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Biomedical subjects

D Zipori

Publications and source records attributed to D Zipori.

At least 37 records · Page 2Linked to original sources
PubMed1996

A hematopoietic organ-specific 49-kD nuclear antigen: predominance in immature normal and tumor granulocytes and detection in hematopoietic precursor cells.

A 49-kD protein was specifically detected in hematopoietic organs by Western blotting with a novel mouse monoclonal antibody (B92) raised against stromal cells. The protein was found in the immunizing cells using a sensitive method. However, its detection in the bone marrow by the B92 antibody seemed to stem from the abundance of p49 in immature cells of the myeloid lineage. Study of the bone marrow following in vivo irradiation or 5-fluorouracil (5-FU) treatment, in vitro culture with differentiation-inducing factors and long-term culture, and cell sorting all pointed in the same direction: the protein was found in early myeloid cells and in hematopoietic precursor cells. These results were in accordance with the specific presence of p49 in primary radiation-induced myeloid leukemia and its absence in spontaneous B lymphoma. Immunofluorescent staining using B92 antibody detected a nuclear antigen forming a dotted pattern in early myeloid cells and day 12 colony-forming units-spleen (CFU-S). Nuclear localization of p49 was further demonstrated by subcellular fractionation followed by Western blotting. We thus identified a nuclear protein that within the hematopoietic population is detected in hematopoietic precursor cells, predominates in early myeloid cells, and is reduced following differentiation. These properties imply that p49 might be involved in the regulation of hematopoietic cell growth or differentiation.

Acute Disease↗
PubMed1996

Interactions between leukemia cells and bone marrow stromal cells: stroma-supported growth vs. serum dependence and the roles of TGF-beta and M-CSF.

Leukemia cell lines that do not proliferate in the absence of serum grow well when cultured with stromal cells. To study this growth dependence on stroma, we selected the M1 myeloblast clone, since its stroma dependence is reminiscent of that exhibited by hematopoietic stem cells. Conditioned medium form a stromal cell line, prepared under serum-free conditions, contained an activity that induced the proliferation of M1 cells and was therefore designated M1 myeloid activity (MMA). Among the various cytokines tested for MMA-like activity, only transforming growth factor-beta (TGF-beta) and macrophage colony-stimulating factor (M-CSF) were found to affect M1 cell survival, and the two cytokines acted synergistically to induce M1 cell growth. Antibodies to both TGF-beta and M-CSF abolished most, but not all, of the MMA in the medium conditioned by stromal cells, indicating that additional factors contribute to MMA. A subclone of M1 cells, M1/M2, selected in medium conditioned by stroma, was found to respond to stromal stimulation but was unable to proliferate in fetal calf serum (FCS). Neutralization experiments indicated that M1/2 cell growth depended mainly on M-CSF and also partially on TGF-beta. By contrast, the same neutralizing antibodies did not affect the ability of serum to support M1 cell growth. The molecules that promoted leukemia cell growth in serum seemed therefore to differ from those provided by stroma. This model system may offer novel information on the interactions of normal and leukemic hematopoietic cells with their stromal microenvironment.

Animals↗
PubMed1996

Selective adhesion of immature thymocytes to bone marrow stromal cells: relevance to T cell lymphopoiesis.

We investigated the interactions between the bone marrow microenvironment and T cell populations at different stages of maturation. Thymocytes were seeded onto confluent layers of bone marrow stromal cell lines (MBA-13 or 14F1.1). Within a few hours two main thymocyte populations were observed; one remained in the liquid phase and the other adhered to the stromal cells. After 24 hours of culture, most of the adhering cells expressed the phenotype of the precursors, double negative (DN) CD4-CD8-, or of immature thymocytes, double positive (DP) CD4+CD8+. The number of adhering DN cells did not change during the time of the culture, whereas that of the DP declined. The CD4+CD8- or CD4-CD8+ cells did not adhere to any significant extent. The expression of CD3 antigen on adherent thymocytes was lower than that on nonadherent ones. Sorted thymocytes at a high level of purification (>96%) were cultured over stromal layer and, after 24 hours, 60% of the DN or 22% of the DP cells were found to adhere to the stroma. The culture medium was replaced every 24 hours or after 48 hours; no significant change was noted in the number of adhering DN and DP cells. The reappearance of immature T cells in the liquid phase suggested proliferation of this cell type. Thus, early thymocytes, phenotypically characterized as DN and DP, preferentially adhere to bone marrow stromal cells. This in vitro phenomenon may represent the function of the BM stroma as an extrathymic site of T cell lymphopoiesis.

