Expression profiling by high throughput sequencing
Summary
Cancer cells can only rarely be reprogrammed to induced pluripotent stem cells (iPSCs) using the overexpression of the transgenes Oct4, Sox2, Klf4, and Myc. Those cancer cells that have been reprogrammed seem to only partially complete the process and are locked in an intermediate state. There are barriers that block cancer cells from reprogramming, but the nature of those barriers is unclear. In this work, we immortalized mouse embryonic fibroblasts (MEFs) using a variety of transgenes and discovered several immortalized cell lines that remain compatible with reprogramming. We utilise the different lines to explore the factors that are compatible with reprogrmming and those that block reprogramming to pluripotent stem cells
Overall design
MEFs were immortalized with combinations of: oncogenes (Myc, Hras, Mef2d, p53DD), viral factors (SV40T, E1A), apoptotic factors (Bcl2), and an engineered epigenetic factor (Hdac7SA; serines 178, 344, and 479 substituted with alanine, to block nuclear export (Luo et al., 2019)). The MEFs were passaged for at least a month to remove untransformed wildtype MEFs, then the cells were reprogrammed using OSKM (Oct4, Sox2, Klf4, c-Myc) transgenes to reprogram. Cells were reprogrammed in serum+ vitamin C and in various inhibitor cocktails against: MEK (PD; PD0325901), GSK3 (CHIR; CHIR99021), ROCK (Y; Y23637), G9a (BIX; BIX-01294), and Histone deacetylases (TSA)). Reprogrammed cells were sorted using GFP driven from the 'OG2' reporter.
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