Cardiovascular diseases remain the major cause of death in the US. Human Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repairs. Recent progress in cellular reprogramming of various somatic cell types into induced pluripotent stem cells opened the door for developing patient-specific, cell-based therapies. However, most SPC-CMs displayed heterogeneous and immature electrophysiological (EP) phenotypes with uncontrollable automaticity. Implanting these electrically immature and inhomogeneous CMs to the hearts would likely be arrhythmogenic and deleterious. Furthermore, as CMs mature, they undergo changes in automaticity and electrical properties. We used human embryonic stem cell-derived CMs (hESC-CMs) as the model system to study the development and EP maturation of CMs in the embryoid body (EB) environment. Elucidating molecular pathways governing EP maturation of early hESC-CMs in EBs would enable engineered microenvironment to create functional pacemaker cells or electrophysiologically compatible hESC-CMs for cell replacement therapies. We have established antibiotic (Abx)-resistant hESC lines conferred by lentiviral vectors under the control of a cardiac-specific promoter. With simple Abx treatment, we easily isolated >95% pure hESC-CMs at various stages of differentiation from EBs. In the first year of this grant support and using the Abx selection system, we found that hESC-CMs isolated at early stages of differentiation without further contacts with non-cardiomyocytes (non-CMs) depicted arrested electrical maturation. The intracellular Ca2+-mediated automaticity developed very early and contributed to dominant automaticity throughout hESC-CM differentiation regardless of the presence or absence of non-CMs. In contrast, sarcolemmal ion channels evolved later upon further differentiation within EBs and their maturation required the interaction with non-CMs. In the second year, we further developed an add-back co-culture system to enable adding non-CMs back to early isolated hESC-CMs, which rescued the arrest of EP maturation. In the third no-cost extension year, we further successfully established the cocultures of human neural crest cell (NCC)-derivatives and early-purified hESC-CMs. We found that peripheral neurons derived from human NCCs exerted strong influences on the development of a specific subset of ion channel populations during early hESC-CM differentiation. Therefore, our study showed for the first time that non-CMs, especially neurons derived from NCCs, exert significant influences on the EP maturation of hESC-CMs during differentiation. Knowledge of this study will allow us to improve functional maturation of primitive hESC-CMs or to create neuro-coupled pacemaker cells before cell transplantation.