Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.     

Increased radioresistance, g(2)/m checkpoint inhibition, and impaired migration of bone marrow stromal cell lines derived from Smad3(-/-) mice

Smad3 protein is a prominent member of the Tgfb receptor signaling pathway. Smad3(-/-) mice display decreased radiation-induced skin fibrosis, suggesting a defect in both Tgfb-mediated fibroblast proliferation and migration. We established bone marrow stromal cell lines from Smad3(-/-) mice and homozygous littermate(+/+) mice. Smad3(-/-) cells displayed a significant increase in radiation resistance with a D(0)=2.25+/- 0.14 Gy compared to Smad3(+/+) cells with a D(0)=1.75+/- 0.03 (P=0.023). Radioresistance was abrogated by reinsertion of the human SMAD3 transgene, resulting in a D(0)=1.49 0.10 (P=0.028) for Smad3(-/-)(3) cells. More Smad3(-/-) cells than Smad3(+/+) cells were in the G(2)/M phase; Smad3(-/-)(3) cells were similar to Smad3(+/+) cells. Smad3(+/+) cells exhibited increased apoptosis 24 h after 5 Gy (15%) or 8 Gy (43%) compared to less than 1% in Smad3(-/-) cells exposed to either dose. The movement of Smad3(-/-) cells, measured in an automated cell tracking system, was slower than that of Smad3(+/+) cells. Smad3(-/-)(3) cells resembled Smad3(+/+) cells. These studies establish concordance of a defective Tgfb signal transduction pathway, an increased proportion of G(2)/M cells, and radioresistance. The decreased migratory capacity of Smad3(-/-) cells in vitro correlates with decreased radiation fibrosis in vivo in mice deficient in Tgfb signaling.     

Multipotent marrow stromal cell line is able to induce hematopoiesis in vivo.

Several murine marrow stromal cells were established from murine bone marrow cultures. Stromal cell lines transfected with a tumor-inducing polyoma virus middle T antigen (MTAg) were inoculated into nude mice subcutaneously. KUSA-MTAg cells, one of these cell lines, led to the rapid local development of bone marrow consisting of trilineage hematopoietic cells and bone; other cell lines produced spindle cell sarcoma or hemangiosarcoma. These results suggested that a single stromal cell line, KUSA-MTAg cells, may induce hematopoietic stem cells or early progenitors of three lineages of hematopoietic cells in vivo. Interestingly, untransfected KUSA cells expressed three new mesenchymal phenotypes, osteocytes, adipocytes, and myotubes, after treatment with 5-azacytidine.     

Oncostatin M regulates mesenchymal cell differentiation and enhances hematopoietic supportive activity of bone marrow stromal cell lines

Bone marrow stromal cell lines (TBR cell lines) established from temperature-sensitive Simian Virus 40 T-antigen gene transgenic mice exhibited myogenic, osteogenic, and adipogenic differentiation. The effect of oncostatin M (OSM) on such mesenchymal cell differentiation of marrow stromal cell lines was examined. One of those stromal cell lines, TBRB, differentiated into skeletal muscle, and its differentiation was stimulated by OSM, whereas differentiation of TBR10-1 into smooth muscle was inhibited by OSM. TBR31-2 is a bipotent progenitor for adipocytes and osteoblasts, and OSM stimulated osteogenic differentiation while inhibiting adipogenic differentiation. On the other hand, TBR cell lines exhibited various potentials for supporting hematopoiesis in culture. When hematopoietic progenitor cells were cocultured with OSM-stimulated stromal cell lines, TBR10-1 and TBR31-2 exhibited enhanced hematopoietic supportive activity. As responsible molecules for stromal cell dependent hematopoiesis, expression of stem cell factor (SCF) (a ligand of c-Kit), vascular cell adhesion molecule (VCAM-1) (a ligand of VLA-4), and secretion of interleukin (IL)-6 were increased by OSM. OSM affected mesenchymal cell differentiation and promoted the hematopoietic supportive activity of marrow stromal cell lines. As OSM production is induced by cytokines from hematopoietic cells, OSM may be a key factor in mutual regulation between hematopoietic cells and stromal cells in the bone marrow. OSM may play a role as a regulator in maintaining the hematopoietic microenvironment in marrow by coordinating mesenchymal differentiation.     

Structure and function of bone marrow environment

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.     

