GENERATION AND CHARACTERIZATION OF DESMINOPATHY PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS FOR PERSONALIZED THERAPY DEVELOPMENT

CASTAÑEDA, SHEILA; LA GRECA, A; MIRIUKA, SG; AMIN, G; LUZZANI, C; MORO, LUCIA; BELLI, CAROLINA; SEVLEVER, GE

Congreso; REUNIÓN DE SOCIEDADES DE BIOCIENCIAS 2020: SAIC; SAI, SAFIS; 2020

SAIC, SAFIS y SAI

Desmin is a type III intermediate filament encoded by the DES gene,mainly expressed in striated muscle,and is involved in contraction, organelle location and gene expression.Mutations in DES generate protein absence or abnormal protein folding disrupting cytoskeletal organization,mitochondrial function and the ubiquitin-proteasome pathway,among others.These phenotypes are known as desminopathies and have an incidence of 1 in 2000 patients.Obtention of patient-specific induced pluripotent stem cells(iPSC) allows the modeling of a desminopathy in vitro.In this work,we aim to generate and characterize a stable iPSC of a male patient with a congenital desminopathy (DES-J).DES-J has nucleotide triplet duplication in DES exon 6 which leads to a Glu353 incorporation.DES mutation was confirmed by Sanger sequencing and in silico analysis revealed the aminoacid incorporation alters desmin folding.To generate DES-J iPSC,a blood sample was taken from the patient and erythroblasts were reprogrammed by infecting with STEMCCA lentiviral vector.A clonal iPSC DES-J colony was amplified for further characterization.STEMCCA silencing was confirmed by RT-qPCR and normal karyotype of DES-J iPSC line was ensured through G-banding.For pluripotency validation,alkaline phosphatase activity was confirmed and flow cytometry analysis revealed positive staining for the pluripotency markers CD326 and TRA-1-81.Expression of NANOG,OCT4 and SOX2 was assessed by immunofluorescence and RT-qPCR.Finally,we proved that DES-J iPSC was capable of differentiating into the three germ layers by embryoid body formation and their characterization.These results enable to conclude that DES-J erythroblasts could be reprogrammed and developed pluripotent characteristics successfully.Our next step is to differentiate validated patient-derived DES-J iPSC into cardiac and skeletal myocytes in order to unravel the desminopathy.Our final purpose is to develop a personalized therapy that reverses the diseased phenotype.