Showing papers on "Cell culture published in 1988"
TL;DR: It is demonstrated that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture and the strict substrate specificity of this reaction suggests that L- arginine is the precursor for NO synthesis in vascular endothelium cells.
Abstract: Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue1 and for the inhibition of platelet aggregation2 and platelet adhesion3 attributed to endothelium-derived relaxing factor4. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay5, chemiluminescence1 or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.
4,803 citations
TL;DR: The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.
Abstract: In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.
3,960 citations
TL;DR: Results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.
Abstract: A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.
3,238 citations
TL;DR: Experiments using radioactive protein show that tat becomes localized to the nucleus after uptake and suggest that chloroquine protects tat from proteolytic degradation, raising the possibility that, under some conditions, tat might act as a viral growth factor to stimulate viral replication in latently infected cells or alter expression of cellular genes.
2,733 citations
TL;DR: In this paper, a recombinant myeloid leukaemia inhibitory factor (LIF) was used to replace DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.
Abstract: Embryonic stem (ES) cells, the totipotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in M1 myeloid leukaemic cells and which we have recently purified, cloned and characterized. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.
2,140 citations
TL;DR: DIA and human interleukin DA/leukaemia inhibitory factor have been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and haemopoetic stem cell systems.
Abstract: Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.
1,984 citations
TL;DR: The predicted carboxy-terminal protein sequence encoded by the pNM23 cDNA clone is novel compared with Genebank animal, bacterial, and viral sequences and may be associated with intrinsic aggressiveness.
Abstract: We describe a gene, NM23, that is associated with the tumor metastatic process. NM23 RNA levels were highest in cells and tumors of relatively low metastatic potential in two experimental systems: (1) murine K-1735 melanoma cell lines, in which the gene was identified, and (2) N-nitroso-N-methylurea-induced rat mammary carcinomas. NM23 RNA levels did not correlate with cell sensitivity to host immunological responses and may, therefore, be associated with intrinsic aggressiveness. The predicted carboxy-terminal protein sequence encoded by the pNM23 cDNA clone is novel compared with Genebank animal, bacterial, and viral sequences.
1,283 citations
01 Jan 1988
TL;DR: In vivo and in vitro studies have shown that CFU-F are a heterogeneous population of stem and progenitor cells and that their differentiation in vitro can be modified at the colony level, which supports the hypothesis that there are stromal stem cells present in the soft connective tissues associated with marrow and bone surfaces.
Abstract: Evidence is discussed for the hypothesis that there are stromal stem cells present in the soft connective tissues associated with marrow and bone surfaces that are able to give rise to a number of different cell lines including the osteogenic line. Fibroblastic colonies, each derived from a single colony-forming unit fibroblastic (CFU-F), are formed when marrow cells are cultured in vitro. In vivo assays of CFU-F have demonstrated that some CFU-F have a high ability for self renewal and multipotentiality whereas some have more limited potential. In vitro studies also support the hypothesis and have shown that CFU-F are a heterogeneous population of stem and progenitor cells and that their differentiation in vitro can be modified at the colony level. Factors added to the medium can activate osteogenesis in a range of multipotential and more committed precursors. Different stromal cell lines can be promoted under different culture conditions. The number and hierarchy of cell lines belonging to the stromal fibroblastic system are not yet fully elucidated and more specific markers for the different lines are required before a better understanding can be achieved.
1,202 citations
TL;DR: Observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.
