BACKGROUND When human induced pluripotent stem cells (hiPSCs) that CCND2-OE (overexpressed cyclin-D2) were differentiated into cardiomyocytes (CCND2-OEhiPSC-CMs) and administered to the infarcted hearts of immunodeficient mice, the cells proliferated after administration and repopulated >50% of the scar. Here, we knocked out human leukocyte antigen class I and class II expression in CCND2-OEhiPSC-CMs (KO/OEhiPSC-CMs) to reduce the cells' immunogenicity and then assessed the therapeutic efficacy of KO/OEhiPSC-CMs for the treatment of myocardial infarction. METHODS KO/OEhiPSC-CM and wild-type hiPSC-CM (WThiPSC-CM) spheroids were differentiated in shaking flasks, purified, characterized, and intramyocardially injected into pigs after ischemia/reperfusion injury; control animals were injected with basal medium. Cardiac function was evaluated via cardiac magnetic resonance imaging, and cardiomyocyte proliferation was assessed via immunostaining and single-nucleus RNA sequencing. RESULTS Measurements of cardiac function and scar size were significantly better in pigs treated with KO/OEhiPSC-CM spheroids than in animals treated with medium or WThiPSC-CM spheroids. KO/OEhiPSC-CMs were detected for just 1 week after administration, but assessments of cell cycle activity and proliferation were significantly higher in the endogenous pig cardiomyocytes of the hearts from the KO/OEhiPSC-CM spheroid group than in those from the other 2 groups. Single-nucleus RNA-sequencing analysis identified a cluster of proliferating cardiomyocytes that was significantly more prevalent in the KO/OEhiPSC-CM spheroid-treated hearts (3.65%) than in the hearts from the medium (0.89%) or WThiPSC-CM spheroid (1.33%) groups at week 1. YAP (Yes-associated protein) protein levels and nuclear localization were also significantly upregulated in pig cardiomyocytes after treatment with KO/OEhiPSC-CM spheroids. Follistatin, which interacts with the HIPPO/YAP pathway, was significantly more abundant in the medium from KO/OEhiPSC-CM spheroids than WThiPSC-CM spheroids (30.29±2.39 versus 16.62±0.83 ng/mL, P=0.0056). Treatment with follistatin increased WThiPSC-CM cell counts by 28.3% over 16 days in culture and promoted cardiomyocyte proliferation in the infarcted hearts of adult mice. CONCLUSIONS KO/OEhiPSC-CM spheroids significantly improved cardiac function and reduced infarct size in pig hearts after ischemia/reperfusion injury by secreting follistatin, which upregulated HIPPO/YAP signaling and proliferation in endogenous pig cardiomyocytes.

中文翻译：

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来自 hiPSC-心肌细胞的卵泡抑素可促进梗死后 LV 重塑猪的肌细胞增殖。


背景 当 CCND2-OE （过表达细胞周期蛋白-D2） 的人诱导多能干细胞 （hiPSC） 分化为心肌细胞 （CCND2-OEhiPSC-CMs） 并施用到免疫缺陷小鼠的梗死心脏时，细胞在给药后增殖并重新填充了 >50% 的疤痕。在这里，我们敲除 CCND2-OEhiPSC-CMs （KO/OEhiPSC-CMs） 中人类白细胞抗原 I 类和 II 类表达，以降低细胞的免疫原性，然后评估 KO/OEhiPSC-CMs 治疗心肌梗死的疗效。方法 在摇瓶中区分 KO/OEhiPSC-CM 和野生型 hiPSC-CM （WThiPSC-CM） 球体，纯化、表征，并在缺血/再灌注损伤后心肌内注射到猪体内;对照动物注射基础培养基。通过心脏磁共振成像评估心脏功能，通过免疫染色和单核 RNA 测序评估心肌细胞增殖。结果 用 KO/OEhiPSC-CM 球体处理的猪的心脏功能和瘢痕大小的测量显著优于用培养基或 WThiPSC-CM 球体处理的动物。给药后仅 1 周即可检测到 KO/OEhiPSC-CMs，但 KO/OEhiPSC-CM 球状体组心脏内源性猪心肌细胞的细胞周期活性和增殖评估显著高于其他 2 组。单核 RNA 测序分析确定了一组增殖心肌细胞，在第 1 周时，在 KO/OEhiPSC-CM 球体处理的心脏 （3.65%） 中明显高于培养基组 （0.89%） 或 WThiPSC-CM 球体 （1.33%） 组的心脏。 用 KO/OEhiPSC-CM 球体处理后，猪心肌细胞中 YAP （Yes-associated protein） 蛋白水平和核定位也显著上调。与 HIPPO/YAP 通路相互作用的卵泡抑素在 KO/OEhiPSC-CM 球体培养基中的丰度显著高于 WThiPSC-CM 球体 （30.29±2.39 vs 16.62±0.83 ng/mL，P = 0.0056）。卵泡抑素治疗在培养 16 天内使 WThiPSC-CM 细胞计数增加 28.3%，并促进成年小鼠梗死心脏中的心肌细胞增殖。结论 KO/OEhiPSC-CM 球体通过分泌卵泡抑素显著改善缺血/再灌注损伤后猪心脏的心脏功能并减小梗死面积，卵泡抑素上调内源性猪心肌细胞的 HIPPO/YAP 信号传导和增殖。