Stem cell-stromal cell interactions.
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Adipose Tissue↗
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In vitro decidualization of human endometrial stromal cells.
Stromal cells isolated from proliferative human endometrium undergo morphologic and biochemical changes when exposed to a mixture of ovarian hormones, acquiring characteristics of decidual cells. In addition to the previously reported progestin-induced secretion of prolactin (PRL) by explants of human proliferative endometrium, and of PRL and laminin by stromal cells in culture, "in vitro" induction of several other decidual cell products was demonstrated in the present study, using cultures of stromal cells isolated from proliferative endometrium. Incubation of stromal cells with a mixture of estradiol, medroxyprogesterone acetate and relaxin, at a concentration reported to yield maximal stimulation of PRL production, resulted in changes from elongated to rounder cells, approx. 90% of which showed immunostaining for PRL under these conditions. Immunocytochemical procedures were carried out on cytospins of decidual cells isolated from decidual tissue adherent to fetal membranes collected at delivery (positive controls), and on stromal cells cultured in Lab-Tek chamber-slides, in the absence (negative controls) or in the presence of added hormones. Antibodies to 24K (a heat-shock protein also named HRP27), desmin (present in intermediate filaments), p29 (a protein associated with the estrogen receptor), and PP12 (an insulin growth factor-1 binding protein), did not react with stromal cells isolated from proliferative endometrium but showed immunostaining of the rounder cells obtained after hormonal treatment when tested with the peroxidase-labeled second antibody complex. In another series of similar experiments, in which the same decidualization end-points were employed, changes in 24K, desmin and PP12 expression were obtained by adding to the insulin-containing medium PRL instead of the hormonal mixture, a finding suggesting sequential steps during the decidualization process.
Culture Techniques↗
Thy-1 antigen-mediated adhesion of mouse lymphoid cells to stromal cells of haemopoietic origin.
Thy-1 antigen is one of the widespread cell surface antigens. The antigen is expressed in the cells of a wide variety of different tissues and species and its pattern varies considerably during development and among species. Although the Thy-1 is one of the members of Ig-superfamily its function(s) remain unknown. Here we show that monoclonal antibodies to mouse Thy-1.2 antigen prevent adhesion of Thy-1.2-positive mouse T-leukemic cells 127 to monolayers of two mouse bone marrow stromal cell lines (14F1.1 and MBA-15). The same antibodies do not prevent adhesion of cell 127 to monolayers of two other mouse bone marrow stromal cell lines (MBA-2.1 and MBA-1.1.1) and to a wide spectrum of different normal and tumor mouse cell lines. A characteristic of the cell line 14F1.1 is the ability to support growth of mouse haemopoietic stem-cells. This data suggest that cells of stromal lines 14F1.1 and MBA-15 contain on their surface a specific ligand for the Thy-1 molecule. Cell-cell interaction via this ligand and the molecule on the cell surface may be an important step for development of certain lineages of cells.
Animals↗
Support of human cord blood progenitor cells on human stromal cell lines transformed by SV40 large T antigen under the influence of an inducible (metallothionein) promoter.
We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony-forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.
Antigens, CD↗
Characterization of heterotypic adherence between transformed human lymphoblastic cells and marrow stromal cells: VCAM-1 is a ligand for one of the leukemia cell adhesion proteins.
