growth factors. These interactions are thought to be essential for hematopoietic cell development and may be dysregulated in chronic myeloid leukemia.
Introduction The control of lineage choice in cellular differentiation is fundamental to developmental biology. At the heart of this question lies the issue of how stable patterns of selective gene expression are induced and regulated in concert with cell proliferation, specialized function and death. The mechanisms involved have been studied in a wide variety of amenable experimental systems includingDictyostelium slime moulds [l], nematodes [2,3], Drosophila [4], sea urchins [J]and, in vertebrate species, in Xenopus embryos [6],rodent muscle [7]and nervous system [8]. Although the processes involved are certainly variable, complex and multifactorial, some consistent themes have emerged. For example, control of gene transcription is regulated at the DNA level by families of transacting DNA-binding proteins, which influence the conformation of DNA itself and the formation of stable transcription complexes [9-l2]and, at the whole cell level, by extracellular signals derived via contact with other cells, extracellular matrix (ECM)or growth Correspondence: Dr. M. Y. Gordon, Leukaemia Research Fund Centre, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, England. Received September 18, 1989; accepted for publication September 18, 1989. 0737-1454/90/$2.00/0 OAlphaMed Press
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factors [13-161. In some systems, genes have been identified and cloned that appear to have a direct role in initiating a cascade of lineage-specific gene expression [3, 12, 17, 181. These controlling or “selector” genes, in the instances in which they have been identified, encode DNA-binding proteins but pose the conundrum of how they themselves are controlled. Even in the simplest systems that are amenable to genetic manipulation and involve only binary choice, no complete explanation of lineage choice is yet at hand. The hematopoietic system has probably been the most exhaustively and successfully investigated mammalian tissue from the perspective of selective gene expression. A great deal has been revealed of the molecular mechanisms controlling the expression of hemoglobin and immunoglobulin genes which have provided valuable paradigms for vertebrate gene control. The developmentally and functionally associated phenotypic properties of different hematopoietic cells have been more thoroughly investigated at the single cell and molecular level than those of any other mammalian tissue. Many of the growth factors that regulate proliferation and differentiationof blood cells have now been isolated and molecularly cloned [19]. Despite these remarkable achievements,the mechanisms involved in lineage choice in hematopoiesis remain unresolved. The development of hematopoiesis (used here to include both myeloid and lymphoid cell production) in the fetus involves the emergence of a stem cell population from mesodermal tissue (Fig. 1) and its migration to the bone marrow (and spleen in mice) from the yolk sac via the liver. This phase is thought to depend on local environmental cues and the selection of a genetic program that is appropriate for further stem cell development. Once formed, it is believed that the hematopoietic stem cell pool is finite and no longer replenished by more pridtive mesodermal cells. The stem cells in adult life are required to sustain hematopoiesis by maintaining homeostatic levels of cell production as well as responding to increased demand caused by infection or damage. These two functions of the stem cell pool appear to be regulated by microenvironmental contact and by exposure to growth factors. The progeny spawned by proliferating stem cells become committed to one of the lymphoid or myeloid lineages-the choice being one out of a total of at least eight lineage options available (Fig 1). We have taken two complementary approaches to this problem. The first has been to assess the conformation or “accessibility” and transcriptional activity of several lineage-specificgenes in multipotenthematopoieticprogenitor cells in order to assess whether such genes are simultaneously, or promiscuously, primed for activation prior to lineage commitment. This possibility was suggested by studies on the multilineage phenotypic properties of some acute leukemia clones [20] and is a prediction of the model of selective gene expression proposed by Cuplan and Orduhl[21]. The second approach has been to investigatethe biochemical nature of the regulatory interaction between primitive progenitor cells and stromal elements in vitro. In particular, we wished to determine whether selective adhesion of progenitor cells to elements of the stroma could provide, in concert with growth factors, a basis for topographically localized regulation of gene expression.
Regulation of Early Hematopoiesis
Fig. 1. Lineage choices involved in the derivation of eight separate hematopoietic cell lineages.
Materials and Methods Cell Lines The cell line KG1 originated from a patient with acute myeloid leukemia and its phenotypic characteristics are described in detail elsewhere [22]. KG1 cells were induced to differentiateinto macrophages by the ionophore ionornycin (1.6 pg/ml) in combinationwith 12-0-tetra decanoyl phorbol-l3-acetate (TPA; 1 x 10-7 M) [23]. The murine cell line A4 originates from a long-term bone marrow culture and is non-leukemic in vivo [24]. These cells are maintained as a suspension culture in the presence of IL3 and can be induced to undergo myeloid differentiation by plating on normal bone marrow, on 3T3 fibroblasts or in the presence of appropriate growth factors (e.g., GM-CSF). DNAse I Hypersensitive Site Analysis Nuclei were prepared and incubated with DNAse I as described previously [23]. After termination ofreactions, DNA was purified and limit restricted with BamHI. Electrophoresis was performed and the DNA was hybridized [23] to a murine J region probe. (a kind gift from G. Yancopoulos). Tmcription Analysis RNA slot blots were performed essentially as described before (231. The TCRg cDNA probe was a kind gift from M. Owen and the genomic actin probe, a kind gift from K. Willison.
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Culture of Blast Colony-Forming Cells The blast colony assay consists of two phases. The first phase encompases the growth of stromal feeder layers from normal bone marrow mononuclear cells. Currently, these s t r o d layers are grown in 0.5 ml volumes held in 4-well plates (Nunc, Roskilde, Denmark). Stromal feeder layer cultures are initiated by plating 2.5 x 105 mononuclear cells per well in a a-medium supplemented with 10% fetal calf serum (FCS), 10% horse serum (both from GIBCO, Grand Island, NY) and 2 X M methylprednisolone (MP;Upjohn, Crawley, UK). The stromal cultures are fed weekly by complete replacement of the medium, serum and MP until confluent layers of adherent stromal cells have formed. For the second phase of the assay,the non-plastic-adherent fraction of 2.5 x lo5marrow mononuclear cells is added to each stromal layer and incubated for 2 h at 37°C in humidified 5% C 0 2 in air. The stromal layers are then washed thoroughly to remove any nonstroma-adherent cells, overlaid with 0.3% agar in a-medium with 15% FCS, incubated for 5-7 days at 3 P C in humidified 5% C 0 2 in air and examined by phase-contrast inverted microscopy for colony growth by stroma-adherent progenitor cells.
