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Abstract
SummaryHuman extended pluripotent stem cells (EPSCs), with bidirectional chimeric ability to contribute to both embryonic and extraembryonic lineages, can be obtained and maintained by converting conventional pluripotent stem cells using chemicals. However, the transition system is based on inactivated mouse fibroblasts, and the underlying mechanism is not clear. Here we report a Matrigel-based feeder-free method to convert human embryonic stem cells and induced pluripotent stem cells into EPSCs and demonstrate the extended pluripotency in terms of molecular features, chimeric ability, and transcriptome. We further identify chemicals targeting glycolysis and histone methyltransferase to facilitate the conversion to and maintenance of feeder-free EPSCs. Altogether, our data not only establish a feeder-free system to generate human EPSCs, which should facilitate the mechanistic studies of extended pluripotency and further applications, but also provide additional insights into the transitions among different pluripotent states.
Highlights
There are two distinct types of cells during mammalian early embryonic development: totipotent cells that harbor superior development potential and are able to give rise to the whole conceptus, including embryonic and extraembryonic tissues, and pluripotent cells that can contribute only to embryonic lineages composed of most of the organs
After testing a panel of chemicals targeting pluripotency and differentiation, we eventually found that LCDM plus another two chemicals, IWR-1-endo and Y27632, with a high concentration of Matrigel, was able to convert human embryonic stem cells (ESCs) into domeshaped extended pluripotent stem cells (EPSCs)
By qRTPCR analysis, we found that feeder-free EPSCs (ffEPSCs) did express significantly higher pre-implantation genes such as DNMT3L and KLF4 but did not express or rarely expressed post-implantation markers, including DUSP6 and ZIC2 (Figure 1C)
Summary
There are two distinct types of cells during mammalian early embryonic development: totipotent cells that harbor superior development potential and are able to give rise to the whole conceptus, including embryonic and extraembryonic tissues, and pluripotent cells that can contribute only to embryonic lineages composed of most of the organs. The extended pluripotent stem cells (EPSCs) exhibited outstanding developmental potential and bidirectional chimeric ability, contributing to both embryonic tissues and extraembryonic tissues, including yolk sac and placenta Another group independently established a chemical cocktail to maintain a mouse single eight-cellstage blastomere in vitro as ‘‘expanded potential’’ stem cells (Yang et al, 2017a) and later succeeded in pig and human species (Gao et al, 2019). These two pioneering works gave rise to irreplaceable cell types compared with naive or primed pluripotent stem cells in terms of both developmental potential and research significance. Both of the conversion conditions were based on feeder cells, which brought uncertain factors that could interfere with further molecular dissection and potential clinical application (Chen et al, 2014)
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