Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Japan.
Division of Stem Cell Research and Drug Development, Center for Development of Advanced Medical Technology, Jichi Medical University, Shimotsuke, Japan.
Sci Rep. 2020 Mar 6;10(1):4249. doi: 10.1038/s41598-020-61163-3.
Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) hold great promise for disease modeling and drug discovery. However, PSC-CMs exhibit immature phenotypes in culture, and the lack of maturity limits their broad applications. While physical and functional analyses are generally used to determine the status of cardiomyocyte maturation, they could be time-consuming and often present challenges in comparing maturation-enhancing strategies. Therefore, there is a demand for a method to assess cardiomyocyte maturation rapidly and reproducibly. In this study, we found that Myomesin-2 (Myom2), encoding M-protein, is upregulated postnatally, and based on this, we targeted TagRFP to the Myom2 locus in mouse embryonic stem cells. Myom2-RFP PSC-CMs exhibited more mature phenotypes than RFP cells in morphology, function and transcriptionally, conductive to sarcomere shortening assays. Using this system, we screened extracellular matrices (ECMs) and identified laminin-511/521 as potent enhancers of cardiomyocyte maturation. Together, we developed and characterized a novel fluorescent reporter system for the assessment of cardiomyocyte maturation and identified potent maturation-enhancing ECMs through this simple and rapid assay. This system is expected to facilitate use of PSC-CMs in a variety of scientific and medical investigations.
多能干细胞衍生的心肌细胞（PSC-CMs）在疾病建模和药物发现方面具有广阔的应用前景。然而，PSC-CMs 在培养中表现出不成熟的表型，成熟度的缺乏限制了它们的广泛应用。虽然物理和功能分析通常用于确定心肌细胞成熟状态，但这些方法可能既耗时又难以比较成熟促进策略。因此，需要一种快速且可重复的方法来评估心肌细胞的成熟度。在这项研究中，我们发现肌球蛋白重链 2（Myom2），编码 M 蛋白，在出生后上调，基于这一点，我们将 TagRFP 靶向到小鼠胚胎干细胞中的 Myom2 基因座。Myom2-RFP PSC-CMs 在形态、功能和转录水平上表现出比 RFP 细胞更成熟的表型，有利于肌节缩短分析。使用该系统，我们筛选了细胞外基质（ECM），并确定了层粘连蛋白 511/521 是心肌细胞成熟的有效增强剂。总之，我们开发并表征了一种用于评估心肌细胞成熟的新型荧光报告系统，并通过这种简单快速的检测方法鉴定了有效的成熟增强 ECM。该系统有望促进 PSC-CMs 在各种科学和医学研究中的应用。
