Differentiation of cultured pre-adipose cells: A probability model

Abstract

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-LI 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 i s just large enough to replace "terminally differentiated" lipidcontaining cells that have been lost, thereby maintaining a constant percentage of fat cells in any given culture of 3T3-Ll.Differentiation i s the process by which cells acquire the ability to express a particular phenotype. Differentiated cells possess specialized properties or functions such as t h e ability t o produce substances l i k e melanin, hemoglobin, crystallin, or fat (Wigley, 1975). At a point in t h e i r life history some change in t h e organization of t h e genome or the expression of certain genes occurs and such cells eventually acquire a differentiated phenotype. This change, assumed for t h e present t o be irreversible, i s referred t o as the commitm e n t point. Commitment and different i a t i o n have been studied in several established cultured cell lines, including F r i e n d erythroleukemia cells (FEL), keratinocytes, muscle cells, and adipocytes (Friend e t al., 1965; F r i e n d e t al., 1971; Dienstman and Holtzer, 1975;Green, 1977;Nadal-Ginard, 1978;Levenson et al., 1979;Pragnell et al., 1980). Many hypotheses based u p o n genetic or epigenetic causes have been offered t o explain how commitment occurs, but a satisfactory explanation has remained elusive.

Stochastic Modeling of Adipogenesis in 3T3-L1 Cultures to Determine Probabilities of Events in the Cell’s Life Cycle

“…Committed cells divide to produce clones of committed cells''. 29 Moreover, they demonstrated that ''(1) there are only two possible outcomes-either the cell commits with probability p or it does not, (2) commitment in any cell is independent of commitment in any other cell, and (3) the probability of commitment is constant during any cell cycle''. 29 In our present modeling, we were consistent with the paper of Steinberg and Brownstein 29 : given their findings with respect to mitosis, if the mother cell is committed to the adipocyte lineage, both daughter cells will be committed as well.…”

“…29 Moreover, they demonstrated that ''(1) there are only two possible outcomes-either the cell commits with probability p or it does not, (2) commitment in any cell is independent of commitment in any other cell, and (3) the probability of commitment is constant during any cell cycle''. 29 In our present modeling, we were consistent with the paper of Steinberg and Brownstein 29 : given their findings with respect to mitosis, if the mother cell is committed to the adipocyte lineage, both daughter cells will be committed as well. If the mother cell is uncommitted, the daughter cells may both be uncommitted, or both be committed, or just one of them may be committed, depending on the stochastic parameter that determines commitment for each such individual daughter cell.…”

“…Otherwise, each cell was considered committed if a raffled number was smaller than p com per division. 29 Afterward, we continued to the next randomly selected cell.…”

“…[12][13][14] It has been previously suggested that cell lifecycle events may be referred to as stochastic events in theoretical modeling. 5,7,16,17,[23][24][25][26][27]29,32,33 For example, Pharr et al developed a stochastic model for mast cell proliferation in a culture. 23 Vogel et al similarly referred to stem cell development as stochastic events.…”

“…[25][26][27] To our knowledge, only Steinberg and Brownstein referred to the differentiation of 3T3-L1 preadipocytes as a stochastic process. 29 Nonetheless, their work was limited and simplified since they only explored the probability of adipose commitment. Song et al mathematically modeled the differentiation kinetics of 3T3-L1 cells over 24 days of maturation using reaction-diffusion differential equations.…”