Abstract: We have defined a signal responsible for the morphological differentiation of human umbilical vein and human dermal microvascular endothelial cells in vitro. We find that human umbilical vein endothelial cells deprived of growth factors undergo morphological differentiation with tube formation after 6-12 wk, and that human dermal microvascular endothelial cells differentiate after 1 wk of growth factor deprivation. Here, we report that morphological differentiation of both types of endothelial cells is markedly accelerated by culture on a reconstituted gel composed of basement membrane proteins. Under these conditions, tube formation begins in 1-2 h and is complete by 24 h. The tubes are maintained for greater than 2 wk. Little or no proliferation occurs under these conditions, although the cells, when trypsinized and replated on fibronectin-coated tissue culture dishes, resume division. Ultrastructurally, the tubes possess a lumen surrounded by endothelial cells attached to one another by junctional complexes. The cells possess Weibel-Palade bodies and factor VIII-related antigens, and take up acetylated low density lipoproteins. Tubule formation does not occur on tissue culture plastic coated with laminin or collagen IV, either alone or in combination, or on an agarose or a collagen I gel. However, endothelial cells cultured on a collagen I gel supplemented with laminin form tubules, while supplementation with collagen IV induces a lesser degree of tubule formation. Preincubation of endothelial cells with antibodies to laminin prevented tubule formation while antibodies to collagen IV were less inhibitory. Preincubation of endothelial cells with synthetic peptides derived from the laminin B1 chain that bind to the laminin cell surface receptor or incorporation of these peptides into the gel matrix blocked tubule formation, whereas control peptides did not. These observations indicate that endothelial cells can rapidly differentiate on a basement membrane-like matrix and that laminin is the principal factor in inducing this change.
1,189 citations
TL;DR: Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, T GF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1 by a novel method for RNA analysis.
Abstract: The presence of macrophages is required for the regeneration of many cell types during wound healing. Macrophages have been reported to express a wide range of mitogenic factors and cytokines, but none of these factors has been shown in vivo to sustain all the wound-healing processes. It has been suggested that transforming growth factor-alpha (TGF-alpha) may mediate angiogenesis, epidermal regrowth, and formation of granulation tissue in vivo. Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, TGF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1. The expression of these transcripts was determined by a novel method for RNA analysis in which low numbers of mouse macrophages were isolated from wound cylinders, their RNA was purified and reverse-transcribed, and the complementary DNA was amplified in a polymerase chain reaction primed with growth factor sequence-specific primers. This single-cell RNA phenotyping procedure is rapid and has the potential for quantification, and mRNA transcripts from a single cell or a few cells can be unambiguously demonstrated, with the simultaneous analysis of several mRNA species. Macrophages from wounds expressed TGF-alpha antigen, and wound fluids contained TGF-alpha. Elicited macrophages in culture also expressed TGF-alpha transcripts and polypeptide in a time-dependent manner after stimulation with modified low-density lipoproteins and lipopolysaccharide endotoxin, which are characteristic of the activators found in injured tissues.
1,136 citations
TL;DR: Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins, and an existing bovine papilloma virus‐based expression vector was modified to allow transformed X63Ag8‐653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector.
Abstract: Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins (mIL2, mIL3, mIL4 or mIL5). An existing bovine papilloma virus-based expression vector, pBV-1MTHA, was modified to allow transformed X63Ag8-653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector, to express the inserted cDNA encoding a single interleukin constitutively, and to secrete the interleukin in high quantities. Cell lines transformed with mIL2 cDNA stably carried 30-100 copies of the plasmid per cell and constitutively secreted biologically active mIL2 in quantities similar to those produced by murine EL4 thymoma cells or rat spleen cells stimulated with mitogens. Deletion of the 3' untranslated region containing AT-rich sequences from the mIL2 cDNA resulted in a 100-fold increase in the constitutive production and secretion of mIL2 by the transformants. Addition of a heavy metal further increased the production 2 to 6-fold. Cells transformed with 3'-deleted mIL3 cDNA constitutively secreted 300-1000 times higher activities of mIL3 than the myelomonocytic leukemia line WEHI3. mIL4 produced by the similar transformants induced [3H]thymidine uptake of a T cell line, a mast cell line and B leukemia cells, and enhanced the production of IgG1 by B cells. IL4 titers were 150 times higher than those produced by the concanavalin A-stimulated T cell line 2.19. mIL5 was secreted by similar transformants at 10-fold higher titers than those produced by concanavalin A-stimulated 2.19 T cells, as judged by the proliferation and maturation of B cell leukemia BCL1. The expression vectors should be useful in establishing eukaryotic cell lines producing proteins from full length cDNA clones at higher rates. The established cell lines secreting IL2, 3, 4 or 5 at high rate should be useful sources for these interleukins in the investigation of their function in the immune system.