Marrow stromal cells are important in normal myelopoiesis and support growth of leukemia/lymphoma (LL) cells in vitro. We have previously described the heterotypic adherence of a human B-lymphoblastic cell line (UTMB-460) to marrow stromal cells (MSC). We have extended these observations to a human T-lymphoblastic cell line (CEM) and characterized the heterotypic adherence of B- and T-lymphoblastic cell lines to human MSC. Electron microscopy demonstrated UTMB-460 cells were in very close apposition to the MSC, but no specific intercellular junctions were noted. Under the conditions employed, these MSC express extracellular fibronectin, collagen types I and IV, intracellular laminin, and vimentin, but no factor VIII-R antigen. In addition, the MSC had receptors for the lectin Ulex europaeus agglutinin I. UTMB-460 and CEM cells do not adhere to extracellular matrix (ECM) proteins secreted by the MSC, i.e., fibronectin, collagen types I, III, or IV, or laminin. Monoclonal antibodies (MoAbs) against CD11a, CD11b, CD18, and CD54 and a polyclonal anti-human fibronectin antibody do not inhibit attachment of either B- or T-lymphoblastic cells to MSC. Peptides GRGES and GRGDS did not inhibit adherence of UTMB-460 and CEM cells to MSC. In contrast, the anti-vascular cell adhesion molecule (VCAM)-1 MoAb (4b9) caused significant inhibition (p < 0.01) of the adherence of both UTMB-460 and CEM cells to normal human MSC monolayers. These data suggest: (1) that MSC to which lymphoblastic cells adhere are specialized mesenchymal cells; (2) that the membrane interactions between T- and B-lymphoblastic cells and MSC involve close apposition of cell membranes of MSC and the lymphoblastic cells; (3) that the heterotypic adherence between B- and T-lymphoblastic cell lines (UTMB-460 and CEM) and MSC does not involve the RGD recognition sequence of the integrin family, the B2 leukocyte integrins, CD44, LAM-1, or the ECM proteins examined; and (4) that VCAM-1 may at least be partially responsible for heterotypic adherence between human MSC and B- and T-lymphoblastic cells.
Amino Acid Sequence↗
Heterotypic adherence between murine leukemia/lymphoma cells and marrow stromal cells involves a recognition mechanism with galactosyl and mannosyl specificities.
We have previously shown that leukemia/lymphoma (LL) cells adhere to marrow stromal cells (MSC), and MSC induce clonal growth of LL cells. Even though CD11a/18 and CD54 are important in leukocyte and endothelial cell interaction, the literature suggested that these adhesion proteins are not involved in adhesion of hematopoietic stem cells to MSC. We therefore utilized a unique ICAM-1- murine MSC line (MLT) to evaluate the mechanisms of adherence of LL cells (L5178Y and L1210) to MLT. Adherence of LL cells to extracellular matrix (ECM) proteins was also examined. L1210 cells attached to collagen types III and IV, laminin, and fibronectin, but not to collagen type I. L5178Y cells did not attach to any of the ECM proteins tested. The adherence of both L1210 and L5178Y to MSC was unaffected by rat monoclonal antibodies to murine CD11a, CD11b, and CD18. Neoglycoprotein probes, mannosyl-bovine serum albumin (BSA) and galactosyl-BSA, inhibited the adherence of L5178Y and L1210 cells to MSC by 34%-63% of controls at concentrations of 10(-3) and 5 x 10(-3) M. In contrast, fucosyl-BSA had no inhibitory effect on LL cell adherence of MLT. These data suggest that 1) LL cells may adhere to MSC by a lectin mechanism with mannosyl and galactosyl specificities; and 2) other mechanisms of adherence, not yet defined, are also important in this system.
Animals↗
Ultrastructural analysis of differentiation of rat endoderm in vitro. Adipose vascular-stromal cells induce endoderm differentiation, which in turn induces differentiation of the vascular-stromal cells into chondrocytes.
Isolated definitive endoderm from 9-day-old rat embryos was cultivated up to 24 days in plastic and glass petri dishes and on developing vascular-stromal cells (mesenchymal cells) from epididymal white and interscapular brown adipose tissue of 4-week-old male rats. Explants were analyzed histologically and ultrastructurally. Endoderm attached to the bottom of the glass or petri dishes degenerated under one week of cultivation. Endoderm free floating in the culture medium developed into unilaminar vesicles whose flat epithelium did not differentiate. However, endoderm inoculated on developing mesenchymal cells differentiated into glandular explants or into ciliated pseudostratified columnar respiratory epithelium. The glandular explants were made up of at least four different kinds of cells whose cytoplasm showed predominantly: a) polyribosomes, b) lysosomes, c) mitochondria or d) cytoskeletal filaments. Endodermal cells differentiated only if, during cultivation, they were in contact with or in close proximity to developing mesenchymal cells. Endoderm differentiating into the respiratory epithelium in turn directed differentiation of the underlying vascular-stromal cells into lamina propria cells and chondrocytes. Cultivated vascular-stromal cells in the upper layers became thicker, ellipsoid in shape and with enlarged intercellular space. They appeared to be lamina propria cells and, together with the respiratory epithelium, built folds of respiratory mucosa. The vascular-stromal cells in the layers close to the bottom developed into chondrocytes; i.e., the cells became oval and agglomerated in nest like structures with a defined extracellular matrix. Their cytoplasm contained abundant cisternae of rough endoplasmic reticulum and numerous vacuoles with PAS positive substance. These observations showed that even developing vascular-stromal cells from adipose tissue from postlactating rats can trigger the process of definitive endoderm differentiation. Once triggered, differentiating endoderm influenced differentiation of the vascular-stromal cells into the cells and tissues of a wall of the respiratory tract.