Adipose Tissue↗
PubMed1996

Control of stroma-dependent hematopoiesis by basic fibroblast growth factor: stromal phenotypic plasticity and modified myelopoietic functions.

It has been suggested that basic fibroblast growth factor (bFGF) affects hematopoietic cells directly and that it may also act indirectly by modulating stromal cell functions. We tested the response of phenotypically and functionally distinct stromal cell clones to this cytokine. We studied cell phenotype, the composition and organization of cytoskeleton and extracellular matrix, the ability to repopulate 'wounded areas', the expression of cytokine genes, and the capacity of the stroma to support long-term hematopoiesis in vitro. Although the impact of bFGF on cell growth was small, it induced a prominent morphological change in three stromal cell types that we tested. We analyzed the molecular basis for this change: bFGF modified the protein expression of alpha-smooth muscle actin (alpha-SMA), tropomyosin, alpha-tubulin, fibronectin and paxillin in a distinct manner characteristic of each of the stromal cell types. Immunofluorescence analysis of these proteins revealed profound changes in the cytoskeleton and extracellular matrix (ECM) networks accompanied by increased ability of the 14F1.1 stromal cells to scatter in in vitro 'wounded' areas. Furthermore, although only limited changes were monitored in the expression of cytokine genes, the ability of the stromal cells to support hematopoiesis was markedly modified. Thus bFGF profoundly changes the cellular organization of stromal cells, their adhesion and their motility properties. These changes are associated with modified capacity to support hematopoiesis in culture.

Adipocytes↗
PubMed1995

Restrictin-P/stromal activin A, kills its target cells via an apoptotic mechanism.

We have recently found that the inhibitor of plasmacytoma cell growth, restrictin-P, is a stroma derived activin A and that it is an antagonist of interleukin-6 and interleukin-11. The present study was aimed at determining the mode by which this cytokine kills its target cells. On addition of the cytokine there was little or no net increase in cell number, depending on the specific target cells. All plasmacytoma cell lines tested exhibited a similar time dependent inhibition of DNA synthesis and a G0/G1 shift in the cell cycle. Electron microscope examination revealed classical apoptotic features i.e. chromatin condensation and membrane blebbing. DNA fragmentation, measured qualitatively and quantitatively, occurred in all cytokine treated plasmacytoma cell lines. Bovine activin A had an identical capacity to reduce cell viability, to induce G0/G1 shift and to cause DNA fragmentation. X-ray microanalysis of intracellular ions revealed an increase in calcium ions, following exposure of plasmacytoma cells to restrictin-P, accompanied by a decrease in phosphor ions. The cytotoxicity of the inhibitor was augmented in an additive manner by cycloheximide (CHX) indicating that the process did not require de novo protein synthesis. This study thus shows that restrictin-P/stromal activin A kills its target cells by inducing apoptosis. This effect was mediated by subnanogram concentrations and therefore may represent one physiological function of this pleiotropic cytokine.

Activins↗
PubMed1994

Bone formation by marrow osteogenic cells (MBA-15) is not accompanied by osteoclastogenesis and generation of hematopoietic supportive microenvironment.

This study was aimed at elucidating the relationship between osteogenic activity of marrow stromal cells and their ability to support hematopoiesis followed by the bone-remodeling process. We used the MBA-15 cell line, which expresses osteoblastic phenotype in vitro and forms bone in diffusion chamber. We have compared bone formation and hematopoietic responses elicited in vivo by these cells with the implantation of freshly isolated bone marrow cells (BMC) or demineralized tooth matrix (DTM). Both MBA-15 cells and BMC, implanted under the kidney capsule, yielded intramembraneous bone, but DTM, implanted subcutaneously, elicited endochondral bone. MBA-15 formed primary bone, mimicking only the initial sequential stages of the ossification process. Neither histologic signs of bone resorption and remodeling nor tartrate-resistant acid phosphatase (TRAP)-positive cells and marrow formation were observed. Bone formation was monitored biochemically. Functions for hematopoietic stem and committed cell content were measured by GM-CFU and BFU-E assays that confirmed the morphologic observations. In both BMC and DTM implantation, bone formation was followed by hematopoietic activity, osteoclastogenesis, and remodeling. We conclude that MBA-15 osteoprogenitor cells, despite their extensive bone formation ability, are unable to form a microenvironment supportive for hematopoiesis and osteoclastogenesis or to initiate bone remodeling.

Animals↗
PubMed1994

Myeloblastic cell line expresses osteoclastic properties following coculture with marrow stromal adipocytes.

