Human PSC-derived sinoatrial node-cardiac plexus assembloids model innervation-associated maturation of pacemaker systems.

Heart rhythm and contraction are initiated by electrical impulses generated by the sinoatrial node (SAN) and modulated by intrinsic cardiac neural inputs. Despite its physiological importance, human in vitro systems that recapitulate neural-SAN interactions are lacking. Here, we develop SAN-plexus assembloids by integrating human pluripotent stem cell-derived SAN organoids (SANOs) with cardiac ganglionated plexus organoids (CGPOs), together with atrial-like cardiac organoids, to model pacemaker-to-atrial conduction in a tri-assembloid system. This platform exhibits molecular, structural, and electrophysiological features of human pacemaker activity and enables functional interrogation of neural control over SAN automaticity, including disease-associated conduction dysfunction. By integrating spatial transcriptomics of human SAN tissue with assembloid-based functional analyses, we identify a neuron-to-pacemaker signaling program in which CGPO-derived prosaposin engages the SAN-enriched receptor GPR37 to promote pacemaker maturation. Together, this work establishes SAN-plexus assembloids as a human platform for studying intrinsic neuro-cardiac interactions in pacemaker development and disease.