GSE137001
Excluding Oct4 from Yamanaka cocktail unleashes the developmental potential of iPSCs
Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). 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. SKM induction could partially activate the pluripotency network even in Oct4-knockout fibroblasts. 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. 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. Overall design: 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). The pMX mCherry+ OG2 MEFs were transduced with polycistronic reprogramming vectors. Samples were collected after 2, 4, 6, 9, and 12 days of dox induction and used for total RNA isolation and sequencing. Only mCherry– cells were used for RNA isolation to reduce heterogeneity and to eliminate contamination by untransduced cells. 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). We also compared the gene expression in all OSKM and SKM iPSC lines tested in 4N complementation assays, including those generated by episomal method, as well as ESC lines—altogether 23 lines. To track the developmental fate of 4N-off OSKM iPSCs, we marked three high quality and four low quality lines (both tet-inducible and episomal) with constitutive mCherry expression. We used these lines to generate 4N-aggregates and examined the embryos at E9.5. At this stage all the tested lines could generate viable embryos with no obvious phenotype (Figure 7D) and the vast majority of the cells are derived from iPSCs (Figure S7E). We sorted mCherry+ iPSC-derived differentiated cells and performed RNA-seq for three embryos per iPSC line, as well 3 control embryos (altogether 24 embryos).