Results and Discussion Accessibility of Lineage-Specific Genes in Progenitor Cells In order for genes to be transcriptionally activated, they and their cis control regions have to be physically accessible to certain DNA binding proteins and RNA polymerase II. Inactive genes are usually restrained within a closed chromatin configuration. These conformational properties of genes are usually controlled by DNA binding proteins and can be assessed by their sensitivity to DNAse I. Expressed genes are DNAse I sensitiveor hypersensitive and, as might be anticipated, in some systems DNAse I sensitivity and accessibility precedes transcriptional activation indicating that genes can be poised or primed for subsequent activation in response to an appropriate environmental signal. We have analyzed the lineage-specificity of DNAse I hypersensitivity in the IgH enhancer-a gene whose transcription is mostly, if not entirely, B lineage-specific and whose activation is a very early event in B cell differentiation, preceeding rearrangement [25]. For this purpose, we used two cell lines; KG1, a human acute leukemic cell line with some phenotypic and response characteristics of primitive multipotent cells [22], and A4, an IL-3-dependent cell line of normal murine multipotent cells which retain the capacity for myeloid differentiation [24]. Details of these data are published elsewhere [23,26,27]. With both cell lines, we found that the IgH enhancer was in an accessible, DNAse I hypersensitiveconformationand that this reverted to a closed DNAse I insensitive form when the cells were induced to differentiate into myeloid (monocyte or granulocyte) cells (Fig. 2). In primitive embryonic stem cells (EScells) and pan-mesodermalprogenitor cells (1oTy2 cells), the IgH enhancer is in a closed configuration. Immature T cells retain an open IgH enhancer, but this is again closed in mature T cells. A similar developmental sequence has been found for the CD36 enhancer (a T cell lineage-specificgene) and, at the level of germline (unrearranged gene) transcription, for the T cell receptor y gene (Fig. 3). Transcription of the T cell
Regulation of Early Hematopoiesis
Fig. 2. DNAse I hypersensitive site analysis of the p gene in myeloid-induced and non-induced A4 cells. Lane 0: nuclei incubated without added DNAse I. Lanes 1-4: nuclei incubated with 1.2, 1.8,2.7 and 4 pglml DNAse I, respectively. Each lane contains 10 pg DNA restricted with BamHI and hybridized to a JH-enhancer intron probe. The Cp intron in the A4 cell line shows a restriction fragment length difference corresponding to allotype and subsequently two germline fragments of different size and seen hybridizing to the J region probe. ehs = enhancer hypersensitive site. Molecular weight markers are given in kb.
receptor y gene is switched off when A4 cells are induced to differentiate into granulocytes (Fig. 3). These features are recapitulated in other IL-3-dependent progenitor cell lines [27]. Some other genes associated with early developmental stages of particular lineages may also be active in these primitive cells, including B220/Ly5-B cell lineage and c-fms gene/M-CSF receptor-monocyte lineage. Most other lineage-specific genes are not, however, active. The Ig kappa enhancer is closed, i.e., not DNAse I hypersensitive. These data suggest that hematopoietic lineage-specificgenes are not generally accessible or transcriptionally active in early embryonic cells or panmesodermal cells. However, under IL-3-induced proliferation, a subset of them can become primed for activation or even transcriptionally active prior to,or independentof, lineage commitment and are then switched off should they be “inappropriate” to the lineage adopted. The model we therefore favor currently is that illustrated in Figure 4. Note that this differs from the earlier model of Cuplun and Ordahl[21] in that the genes concerned are not generally accessible or active in early embryonic stem cells. The model differs also from the predictions of Ell [28] and Bern*& [29] in that the “priming”
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Fig. 3. RNA slot blot analysis of cytoplasmic RNA extracted from induced and noninduced A4 cells. Slots 1-5 contain samples of 2, 1.0,0.5,0.25 and 0.125 pg total cellular RNA, hybridized to murine TCRy and actin probes, respectively.
of lineage-specific genes in stem cells is considered to be a feature of a particular subset of genes, i.e., those that are programmed to be expressed early in the developmental cascade of phenotypic maturation. Implicit in this type of model is the idea that extracellular signals play a crucial role in inducing lineage commitment and restriction of gene expression, and the likelihood is that this operates by modifications that result in stable transcription complexes. The developmental regulation of the transcription factors themselves is now amenable to investigation. Bone Marrow Stromal Matrices Provide a Local Environmentfor Progenitor Cell Regulation Although gene expression in hematopoietic progenitor cells can clearly be regulated by soluble growth factors alone in colony assay systems in semi-solid culture medium, it seems likely that long-term bone marrow culture systems that facilitate interactions between progenitor/stem cells, bone marrow stromal cells and extracellular matrix [30]approximate more closely to the in vivo situation in the bone marrow. There are of course many embryological precedents for developmental regulation that are dependent upon a structured microenvironment of interacting cell types and associated matrices [31,321. As a model for the regulation of adult stem cells by microenvironmental influences in the bone marrow, we have used a culture system involvingthe binding of progenitor cells to preformed bone marrow stromal layers and subsequent development of colonies of blast cells. The responding cells have characteristics of primitive progenitor cells or stem cells (Table I) and proliferate in the absence of added growth factors. This system is therefore a colony-forming assay equiva-
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Fig. 4. Developmental accessibility of heage-specific genes. Closed symbols: the lineage specific genes are in a closed, inaccessible configuration. Open symbols: genes are in an accessible configuration. lent of the murine long-term bone marrow culture system developed by Darer [30], and the cells assayed are probably closely related to those identified in the Oguwa culture system [33]. Two key questions relating to this in vitro system of hematopoietic regulation are the biochemical basis and specificity of the adhesion reaction of progenitor cells to stroma and the nature of the proliferative signal that initiates colony formation. The model illustrated in Figure 5 best fits the data we have accumulated over the past three years. We have shown that the cell adhesion molecule (CAM) expressed by the blast colony-forming cells (Bl-CFC) is unusual in its functional requirements in that binding occurs in the absence of divalent cations. In this respect, the structure resembles the neural CAM, (N-CAM) [34, 351. Also, like N-CAM [36], heparan sulphate (which is present in the ECM generated by the stromal cells) is necessary for binding to take place [37]. However, it is distinct from N-CAM because N-CAM antibodies do not block binding [38]. Treatment of the colony-forming cells with trypsin or phosphatidylinositol-specific phospholipase C (PI-PLC) reduces binding significantly, but incompletely (Table II). These results suggest fmt that the CAM is glycosylated and, hence, protected from total proteolytic degradation [39, 401 and, second, that it can be expressed either in a phospha-
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’Igble I. Characteristics of blast colony-forming cells Specific characteristic
Reference No.
