Abstract P1136: Amino Acid Availability Regulates Differentiation Of HIPSC-derived Cardiomyocytes In A Protein-free System

Abstract

Traditional protocols for the differentiation of hiPSC-derived cardiomyocytes (hiPSC-CMs) rely on complex media and the use of animal or recombinant proteins either directly or in supplements, such as B27, to obtain tissue monolayers. This fact decreases their reproducibility, while potentially increasing their costs and limiting higher-throughput production of cardiomyocytes for applications in both regenerative medicine, disease modeling, and pharmacological screenings. Albumin/serum-free protocols have been developed but produce cells with limited functionality and at lower yields, despite them being TNNT2-positive. We previously demonstrated it is possible to simplify the differentiation protocol utilizing a stepwise approach and combining time-point interventions with small molecules or nutritional inputs. Here, we performed a thorough screening of commercial media and their suitability for differentiation of hiPSC-CMs. We identified suitable basal media formulations and developed a novel protein-free differentiation protocol (PFDM) for generating hiPSC-CMs that preserves purity, yield, morphology, and functionality while reducing the cost of the differentiation process. PFDM also enabled the identification of nutritional requirements essential for cardiac differentiation and allowed a detailed investigation into how some common components supplemented during hiPSC-CM differentiation impact tissue morphology and contractility. We showed that non-essential amino acids regulate differentiation in a time-dependent manner. Similarly, antioxidant agents such as sodium pyruvate and ascorbic acid, while essential for differentiation, are potentially harmful during early cardiac mesoderm induction. Lastly, we validated this PFDM methodology in 35 control and patient-derived hiPSC lines, with monolayer differentiations in 6- and 12-well plates, 15 cm dishes, in addition to differentiations in 3D using 100- and 250-mL spinner flasks.