Feeney Austin K, Simmons Aaron D, Peplinski Claire J, Zhang Xiaotian, Palecek Sean P
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.
iScience. 2025 Apr 16;28(5):112452. doi: 10.1016/j.isci.2025.112452. eCollection 2025 May 16.
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 + mesoderm and +/+ cardiac progenitor stages. Moreover, we demonstrate that + mesoderm and +/+ 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 + mesoderm and +/+ 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.
人多能干细胞衍生的心肌细胞（hPSC-CMs）有潜力改变人们对心脏发育和心力衰竭治疗的理解。然而，hPSC-CM的分化效率受到批次间和细胞系间差异的困扰。在此，我们描述了一种方法，通过在中胚层阳性和双阳性心脏祖细胞阶段分离并重新接种祖细胞，可将CM纯度提高10%-20%（绝对值），且不会对收缩性、肌节结构、多核化、连接蛋白Cx43或CM数量产生负面影响。此外，我们证明中胚层阳性和双阳性心脏祖细胞可冷冻保存，恢复分化后CM纯度有类似提高，便于储存大批hPSC-CM祖细胞以按需生产CM。分化过程中的重新接种还能过渡到特定的细胞外基质，包括纤连蛋白、玻连蛋白和层粘连蛋白-111，所有这些都支持hPSC衍生的中胚层阳性和双阳性心脏祖细胞分化为CM。总之，我们提出了一种提高hPSC-CM分化纯度的方法，并证明特定的CM祖细胞适合冷冻保存。
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