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

Mathiesen, S. B.; Bjerre, F. A.; Terp, A. K. S.; Ellman, D. G.; Larsen, J. H.; Horn, P. B.; Svenningsen, P.; Poon, E. N.-Y.; Jensen, C. H.; Andersen, D. C.

doi:10.64898/2026.09.16.750048

bioRxiv · 10.64898/2026.09.16.750048

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

Mathiesen, S. B.·Bjerre, F. A.·Terp, A. K. S.·Ellman, D. G.·Larsen, J. H.·Horn, P. B.·Svenningsen, P.·Poon, E. N.-Y.·Jensen, C. H.·Andersen, D. C.

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Abstract

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.

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Mathiesen, S. B., Bjerre, F. A., Terp, A. K. S., Ellman, D. G., Larsen, J. H., Horn, P. B., Svenningsen, P., Poon, E. N.-Y., Jensen, C. H., Andersen, D. C.. 2026-09-21. Sustained 10% Oxygen Promotes Atrial Rather Than Ventricular Specification During Human iPSC-Cardiomyocyte Differentiation. https://doi.org/10.64898/2026.09.16.750048

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