Critical Functions of MicroRNAs during Embryonic Stem Cell Derivation

Background: Embryonic stem cells (ESCs), derived from in-ner cell mass (ICM) of pre-implantation embryos, exhibit un-limited self-renewal and multi-lineage differentiation potential, which is due to activity of a variety of biomolecules, including microRNAs. Here, for the first time, we functionally analyzed specific candidate microRNAs during ESC derivation under R2i condition (dual inhibition of Fgf/Erk and TGF-β pathways). Materials and Methods: Immunosurgery was performed to isolate ICMs from blastocysts and culture them under R2i con-dition to generate ESC lines. MicroRNA profiles were obtained using small-RNA sequencing during ICM-ESC transition (Mo-radi et al, unpublished data). MicroRNA mimics were transfect-ed into ICM to explore their effects on ESC derivation, or into ESCs to examine if they influence ESC self-renewal. Candidate upregulated microRNAs were analyzed in the presence of Fgf/ Erk inhibitor (PD032, with a low ESC derivation efficiency) to assess if they promote ESC generation, while downregulated candidates were studied in R2i culture (with ~100% derivation efficiency) to examine if they impair ESC derivation. Alkaline phosphatase (AP) staining was performed to analyze ESC self-renewal.Results: Small-RNA sequencing revealed that microRNAs ex-hibited dynamic expression patterns. We considered certain cri-teria e.g. dynamic and abundant expression, conservation, and previously documented functions to shortlist the microRNAs down to 25 candidates, mostly reported to be involved in regu-lating pluripotency. Among these, miR-212-5p and let-7d-3p (with downregulated patterns) and miR-183-5p and miR-363-3p (with upregulated patterns) appeared to be the best candi-dates. Then, we examined the impact of these microRNAs on the efficiency of ESC derivation, and found interestingly that miR-212-5p and let-7d-3p significantly decreased the number of ESC lines derived in R2i culture compared to the scrambled control. Notably, the majority of ESC colonies in miR-212-5p and let-7d-3p-treated groups were partially differentiated. On the other hand, miR-183-5p and miR-363-3p considerably enhanced the efficiency of ESC derivation in the presence of PD032, compared to the scrambled oligo.Conclusion: MicroRNAs downregulated during ICM-ESC transition impair ESC derivation and those upregulated during this process improve the efficiency of ESC formation.

کلیدواژه ها:

ESC ، Inner Cell Mass ، Embryonic Stem Cell ، Micro-rna ، Ground State Pluripotency