摘要
目的通过使用标准的化学定义和基于小分子诱导方案(chemically defined medium,3 components,CDM3)获得人诱导多能干细胞分化来源的心肌细胞(human induced pluripotent stem cell-derived cardiomyocytes,hiPSC-CMs),进一步对其施加机械牵张,探讨机械牵张对hiPSC-CMs成熟的影响及潜在机制。方法复苏、培养和鉴定hiPSCs后,将hiPSCs接种到覆有基质胶的传统培养皿上。24 h后在荧光显微镜下通过DAPI荧光观察细胞生长情况,并通过八聚体结合转录因子4(octamer-binding transcription factor 4,OCT4)荧光进行hiPSCs干性鉴定,在细胞汇合度达到80%时更换CDM3分化培养基,分化6 d后将获得的hiPSC-CMs分为对照组和机械牵张组,拉伸结束后重新种板培养24 h后在荧光显微镜下通过DAPI荧光观察细胞生长情况,通过心肌肌钙蛋白T(cardiac troponin T,cTnT)和肌球蛋白轻链2V(myosin light chain 2V,MLC2V)荧光进行hiPSC-CMs心肌细胞标志物鉴定,Piezo1荧光进行潜在机制研究。结果hiPSCs培养24 h免疫荧光显示:OCT4发出绿色荧光,hiPSCs保持干性。DAPI发出蓝色荧光:细胞呈克隆生长,细胞形态均一。机械牵张结束后重新种板培养,24 h后免疫荧光显示机械牵张组较对照组心肌标志物cTnT和MLC2V表达增高(P<0.05);机械牵张组较对照组机械敏感型离子通道Piezo1蛋白表达增高(P<0.05)。结论机械牵张可以促进人诱导多能干细胞分化来源心肌细胞的成熟,这可能与Piezo1蛋白表达增高有关。
Objective Human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)were obtained using standard chemical definitions and small molecule induction protocol,and further mechanical stretching was applied to them to explore the effect of mechanical stretch on hiPSC-CMs maturation and the potential mechanism.Methods After recovery,culture,and identification of hiPSCs,hiPSCs were inoculated on traditional culture dishes covered with matrigel.After 24 h,the cell growth was observed by DAPI fluorescence under a fluorescence microscope,and dry identification of hiPSCs was performed by octamer-binding transcription factor 4(OCT4)fluorescence.CDM3 differentiation medium was replaced when the cell connexion reached 80%.After 6 days of differentiation,hiPSC-CMs obtained were divided into control group and mechanical stretch group.After the stretching,Piezo1 fluorescence was used to investigate the potential mechanism of Piezo1 fluorescence.The growth of the cells was observed by DAPI fluorescence under a fluorescence microscope.The hiPSC-CMs myocardial markers were identified by cardiac troponin T(cTnT)and myosin light chain 2V(MLC2V)fluorescence.Results Immunofluorescence of hiPSCs co-cultured with traditional medium for 24 h showed that OCT4 emitted green fluorescence and hiPSCs remained dry.DAPI gave off blue fluorescence:the cells were cloned and homogeneous.After the mechanical stretching was over,the plates were re-cultured.After 24 h,immunofluorescence showed that the expressions of cTnT and MLC2V in mechanical stretch group were higher than those in control group(P<0.05).The protein expression of Piezo1 in the mechanically sensitive ion channel increased in the mechanical stretch group compared with that in the control group(P<0.05).Conclusions Mechanical extension may stimulate the maturation of human induced pluripotent stem cell-derived cardiomyocytes by facilitating the increased expression of the protein Piezo1.
作者
戴越
周帆
郑建伟
穆军升
伯平
尤斌
DAI Yue;ZHOU Fan;ZHENG Jianwei;MU Junsheng;BO Ping;YOU Bin(Departemnt of Cardiac Surgery,Beijing Anzhen Hospital,Capital Medical University,Beijing 100029;Department of Ultrasound,The Third Medical Center of PLA General Hospital,Beijing 100039;Heart Center of Sunshine Union Hospital,Weifang,Shandong Province 261205)
出处
《北京生物医学工程》
2023年第2期130-137,共8页
Beijing Biomedical Engineering
基金
国家自然科学基金(81870181、82270255)资助。
