Human interleukin-4 enhances stromal cell-dependent hematopoiesis: costimulation with stem cell factor

Interleukin-4 (IL-4) has distinct hematopoietic activities, primarily as a costimulant with other cytokines to enhance colony formation of hematopoietic progenitors. We investigated the influence of IL-4 on stromal cell-supported long-term cultures (LTCs) of normal human bone marrow. Addition of IL-4 to LTCs of unseparated bone marrow or highly enriched CD34+ cells resulted in a significant increase of myeloid progenitors in the nonadherent, as well as in the stromal cell-adherent cell populations. In contrast, the total cell number was not influenced by IL-4, suggesting a selective effect on primitive progenitor cells. Cord blood cells or CD34+ bone marrow cells were incubated with stem cell factor (SCF) and/or IL-4 in stromal cell-free cultures. In these experiments, a twofold to fivefold increase of myeloid progenitor cells was observed in the presence of SCF and IL-4 as compared with SCF alone. Preincubation of the stromal cell cultures with IL-4 resulted in an enhanced adherence of CD34+ cells to the stromal layer. Secretion of hematopoietic growth factors produced by the stromal cells, such as granulocyte-macrophage colony-stimulating factor (G-CSF), and IL-1, was inhibited by IL-4. Thus, the increase of hematopoietic progenitors in LTCs, as observed in the presence of IL-4, can be at least partially explained by a costimulation of SCF and IL-4 on primitive progenitor cells and by an enhancement of hematopoietic cells to stroma. The downregulation of CSFs by IL-4 might prevent the expansion of the mature hematopoietic cell compartment.     

Bone marrow stromal cell regulation of B lymphopoiesis: interleukin-1 (IL-1) and IL-4 regulate stromal cell support of pre-B cell production in vitro

Bone marrow stromal cells appear to be key regulatory elements in hematopoiesis and lymphopoiesis. These stromal cells respond to cytokine exposure and alter their pattern of hematopoietic growth factor production, suggesting a degree of functional plasticity. We examined the effect of two cytokines, interleukin-1 (IL-1) and IL-4, on stromal cell regulation of pre-B cell generation using the bone marrow stromal cell line, S17. Neither lymphokine potentiated pre-B cell generation in the absence of stromal cells. However, addition of either 10 U/mL rIL-1 alpha or 50 U/mL rIL-4 to cultures of bone marrow cells containing S17 cells dramatically suppressed subsequent pre-B cell formation. Preculture of S17 stromal cells with either rIL-1 or rIL-4 completely abrogated their ability to support pre-B cell generation in subsequent coculture with freshly explanted bone marrow cells. Conditioned medium from IL-1- or IL-4-treated S17 cells also suppressed pre-B-cell generation in culture. Although it is not yet known which induced stromal cell factors are responsible for failure of pre-B-cell generation in treated cultures, these data do clearly demonstrate that local levels of IL-1 and IL-4 in the hematopoietic microenvironment may play a significant role in regulation of bone marrow stromal cell function. These data also demonstrate that fibroblastic stromal cells are primary target cells that respond to cytokine concentration and affect lymphopoietic cell development.     

The hematopoietic microenvironment in the elderly: defects in IL-1-induced CSF expression in vitro

The expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) genes by stromal cells of the hematopoietic microenvironment is regulated, in vitro, by interleukin 1 alpha (IL-1 alpha) and IL-1 beta. We reasoned that malfunction of this inductive mechanism in vivo might contribute to the intolerance of the aged to myelosuppressive therapy. We found that bone marrow fibroblasts from healthy elderly (ages 65-75 years) volunteers were less sensitive to the inductive effects of recombinant IL-1 beta than were marrow fibroblasts from younger volunteers, and we proposed that an age-related decline in sensitivity of marrow stroma to IL-1 may impair the ability of the elderly to increase production of hematopoietic growth factors under conditions of physiologic stress.     