Osteoclasts are derived from hemopoietic precursors in the marrow. Their differentiation pathway is still undefined, but an important role was observed for the marrow microenvironment in the regulation of osteoclastogenesis. Various marrow stromal cell subtypes were used to study their possible role in the formation of osteoclasts from myeloblast (M1) cells. Interactions between M1 cells and the 14F1.1 endothelial-adipocyte stromal cell line were demonstrated in a coculture model. M1 cells attached to the adherent layer of 14F1.1 cells and formed distinct foci reminiscente of "cobblestone areas." Following these interactions, M1 cells developed specific enzymatic activities and became multinucleated. Both mononuclear and multinuclear M1 cells became positive to tartrate-resistant acid phosphatase (TRaP) and ATPase, a feature characteristic of osteoclasts, and were also responsive to calcitonin. Furthermore, they attached to mineralized bone particles and their membrane changed into a ruffled border at the zone of interaction with the bone matrix. We thus demonstrated that marrow endothelial-adipocytes may play a role in regulating the differentiation of myeloblasts into osteoclasts.

Acid Phosphatase↗
PubMed1994

Expression of the c-kit ligand and interleukin 6 genes in mouse bone marrow stromal cell lines.

The expression of c-kit ligand and interleukin 6 (IL-6) genes in mouse bone marrow-derived stromal cell lines was examined using quantitative polymerase chain reaction (PCR) analysis based on the design of an internal DNA control. The stromal cells studied included the 14F1.1 endothelial-adipocytes that support long-term hemopoiesis and two additional cell lines (MBA-1, MBA-13) which do not have this function. All the cell lines expressed c-kit ligand gene constitutively, and this expression was not increased by lectins. On the other hand, the expression of the IL-6 gene was markedly induced in all the lines by lipopolysaccharide (LPS) and by phorbol 12-myristate 13 acetate (PMA). The constitutive expression of c-kit ligand in 14F1.1 cells was the lowest among the three cell lines studied and could be increased by stimulation with IL-4. Thus, we observed some quantitative differences among the cell lines in their expression of cytokine genes. However, the unique capacity of 14F1.1 cells to support in vitro hemopoiesis cannot thus far be explained solely on the basis of the ability of these cells to secrete cytokines which are not produced by other stromal cell lines. c-kit ligand may be necessary, but its presence alone is not sufficient for 14F1.1 cells to support prolonged hemopoiesis.

Adipose Tissue↗
PubMed1994

Mineralization of marrow-stromal osteoblasts MBA-15 on three-dimensional carriers.

The present study describes a new three-dimensional (3-D) culture system that enables the maintenance and phenotypic expression of bone marrow stromal osteoblasts. This culture substratum is advantageous in that it provides suitable conditions for attachment, growth, and differentiation of cells forming 3-D layers. The MBA-15 cell line was grown in unlimited quantities on 3-D Fibro-Cel carriers. These cells mineralized when exposed to ascorbic acid and beta-glycerophosphate (beta GP). Under these mineralization conditions, mRNA expressions of procollagen alpha 2(I) and [3H]-proline-labeled protein were increased. The expression of mRNA for osteonectin and to a lesser extent, for osteopontin was increased, whereas alkaline phosphatase and biglycan remained unaffected under similar conditions. Exposure of mineralizing cultures to dexamethasone reduced mRNA of procollagen alpha 2 (I) and osteonectin to control level. Scanning electron microscopy revealed that cells were grown along the fabric's fibers and produced collagen fibrils. Under appropriate conditions, extensive mineralization had taken place. The mineralization process involves the formation of calcospherites, and correlates with an increase in calcium content. The Fibro-Cel carriers enable formation of 3-D architecture and mineralized tissue in vitro.

Animals↗
PubMed1993

Marrow adipocytes regulate growth and differentiation of osteoblasts.

Conditioned media (CM) from various marrow subpopulations (MBA series) were examined for their effects on growth and maturation of marrow-derived osteoblastic cells, MBA-15. CM harvested from a stromal adipocytes, 14F1.1, stimulated cell growth, collagen but not non-collagenous proteins synthesized by MBA-15 cells. Alkaline phosphatase activity was inhibited under the same culture conditions. These results were associated with morphological changes, i.e., the large cuboidal MBA-15 cells acquired a fibroblast-like appearance. Exposure of MBA-15 cells to known growth factors: BMP-2, TGF beta, IGF-I and PDGF or combined with 14F1.1 CM resulted in a dominant effect of the latter. This may imply that marrow adipocytes produce factor/s that participate in the stromal regulation of osteoblastic functions.

