Analysis of gene expression dynamics during iPS cell derivation from mouse embryonic fibroblasts using reprogramming systems with different Klf4 stoichiometry

The forced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc) reprograms cells into induced pluripotent stem cells (iPSCs) through a series of sequential cell fate conversions. The order and robustness of gene expression changes are highly depended on the Yamanaka factor stoichiometry. We specifically focused on two different reprogramming paths induced by high- and low-Klf4 stoichiometry, which were accomplished by introducing OK+9MS or OKMS polycistronic cassettes, respectively, into mouse embryonic fibroblasts. By comparing these reprograming intermediates with embryonic stem cells (ESCs) and primary keratinocytes, we identified high-Klf4 specific, transiently up-regulated epithelial genes. We found that expression of these epithelial genes was enriched in a TROP2-positive cell population. Moreover, we identified a set of transcription factors which are candidates for the regulation of transiently expressed epithelial genes, and revealed their connection to high-Klf4-specific reprogramming hallmarks. 20 samples were analyzed. MEFs were transfected with OKMS (Low-Klf4) or OK+9MS (High-Klf4), followed by FACS sorting for transgene-positive mCherry+ cells (d2, d4, d8, d18) or TROP2+ cells (d8). FACS sorted mCherry+ cells (d6) were submitted previously (GSE65468). A mouse embryonic stem cell, iPS clones derived from each reprogramming methods (OKMS: A7 and A9, OK+9MS: A10 and H10) and primary keratinocyte were used as references for intermediate reprogramming populations.