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.     

Correlation between nicotine-induced inhibition of hematopoiesis and decreased CD44 expression on bone marrow stromal cells.

This study demonstrates that in vivo exposure to cigarette smoke (CS) and in vitro treatment of long-term bone marrow cultures (LTBMCs) with nicotine, a major constituent of CS, result in inhibition of hematopoiesis. Nicotine treatment significantly delayed the onset of hematopoietic foci and reduced their size. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) within an adherent layer of LTBMCs was significantly reduced in cultures treated with nicotine. Although the production of nonadherent mature cells and their progenitors in nicotine-treated LTBMCs was inhibited, this treatment failed to influence the proliferation of committed hematopoietic progenitors when added into methylcellulose cultures. Bone marrow stromal cells are an integral component of the hematopoietic microenvironment and play a critical role in the regulation of hematopoietic stem cell proliferation and self-renewal. Exposure to nicotine decreased CD44 surface expression on primary bone marrow-derived fibroblastlike stromal cells and MS-5 stromal cell line, but not on hematopoietic cells. In addition, mainstream CS altered the trafficking of hematopoietic stem/progenitor cells (HSPC) in vivo. Exposure of mice to CS resulted in the inhibition of HSPC homing into bone marrow. Nicotine and cotinine treatment resulted in reduction of CD44 surface expression on lung microvascular endothelial cell line (LEISVO) and bone marrow-derived (STR-12) endothelial cell line. Nicotine treatment increased E-selectin expression on LEISVO cells, but not on STR-12 cells. These findings demonstrate that nicotine can modulate hematopoiesis by affecting the functions of the hematopoiesis-supportive stromal microenvironment, resulting in the inhibition of bone marrow seeding by LTC-ICs and interfering with stem cell homing by targeting microvascular endothelial cells.     

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.     

Increased longevity of hematopoiesis in continuous bone marrow cultures derived from NOS1 (nNOS, mtNOS) homozygous recombinant negative mice correlates with radioresistance of hematopoietic and marrow stromal cells

OBJECTIVE: Neuronal nitric oxide synthase (NOS1, mitochondrial NOS, neuronal NOS) homozygous deletion recombinant negative mice demonstrate ionizing irradiation resistance in vivo, attributable to the decrease in mitochondrial-localized production of peroxynitrite, a potent lipid toxic free radical species resulting from the combination of nitric oxide and superoxide. The present studies were designed to determine whether reduced mitochondrial generation of toxic radical oxygen species in NOS1-/- mice also increased the longevity of hematopoiesis in continuous bone marrow cultures and conferred radioresistance to cells in vitro. MATERIALS AND METHODS: Long-term bone marrow cultures (LTBMCs) were established from NOS1-/- and NOS1+/+ littermate mice. Radiation resistance of hematopoietic and marrow stromal cells was measured. Cell cycle analysis and measurement of glutathione and glutathione peroxidase were carried out on irradiated clonal bone marrow stromal cell lines. RESULTS: A significant increase in longevity of hematopoiesis was detected in NOS1-/- mouse LTBMCs for over 64 weeks in culture compared to 20 weeks for NOS1+/+ mouse LTBMCs (p < 0.001). Permanent bone marrow stromal cell lines derived from NOS1-/- mouse LTBMCs demonstrated increased radioresistance in vitro reflected by an increased shoulder on the survival curve with n = 32.15 +/- 1.21 compared to NOS1+/+ cells n = 10.47 +/- 3.2 (p = 0.0026), interleukin-3-dependent NOS1-/- hematopoietic progenitor cell lines also demonstrated decreased apoptosis after 10 Gy irradiation. Both pre- and postirradiation stabilization of the cellular antioxidant pool was detected in NOS1-/- cells. NOS1-/- cells showed a prolonged G1 cell cycle arrest after 10 Gy. CONCLUSIONS: Prolonged hematopoiesis in LTBMCs correlates with intrinsic radioresistance of hematopoietic and marrow stromal cells from NOS1-/- mice. The data confirm the importance to hematopoiesis of mitochondrial localized nitric oxide in both radioresistance and longevity of hematopoiesis in continuous bone marrow cultures.     

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.     

