Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China.
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Theranostics. 2020 Aug 1;10(21):9686-9701. doi: 10.7150/thno.44146. eCollection 2020.
Cardiomyocytes differentiated from human embryonic stem cells (hESCs) represent a promising cell source for heart repair, disease modeling and drug testing. However, improving the differentiation efficiency and maturation of hESC-derived cardiomyocytes (hESC-CMs) is still a major concern. Retinoic acid (RA) signaling plays multiple roles in heart development. However, the effects of RA on cardiomyocyte differentiation efficiency and maturation are still unknown. RA was added at different time intervals to identify the best treatment windows for cardiomyocyte differentiation and maturation. The efficiency of cardiomyocyte differentiation was detected by quantitative real-time PCR and flow cytometry. Cardiomyocytes maturation was detected by immunofluorescence staining, metabolic assays and patch clamp to verify structural, metabolic and electrophysiological maturation, respectively. RNA sequencing was used for splicing analysis. We found that RA treatment at the lateral mesoderm stage (days 2-4) significantly improved cardiomyocyte differentiation, as evidenced by the upregulation of , and on day 10 of differentiation. In addition, flow cytometry showed that the proportion of differentiated cardiomyocytes in the RA-treated group was significantly higher than that in control group. RA treatment on days 15-20 increased cardiomyocyte area, sarcomere length, multinucleation and mitochondrial copy number. RNA sequencing revealed RA promoted RNA isoform switch to the maturation-related form. Meanwhile, RA promoted electrophysiological maturation and calcium handling of hESC-CMs. Importantly, RA-treated cardiomyocytes showed decreased glycolysis and enhanced mitochondrial oxidative phosphorylation, with the increased utilization of fatty acid and exogenous pyruvate but not glutamine. Our data indicated that RA treatment at an early time window (days 2-4) promotes the efficiency of cardiomyocyte differentiation and that RA treatment post beating (days 15-20) promotes cardiomyocyte maturation. The biphasic effects of RA provide new insights for improving cardiomyocyte differentiation and quality.
人胚胎干细胞（hESC）分化而来的心肌细胞代表了一种很有前途的心脏修复、疾病建模和药物测试的细胞来源。然而，提高 hESC 分化而来的心肌细胞（hESC-CM）的分化效率和成熟度仍然是一个主要关注点。视黄酸（RA）信号在心脏发育中发挥多种作用。然而，RA 对心肌细胞分化效率和成熟度的影响尚不清楚。我们在不同的时间间隔添加 RA，以确定对心肌细胞分化和成熟的最佳治疗窗口。通过定量实时 PCR 和流式细胞术检测心肌细胞分化效率。通过免疫荧光染色、代谢测定和膜片钳技术分别检测心肌细胞成熟度，以验证结构、代谢和电生理成熟度。进行 RNA 测序进行剪接分析。我们发现，RA 在侧中胚层期（第 2-4 天）的处理显著提高了心肌细胞的分化效率，在第 10 天分化时上调了 、 和 。此外，流式细胞术显示，RA 处理组分化的心肌细胞比例明显高于对照组。RA 在第 15-20 天的处理增加了心肌细胞面积、肌节长度、多核化和线粒体拷贝数。RNA 测序显示 RA 促进了 RNA 异构体向成熟相关形式的转变。同时，RA 促进了 hESC-CM 的电生理成熟和钙处理。重要的是，RA 处理的心肌细胞表现出降低的糖酵解和增强的线粒体氧化磷酸化，增加了脂肪酸和外源性丙酮酸的利用，但不是谷氨酰胺。我们的数据表明，RA 在早期时间窗口（第 2-4 天）处理可提高心肌细胞的分化效率，而在搏动后（第 15-20 天）处理可促进心肌细胞的成熟。RA 的双相作用为提高心肌细胞分化和质量提供了新的见解。