TL;DR: It is concluded that both 70Z/3 and HeLa cells contain apparently cytosolic NF-kappa B in a form with no evident DNA-binding activity, and phorbol esters both release the inhibition of binding and cause a translocation to the nucleus.
TL;DR: A model is proposed in which a ligand-dependent conformational change in a heme protein accounts for the mechanism by which hypoxia as well as cobalt and nickel stimulate the production of Epo.
Abstract: Erythropoietin (Epo), the hormone that stimulates red blood cell production, is synthesized in the kidney and liver in response to hypoxia. The human hepatoma cell line Hep3B regulates its production of Epo in a physiologic manner. Either hypoxia or cobalt chloride markedly increases expression of Epo mRNA as well as production of biologically active and immunologically distinct Epo protein. New protein synthesis is required before the induction of increased levels of hypoxia- or cobalt-induced Epo mRNA. Hypoxia, cobalt chloride, and nickel chloride appear to stimulate Epo production through a common pathway. The inhibition of Epo production at low partial pressures of oxygen by carbon monoxide provides evidence that a heme protein is integrally involved in the oxygen-sensing mechanism. This hypothesis is further supported by the finding that when heme synthesis is blocked, hypoxia-, cobalt-, and nickel-induced Epo production are all markedly inhibited. A model is proposed in which a ligand-dependent conformational change in a heme protein accounts for the mechanism by which hypoxia as well as cobalt and nickel stimulate the production of Epo.
TL;DR: Transforming growth factor-α (TGF-α) was first detected in the culture medium of certain retrovirus-transformed cell lines as an activity which binds to the receptor for epidermal growth factor (EGF) and thereby inhibits the binding of EGF as mentioned in this paper.
TL;DR: The kinetics of induction after serum starvation and density-dependent inhibition of two growth-arrest-specific (gas) genes were investigated in more detail and two cell lines transformed by viral onc genes did not express the two gas genes.
TL;DR: Hemolytic-positive revertants were selected after passage of the hly- mutants through monolayers of J774 cells, and in each case, the hemolytic revertants possessed the 58-kD polypeptide, were capable of intracellular growth in tissue culture monolayer and were virulent for mice.
Abstract: Listeria monocytogenes insertion mutants defective in hemolysin production were generated using the conjugative transposons Tn916 and Tn1545. All of the nonhemolytic mutants (hly-) lacked a secreted 58-kD polypeptide, presumedly hemolysin, and were avirulent in a mouse model. An intracellular multiplication assay was established in monolayers of mouse bone marrow-derived macrophages, the J774 macrophage-like cell line, the CL.7 embryonic mouse fibroblast cell line, and the Henle 407 human epithelial cell line. The hly+ strain grew intracellularly in all of the tissue culture cells with a doubling time of approximately 60 min. In contrast, the hly- mutants failed to grow in the murine-derived tissue culture cells, but retained the ability to grow in the human tissue culture cells examined. Hemolytic-positive revertants were selected after passage of the hly- mutants through monolayers of J774 cells. In each case, the hemolytic revertants possessed the 58-kD polypeptide, were capable of intracellular growth in tissue culture monolayers and were virulent for mice.
TL;DR: Osteoclasts and activated macrophages in culture were shown to generate an acidic microenvironment specifically in the attachment zone between the cell and the base of the culture dish, which would favor the direct extracellular action of secreted lysosomal proteinases in the degradation of collagen by both cell types in vivo.
TL;DR: The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo and is associated with the preferential affinity of the mutant protein for GDP.
Abstract: Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.
TL;DR: Evidence for the hypothesis that there areStromal stem cells present in the soft connective tissues associated with marrow and bone surfaces that are able to give rise to a number of different cell lines is reviewed and the possibility that the marrow stromal system is part of a wider stromAL cell system of the body is proposed.
