Xeno-Free Peptide-Functionalized Hydrogels Support hiPSC Encapsulation and In Situ Differentiation into Cardiomyocytes with Enhanced Structural Maturation

While defined synthetic substrates can replace Matrigel for human induced pluripotent stem cell (hiPSC) culture and hiPSC-derived cardiomyocyte (hiPSC-CM) production, existing approaches culture cells on two-dimensional surfaces and yield structurally immature cardiomyocytes, limiting their use in disease modeling and regenerative medicine. Here, we developed a xeno-free, fully-defined cyclic RGD (cRGD)-functionalized alginate platform in which we encapsulated hiPSCs to support their expansion and in situ cardiac differentiation. cRGD functionalization was essential for hiPSC survival and pluripotency, with maximal support achieved at a low ligand density (25 M). In the presence of cRGD, hiPSC encapsulation into softer gels made from lower molecular weight alginates led to enhanced hiPSC expansion and improved cardiogenesis. Strikingly, differentiation in situ with 3D gels led to hiPSC-CM with higher structural maturity, including a markedly increased proportion of cardiomyocytes positive for the structural maturation markers Desmin and MYH7. Finally, after enzymatic retrieval from hydrogels, cardiomyocytes derived from softer gels formed tissue-engineered myocardium with superior contractile stress compared to tissue fashioned from hiPSC-CM derived from more rigid gels. Together, these results demonstrate the promise of this defined, tunable platform for biomanufacturing of structurally more mature cardiomyocytes from hiPSC.