Stromal cell-associated hematopoiesis: immortalization and characterization of a primate bone marrow-derived stromal cell line

An elucidation of the interaction between the bone marrow microenvironment and hematopoietic stem cells is critical to the understanding of the molecular basis of stem cell self renewal and differentiation. This interaction is dependent, at least in part, on direct cell to cell contact or cellular adhesion to extracellular matrix proteins. Long-term bone marrow cultures (LTMC) provide an appropriate microenvironment for maintenance of primitive hematopoietic stem cells and a means of analyzing this stem cell-stromal cell interaction in vitro. Although LTMC have been successfully generated from murine and human bone marrow, only limited success has been reported in a primate system. In addition, few permanent stromal cell lines are available from nonmurine bone marrow. Because the primate has become a useful model for large animal bone marrow transplant studies and, more specifically, retroviral-mediated gene transfer analysis, we have generated immortalized bone marrow stromal cell lines from primate bone marrow using gene transfer of the Simian virus large T (SV40 LT) antigen. At least one stromal cell line has demonstrated the capacity to maintain early hematopoietic cells in long-term cultures for up to 4 weeks as measured by in vitro progenitor assays. Studies were undertaken to characterize the products of extracellular matrix biosynthesis and growth factor synthesis of this cell line, designated PU-34. In contrast to most murine bone marrow-derived stromal cell lines capable of supporting hematopoiesis in vitro that have been examined, the extracellular matrix produced by this primate cell line includes collagen types I, laminin. Growth factor production analyzed through RNA blot analysis, bone marrow cell culture data, and factor-dependent cell line proliferation assays includes interleukin-6 (IL-6), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, leukemia inhibitory factor, and a novel cytokine designated IL-11. This immortalized primate bone marrow stromal cell line may be useful in maintaining early progenitor cells for experimental manipulation without the loss of reconstituting capacity and as a potential source of novel hematopoietic growth factors.     

Interleukin-1 induces human bone marrow-derived fibroblasts to produce multilineage hematopoietic growth factors

Interleukin-1 (IL-1) has been shown to induce stromal cells, including endothelial cells and fibroblasts, to produce multilineage hematopoietic growth factors. Although both of these cell types are well-described elements of the hematopoietic microenvironment, previous studies of IL-1-inducible colony-stimulating factor responses have utilized fibroblasts and endothelial cells from nonhematopoietic sites. Since we hypothesize that this intercellular growth network is active in the hematopoietic microenvironment, we sought to determine the responsiveness of bone marrow fibroblasts to IL-1. We demonstrate here that recombinant human IL-1 alpha and beta stimulate the dose-dependent release of granulocyte-macrophage colony-stimulating activity (GM-CSA) and burst-promoting activity (BPA) by cultured human bone marrow fibroblasts. We conclude that bone marrow fibroblasts produce hematopoietic growth factors in response to interleukin-1, and that this may be a mechanism by which stromal cells regulate cellular growth and differentiation within the hematopoietic microenvironment.     

Hepatocyte growth factor plays roles in the induction and autocrine maintenance of bone marrow stromal cell IL-11, SDF-1 alpha, and stem cell factor

OBJECTIVE: Bone marrow (BM) stroma provides the microenvironment required for long-term hematopoiesis, and this is supported by direct interaction between stromal cells and hematopoietic cells, mediated by adhesion molecules, and through cytokine releases from the BM stroma. In a previous study, we demonstrated that hepatocyte growth factor (HGF) is one of the cytokines constitutively produced from BM stromal cells, promoting hematopoiesis mainly in an indirect way. We also showed that stromal cells themselves express HGF receptor c-MET. It was therefore postulated that HGF exerts its effect on hematopoiesis and maintenance of the hematopoietic microenvironment in a paracrine and autocrine manner. METHODS: The effect of HGF on stromal cells was analyzed by neutralizing intrinsic HGF. RESULTS: Addition of neutralizing anti-HGF antibody inhibited the ability of BM stromal cells to support colony formation from CD34(+) cells and reduced production of significant cytokines from stromal cells, interleukin-11 (IL-11), stromal cell-derived factor-1 alpha (SDF-1 alpha), and to a lesser extent, stem cell factor (SCF). Furthermore, this neutralizing antibody reduced proliferation of stromal cells and inhibited adhesion of stromal cells to collagen type IV and fibronectin. Inhibition of adhesion to fibronectin was mediated by inhibition of alpha(5)beta(1)-integrin. CONCLUSION: These findings indicate that HGF constitutively produced from BM stromal cells is an autocrine regulator, which is able to maintain the hematopoietic microenvironment through stimulating proliferation and adhesion to the extracellular matrix and promoting hematopoiesis through inducing constitutive production of IL-11, SDF-1 alpha, and SCF by stromal cells themselves.     

Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation

We have recently shown that conservation and differentiation of primitive human hematopoietic progenitors in in vitro long-term bone marrow cultures (LTBMC) occurs to a greater extent when hematopoietic cells are grown separated from the stromal layer than when grown in direct contact with the stroma. This finding suggests that hematopoiesis may depend mainly on soluble factors produced by the stroma. To define these soluble factors, we examine here whether a combination of defined early-acting cytokines can replace soluble stroma-derived biologic activities that induce conservation and differentiation of primitive progenitors. Normal human Lineage- /CD34+/HLA-DR- cells (DR-) were cultured either in the absence of a stromal layer ("stroma-free") or in a culture system in which DR- cells were separated from the stromal layer by a microporous membrane ("stroma-noncontact"). Both culture systems were supplemented three times per week with or without cytokines. These studies show that culture of DR- cells for 5 weeks in a "stroma-free" culture supplemented with a combination of four early acting cytokines (Interleukin-3 [IL-3], stem cell factor [SCF], leukemia-inhibitory factor [LIF], and granulocyte colony-stimulating factor [G-CSF]) results in a similar cell expansion as when DR- cells are cultured in "stroma-noncontact" cultures supplemented with the same cytokines. However, generation of committed progenitors and conservation of the more primitive long-term bone marrow culture initiating cells (LTBMC- IC) was far superior in "stroma-noncontact" cultures supplemented with or without IL-3 than in "stroma-free" cultures supplemented with IL-3 alone or a combination of IL-3, LIF, G-CSF, and SCF. These studies indicate that human BM stroma produces soluble factors that can either alone or in synergy with defined cytokines (1) conserve primitive LTBMC- IC, (2) induce early differentiation of a fraction of the primitive progenitors, and (3) prevent their terminal differentiation. We show here that these stroma-derived factors are not likely to be the known early acting cytokines IL-3, SCF, LIF, or G-CSF. Characterization of the stroma-derived factor(s) may have important implications for clinically relevant studies, such as in vitro stem cell expansion in cancer treatment and gene therapy.     

Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.     

Maintenance of high levels of pluripotent hematopoietic stem cells in vitro: effect of stromal cells and c-kit

We show here that mouse pluripotent hematopoietic stem cells can be maintained in vitro on stroma for at least 3 weeks at levels close to those found in bone marrow. The extent of stem cell maintenance is affected by the nature of the stromal cells. The stromal cell line S17 supported stem cells significantly better than heterogeneous, primary stromal layers or the stromal cell line Strofl-1. Stem cells cultured on S17 repopulated all hematopoietic lineages in marrow-ablated hosts for at least 10 months, indicating that this culture system maintained primitive stem cells with extensive proliferative capacity. Furthermore, we demonstrate that, while pluripotent stem cells express c-kit, this receptor appears to play only a minor role in stem cell maintenance in vitro. The addition of an antibody that blocks the interaction of c-kit with its ligand essentially abrogated myelopoiesis in cultures. However, the level of stem cells in antibody-treated cultures was similar to that found in untreated cultures. Thus, it seems likely that the maintenance of primitive stem cells in vitro depends on yet unidentified stromal cell-derived factor(s).     