Adipocytes↗
PubMed1993

Osteoblasts release a soluble factor that modulates immunoglobulin secretion by mature B cells.

An osteogenic member, MBA-15 cell line, from marrow stroma compartment, was examined for its ability to regulate mature lymphoma B cell line functions. The effect of MBA-15 cell secreting factor/s on cell proliferation, immunoglobulin secretion and isotype switch of 29M4.1 IgM+ mature B cell line was examined. The factor causes temporal arrest in cell proliferation, augments immunoglobulin secretion and has no effect on isotype switching. The results revealed a direct effect of the MBA-15 factors on the B cell functions. Factors secreted by other osteoblastic cells (ROS 17/2.8 and MC-3T3-E1) possess the same biological activities. In contrast, factors secreted by an endothelial adipocyte stromal cell line 14F1.1 lack these properties.

Analysis of Variance↗
PubMed1993

Transfection of interferon-gamma gene in a mouse bone marrow stromal preadipocyte cell line causes apoptotic cell death.

It has been reported that bone marrow and serum of patients with aplastic anemia or chronic myeloproliferative disorders contain an abnormal concentration of cytokines. In the present study, we tried to isolate mouse bone marrow stromal cell lines that were stably transformed with a variety of cytokine genes and that expressed them constitutively. From mouse bone marrow stromal cell lines MBA-1, MBA-13, and 14F1.1, we isolated clones secreting interleukin-3 (IL-3), IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte (G)-CSF. Interferon-gamma (IFN-gamma)-producing stable transformants could not be established from 14F1.1 cells in spite of repeated transfection trials. At early stages of transfection, 14F1.1 cells did secrete IFN-gamma; however, exogenously added mouse IFN-gamma could not inhibit 14F1.1 cell growth. We discovered that chromosomal DNA isolated from 14F1.1 after transfection with the mouse IFN-gamma gene was fragmented. This is characteristic of cells undergoing apoptotic cell death. DNA fragmentation was also observed in 14F1.1 cells transfected with the human IFN-gamma gene. These results indicate that intracellular IFN-gamma induces apoptotic cell death of 14F1.1 stromal cells.

Adipocytes↗
PubMed1993

Hematopoiesis on cellulose ester membranes. XIII. A combination of cloned stromal cells is needed to establish a hematopoietic microenvironment supportive of trilineal hematopoiesis.

A mixture of stromal cells from murine bone marrow placed upon cellulose ester membranes (CEM) and then implanted intraperitoneally (i.p.) in mice results in a regenerated hematopoietic microenvironment which supports trilineal hematopoiesis. We used this model to study the capacity of 5 cloned murine stromal cell lines of marrow origin to support hematopoiesis in vivo: MBA-1 (fibroblast); MBA-2 (endothelial); MBA-13 (fibroendothelial); 14F1.1 (endothelial-adipose); and 14M1.4 (macrophage).10(7) stromal cells of a single cell line were applied to 1.5 cm2 CEM, which were folded into tubes and implanted i.p. into mice. Similarly, combinations of 4, 3 and 2 stromal cell lines were applied to CEM and implanted i.p. Single lines were implanted into syngeneic hosts of the same murine strain from which the clone was derived and into nude mice. Combinations of stromal cells were implanted only in nude mice to avoid allogeneic incompatibility. CEM implants were removed after intervals of 5 to 36 weeks and examined histologically. 1) Stromal cells of a single phenotype did not develop hematopoiesis. 2) A combination of 4 stromal phenotypes (MBA-1, MBA-2, MBA-13 and 14F1.1) formed a hematopoietic microenvironment supportive of trilineal hematopoiesis and bone. 3) The combination of 14F1.1 (endothelial adipose) + a second stromal phenotype--MBA-1 (fibroblast) or MBA-2 (endothelial) or MBA-13 (fibroendothelial) also supported trilineal hematopoiesis and bone. 4) CEM coated with MBA-13 or MBA-1 developed bone but no hematopoiesis. The endothelial-adipose phenotype appears to be essential to support hematopoiesis but requires other types of stromal cells--fibroblast, fibroendothelial or endothelial phenotype.

Animals↗
PubMed1993

Potentiation of myeloid colony-formation in bone marrow of intact and neonatally thymectomized mice by the thymic hormone THF-gamma 2.