Adipose Tissue↗
Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation.
Endometrial stromal cells undergo decidual transformation, in response to epidermal growth factor (EGF) and progesterone. Since insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are believed to be involved in endometrial differentiation, and insulin regulates IGFBP production in a variety of cells, we have investigated the modulatory roles of EGF, progesterone, and insulin on IGFBP secretion by long term cultures of human endometrial stromal cells. Without insulin, the principal IGFBP secreted into conditioned medium, detected by Western ligand blotting, was a 28-kilodalton (kDa) IGFBP, identified by immunoprecipitation as IGFBP-1. This was observed only when the stromal cells were decidualized. With increasing insulin, IGFBP-1 decreased to undetectable levels. Concomitantly, IGFBP-2 increased, as did a 24-kDa IGFBP (believed to be IGFBP-4) and a 28-kDa IGFBP, shown to be a glycoprotein by endoglycosidase sensitivity (and believed to be glycosylated IGFBP-4). In the nondecidualized state, insulin increased the secretion of IGFBP-3, IGFBP-2, and the 24-kDa IGFBP, which were slightly inhibited by EGF and relatively unaffected by progesterone alone. In the absence of insulin, progesterone weakly stimulated IGFBP-1 secretion, which increased markedly when the cells were decidualized by combined treatment with EGF and progesterone. These data show that IGFBP-3, IGFBP-2, IGFBP-1, and presumably IGFBP-4 and its glycosylated form are differentially regulated by peptide and steroid hormones in endometrial stromal cells and that their regulation is a function of stromal differentiation.
Carrier Proteins↗
Production of matrix metalloproteinases 2 and 3 (stromelysin) by stromal cells of giant cell tumor of bone.
Matrix metalloproteinases play a central role in the catabolism of extracellular matrix macromolecules. Here the authors report that giant cell tumor of bone (GCT) produces two matrix metalloproteinases (MMPs) in zymogen form, which have been identified as proMMP-2 (also known as "72-kDa-progelatinase/type IV procollagenase") and proMMP-3 (prostromelysin). Giant cell tumor is known to consist of two major cell populations, multinucleated giant cells and stromal cells. On several passages of the tumor cells in culture, only stromal cells proliferated. These stromal cells produced proMMP-2 but not proMMP-3. Addition of the conditioned medium of primary GCT culture or human macrophage-conditioned medium to the passaged stromal cells induced the production of proMMP-3. The production of proMMP-3 was also induced by interleukin 1 (IL-1), but not by tumor necrosis factor alpha (TNF alpha). ProMMP-1 (tissue procollagenase) was not detected even after treatment with these stimuli. Immunohistochemical studies have demonstrated that multinucleated giant cells in GCT both produce IL-1 and TNF alpha, suggesting that IL-1 secreted by multinucleated giant cells may be responsible for in vivo production of proMMP-3 by the stromal cells. The authors propose that GCT has a self-stimulatory system for the production of matrix-degrading proteinases and that the ability of the passaged stromal cells to synthesize and secrete proMMP-3 with appropriate stimuli may contribute the malignant behavior of GCT.
Adult↗
Phorbol ester-induced promyelocytic leukemia cell adhesion to marrow stromal cells involves fibronectin specific alpha 5 beta 1 integrin receptors.
The human promyelocytic cell line NB4 exhibited a weak adhesion capacity for bone marrow-derived stromal cells and their extracellular matrices (5-15% of adherent cells). Adhesion was enhanced by pulse-treatment of cells with phorbolester (PMA 10(-7) M). Adhesion was induced within minutes, was fibronectin-specific, and affected up to 100% of the treated cells. This biological response to PMA resulted from the activation of protein kinase C (PKC), since PKC inhibitors (staurosporine, sphingosine, CGP 41251, and calphostin C) prevented the phenomenon. Phenotypical analysis of integrin receptor expression (particularly FN receptors VLA-4 and VLA-5) at the membrane of untreated or PMA-treated cells revealed that PMA induced no significant modification of the level of expression of these receptors. However, inhibition studies carried out with anti-VLA monoclonal antibodies demonstrated that the FN-specific adhesion triggered by PKC involved the alpha 5 beta 1 FN-specific receptors (VLA-5). We showed that the binding of NB4 cells to fibronectin was RGD-dependent. PMA-induced adhesion was not correlated to phosphorylation of the VLA-5 receptor. These findings may partially explain the malignant behaviour of these cells: The loss of their capacity to adhere to stromal cells may arrest differentiation and explain the large number of leukemic cells in the circulation.
