OPTIMIZED IN VITRO DIFFERENTIATION PROTOCOL OF HUMAN PLURIPOTENT STEM CELLS TO SKELETAL MUSCLE CELLS FOR MYOPATHY MODELING

SHEILA CASTAÑEDA; FEDERICO ZABALEGUI; GUADALIPE AMÍN; GUSTAVO E. SEVLEVER; SANTIAGO MIRIUKA; ARIEL WAISMAN; LUCÍA MORO

Congreso; LXVIII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2023

Myopathies are skeletal muscle disorders mainly characterized by muscle weakness,determined by failure of cytoskeletal structure, mitochondrial physiology, glycogen or lipidmetabolism, or ion channel function. Skeletal myocytes (SMs) differentiated from myopathypatient-derived induced pluripotent stem cells (PSCs) represent a source of study for diseasemodeling, drug discovery and personalized therapy development. The aim of this work was toestablish an in vitro two dimensional (2D) differentiation protocol of human PSCs to SMs thatwill be used for myopathies in vitro modeling. In order to exit pluripotent state and inducepresomitic mesoderm we tried CHIR99021 (CHIR) incubation with 2 or 7 days of LDN193189(LDN) combined with and without SB431542 (SB), reaching the lowest expression of NANOGand OCT4, and the highest expression of MSGN, MYF5, TBX6 and TBXT, at day 3 by qPCRwith 7 days of LDN without SB and 2 days of LDN with SB conditions. Next step was to obtainpremyogenic progenitors and both conditions were followed by 1 week culture with fibroblastgrowth factor (GF), hepatocyte GF and insulin GF. We obtained the highest expression of PAX3at day 13 by qPCR with 7 days of LDN without SB. We terminally differentiated myoblasts toSMs following the culture with horse serum or a combination of CHIR, prednisolone and SB,obtaining with the latter one the highest expression of MYOD, MYOG and PAX7 at day 30 byqPCR. As culture replating expands and purifies myoblasts, we tried this by replating at day 12or 30 and sampling 2 weeks later. We got higher DES, MYOG and TTN expression at day 12condition, which significantly reduces the protocol duration. Lastly, we detected NANOG andOCT4, TBX6 and TBXT, and DES expression at days 0, 3 and 26 respectively of the optimizedprotocol by immunofluorescence staining. In conclusion, we successfully differentiatedNANOG/OCT4+ PSCs to DES+ SMs by an optimized 2D protocol by which myopathies will bestudied in vitro.