胚胎干细胞向心肌细胞分化过程中能量代谢变化的研究

Metabolic Phenotyping to Identify Cellular Transitions During Cardiomyocytes Differentiation From Human Embryonic Stem Cells

本文通过定向诱导人胚胎干细胞分化为心肌细胞,对分化过程中胚胎干细胞、心肌祖细胞和心肌细胞糖酵解能力和线粒体氧化磷酸化能力进行实时定量检测,旨在探索分化过程中细胞能量代谢表型的转换机制.用GSK3抑制剂CHIR99021和Wnt信号通路小分子抑制剂IWP2的方法定向分化人胚胎干细胞为心肌祖细胞和心肌细胞;细胞免疫荧光检测人胚胎干细胞标志物,流式细胞术检测人心肌祖细胞和心肌细胞标志物;应用细胞外流量分析(extracellular flux analysis)方法检测人胚胎干细胞、心肌祖细胞和心肌细胞能量代谢情况.研究发现,人胚胎干细胞干性保持稳定,均表达Nanog、OCT4、SOX2细胞标志物;在向心肌分化过程中,第7 d心肌祖细胞标志物Isl1表达99%以上,分化第14 d心肌细胞标志物cTnT表达83%以上;人胚胎干细胞糖酵解代谢能力最强,心肌细胞线粒体功能最强,心肌祖细胞处于两种代谢方式的过度阶段.因此推断,在人胚胎干细胞向心肌细胞分化的过程中,细胞糖酵解能力逐渐减弱,线粒体氧化磷酸化能力逐渐增强,细胞的能量代谢类型发生转变.本研究旨在优化人胚胎干细胞定向分化为心肌细胞的方法,揭示分化依赖的细胞能量代谢类型的转变,为心血管疾病的理论研究和临床治疗提供基础.

We performed directional differentiation of human embryonic stem cells into cardiomyocytes.In order to explore the mechanisms of cell metabolic phenotype conversion during cardiac lineage differentiation,we conducted real-time quantitative detection of glycolytic and mitochondrial oxidative phosphorylation capabilities of embryonic stem cells,cardiac progenitor cells,and cardiomyocytes during differentiation.GSK3 inhibitor CHIR99021 and Wnt signaling pathway inhibitor IWP2 were used to differentiate human embryonic stem cells into cardiac progenitor cells and cardiomyocytes.Immunocytochemistry was used to detect the expression of human embryonic stem cell markers.Flow cytometry was used to detect the markers of human cardiomyocytes and cardiac progenitor cells.Extracellular flux analysis was used to test the energy metabolic phenotype of human embryonic stem cells,cardiac progenitor cells,and cardiomyocytes.The stemness of human embryonic stem cells remains stable and all express Nanog,OCT4 and SOX2 cell markers.During the differentiation,more than 99%cells expressed cardiac progenitor cell marker Isl1 on the 7th day,and more than 83%of cells expressed the cardiomyocytes marker cTnT on the 14th day.Human embryonic stem cells have the strongest glycolytic metabolism capacity,while cardiomyocytes have the strongest mitochondrial oxidative phosphorylation capability.Cardiac progenitor cells are in the transition stage of the two ways of metabolism.During the differentiation of human embryonic stem cells into cardiomyocytes,cells gradually loss the glycolytic capacity,while the mitochondrial oxidative phosphorylation capacity gradually increases,followed by the cell metabolic phenotype conversion.This research aims to optimize the method of directional differentiation of human embryonic stem cells into cardiomyocytes,reveal the transformation of differentiation-dependent cell energy metabolism,and provide a basis for theoretical research and clinical treatment of cardiovascular diseases.