Heterogeneity amongst splenic stromal cell lines which support dendritic cell hematopoiesis

Summary Long-term cultures (LTC) producing dendritic cells (DC) have been previously established from spleen. LTC support the development of nonadherent cells comprising small DC progenitors and immature DC. Similarly, the splenic stroma STX3, derived from a LTC which ceased DC production, can support DC development from precursors in overlaid bone marrow. The STX3 stroma is an immortalised mixed population of endothelial cells and elongated spindle-shaped cells, thought to be fibroblasts. The stromal cell components of STX3 have been studied here. A panel of 102 cell lines was established by single-cell sorting. A range of clone morphology, including cobblestone cells and elongated spindle-shaped cells, was reflective of heterogeneity in STX3. However, similar expression levels for the endothelial genes ACVRL1/ALK1, COL18A1, and MCAM in 13 splenic stromal cell lines suggested that both cell types had endothelial origin. The hematopoietic support function of stromal clones was tested in coculture assays with a bone marrow cell overlay. Splenic stromal cell lines with different morphology were both supporters and nonsupporters of hematopoiesis, in terms of foci formation or release of suspension cells. Cloning of STX3 led to the isolation of a panel of splenic endothelial cell lines heterogeneous in terms of morphology and hematopoietic support function.     

Phenotypic and functional characterization of human thymic stromal cell lines.

To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.     

Cell density-dependent proliferation of the murine bone marrow-derived stromal cell lines

Proliferation of three murine marrow-derived stromal cell lines, LC1, LC2, and LC3, depended on initial cell density. For LC2 and LC3, the cell density-dependence was negated by conditioned-media, indicating growth dependence on a soluble growth factor. For LC1, conditioned-media failed to stimulate proliferation, suggesting growth dependence on direct cell-cell contact.     

Functional characterization of two stromal cell lines that support B lymphopoiesis.

Bone marrow stromal cells have well documented effects on the production of B lymphocytes, but whether or not stromal cell signals are involved in the pre-B to B cell transition is unclear. The potential of two stromal cell lines, S10 and S17, in this process was examined. Initial experiments, using a short term liquid culture, indicated that S10 and S17 stroma efficiently supported the generation of clonable B cells (B lymphocyte CFU) from their immediate precursors in fresh bone marrow. The contribution of macrophages and other accessory cells in those experiments was minimized through use of a colony assay system that permits the direct effects of stromal cell signals on single B cell progenitors to be evaluated. The results indicated that soluble mediators from the S10 and S17 lines could support colony formation from fresh or cultured surface Ig- bone marrow cells. Colonies supported by S17 stroma appeared on day 15 and contained cells that expressed the B220 Ag; surface IgM expression was never observed. S10 supported colonies appeared on day 7 and routinely included surface IgM+ cells. Individual colonies were capable of undergoing additional growth when picked and replated directly onto the different stroma. Those colonies replated onto S10 stroma generated surface IgM expressing cells in up to 60% of experiments, but colonies transferred onto the S17 cell line included B cells only 10% of the time. These data demonstrate that stromal cells alone can provide the signals necessary for generating a surface IgM+ B cell from precursors but that not all stromal cell lines are equally efficient at doing so.     

Thymus-derived Striated Muscle Clones

Myogenic clones can be induced in thymus reticulum cultures from adult mice. We investigated the differentiation and maturation of myogenic cells by combined ultrathin section and freeze etching analyses. New insights into the development of cell membranes and cytoplasmic organelles will be discussed in the light of previous investigations of embryonic muscle cell cultures.     

Prostate stromal and urogenital sinus mesenchymal cell lines for investigations of stromal-epithelial interactions

Abstract Bidirectional signaling between the urogenital sinus epithelium and mesenchyme is an essential element of prostate development that regulates ductal morphogenesis, growth, and differentiation. Comparable interactions between the epithelium and stroma in the adult prostate appear to regulate normal growth homeostasis. Alterations in the stromal–epithelial dialogue that recapitulate features of the mesenchymal–epithelial interactions of development may play a critical role in the development of benign prostatic hyperplasia and in the progression of prostate cancer. For this reason, the mesenchymal–epithelial interactions of development are of considerable interest. In this review, we provide an overview of the mesenchymal contribution to rodent prostate development with an emphasis on the stage just before ductal budding (embryonic day 16; E16) and describe the isolation, characterization and utility of a newly established E16 urogenital sinus mesenchymal cell line.     

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.     

Effect of oestradiol on cytokine production in immortalized human marrow stromal cell lines.