The construction of high efficiency human bone marrow tissue ex vivo

The successful ex vivo reconstruction of human bone marrow is an extraordinarily important basic scientific and clinical goal. Fundamentally, the system is the paradigm of a complex interactive tissue, in which the proliferation and regulated differentiation of one parenchymal cell type (the hematopoietic stem cell) is governed by the surrounding stromal cells. Understanding and reproducing the molecular interactions between bone marrow stromal cells and stem cells in tissue culture models is therefore the critical step in successful bone marrow tissue culture. Clinically, successful reconstruction of human bone marrow would permit the controlled production of mature blood cells for transfusion therapy, and immature bone marrow stem cells for bone marrow transplantation. In approaching the bone marrow culture system, we recognize the critical role that hematopoietic growth factors (HGFs) play in hematopoiesis. Since stromal cells in traditional human bone marrow cultures produce little HGFs, we have begun by asking whether local supplementation of hematopoietic growth factors via genetically engineered stromal cells might augment hematopoiesis in liquid cultures. The results indicate that locally produced GM-CSF and IL-3 do augment hematopoiesis for several weeks in culture. In combination with geometric and dynamic approaches to reconstructing physiological bone marrow microenvironments, we believe that this approach has promise for reconstructing human bone marrow ex vivo, thereby permitting its application to a variety of basic and clinical problems.     

Deficiency of Trp53 rescues the male fertility defects of Kit(W-v) mice but has no effect on the survival of melanocytes and mast cells.

Mutations of the receptor tyrosine kinase, Kit, or its ligand, mast growth factor (Mgf), affect three unrelated cell populations: melanocytes, germ cells, and mast cells. Kit signaling is required initially to prevent cell death in these lineages both in vitro and in vivo. Mgf appears to play a role in the survival of some hematopoietic cells in vitro by modulating the activity of p53. Signaling by Mgf inhibits p53-induced apoptosis of erythroleukemia cell lines and suppresses p53-dependent radiation-induced apoptosis of bone marrow cells. We tested the hypothesis that cell survival in Kit mutant mice would be enhanced by p53 deficiency in vivo. Double-mutant mice, which have greatly reduced Kit receptor tyrosine kinase activity and also lack Trp53, were generated and the affected cell lineages examined. Mast cell, melanoblast, and melanocyte survival in the double Kit(W-v/W-v):Trp53(-/-) mutants was not increased compared to the single Kit(W-v/W-v):Trp53(+/+) mutants. However, double-mutant males showed an increase in sperm viability and could father litters, in contrast to their homozygous Kit mutant, wild-type p53 littermates. This germ cell rescue appears to be male specific, as female ovaries were similar in mice homozygous for the Kit mutant allele with or without p53. We conclude that defective Kit signaling in vivo results in apoptosis by a p53-independent pathway in melanocyte and mast cell lineages but that in male germ cells apoptosis in the absence of Kit is p53-dependent.     

Decreased pulmonary radiation resistance of manganese superoxide dismutase (MnSOD)-deficient mice is corrected by human manganese superoxide dismutase-Plasmid/Liposome (SOD2-PL) intratracheal gene therapy

The pulmonary ionizing radiation sensitivity of C57BL/6 Sod2(+/-) mice heterozygous for MnSOD deficiency was compared to that Sod2(+/+) control littermates. Embryo fibroblast cell lines from Sod2(-/-) (neonatal lethal) or Sod2(+/-) mice produced less biochemically active MnSOD and demonstrated a significantly greater in vitro radiosensitivity. No G(2)/M-phase cell cycle arrest after 5 Gy was observed in Sod2(-/-) cells compared to the Sod2(+/-) or Sod2(+/+) lines. Subclonal Sod2(-/-) or Sod2(+/-) embryo fibroblast lines expressing the human SOD2 transgene showed increased biochemical activity of MnSOD and radioresistance. Sod2(+/-) mice receiving 18 Gy whole-lung irradiation died sooner and had an increased percentage of lung with organizing alveolitis between 100 and 160 days compared to Sod2(+/+) wild-type littermates. Both Sod2(+/-) and Sod2(+/+) littermates injected intratracheally with human manganese superoxide dismutase-plasmid/liposome (SOD2-PL) complex 24 h prior to whole-lung irradiation showed decreased DNA strand breaks and improved survival with decreased organizing alveolitis. Thus underexpression of MnSOD in the lungs of heterozygous Sod2(+/-) knockout mice is associated with increased pulmonary radiation sensitivity and parallels increased radiation sensitivity of embryo fibroblast cell lines in vitro. The restoration of cellular radioresistance in vitro and in lungs in vivo by SOD2-PL transgene expression supports a potential role for SOD2-PL gene therapy in organ-specific radioprotection.     

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.     

