CHARACTERIZATION OF IPSCS DERIVED CARDIOMYOCYTES FOR THEIR APPLICATION IN DISEASE MODELLING

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

Buenos Aires

Congreso; Reunion Conjunta de Sociedades de Biociencias; 2017

SAIC - Sociedad Argentina de Investigacion Clinica

Cardiomyocyte differentiation is a powerful tool for disease modeling. One cardiomyopathy we are interested in modeling by CRISPR is the arrythmogenic right ventricular dysplasia (C/DAVD), caused by mutations in desmosomal genes including desmoplakin (Dsp), plakoglobin (Pkg), plakophilin 2 (Pkp2), desmoglein 2 (Dsg 2) and desmocolin 2 (Dsc2). The aim of this work was to characterize the cardiomyocyte differentiation protocol from induced pluripotent stem cells (iPSCs) and to analyze the expression of these genes. The differentiation protocol consisted on culturing 3x105 iPSCs in a 24 well with mTeSR medium for 3 days, until when cells were incubated with 9uM CHIR99021 (GSK3 pathway inhibitor) in RPMI+B27 basal medium for 24h (day 0). On day 3, cells were incubated with 5uM IWP2 (Wnt pathway inhibitor) for 48h and on day 7 the medium was changed to RPMI+B27+insulin. To enrich the cardiomyocyte population, the medium was changed to RPMI-glucose+7uM lactate (days 12-18). After that, the cardiomyocytes were maintained with RPMI+ B27+insulin. Trizol samples were taken on day 0, day 3.5, day 7 and day 21, from 3 independent experiments. After RNA isolation and RT-PCR, qPCRs were performed for pluripotency genes (Oct4 and Nanog), mesoderm-cardiac differentiation genes (Brachyury, NKX2.5 and cTnT), and desmosomal genes (Dsp, Pkg, Pkp2, Dsg2 and Dsc2). Student?s T test was used for statistical analysis (p