Abstract: Summary Evidence for the hypothesis that there are stromal stem cells present in the soft connective tissues associated with marrow and bone surfaces that are able to give rise to a number of different cell lines is reviewed. The lines are currently designated fibroblastic, reticular, adipocytic and osteogenic. Fibroblastic colonies, each derived from a single colony-forming unit fibroblastic (CFU-F), are formed when marrow cells are cultured in vitro. In vivo assays of tissue formed by CFU-F in open transplant or in diffusion chambers, have demonstrated that some CFU-F have a high ability for self renewal and multipotentiality whereas some have more limited potential. Preliminary investigations in vitro also support the hypothesis and have shown that CFU-F are a heterogeneous population of stem and progenitor cells and that their differentiation in vitro can be modified at the colony level. The stromal cells which survive and proliferate in vitro are highly dependent on culture conditions. The number and hierarchy of cell lines belonging to the stromal fibroblastic system are not yet fully elucidated and more specific markers and better assays for the different phenotypes are required before a greater understanding can be achieved. The possibility that the marrow stromal system is part of a wider stromal cell system of the body is proposed.
TL;DR: The role of recombinant B cell stimulatory factor 2 (BSF-2/IL-6) in the regulation of growth and differentiation of B cells was investigated and BSF-2 is one of the essential late-acting factors in PWM-induced Ig production.
Abstract: The role of recombinant B cell stimulatory factor 2 (BSF-2/IL-6) in the regulation of growth and differentiation of B cells was investigated. rBSF-2 at 200 pg/ml could induce 50% of the maximum Ig production in B lymphoblastoid cell lines, the specific activity being estimated as 5 X 10(6) U/mg. rBSF-2 augmented PWM-induced IgM, IgG, and IgA production in mononuclear cells (MNC); the effect was exerted by directly acting on PWM-induced B blast cells to induce Ig production. However, rBSF-2 did not induce any growth of activated B cells. In contrast, rBSF-2 showed a potent growth activity on a murine hybridoma clone, MH60.BSF2. The concentration required for half-maximal [3H]TdR uptake was approximately 5 pg/ml, which was 40 times less than that required for Ig induction in a B cell line. Anti-BSF-2 antibody inhibited PWM-induced Ig production in MNC, but not PWM-induced proliferation. The antibody was effective even when added on day 4 of an 8-d culture, indicating that BSF-2 is one of the essential late-acting factors in PWM-induced Ig production.
TL;DR: TRACP-positive multinucleated cells formed in response to osteotropic hormones in mouse marrow cultures satisfy most of the criteria of osteoclasts, and osteoblasts may play an important role in osteoclast formation.
Abstract: We developed a mouse bone marrow culture system to examine the process of osteoclast-like multinucleated cell formation from its progenitors. When mouse marrow cells were cultured for 8 days with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3, 10(-10) to 10(-7) M] or human PTH (1-34) (25-100 ng/ml), tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells formed. No TRACP-positive multinucleated cells appeared in the absence of these hormones. 1 alpha,25-(OH)2D3 and PTH also increased the number of the clusters of TRACP-positive mononuclear cells. Time course studies showed that these TRACP-positive mononuclear cell clusters appeared before the formation of TRACP-positive multinucleated cells, suggesting that the TRACP-positive mononuclear cells are precursors of the multinucleated cells. Salmon calcitonin markedly inhibited the formation of TRACP-positive multinucleated cells but not TRACP-positive mononuclear cell clusters induced by 1 alpha,25-(OH)2D3 or PTH. TRACP-positive mononuclear cells and multinucleated cells were rarely stained for nonspecific esterase, but some mononuclear cells were positively stained for both nonspecific esterase and TRACP. More that 90% of the TRACP-positive mononuclear cell clusters and multinucleated cells were found near colonies of alkaline phosphatase-positive mononuclear cells (possibly osteoblasts). When marrow mononuclear cells were cultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 or PTH, numerous resorption lacunae were formed. These results suggest that 1) TRACP-positive multinucleated cells formed in response to osteotropic hormones in mouse marrow cultures satisfy most of the criteria of osteoclasts, and 2) osteoblasts may play an important role in osteoclast formation.
