CHROMOSOME 19 MICRORNA CLUSTER (C19MC) REGULATES EARLY HUMAN PLURIPOTENT STEM CELL CARDIAC DIFFERENTIATION

ALAN MIQUEAS MÖBBS; XIMENA GARATE; MARIA AGUSTINA SCARAFIA; CAROLINA COLLI; NATALIA LUCÍA SANTÍN VELAZQUE; GABRIEL NEIMAN; ARIEL WAISMAN; MARIA SOLEDAD RODRIGUEZ VARELA; LEONARDO ROMORINI; MARIA ELIDA SCASSA; GUSTAVO EMILIO SEVLEVER; CARLOS DANIEL LUZZANI; LUCIA NATALIA MORO; ALEJANDRO DAMIÁN LA GRECA; SANTIAGO GABRIEL MIRIUKA

Mar del Plata

Congreso; LXIII Annual Meeting of the Argentine Society for Clinical Investigation (SAIC); 2018

SAIC - Sociedad Argentina de Investigacion Clinica

Humanpluripotent stem cells (PSC) have the capacity to self-renewindefinitely in-vitro and to differentiate into any cell type of thethree germ layers. The primate-specific Chromosome 19 microRNAsCluster (C19MC) is composed of 56 microRNAs located within a 100kbp-long locus. We previously found that C19MC microRNA expressionrapidly falls upon PSC differentiation. However, little is knownabout C19MC function. We then investigated the role of thesemicroRNAs in the pluripotency state. Analysis of different smallRNA-seq data confirmed that C19MC is downregulated in PSC upondifferentiation. We performed Ago-IP-seq for mir-520a-3p in order tostudy gene targets and identified more than a hundred genes. GeneOntology (GO) analysis revealed cell proliferation and embryonicdifferentiation pathways, in particularly cardiac differentiation(CD), as main targets. Two strategies for microRNA downregulationfailed: LNAs for a few microRNAs and CRISPR-OFF techniques proved notto be effective. We then generated a clone by CRISRP/Cas9 to knockoutthe whole C19MC and confirmed undetectable microRNA expression. Fiftyclones were picked and one of them turned out to be positive for theC19MC deletion. C19MC(-)-PSC grew as usual and major features ofundifferentiation were mostly unchanged. Minor increased in G1 wasdetected and slight changes in the expression of pluripotencyfactors, D1 and D2 Cyclines, FZD3 and MAPK genes were observed. Allof them showed up to four-fold change upregulation compared to wildtype cells. Cardiac differentiation was dramatically compromised inC19MC(-) cells (74% vs 1%). MESP1, a master gene for CD, was notupregulated in C19MC(-) cells. A lower increased in Zeb1 was alsoseen. Of note, neural differentiation was not compromised. Theseresults suggest that C19MC is a master regulator of early cardiacdifferentiation.