Study of lncRNAs transcriptomic profile in cardiac differentiation fromhuman pluripotent stem cells

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

Bernal, Quilmes

Jornada; II Reunión Argentina de Biología de ARNs no codificantes; 2018

Universidad Nacional de Quilmes

Long non-coding RNAs (lncRNAs), a heterogeneous group of transcripts defined as being over 200 nucleotides long and lacking protein coding potential, have emerged as key transcriptional regulators in the past few years. They exert their biological function through epigenetic, transcriptional and post-transcriptional regulation of gene expression. lncRNAs are known to orchestrate a myriad of cellular pathways, such as cell differentiation, lineage specification and cell fate determination amongst others, though their functions are yet to be fully clarified. Previous reports have demonstrated the modulatory role of some lncRNAs in cardiac differentiation, including lincROR, lncRNA_ES1, MALAT1 and CARMN. this work was to study the expression profiles of lncRNAs along cardiac differentiation. CMs were generated from an iPSC line developed in our laboratory (FN2.1) through the implementation and optimization of a previously reported cardiac protocol (Lian et al. 2012). The protocol focuses on three stages: a) pluripotent state (day 0), confirmed by immunofluorescent staining and qRT-PCR of specific pluripotency marker NANOG; b) early mesoderm progenitor (day3.5), population isolated by CD56/CD326-based cell sorting as well as qRT-PCR of mesoderm marker MESP1; and c) immature cardiomyocyte (day 21), purified using a metabolic selection medium, confirmed by spontaneous contractile activity,immunofluorescence staining and qRT-PCR of TNNT2 (cardiac marker). Weanalyzed the expression levels of known lncRNAs by qRT-PCR in threeindependent experiments on the three populations mentioned above. Wewere able to see a similar tendency as reported literature.Pluripotency lncRNAs lincROR and lncRNA_ES1 were highly downregulatedalong differentiation. Early mesoderm commitment lncRNAs MALAT1, HoxBlinc and HOTAIR showed a strong tendency to increase their levels compared to the pluripotent population, although it was not statistically significant. Cardiac lineage specification lncRNA CARMN was tested for two of its isoforms: CARMN1 and CARMN2. They showed 400 and 60 fold increase (vs. pluripotent state), respectively. Finally, total RNA prepared as before was subjected to deep sequencing (~50 million 100bp-long pair-end reads) on Illuminaplatform to characterize genome-wide expression profiles of lncRNAs (pending results). In conclusion, derivation of cardiomyocytes (CMs) from human induced pluripotent stem cells (iPSC) is a resourceful approach to investigate diverse aspects of clinical and biologicalinterest. Studying the transcriptomic networks that control cardiac commitment and development will provide a better understanding of how the process is regulated, to help address the research of diseases and enhance the design of treatment strategies.