Regulation of cytokine expression by interferon-alpha in human bone marrow stromal cells: inhibition of hematopoietic growth factors and induction of interleukin-1 receptor antagonist

We investigated the effects of interferon-alpha (IFN-alpha) on the expression of cytokines by human bone marrow stromal cells. Production of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), and interleukin-1 beta (IL-1 beta) in stromal cell layers was induced by incubation with IL-1 alpha, tumor necrosis factor (TNF), or lipopolysaccharide (LPS). Addition of IFN-alpha to such stimulated cultures resulted in a strong downregulation of mRNA expression of GM-CSF and IL-1 beta. Similarly, the protein levels of GM- CSF and IL-1 beta were significantly reduced by IFN-alpha, whereas G- CSF production was only moderately inhibited. In contrast, IFN-alpha markedly stimulated the production of IL-1 receptor antagonist (IL-1RA) by stromal cells. The inhibition of cytokine expression resulted in a reduced hematopoietic activity of stromal cells, indicated by a reduced proliferation of the factor dependent cell line MO7e on IFN-alpha- treated stromal cells. In the presence of cycloheximide (CHX), IFN- alpha failed to inhibit IL-1 mRNA expression, whereas the regulation of GM-CSF and IL-1RA by IFN-alpha was not affected. Our results indicate that the myelosuppressive effects of IFN-alpha, as observed in therapeutic applications or associated with viral infections, are, in part, indirectly mediated by inhibition of the paracrine production of hematopoietic growth factors.     

Hematopoietic growth factors released by marrow stromal cells from patients with aplastic anemia.

We studied the production of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6) by stromal cells from 33 patients with aplastic anemia (AA). Complete, confluent stromal layers were produced by 29 of the 33 samples using the long-term bone marrow culture (LTBMC) system. The concentration of G-CSF, GM-CSF, and IL-6 in culture media with or without interleukin-1 (IL-1) stimulation was determined by an enzyme-linked immunoadsorbent assay (ELISA). The spontaneous production of G-CSF, GM-CSF, and IL-6 did not differ significantly between normal controls and the patients with AA. The ability of stromal cells to release the three hematopoietic growth factors in response to IL-1 was either normal or elevated in all but one patient. We also studied the change in production of G-CSF, GM-CSF, and IL-6 by stromal cells before and after antilymphocyte globulin (ALG) therapy in 16 patients with AA. There was no correlation between the change in production of these cytokines and the response to ALG. In contrast to previous studies that showed a defect in the production of hematopoietic growth factors by stromal cells from patients with AA, the results indicated a normal or elevated production of G-CSF, GM-CSF, and IL-6 by marrow stromal cells in patients with AA.     

Interleukin-6 deficiency affects bone marrow stromal precursors, resulting in defective hematopoietic support

Interleukin-6 (IL-6) is a critical factor in the regulation of stromal function and hematopoiesis. In vivo bromodeoxyuridine incorporation analysis indicates that the percentage of Lin(-)Sca-1(+) hematopoietic progenitors undergoing DNA synthesis is diminished in IL-6-deficient (IL-6(-/-)) bone marrow (BM) compared with wild-type BM. Reduced proliferation of IL-6(-/-) BM progenitors is also observed in IL-6(-/-) long-term BM cultures, which show defective hematopoietic support as measured by production of total cells, granulocyte macrophage-colony-forming units (CFU-GMs), and erythroid burst-forming units (BFU-Es). Seeding experiments of wild-type and IL-6(-/-) BM cells on irradiated wild-type or IL-6-deficient stroma indicate that the hematopoietic defect can be attributed to the stromal and not to the hematopoietic component. In IL-6(-/-) BM, stromal mesenchymal precursors, fibroblast CFUs (CFU-Fs), and stroma-initiating cells (SICs) are reduced to almost 50% of the wild-type BM value. Moreover, IL-6(-/-) stromata show increased CD34 and CD49e expression and reduced expression of the membrane antigens vascular cell adhesion molecule-1 (VCAM-1), Sca-1, CD49f, and Thy1. These data strongly suggest that IL-6 is an in vivo growth factor for mesenchymal precursors, which are in part implicated in the reduced longevity of the long-term repopulating stem cell compartment of IL-6(-/-) mice.     

Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer.