Bind to stroma grown with methylprednisolone (MP) -not to MP- stroma or plastic Resistant to 4-hydroperoxycyclophosphamide exposure Ancestral to GEMM-CFC. BFU-eand GM-CFC Self renew in vitro Immunophenotype: HLA.DR: CD34: CD33’
53 75 76 76 77;
CR Dowdingunpublished observations
Table 11. Sensitivity to phosphatidylinositol-specificphospholipaseC (PI-PLC) in normal marrow and CML blood or marrow % PI-PLC-sensitive
N o d
61.5 f 14.6’
% Trypsin-sensitive 49.4 f 5.9
n=6
CML n = 3b
-3.3 f 1.9
71.0 f 3.0
‘mean f SD CML marrow and two CML blood samples tested
tidylinositol-glycan (PI-G)-linked form or in a transmembraneform. Several other PI-G-linked structures have been shown to be alternatively expressed as transmembrane proteins [41, 42, 431. It is likely that the PI-G-linked form of the CAM is instrumental in mediating efficient binding by B1-CFC to stromal layers because of its assumed capacity to migrate laterally in the cell membrane and accumulate at focal adhesion sites [44]. This property has been demonstrated for other PI-G-linked molecules using fluorescence photobleaching and recovery [45,46]. It is unlikely that occupation of the CAM results in stimulation of the B1-CFC to divide because PI-G-linked structures do not appear to possess signal-transducing properties [42,47-49]. We suggest, therefore, that the Bl-CFC express a separate receptor for a known and/or undiscovered growth factor@).It can be assumed that this growth factor(s) is produced by the stromal cells in our culture system because B1-CFC proliferate in the absence of added growth factors. Our model (Fig. 5 ) proposes that growth factors bind to the ECM in the stromal feeder layers where they are protected from proteolysis [50] and “presented” to immobilized, adherent progenitor cells. The adherence of progenitor cells to stromal matrix thus serves a docking function enabling cells to be locally regulated by
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Fig. 5. A model for the regulation of hematopoietic progenitor cells in the microenvironment of the bone marrow. CAM = cell adhesion molecule; HS-PG = heparan sulphate proteoglycan; ECM = extracellular matrix.
bound growth factors and, under some circumstances,subjected to negative regulation by stromal cells. In support of this idea, we have shown that a hematopoietic colony-stimulatingactivity can be extracted from s t r o d layers [16] and that GMCSF and IL-3 bind to ECM glycosaminoglycans[16,51,52]. Thus, both locally produced and exogenous growth factors can be sequestered in the hematopoietic microenvironment and interact with target progenitors. Since hematopoieticcells at different stages of differentiation exhibit different binding properties [53-571, the regulated expression of CAMS can dictate the migration and localization of progenitor cells in distinct microenvironments and, consequently, the signals they receive. Implicit in this view is the assumptionthat the patterns of lineage-specific gene transcription that are elicited during hematopoiesis are determined at least in part by the positioning of cells and the extracellular signals they are exposed to. Studies with colony-formingcells exposed to soluble growth factors in vitro have supported a very different, stochastic model of lineage choice [58]. Localized regulation of proliferation and differentiation by matrix-bound growth factors has also been described in other cellular systems [59,60],and there is accumulatingevidence that cell adhesion to matrix molecules has profound effects on the pattern of gene expression and cellular phenotypes [16,61,62]. The type of model we favor for the regulation of gene expressionin early hematopoietic differentiation therefore has much in common with other developmentalsystems where heterotypic cell interaction is required [63].
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The localized regulation of hematopoietic proliferation and differentiation will certainly be more complex than our minimal model predicts. In particular, it is likely that synthesis of growth factors and extracellular matrix proteins and other functional properties of stromal cells will themselves be subject to both local and systemic regulatory signals [64,651. The dysregulated expression of cell surface adhesion molecules or ECM molecules involved in adhesion could have important developmentalor pathological consequences. Morrison-Graham and Weston [66] recently summarized a number of embryological developmentaldefects in mice that might be ascribed to mutations in such genes including Steel defect (Sld/Sld) in which defective hematopoiesis is linked to altered composition and function of the bone marrow microenvironment in heterozygotes [67]. An inherited leukocyte adhesion deficiency (LAD) involves CAM dysfunctionand has now been characterized in more than 30 patients. These individuals are deficient in the cell surface adhesion molecules LFA-1, Mac-1 and ~15495, suffer life-threatening bacterial and fungal infections, progressive periodontitis, lack of pus formation and, if severely affected, rarely survive beyond childhood [68]. We have demonstrated [69] that the apparent defect in chronic myeloid leukemia (CML) progenitor cell adhesion to stroma [70] is associated with an increased rate (over normal cells) of detachment from stromal cell-binding sites. Preliminary results indicate that there may be a deficiency of the PI-G-linked form of the CAM leading to inefficient binding to stromal matrix (Table II). This abnormality could explain the presence of very high numbers of Bl-CFC in the circulation in CML [71]. The crucial biochemical change in CML stem cells is the activation by bcr-ubl gene fusion of abl protein tyrosine kinase activity (reviewed by Kunrock, [72]). It may be relevant in this respect that phosphotyrosine-containing proteins are concentrated in focal adhesions of normal cells [73] and that the fibronectin receptor complex is abnormally phosphorylated in cells transformed by oncogenes that encode tyrosine kinases [74]. The abl tyrosine kinase might, therefore, directly influence the progenitor cell adhesion, but other less-direct mechanisms are certainly possible.
