The ability to reprogram human somatic cells into human induced pluripotent stem cells (hiPSCs) has enabled researchers to generate cell types in vitro that have the potential to faithfully recapitulate patient-specific disease processes and phenotypes. hiPSC-derived cardiomyocytes (hiPSC-CMs) offer the promise of in vitro patient- and disease-specific models for drug testing and the discovery of novel therapeutic approaches for treating cardiovascular diseases. While methods to differentiate hiPSCs into cardiomyocytes have been demonstrated, the heterogeneity and immaturity of these differentiated populations have restricted their potential in reproducing human disease and the associated target cell phenotypes. These barriers may be overcome through comprehensive single-cell characterization to dissect the rich heterogeneity of hiPSC-CMs and to study the source of varying cell fates. In this study, we optimized and validated a new Single-Cell Western method to assess protein expression in hiPSC-CMs. To better understand distinct subpopulations generated from cardiomyocyte differentiations and to track populations at single-cell resolution over time, we measured and quantified the expression of cardiomyocyte subtype-specific proteins (MLC2V and MLC2A) using Single-Cell Westerns. By understanding their heterogeneity through single-cell protein expression and quantification, we may improve upon current cardiomyocyte differentiation protocols, generate hiPSC-CMs that are more representative of in vivo derived cardiomyocytes for disease modeling, and utilize hiPSC-CMs for regenerative medicine purposes. Single-Cell Westerns provide a robust platform for protein expression analysis at single-cell resolution.

将人类体细胞重编程为人类诱导多能干细胞 (hiPSC) 的能力使研究人员能够在体外生成细胞类型，这些细胞类型有可能忠实地概括患者特定的疾病过程和表型。hiPSC 衍生的心肌细胞 (hiPSC-CMs) 提供了体外用于药物测试的患者和疾病特异性模型以及治疗心血管疾病的新治疗方法的发现。虽然已经证明了将 hiPSC 分化为心肌细胞的方法，但这些分化群体的异质性和不成熟性限制了它们在复制人类疾病和相关靶细胞表型方面的潜力。这些障碍可以通过全面的单细胞表征来克服，以剖析 hiPSC-CM 的丰富异质性并研究不同细胞命运的来源。在这项研究中，我们优化并验证了一种新的单细胞西方方法来评估 hiPSC-CM 中的蛋白质表达。为了更好地了解心肌细胞分化产生的不同亚群，并随着时间的推移以单细胞分辨率跟踪种群，我们使用 Single-Cell Westerns 测量并量化了心肌细胞亚型特异性蛋白（MLC2V 和 MLC2A）的表达。通过单细胞蛋白表达和量化了解它们的异质性，我们可以改进当前的心肌细胞分化方案，生成更具有代表性的 hiPSC-CM体内衍生的心肌细胞用于疾病建模，并将 hiPSC-CM 用于再生医学目的。Single-Cell Westerns 为单细胞分辨率的蛋白质表达分析提供了强大的平台。