A murine stromal cell line allows the proliferation of very primitive human CD34++/CD38- progenitor cells in long-term cultures and semisolid assays

Analysis of molecular mechanisms associated with stem cell commitment and differentiation requires an in vitro assay that identifies the most primitive hematopoietic stem cells in human bone marrow. Such primitive stem cells usually do not form colonies in short-term semisolid assays and are best identified by their ability to initiate sustained hematopoiesis when they are cocultured with competent stromal cells. In this study, we investigated whether a murine marrow stromal cell line (MS-5) that supports colony-forming unit-spleen (CFU-S) maintenance would permit, both in short-term colony assays and long-term cultures, the development of primitive human stem cells sorted on the basis of their high expression of CD34 and lack of expression of CD38 antigen. In short-term colony assays, this population included almost exclusively primitive progenitor cells. MS-5 cells synergized with any combination of interleukin-3, Steel factor, granulocyte colony- stimulating factor, agar-leukocyte conditioned medium, and erythropoietin and increased at least twofold both the cloning efficiency of CD34++/CD38- cells and the size of the colonies. Furthermore, MS-5 cells triggered the development of multipotent blast cell progenitors with a high proliferative potential, which in these conditions represented 1% to 2% of CD34++/CD38- cells. When MS-5 cells were substituted by human stromal cells or when growth factor combinations were used in the absence of stromal cells, much lower numbers of CFU-blast were detected. This selective action of MS-5 on early progenitors was also observed when MS-5 cells were used as feeders in long-term cultures of CD34++/CD38- cells. Murine cells promoted the expansion of high proliferative potential primitive progenitor cells up to 3 months, although they did not support their differentiation in mature clonogenic progenitors or terminally differentiated cells. Sustained hematopoiesis in these longterm cultures was accounted for by 2% to 5% of initial CD34++/CD38- cells as estimated by limiting dilution experiments. Mechanisms by which murine stromal cells act specifically on human primitive stem cells are unclear, but from our data this effect is unlikely to be explained solely by known species cross-reactive growth factors. Further manipulation of this long-term coculture system should prove useful in identifying stromal molecules regulating commitment and differentiation of early human progenitor cells.     

CCL18/PARC stimulates hematopoiesis in long-term bone marrow cultures indirectly through its effect on monocytes

Chemokines play a role in regulating hematopoietic stem cell function, including migration, proliferation, and retention. We investigated the involvement of CCL18 in the regulation of bone marrow hematopoiesis. Treatment of human long-term bone marrow cultures (LTBMCs) with CCL18 resulted in significant stimulation of hematopoiesis, as measured by the total number of hematopoietic cells and their committed progenitors produced in culture. Monocytes/macrophages, whose survival was almost doubled in the presence of CCL18 compared with controls, were the primary cells mediating this effect. Conditioned media from CCL18-treated mature monocytes fostered colony-promoting activity that increased the number of colonies formed by hematopoietic progenitor cells. Gene expression profiling of CCL18-stimulated monocytes demonstrated more than 200 differentially expressed genes, including those regulating apoptosis (caspase-8) and proliferation (IL-6, IL-15, stem cell factor [SCF]). Up-regulation of these cytokines was confirmed on the protein expression level. The contribution of SCF and IL-6 in CCL18-mediated stimulatory activity for hematopoiesis was confirmed by SCF- and IL-6-blocking antibodies that significantly inhibited the colony-promoting activity of CCL18-stimulated conditioned medium. In addition to the effect on monocytes, CCL18 facilitated the formation of the adherent layer in LTBMCs and increased the proliferation of stromal fibroblast-like cells.     

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.     

Extracellular matrix production by the adherent cells of long-term murine bone marrow cultures

We have studied the deposition of extracellular matrix proteins in the adherent stroma of long-term murine bone marrow cultures. Stable hematopoiesis was maintained for greater than 12 wk. At selected intervals, culture dishes were sacrificed by removing all nonadherent cells and air drying the dishes. The adherent stromal layer was analyzed for the presence of intracellular and extracellular collagen, fibronectin, and laminin using double immunofluorescent staining with specific antisera against these matrix components. In cultures examined during the first 2 wk, large numbers of stromal cells contained collagen, fibronectin, and laminin. Over the next 2 wk, an extensive extracellular network of fibronectin, laminin, and collagen was deposited on the dishes, which persisted throughout the life of the cultures. In contrast to a previous report, we detected substantial numbers of endothelial cells by means of immunofluorescent staining of stromal cells with antisera to type IV collagen, laminin, and factor VIII antigen. Although deposition of these extracellular matrix proteins coincides with onset of active hematopoietic cell production, the relative roles of the stromal cells and the extracellular matrix in supporting hematopoiesis in murine bone marrow cell cultures remain to be determined.     

Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6)

Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively gamma-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be gamma-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells.     

