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

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.