Adipocyte Conversion of CHEF Ceils in Serum-free Medium

When grown in the presence of serum with added insulin, Chinese hamster embryonic fibroblasts (CHEF/18) cells can be induced to become preadipocytes that are committed to the adipocyte pathway of terminal differentiation (Sager, R., and P. Kovac, 1982, Proc. Natl. Acad. Sci. USA, 79:480-484). We found that commitment to the adipocyte pathway, as well as terminal differentiation to form mature adipocytes, can occur in a defined serumfree medium containing insulin. When CHEF/18 cells are plated in serum-containing medium, only 5-10% of cells in each colony undergo terminal differentiation, whereas in serum-free medium, >90% of the cells became adipocytes. These and other results show that CHEF/18 cells require no adipogenic factors in addition to insulin and the other components of the serum-free medium (transferrin, epithelial growth factor, thrombin) to form adipocytes, and furthermore, that serum inhibits the rate of terminal adipocyte differentiation of these cells. As little as 10 ng/ml insulin added to serum-containing medium can induce adipogenesis, suggesting that insulin rather than an insulinlike growth factor is the active agent. The results further demonstrate that virtually every CHEF/18 cell can be induced into the adipocyte pathway. In embryonic development, secondary stem cells of mesenchymal origin give rise to a number of differentiated cell types including adipocytes, myoblasts, myotubes, chondrocytes, osteocytes, and adult fibroblasts. This process has been mimicked in cell culture by treating fibroblastic cells of embryonic origin with 5-azacytidine for as short a time as a single cell cycle (1-3). Indeed, the response of embryonic fibroblastic cell lines to this drug has provided the basis for their identification as secondary mesenchymal stem cells. Since 5-azacytidine decreases the extent of cytosine methylation in DNA, its effect on stem cells has suggested that decreased DNA methylation is involved in the differentiation process. The results reported here are part of an ongoing investigation of molecular mechanisms underlying specific steps in development and the distribution of stem cells into alternative pathways of terminal differentiation. In a previous report, we described the ability of 5-azacytidine to induce Chinese hamster embryonic fibroblastic (CHEF/18) ~ stem cells to become preadipocytes, myoblasts, or chondrocytes, and showed further that the same clonal CHEF/18 cell populations could be directed exclusively into the adipocyte pathway if the cells were grown in the presence J Abbreviations used in this paper: CHEF cells, Chinese hamster embryonic fibroblastic cells; FBS, fetal bovine serum; GPDH, snglycerol-3-phosphate dehydrogenase. of added insulin without 5-azacytidine (1). Since insulin was added to a medium containing 10% fetal bovine serum (FBS), it was possible that the added insulin was interacting with some other adipogenic serum factor in eliciting the adipogenic response. The action of adipogenic factors in serum has been described in the maturation of adipocytes from 3T3 preadipocytes (4). In this paper, we describe the differentiation of CHEF/18 stem cells into adipocytes in serum-free medium. This medium, originally described by Cherington et al. (5), and subsequently improved by Sager et al. (6), supports continuous growth of CHEF/18 cells at a rate similar to that in serum (6). The medium contains epithelial growth factor, transferrin, and thrombin, in addition to 10 #g/ml insulin, a concentration required for optimal growth. The high concentration of insulin that is required suggests that it may be substituting for an insulinlike growth factor (7, 8) for the growth response, but this possibility has not been tested due to the unavailability of purified somatomedins or multiplication stimulating activity for growth studies. It is likely that insulin rather than an insulinlike growth factor is required for the adipocyte conversion in view of the fact that adipocyte differentiation can be induced by as little as 10 ng/ml insulin in serumcontaining medium as described in this paper. Commercial serum itself contains very low levels of insulin, and does not induce adipogenesis in CHEF/18 cells. THE JOURNAL OF CELL BIOLOGY • VOLUME 100 FEBRUARY 1985 429-434 © The Rockefeller University Press . 0021-9525185/02/0429/06 $1.00 429 on A ril 5, 2017 D ow nladed fom Published February 1, 1985