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

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have the potential to transform understanding of heart development and treatment of heart failure. 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.人类多能干细胞衍生心肌细胞（human pluripotent stem cell-derived cardiomyocytes，hPSC-CMs）有望革新心脏发育的研究认知并优化心力衰竭的治疗手段。然而，hPSC-CM的分化效率始终受批次间与细胞系间的异质性困扰。本研究报道了一种可将心肌细胞纯度绝对提升10%~20%的方法：通过在EOMES阳性中胚层与ISL1+/NKX2-5阳性心脏祖细胞阶段之间对祖细胞进行解离并重新接种，该方法不会对心肌细胞的收缩功能、肌节结构、多核化状态、连接蛋白Cx43表达以及心肌细胞数量产生负面影响。此外，本研究证实，EOMES阳性中胚层与ISL1+/NKX2-5阳性心脏祖细胞均可实现低温保存，且在复苏后继续分化时仍可获得类似的心肌细胞纯度提升效果，这为大规模储存hPSC-CM祖细胞以按需生产心肌细胞提供了便利。分化过程中的重新接种操作还可支持向化学成分限定的细胞外基质体系过渡，包括纤连蛋白、玻连蛋白与层粘连蛋白-111，上述三种基质均能支持hPSC来源的EOMES阳性中胚层及ISL1+/NKX2-5阳性心脏祖细胞向心肌细胞分化。综上，本研究提出了一种可提升hPSC-CM分化纯度的方法，并证实特定心脏祖细胞可适用于低温保存操作。