OBTAINING IN VITRO HUMAN-INDUCED PLURIPOTENT STEM CELLS-DERIVED CARDIOMYOCYTES: AN ALTERNATIVE TO PERSONALIZED MEDICINE IN CARDIO-ONCOLOGY

Background Anthracyclines are highly effective chemotherapeutic drugs commonly used to treat various types of tumors, however, they induce cardiotoxicity in about 30% of patients. So far, it is uncertain what induces individuals to develop anthracycline-induced cardiotoxicity (AIC), but it is well known that the damage is proportional to the cumulative dose used. In this context, the use of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) represents an alternative for in vitro disease modeling. However, most differentiation protocols use the ‘B27’ supplement, a compound rich in antioxidant molecules. It's likely that the usage of this supplement results in a cardioprotective effect on iPSC-CMs derived from AIC patients, possibly altering their ability to mimic the sensitive phenotype. This work aimed to establish a cardiac differentiation protocol for patient-specific iPSCs using a chemically defined medium (CDM) without antioxidant molecules. Methods A patient who was treated with anthracycline (240 mg/m2 doxorubicin, 300 mg/m2 epirubicin) and developed heart failure (ejection fraction = 33%) 7 years after the end of treatment was recruited, as well as a healthy unrelated donor. Patient-specific iPSCs underwent cardiac differentiation using two distinct protocols that modulate the Wnt canonical pathway: P1 - using B27 (RPMI 1640, B27 serum free supplement 1x or B27 minus insulin 1x) and P2 - CDM (RPMI 1640, 213 μg/mL L-ascorbic acid 2-phosphate, 500 μg/mL bovine serum albumin). After thirty days of culture (D30), the efficiency of differentiation was evaluated and compared between P1 and P2 through the expression of cardiac troponin T (cTnT) via flow cytometry. Results iPSCs derived from the sensitive patient (SP) and the healthy donor (HD) were successfully differentiated into CMs using both protocols (starting cell density SP: P1 and P2 = 4.2 × 105 cells/cm2; HD: P1 = 4.7 × 105 cells/cm2, P2 = 4.4 × 105 cells/cm2; CHIR99021 concentration SP: P1=9μM, P2=6μM; HD: P1=9μM, P2=7μM). CMs presented spontaneous contractions in vitro from D8. Both protocols attained high differentiation efficiencies, with no significant difference between them (cTnT SP: P1 = 82.45±14.09%, n=6; P2 = 81.13±12.99%, n=7, p=0.8635; HD: P1 = 90.45±4.99%, n=6; P2 = 91.80±3.68%, n=2). Conclusion A protocol for differentiating SP- and HD-iPSCs into CMs without using antioxidants has been successfully established, thus enabling its use in future drug screening experiments.