Survival of erythroid burst-forming units and erythroid colony-forming units in canine bone marrow cells exposed in vitro to 1 MeV neutron radiation or X rays

Conditioned media (CM) from allogeneic stimulated cultures of light density cells (less than 1.08 g/cm3) from the peripheral blood of normal dogs were used to stimulate the growth of erythroid burst-forming units (BFU-E) in bone marrow from normal dogs. Maximum numbers of BFU-E were obtained when 5% (vol/vol) 3 X CM and 2 U/ml erythropoietin were added to plasma clot cultures of bone marrow cells. In addition, the radiation sensitivity (D0 value) was determined for CFU-E and for BFU-E in bone marrow cells exposed in vitro to 1 MeV fission neutron radiation or 250 kVp X rays. BFU-E were more sensitive than CFU-E to the lethal effects of both types of radiation. For bone marrow cells exposed to 1 MeV neutron radiation, the D0 for CFU-E was 0.27 +/- 0.01 Gy, and the D0 for BFU-E was 0.16 +/- 0.03 Gy. D0 values for CFU-E and BFU-E were, respectively, 0.61 +/- 0.05 Gy and 0.26 +/- 0.09 Gy for cells exposed to X rays. The neutron RBE values for the culture conditions described were 2.3 +/- 0.01 for CFU-E and 1.6 +/- 0.40 for BFU-E.     

Sl/Sld mice have an increased number of gap junctions in their bone marrow stromal cells

To study the defect of the hematopoietic inductive microenvironment (HIM) in Sl/Sld mice, femoral bone marrow tissue of 10 of each mutant, (Sl/Sld and W/Wv) their normal littermates (Sl+/Sl+ and W+/W+), and 20 normal C57BL mice were examined by electron microscopy using morphometric and statistical methods. Gap junctions were observed in all strains of mice, in the following stromal cell types: 1) reticular cells, 2) between reticular cells and periarterial adventitial cells, and 3) between periarterial adventitial cells. The frequency of gap junctions in bone marrow stromal cells of Sl/Sld mice (mean = 2.2/9.4 X 10(-3) mm2) was significantly higher than in control mice. It is suggested that there is a relationship between the increased numbers of gap junctions in bone marrow stromal cells of Sl/Sld mice and the defect in HIM function in these genetically anemic animals.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part I

The spontaneous stratification in long-term bone marrow cultures was illustrated and quantified. The cultures were separated into three hematopoietic layers: nonadherent cells in the supernatant medium, lightly adherent cells on top of the stromal layer, and remaining cells buried within the stromal layer. The cells of each layer were subcultured for 10 days in plastic tubes that inhibit the formation of a stromal layer. Daily samplings with absolute and differential cell counts were obtained. We identified three families of cell disappearance curves and cell types: CFU-s, hemocytoblasts, myeloblasts, and promyelocytes (G1, 2); myelocytes (G3); and postmitotic granulocytes (G4). Also, the numbers of mitotic and necrotic cells were determined. The longest half-time of CFU-s was 2.5 days. Lacking stromal support, CFU-s disappeared faster than other differentiated cells. Generally, these cells maintained their numbers for the first week of subcultures, which was attributable to a temporarily maintained balance of cell death and fresh cell production. After more than 7 days, there was a rapid decline of all differentiated cell types.     

Bone marrow stromal proteoglycan heterogeneity: phenotypic variability between cell lines and the effects of glucocorticoid

Hematopoiesis in vivo is dependent upon the interaction of hematopoietic stem cells with a complex microenvironment, of which stromal proteoglycans are an important functional component. Certain bone marrow stromal cell lines provide a microenvironment that supports hematopoiesis in vitro, a function that is dependent upon glucocorticoid supplementation. Proteoglycan synthesis in the hematopoietic-supportive D2XRII, Bl6 and 14F1 bone marrow stromal cell lines was studied by 35S-sulfate precursor labelling and ion-exchange separation, followed by isopyknic CsCl density centrifugation and gel filtration HPLC. The effects of glucocorticoid were also investigated. A similar pattern of proteoglycan heterogeneity was observed in all three cell lines, although there was considerable quantitative variation. All cultures synthesized three species of chondroitin/dermatan sulfate (CS/DS) proteoglycans: DS1, excluded from a Bio-Sil TSK-400 HPLC column, and DS2, eluting at Kd = 0.31, were present mainly in the culture media. The smallest (DS3) eluted at Kd = 0.63 and was present mainly in the cell layers. CS/DS species were the major proteoglycans in all cultures. Hydrocortisone-free cultures also synthesized heparan sulfate (HS) proteoglycans, including a cell-associated form (HS1), partially excluded from the TSK-400 column, and a secretory form (HS2), eluting at Kd = 0.15. D2XRII cells also secreted an apparently-unique, high-density proteoglycan, Kd = 0.65, into the culture medium. Hydrocortisone at 10(-6) M virtually abolished HS proteoglycan synthesis in all three cell lines, and altered the pattern of CS/DS proteoglycans in the culture media, increasing the quantity of DS1 and DS3, and reducing the quantity of DS2.     

