OCT4 Activity during Conversion of Human Intermediately Reprogrammed Stem Cells to iPS Cells through MET

Abstract

To facilitate understanding the mechanisms of somatic reprogramming to human induced pluripotent stem cells (iPSCs), we have established intermediately reprogrammed stem cells (iRSCs), human mesenchymal cells that express exogenous Oct4, Sox2, Klf4 and c-Myc (OSKM) and endogenous SOX2 and NANOG. iRSCs can be stably maintained at low density. At high density, however, they are induced to enter mesenchymal-epithelial transition (MET), resulting in reprogramming to an iPSC state. Morphological changes through MET correlate with silencing of exogenous OSKM, and upregulation of endogenous OCT4. A CRISPR/Cas9-mediated GFP knock-in visualized the temporal regulation of endogenous OCT4 in cells converting from iRSC to iPSC state. OCT4 activation coincident with silencing of OSKM occurred prior to entering MET. Notably, OCT4 instability was frequently observed in cells of developing post-MET colonies until a late stage (>200 cells), demonstrating that OCT4-activated post-MET cells switched from asymmetric to symmetric cell division in late stage reprogramming.

TGF-β Family Signaling in Embryonic and Somatic Stem-Cell Renewal and Differentiation

“…However, epithelial character is a defining feature of hESCs and, as in mouse reprogramming, MET is important for conversion to the pluripotent state. For example, intermediary reprogrammed cells undergo MET in transition to stable human iPSCs (Teshigawara et al, 2016), and promotion of TGF-β signaling by Ezh2 during reprogramming promotes the mesenchymal phenotype and inhibits generation of iPSC (Rao et al, 2015). Collectively, these studies indicate that during the transition to transgene-independent hiPSCs, TGF-β-dependent epithelial-to-mesenchymal transition (EMT) pathways must be circumvented to allow TGF-β-dependent stabilization of the pluripotent state.…”

Visualization of sequential conversion of human intermediately reprogrammed stem cells into iPS cells

“…iRSCs and genome-edited iRSCs were maintained and reprogrammed to iPSCs as previously described (Teshigawara et al, 2016).…”

“…The expression level of LIN28 was expected to change after OCT4 activation. In OCT4 (−) cells, SOX2 was maintained (Teshigawara et al, 2016). In OCT4 (−) cells, SOX2 was maintained (Teshigawara et al, 2016).…”

“…In our previous report (Teshigawara, Hirano, Nagata, Ainscough, & Tada, 2016), a novel type of human pre-iPSCs called intermediately reprogrammed stem cell "iRSCs" were reported as stable cell lines. Genome editing-mediated knock-in of GFP (Green fluorescence protein) into the endogenous OCT4 locus in iRSCs visualized gene activation of OCT4 in reprogramming through MET (Teshigawara et al, 2016). Notably, iRSCs efficiently and reproducibly resume the reprogramming process toward the full iPSC under culture conditions at high cell density (1 x 10 6 cells/ 3.5 cm culture dish) in no need of additional treatment with inhibitors or transgenes.…”

“…In OCT4 (−) cells, SOX2 was maintained (Teshigawara et al, 2016). SOX2 (+) and a high expression of LIN28 indicated differentiation of OCT4 (−) cells into neural ectodermal cells (Teshigawara et al, 2016). SOX2 (+) and a high expression of LIN28 indicated differentiation of OCT4 (−) cells into neural ectodermal cells (Teshigawara et al, 2016).…”