Differentiation of cultured pre-adipose cells: a probability model

Some cells of the established preadipose cell line, 3T3-L1, synthesize triglyceride after becoming confluent and quiescent. An analysis of the distribution of clusters of lipid-containing cells was consistent with a commitment event during exponential growth followed by clonal growth of committed cells. Experiments were designed to determine if the final clonal pattern of fat among nonfat cells could be described by a probability model. Undifferentiated cells (fibroblastic cells with no detectable accumulation of triglycerides) were plated at various cell numbers so that the total number of cell divisions to confluence could be controlled. Cells were passaged by trypsinization and replating, or trypsinization followed by passage through a narrow-bore needle before replating. Passing cells through a 22G needle seems to eliminate already committed cells from the population. We determined the percentage of fat cells and the range of clone sizes in cultures in which clone sizes depended upon the number of allowed cell divisions. Patterns of clone sizes in experimental cultures were compared to expected patterns obtained by computer simulations of several programmed and stochastic commitment models. Both the observed range of clone sizes and pattern of clones can be approximated by a simple stochastic model, suggesting that commitment to fat production in 3T3-L1 cells is a random process occurring with a fixed probability in single cells in exponential growth, followed by division of both committed and uncommitted cells. The probability of commitment was essentially constant at each cell division. The number of cells committed during each passage is just large enough to replace "terminally differentiated" lipid-containing cells that have been lost, thereby maintaining a constant percentage of fat cells in any given culture of 3T3-L1.