FHL1-LINKED MUSCULAR DYSTROPHY: PATIENT DERIVED IPSCS AND DIFFERENTIATION TO CARDIOMYOCYTES FOR IN VITRO DISEASE MODELING

FEDERICO ZABALEGUI; SHEILA CASTAÑEDA; GUADALIPE AMÍN; CAROLINA BÁRBARA BELLI; ARIEL WAISMAN; GUSTAVO SEVLEVER; SANTIAGO MIRIUKA; LUCÍA MORO

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

The FHL1 gene locates in the Xq26 region and encodes for four and half LIM domainprotein 1, which is mainly expressed in skeletal and cardiac muscle tissues. This geneplays a crucial role in cytoskeletal remodeling, myoblasts differentiation, sarcomereassembly, and autophagy regulation. Unfortunately, mutations in FHL1 have beenlinked to muscular dystrophy (MD) and associated with limited life expectancy. Theaim of this research was to generate and characterize induced pluripotent stem cells(iPSCs) derived from a patient with MD as consequence of a hemizygous missensemutation in FHL1 (c.377G>A, p.C126Y) in order to facilitate in vitro disease modelingand personalized therapy development. To generate MD-iPSC, a blood sample wastaken from the patient for erythroblasts expansion and reprogramming by transductionwith STEMCCA lentiviral vector carrying the reprogramming factors Oct4-Sox2-Klf4-cMyc. After MD-iPSCs isolation and expansion we confirmed the presence of thec.377G>A mutation in FHL1 by Sanger sequencing, STEMCCA silencing, and normalkaryotype. The pluripotency of the MD-iPSCs cell line was validated throughassessments of alkaline phosphatase activity and the expression of pluripotencygenes (OCT4, NANOG, SOX2) by RT-qPCR and immunohistochemistry. Additionally,MD-iPSCs were capable of differentiating into cells representing the three germ layersusing embryoid body formation. Furthermore, MD-iPSCs were successfullydifferentiated into cardiomyocytes, observing high expression levels of cardiacmarkers (cTNT, NKX2.5) on day 21 of differentiation, using RT-qPCR andimmunohistochemistry analyses.In conclusion, an iPSCs line derived from a patient with MD was successfullygenerated and differentiated into muscle cells (cardiomyocytes). It remains todetermine the molecular and cellular consequences of the mutation, which will beelucidated through the differentiation and study of this cell line, as well as thedevelopment of a personalized therapy.