The effect of the thymic hormone THF-gamma 2 on committed stem cells of bone marrow (BM) origin was determined using the myeloid progenitor cell clonal assay. Preincubation of normal BM cells with THF-gamma 2 for 1 hour or 18 hours caused a 2- to 6-fold increase in the number of myeloid colonies in the presence of suboptimal concentrations of colony-stimulating factor (CSF). The optimal dose of THF-gamma 2 causing this enhancement was in the range of 25 to 100 ng/mL. THF-gamma 2 was not able to replace CSF as an inducer in these experiments. THF-gamma 2 neither induced IL-6 activity upon 24-hour incubation with bone marrow cells nor enhanced LPS-induced IL-6 secretion by bone marrow cells in vitro. Neonatal thymectomy (NTx) of Balb/c mice caused a decrease in myeloid progenitors, which was repaired by serial injections of THF-gamma 2. The repair of the stem cell compartment in the bone marrow correlated with an increased percentage of Thy1+ cells in the spleen of THF-gamma 2-treated NTx mice. These findings indicate that THF-gamma 2 is able to regulate committed stem cell functions in the bone marrow of immune-deprived NTx and of normal mice.

Animals↗
PubMed1992

Long-term survival of human myeloid progenitor cells induced by a mouse bone marrow stromal cell line.

Mouse endothelial-adipocyte cell line (14F1.1), which induces proliferation of mouse stem cells in culture, is also capable of supporting long-term survival in culture of human myeloid progenitor cells; colony forming unit-granulocyte/macrophage (CFU-GM) was recovered from cultures incubated with the 14F1.1 cell line after over a month of incubation. The CFU-GM population increased beyond the input number, whereas, in control cultures initiated without stromal cells, the number of progenitors gradually declined. Addition of a relatively low concentration of human colony-stimulating factors (CSFs) into the cultures promoted the formation of "cobblestone areas," where mouse stroma and human hemopoietic cells closely interacted. 14F1.1 supernatant alone did not support the survival of human CFU-GM but synergized with the function of human granulocyte-macrophage colony-stimulating factor (GM-CSF) to stimulate adherent macrophage proliferation.

Animals↗
PubMed1992

Dynamic changes in cytokine secretion by stromal cells during prolonged maintenance under protein-free conditions.

Stromal cells of bone marrow origin produce a variety of known cytokines and some factors exhibiting apparently new biological activities. Several of these were identified by the study of cell to cell interactions and were not found in detectable amounts in media conditioned by the cells. We describe here a culture system that enables the release of stromal cytokines into medium free of any added proteins and supplemented with peptides from casein hydrolysate (0.1%). The absence of serum proteins allows extensive concentration and monitoring of activities that are otherwise undetectable. Stromal cells of the MBA-2.1 clonal cell line were seeded in a stationary bed reactor packed with a carrier of non-woven fabric matrix. After a proliferation phase with serum containing medium, the cells were maintained for over 10 months in protein-free medium. Throughout this extended incubation in the absence of serum or serum replacing proteins, stromal cells retained their viability and continuously released transforming growth factor-beta (TGF-beta), macrophage-colony stimulating factor (M-CSF) and restrictin-P, a cytotoxic factor that specifically arrested the growth of plasmacytoma cells. In addition, interleukin-6 (IL-6) was first undetectable, and later in culture its titer reached a maximum of 180,000 international units (IU)/ml. Concomitantly, the production of restrictin-P diminished and reached its lowest levels at the end of 10 months. The results may imply a possible causal relationship between the expression of IL-6 and restrictin-P, since no similarly significant changes were observed in the titers of M-CSF and TGF-beta. This novel bioreactor system may be adaptable for efficient production of different cytokines under absolute serum-free conditions.

Animals↗
PubMed1992

Hemopoietic functions of marrow-derived osteogenic cells.

Osteoblasts, members of the marrow stromal cellular network, may play an active role in the hemopoietic microenvironment as well as in bone remodeling. In this study, we examined the extent to which marrow-derived osteogenic cells (MBA-15) possess various stromal functions. This marrow stromal-derived cell line was shown by us to exhibit osteoblastic characteristics in culture and to form bone in vivo. These cells are shown here to constitutively produce and secrete cytokines identified as M-CSF, GM-CSF, and IL-6. MBA-15 cells modulate growth of normal and malignant myeloid and lymphoid cells as well as leukemia cell lines in vitro. Cell-cell interactions were studied in co-cultures with adherent MBA-15 cells and the target hemopoietic cells. Growth inhibition effects, observed under various experimental conditions, can be attributed to the presence of different soluble and membrane-bound inhibitory activities produced by MBA-15 cells. Thus, MBA-15 cells spontaneously produce both stimulators and inhibitors that can affect myeloid and lymphoid cell growth. Marrow osteogenic cells may therefore participate in the stromal regulation of hemopoiesis.

Alkaline Phosphatase↗