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67 McCuskey RS,Meincke HA. Studies of the hemopoietic microenvironmentEl.Differences in the splenic microvasculature system between Sl/Sld and Wlw" anemic mice. Am J Anat 1973;137:187-198. 68 Springer TA, Dustin MC, Kishimoto TK, Marlin SD. The lymphocyte functionassociated LFA-1, CD2 and LFA-3 molecules: cell adhesion receptors of the immune system. AM Rev Immunol 1987,5:223-252. 69 Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF. Adhesive defects in chronic myeloid leukemia. In: Shen-Ong GLC, Potter M, Copeland NG, eds. Current Topics in Microbiology and Immunology Vol 149. Berlin: Springer-Verlag,l989:151-155. 70 Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF. Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia. Nature 1987;328:342-344. 71 Dowding CR, Gordon MY, Goldman JM. Primitive progenitor cells in the blood of patients with chronic granulocytic leukemia. Int J Cell Cloning 1986;4:331-340. 72 Kuxzrock R, Gutterman JLJ, Talpaz M. The molecular genetics of Philadelphia chromosome-positive leukemias. N Eng J Med 1988;319:990-998. 73 Maher PA, Pasquale EB, Wang JYJ, Singer SJ. Phosphotyrosine-containingproteins are concentrated in focal adhesions and intercellular junctions in normal cells. Proc Natl Acad Sci USA 1985;82:6576-6580. 74 Hirst R, Horwitz A, Buck C, Rohrschneider L. Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. Proc Natl Acad Sci USA 1986;83:6470-6474. 75 Gordon MY, Goldman JM, Gordon-Smith EC. 4-Hydroperoxycyclophosphamideinhibits proliferation by human granulocytemacrophage colony-formingcells (GM-CFC) but spares more primitive progenitor cells. Leuk Res 1985;9:1017-1021. 76 Gordon MY, Dowding CR, Riley GP, Greaves MF. Characterisation of stromadependent blast colony-forming cells in human manow. J Cell Physiol 1987;130:150-156. 77 Watt SM, Katz FE, Davis L, et al. Expression of HPCA-I and HLA-DR antigens on growth factor- and stroma-dependent colony-forming cells. Br J Haematol 1987;66: 153-159.
Discussion DiF'ersio: Have you ever incubated CML cells with G-CSF, since previous data indicate that the level of alkaline phosphate in these cells may increase after pre-incubation with G-CSF and whether that alters binding to stromal layers? Gordon: No, we haven't done that, but clearly we're going to do experiments which are designed to alter the expression of PI-linked molecules.
Broxmeyer: I have a question and a comment. Malcolm Moore, myself and our colleagues originally showed that the cell that generates hematopoiesis in long-term culture was HLA-DR negative, although we could not allow that there was a low density of HLA-DR. The S cell that Makio Ogawa studies, has been studied by Ron Hoffman of Indianapolis and he says that this S cell is either HLA-DR negative or a very low HLA-DR positive cell. Where does your cell fit in? You mention that they are HLA-DR positive, but do you have any idea about the density distribution of HLA-DR per cell?
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Gordon: I can’t answer that question in detail. I mentioned that we now had evidence that we had a CD33 negative component in that cell population. That evidence was obtained by preparing the cell population for adherence to the stmma in a different way, and we did not do experiments to assess DR expression on that population. So I don’t think that I can answer that question precisely. We have not looked for DR positivity or negativity on those particular cells. Murphy: Have you been able to coax your blast colony-forming cells into forming megakaryocytes in response to Meg-CSF or a thrombopoietin?
Gordon: We haven’t actually looked for that. O’ReiUy: Have you looked at the differential in terms of stromal binding of the C33 positive and negative cells? Gordon: We haven’t looked at them separately.
Cell Interactions and Gene Expression in Early Hematopoiesis Myrtle Z Gordon, Anthony M . Ford, Melvyn E Greaves Leukaemia Research Fund Centre, Institute of Cancer Research, London, England
Key Words. Gene expression
Stem cells
Microenvironment
Cell adhesion
Abstract. As part of an investigation of the mechanisms controlling gene expression during lineage commitment, we have investigated the transcriptional status of hematopoietic lineage-specific genes and the interactions of early hematopoietic progenitor cells with stromal cells of the marrow microenvironment. The results indicate that a subset of otherwise lineage-restrictedgenes are transcriptionally active and/or DNAse I hypersensitive (i.e., “primed” for transcription) in multipotent, interleukin 3-dependent hematopoietic cells, and that they may become inaccessible and transcriptionallysilent when cells are induced to adopt a single lineage during commitment. The external influences regulating gene expression in hematopoietic cells include binding interactionswith stromal cells and exposure to locally presented growth factors. These interactions are thought to be essential for hematopoietic cell development and may be dysregulated in chronic myeloid leukemia.
