Generation of self-assembling heart organoids from human pluripotent stem cells

Abstract Studying the development and disease etiology in the developing human heart is limited by the ability to recapitulate the complexity of the heart in vitro. This protocol introduces a method to generate complex individually contained heart organoids derived from human pluripotent stem cells (hPSCs). Embryoid bodies (EBs) are formed in a 96 well plate generating 1 EB per well, allowing for 96 individualized experiments with each plate. EBs are differentiated into human heart organoids in a 3-step Wnt pathway modulation strategy including an activation, inhibition and a second activation step corresponding to mesoderm, cardiac mesoderm and proepicardial organ inductions, respectively. The resulting human heart organoids demonstrated cellular, molecular, structural, and transcriptomic features that closely resemble the developing fetal heart.