Small RNA Sequencing Reveals Dlk1-Dio3 Locus-Embedded MicroRNAs as Major Drivers of Ground-State Pluripotency

Ground-state pluripotency is a cell state in which pluripotency is established and maintained through efficient repression of endogenous differentiation pathways. Self-renewal and pluripotency of embryonic stem cells (ESCs) are influenced by ESC-associated microRNAs (miRNAs). Here, we provide a comprehensive assessment of the miRNome'' of ESCs cultured under conditions favoring ground-state pluripotency. We found that ground-state ESCs express a distinct set of miRNAs compared with ESCs grown in serum. Interestingly, most ground-state miRNAs'' are encoded by an imprinted region on chromosome 12 within the Dlk1-Dio3 locus. Functional analysis revealed that ground-state miRNAs embedded in the Dlk1-Dio3 locus (miR-541-5p, miR-410-3p, and miR-381-3p) promoted pluripotency via inhibition of multi-lineage differentiation and stimulation of self-renewal. Overall, our results demonstrate that ground-state pluripotency is associated with a unique miRNA signature, which supports ground-state self-renewal by suppressing differentiation.