TL;DR: A cell line showing many characteristics of immature mast cells has been established from the peripheral blood of a patient with mast cell leukemia, and solid tumors of metachromatically positive cells have been successfully induced and serially passed in nude mice using 5-azacytidine transformed cells.
TL;DR: The data suggest that this clonal cell line contains subpopulations of mesenchymal progenitor cells which can, under the influence of glucocorticoid hormones, differentiate in vitro into four distinct cell types.
Abstract: RCJ 3.1, a clonally derived cell population isolated from 21-d fetal rat calvaria, expresses the osteoblast-associated characteristics of polygonal morphology, a cAMP response to parathyroid hormone, synthesis of predominantly type I collagen, and the presence of 1,25-dihydroxyvitamin D3-regulated alkaline phosphatase activity. When cultured in the presence of ascorbic acid, sodium beta-glycerophosphate, and the synthetic glucocorticoid dexamethasone, this clone differentiated in a time-dependent manner into four morphologically distinct phenotypes of known mesenchymal origin. Multinucleated muscle cells were observed as early as 9-10 d in culture, lipid-containing adipocytes formed after 12 d, chondrocyte nodules were observed after 16 d, and mineralized bone nodules formed after 21 d in culture. The differentiated cell types were characterized morphologically, histochemically, and immunohistochemically. The formation of adipocytes and chondrocytes was dependent upon the addition of dexamethasone; the muscle and bone phenotypes were also expressed at low frequency in the absence of dexamethasone. The sex steroid hormones progesterone and 17 beta-estradiol had no effect on differentiation in this system, suggesting that the effects of dexamethasone represent effects specific for glucocorticosteroids. Increasing concentrations of dexamethasone (10(-9)-10(-6) M) increased the numbers of myotubes, adipocytes, and chondrocytes; however, when present continuously for 35 d, the lower concentrations appeared to better maintain the muscle and adipocyte phenotypes. Bone nodules were not quantitated because the frequency of bone nodule formation was too low. Single cells obtained by plating RCJ 3.1 cells at limiting dilutions in the presence of dexamethasone, were shown to give rise to subclones that could differentiate into either single or multiple phenotypes. Thus, the data suggest that this clonal cell line contains subpopulations of mesenchymal progenitor cells which can, under the influence of glucocorticoid hormones, differentiate in vitro into four distinct cell types. It is, therefore, a unique cell line which will be of great use in the study of the regulation of mesenchymal stem cell differentiation.
TL;DR: These results provide the first direct evidence that the differential expression of cadherins can play a role in cell sorting in heterogeneous cell populations.
TL;DR: This method of cell transplantation, which is termed "chimeric neomorphogenesis," is an alternative to current methods and requires further study.
TL;DR: It is found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic fibroblast growth factor (bFGF).
Abstract: We have found that the spontaneous migration of bovine aortic endothelial cells from the edge of a denuded area in a confluent monolayer is dependent upon the release of endogenous basic fibroblast growth factor (bFGF). Cell movement is blocked by purified polyclonal rabbit IgG to bFGF as well as affinity purified anti-bFGF IgG and anti-bFGF F(ab')2 fragments. The inhibitory effect of the immunoglobulins is dependent upon antibody concentration, is reversible, is overcome by the addition of recombinant bFGF, and is removed by affinity chromatography of the antiserum through a column of bFGF-Sepharose. Cell movement is also reversibly inhibited by the addition of protamine sulfate and suramin; two agents reported to block bFGF binding to its receptor. The addition of recombinant bFGF to wounded monolayers accelerates the movement of cells into the denuded area. Transforming growth factor beta which has been shown to antagonize several other effects of bFGF also inhibits cell movement. The anti-bFGF IgG prevents the movement of bovine capillary endothelial cells, BHK-21, NIH 3T3, and human skin fibroblasts into a denuded area. Antibodies to bFGF, as well as suramin and protamine sulfate also suppress the basal levels of plasminogen activator and DNA synthesis in bovine aortic endothelial cells.