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified, but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved, we performed Northern analyses on RNA extracted from human marrow LTC adherent layers, or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta), IL-6, and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF, GM-CSF, and IL-6 showed significant elevation in growth factor levels 24 hours after IL-1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast, transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC, and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures, respectively. Together, these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC, with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF, GM-CSF, and IL-6. Given the similarities between the LTC system and the marrow microenvironment, it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells.     

A tumor necrosis factor-responsive long-term-culture-initiating cell is associated with the stromal layer of mouse long-term bone marrow cultures.

Long-term bone marrow cultures provide a model for the study of hematopoiesis. Both an intact, adherent stromal layer and hematopoietic stem cells are necessary components in these cultures. Mycophenolic acid treatment of mouse long-term bone marrow cultures depletes them of all assayable hematopoietic precursors. The residual stromal cells are functional and support hematopoiesis if new progenitor cells are supplied. We now show that these mycophenolic acid-treated stromal cell cultures contain cells capable of hematopoietic differentiation without the addition of new progenitors. When treated with tumor necrosis factor alpha (20-200 units/ml), the apparently pure stromal cultures undergo an intense burst of hematopoietic activity. After 4 days such cultures contain approximately 2 x 10(6) hematopoietic cells and, by 1 week, they are indistinguishable from control long-term cultures that were not treated with mycophenolic acid. These results suggest that the stromal cultures either contain hematopoietic stem cells that are maintained quiescent and mycophenolic acid-resistant, perhaps by intimate contact with the stroma, or contain adherent cells that can be induced to differentiate into hematopoietic stem cells. These stem cells are primitive, in that they are capable of multilineage development in the long-term cultures, but are unable to form spleen colonies or myeloid colonies in semisolid medium. These data demonstrate that the adherent fraction of cultured bone marrow contains very primitive hematopoietic cells and that tumor necrosis factor alpha activates their proliferation and differentiation. They also suggest a strategy for obtaining the earliest progenitors free of other, more mature cell types.     

B lymphocyte precursors and myeloid progenitors survive in diffusion chamber cultures but B cell differentiation requires close association with stromal cells

The aim of this study was to investigate the relative contribution of direct contact with stromal cells v stromal cell-derived soluble mediators to the differentiation of B lymphocytes and cells from other hematopoietic lineages. This was investigated by making a comparison between hemopoietic cells grown in direct contact with stroma to those in diffusion chambers (DCs) placed over purified populations of stroma. The source of stromal cells was adherent layers from myeloid or lymphoid long-term bone marrow cultures that had been treated with mycophenolic acid, an antibiotic that depletes hemopoietic cells from the cultures but retains a functional stroma. The cells seeded into the chambers were fresh marrow cells that had been passed through two consecutive nylon wool columns to deplete cell populations capable of forming an adherent cell layer in vitro. DCs were placed in wells in which the adherent stroma, growing under myeloid or lymphoid conditions, was present. The results indicate that progenitors of granulocytes and macrophages survived and differentiated in DCs under myeloid culture conditions, as the number of cells and absolute number of CFU-GM increased over that present in the reseed population. These levels, however, were markedly less than in parallel cultures in which the cells were seeded directly onto stroma. Hematopoiesis in DCs placed over hemopoietically active stroma was not optimal, suggesting that factors were used by those hemopoietic cells closest to the stroma. A B lymphocyte precursor survived in DCs under myeloid but not lymphoid conditions, and its differentiation into B lymphocytes was dependent on close association with stromal cells; B lymphopoiesis initiated when cells from DCs grown under myeloid conditions were harvested from the chambers and seeded directly onto stroma initiated and maintained under lymphoid bone marrow culture conditions. B lymphopoiesis did not initiate if the DC from the myeloid conditions was left intact and placed directly over a lymphoid stromal cell layer in lymphoid conditions.     

Engraftment of a clonal bone marrow stromal cell line in vivo stimulates hematopoietic recovery from total body irradiation.