Oestrogen deficiency enhances bone osteoclastogenesis and bone resorption. Evidence of cooperation between stromal cells and osteoclast precursors in mice suggests that oestradiol acts by regulating cytokine release from stromal cells. Bone marrow stroma contains multipotent progenitors that give rise to many mesenchymal lineages, including osteoblasts that may regulate osteoclast differentiation. We immortalized and characterized six human bone marrow stromal cell lines (presence of Stro1, secretion of alkaline phosphatase, osteocalcin, formation of lipid droplets, and presence of alpha and beta oestrogen receptors). The response of cytokines to oestradiol was then evaluated in vitro, as were the phorbol myristate acetate (PMA)-stimulated cytokine levels. Cells had the characteristics of undifferentiated stromal cells (Stro1+, RANK-L+), and expressed alpha-oestrogen receptors. The osteoblast phenotype (amounts of alkaline phosphatase and osteocalcin) was weak and there was a poor capacity to differentiate into adipocytes. These cell lines did not respond to oestradiol by producing interleukin 6 (IL-6), IL-1 or tumour necrosis factor alpha (TNF-alpha) either constitutively or after stimulation with PMA. Moreover, RANK-L and osteoprotegerin expressions were not regulated by oestradiol in vitro. Thus, modulation of these cytokines by stromal cells do not appear to be the mechanism by which oestradiol regulates bone resorption in humans.     

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.     

Human bone marrow stromal cell lines from myeloma and rheumatoid arthritis that can support murine pre-B cell growth.

In order to elucidate the pathologic significance of the bone marrow (BM) microenvironment in multiple myeloma (MM) and rheumatoid arthritis (RA), we established patient- or healthy donor (HD)-derived BM stromal cell lines by transfecting the plasmid for expression of SV40 large T Ag and examined their ability to support the stromal cell-dependent growth of a pre-B cell line, DW34. The means of recovered cell numbers of DW34 co-cultured with MM- and RA-derived BM stromal cell lines ranged from 6- to 10-fold more than those with HD-derived ones. Their enhanced ability to support DW34 cell growth was not caused by cytokines, including IL-6, IL-7, and c-kit ligand, although exogenous IL-7 could augment the growth-supporting ability. DW34 cell growth on the stromal cell lines was abolished by inhibiting cell-to-cell interaction with a membrane filter. FACS analysis revealed that the stromal cell lines did not express LFA-1 alpha, beta, NCAM, or ELAM-1. Both patient and HD BM stromal cell lines variably expressed ICAM-1, VCAM-1, and CD44. However, surface expression levels of these molecules did not correlate with the ability of the stromal cell lines to support DW34 cell growth. Taken together, these results suggested that BM microenvironment might play important roles in the pathogenesis of MM and RA.     

Different growth factors stimulate cell division of rat mammary epithelial, myoepithelial, and stromal cell lines in culture

A number of single-cell-cloned cell lines have been used to examine the growth-promoting effects of putative mammotrophic agents on the various cell types in normal and neoplastic rat mammary glands. A partially purified novel pituitary-derived growth factor stimulates only cuboidal epithelial cells to divide whereas fibroblast growth factor (FGF) stimulates the growth of stromal and myoepithelial-like cells. Epidermal growth factor (EGF) has a widespread but variable growth-stimulating action, but prolactin and growth hormone are essentially inactive when added alone at a concentration of 5 micrograms/ml. Phosphoethanolamine stimulates the growth of one epithelial cell line and a derivative myoepithelial-like cell line, but is inactive on the other cell lines tested. The use of defined cloned cell lines provides a direct and reproducible assay for the identification and purification of inducers of mammary growth.     

Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule

Bone marrow stromal cell lines have been isolated that directly support B lymphopoiesis in vitro. Single B-lineage precursors proliferate and differentiate on certain of these stromal cell lines to establish long-term B-lineage cultures. These lymphopoietic stromal cells produce novel soluble factors that support proliferation of in vitro established pre-B cell populations. Lymphoid populations established on lymphopoietic stromal cell lines lack surface Ig-bearing cells, but give rise to surface Ig+ cells when transferred to mixed bone marrow feeder layers. Several stromal lines expressed a B-lineage neoplasia marker detected by the monoclonal antibody MAb6C3. Remarkably, only the 6C3Aghi stromal lines supported long-term proliferation of B-lineage cells. We propose that the 6C3 antigen-bearing molecule may play a role in stromal cell-dependent, pre-B cell proliferation, as well as in neoplastic proliferation of pre-B leukemias.     