TL;DR: A set of procedures for patterning the outgrowth of cells cultured on 2-dimensional substrates showed that glia are patterned along with the associated granule cells, and Interestingly, the GFAP-positive glia that proliferated on surfaces bound with amine derivatives attained primarily a tile-shaped, fibroblast-like morphology, while those proliferating on glass coated with poly(D-lysine) developed primarily a spindle- shaped, process-bearing morphology.
Abstract: The cytoarchitecture of nervous tissue is lost during the dissociation procedures used to form primary cell cultures. As a first step toward reestablishing an ordered arrangement of these cells in vitro, we developed a set of procedures for patterning the outgrowth of cells cultured on 2-dimensional substrates. These procedures used a combination of surface chemistry and photolithographic techniques. The adhesive properties of either silicon or silicon dioxide (quartz) surfaces were controlled by covalently binding small organic molecules to the surface with silane coupling agents. The attachment and growth of either embryonic mouse spinal cells or perinatal rat cerebellar cells were found to be promoted by binding certain amine derivatives to the surface. In particular, cells grown on surfaces bound with diamines and triamines, but not with monoamines, formed cultures whose morphology was similar to that of cells cultured on conventional substrates, i.e., glass coated with poly(D-lysine). The attachment of cells to a substrate was inhibited by binding alkane chains (e.g., n- tetradecane) to the surface and plating the cells in media containing 5– 10% (vol/vol) serum. Patterns of selected adhesivity were formed using photochemical resist materials and lithographic masking techniques compatible with the silane chemistry. Cultures of either spinal cord cells or cerebellar cells could be confined to square regions on the scale of 50 micron. Cerebellar cells could be confined to grow on lines with widths less than 10 micron. This width is comparable to the diameter of granule cell somata. The patterned growth of cerebellar cells was maintained up to 12 d in vitro. Over this time period the granule cells were observed to develop electrical excitability and immunoreactivity for neuron-specific enolase. Purkinje neurons also developed electrical excitability when grown on the chemically modified surfaces. Immunochemical reactivity of the patterned cultures for glial fibrillary acid protein (GFAP) showed that glia are patterned along with the associated granule cells. Interestingly, the GFAP-positive glia that proliferated on surfaces bound with amine derivatives attained primarily a tile-shaped, fibroblast-like morphology, while those proliferating on glass coated with poly(D-lysine) developed primarily a spindle-shaped, process-bearing morphology. Granule cells preferentially associated with the spindle-shaped glia.
TL;DR: NGFI-B, as a possible ligand-dependent transcriptional activator induced by NGF, may play a role in initiating NGF-induced differentiation.
Journal Article•
TL;DR: Different mechanisms in the regulation and expression of ICAM-1 on the various cell types are indicated and some insight is provided into the anti-inflammatory effects of some pharmacologic agents.
Abstract: The expression of intercellular adhesion molecule-1 (ICAM-1) on primary human fibroblasts, a human fibrosarcoma, chondrosarcoma, and adenocarcinoma cell line in response to IL-1, TNF-alpha, or IFN-gamma was studied using an ELISA with anti-ICAM-1 mAb. The induction of ICAM-1 by these cytokines was neutralized by cytokine-specific antisera as well as some steroids and the glycosylation inhibitor, tunicamycin. Cyclohexamide up-regulated the expression of ICAM-1 on chondrosarcoma cells but had little or no effect on carcinoma cells. These data indicate different mechanisms in the regulation and expression of ICAM-1 on the various cell types and provide some insight into the anti-inflammatory effects of some pharmacologic agents.
TL;DR: It is reported that TGF-beta 1 increases steady-state levels of its own message in six different normal and transformed cells in culture.