Wei Yuhua, Walcott Gregory, Nguyen Thanh, Geng Xiaoxiao, Guragain Bijay, Zhang Hanyu, Green Akazha, Rosa-Garrido Manuel, Rogers Jack M, Garry Daniel J, Ye Lei, Zhang Jianyi
Department of Biomedical Engineering, School of Medicine and School of Engineering, University of Minnesota, Minneapolis (Y.W., G.W., T.N., X.G., B.G., H.Z., A.G., M.R.-G., J.M.R., L.Y., J.Z.).
Department of Medicine, Division of Cardiovascular Disease, School of Medicine, University of Minnesota, Minneapolis (G.W., J.Z.).
Circ Res. 2025 Jan 17;136(2):161-176. doi: 10.1161/CIRCRESAHA.124.325562. Epub 2024 Dec 18.
When human induced pluripotent stem cells (hiPSCs) that CCND2-OE (overexpressed cyclin-D2) were differentiated into cardiomyocytes (hiPSC-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 hiPSC-CMs (hiPSC-CMs) to reduce the cells' immunogenicity and then assessed the therapeutic efficacy of hiPSC-CMs for the treatment of myocardial infarction.
hiPSC-CM and wild-type hiPSC-CM (hiPSC-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.
Measurements of cardiac function and scar size were significantly better in pigs treated with hiPSC-CM spheroids than in animals treated with medium or hiPSC-CM spheroids. hiPSC-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 hiPSC-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 hiPSC-CM spheroid-treated hearts (3.65%) than in the hearts from the medium (0.89%) or hiPSC-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 hiPSC-CM spheroids. Follistatin, which interacts with the HIPPO/YAP pathway, was significantly more abundant in the medium from hiPSC-CM spheroids than hiPSC-CM spheroids (30.29±2.39 versus 16.62±0.83 ng/mL, =0.0056). Treatment with follistatin increased hiPSC-CM cell counts by 28.3% over 16 days in culture and promoted cardiomyocyte proliferation in the infarcted hearts of adult mice.
hiPSC-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.
当将过表达细胞周期蛋白 D2（CCND2 - OE）的人诱导多能干细胞（hiPSC）分化为心肌细胞（hiPSC - CM）并将其注入免疫缺陷小鼠的梗死心脏时，细胞在注入后会增殖并重新填充超过 50%的瘢痕组织。在此，我们敲除了 hiPSC - CM 中的人类白细胞抗原 I 类和 II 类表达以降低细胞的免疫原性，然后评估 hiPSC - CM 治疗心肌梗死的疗效。
hiPSC - CM 和野生型 hiPSC - CM（hiPSC - CM）球体在摇瓶中分化、纯化、鉴定，并在缺血/再灌注损伤后经心内膜注射到猪体内；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，并通过免疫染色和单核 RNA 测序评估心肌细胞增殖。
用 hiPSC - CM 球体治疗的猪的心脏功能和瘢痕大小测量结果明显优于用培养基或 hiPSC - CM 球体治疗的动物。给药后仅 1 周就检测到 hiPSC - CM，但 hiPSC - CM 球体组心脏的内源性猪心肌细胞的细胞周期活性和增殖评估结果明显高于其他两组。单核 RNA 测序分析确定了一组增殖心肌细胞，在第 1 周时，hiPSC - CM 球体治疗的心脏中该组细胞（3.65%）比培养基组（0.89%）或 hiPSC - CM 球体组（1.33%）的心脏中更为普遍。用 hiPSC - CM 球体治疗后，猪心肌细胞中的 Yes 相关蛋白（YAP）蛋白水平和核定位也明显上调。与 Hippo/YAP 信号通路相互作用的卵泡抑素在 hiPSC - CM 球体的培养基中比 hiPSC - CM 球体中含量明显更高（30.29±2.39 对 16.62±0.83 ng/mL，P = 0.0056）。在培养 16 天内，用卵泡抑素治疗使 hiPSC - CM 细胞计数增加了 28.3%，并促进了成年小鼠梗死心脏中的心肌细胞增殖。
hiPSC - CM 球体通过分泌卵泡抑素显著改善了猪心脏缺血/再灌注损伤后的心脏功能并减小了梗死面积，卵泡抑素上调了内源性猪心肌细胞中的 Hippo/YAP 信号传导和增殖。
