IN VITRO CARDIAC DIFFERENTIATION OF IPSCS DERIVED FROM PATIENTS WITH A NOVEL DESMIN MUTATION FOR DESMINOPATHY MODELLING

SHEILA LUCIA CASTAÑEDA; GUADALUPE AMIN; CAROLINA BARBARA BELLI; JOAQUIN SMUCLER; ALAN MIQUEAS MÖBBS; MARIA AGUSTINA SCARAFIA; ARIEL WAISMAN; ALEJANDRO DAMIÁN LA GRECA; GUSTAVO EMILIO SEVLEVER; SANTIAGO GABRIEL MIRIUKA; LUCIA NATALIA MORO

San Francisco

Congreso; ISSCR 2022 Annual Meeting; 2022

International Society for Stem Cell research

Desmin (DES) is a type III intermediate filament that attaches organelles and nucleus to contractile apparatus. Aberrant DES isoforms generate protein cytoplasmic aggregates that cause cardiomyopathy. Our main purpose is to model a desminopathy-associated cardiomyopathy in vitro by differentiating human induced pluripotent stem cell (hiPSC) lines to cardiomyocytes (CMs). We generated hiPSC lines after reprogramming peripheral blood mononuclear cells (PBMCs) from one healthy (DES-S) and two diseased (DES-J, DES-Y) siblings that have a non-reported heterozygous mutation in exon 6 of DES gene that generates Glu353 incorporation. After determining normal karyotypes, we detected NANOG, OCT4 and SOX2 expression by RT-qPCR and ability to differentiate to the three germ layers of all hiPSC lines. Once DES-S, DES-J and DES-Y hiPSC lines were validated, we proceeded to differentiate them to CMs using a monolayer protocol. First, we observed that DES-J and DES-Y CMs expressed wild type and mutated DES alleles in similar proportions along cardiac differentiation by RT-qPCR with specific primers. Then, we confirmed that pluripotency genes (NANOG, OCT4) were downregulated from day 3 while mesoderm (MESP1, NKX2.5, TBXT), cardiac (cTnT, VIM, CX43) and desmosomal genes (DSC2, DSG2, DSP, PKG, PKP2) were upregulated at days 3, 7 and 14 within the three cell lines by RT-qPCR. Flow cytometry analysis showed that ~80% of day 3 cells were CD56+ (mesoderm) and ~35% of day 14 cells were cTnT+ forDES-S, DES-J and DES-Y cell lines. CMs MitoTracker incubation revealed higher mitochondrial staining forDES-J and DES-Y respect to DES-S. We also analyzed DES, PKG and PKP2 protein expression in CMs derivedfrom the three hiPSC lines. Lastly, in vitro cardiac contraction was characterized by Contraction Wave softwareobserving higher beating frequency of DES-J and DES-Y CMs comparing to DES-S CMs, among other differentialparameters. In conclusion, we differentiated DES-S, DES-J and DES-Y validated hiPSC lines to CMs and weobserved differences that could be attributed to a cardiomyopathy of a non-reported desminopathy. We expect todeepen in cardiomyopathy modelling and study skeletal muscle desminopathy-associated implications in vitro.