Additionally, we sequenced subpopulations of reprogramming intermediates that underwent mesenchymal to epithelial transition (MET) on day 6 (mCherry–/GFP–/Epcam+) or activated endogenous Oct4 (mCherry–/GFP+) on days 9 and 12 (2 days after dox withdrawal) (Figure S4E).
Only mCherry– cells were used for RNA isolation to reduce heterogeneity and to eliminate contamination by untransduced cells. Samples were collected after 2, 4, 6, 9, and 12 days of dox induction and used for total RNA isolation and sequencing. The pMX mCherry+ OG2 MEFs were transduced with polycistronic reprogramming vectors. To gain deeper insight into the mechanism of SKM reprogramming, we performed RNA-sequencing (RNA-seq) over the course of reprogramming under both SKM and OSKM conditions (Figure 4E).
Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs, and thereby bear major implications for further development and application of iPSC technology. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. SKM induction could partially activate the pluripotency network even in Oct4-knockout fibroblasts. Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors.
Here we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. GEO help: Mouse over screen elements for information.Įxcluding Oct4 from Yamanaka cocktail unleashes the developmental potential of iPSCsĮxpression profiling by high throughput sequencing