Increased longevity of hematopoiesis in continuous bone marrow cultures and adipocytogenesis in marrow stromal cells derived from Smad3(-/-) mice

OBJECTIVE: To determine the role of Smad3 in modulating hematopoiesis, continuous bone marrow cultures were established from Smad-/- mice, and the longevity of hematopoiesis and extent of adipogenesis in the supportive hematopoietic microenvironment were compared to those from cultures of control, Smad3+/+ or heterozygous Smad3+/- mice. MATERIALS AND METHODS: Long-term bone marrow cultures (LTBMCs) were established from Smad3+/+, Smad3+/-, or Smad3-/- mice. On a weekly basis, the number of cobblestone islands, number of nonadherent cells, confluence of the adherent cells, or CFU-GEMM colonies was determined. Bone marrow stromal cell lines were established and cobblestone island production on these cell lines determined in the presence of nonadherent cells from week-42 Smad3-/- or week-4 C57BL/6J LTBMCs. RESULTS: Initial proliferative capacity of the LTBMCs was similar in all groups through week 20, at which time there was an increase in cobblestone islands and production of nonadherent cells and CFU-GEMM colonies in the Smad3-/- group. By week 28, only the Smad3-/- LTBMCs had significantly maintained increased production of these parameters. Maintenance of cobblestone islands indicative of the most primitive hematopoietic progenitor cells persisted past 45 weeks in Smad3-/- cultures. The Smad3-/- stromal cell line also demonstrated increased support of cobblestone island production when incubated with nonadherent cells from week-42 Smad3-/- or week-4 C57BL/6J LTBMCs. Evaluation of adipocytogenesis in stromal cells showed significantly greater accumulation of adipocytes in lines from Smad3-/- than from Smad3+/+ mice. CONCLUSIONS: These data provide evidence for a significant effect of deletion of the Smad3 signaling pathway in increased hematopoiesis in LTBMCs and support the negative regulatory influence of TGFbeta signaling on adipocytogenesis and long-term hematopoiesis in vitro.     

Self-renewal and differentiation of a novel bipotent myeloid progenitor clone in the stroma-dependent culture

To understand regulation of myeloid development, it is necessary to obtain the myeloid progenitor cell lines with self-renewal and differentiation capacities. Because prolonged hematopoiesis occurs with the production of myeloid cells at all stages of differentiation in the Dexter-type long-term bone marrow cultures, we tried to obtain stroma-dependent myeloid progenitor cells starting from the long-term bone marrow culture. Murine cobblestone areas generated in long-term bone marrow cultures were serially passaged every 10 days. After 4 months, the resultant hematopoietic cells, designated as DFC, were passaged on a monolayer of established spleen stromal cell line, MSS62. After 10-12 passages of DFC cells on MSS62, several clones were obtained by colony formation on MSS62 cell layer. Among these clones, DFC-a cells could be maintained for a long period by coculturing with the established stromal cell line, MSS62.DFC-a cells proliferated by forming cobblestones and contained blast cells, granulocytes, and macrophages. Cell sorting and coculture experiments indicated that the blast type cells exhibiting c-Kit(+) Gr-1(-) Mac-1(-), stroma-dependently self-renewed, and spontaneously differentiated toward granulocytes (c-Kit(+) Gr-1(+) Mac-1(+)) and macrophages (c-Kit(low/+) Gr-1(-) Mac-1(high)). Although most of DFC-a cells expressed c-Kit, SCF-c-Kit interaction was not always necessary for their growth. In the presence of stromal cells, growth and differentiation of DFC-a cells were stimulated by GM-CSF or IL-3. Without stromal cells, DFC-a was transiently expanded by GM-CSF or IL-3 but could not be maintained constantly by these cytokines.The present study demonstrated that DFC-a is a novel bipotent myeloid progenitor cell clone as a simple model system of stroma-dependent myeloid development. It may reflect distinct properties for the earliest myeloid progenitor cells in vivo. It is of interest to know what signals are provided by MSS62 stromal cells to maintain the myeloid progenitor cells.     

Bone marrow dysfunction in mice lacking the cytokine receptor gp130 in endothelial cells

In vitro studies suggest that bone marrow endothelial cells contribute to multilineage hematopoiesis, but this function has not been studied in vivo. We used a Cre/loxP-mediated recombination to produce mice that lacked the cytokine receptor subunit gp130 in hematopoietic and endothelial cells. Although normal at birth, the mice developed bone marrow dysfunction that was accompanied by splenomegaly caused by extramedullary hematopoiesis. The hypocellular marrow contained myeloerythroid progenitors and functional repopulating stem cells. However, long-term bone marrow cultures produced few hematopoietic cells despite continued expression of gp130 in most stromal cells. Transplanting gp130-deficient bone marrow into irradiated wild-type mice conferred normal hematopoiesis, whereas transplanting wild-type bone marrow into irradiated gp130-deficient mice did not cure the hematopoietic defects. These data provide evidence that gp130 expression in the bone marrow microenvironment, most likely in endothelial cells, makes an important contribution to hematopoiesis.     

