The cytoskeleton of stromal cells from human bone marrow cultures resembles that of cultured smooth muscle cells

The cytoskeleton of stromal cells from the adherent layer of human Dexter-type cultures has been studied. It was found that the stress fibers contained actin specific for smooth muscle, mainly the alpha SM actin isoform. The intermediate filaments consisted of vimentin, and there were no desmin filaments. This pattern was similar to that of cultured vascular smooth muscle cells. The detectability of the alpha SM actin isoform is coincident with the appearance of stromal cells in long-term marrow cultures and may provide a useful marker for stromal cells. The potential in vivo cellular counterpart for stromal cells generated in the Dexter-type culture system is discussed.     

Role of stromal cells and macrophages in fibronectin biosynthesis and matrix assembly in human long-term marrow cultures

Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.     

CGA-7 and HHF, two monoclonal antibodies that recognize muscle actin and react with adherent cells in human long-term bone marrow cultures

The CGA-7, a monoclonal antibody that reacts with smooth muscle cell actin but not with endothelial cell or fibroblast actin, and HHF, a monoclonal antibody that reacts with smooth muscle, skeletal muscle, and cardiac muscle actin, both recognize microfilaments present within adherent cells from actively hematopoietic human long-term marrow cultures. Macrophages, monocytes, and cultured marrow fibroblasts do not react with either antibody. These data suggest that the anti-actin antibodies may serve as useful markers for in vitro microenvironmental cells and lend support to the hypothesis that stromal cells from long- term marrow cultures are different from marrow fibroblasts and may constitute a unique cell lineage.     

Modification of human long-term bone marrow cultures: establishment of a functional stromal microenvironment devoid of myeloid progenitors.

Differences in the plastic adhesive properties of bone marrow (BM) cells were used to initiate modified stromal layers (MSL) from long-term cultures by removing non-adherent cells shortly (4 to 18 hours) after initial seeding. Following this early modification, adherent cells generated a confluent layer after 21 days of culture. Cellular characteristics of volume and spontaneous fluorescence determined by flow cytometry showed that the MSL included 82% fibroblastic stromal cells, 8% macrophages and 10% myelomonocytic cells. Furthermore, clonogenic assays revealed that the MSL were devoid of hematopoietic progenitor cells. MSL were found to sustain long-term myelopoiesis for at least 7 weeks from exogenously added hematopoietic progenitors isolated from bone marrow (CD34+ cells), thereby demonstrating their functionality. The present experimental model appears of interest for the study of interactions between defined populations of hematopoietic cells and cells of the adherent layer. Of importance, our present modifications of human long-term bone marrow culture are technically simple and do not involve manipulation of the stromal cells.     

The origin of stromal cells in patients treated by bone marrow transplantation

The source of stromal cells following bone marrow transplantation has been investigated by culturing marrow cells obtained from patients who have a graft cells from a donor of the opposite sex. Two culture systems have been used for these studies. The first supports the proliferation of fibroblast colony-forming cells (F-CFC) and the formation of confluent layers of fibroblastoid cells; the second (the long-term bone marrow culture system) results in the formation of a complex and heterogeneous layer of adherent cells composed of fibroblasts, fat cells, endothelial cells, macrophages and 'blanket' cells. A survey of the work done using these culture systems shows that, although the issue of the transplantability of stromal cells remains controversial, it now seems reasonably certain that repopulation of the fibroblast component by donor cells is rare. In contrast, one study has shown that endothelial-like cells can engraft in transplant recipients. Another study has demonstrated a donor origin for the macrophages found in long-term cultures of marrow from transplanted patients. This finding might have been predicted because macrophages are derived from haemopoietic stem cells.     

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1- stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.     

Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of CD34++/CD38- long-term culture-initiating cells

Very primitive human hematopoietic progenitor cells are identified indirectly by their ability to give rise to clonogenic progenitors in the presence of either human or murine stromal cells. These long-term culture-initiating cell (LTC-IC) assays are usually performed in the presence of hydrocortisone based on the initial observation that hydrocortisone was required for prolonged hematopoiesis in standard long-term bone marrow cultures. In this report, we investigated the role of hydrocortisone in LTC-IC assays initiated with CD34++/CD38- cells seeded onto either human bone marrow LTC-derived adherent cells or a murine marrow-derived stromal cell line, MS-5. It was found that weekly addition of hydrocortisone to the cultures reduced the frequency of LTC-IC (from 1/5 to 1/20) calculated from limiting dilution experiments and also reduced fivefold to 10-fold the number of their progeny clonogenic cells detected after 4 to 5 weeks. In contrast, the frequency and differentiative potential of CD34++/CD38- grown in the presence of human marrow feeders was unaltered by the addition of glucocorticoids. Data are consistent with the hypothesis that hydrocortisone inhibited LTC-IC differentiation by downregulating the expression of a synergistic factor produced by MS-5 cells. (1) In the absence of hydrocortisone, the number of clonogenic progenitors generated by LTC-IC was much higher in cultures seeded on MS-5 than in cultures seeded on human marrow adherent cells, which was also true when cytokines were added to the cocultures. However, based on the phenotype of the colonies, progenitors produced in MS-5 cocultures were more mature than those generated on human marrow adherent cells. (2) Hydrocortisone counteracted the stimulatory effect of recombinant human cytokines (interleukin-3, interleukin-6, and steel factor) in assays performed on MS-5 but not on human marrow feeders. (3) Hydrocortisone led to a 50% decrease in the numbers of colony-forming units- granulocyte-macrophage found in methycellulose colony assays of CD34++/CD38- cells performed in the presence of MS-5 cells. Taken together, our results indicate that hydrocortisone acts differently on a murine stromal cell line and on marrow-derived human stromal cells and may suppress the expression by MS-5 cells of an activity selectively promoting amplification of clonogenic cells derived from primitive LTC-IC.     

Effects of marrow storage at 4 degrees C on the subsequent generation of long-term marrow cultures

The present study was undertaken to examine the effect of marrow preservation at 4 degrees C on subsequent long-term culture, which evaluates both hematopoietic precursor cells and hematopoietic microenvironmental cells. Storage of unfractionated marrow was superior to storage of buffy-coat cells in tissue culture medium with 20% fetal calf serum. CFU-C recovery in unfractionated marrow was 48.4% at four days and 21.4% at seven days. Long-term marrow cultures from cells stored at 4 degrees C for up to seven days produced CFU-C for up to seven weeks and established confluent marrow stromal cell layers. Suspension cultures of marrow cells preserved at 4 degrees C for seven days cultured with irradiated allogeneic marrow stromal cell layers from normal long-term marrow cultures showed significantly increased CFU-C production from week 2 to week 5 when compared with the control cultures without adherent cell layers. These data suggest that marrow storage at 4 degrees C for up to seven days preserves early hematopoietic precursor cells and microenvironmental cells and may be used for autologous rescue from marrow ablative therapy.     

Expression of alpha-smooth muscle actin in murine bone marrow stromal cells

Human fibrotic bone marrow (BM) stroma has been shown to contain alpha-smooth muscle actin (alpha-SMA)-positive cells. These closely resemble myofibroblasts that were described in other fibrotic tissues. We studied the expression of alpha-SMA in a series of murine BM-derived stromal cell lines to investigate the cellular origin and functional significance of myofibroblast-like cells in hematopoietic tissues. Although these cell lines differed in their biologic properties, most of them expressed alpha-SMA under certain conditions. Cells expressing alpha-SMA constituted a minor population in post-confluent, growth-arrested cultures. However, the incidence of cells expressing alpha-SMA increased significantly when cultures were transferred to nonconfluent conditions. A similar increase in alpha-SMA-positive cells occurred after a strip of cells was scraped away from the confluent cell layer; the cells of the affected area acquired alpha-SMA-positive contractile phenotype. The relationship between alpha-SMA expression and hematopoietic activity was studied using a cloned cell line of BM origin (14F1.1). The ability of these endothelial-adipocyte cells to support hematopoiesis in vitro was maximal under confluent conditions, whereas their expression of alpha-SMA under such conditions was residual. Moreover, in long-term BM cultures supported by confluent 14F1.1 cells, stromal areas associated with proliferating hematopoietic precursors, known as "cobblestone areas," were devoid of alpha-SMA-positive cells. These observations suggest that the expression of alpha-SMA is reversible and inversely related to hematopoietic activity.     

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.     

Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins

This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.     

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.     

