OCT4 activity during conversion of human intermediately reprogrammed stem cells to iPSCs through mesenchymal-epithelial transition

• Published in: Development (Cambridge) Vol. 143; no. 1; pp. 15 - 23
• Main Authors: Teshigawara, Rika, Hirano, Kunio, Nagata, Shogo, Ainscough, Justin, Tada, Takashi
• Format: Journal Article
• Language: English
• Published: England 01.01.2016
• Subjects: Cell Division - physiology; Cell Line; Cell Transdifferentiation - physiology; Cellular Reprogramming - physiology; Clustered Regularly Interspaced Short Palindromic Repeats; Enzyme Activation; Epithelial Cells - cytology; Green Fluorescent Proteins - genetics; Homeodomain Proteins - metabolism; Humans; Induced Pluripotent Stem Cells - cytology; Kruppel-Like Transcription Factors - genetics; Kruppel-Like Transcription Factors - metabolism; Mesenchymal Stromal Cells - cytology; Mesenchymal Stromal Cells - metabolism; Nanog Homeobox Protein; Octamer Transcription Factor-3 - genetics; Octamer Transcription Factor-3 - metabolism; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - metabolism; SOXB1 Transcription Factors - genetics; SOXB1 Transcription Factors - metabolism; Human; OCT4; CRISPR; Reprogramming; IPS; MET
• ISSN: 1477-9129
• DOI: 10.1242/dev.130344

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.