CARDIAC DIFFERENTIATION OF INDUCED PLURIPOTENT STEM CELLS DERIVED FROM PATIENTS WITH HUTCHINSON-GILFORD PROGERIA SYNDROME

Background/Objective Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by a single point mutation in the lamin A gene that leads to the production of a truncated protein, Progerin, and accelerated aging, which culminates in the patient\u0027s death mainly due to cardiovascular complications. Since the pathogenesis of HGPS shares some mechanisms with normal aging, it gives an opportunity to understand the cellular and molecular mechanisms of this natural phenomenon. Therefore, the aim of the present study was to generate cardiomyocytes from induced pluripotent stem cells of HGPS patients (HGPS-CM) in order to provide a powerful in vitro platform for the study of age-related cardiac disease. Methods Induced pluripotent stem cell line was obtained from the Progeria Research Foundation Cell and Tissue Bank (HGPS-iPSC). HGPS-iPSC pluripotency was characterized by RT-PCR. G-banding patterns were evaluated so as to ensure that these cells have a normal karyotype. For cardiomyocyte differentiation, 2,1 × 105/cm2 and 4,2 × 105/cm2 HGPS-iPSC were cultured for 30 days in RPMI 1640 medium supplemented with recombinant human albumin and L-ascorbic acid 2-phosphate. Forty-eight hours later (day 0), 5, 6, or 7 µM CHIR99021 was added to induce mesoderm formation. On day 2, 2 µM Wnt-C59 was used to specify in cardiogenic mesoderm. Progerin and SERCA2A expression in HGPS-iPSC and HGPS-CM were analyzed by RT-PCR. Cardiac differentiation efficiency was analyzed by the percentage of positive cells for cardiac troponin T using flow cytometry. Moreover, immunostaining for the same protein was performed to evaluate the contractile structure. Results HGPS-iPSC expressed OCT3/4, NANOG, SOX2, KLF4, confirming the pluripotent state and exhibited normal karyotype after fifty passages. After 10 days of differentiation, it was possible to visualize contractile cells only in 5 µM CHIR99021 condition, for both cell concentrations. On the thirtieth day of differentiation, progerin and SERCA2A mRNA were only detected in HGPS-CM and approximately seventy percent were positive to cardiac troponin T. Moreover, the presence of troponin T revealed that HGPS-iPSC derived cardiomyocytes showed a striated pattern, resembling sarcomere structures. Conclusions In this work, we established an efficient protocol to obtain cardiomyocytes using iPSC from HGPS patients, which can be used as an important tool for the study of novel mechanisms underlying cardiac aging.