Enhancing human pluripotent stem cell differentiation to cardiomyocytes through cardiac progenitor reseeding and cryopreservation

Austin K. Feeney; Aaron D. Simmons; Claire J. Peplinski; Xiaotian Zhang; Sean P. Palecek

doi:10.1016/j.isci.2025.112452

iScience (May 2025)

Enhancing human pluripotent stem cell differentiation to cardiomyocytes through cardiac progenitor reseeding and cryopreservation

Austin K. Feeney,
Aaron D. Simmons,
Claire J. Peplinski,
Xiaotian Zhang,
Sean P. Palecek

Affiliations

Austin K. Feeney: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA; Medical Scientist Training Program, University of Wisconsin School of Medicine and Public Health, Madison, WI 53726, USA
Aaron D. Simmons: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
Claire J. Peplinski: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
Xiaotian Zhang: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA
Sean P. Palecek: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706, USA; Corresponding author

DOI: https://doi.org/10.1016/j.isci.2025.112452
Journal volume & issue: Vol. 28, no. 5
p. 112452

Abstract

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Summary: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have the potential to transform the understanding of heart development and heart failure treatment. However, hPSC-CM differentiation efficiency is plagued by batch-to-batch and line-to-line variability. Here, we describe a method to improve CM purity by 10–20% (absolute) without negatively affecting contractility, sarcomere structure, multinucleation, junctional Cx43, or CM number by detaching and reseeding progenitors between the EOMES+ mesoderm and ISL1+/NKX2-5+ cardiac progenitor stages. Moreover, we demonstrate that EOMES+ mesoderm and ISL1+/NKX2-5+ cardiac progenitors are cryopreservable with similar improvements in CM purity after resuming differentiation, facilitating storage of large batches of hPSC-CM progenitors for on-demand CM production. Reseeding during differentiation also enables transition to defined extracellular matrices, including fibronectin, vitronectin, and laminin-111, which all supported hPSC-derived EOMES+ mesoderm and ISL1+/NKX2-5+ cardiac progenitor differentiation to CMs. In summary, we present a method to increase hPSC-CM differentiation purity and demonstrate that specific CM progenitors are amenable to cryopreservation.

Published in iScience

ISSN: 2589-0042 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science
Website: https://www.cell.com/iscience/home

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