Project description:Mouse embryonic stem cells (mESCs) are in naive pluripotency that represents the ground state of development, from which all cells in the mouse embryo are derived. In contrast, human embryonic stem cells (hESCs) are in a primed state of pluripotency with many different properties. Despite intense efforts to generate naive human pluripotent stem cells (hPSCs), it has not been possible to derive naive hPSCs without relying on transgene overexpression or chemicals. Here, we show that a transient treatment with Torin1, a selective inhibitor of mTOR, converted hPSCs from primed to naive pluripotency. The naive hPSCs were maintained in the same condition as mESCs in defined media with 2iLI (MEK inhibitor, GSK3b inhibitor, LIF and Insulin). Like mESCs, they exhibited high clonal efficiency, rapid cell proliferation, active mitochondrial respiration, X chromosome activation, DNA hypomethylation, and transcriptomes similar to those of human blastocysts than primed hESCs. Most importantly, the naive hPSCs significantly contributed to mouse embryos when transferred to mouse blastocysts. mTor inhibition induced nuclear translocation of TFE3, a critical transcription factor at the interplay of autophagy and pluripotency. TFE3 with mutated nuclear localization signal blocked the conversion from primed to naive pluripotency. It appears that by mimicking diapause at the cellular level, naive pluripotency in human can be readily attained from primed hPSCs, thus establishing the unified ground state of pluripotency in mammals.
Project description:Mouse embryonic stem cells (mESCs) are in naive pluripotency that represents the ground state of development, from which all cells in the mouse embryo are derived. In contrast, human embryonic stem cells (hESCs) are in a primed state of pluripotency with many different properties. Despite intense efforts to generate naive human pluripotent stem cells (hPSCs), it has not been possible to derive naive hPSCs without relying on transgene overexpression or chemicals. Here, we show that a transient treatment with Torin1, a selective inhibitor of mTOR, converted hPSCs from primed to naive pluripotency. The naive hPSCs were maintained in the same condition as mESCs in defined media with 2iLI (MEK inhibitor, GSK3b inhibitor, LIF and Insulin). Like mESCs, they exhibited high clonal efficiency, rapid cell proliferation, active mitochondrial respiration, X chromosome activation, DNA hypomethylation, and transcriptomes similar to those of human blastocysts than primed hESCs. Most importantly, the naive hPSCs significantly contributed to mouse embryos when transferred to mouse blastocysts. mTor inhibition induced nuclear translocation of TFE3, a critical transcription factor at the interplay of autophagy and pluripotency. TFE3 with mutated nuclear localization signal blocked the conversion from primed to naive pluripotency. It appears that by mimicking diapause at the cellular level, naive pluripotency in human can be readily attained from primed hPSCs, thus establishing the unified ground state of pluripotency in mammals.
Project description:We have pioneered human pluripotent stem cell (hPSC) manufacturing in stirred suspension bioreactors. Cell therapies require large numbers of quality-controlled hPSCs yet technologies are limited in their ability to efficiently grow and scale clinically-viable hPSCs. We report here that naive hPSCs exhibit superior growth in suspension bioreactors compared to their primed counterpart. Naive hPSCs exhibited a shorter lag phase, and grew into more uniform, homogenous aggregates. Compared to static culture, gene expression analyses revealed that the bioreactor environment promoted the upregulation of naïve- and downregulation of primed-associated transcripts in both primed and naive hPSCs. Bioreactor-cultured naive hPSCs similarly showed more hypomethylated DNA and less primed hPSC-associated surface protein marker compared to statically-cultured naive hPSCs. Gene expression, epigenetic, and cell surface protein marker analyses all suggest that the bioreactor environment promotes the transition from primed-to-naive pluripotent state. Our research shows that reprogramming conventional hPSCs to the naive pluripotent state enhances hPSC manufacturing.
Project description:The extent of naive characteriztics of recently reported naive human pluripotent stem cells (hPSCs) obtained in different naive-permissive media, is unclear. Moreover, these naive hPSCs were mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. Here, we derived genetically matched human naive hPSCs by direct reprogramming of fibroblasts as well as primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Comprehensive characterization showed that naive hPSCs obtained in these different conditions represent a spectrum of naive characteriztics irrespective of whether they were derived by conversion or reprograming. Importantly, only t2iLGöY hPSCs displayed a similar transcriptome to human cells from the inner cell mass, karyotypic stability and require re-priming for trilineage differentiation. Furthermore, our analyses identified KLF4 as a key reprogramming factor which enables conversion of primed hPSCs into naive t2iLGöY hPSCs. These findings underscore the role that reprogramming factors can play for the derivation of bona fide naive hPSCs and provide a molecular and functional reference for all the analysed conditions, which will help accelerate the downstream applications of naive hiPSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:scRNAseq of day 30 of trophoblast conversion together with naïve and primed hPSCs. Cells were mixed using the following ratio (H9 primed: Sigma primed: H9 naive: Sigma naive: day 30 ASECRiAV conversion = 1:1:1:1:2)
Project description:A detailed understanding of the developmental substates of human pluripotent stem cells (hPSCs) is needed to optimize their use in cell therapy and models of early development. Genetic instability and risk of tumorigenicity of primed hPSCs are well documented, but a systematic isogenic comparison between primed and naive substates has not been previously performed. We derived four hESC lines in naïve conditions and generated isogenic pairs of naïve and primed cultures. Through phenotypic, mRNA, miRNA, and DNA methylation profiling, we identified changes that arose during the naïve-primed transition and over extended culture. Although early naïve hESC cultures showed greater proliferation and clonogenic potential compared to primed cultures, they drifted toward a more primed-like substate over time, including accumulation of genetic abnormalities. Overall, we show that transcriptomic and epigenomic profiling can be used to place naïve/primed cultures along a developmental continuum, and may inform their utility for clinical and research applications.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.