CARDIAC DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS DERIVED FROM A DESMINOPATHY PATIENT FOR IN VITRO DISEASE MODELING

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

Virtual

Congreso; ISSCR 2021 Virtual Annual Meeting; 2021

International Society for Stem Cell research

Desmin (DES) is a type III intermediate filament of adult striated muscle that regulates geneexpression, organelles performance and fiber contraction. Desmin-related diseases are termeddesminopathies and involve cytoplasmic protein aggregates that generate cardiomyopathy. Wepropose to model this disease in vitro by differentiating to cardiomyocytes a pluripotent stem cell(iPSC) line derived from a patient with a desminopathy (DES-J). DES-J has heterozygous tripletduplication in DES exon 6 (Glu353 incorporation) not previously described that impairs normaldesmin structure. DES-J iPSC line was characterized with a normal karyotype, expression ofpluripotency genes and ability to differentiate to the three germline layers in embryoid bodiesanalyzed by RT-qPCR and immunofluorescence. DES-J iPSC line was then differentiated tocardiomyocytes using a monolayer protocol. In order to associate the mutation to disease, weanalyzed DES alleles expression during cardiac differentiation and we determined that both normaland mutated alleles are expressed in iPSCs and after cardiac mesoderm induction. Afterwards, weconfirmed by RT-qPCR that pluripotency genes (NANOG, OCT4) are downregulated whereasmesendoderm (TBXT), mesoderm (NKX2.5, MESP1) and cardiac genes (TNNT, VIM, CX43) areupregulated at days 3.5 and 14 of cardiac protocol. Flow cytometry analysis revealed that 75% ofthe cells were CD56+ (mesoderm) by day 3.5 and 35% were TNNT+ (cardiomyocytes) by day 14of differentiation, similar to control iPSCs. Moreover, we analyzed gene expression of desmosomal,mitochondrial, intermediate filaments, ubiquitin and other desmin interacting proteins throughoutdifferentiation protocol of DES-J and control cells. Interestingly, we observed different geneexpression profiles between both cell lines including lower expression of desmosomal andintermediate filaments genes in DES-J respect to control from day 0 to day 23 of differentiation. Inconclusion, we generated an iPSC line from a desminopathy patient with a novel mutation, whichwas able to differentiate to cardiomyocytes. Despite preliminary results showed differences in thecardiac differentiation process respect to normal cells, we could obtain cardiomyocytes which willbe studied in order to model the disease in vitro.