Sustained 10% Oxygen Promotes Atrial Rather Than Ventricular Specification During Human iPSC-Cardiomyocyte Differentiation

Background: Induced pluripotent stem cell-derived ventricular cardiomyocytes (iPSC-vCMs) hold great promise for replacing ventricular cardiomyocytes lost after myocardial infarction. However, their immature phenotype limits successful engraftment by increasing the risk of post-transplant arrhythmias. In contrast to the atmospheric O2 used during most iPSC-vCM differentiations, O2 levels inside the developing heart remain low, but very little is known on the effect on O2 on iPSC-vCM differentiation. Methods: We used a GMP compliant Quad Physoxia glovebox platform providing continuous stable specified O2 tensions during all processes to simulate the in vivo O2 conditions more closely with the aim of improving iPSC-vCM maturation. Results: We demonstrate by single cell RNA sequencing, and data integration with datasets for human cardiomyocytes from the different heart chambers as well as cell morphology-, ploidy-, and functional studies, that sustained 10% O2 throughout iPSC-CM differentiation promotes atrial- instead of ventricular iPSC-CM subtype specification. Conclusions: While this rejects our original hypothesis and forces some concerns to iPSC-vCM manufacturing by sphere technology, these unexpected data may serve as an attractive and easy approach to refine atrial iPSC-CM specification to benefit their exponentially growing diagnostic-, cytotoxic-, and regenerative use.