The combinatorial effects of small molecules and ECM biophysical cues on cardiac reprogramming in 3D

The cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCM) represents a promising approach to cardiac regeneration. Research has shown that cardiac reprogramming is enhanced in vivo and that the extracellular matrix’s (ECM) biophysical properties and the addition of small molecules boost reprogramming outcomes in two-dimensional cell cultures. However, little is known about how three-dimensional (3D) ECM biophysical cues impact reprogramming. We investigated how a small molecule cocktail (CHIR99021, forskolin, SB431542, and tranylcypromine) and 3D ECM biophysical cues impact Yamanaka-factor-driven cardiac reprogramming in both Matrigel-coated wells and 3D poly(ethylene glycol) (PEG) hydrogels. We found that small molecules increased iCM colony formation in both Matrigel and PEG and boosted mesoderm and early cardiac gene expression. Early colony formation in PEG was impacted by the gels’ biophysical properties, with colonies preferentially forming in softer, more rapidly degradable hydrogels. Despite increased colony formation due to small molecules, colonies in PEG were rarely contractile. Colonies removed from PEG after different reprogramming phases and transferred to Matrigel-coated wells formed contractile colonies with characteristics similar to cells reprogrammed exclusively on Matrigel. These results highlight the beneficial effects of small molecule supplementation and demonstrate that ECM biophysical cues can be tuned for different stages of reprogramming.