This has led to studies aimed at converting cultured human pluripotent cells into a naive state by modifying growth conditions that support self-renewal of hESCs and hiPSCs to make them akin to human preimplantation embryos. development. Here, we successfully converted three in-house-derived primed hPSC lines (H10, H24, and iPS) to a naive state and an expanded pluripotent stem cell (EPS) state. Primed, naive and EPS cells displayed state-specific morphologies and expressed pluripotent markers. The expression of SSEA4 and TRA-1-60 was downregulated in the conversion process. The H3K27me3 expression level also decreased, indicating that global methylation was reduced and that the X chromosome started to reactivate. RNA-sequencing analysis results revealed that differentially expressed genes (DEGs) were significantly enriched in both naive hPSCs and EPS cells when compared to the primed state. However, imprinted gene expression barely changed before and after state reversion. Gene ontology (GO) analyses showed that the upregulated DEGs were mostly enriched in RNA processing, DNA replication and repair, and regulation of cell cycle process, while downregulated DEGs were related to extracellular adhesion and various tissue developmental processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that EPS cells were enriched in the PI3K-Akt and Wnt signaling KIRA6 pathways. Analysis of the lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network between primed, naive hPSCs and EPS cells revealed that hsa-miR-424-5p, has-miR-16-5p, has-miR-27b-3p, has-miR-29c-3p, and KCNQ1OT1 were crucial nodes with high degrees of connectivity. Our work may represent new insight into the intrinsic molecular features of different hPSC states. Introduction Conventional human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are pluripotent cell types with the capacity to proliferate and differentiate, which makes them a critical platform for studying mechanisms for human embryo development, drug development, genome screening, cell therapies, etc. Although hESCs are derived from preimplantation human blastocysts, they typically exhibit primed pluripotency, in which they are morphologically and transcriptionally similar to stem cells derived from the mouse postimplantation epiblast (mEpiSCs) [1,2]. However, mouse ESCs derived from the preimplantation blastocyst can differentiate into all embryonic cell lineages and in a chimera model, exhibiting a naive state corresponding to an stage of development KIRA6 that is earlier than the postimplantation epiblast [1,3]. This has led to studies aimed at converting cultured human pluripotent cells into a naive state by modifying growth conditions that support self-renewal of hESCs and hiPSCs to make them akin to human preimplantation embryos. Previous studies have yielded multiple, distinct conditions and transgene-free interconversion to induce and maintain naive pluripotency [4C11]. Recently, studies have reported the establishment of human extended pluripotent stem (hEPS) cell lines featuring the molecular characteristics of blastomeres and ZNF346 possessing developmental potency for all embryonic and extraembryonic cell lineages KIRA6 [12C14]. At the same time, omics technologies have provided unprecedented insights into the molecular complexity and heterogeneity of the human naive and primed pluripotent state [9,15C22], but to date, the extent to which the resulting cells recapitulate the situation and a comprehensive investigation of the expression changes in mRNAs, microRNAs (miRNAs) and long noncoding (lnc)RNAs as KIRA6 competing endogenous RNA (ceRNA) networks of pluripotency still await full elucidation. Here, we adopt commercially available defined medium (RSeT?) and a chemical cocktail medium (N2B27-LCDM) to revert primed hPSCs to a naive state and EPS cells with extended developmental potency. RNA-Seq and bioinformatics approaches were used to comprehensively investigate the differentially expressed genes and screen crucial ceRNA interaction axes to identify different states of pluripotent stem cells. The results of this study may improve the current understanding of the molecular mechanisms involved in the transition of KIRA6 primed towards naive states and provide insight for stem cell studies. Materials and methods Cell culture Human embryonic stem cell lines H10, H24 and human induced pluripotent stem cell line iPS-46 were independently established in our laboratory [23,24]. MTeSR?1 medium (STEMCELL Technologies), containing recombinant human basic fibroblast growth factor (rh-bFGF) and recombinant human transforming growth factor (rh.
This has led to studies aimed at converting cultured human pluripotent cells into a naive state by modifying growth conditions that support self-renewal of hESCs and hiPSCs to make them akin to human preimplantation embryos