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.     

Human bone marrow stromal cell: coexpression of markers specific for multiple mesenchymal cell lineages

The role of hematopoietic stem cells in blood cell development is reasonably understood, whereas the identity and the function of bone marrow stromal cells are much less clear. Using stromal cells in bone marrow cultures of the Dexter type, a favorite medium for the study of hematopoiesis, we show that stromal cells actually represent a unique cell type. Conventional wisdom has held that stromal cells in Dexter cultures comprise a mixture of macrophages, hematopoietic cells, adipocytes, osteoblasts, fibroblasts, muscle cells, and endothelial cells. Our findings demonstrate that Dexter cultures consist of three cell types: macrophages ( approximately 35%), hematopoietic cells ( approximately 5%), and nonhematopoietic cells ( approximately 60%). We have purified the nonhematopoietic cells free of macrophages and hematopoietic cells to produce compelling evidence that they in fact represent a single cell type (multidifferentiated mesenchymal progenitor cell, MPC) which coexpresses genes specific for various mesenchymal cell lineages including adipocytes, osteoblasts, fibroblasts, and muscle cells. We further show that these multi- or pluridifferentiated MPCs are capable of supporting hematopoiesis by demonstrating the expression of several hematopoietic growth factors and extracellular matrix receptors including G-CSF, SCF, VCAM-1, ICAM-1, and ALCAM. Since the MPCs can be easily purified to near homogeneity (95%), they can be of value in enhancing engraftment of hematopoietic stem cells. Also, this new understanding of bone marrow stromal cells as "one cell with many different faces" promises to advance our knowledge of regulatory cellular interactions within bone marrow.     

Study of possible correlations between in vitro growth of bone marrow stromal and hematopoietic precursor cells early after allogeneic bone marrow transplantation.

Growth characteristics of stromal cells, assessed as adherent cells in long-term bone marrow cultures, and of hematopoietic progenitor cells, prior to and shortly after allogeneic bone marrow transplantation (BMT), were investigated, more specifically with regard to their possible correlations. The main constituent cells of the bone marrow stroma, that is, endothelial cells, reticular cells/fibroblasts, and monocytes/macrophages, showed an as yet inexplicable increased growth in samples taken from recipients prior to BMT, as compared with the growth in samples from their healthy donors and those taken after BMT. In the third week after BMT the in vitro outgrowth of hematopoietic precursors was severely depressed, but cell numbers in the adherent layer were normal. No relationship between in vitro growth of hematopoietic precursor cells and stromal cells was observed at that time. Probably the precursor cells growing in vitro are committed progenitor cells, relatively independent of stromal influences. In the eight week after grafting, endothelial cell outgrowth in vitro was highly correlated with granulocyte-macrophage colony-forming unit (CFU-GM) colony formation and to a lesser extent with mixed-lineage colony-forming unit (CFU-Mix) colony formation. This may indicate the reappearance of cytokine-mediated influences or the reappearance of a direct interaction, for example, by cell-cell contact between stromal cells and hematopoietic progenitor cells at that time.     

Normal bone marrow hematopoietic stem cell reserves and normal stromal cell function support the use of autologous stem cell transplantation in patients with multiple sclerosis

Bone marrow (BM) stem cell reserves and function and stromal cell hematopoiesis supporting capacity were evaluated in 15 patients with multiple sclerosis (MS) and 61 normal controls using flow cytometry, clonogenic assays, long-term BM cultures (LTBMCs) and enzyme-linked immunosorbent assays. MS patients displayed normal CD34+ cell numbers but a low frequency of colony-forming cells (CFCs) in both BM mononuclear and purified CD34+ cell fractions, compared to controls. Patients had increased proportions of activated BM CD3+/HLA-DR+ and CD3+/CD38+ T cells that correlated inversely with CFC numbers. Patient BM CD3+ T cells inhibited colony formation by normal CD34+ cells and patient CFC numbers increased significantly following immunomagnetic removal of T cells from BMMCs, suggesting that activated T cells may be involved in the defective clonogenic potential of hematopoietic progenitors. Patient BM stromal cells displayed normal hematopoiesis supporting capacity indicated by the CFC number in the nonadherent cell fraction of LTBMCs recharged with normal CD34+ cells. Culture supernatants displayed normal stromal derived factor-1 and stem cell factor/kit ligand but increased flt-3 ligand levels. These findings provide support for the use of autologous stem cell transplantation in MS patients. The low clonogenic potential of BM hematopoietic progenitors probably reflects the presence of activated T cells rather than an intrinsic defect.     

