Chemically Defined Production of Tri‐Lineage Human iPSC‐Derived Cardiac Spheroids

Cardiac spheroids derived from human induced pluripotent stem cells (hiPSC-cardiac spheroids) represent a powerful three-dimensional (3D) model for examining cardiac physiology and for drug toxicity screening. Recent advances with self-organizing, multicellular cardiac organoids highlight the capability of directed stem cell differentiation approaches to recapitulate the composition of the human heart in vitro. Using hiPSC-derived cardiomyocytes (hiPSC-CMs), hiPSC-derived endothelial cells (hiPSC-ECs), and hiPSC-derived cardiac fibroblasts (hiPSC-CFs) is advantageous for enabling tri-cellular crosstalk within a multilineage system and for generating patient-specific models. Chemically defined medium containing factors needed to simultaneously maintain hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs is used to produce the spheroid system. In this article, we present protocols to illustrate the methods for conducting small-molecule-mediated differentiations of hiPSCs into cardiomyocytes, endothelial cells, and cardiac fibroblasts, as well as to assemble the fully integrated cardiac spheroids. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Maintenance and expansion of hiPSCs Basic Protocol 2: Differentiation of hiPSCs into cardiomyocytes Basic Protocol 3: Differentiation of hiPSCs into vascular endothelial cells Basic Protocol 4: Differentiation of hiPSCs into cardiac fibroblasts Basic Protocol 5: Production of hiPSC-derived cardiac spheroids.