5-Azacytidine Induction of Stable Mesodermal Stem Cell Lineages from 10T1/2 Cells: Evidence for Regulatory Genes Controlling Determination

5-Azacytidine converts the mouse embryonic cell line C3H 10T1/2 into differentiated chondrocytes, adipocytes, and skeletal muscle. Clonal and 2D protein gel analyses demonstrate that 5-azacytidine converts 10T1/2 cells into three stably determined, but undifferentiated, stem cell lineages which can differentiate into myofibers, chondrocytes, and adipocytes. Conversion of 10T1/2 cells is accompanied by specific changes in protein synthetic patterns unique for each cell lineage. We propose that 5-azacytidine converts 10T1/2 cells by hypomethylation of "determination" regulatory loci which establish lineages of stem cells with a restricted potential to differentiate into muscle, cartilage, or fat cells. Our results suggest that these three lineages are specified by separate regulatory loci and that as few as 1-3 hypomethylation events per cell are sufficient to activate the hypothesized muscle regulatory locus. Conversion of 10T1/2 cells by 5-azacytidine provides a model for studying regulatory genes involved in cell lineage determination.