Engineered cardiac tissue microsphere production through direct differentiation of hydrogel-encapsulated human pluripotent stem cells

Engineered cardiac tissues that can be directly produced from human induced pluripotent stem cells (hiPSCs) in scalable, suspension culture systems are needed to meet the demands of cardiac regenerative medicine. Here, we demonstrate successful production of functional cardiac tissue microspheres through direct differentiation of hydrogel encapsulated hiPSCs. To form the microspheres, hiPSCs were suspended within the photocrosslinkable biomaterial, PEG-fibrinogen (25 million cells/mL), and encapsulated at a rate of 420,000 cells/minute using a custom microfluidic system. Even at this high cell density and rapid production rate, high intra-batch and batchto-batch reproducibility was achieved. Following microsphere formation, hiPSCs maintained high cell viability and continued to grow within and beyond the original PEG-fibrinogen matrix. These initially soft microspheres ( 250 Pa) supported efficient cardiac differentiation; spontaneous contractions initiated by differentiation day 8, and the microspheres contained 75% cardiomyocytes (CMs). CMs responded appropriately to pharmacological stimuli and exhibited 1:1 capture up to 6.0 Hz when electrically paced. Over time, cells formed cell-cell junctions and aligned myofibril fibers; engineered cardiac microspheres were maintained in culture over 3 years. The capability to rapidly generate uniform cardiac microsphere tissues is critical for advancing downstream applications including biomanufacturing, multi-well plate drug screening, and injection-based regenerative therapies.

Automated summary

The study demonstrates successful production of functional cardiac tissue microspheres through direct differentiation of hydrogel encapsulated hiPSCs, with high reproducibility and cell viability. These microspheres support efficient cardiac differentiation and have potential applications in biomanufacturing, drug screening, and regenerative therapies.