Research ArticlesDevelopment

Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells

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Science  10 Feb 2017:
Vol. 355, Issue 6325, eaag1927
DOI: 10.1126/science.aag1927

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Limiting potential for totipotency

Biological roles for microRNAs are not limited to RNA silencing and posttranscriptional regulation; they have now been shown to also regulate cell pluripotency. Choi et al. eliminated miR-34a from mouse embryonic stem cells and found that the cells exhibited a bidirectional cell fate potential, generating both embryonic and extraembryonic lineages (see the Perspective by Hasuwa and Siomi). During miR-34a deficiency, an endogenous retrovirus was induced, at least in part through Gata2-dependent transcriptional activation. Thus, the interplay of protein-coding genes, noncoding RNAs, and endogenous retroviruses can change cell fate plasticity and the developmental potential of pluripotent stem cells.

Science, this issue p. eaag1927; see also p. 581

Structured Abstract

INTRODUCTION

Mouse zygotes and early blastomeres have totipotent cell fate potential, generating both embryonic and extraembryonic cell lineages during normal development. This totipotent potential is gradually restricted during development, with the first cell fate specification event being completed by the blastocyst stage. Mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) exhibit a pluripotent potential similar to that of the epiblast in blastocysts, efficiently generating all embryonic cell types but rarely contributing to extraembryonic lineages in the placenta and yolk sac. Experimentally, ESCs and iPSCs can be induced into cells with expanded developmental potential, albeit with low efficiency. Such pluripotent stem cells are characterized by their bidirectional developmental potential (contributing to both embryonic and extraembryonic lineages) and their strong induction of the MuERV-L (MERVL) endogenous retroviruses (ERVs), both of which are features of totipotent two-cell (2C) blastomeres. The low efficiency in generating bipotential ESCs reflects the existence of multiple cellular and molecular impediments that restrict the pluripotent cell fate potential.

RATIONALE

We identified miR-34a microRNA (miRNA) as a potent regulator that restricts the cell fate potential of ESCs and iPSCs to a pluripotent state. miR-34a−/− pluripotent stem cells exhibit a bidirectional cell fate potential, generating both embryonic and extraembryonic cell lineages in multiple functional assays. Hence, the miR-34a−/− pluripotent stem cells provide a powerful experimental system to dissect the molecular mechanisms that restrict cell fate potential in ESCs and iPSCs.

RESULTS

miR-34a−/− ESCs and iPSCs exhibited an expanded cell fate potential, generating both embryonic and extraembryonic lineages in teratomas, embryoid bodies, and chimeric embryos. In particular, a single miR-34a−/− ESC injected into a recipient morula could yield progenies in both inner cell mass (ICM) and trophectoderm. Expression profiling studies comparing wild-type and miR-34a−/− pluripotent stem cells revealed a strong and specific induction of the MERVL ERVs, together with many MERVL-proximal genes, in miR-34a−/− ESCs and iPSCs. Whereas wild-type ESCs and iPSCs almost exclusively expressed Oct4, miR-34a−/− ESCs and iPSCs were heterogeneous, containing mutually exclusive populations with either Oct4 expression or MERVL induction. Because MERVL is a specific and highly expressed molecular marker for totipotent 2C blastomeres and for bipotential ESCs, we investigated the mechanism by which miR-34a regulates MERVL expression. We demonstrated that MERVL induction in miR-34a–deficient pluripotent stem cells is regulated at the transcriptional level, at least in part because of an increase of the transcription factor Gata2, a direct target of miR-34a. Knockdown of gata2 in miR-34a–deficient pluripotent stem cells phenocopied miR-34a overexpression, not only down-regulating the expression of MERVL but also abolishing their bipotential cell fate. Thus, miR-34a restricts cell fate potential and represses MERVL induction in pluripotent stem cells, at least in part through down-regulation of Gata2.

CONCLUSION

We have identified miR-34a as a noncoding RNA that restricts the cell fate potential of ESCs and iPSCs to a pluripotent state. The miR-34a/gata2/MERVL axis plays an essential role in modulating the transition between pluripotent stem cells and bipotential stem cells in culture. Thus, an intricate network of protein-coding genes, noncoding RNAs, and endogenous retroviruses could act cooperatively to define cell fate plasticity and developmental potential in pluripotent stem cells.

The miR-34a/Gata2 pathway restricts the cell fate potential of ESCs and iPSCs to a pluripotent state.

Pluripotent stem cell cultures contain mutually exclusive populations of pluripotent MERVLlow/Oct4high cells and bipotent MERVLhigh/Oct4low cells. In wild-type ESC and iPSC culture, this equilibrium strongly favors the MERVLlow/Oct4high population at the expense of the MERVLhigh/Oct4low population. miR-34a deficiency increases Gata2-dependent transcription of MERVL endogenous retroviruses, shifting the equilibrium to enable more cells to acquire a bipotential cell fate that yields both embryonic and extraembryonic cell lineages.

Abstract

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) efficiently generate all embryonic cell lineages but rarely generate extraembryonic cell types. We found that microRNA miR-34a deficiency expands the developmental potential of mouse pluripotent stem cells, yielding both embryonic and extraembryonic lineages and strongly inducing MuERV-L (MERVL) endogenous retroviruses, similar to what is seen with features of totipotent two-cell blastomeres. miR-34a restricts the acquisition of expanded cell fate potential in pluripotent stem cells, and it represses MERVL expression through transcriptional regulation, at least in part by targeting the transcription factor Gata2. Our studies reveal a complex molecular network that defines and restricts pluripotent developmental potential in cultured ESCs and iPSCs.

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