Bone Marrow↗
Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures.
The differentiation of adipocytic and osteogenic cells has been investigated in cultures of adult rat marrow stromal cells. Adipocytic differentiation was assessed using morphological criteria, changes in expression of procollagen mRNAs, consistent with a switch from the synthesis of predominantly fibrillar (types I and III) to basement membrane (type IV) collagen, and the induction of expression of aP2, a specific marker for differentiation of adipocytes. Osteogenic differentiation was assessed on the basis of changes in the abundance of the mRNAs for the bone/liver/kidney isozyme of alkaline phosphatase and the induction of mRNAs for bone sialoprotein and osteocalcin. In the presence of foetal calf serum and dexamethasone (10(-8) M) there was always differentiation of both adipocytic and osteogenic cells. When the steroid was present throughout primary and secondary culture the differentiation of osteogenic cells predominated. Conversely, when dexamethasone was present in secondary culture only, the differentiation of adipocytes predominated. When marrow stromal cells were cultured in the presence of dexamethasone in primary culture and dexamethasone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10(-8) M) in secondary culture, the differentiation of adipocytes was inhibited whereas the differentiation of osteogenic cells was enhanced, as assessed by an increase in expression of osteocalcin mRNA. The results, therefore, demonstrate an inverse relationship between the differentiation of adipocytic and osteogenic cells in this culture system and are consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.
Adipose Tissue↗
Requirement for VLA-4 and VLA-5 integrins in lymphoma cells binding to and migration beneath stromal cells in culture.
Physical interaction between human lymphomas and murine bone marrow derived stromal cells were studied. Nalm-6 pre-B cells adhered to BMS2 stromal cells and subsequently migrated beneath them, while Ramos Burkitt lymphoma cells, adhered but did not migrate. Four mAbs were established against Nalm-6 cells, which were able to block initial adhesion of Nalm-6 cells. Two of them were directed against the alpha 4 chain of VLA-4, and other two recognized the beta 1 chain of VLA integrins. Therefore, the initial adhesion of Ramos and Nalm-6 cells to BMS2 was largely mediated by the VLA-4 integrin expressed on lymphocytes. The corresponding ligand on stromal cells appears to be VCAM-1, because antibodies against murine VCAM-1 blocked the adhesion. However, antibodies against the alpha chain of VLA-4 were not capable of blocking subsequent migration beneath stromal cells. In contrast, antibodies against the beta chain of VLA integrins blocked the migration beneath stromal cells as well as the initial adhesion. Because a common beta chain can be shared among integrins, the role of other VLA integrins in Nalm-6 cells migration was investigated. VLA-5 and VLA-6 as well as VLA-4 were expressed on Nalm-6 cells, but not on Ramos cells. Additional blocking experiments revealed that VLA-4 and VLA-5 are likely to work in concert to mediate the migration of Nalm-6 cells beneath stromal cells. Thus, particular VLA integrins appear to be responsible not only for lymphocyte adhesion but also for migration with respect to stromal cells. These findings may have implications for cell-cell interactions and directed migration of lymphocytes in bone marrow and other tissues.
Animals↗
Expression of M-CSF and its receptor (C-FMS) during factor-independent cell line evolution from hematopoietic progenitor cells cocultivated with gamma irradiated marrow stromal cell lines.
