CHARACTERIZATION OF lncRNAs EXPRESSION LEVELS ALONG CARDIAC DIFFERENTIATION

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

Buenos Aires

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

SAIC - Sociedad Argentina de Investigacion Clinica

Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are extensively used for the study and modelling of diseases. Long non-coding RNAs (lncRNAs), a diverse group of RNA that have no coding potential, have been shown to actively participate in cardiac differentiation through the modulation of key gene expression networks. This study is focused on three main goals: a) generation of hPSC-derived CMs by the implementation and optimization of a previously reported protocol (Lian et al. 2012); b) evaluation of lncRNAs expression levels at three stages of the differentiation protocol -pluripotent (day 0), early mesoderm progenitor (day 3.5) and immature cardiomyocyte (day 21)- by quantitative real-time PCR (qPCR); and c) study the effect of manipulating lncRNA expression levels by CRISPR/Cas9 technology on cardiac differentiation. CMs were generated from two cell lines, an induced hPSCs (hiPSC) line developed in our laboratory (FN 2.1) and an embryonic hPSC (hESC) line (HES3). qPCR analysis was performed with samples collected at the times indicated above. Throughout the differentiation we were able to see a downregulation of the lncRNAs associated with pluripotency (linc-ROR and lncRNA_ES1). Correspondingly, cardiac differentiation lncRNAs markers showed an upregulation: HOTAIR and MALAT1 (epithelial-to-mesenchymal transition); HoxBlinc (early mesoderm) and CARMN (immature CM commitment). The next step was to induce or repress these lncRNAs using CRISPR/Cas9 with the purpose of gaining insight in their roles during thecardiac differentiation. Although no results have been produced yet, preliminary data in our group indicates that the strategy is functional.In conclusion, this work provides a simple, efficient protocol for the generation of hPSC-derived CMs, a characterization of lncRNAs expression levels through the differentiation process and contributes to define a clear strategy for modulating these lncRNAs by (epi)genome editing technology.