A Novel Fluorescent Reporter System Identifies Laminin-511/521 as Potent Regulators of Cardiomyocyte Maturation
A Novel Fluorescent Reporter System Identifies Laminin-511/521 as Potent Regulators of Cardiomyocyte Maturation
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DOI：
10.1038/s41598-020-61163-3
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发表时间：
2020-03-06
影响因子：
4.6
通讯作者：
Uosaki, Hideki
中科院分区：
文献类型：
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作者：
Chanthra, Nawin;Abe, Tomoyuki;Uosaki, Hideki
多能干细胞衍生的心肌细胞（PSC-CM）在疾病建模和药物发现方面具有巨大的前景。然而，PSC-CM在培养中表现出不成熟的表型，缺乏成熟度限制了其广泛的应用。虽然物理和功能分析通常用于确定心肌细胞成熟的状态，但它们可能很耗时，并且在比较成熟促进策略时经常存在挑战。因此，需要一种快速且可重复地评估心肌细胞成熟的方法。在这项研究中，我们发现Myomesin-2（Myom 2），编码M蛋白，是在出生后上调，并在此基础上，我们在小鼠胚胎干细胞中的Myom 2位点的TagRFP。Myom 2-RFP+ PSC-CM在形态、功能和转录方面比RFP-细胞表现出更成熟的表型，有助于肌节缩短测定。使用这个系统，我们筛选细胞外基质（ECM），并确定层粘连蛋白-511/521作为心肌细胞成熟的有效增强剂。我们共同开发并表征了一种新的荧光报告系统，用于评估心肌细胞成熟，并通过这种简单快速的测定法鉴定了有效的成熟增强ECM。预计该系统将有助于在各种科学和医学调查中使用PSC-CM。
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.