Gamma irradiation of plateau-phase clonal bone marrow stromal cell lines produces factor-independent growth of cocultivated clonal interleukin-3/granulocyte-macrophage colony-stimulating factor-dependent hematopoietic progenitor cell lines. The process is associated with three biologic changes including: (i) adherence of hematopoietic cells to stromal cells forming 'cobblestone islands'; (ii) an intermediate stage [during which the cells show proliferation in suspension in the presence in leukemogenic stromal factor (LSF), a factor similar to macrophage colony-stimulating factor (M-CSF) released by irradiated stromal cells, and transient hematopoietic cell surface expression of MAC-1, and c-fms (M-CSF receptor)]; and (iii) a third stage of factor-independence. A monoclonal antibody to M-CSF receptor inhibited proliferation of intermediate stage but not all factor-independent cell subclones. In the present studies, a subclonal factor-independent malignant subline of FDC-P1JL26 derived by cocultivation with gamma-irradiated stromal cells as well as the parent clone and intermediate stage cells were shown to express significant levels of M-CSF polyA+ mRNA and M-CSF of at least two sizes (23 and 15 kDa) as detected by 35S-methionine labelling and immunoprecipitation with polyclonal anti-M-CSF antiserum. There was no significant difference in intracellular M-CSF protein size between cells at each of the three stages of biologic change. This M-CSF was not detected on the cell surface by fluorescence-activated cell sorting (FACS). In contrast, c-fms expression at the cell surface was detected by FACS analysis and c-fms polyA+ mRNA was only detected during the intermediate stage of induction of factor-independence. FDC-P1JL26 parent cells, the subclone stimulated by LSF, and the factor-independent subclone, showed little or no detectable autophosphorylation of the c-fms receptor at tyrosine. There was no detectable rearrangement of the M-CSF or c-fms genes by Southern analysis between clonal lines during the three stages. While we cannot rule out an autocrine mechanism or mutated c-fms receptor mechanism, the data also suggest that evolution of hemopoietic cell factor-independence during cocultivation with irradiated stromal cells may involve a mechanism distal to the c-fms receptor/M-CSF interaction.
Animals↗
Enrichment of primary lymphocyte-supporting stromal cells and characterization of associated B lymphocyte progenitors.
Studies of Whitlock/Witte long-term bone marrow cultures have revealed the necessity of two cell types for B lymphopoiesis, a stem cell and the stromal cell. While a number of stromal cell lines exist they have been found to be heterogeneous with respect to cell surface marker expression and growth factor production. Separation and analysis of fresh bone marrow stromal cells is, therefore, necessary to understand the regulation of lymphopoiesis in vivo. Here we report the early stages of such studies. We demonstrate that stromal cells, as assessed by morphology and alkaline phosphatase reactivity after short-term culture, are enriched in cellular aggregates that can be separated from bone marrow suspensions. Stromal cells are present in aggregates at a frequency of one per thousand cells, whereas marrow from which the aggregates have been removed contains only one stromal cell per fifty-thousand cells. These aggregates are able to form Whitlock cultures from greatly reduced numbers of initiating cells, indicating that they contain culturable B lineage precursors as well as stromal cells capable of supporting B lymphopoiesis. The aggregates appear to be naturally formed and provide a means to examine native B cell precursor-stromal cell contacts. We find little evidence for sequestering of late-stage B cell precursors within the aggregates. Terminal deoxynucleotidyl transferase-positive cells, on the other hand, are approximately three times more frequent in bone marrow aggregates, suggesting close contact between very early B cell progenitors and stromal cells within the aggregates. The finding that stromal cells are enriched in cellular aggregates is an important first step in the ultimate isolation of these cells from marrow suspensions, which is vital to understanding stromal cell function in vivo.
Animals↗
Binding of primitive hematopoietic progenitor cells to marrow stromal cells involves heparan sulfate.
Blast colony-forming cells (BI-CFC) and pre-colony-forming unit-granulocyte, monocyte (CFU-GM) in human bone marrow bind to marrow-derived stromal layers grown in the presence of methylprednisolone (MP+), but do not bind to stroma grown without MP (MP-). The BI-CFC bind to stroma and form colonies when overlaid with agar; the pre-CFU-GM bind to stroma and release CFU-GM into the supernatant culture medium (delta assay). These two classes of progenitor may represent similar stages of hematopoietic cell development. Their binding to stroma depends on the presence of heparan sulfate proteoglycan (HS-PG) in the extracellular matrix secreted by the stromal cells. Here, we have analyzed the functional and biochemical properties of HS-PG isolated from MP+ and MP- stromal cultures. HS-PG or isolated HS glycosaminoglycan (GAG) side chains partially blocked progenitor cell binding when they were added to the 2-hour binding phase of the BI-CFC or delta assays. Gel electrophoresis of HS-PG resolved more bands in matrix preparations from MP+ cultures than in preparations from MP- cultures. The blocking activity of the eluted MP+ HS-PG bands depended partly on the amount of GAG attached to the protein core and presumably partly on the structure of the core itself. Time course studies demonstrated that the HS-dependent phase of the binding interaction was limited to the first 30 to 60 minutes of the 2-hour binding phase. The different blocking effects of MP+ and MP- HS indicate that they have different biochemical properties. The HS-GAG in MP+ stroma has a higher degree of sulfation and a greater negative charge to mass ratio compared with MP- HS-GAG. Variations in HS may determine specific binding by hematopoietic progenitor cells and a heparan sulfate receptor is envisaged as acting in concert with further cell adhesion molecules (CAMs) on the progenitor cell surface.
