A critical pathway in human development: C19MC micrornas regulates FGF2 response in a model of human pluripotent stem cells cardiac differentiation

Abstract

Human pluripotent stem cells (hPSC) have the capacity to self-renew and differentiate in vitro into all cell types of the organism, and it is an established model for early human embryo development. Recently, we found a 56-miRNA- cluster located at human chromosome 19 (C19MC) that downregulates during hPSC cardiac differentiation (CD). To ascertain the role of this primate-specific microRNA cluster, a hPSC-C19MC(-/-) line was generated with CRISPR/Cas9. C19MC(-/-) cells displayed no evident changes in the cell cycle, apoptosis or differentiation markers compared to wild type. Contrarily, C19MC(-/-) cells were significantly impaired to differentiate into cardiomyocytes. Early mesoderm and cardiac RNA markers, like EOMES, TBX6, MESP1, were found altered. In order to further explore the early steps of differentiation, we performed RNA-seq of the cells at the gastrulation stage (0 and 24hs after CHIR99021 incubation). Gene ontology analysis revealed altered signaling pathways, including PI3K-Akt, MAPK and Wnt, and FGF2. As FGF2 is a key pathway in pluripotency, we address its role through two different approaches. First, both wild type and mutant cells were treated with FGF2 for 3 hours before gastrulation. Wild-type phenotype was partly recovered, as evidenced by the presence of contractile cardiomyocytes at day 15. Second, given that FGF2 is an important activator of RAS cascade that phosphorylates ERK1/2 (pERK), we incubated the cells with FGF2 for up to 5 hs in pluripotency media. Mutant cells exhibited an elevated pERK mark in ground conditions, and it was noticeable that the phosphorylation took place faster when they were treated. In summary these findings support a critical role of the C19MC microRNA cluster in early stages of primate differentiation.