Direct Production of Human Cardiac Tissues by Pluripotent Stem Cell Encapsulation in Gelatin Methacryloyl

Direct stem cell encapsulation and cardiac differentiation within supporting biomaterial scaffolds are critical for reproducible and scalable production of the functional human tissues needed in regenerative medicine and drug-testing applications. Producing cardiac tissues directly from pluripotent stem cells rather than assembling tissues using pre-differentiated cells can eliminate multiple cell-handling steps that otherwise limit the potential for process automation and production scale-up. Here we asked whether our process for forming 3D developing human engineered cardiac tissues using poly(ethylene glycol)-fibrinogen hydrogels can be extended to widely used and printable gelatin methacryloyl (GelMA) hydrogels. We demonstrate that low-density GelMA hydrogels can be formed rapidly using visible light (<1 min) and successfully employed to encapsulate human induced pluripotent stem cells while maintaining high cell viability. Resulting constructs had an initial stiffness of approximately 220 Pa, supported tissue growth and dynamic remodeling, and facilitated high-efficiency cardiac differentiation (>70%) to produce spontaneously contracting GelMA human engineered cardiac tissues (GEhECTs). GEhECTs initiated spontaneous contractions on day 8 of differentiation, with synchronicity, frequency, and velocity of contraction increasing over time, and displayed developmentally appropriate temporal changes in cardiac gene expression. GEhECT-dissociated cardiomyocytes displayed well-defined and aligned sarcomeres spaced at 1.85 ± 0.1 μm and responded appropriately to drug treatments, including the β-adrenergic agonist isoproterenol and antagonist propranolol, as well as to outside pacing up to 3.0 Hz. Overall results demonstrate that GelMA is a suitable biomaterial for the production of developing cardiac tissues and has the potential to be employed in scale-up production and bioprinting of GEhECTs.