A CRITICAL PATHWAY IN HUMAN DEVELOPMENT: C19MC MICRORNAS REGULATES FGF2 RESPONSE IN A MODEL OF HUMAN PLURIPOTENT STEM CELLS CARDIAC DIFFERENTIATION Alan Miqueas Möbbs, Ximena

ALAN MIQUEAS MÖBBS; XIMENA GARATE; MARIA AGUSTINA SCARAFÍA; CAROLINA COLLI; LUCÍA NATALIA MORO; CARLOS DANIEL LUZZANI; ARIEL WAISMAN; ALEJANDRO DAMIÁN LA GRECA; SANTIAGO GABRIEL MIRIUKA

Congreso; REUNIÓN ANUAL DE SOCIEDADES DE BIOCIENCIA 2021; 2021

Human pluripotent stem cells (hPSC) have the capacity to self-renewand differentiate in vitro into all cell types of the organism, andit is an established model for early human embryo development.Recently, we found a 56-miRNA-cluster located at human chromosome19 (C19MC) that downregulates during hPSC cardiac differentiation(CD). To ascertain the role of this primate-specific microRNAcluster, 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 intocardiomyocytes. Early mesoderm and cardiac RNA markers, likeEOMES, TBX6, MESP1, were found altered. In order to further explorethe early steps of differentiation, we performed RNA-seq of thecells 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 keypathway in pluripotency, we address its role through two differentapproaches. First, both wild type and mutant cells were treated withFGF2 for 3 hours before gastrulation. Wild-type phenotype was partlyrecovered, as evidenced by the presence of contractile cardiomyocytesat day 15. Second, given that FGF2 is an important activatorof RAS cascade that phosphorylates ERK1/2 (pERK), we incubatedthe cells with FGF2 for up to 5 hs in pluripotency media. Mutantcells exhibited an elevated pERK mark in ground conditions, and itwas noticeable that the phosphorylation took place faster when theywere treated. In summary these findings support a critical role of theC19MC microRNA cluster in early stages of primate differentiation.