Friend leukemia virus infection enhances DNA damage-induced apoptosis of hematopoietic cells,causing lethal anemia in C3H hosts

Exposure of hematopoietic progenitors to gamma irradiation induces p53-dependent apoptosis. However, host responses to DNA damage are not uniform and can be modified by various factors. Here, we report that a split low-dose total-body irradiation (TBI) (1.5 Gy twice) to the host causes prominent apoptosis in bone marrow cells of Friend leukemia virus (FLV)-infected C3H mice but not in those of FLV-infected DBA mice. In C3H mice, the apoptosis occurs rapidly and progressively in erythroid cells, leading to lethal host anemia, although treatment with FLV alone or TBI alone induced minimal apoptosis in bone marrow cells. A marked accumulation of P53 protein was demonstrated in bone marrow cells from FLV-infected C3H mice 12 h after treatment with TBI. Although a similar accumulation of P53 was also observed in bone marrow cells from FLV-infected DBA mice treated with TBI, the amount appeared to be parallel to that of mice treated with TBI alone and was much lower than that of FLV- plus TBI-treated C3H mice. To determine the association of p53 with the prominent enhancement of apoptosis in FLV- plus TBI-treated C3H mice, p53 knockout mice of the C3H background (C3H p53(-/-)) were infected with FLV and treated with TBI. As expected, p53 knockout mice exhibited a very low frequency of apoptosis in the bone marrow after treatment with FLV plus TBI. Further, C3H p53(-/-) --> C3H p53(+/+) bone marrow chimeric mice treated with FLV plus TBI survived even longer than the chimeras treated with FLV alone. These findings indicate that infection with FLV strongly enhances radiation-induced apoptotic cell death of hematopoietic cells in host animals and that the apoptosis occurs through a p53-associated signaling pathway, although the response was not uniform in different host strains.     

Blast colony-forming cell binding from CML bone marrow, or blood, on stromal layers pretreated with G-CSF or SCF

Blast colony-forming cells (CFU-BL) represent a specific subpopulation of special primitive progenitors characterized by colony formation only in close contact with a preformed stromal layer. CFU-BL derived from bone marrow of chronic myeloid leukaemia (CML) patients have been proved to adhere poorly to bone marrow derived stromal layers suggesting that the appearance of progenitors and precursors in the circulation is due to a defective adhesion of these cells to the bone marrow microenvironment. In the present experiments the effect of short-term incubation of preformed normal bone marrow stroma on the adherence of CML derived CFU-BL was studied. For stroma cultures bone marrow cells were cultured in microplates in the presence of hydrocortisone. Cultures were used when stromal layers became confluent and no sign of haemopoiesis could be observed. CFU-BL were studied by panning plastic non-adherent mononuclear (PNAMNC) bone marrow or blood cells. 8.9 +/- 2.4 colonies/103 PNAMNC (six experiments) were formed from normal bone marrow on stromal layers and 4.8 +/- 2.1 colonies/103 PNAMNC (five experiments) from CML bone marrow. Colony formation from normal bone marrow was not increased if stromal layers were incubated with 100 ng/mL granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF). Incubation of stroma with G-CSF or SCF, however, increased the colony formation of PNAMNC from CML bone marrow or blood significantly. These findings suggest that local concentration of haemopoietic growth factors at the time of panning may influence the attachment of CML progenitors to the stroma.     

Pre CFU-F in bone marrow cultures from children with hematopoietic disease including acute leukemia

Stromal precursor cells from bone marrow aspirates of children have been studied in culture. In 7 day liquid cultures normal individuals and patients with acute leukemia in remission grew 110 +/- 50 CFU-F and 100 +/- 40 CFU-F (colony forming unit--fibroblasts) respectively, per 6 X 10(5) buffy coat mononuclear cells. Staining with monoclonal antibodies suggests that stromal cells from CFU-F colonies are fibroblasts. CFU-F colony growth from the bone marrow of patients with active leukemia was low. After cultivation periods of more than 21 days, we observed, in addition, still more immature, clonogenic fibroblast precursor cells, "pre CFU-F", and round cells attached to stromal cells from pre CFU-F colonies. From the round cells, we have passaged pre CFU-F and CFU-GM (colony forming unit--granulocytic, monocytic) in secondary cultures. Our observations are in agreement with the concept that the bone marrow stromal cell matrix serves as a sanctuary for reversibly attached clonogenic cells of both the hematopoietic and fibroblast lineages.     

