Retinoic acid promotes metabolic maturation of human Embryonic Stem Cell-derived Cardiomyocytes
Retinoic acid promotes metabolic maturation of human Embryonic Stem Cell-derived Cardiomyocytes
复制标题
视黄酸促进人胚胎干细胞来源的心肌细胞的代谢成熟
DOI：
10.7150/thno.44146
复制
发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Hu, Shijun
中科院分区：
文献类型：
--
作者：
Miao, Shumei;Zhao, Dandan;Hu, Shijun
从人胚胎干细胞（hESC）分化的心肌细胞代表了心脏修复，疾病建模和药物测试的有前途的细胞来源。然而，提高hESC衍生的心肌细胞（hESC-CM）的分化效率和成熟仍然是一个主要的问题。维甲酸（RA）信号在心脏发育中起着多种作用。然而，RA对心肌细胞分化效率和成熟的影响仍不清楚。方法：在不同的时间间隔加入RA，以确定心肌细胞分化和成熟的最佳治疗窗口。实时荧光定量PCR和流式细胞仪检测心肌细胞分化效率。通过免疫荧光染色、代谢分析和膜片钳技术分别检测心肌细胞的结构、代谢和电生理成熟。RNA测序用于剪接分析。结果如下：我们发现，RA治疗在侧中胚层阶段（第2-4天）显着改善心肌细胞分化，如TNNT 2，NKX2.5和MYH 6在分化第10天的上调所证明的。流式细胞仪检测结果显示，RA处理组心肌细胞分化比例明显高于对照组。RA治疗15-20天增加心肌细胞面积、肌节长度、多核化和线粒体拷贝数。RNA测序显示RA促进RNA异构体转换为成熟相关形式。同时，RA促进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糖酵解减少和线粒体氧化磷酸化增强，增加了脂肪酸和外源性丙酮酸的利用，但没有谷氨酰胺。结论：我们的数据表明，在早期时间窗（第2-4天）的RA处理促进心肌细胞分化的效率，并且在搏动后（第15-20天）的RA处理促进心肌细胞成熟。RA的双相效应为改善心肌细胞分化和质量提供了新的见解。
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. Methods: 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. Results: We found that RA treatment at the lateral mesoderm stage (days 2-4) significantly improved cardiomyocyte differentiation, as evidenced by the upregulation of TNNT2, NKX2.5 and MYH6 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. Conclusion: 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.