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Generation of Cardiac Organoids Using Cardiomyocytes, Endothelial Cells, Epicardial Cells, and Cardiac Fibroblasts Derived From Human Induced Pluripotent Stem Cells

Two‐dimensional (2D) cell culture models fail to recapitulate the complex tissue architectures, extracellular matrices, and cellular crosstalk that occur in vivo. Three‐dimensional models (3D), including scaffold‐free organoids, derived from a single human induced pluripotent stem cell (hiPSC) cell source can more accurately model cell‐cell and cell‐matrix interactions to study specific genetic and organ‐level diseases that are difficulty to study in vitro. Additionally, 3D culture methods utilizing simulated microgravity environments have been shown to improve (iPSC)‐derived cardiomyocyte (CM) maturity, differentiation efficiency, and function, while promoting self‐organization in models that include multiple cardiac cell types. However, current published models have incorporated only one or two cell types and have used cells from a mixture of primary and hiPSC‐derived sources, primarily due to inefficient hiPSC differentiation protocols, especially for epicardial cells (epiC) and cardiac fibroblasts (CF). This study investigates and compares the cellular organization and function of cardiac organoids consisting of all four predominant cardiac cell types (CM, epiC, endothelial, and CF) differentiated from a single hiPSC source using two differentiation methods for cardiac fibroblasts and two 3D culture techniques. hiPSCs were differentiated into cardiomyocytes, endothelial cells (EC), and epiC using established methods. hiPSC‐CF were differentiated two ways: differentiation of hiPSC‐epiC to hiPSC‐CF and isolation of vimentin‐positive, cardiac troponin t‐negative cells from standard CM differentiation. All cells were singularized with Accutase, mixed at a 5:2:2:1 ratio (CM:CF:epiC:EC) as proposed by the developing cardiac model, and cultured in a non‐adherent spheroid plate (Aggrewell, StemCell Tech) for two days. The spheroids were then cultured for seven days in standard gravity (suspension) or simulated microgravity (rotating bioreactor) environment, with media change every two days. After seven days the organoids were analyzed for organization through immunofluorescence, maximum pacing frequency, calcium transients, and motion contraction velocities. Cardiac organoids containing cells from both hiPSC‐CF differentiation methods were compact and viable, but organoids did not form when no CF was included. This suggests non‐CM cells (cardiac troponin t negative) from CM differentiations are functional cardiac fibroblasts for organoid formation and ECM production. Organoid formation was further supported by the positive staining of cTnT, CD31, WT1, Cx‐43, and alpha‐SMA. Therefore, this study demonstrates a method of generating cardiac organoids containing all four cardiac cell types derived from the same hiPSC with tissue‐level organization and function for the in vitro study of genetic diseases targeting cardiac tissue.