Derivation of feeder-free human extended pluripotent stem cells

Abstract

Summary Human 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.

Active endogenous retroviral elements in human pluripotent stem cells play a role in regulating host gene expression

“…LTR5_Hs/LTR5 is expressed from the 8-cell stage of human embryos and continues to the inner cell mass in blastocyst stage (Figure 5B ), and this process is correlated with the up-regulation of pluripotent factors (Figure 5C ). Since conventional human ESCs are considered as epiblast stage (late blastocyst) ( 31 , 67 ), and LTR5_Hs/LTR5-bound transcription factors and the HERVK transcript exhibited the higher expression levels in early 8-cell or morula stage, we wondered whether the activity of LTR5_Hs/LTR5 elements are higher in pluripotent cell with early embryonic characteristics, such as naïve ESCs and extended pluripotent stem cells (EPSCs) ( 68 ). We detected the activity of LTR5_Hs/LTR5/HERVK in naïve ESCs and EPSCs, truly higher than conventional human ESCs (Figure 5D , Supplementary Table S7 ).…”

“…LTR5_Hs/LTR5 elements exhibit higher activity in EPSCs, so we focused on the regulation in feeder-free EPSCs (ffEPSCs) previously established in our laboratory ( 31 ). We first validated the expression pattern by RT-qPCR showing HERVK is much higher in ffEPSCs than the parental ESCs (Figure 6A ).…”

“…Culture medium was refreshed daily, and the cells were passaged with accutase (Stemcell Technologies, # A1110501) every 4–5 days. The maintenance and conversion of ffEPSCs from ESCs were previously described ( 31 ). HEK293T cells were cultured with DMEM containing 10% FBS and 1% penicillin-streptomycin.…”

Stepwise differentiation of functional pancreatic β cells from human pluripotent stem cells

“…For instance, naïve PSCs or even totipotent-like stem cells have recently been established (Guo et al, 2016;Geng et al, 2019;Mazid et al, 2022). The human expanded potential stem cells or extended pluripotent stem cells (EPSCs) exhibit both embryonic and extraembryonic bidirectional developmental potential (Yang et al, 2017;Gao et al, 2019;Zheng et al, 2021), possibly providing an alternative cell source. Thus, human PSCs could be feasible to manufacture limitless numbers of the human cells with given types in vitro for transplantation therapies, including pancreatic β cells and other diabetes-relevant cells (Kim et al, 2020).…”

Comparative functional genomics identifies unique molecular features of EPSCs

“…EPSCs have been derived from various sources, including four- or eight-cell mouse embryos, human fibroblast-derived induced pluripotent stem cells (iPSCs), mouse and human ESCs ( Yang et al, 2017a , 2017b ), pig ( Gao et al, 2019 ), and bovine ( Zhao et al, 2021 ) early blastocysts. These cells can be further adapted to grow in feeder-free ( Zheng et al, 2021 ) and xeno-free ( Liu et al, 2021 ) conditions, thus opening new avenues for their molecular dissection and clinical applications. Compared with ESCs, EPSCs display superior developmental potential as they can generate both embryonic and extraembryonic tissues, including yolk sac and placenta ( Yang et al, 2017a , 2017b ).…”