The differentiation of proadipocytes in vitro typically required prolonged culture of cells as a high density in high concentrations of serum and added hormones. With such culture conditions it is difficult to design experiments to determine the mechanisms that control the differentiation process. We now describe the rapid and parasynchronous growth arrest and differentiation of low density murine proadipocytes in heparinized medium containing only human plasma. When low density cells are cultured under these conditions, growth arrest at a distinct state in the G1 phase of the cell cycle occurs within 2 d and the differentiation of 80-100% of the cell population occurs within 4 d thereafter. The factors in human plasma which promote growth arrest and differentiation are heat labile and can be separated by barium adsorption. In the following paper we have used these methods to show that there are five separate phases which regulate the coupling of proadipocyte growth arrest and differentiation. The data reported in this paper establish that: (a) high cell density and extensive cell-to-cell contact are not required for adipocyte differentiation, (b) prolonged culture is not required for adipocyte differentiation, and (c) high concentrations of serum and/or added hormones are not required for adipocyte differentiation.

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