Whether bone marrow stromal cells of donors contribute physiologically to hematopoietic stem cell reconstitution after marrow transplantation is unknown. To determine the transplantability of nonhematopoietic marrow stromal cells, stable clonal stromal cell line (GB1/6) expressing the a isoenzyme of glucose-6-phosphate isomerase (Glu6PI-a, D-glucose-6-phosphate ketol-isomerase; EC 5.3.1.9) was derived from murine long-term bone marrow cultures and made resistant to neomycin analogue G418 by retroviral gene transfer. GB1/6 cells were fibronectin+, laminin+, and collagen-type IV+ and collagen type I-; these GB1/6 cells supported in vitro growth of hematopoietic stem cells forming colony-forming units of spleen cells (CFU-S) and of granulocytes, erythrocytes, and macrophage/megakarocytes (CFU-GEMM) in the absence of detectable growth factors interleukin 3 (multi-colony-stimulating factor), granulocyte/macrophage colony-stimulating factor, granulocyte-stimulating factor, or their poly(A)+ mRNAs. The GB1/6 cells produced macrophage colony-stimulating factor constitutively. Recipient C57BL/6J (glucose-6-phosphate isomerase b) mice that received 3-Gy total-body irradiation and 13 Gy to the right hind limb were injected i.v. with GB1/6 cells. Engrafted mice demonstrated donor-originating Glu6PI-a+ stromal cells in marrow sinuses in situ 2 mo after transplantation and a significantly enhanced hematopoietic recovery compared with control irradiated nontransplanted mice. Continuous (over numerous passages) marrow cultures derived from transplanted mice demonstrated G418-resistant, Glu6PI-a+ stromal colony-forming cells and greater cumulative production of multipotential stem cells of recipient origin compared with cultures established from irradiated, nontransplanted control mice. These data are evidence for physiological function in vivo of a transplanted bone marrow stromal cell line.     

Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages

The bone marrow microenvironment supports and regulates the proliferation and differentiation of hematopoietic cells. Dysregulated hematopoiesis in chronic myelogenous leukemia (CML) is caused, at least in part, by abnormalities in CML hematopoietic progenitors leading to altered interactions with the marrow microenvironment. The role of the microenvironment itself in CML has not been well characterized. We examined the capacity of CML stroma to support the growth of long-term culture-initiating cells (LTC-IC) obtained from normal and CML marrow. The growth of normal LTC-IC on CML stroma was significantly reduced compared with normal stroma. This did not appear to be related to abnormal production of soluble factors by CML stroma because normal LTC- IC grew equally well in Transwells above CML stroma as in Transwells above normal stroma. In addition, CML and normal stromal supernatants contained similar quantities of both growth-stimulatory (granulocyte colony-stimulating factor (CSF), interleukin-6, stem cell factor, granulocyte-macrophage CSF, and interleukin-1 beta) and growth- inhibitory cytokines (transforming growth factor-beta, macrophage inflammatory protein-1 alpha, and tumor necrosis factor-alpha). The relative proportion of different cell types in CML and normal stroma was similar. However, polymerase chain reaction and fluorescence in situ hybridization studies showed the presence of bcr-abl-positivo cells in CML stroma, which were CD14+ stromal macrophages. To assess the effect of these malignant macrophages on stromal function, CML and normal stromal cells were separated by fluorescence-activated cell sorting into stromal mesenchymal cell (CD14-) and macrophage (CD14+) populations. CML and normal CD14- cells supported the growth of normal LTC-IC equally well. However, the addition of CML macrophages to normal or CML CD14- mesenchymal cells resulted in impaired progenitor support. This finding indicates that the abnormal function of CML bone marrow stroma is related to the presence of malignant macrophages. In contrast to normal LTC-IC, the growth of CML LTC-IC on allogeneic CML stromal layers was not impaired and was significantly better than that of normal LTC-IC cocultured with the same CML stromal layers. These studies demonstrate that, in addition to abnormalities in CML progenitors themselves, abnormalities in the CML marrow microenvironment related to the presence of malignant stromal macrophages may contribute to the selective expansion of leukemic progenitors and suppression of normal hematopoiesis in CML.     

Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells.

Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell-free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.