Partial pluripotent reprogramming in C2C12 myoblasts

Partial pluripotent reprogramming has been reported to reverse some features of aging in mammalian cells and tissues. However, the impact of partial reprogramming on somatic cell identity programs and the necessity of individual pluripotency factors remain unknown. Here, we mapped trajectories of partial reprogramming in young and aged cells from multiple murine cell types using single cell transcriptomics to address these questions. We found that partial reprogramming restored youthful gene expression in adipogenic cells and mesenchymal stem cells but also temporarily suppressed somatic cell identity programs. We further screened Yamanaka Factor subsets and found that many combinations had an impact on aging gene expression and suppressed somatic identity, but that these effects were not tightly entangled. We also found that a partial reprogramming approach inspired by amphibian regeneration restored youthful gene expression in aged myogenic cells. Our results suggest that partial pluripotent reprogramming poses a neoplastic risk, but that restoration of youthful gene expression can be achieved with alternative strategies. C2C12 myoblasts were transduced with lentiviral vectors harboring CMV>Tet3G-T2A-Hygro alleles and positive cells were selected by culture in hygromycin B. Selected clones were then transduced with lentiviral vectors harboring TRE>{Sox2,Oct4,Klf4,Myc}-EFS>eGFP alleles and selected by FACS for eGFP expression. Cells were cultured for eight days in three experimental groups. (1) Control cells were not treated with Dox, (2) partially reprogrammed cells were treated with Dox for the first three days, and (3) positive control cells were treated with Dox for all eight days. RNA was isolated from triplicate wells of each condition on days 1, 3, 4, 5, 6, and 8. Only partially reprogrammed cells were collected on days 4, 5, and positive controls were only collected on day 8.