Practical clinical applications for current induced pluripotent stem cell (iPSC) technologies are hindered by very low generation efficiencies. Here, we demonstrate that newborn human (h) and mouse (m) extra-embryonic amnion (AM) and yolk-sac (YS) cells, in which endogenous KLF4 ⁄ Klf4, c-MYC ⁄ c-Myc and RONIN ⁄ Ronin are expressed, can be reprogrammed to hiPSCs and miPSCs with efficiencies for AM cells of 0.02% and 0.1%, respectively. Both hiPSC and miPSCs are indistinguishable from embryonic stem cells in colony morphology, expression of pluripotency markers, global gene expression profile, DNA methylation status of OCT4 and NANOG, teratoma formation and, in the case of miPSCs, generation of germline transmissible chimeric mice. As copious amounts of human AM cells can be collected without invasion, and stored long term by conventional means without requirement for in vitro culture, they represent an ideal source for cell banking and subsequent 'on demand' generation of hiPSCs for personal regenerative and pharmaceutical applications.
Induced pluripotent stem cells (iPSCs) are generated by directly reprogramming somatic cells by forcing them to express the exogenous transcription factors, Oct4, Sox2, Klf4 and c-Myc (OSKM). These cells could potentially be used in clinical applications and basic research. Here, we explored the molecular role of Sox2 by generating iPSCs that expressed Sox2 at various levels. Low Sox2 (LS) expression increased the efficiency of generating partially reprogrammed iPSCs in combination with OKM. Notably, we detected a significant increase in the number of fully reprogrammed iPSCs with three factors of OK and LS. LS expression was linked with the reduced expression of ectoderm and mesoderm marker genes. This indicates that cell differentiation into the ectoderm and mesoderm lineages was impeded during reprogramming. The quality of the iPSCs that was generated by using OK and LS was comparable to that of iPSCs that were produced via conventional OSK as seen by pluripotent marker gene expression and chimera formation. We conclude that Sox2 plays a crucial role in a dose-dependent manner in direct reprogramming of somatic cells to iPSCs.
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.
Human pluripotent stem cells (hPSCs) are thought to be a promising cell-source solution for regenerative medicine due to their indefinite proliferative potential and ability to differentiate to functional somatic cells. However, issues remain with regard to achieving reproducible differentiation of cells with the required functionality for realizing human transplantation therapies and with regard to reducing the potential for bacterial or fungal contamination. To meet these needs, we have developed a closed-channel culture device and corresponding control system. Uniformly-sized spheroidal hPSCs aggregates were formed inside wells within a closed-channel and maintained continuously throughout the culture process. Functional islet-like endocrine cell aggregates were reproducibly induced following a 30-day differentiation protocol. Our system shows an easily scalable, novel method for inducing PSC differentiation with both purity and functionality.
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