CD34 expression by stromal precursors in normal human adult bone marrow.

Normal bone marrow cells were isolated by fluorescence-activated cell sorting (FACS) on the basis of CD34 antigen expression and then assayed in vitro for colonies of fibroblastic cells (fibroblast colony-forming units [CFU-F]). Greater than 95% of detectable CFU-F were recovered in the CD34+ population, while their numbers were markedly depleted in the CD34- population. Additional experiments showed that the majority of CFU-F exhibited high forward and perpendicular light scatter and low-density CD34 antigen. Growth of sorted cells in medium optimized for long-term marrow culture (LTMC) produced a complex mixture of adherent stromal elements including fibroblasts, adipocytes, smooth muscle cells, and macrophages. Monoclonal antibody STRO-1, which identifies bone marrow stromal cells, reacted with approximately 5% of CD34+ cells, which included all CFU-F and stromal precursors in LTMC. Experiments using soybean agglutinin (SBA) further showed that these stromal elements were restricted to a population of bone marrow cells with the phenotype CD34+/SBA+. These properties of stromal precursors are quite distinct from those of primitive hematopoietic progenitors, showing that although the precursors of the hematopoietic and stromal systems share expression of CD34, they are otherwise phenotypically distinct cell types.     

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.     

Human B-lymphopoiesis is supported by bone marrow-derived stromal cells

We have recently reported an in vitro culture system that allows the clonal growth and differentiation of normal human bone marrow B-lineage cells. In the report presented here, we have used this B-cell colony assay to study the influence of cellular components of the human bone marrow microenvironment on B-lymphopoiesis. It is demonstrated that bone marrow stromal cells were able to provide all the necessary requirements for the growth and differentiation of B-lineage cells under the conditions of the B-cell colony assay. These stromal cells were obtained from long-term bone marrow cultures (LTBMC) that had been established from the spicules in human bone marrow. When these stromal cells were plated as an adherent underlayer in the double-agar B-cell colony assay, both immature and mature B-lineage cells were induced to differentiate into colonies containing cells that secreted immunoglobulin. The stromal cells from these spicule-derived LTBMCs maintained the capacity to support B-cell colony formation for up to 9 months.     

Origin of hemopoietic stromal progenitor cells in chimeras

Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACL in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice.     

Granulopoiesis-supporting effects of marrow stromal cells in continuous culture from patients with primary myelofibrosis

The granulopoiesis-supporting effects of marrow adherent cells from seven patients with primary myelofibrosis (PMF) were studied by a continuous allogeneic co-culture system in which the survival of donor granulocyte-macrophage colony-forming cells (CFU-GM) depends upon the supporting ability of adherent cells. Marrow adherent cells from all these seven patients were able to sustain the same number of CFU-GM as those from control subjects. Cytochemical studies showed that colonies grown from cells sustained on fibrous marrow adherent cell layers were predominantly neutrophilic, as were those in control cultures, although many eosinophil colonies grew from patients' bone marrow. These results indicate that marrow stromal cells from PMF patients function normally in their ability to support granulopoiesis.     

CFU-C proliferation and granulopoiesis in suspension cultures of bone marrow,cord blood and adult blood

Summary. Normal bone marrow, cord blood and adult blood cells were separated on ficoll-hypaque density gradients and cultured in MCoy 5A medium with 15% human serum in 25 cm² flasks for up to 14 days to characterize their growth. The system permits the study of granulopoiesis from the generation in culture of new CFU-C to the emergence of neutrophils and monocyte-macrophage cells at the end of the culture period. Bone marrow, cord blood and adult blood, showed 2-3-fold increases in CFU-C, but characteristically only small numbers of CFU-C were detected in adult blood, and only small numbers of granulocytes were produced. The total nucleated cell count only rose consistently in bone marrow cultures. Adherent cell layers formed in all cultures with confluent or near confluent growth of macrophages after 14 days. Some macrophages were multinucleated giant cells; these cells were found most commonly in adult blood cultures. Exclusively in bone marrow cultures macroscopic fibroblast colonies and smaller fibroblast clusters of about 20-50 cells occurred. CFU-C did not proliferate if they were deprived of adherent cells, but some growth did occur when bone marrow depleted of adherent cells was cultured on preformed marrow or fibroblast-free peripheral blood adherent layers. These allogeneic adherent cells were, however, less stimulatory than the autologous cells. It is concluded that CFU-C progenitors are detectable in blood and bone marrow, while fibroblast colony forming cells do not circulate. It appears that macrophages in the absence of fibroblasts will support some CFU-C generation and granulopoiesis in suspension culture. This system is a useful model for examining the cellular and humoral regulation of granulopoiesis in vitro and further studies are in progress.     

