Fibroblast feeder layers inhibit differentiation of retinoic acid-treated embryonal carcinoma cells by increasing the probability of stem cell renewal.
The appearance of differentiated cells in embryonal carcinoma (EC) cultures can be inhibited by culturing the cells on fibroblast feeder layers. To determine whether or not feeder layers act by increasing the probability of stem cell renewal, growth and differentiation were monitored in cultures of F9 (subclone OTF9 -63) EC cells exposed to retinoic acid (RA) in either the presence or absence of feeder layers. By measuring the fraction of laminin-positive TROMA 1-positive or alkaline phosphatase-negative cells, it was determined that the frequency of differentiated cells in RA-treated F9 cultures was reduced by 70-80% when cells were cultured on fibroblast feeder layers instead of gelatin-coated dishes. Experiments in which EC cells were cultured in close proximity to a feeder layer demonstrated that cell-cell contact was required for maximal inhibition of differentiation. The probability of stem cell renewal was determined by measuring the number of colony-forming cells in RA-treated cultures as a function of time. Analysis of the data demonstrated that the probabilities of stem cell renewal were 0.5 and 0.25 during the first and second 48 h periods, respectively, following addition of RA for cells cultured without feeder layers. Cultures maintained on feeder layers exhibited a stem cell renewal probability of 0.72. Thus, feeder layers reduce the frequency of differentiated cells in RA-treated cultures by increasing the probability of stem cell renewal. Determining the mechanism by which feeder layers counteract the effect of a chemically defined differentiation inducer should help to uncover the processes that regulate the probability of stem cell renewal.
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D054278:Embryonal Carcinoma Stem Cells; D051379:Mice; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000473:pathology*; D013234:Stem Cells / Q000473:pathology*; D013724:Teratoma / Q000473:pathology*; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology*
D014212:Tretinoin