Single-cell transcriptomics, scRNA-Seq and C1 CAGE discovered distinct phases of pluripotency during naïve-to-primed conversion in mice

Background Two types of mammalian pluripotent stem cells (PSC), i.e. naïve and primed possess distinct cellular characteristics. It is largely unknown how these differences are generated during naïve-to-primed transition process. We have established a robust in vitro transition system using a Wnt inhibitor for the first time and analyzed dynamic changes in cellular status via single-cell RNA-sequencing and C1 CAGE analyses. Results Analysis of known marker genes suggested that the cell transition process progresses as expected. However, cluster analyses revealed a sudden increase in expression profile diversities three and four days after induction of the transition. These expression diversities can be reconciled by the presence of two subpopulations with distinct transcription profiles emerging at these time points. One of the subpopulations appears transiently, and surprisingly these cells showed a global downregulation of gene expression. Moreover, initiation of random X chromosome inactivation (XCI) coincides with the appearance of these transient cells. The other subpopulation can be maintained as a stem cell line and possesses expression profiles more similar to those of primed epiblast stem cells (EpiSC) than embryonic stem cells (ESC). However, there are important differences in gene expression related to epithelial-mesenchymal transition (EMT), suggesting that this subpopulation may represent a novel pluripotent state that has an intermediate cellular phenotype between ESC and EpiSC. Conclusions These findings should contribute to our understanding of the establishment and maintenance of distinct differentiation statuses of mammalian PSCs and provide new insights into the pluripotency spectrum in general. ### Competing Interest Statement The authors have declared no competing interest. * PSC : pluripotent stem cell XCI : X chromosome inactivation EpiSC : epiblast stem cell eRNA : enhancer RNA ESC : embryonic stem cell EMT : epithelial-mesenchymal transition iPSC : induced pluripotent stem cell EpiLC : epiblast-like cell scRNA-Seq : single-cell RNA-Seq C1 CAGE : single-cell Cap Analysis of Gene Expression GMEM : Glasgow-Minimal Essential Medium KSR : knockout serum replacement FCS : fetal calf serum NEAA : non-essential amino acid MEF : mouse embryonic fibroblast ERCC : External RNA Controls Consortium t-SNE : t-Distributed Stochastic Neighbor Embedding SNP : single nucleotide polymorphism GATK : Genome Analysis Toolkit NAST : non-annotated stem cell transcript rXCI : random X chromosome inactivation snRNP : small nuclear ribonucleoprotein