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Mouse totipotent stem cells captured and maintained through spliceosomal repression

ABSTRACT: Since the establishment of the first embryonic stem cells (ESCs), in vitro culture of totipotent cells functionally and molecularly comparable to in vivo blastomeres with embryonic and extraembryonic developmental potency is unviable. Spliceosomes are responsible for mRNA splicing and maturation. Here, we report that spliceosomal repression in mouse ESCs drives pluripotent-to-totipotent state transition. Using the splicing inhibitor Pladienolide B, we realize in vitro culturing of totipotent ESCs comparable to 2- and 4-cell blastomeres at molecular levels for long-time passages, which are therefore termed as totipotent blastomere-like cells (TBLCs). Mouse chimeric assays combined with single-cell RNA-seq technology demonstrate that TBLCs own a robust bidirectional development capability to generate multiple embryonic and extraembryonic cell lineages. Mechanically, spliceosomal repression causes widespread splicing inhibition of pluripotent genes, whereas the totipotent genes featured with few short introns are efficiently spliced and transcriptionally activated. Our study provides a principle for capturing and maintenance of totipotent stem cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE168728 | GEO | 2021/05/15

REPOSITORIES: GEO

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Project description:Mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) exhibit a pluripotent developmental potential, contributing to all embryonic cell types, though rarely to extra-embryonic lineages. Unexpectedly, rare, totipotent-like stem cells have been identified in cultured ESC populations, suggesting the existence of a discrete molecular pathway that regulates the transition between totipotency and pluripotency in vitro. Here, we identify a single miRNA, miR-34a, whose deficiency in mouse pluripotent stem cells expands cell fate potential, giving rise to both embryonic and extra-embryonic lineages in vitro and in vivo. The expression profiles of the totipotent-like miR-34a-knockout murine pluripotent stem cells are characterized by a strong induction of MERVL endogenous retroviruses, a key molecular hallmark shared with totipotent mouse 2-cell blastomeres and totipotent-like mouse ESCs. In all three cell types, a subset of MERVL elements promotes the expression of specific isoforms of the proximal protein-coding genes. We demonstrate that miR-34a represses MERVL expression through transcriptional regulation, at least in part, by directly targeting the transcription factor GATA-binding protein 2 (Gata2). Since MERVL activation correlated precisely with the totipotent-like state, we hypothesized that the miR-34a/Gata2 pathway that regulates MERVL expression in ESCs/iPSCs also regulates the acquisition of totipotency in culture. Consistent with this hypothesis, gata2 knock-down in miR-34a-knockout mouse pluripotent stem cells not only reduced MERVL expression, but also abolished the expanded cell fate potential of these cells both in vitro and in vivo. Taken together, our findings not only provide key insights into the functional importance of miR-34a in restricting the totipotent cell fate potential of pluripotent stem cells, but also elucidate the underlying molecular basis by which miR-34a regulates the developmental potentials of ESCs/iPSCs.

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Mouse totipotent stem cells captured and maintained through spliceosomal repression

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