Introduction The control of lineage choice in cellular differentiation is fundamental to developmental biology. At the heart of this question lies the issue of how stable patterns of selective gene expression are induced and regulated in concert with cell proliferation, specialized function and death. The mechanisms involved have been studied in a wide variety of amenable experimental systems includingDictyostelium slime moulds [l], nematodes [2,3], Drosophila [4], sea urchins [J]and, in vertebrate species, in Xenopus embryos [6],rodent muscle [7]and nervous system [8]. Although the processes involved are certainly variable, complex and multifactorial, some consistent themes have emerged. For example, control of gene transcription is regulated at the DNA level by families of transacting DNA-binding proteins, which influence the conformation of DNA itself and the formation of stable transcription complexes [9-l2]and, at the whole cell level, by extracellular signals derived via contact with other cells, extracellular matrix (ECM)or growth Correspondence: Dr. M. Y. Gordon, Leukaemia Research Fund Centre, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, England. Received September 18, 1989; accepted for publication September 18, 1989. 0737-1454/90/$2.00/0 OAlphaMed Press
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factors [13-161. In some systems, genes have been identified and cloned that appear to have a direct role in initiating a cascade of lineage-specific gene expression [3, 12, 17, 181. These controlling or “selector” genes, in the instances in which they have been identified, encode DNA-binding proteins but pose the conundrum of how they themselves are controlled. Even in the simplest systems that are amenable to genetic manipulation and involve only binary choice, no complete explanation of lineage choice is yet at hand. The hematopoietic system has probably been the most exhaustively and successfully investigated mammalian tissue from the perspective of selective gene expression. A great deal has been revealed of the molecular mechanisms controlling the expression of hemoglobin and immunoglobulin genes which have provided valuable paradigms for vertebrate gene control. The developmentally and functionally associated phenotypic properties of different hematopoietic cells have been more thoroughly investigated at the single cell and molecular level than those of any other mammalian tissue. Many of the growth factors that regulate proliferation and differentiationof blood cells have now been isolated and molecularly cloned [19]. Despite these remarkable achievements,the mechanisms involved in lineage choice in hematopoiesis remain unresolved. The development of hematopoiesis (used here to include both myeloid and lymphoid cell production) in the fetus involves the emergence of a stem cell population from mesodermal tissue (Fig. 1) and its migration to the bone marrow (and spleen in mice) from the yolk sac via the liver. This phase is thought to depend on local environmental cues and the selection of a genetic program that is appropriate for further stem cell development. Once formed, it is believed that the hematopoietic stem cell pool is finite and no longer replenished by more pridtive mesodermal cells. The stem cells in adult life are required to sustain hematopoiesis by maintaining homeostatic levels of cell production as well as responding to increased demand caused by infection or damage. These two functions of the stem cell pool appear to be regulated by microenvironmental contact and by exposure to growth factors. The progeny spawned by proliferating stem cells become committed to one of the lymphoid or myeloid lineages-the choice being one out of a total of at least eight lineage options available (Fig 1). We have taken two complementary approaches to this problem. The first has been to assess the conformation or “accessibility” and transcriptional activity of several lineage-specificgenes in multipotenthematopoieticprogenitor cells in order to assess whether such genes are simultaneously, or promiscuously, primed for activation prior to lineage commitment. This possibility was suggested by studies on the multilineage phenotypic properties of some acute leukemia clones [20] and is a prediction of the model of selective gene expression proposed by Cuplan and Orduhl[21]. The second approach has been to investigatethe biochemical nature of the regulatory interaction between primitive progenitor cells and stromal elements in vitro. In particular, we wished to determine whether selective adhesion of progenitor cells to elements of the stroma could provide, in concert with growth factors, a basis for topographically localized regulation of gene expression.
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Fig. 1. Lineage choices involved in the derivation of eight separate hematopoietic cell lineages.
Materials and Methods Cell Lines The cell line KG1 originated from a patient with acute myeloid leukemia and its phenotypic characteristics are described in detail elsewhere [22]. KG1 cells were induced to differentiateinto macrophages by the ionophore ionornycin (1.6 pg/ml) in combinationwith 12-0-tetra decanoyl phorbol-l3-acetate (TPA; 1 x 10-7 M) [23]. The murine cell line A4 originates from a long-term bone marrow culture and is non-leukemic in vivo [24]. These cells are maintained as a suspension culture in the presence of IL3 and can be induced to undergo myeloid differentiation by plating on normal bone marrow, on 3T3 fibroblasts or in the presence of appropriate growth factors (e.g., GM-CSF). DNAse I Hypersensitive Site Analysis Nuclei were prepared and incubated with DNAse I as described previously [23]. After termination ofreactions, DNA was purified and limit restricted with BamHI. Electrophoresis was performed and the DNA was hybridized [23] to a murine J region probe. (a kind gift from G. Yancopoulos). Tmcription Analysis RNA slot blots were performed essentially as described before (231. The TCRg cDNA probe was a kind gift from M. Owen and the genomic actin probe, a kind gift from K. Willison.
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Culture of Blast Colony-Forming Cells The blast colony assay consists of two phases. The first phase encompases the growth of stromal feeder layers from normal bone marrow mononuclear cells. Currently, these s t r o d layers are grown in 0.5 ml volumes held in 4-well plates (Nunc, Roskilde, Denmark). Stromal feeder layer cultures are initiated by plating 2.5 x 105 mononuclear cells per well in a a-medium supplemented with 10% fetal calf serum (FCS), 10% horse serum (both from GIBCO, Grand Island, NY) and 2 X M methylprednisolone (MP;Upjohn, Crawley, UK). The stromal cultures are fed weekly by complete replacement of the medium, serum and MP until confluent layers of adherent stromal cells have formed. For the second phase of the assay,the non-plastic-adherent fraction of 2.5 x lo5marrow mononuclear cells is added to each stromal layer and incubated for 2 h at 37°C in humidified 5% C 0 2 in air. The stromal layers are then washed thoroughly to remove any nonstroma-adherent cells, overlaid with 0.3% agar in a-medium with 15% FCS, incubated for 5-7 days at 3 P C in humidified 5% C 0 2 in air and examined by phase-contrast inverted microscopy for colony growth by stroma-adherent progenitor cells.
