Somatic reprogramming to induced pluripotent stem cells (iPSC) was realized in the year 2006 in mice, and in 2007 in humans, by transiently forced expression of a combination of exogenous transcription factors. Human and mouse iPSCs are distinctly reprogrammed into a 'primed' and a 'naïve' state, respectively. In the last decade, puzzle pieces of somatic reprogramming have been collected with difficulty. Collectively, dissecting reprogramming events and identification of the hallmark of sequentially activated/silenced genes have revealed mouse somatic reprogramming in fragments, but there is a long way to go toward understanding the molecular mechanisms of human somatic reprogramming, even with developing technologies. Recently, an established human intermediately reprogrammed stem cell (iRSC) line, which has paused reprogramming at the endogenous OCT4-negative/exogenous transgene-positive pre-MET (mesenchymal-to-epithelial-transition) stage can resume reprogramming into endogenous OCT4-positive iPSCs only by change of culture conditions. Genome-editing-mediated visualization of endogenous OCT4 activity with GFP in living iRSCs demonstrates the timing of OCT4 activation and entry to MET in the reprogramming toward iPSCs. Applications of genome-editing technology to pluripotent stem cells will reshape our approaches for exploring molecular mechanisms.

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.

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Adiponectin secreted from adipocytes into plasma has anti‐aging, anti‐obesity and anti‐inflammation effects. Here, we detected intracellular adiponectin localized in the nuclei of human and mouse pluripotent stem cells, mouse germ cells and some somatic cells. Nucleus‐localized (Nu) adiponectin protein is characterized by an N‐terminal truncated monomer form in a native state, compared with intact multimer forms of cytoplasm‐localized (Cy) adiponectin protein. Doxycycline‐induced over‐expression of ADIPONECTIN caused cell death in human and mouse Nu‐type pluripotent stem cells. Genome‐wide gene expression analysis indicated that apoptosis by ADIPONECTIN over‐expression was induced in accompany with upregulation of AIFM2 and MEG3. Upregulation of AIFM2 and MEG3 and down‐regulation of miR‐214‐3p verified by qPCR analyses after ADIPONECTIN over‐expression indicated that the MEG3/miR‐214/AIFM2 pathway played a role in the apoptotic cell death of pluripotent cells. Adiponectin‐induced cell death was rescued by the treatment with miR‐214‐3p mimic. Global data analysis shows that Nu adiponectin has a role in microRNA‐mediated post‐transcription regulation, cell–cell interactions and chromatin remodeling as a survival gatekeeper.

Analysis of gene expression in single cells is required to understand somatic cell reprogramming into human induced pluripotent stem cells (iPSCs). To facilitate this, we established intermediately reprogrammed stem cells (iRSCs), pre‐iPSC lines. The iRSC‐iPSC conversion system enables the reproducible monitoring of reprogramming events and the analysis of progressive gene expression profiles using single‐cell microarray analysis and genome editing. Here, single‐cell microarray analysis showed the stage‐specific sequential gene activation during the conversion of iRSCs into iPSCs, using OCT4, TDGF1 and E‐CADHERIN as marker genes. Out of 75 OCT4‐related genes, which were significantly up‐regulated after the activation of OCT4, and entry into the mesenchymal‐to‐epithelial transition (MET), LIN28 (LIN28A) and FOXO1 were selected for applying to gene expression visualization. Multicolored visualization was achieved by the genome editing of LIN28 or FOXO1 with mCherry into OCT4‐GFP iRSCs. Fluorescent analysis of gene activity in individual cells showed that OCT4 was dispensable for maintenance, but required for activation, of the LIN28 and FOXO1 expression in reprogramming.