Interleukin-4 induces a substance in bone marrow stromal cells that reversibly inhibits factor-dependent and factor-independent cell proliferation

Bone marrow-derived stromal cell monolayers pretreated with recombinant interleukin-4 (IL-4) inhibit the growth of hematopoietic cells. This was demonstrated by inhibition of fresh bone marrow-derived, IL-3- induced soft agar colonies as well as by inhibition of proliferation of IL-3-dependent cell lines and of a Friend virus-transformed erythroleukemic cell line. Pretreatment of stromal cells with IL-4 for five to seven days induced the inhibitory activity. IL-4 could then be removed before "plating" the bone marrow cells in soft agar, indicating that the inhibitory activity did not depend on the action of IL-4 on the precursors of the soft agar colonies. The inhibitory activity appears to be mediated by a soluble factor since inhibition was achieved even if the stromal cell layer was separated from the colony forming cells by an "empty" agar layer. However, supernatants of IL-4- induced stromal cell layers had no detectable inhibitory activity. The inhibitory action of the IL-4-pretreated stromal cell lines was not the result of killing of the precursor cells since it could be reversed if the agar layer containing the colony-forming cells was removed from the stromal cell layer and cultured with IL-3. Hydrocortisone (HC) blocked the inhibitory effect if added either in the IL-4 preincubation phase or during the colony formation stage, implying that HC blocked both induction of the inhibitory activity and its release or its effector function. A homogenous long-term stromal cell line could not be induced to exert the inhibitory activity; partial inhibition could be achieved with pure macrophages stimulated with IL-4 and CSF-1, suggesting that the inhibitory activity induced by IL-4 in mixed stromal cell layers may depend on a complex mechanism involving more than one cell type. Northern analysis of RNA from IL-4-induced and uninduced stromal cells indicated that IL-4 did not upregulate expression of CSF-1 or transforming growth factor-beta (TGF-beta) and only modestly increased expression of tumor necrosis factor, suggesting that these cytokines were not responsible for the inhibitory activity. The capacity of IL-4 to induce inhibitory activity in stromal cell layers suggests that IL-4 may play a role in the regulation of hematopoiesis.     

The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence- activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38- , HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.     

Inhibition of hematopoiesis in normal human long-term marrow cultures treated with recombinant human macrophage colony-stimulating factor

The effects of recombinant human macrophage colony-stimulating factor (rhCSF-1) in long-term marrow cultures (LTMC) established from normal bone marrow cells were examined. When added during the first 3 weeks of culture (every second day, at 15 ng/mL), rhCSF-1 strongly inhibited the growth of all hematopoietic progenitors analyzed (colony-forming unit-MIX [CFU-MIX], CFU-granulocyte macrophage [CFU-GM], CFU-M, CFU-G, burst-forming unit-erythroid). Paralleling the inhibition of progenitors was the complete loss of adipocytes from the stromal layer of rhCSF-1-treated cultures. The inhibitory effect of rhCSF-1 correlated in all instances with the accumulation in the supernatants of these cultures of an activity (different from CSF-1) that inhibited colony formation in semisolid cultures. When addition of rhCSF-1 was delayed 3 weeks, its inhibitory effects were significantly reduced, which correlated with reduced inhibitory activity detected in the supernatants. Analysis of CSF-1 concentration by radioreceptor assay confirmed that added rhCSF-1 increased culture CSF-1 levels and showed that the decreased inhibition observed when rhCSF-1 is added later in culture was not due to decreased CSF-1 levels at that point. In contrast, the ability of rhCSF-1 to inhibit hematopoiesis and accumulate inhibitory activity in LTMC correlated with its rate of utilization, much higher in the first 2 weeks of culture, when the stromal layer was being established, than later. These observations document the inhibitory effect of rhCSF-1 on all aspects of hematopoiesis conducted in cultures that simulate the hematopoietic microenvironment, demonstrate the importance of accessory/stromal cells in mediating the effects of rhCSF-1 in LTMC, and point to an inhibitory activity as the mediating agent.