Results and Discussion Accessibility of Lineage-Specific Genes in Progenitor Cells In order for genes to be transcriptionally activated, they and their cis control regions have to be physically accessible to certain DNA binding proteins and RNA polymerase II. Inactive genes are usually restrained within a closed chromatin configuration. These conformational properties of genes are usually controlled by DNA binding proteins and can be assessed by their sensitivity to DNAse I. Expressed genes are DNAse I sensitiveor hypersensitive and, as might be anticipated, in some systems DNAse I sensitivity and accessibility precedes transcriptional activation indicating that genes can be poised or primed for subsequent activation in response to an appropriate environmental signal. We have analyzed the lineage-specificity of DNAse I hypersensitivity in the IgH enhancer-a gene whose transcription is mostly, if not entirely, B lineage-specific and whose activation is a very early event in B cell differentiation, preceeding rearrangement [25]. For this purpose, we used two cell lines; KG1, a human acute leukemic cell line with some phenotypic and response characteristics of primitive multipotent cells [22], and A4, an IL-3-dependent cell line of normal murine multipotent cells which retain the capacity for myeloid differentiation [24]. Details of these data are published elsewhere [23,26,27]. With both cell lines, we found that the IgH enhancer was in an accessible, DNAse I hypersensitiveconformationand that this reverted to a closed DNAse I insensitive form when the cells were induced to differentiate into myeloid (monocyte or granulocyte) cells (Fig. 2). In primitive embryonic stem cells (EScells) and pan-mesodermalprogenitor cells (1oTy2 cells), the IgH enhancer is in a closed configuration. Immature T cells retain an open IgH enhancer, but this is again closed in mature T cells. A similar developmental sequence has been found for the CD36 enhancer (a T cell lineage-specificgene) and, at the level of germline (unrearranged gene) transcription, for the T cell receptor y gene (Fig. 3). Transcription of the T cell
Regulation of Early Hematopoiesis
Fig. 2. DNAse I hypersensitive site analysis of the p gene in myeloid-induced and non-induced A4 cells. Lane 0: nuclei incubated without added DNAse I. Lanes 1-4: nuclei incubated with 1.2, 1.8,2.7 and 4 pglml DNAse I, respectively. Each lane contains 10 pg DNA restricted with BamHI and hybridized to a JH-enhancer intron probe. The Cp intron in the A4 cell line shows a restriction fragment length difference corresponding to allotype and subsequently two germline fragments of different size and seen hybridizing to the J region probe. ehs = enhancer hypersensitive site. Molecular weight markers are given in kb.
receptor y gene is switched off when A4 cells are induced to differentiate into granulocytes (Fig. 3). These features are recapitulated in other IL-3-dependent progenitor cell lines [27]. Some other genes associated with early developmental stages of particular lineages may also be active in these primitive cells, including B220/Ly5-B cell lineage and c-fms gene/M-CSF receptor-monocyte lineage. Most other lineage-specific genes are not, however, active. The Ig kappa enhancer is closed, i.e., not DNAse I hypersensitive. These data suggest that hematopoietic lineage-specificgenes are not generally accessible or transcriptionally active in early embryonic cells or panmesodermal cells. However, under IL-3-induced proliferation, a subset of them can become primed for activation or even transcriptionally active prior to,or independentof, lineage commitment and are then switched off should they be “inappropriate” to the lineage adopted. The model we therefore favor currently is that illustrated in Figure 4. Note that this differs from the earlier model of Cuplun and Ordahl[21] in that the genes concerned are not generally accessible or active in early embryonic stem cells. The model differs also from the predictions of Ell [28] and Bern*& [29] in that the “priming”
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Fig. 3. RNA slot blot analysis of cytoplasmic RNA extracted from induced and noninduced A4 cells. Slots 1-5 contain samples of 2, 1.0,0.5,0.25 and 0.125 pg total cellular RNA, hybridized to murine TCRy and actin probes, respectively.
of lineage-specific genes in stem cells is considered to be a feature of a particular subset of genes, i.e., those that are programmed to be expressed early in the developmental cascade of phenotypic maturation. Implicit in this type of model is the idea that extracellular signals play a crucial role in inducing lineage commitment and restriction of gene expression, and the likelihood is that this operates by modifications that result in stable transcription complexes. The developmental regulation of the transcription factors themselves is now amenable to investigation. Bone Marrow Stromal Matrices Provide a Local Environmentfor Progenitor Cell Regulation Although gene expression in hematopoietic progenitor cells can clearly be regulated by soluble growth factors alone in colony assay systems in semi-solid culture medium, it seems likely that long-term bone marrow culture systems that facilitate interactions between progenitor/stem cells, bone marrow stromal cells and extracellular matrix [30]approximate more closely to the in vivo situation in the bone marrow. There are of course many embryological precedents for developmental regulation that are dependent upon a structured microenvironment of interacting cell types and associated matrices [31,321. As a model for the regulation of adult stem cells by microenvironmental influences in the bone marrow, we have used a culture system involvingthe binding of progenitor cells to preformed bone marrow stromal layers and subsequent development of colonies of blast cells. The responding cells have characteristics of primitive progenitor cells or stem cells (Table I) and proliferate in the absence of added growth factors. This system is therefore a colony-forming assay equiva-
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Fig. 4. Developmental accessibility of heage-specific genes. Closed symbols: the lineage specific genes are in a closed, inaccessible configuration. Open symbols: genes are in an accessible configuration. lent of the murine long-term bone marrow culture system developed by Darer [30], and the cells assayed are probably closely related to those identified in the Oguwa culture system [33]. Two key questions relating to this in vitro system of hematopoietic regulation are the biochemical basis and specificity of the adhesion reaction of progenitor cells to stroma and the nature of the proliferative signal that initiates colony formation. The model illustrated in Figure 5 best fits the data we have accumulated over the past three years. We have shown that the cell adhesion molecule (CAM) expressed by the blast colony-forming cells (Bl-CFC) is unusual in its functional requirements in that binding occurs in the absence of divalent cations. In this respect, the structure resembles the neural CAM, (N-CAM) [34, 351. Also, like N-CAM [36], heparan sulphate (which is present in the ECM generated by the stromal cells) is necessary for binding to take place [37]. However, it is distinct from N-CAM because N-CAM antibodies do not block binding [38]. Treatment of the colony-forming cells with trypsin or phosphatidylinositol-specific phospholipase C (PI-PLC) reduces binding significantly, but incompletely (Table II). These results suggest fmt that the CAM is glycosylated and, hence, protected from total proteolytic degradation [39, 401 and, second, that it can be expressed either in a phospha-
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’Igble I. Characteristics of blast colony-forming cells Specific characteristic
Reference No.
