Preadipocyte screening by laminin in porcine stromal vascular cell cultures.

OBJECTIVE To determine if subpopulations of cells in stromal vascular (S-V) cultures could be segregated and separated based on affinity for laminin substratum. RESEARCH METHODS AND PROCEDURES S-V cells were seeded and allowed to attach for various times; 4 hours was found to be optimal for cell attachment. Cultures were rinsed after 4 hours of seeding, and S-V cells were divided into three subpopulations based on affinity for laminin: (1) cells that did not attach to laminin; (2) cells that had a low affinity for laminin; and (3) cells that had a high affinity for laminin. After 24 hours, cultures were either stained for the AD-3 antigen (a marker for preadipocytes), C/EBP-alpha (a terminal differentiation marker), or C/EBP-delta (an early preadipocyte marker). Companion cultures were treated with various media for 9 days and stained with oil red-O. RESULTS Cells with a high affinity for laminin had the highest proportion of AD-3 and C/EBP-alpha positive cells and the highest proportion of fat cells after treatment with insulin +/- dexamethasone. Cells with a low affinity for laminin had the highest proportion of C/EBP-delta cells and the highest proportion of fat cells after treatment with fetal bovine serum+dexamethasone, followed by insulin. DISCUSSION These results indicate that differentiating preadipocytes adhere to laminin to a much greater degree than do non-preadipocytes. Therefore, laminin-coated dishes can be used to screen S-V cells to produce preadipocyte or fibroblast-enriched S-V cultures.

[1]  R. L. Richardson,et al.  The influence of extracellular matrix substrata on preadipocyte development in serum-free cultures of stromal-vascular cells. , 1996, Journal of animal science.

[2]  G. Hausman,et al.  Cytochemical studies of adipose tissue-associated blood vessels in untreated and thyroxine-treated hypophysectomized pig fetuses. , 1996, Journal of animal science.

[3]  E. Schleicher,et al.  Distribution of basement membrane components in normal adipose tissue and in benign and malignant tumors of lipomatous origin. , 1996, Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc.

[4]  K. Wong,et al.  Extracellular matrix components secreted by microvascular endothelial cells stimulate preadipocyte differentiation in vitro. , 1994, Metabolism: clinical and experimental.

[5]  J. T. Wright.  Flow cytometric analysis of porcine preadipocytes , 1992, Journal of cellular biochemistry.

[6]  R. Mecham.  Receptors for laminin on mammalian cells , 1991, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[7]  Wright Jt,et al.  Monoclonal antibodies against cell surface antigens expressed during porcine adipocyte differentiation. , 1990 .

[8]  Y. Aratani,et al.  Ascorbic acid phosphate stimulates type IV collagen synthesis and accelerates adipose conversion of 3T3-L1 cells. , 1990, Experimental cell research.

[9]  G. Hausman.  The influence of insulin, triiodothyronine (T3) and insulin-like growth factor-I (IGF-1) on the differentiation of preadipocytes in serum-free cultures of pig stromal-vascular cells. , 1989, Journal of animal science.

[10]  H. Kleinman,et al.  Laminin: structure, functions and receptors. , 1989, Current opinion in cell biology.

[11]  Y. Aratani,et al.  Enhanced synthesis and secretion of type IV collagen and entactin during adipose conversion of 3T3-L1 cells and production of unorthodox laminin complex. , 1988, The Journal of biological chemistry.

[12]  R. Timpl,et al.  The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites. , 1987, The Journal of biological chemistry.

[13]  D. Zipori,et al.  Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells. , 1985, Blood.

[14]  D. Gospodarowicz,et al.  Respective roles of laminin and fibronectin in adhesion of human carcinoma and sarcoma cells , 1981, Nature.

[15]  G. Martin,et al.  Role of laminin in the attachment of PAM 212 (epithelial) cells to basement membrane collagen , 1980, Cell.

[16]  R. L. Richardson,et al.  Newly recruited and pre-existing preadipocytes in cultures of porcine stromal-vascular cells: morphology, expression of extracellular matrix components, and lipid accretion. , 1998, Journal of animal science.

[17]  G. Hausman,et al.  Preadipocyte recruitment in stromal vascular cultures after depletion of committed preadipocytes by immunocytotoxicity. , 1997, Obesity research.

[18]  G. Hausman,et al.  Vascular and cellular development in fetal adipose tissue: lectin binding studies and immunocytochemistry for laminin and type IV collagen. , 1991, Microvascular research.

[19]  H. Kleinman,et al.  Nerve growth factor, laminin, and fibronectin promote neurite growth in human fetal sensory ganglia cultures , 1982, Journal of neuroscience research.