ANALYSIS OF CHROMOSOME 19 MICRORNAS CLUSTER (C19MC) IN HIPSC CARDIAC DIFFERENTIATION

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

Mar del Plata

Congreso; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2019

Latin American Society for Developmental Biology (LASDB)

Human pluripotent stem cells (hPSC) have the capacity to self-renew and differentiate in vitro into any cell type of the three germ layers. Our lab performed a small-RNAseq and described the MIRNOME of hPSC cardiac differentiation (CD). Peculiarly, a 56-microRNA-group was found to be transcribed in pluripotency and its expression decayed over the mesoderm stage. These microRNAs were clustered in a 100 kb-long chromosome 19 sequence, known as C19MC. However, little is known about them in hPSC. Herefore, we decided to study their role in pluripotency and CD. A C19MC deficient human induced PSC line was generated by CRISPR/Cas9 technology. Two sgRNA were designed to flank both 5? and 3? extremes of the C19MC. After clonal selection for the mutant, lack of microRNA expression was confirmed by RT-qPCR. Chromatin immunoprecipitation assay (ChIP) was optimized in order to evaluate whether nearby histones modifications were affected by the big deletion. Eleven loci, ranging from -70 kb to +100 kb from the cluster, were selected: five for H3K27ac, three for H3K9ac, three for H3K4me2, in addition to positive controls, nearby OCT4 and RPL7 genes, and a negative control. ChIP results showed that the selected loci conserved their characteristics in the mutant line. Further, C19MC upstream protein coding gene DRPX and downstream microRNAs mir-371, -372 and -373 expression were evaluated by RT-qPCR, showing no differences. Next, pluripotency genes OCT4, NANOG and SOX2 expression were measured and showed slight upregulation. Although mutant cell line culture was not affected, cardiac differentiation in vitro was dramatically compromised, 74 % of cardiomyocytes (wt) compared to 1 % (mutant) (n= 3). In concordance, MESP1, the master gene for CD, was downregulated. We hypothesize that pluripotency genes balance is altered, and that inhibits OCT4/LEF1 complex formation at MESP1 promoter, important for its normal activation. However, more experiments are necessary to confirm it.