Bind to stroma grown with methylprednisolone (MP) -not to MP- stroma or plastic Resistant to 4-hydroperoxycyclophosphamide exposure Ancestral to GEMM-CFC. BFU-eand GM-CFC Self renew in vitro Immunophenotype: HLA.DR: CD34: CD33’
53 75 76 76 77;
CR Dowdingunpublished observations
Table 11. Sensitivity to phosphatidylinositol-specificphospholipaseC (PI-PLC) in normal marrow and CML blood or marrow % PI-PLC-sensitive
N o d
61.5 f 14.6’
% Trypsin-sensitive 49.4 f 5.9
n=6
CML n = 3b
-3.3 f 1.9
71.0 f 3.0
‘mean f SD CML marrow and two CML blood samples tested
tidylinositol-glycan (PI-G)-linked form or in a transmembraneform. Several other PI-G-linked structures have been shown to be alternatively expressed as transmembrane proteins [41, 42, 431. It is likely that the PI-G-linked form of the CAM is instrumental in mediating efficient binding by B1-CFC to stromal layers because of its assumed capacity to migrate laterally in the cell membrane and accumulate at focal adhesion sites [44]. This property has been demonstrated for other PI-G-linked molecules using fluorescence photobleaching and recovery [45,46]. It is unlikely that occupation of the CAM results in stimulation of the B1-CFC to divide because PI-G-linked structures do not appear to possess signal-transducing properties [42,47-49]. We suggest, therefore, that the Bl-CFC express a separate receptor for a known and/or undiscovered growth factor@).It can be assumed that this growth factor(s) is produced by the stromal cells in our culture system because B1-CFC proliferate in the absence of added growth factors. Our model (Fig. 5 ) proposes that growth factors bind to the ECM in the stromal feeder layers where they are protected from proteolysis [50] and “presented” to immobilized, adherent progenitor cells. The adherence of progenitor cells to stromal matrix thus serves a docking function enabling cells to be locally regulated by
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Fig. 5. A model for the regulation of hematopoietic progenitor cells in the microenvironment of the bone marrow. CAM = cell adhesion molecule; HS-PG = heparan sulphate proteoglycan; ECM = extracellular matrix.
bound growth factors and, under some circumstances,subjected to negative regulation by stromal cells. In support of this idea, we have shown that a hematopoietic colony-stimulatingactivity can be extracted from s t r o d layers [16] and that GMCSF and IL-3 bind to ECM glycosaminoglycans[16,51,52]. Thus, both locally produced and exogenous growth factors can be sequestered in the hematopoietic microenvironment and interact with target progenitors. Since hematopoieticcells at different stages of differentiation exhibit different binding properties [53-571, the regulated expression of CAMS can dictate the migration and localization of progenitor cells in distinct microenvironments and, consequently, the signals they receive. Implicit in this view is the assumptionthat the patterns of lineage-specific gene transcription that are elicited during hematopoiesis are determined at least in part by the positioning of cells and the extracellular signals they are exposed to. Studies with colony-formingcells exposed to soluble growth factors in vitro have supported a very different, stochastic model of lineage choice [58]. Localized regulation of proliferation and differentiation by matrix-bound growth factors has also been described in other cellular systems [59,60],and there is accumulatingevidence that cell adhesion to matrix molecules has profound effects on the pattern of gene expression and cellular phenotypes [16,61,62]. The type of model we favor for the regulation of gene expressionin early hematopoietic differentiation therefore has much in common with other developmentalsystems where heterotypic cell interaction is required [63].
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The localized regulation of hematopoietic proliferation and differentiation will certainly be more complex than our minimal model predicts. In particular, it is likely that synthesis of growth factors and extracellular matrix proteins and other functional properties of stromal cells will themselves be subject to both local and systemic regulatory signals [64,651. The dysregulated expression of cell surface adhesion molecules or ECM molecules involved in adhesion could have important developmentalor pathological consequences. Morrison-Graham and Weston [66] recently summarized a number of embryological developmentaldefects in mice that might be ascribed to mutations in such genes including Steel defect (Sld/Sld) in which defective hematopoiesis is linked to altered composition and function of the bone marrow microenvironment in heterozygotes [67]. An inherited leukocyte adhesion deficiency (LAD) involves CAM dysfunctionand has now been characterized in more than 30 patients. These individuals are deficient in the cell surface adhesion molecules LFA-1, Mac-1 and ~15495, suffer life-threatening bacterial and fungal infections, progressive periodontitis, lack of pus formation and, if severely affected, rarely survive beyond childhood [68]. We have demonstrated [69] that the apparent defect in chronic myeloid leukemia (CML) progenitor cell adhesion to stroma [70] is associated with an increased rate (over normal cells) of detachment from stromal cell-binding sites. Preliminary results indicate that there may be a deficiency of the PI-G-linked form of the CAM leading to inefficient binding to stromal matrix (Table II). This abnormality could explain the presence of very high numbers of Bl-CFC in the circulation in CML [71]. The crucial biochemical change in CML stem cells is the activation by bcr-ubl gene fusion of abl protein tyrosine kinase activity (reviewed by Kunrock, [72]). It may be relevant in this respect that phosphotyrosine-containing proteins are concentrated in focal adhesions of normal cells [73] and that the fibronectin receptor complex is abnormally phosphorylated in cells transformed by oncogenes that encode tyrosine kinases [74]. The abl tyrosine kinase might, therefore, directly influence the progenitor cell adhesion, but other less-direct mechanisms are certainly possible.