Cell cycle progression and apoptosis after irradiation in an acidic environment

We investigated the effect of an acidic environment on the radiation-induced G2/M arrest and apoptosis using RKO.C human colorectal cancer cells expressing wild-type p53 and RC10.1 cells, a subline of RKO.C cells deficient in p53 as well as p53+/+ MEFs and p53-/- MEFs (mouse embryonic fibroblasts). The cells were irradiated with 4 Gy or 12 Gy of gamma-rays in pH 7.5 medium or pH 6.6 medium. p53 accentuated the progression of cells from radiation-induced G2/M arrest to apoptosis and the pH 6.6 environment suppressed the progression of cells through G2/M-phase to apoptosis after irradiation. Further analysis indicated that the radiation-induced G2/M arrest was due mainly to G2 arrest in both pH 7.5 and pH 6.6. Therefore, it was concluded that p53 enhances, and an acidic environment suppresses, the exit of cells from radiation-induced G2 arrest by altering cyclin B1-Cdc2 kinase activity.     

Isolation of a novel PDZ-containing myosin from hematopoietic supportive bone marrow stromal cell lines

Stromal cells in bone marrow provide an optimal microenvironment for hematopoiesis. The established stromal cell lines from bone marrow showed various cellular heterogeneities and differed in their hematopoietic supportive ability. By a differential display method, we cloned a gene whose expression levels were correlated with the hematopoietic supportive ability of stromal cells. Its deduced amino acid sequence shows a structure similar to myosins, except that it lacks an actin binding site. Interestingly, it contains a KE-rich sequence and a PDZ domain in the NH(2)-terminal, which are protein-protein interaction domains; therefore we termed this novel myosin Myosin containing PDZ domain (MysPDZ). Western blot analysis showed that its protein levels positively correlated with the supportive ability of stromal cells and immunostaining suggested that MysPDZ was present at cytoskeleton in a filamentous and/or network form. Thus MysPDZ may be involved in the maintenance of the stromal cell architectures required for cell to cell contact.     

Defective lymphopoiesis in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mutant mice. II. Description of a microenvironmental defect for the generation of terminal deoxynucleotidyltransferase-positive bone marrow cells in vitro

We have presented evidence in a previous paper that the development of prothymocytes, pre-B cells, and TdT+ lymphoid precursor cells in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mice is defective. In the present study, we have used a selective culture system that supports the generation of rat- and mouse-origin TdT+ bone marrow lymphoid cells in vitro to further investigate the early stages of lymphopoiesis in me/me and mev/mev mice. The results demonstrate that bone marrow stromal cell feeder layers derived from me/me and mev/mev mice do not support the growth of rat TdT+ cells in vitro, whereas stromal cell feeder layers from heterozygous (+/-) littermates and wild type (+/+) control mice do. Moreover, composite feeder layers formed by mixing as few as one part me/me and mev/mev bone marrow cells with 7 to 9 parts +/- littermate bone marrow cells also fail to effectively support the generation of TdT+ cells in vitro. In contrast to me/me and mev/mev mice, other mutant mouse models of autoimmune (NZB, NZB/W), immunodeficient (nu/nu), and hemopoietic (W/Wv, Sl/Sld) disorders form feeder layers that support normal to elevated levels of TdT+ cell growth in vitro. Thus, to date, only the me/me and mev/mev mutant mice have been found to lack the appropriate microenvironment for the generation of TdT+ bone marrow cells. Histologic analysis of the stromal cell feeder layers that are formed in our culture system shows that multilayered cellular patches, which normally are the most active sites of TdT+ cell development in vitro, are absent in feeder layers of me/me and mev/mev cells. Moreover, feeder layers from mev/mev mice contain a population of MAC 1+, basophilic, nonvacuolated, macrophage-like cells; whereas feeder layers from control mice contain MAC 1+, eosinophilic, vacuolated macrophage-like cells. Stromal cell feeder layers formed by mixtures of me/me or mev/mev and control mouse bone marrow cells contain numerous multilayered cellular patches and vacuolated mononuclear cells, but also contain large numbers of basophilic mononuclear cells. These composite feeder layers have a disproportionately reduced capacity to support the generation of TdT+ cells in vitro. Although the stromal microenvironment of me/me and mev/mev bone marrow does not support the growth of TdT+ cells in vivo or in vitro, the bone marrow from these mutant mice contains detectable numbers of pre-TdT+ cells. Thus, when cultured on normal mouse feeder layers, mutant mouse bone marrow rapidly generates TdT+ cells in vitro, albeit at significantly reduced levels as compared to +/- littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)