Directed Self-Organization of Human and Non-Human Primate Heart Muscle Organoids from Pluripotent Stem Cells

Pluripotent stem cells (PSCs) can differentiate into all somatic cell lineages, but fail to form functional macro-scale heart muscle organoids in monolayer, suspension, or aggregate cultures. Here we show that human and non-human primate (NHP) PSCs have the capacity to self-organize into bioengineered heart muscle (BHM) if exposed to a serum-free cardio-instructive collagen-hydrogel environment with defined temporarily restricted growth factor, small molecule, and mechanical stimulation. BHMs traversed through distinct developmental stages with molecular signatures representative for: (i) mesoderm induction (3 days), (ii) cardiac specification (+10 days), and (iii) cardiac maturation (+9 days). Matured BHM responded to electrical pacing, preloading, inotropic stimuli and pharmacological agents similar to bona fide myocardium. We further demonstrate the applicability of BHM in modelling of Noonan Syndrome associated with Raf1 mutations. Collectively, we provide proof-of-concept for directed assembly of human and NHP PSCs into force-generating macro-scale myocardial organoids for applications in simulations of heart development and disease.