Characterization of human intestinal stromal cell lines: response to cytokines and interactions with epithelial cells

The maintenance of the physiological homeostasis of the gut mucosa characterized by continuous proliferation and differentiation processes results from epithelial-mesenchymal cell cross-talk. To set out stable and homogeneous models for the study of the (dys)regulation of various morphofunctional aspects, we established and characterized three clonal cell lines (C9, C11, and C20) derived from human duodenal mucosal connective tissue. We defined the expression of (i) cytoskeletal proteins; (ii) basement membrane molecules (laminins, collagen IV, nidogen) which have been shown formerly to be deposited at the epithelial/mesenchymal interface in situ by the mesenchymal compartment; and (iii) soluble factors, HGF, and TGFbeta1. The three cell lines display common but also specific proliferative responses to cytokines (IL1beta, IL2, IL8, TNFalpha, IFNgamma, TGFbeta1, and HGF). When cocultured with embryonic intestinal endoderms or with human colonic Caco2 or HT29 cancer cells, C9 versus C11 and C20 cell lines induced limited versus extensive growth of the associated epithelial cells. In addition C20 cells allowed spreading of HT29 cells with the formation of a basement membrane at the heterologous interface. Morphogenesis obtained by intracoelomic grafts of associations comprising the mesenchymal cell lines and intestinal endoderms was also different among those composed of C9 cells or of C11 or C20 cells. In conclusion, these data indicate that the mucosal connective tissue is heterogeneous and comprises several phenotypically different mesenchyme-derived cells whose equilibrium may be important in the gut homeostasis. These cells can now be used to define tissue-specific factors which may be involved in the physiopathology of the intestinal epithelium.     

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.     

Thymus-derived Cells in Mouse Thoracic Duct Lymph

Thymus lymphocytes injected into neo-natally thymectomized mice were identified in the thoracic duct lymph by their θ antigens and shown to form part of the recirculating lymphocyte pool.     

Heterologous Antiserum to Thymus-derived Cells in the Guinea-pig

USEFUL antisera specific for thymocytes and peripheral T lymphocytes have only been widely available in the form of antisera to thymic isoantigens of the mouse¹. We describe here the preparation and properties of a heterologous antiserum to guinea-pig thymocytes which is rendered specific for T lymphocytes after absorption with a pure population of B lymphocytes. We have already described^2,3 the properties of the transplantable acute lymphatic leukaemia L₂C of inbred strain two guinea-pigs. The L₂C leukaemia cell is characterized as a B cell by the presence of surface immunoglobulin of the λ₂ class, the secretion of a small amount of λ₂ immunoglobulin and the presence of a receptor for antigen-antibody-complement (C3) complexes characteristic of the B cell population⁴. Because, as will be shown, the antiserum is specific both for thymocytes and thymus derived lymphocytes, it will be referred to as anti-thymus derived cell (anti-TDC) serum. The availability of such an antiserum for a species in which the in vivo and in vitro manifestations of delayed hyper-sensitivity are so easily demonstrated may prove to be highly advantageous. illustration

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The effect of bone marrow stromal cells on the cloning of human leukemia/lymphoma cell lines

The cloning of established human leukemia and lymphoma cell lines at limiting dilutions in microwells and soft agar is described. Growth of most cell lines was improved by the addition of human AB serum and irradiated human bone marrow stromal cells. In general, the cloning efficiency in microwells was greater than in soft agar. The effect of bone marrow stromal cells appeared to be caused by a diffusable factor(s), but close cellular interaction could not be excluded since cloning in microwells produced consistent and optimal cell growth compared to growth in soft agar. It was concluded that cloning of leukemia and lymphoma cell lines in microwells was the preferred method and that similar techniques could improve the cloning of fresh leukemia and lymphoma cells.     

Biochemical and functional characterization of proteoglycans produced by Sl/Sld murine bone marrow stromal cell lines

The Steel anemia of mice results from an inherited defect in the hematopoietic microenvironment. Proteoglycans synthesized by bone marrow stromal cells are an important functional component of the hematopoietic microenvironment in normal animals. It is thus possible that Steel anemia results from a molecular abnormality involving bone marrow stromal proteoglycans. To investigate this possibility, we studied proteoglycan synthesis in three stromal cell lines from Steel anemic (Sl/Sld) animals and two control stromal cell lines, one (+/+2.4) from a non-anemic littermate, and one (GBl/6) from a normal mouse. Proteoglycans were precursor labelled with 35S sulfate and separated by ion exchange HPLC, CsCl density gradient centrifugation, and molecular sieve HPLC. Glycosaminoglycan (GAG) moieties were characterized by molecular sieve HPLC and enzyme sensitivity. There were no consistent differences in total proteoglycan synthesis, proteoglycan heterogeneity, GAG hydrodynamic size, or enzyme sensitivity among the cell lines studied. Growth factor binding to stromal extracellular matrix (ECM) was studied by co-culture of an IL-3-dependent cell line (FDC-P1) with cell-free ECM preparations from an Sl/Sld and a control (GBl/6) stromal cell line, with and without pre-incubation with IL-3. Cell-free ECM preparations from Sl/Sld and control cell lines supported FDC-P1 growth to an approximately equal extent after pre-incubation with IL-3. FDC-P1 growth support by ECM preparations from both cell lines was also observed without IL-3 pre-incubation, although to a lesser extent, suggesting ECM binding of endogenous growth factors synthesized by the stromal cells.