Regulation of erythropoiesis in long-term hamster marrow cultures: role of bone marrow-adherent cells

The initial establishment of hamster long-term bone marrow (LTBM) cultures requires formation of an adherent stromal layer, but continued long-term proliferation of these cultures is best accomplished by removal of the suspension cells from the adherent layer and subsequent incubation in liquid suspension culture. Continued maintenance of bone marrow cells in the presence of the adherent layer for more than four to six weeks leads to a decline and eventual disappearance of erythroid and multipotent colony-forming cells. Addition of erythropoietin to LTBM suspension cultures produces mature, hemoglobinized erythrocytes. Incubation of the same cells plus erythropoietin in the presence of autologous parental adherent layers markedly inhibits both terminal erythroid differentiation and the number of detectable erythroid burst- forming units (BFU-Es). This erythroid inhibition occurs primarily within the first 24 hours with little or no effect on CFU-GEMMs or granulocyte-macrophage colony-forming units (GM-CFUs). However, continued incubation for seven days produces a reduction in all parameters. Removal of suspension cells from the adherent layer and restimulation with erythropoietin allows regeneration of erythropoiesis. Pretreatment of suspension cells with erythropoietin for 96 hours before exposure to the adherent culture only slightly inhibits erythropoiesis, suggesting that more mature erythroid progenitors are unaffected. Conditioned medium from the marrow adherent layer (ALCM) produces similar erythroid inhibitory effects in LTBM cultures with as little as two hours of incubation. The inhibition is actively produced by the adherent cells, since cycloheximide abolishes its production, while indomethacin has no apparent effect. Adherent marrow stromal cells may regulate hemopoiesis through negative as well as positive humoral signals, and they are particularly effective in erythroid regulation.     

Impaired ability of bone marrow cells from immunodeficient mice to establish long-term cultures

Summary Haemopoiesis is often depressed in patients suffering from acquired immune deficiency syndrome (AIDS). Although several mechanisms have been postulated to be responsible for depressed haemopoiesis in AIDS patients, the aetiology of this disorder is still unknown. We hypothesized that failure of the stromal microenvironment may account for part of the haemopoietic defect observed in patients with AIDS. We therefore studied a murine model of AIDS (MAIDS) caused by infection with LP-BM5 virus to determine the ability of bone marrow cells from immunodeficient mice to establish long-term stromal cultures. In addition, normal and MAIDS mice received AZT (2 mg/ml) in their drinking water for up to 1 month to determine the effects of AZT treatment in vivo on the ability of bone marrow cells to support haemopoiesis in long-term cultures. Decreased numbers of non-adherent cells were observed in long-term bone marrow cultures (LTBMC) of MAIDS mice when compared to cultures derived from normal mice. Decreased numbers of non-adherent cells were observed in cultures of bone marrow cells from AZT-treated normal mice, when compared to untreated normal controls. Cells from AZT-treated MAIDS mice produced the smallest number of non-adherent cells. BFU-E and CFU-G/M were decreased in cultures of MAIDS mice when compared to those of normal mice. AZT-treatment further decreased the number of colony-forming cells in both MAIDS and normal cultures. Stromal cell function of MAIDS mice was also assessed by inoculating non-adherent cells from normal mice onto confluent irradiated MAIDS LTBMC. Stroma from MAIDS mice was unable to support haemopoietic function of normal bone marrow cells. Polymerase chain reaction (PCR) analysis of steady state levels of cytokine mRNAs of cells from confluent cultures revealed that levels of interleukin-6 mRNA were unchanged in MAIDS mice, as compared to normal controls, but the levels of GM-CSF were decreased in MAIDS mice. These data suggest that LP-BM5 MuLV infection alters the functioning of the haemopoietic stroma and that one mechanism of this depression in haemopoiesis may be via alterations of cytokine production.