Adult↗
Generation of TWO iPSC lines (CRICKi025-A and CRICKi026-A) from healthy donor bone marrow mesenchymal stromal cells.
Mesenchymal stromal cells (MSCs) are key components of the bone marrow (BM), providing structural support and paracrine signals that regulate haematopoietic stem cell maintenance, self-renewal and differentiation. However, primary BM MSCs are rare, heterogeneous, and subject to donor variability and have limited ex-vivo expansion capacity, restricting their utility. Here, we describe two human induced pluripotent stem cells lines, CRICKi0025-A and CRICKi0026-A, reprogrammed from adult BM-derived MSCs using non-integrating Sendai virus vectors. Both lines showcase grade-A morphology, are genomically stable, upregulate essential pluripotent markers and can differentiate into the three germ layers. These lines are a well-characterised resource for generating MSCs.
Journal Article↗
Mouse uterine stromal cells secrete a 30-kilodalton protein in response to coculture with uterine epithelial cells.
While uterine stromal cells (USC) appear to modify the function of uterine epithelial cells (UEC) under certain conditions in vivo, relatively little is known about the effect of epithelial cells on stromal cell differentiation and function. To determine if UEC modulate USC function in vitro, highly enriched (> 95%) cultures of polarized UEC were first cultured on Matrigel-coated filters in serum-free medium until confluent, then cocultured with USC for up to 120 h. Subsequently, while maintaining both cell types in physically separate compartments, filters containing UEC were removed, and USC phenotypic markers assayed. Coculture with UEC did not affect the expression of two markers of USC differentiation (desmin and laminin), USC DNA content, [35S]methionine uptake, or total protein synthesis or secretion. However, coculture of USC with UEC or medium conditioned by UEC induced the secretion of a 30-kilodalton protein (p30) from USC as early as 24 h of coculture and through 120 h of coculture. In addition, secretion of a 60-kilodalton protein by USC was frequently observed in response to coculture with UEC. Neither the hormonal stage from which uterine cells were recovered, nor the addition of exogenous progesterone or estradiol modulated UEC-induced p30 secretion. Several purified growth factors (transforming growth factor-beta, epidermal growth factor, interleukin-1 alpha, and fibroblast growth factor) added to the serum-free culture medium failed to induce p30 secretion by USC. The p30-inducing activity in UEC-conditioned medium could not be abolished by either heat or trypsin treatment, suggesting that it is not a protein. Purified prostaglandin E2 or F2 alpha or platelet-activating factor did not induce p30 secretion by isolated USC. Of several epithelial and fibroblastic cell lines tested, UEC and a human uterine adenocarcinoma cell line (RL95-2) were the most effective in inducing p30 secretion by USC. Moreover, UEC also were able to modulate protein secretion by nonuterine murine fibroblast cell lines. Collectively, these data demonstrate that UEC can modulate USC function in vitro via a soluble factor(s).
Animals↗
Regulation of B cell differentiation by bone marrow stromal cells.
B lymphocyte development occurs in the intersinusoidal spaces of bone marrow in association with a sessile population of stromal cells. Development of long-term bone marrow culture systems that support B lymphopoiesis has allowed the isolation of stromal cells that support that process and permitted analysis of their role in controlling the growth and differentiation of B lineage cells in culture. In addition to direct interactions with developing lymphocytes, stromal cells secrete a variety of cytokines that affect lymphocyte growth and differentiation, and many of these molecules have been expressed in recombinant form. These relatively recent advances have made it possible to formulate a working model of the cells and molecules involved in regulating primary B cell production.
Animals↗