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48 Simmons D, Seed B. The Fcy receptor of natural killer cells is a phospholipid-linked membrane protein. Nature 1988;333:568-570. 49 Huizinga TWJ, van der Schoot CE, Jost C, et al. The PI-linked receptor FcFUII is released on stimulation of neutrophils. Nature 1988;333:667-669. 50 Saksela 0,Moscatelli D, Sommer A, Rifkin DB. Endothelial cell-derived heparan sulphate binds basic fibroblast growth factor and protects it from proteolytic degradation. J Cell Biol l988;107:743-751. 51 Gordon MY, Bearpark AD, Clarke D, Healy L. Matrix glycoproteins may locally regulate the relative concentrations of different hemopoietic growth factors. In: Baum SJ, Dicke KA,Lotzova E, Pluznik DH, eds. Experimental Hematology Today 1989. New York: Springer-Verlag, 1989:31-35. 52 Roberts R,Gallagher J, Spooncer E, Allen TD, Bloomfield F, Dexter TM. Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis. Nature 1988;332:376-378. 53 Gordon MY, Hibbin JA, Dowding C, Gordon-Smith EC, Goldman JM. Separation of human blast progenitors from granulocytic, erythroid, megakaryocytic and mixed colony-forming cells by panning on cultured marrowderived stromal layers. Exp Hematol 1985;13:937-940. 54 Coulombel L, Vuillet MH, Leroy C, Tchernia G.Lineage- and stage-specificadhesion of human hematopoietic progenitor cells to extracellular matrices from marrow fibroblasts. Blood 1988;71:329-334. 55 Patel VP, Lodish H. A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes. J Cell Biol 1987,105:3105-3118. 56 Dustin ML, Sanders ME, Shaw S, Springer TA. Purified lymphocyte functionassociated antigen 3 binds to CD2 and mediates T lymphocyte adhesion. J Exp Med 1987,165~677-692. 57 Mizutani S, Watt SM, Robertson D,et al. Cloning of human thymic subcapsularcortex epithelial cells with T-lymphocyte binding sites and hemopoietic growth factor activity. Proc Natl Acad Sci USA 1987,84:4999-5003. 58 Ogawa M, Porter PN, Nakahata T. Renewal and commitment to differentiation of hemopoietic stem cells (an interpretive review). Blood 1983;61:823-831. 59 Rogelj S, Klagsbrun M. Atvnon R, et al. Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiaton of PC12 cells. J Cell Biol 1989;109:823-831. 60 Moscatelli D. Metabolism of receptor-bound and matrix-bound basic fibroblast growth factor by bovine capillary endothelial cells. J Cell Biol 1988;107:753-759. 61 Perris R, von Boxberg Y, Lofberg J. Local embryonic matrices determine regionspecific phenotypes in neural crest cells. Science 1988;241:86-89. 62 Ben-Ze'ev A, Robinson GS, Bucher NLR, Farmer SR. Cell-cell and cell-matrix interactions differentially regulate the expression of hepatic and cytoskeletal genes in primary cultures of rat hepatocytes. Proc Natl Acad Sci USA 1988;85:2161-2165. 63 Torok-Storb B. Cellular interactions. Blood 1988;72:373-385. 64 Kimura A, Katoh 0,Hyodo H, Kuramoto A. Transforming growth factor-0 regulates growth as well as collagen and fibronectin synthesis of human marrow fibroblasts. Brit J Haematol 1989;72:486-491. 65 Gimble JM, Pietrangeli C, Henley'A, et al. Characterizationof murine bone marrow and spleenderived stromal cells: analysis of leukccyte marker and growth Eictor mRNA transcript levels. Blood l989;74:303-311. 66 Momson-Graham K, Weston JA. Mouse mutants provide new insights into the role of extracellular matrix in cell migration and differentiation. Trends Genet 1989;5:ll6-120.
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67 McCuskey RS,Meincke HA. Studies of the hemopoietic microenvironmentEl.Differences in the splenic microvasculature system between Sl/Sld and Wlw" anemic mice. Am J Anat 1973;137:187-198. 68 Springer TA, Dustin MC, Kishimoto TK, Marlin SD. The lymphocyte functionassociated LFA-1, CD2 and LFA-3 molecules: cell adhesion receptors of the immune system. AM Rev Immunol 1987,5:223-252. 69 Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF. Adhesive defects in chronic myeloid leukemia. In: Shen-Ong GLC, Potter M, Copeland NG, eds. Current Topics in Microbiology and Immunology Vol 149. Berlin: Springer-Verlag,l989:151-155. 70 Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF. Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia. Nature 1987;328:342-344. 71 Dowding CR, Gordon MY, Goldman JM. Primitive progenitor cells in the blood of patients with chronic granulocytic leukemia. Int J Cell Cloning 1986;4:331-340. 72 Kuxzrock R, Gutterman JLJ, Talpaz M. The molecular genetics of Philadelphia chromosome-positive leukemias. N Eng J Med 1988;319:990-998. 73 Maher PA, Pasquale EB, Wang JYJ, Singer SJ. Phosphotyrosine-containingproteins are concentrated in focal adhesions and intercellular junctions in normal cells. Proc Natl Acad Sci USA 1985;82:6576-6580. 74 Hirst R, Horwitz A, Buck C, Rohrschneider L. Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. Proc Natl Acad Sci USA 1986;83:6470-6474. 75 Gordon MY, Goldman JM, Gordon-Smith EC. 4-Hydroperoxycyclophosphamideinhibits proliferation by human granulocytemacrophage colony-formingcells (GM-CFC) but spares more primitive progenitor cells. Leuk Res 1985;9:1017-1021. 76 Gordon MY, Dowding CR, Riley GP, Greaves MF. Characterisation of stromadependent blast colony-forming cells in human manow. J Cell Physiol 1987;130:150-156. 77 Watt SM, Katz FE, Davis L, et al. Expression of HPCA-I and HLA-DR antigens on growth factor- and stroma-dependent colony-forming cells. Br J Haematol 1987;66: 153-159.
Discussion DiF'ersio: Have you ever incubated CML cells with G-CSF, since previous data indicate that the level of alkaline phosphate in these cells may increase after pre-incubation with G-CSF and whether that alters binding to stromal layers? Gordon: No, we haven't done that, but clearly we're going to do experiments which are designed to alter the expression of PI-linked molecules.
Broxmeyer: I have a question and a comment. Malcolm Moore, myself and our colleagues originally showed that the cell that generates hematopoiesis in long-term culture was HLA-DR negative, although we could not allow that there was a low density of HLA-DR. The S cell that Makio Ogawa studies, has been studied by Ron Hoffman of Indianapolis and he says that this S cell is either HLA-DR negative or a very low HLA-DR positive cell. Where does your cell fit in? You mention that they are HLA-DR positive, but do you have any idea about the density distribution of HLA-DR per cell?
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Gordon: I can’t answer that question in detail. I mentioned that we now had evidence that we had a CD33 negative component in that cell population. That evidence was obtained by preparing the cell population for adherence to the stmma in a different way, and we did not do experiments to assess DR expression on that population. So I don’t think that I can answer that question precisely. We have not looked for DR positivity or negativity on those particular cells. Murphy: Have you been able to coax your blast colony-forming cells into forming megakaryocytes in response to Meg-CSF or a thrombopoietin?
Gordon: We haven’t actually looked for that. O’ReiUy: Have you looked at the differential in terms of stromal binding of the C33 positive and negative cells? Gordon: We haven’t looked at them separately.
