Yan Ying, Liu Feng, Hou Xiaojie, Wan Juyi, Xiong Qi, Zhou Rui, Liao Bin
Clinical Medicine College of Chengdu University of Traditional Chinese Medicine, Chengdu Sichuan, 610075, P.R.China;College of Integrative Chinese and Western Medicine, Southwest Medical University, Luzhou Sichuan, 646000, P.R.China.
Department of Cardiac Macrovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou Sichuan, 646000, P.R.China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2020 Oct 15;34(10):1313-1321. doi: 10.7507/1002-1892.201912087.
To explore the role of joint regulation of Wnt and bone morphogenetic protein (BMP) signaling pathways in the differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes.
HiPSCs were cultured and observed under inverted phase contrast microscope. Immunofluorescence staining was used to observe the expressions of hiPSCs pluripotent markers (OCT3/4, NANOG, and TRA-1-60). HiPSCs were passaged which were taken for subsequent experiments within the 35th passage. When the fusion degree of hiPSCs was close to 100%, the CHIR99021 (Wnt pathway activator) was added on the 0th day of differentiation. Different concentrations of IWP4 (inhibitor of Wnt production) were added on the 3rd day of differentiation, and the best concentration of IWP4 was added at different time points. The optimal concentration and the best effective period of IWP4 were obtained by detecting the expression of troponin T (TNNT2) mRNA by real-time fluorescence quantitative PCR. Then, on the basis of adding CHIR99021 and IWP4, different concentrations of BMP-4 were added on the 5th day of differentiation, and the best concentration of BMP-4 was added at different time points. The optimal concentration and best effective period of BMP-4 were obtained by detecting the expression of TNNT2 mRNA. Finally, hiPSCs were divided into three groups: Wnt group, BMP group, and Wnt+BMP group. On the basis of adding CHIR99021 on the 0th day of differentiation, IWP4, BMP-4, and IWP4+BMP-4 were added into Wnt group, BMP group, and Wnt+BMP group respectively according to the screening results. Cells were collected on the 7th and the 15th days of differentiation. The expressions of myocardial precursor cell markers [ISL LIM homeobox 1 (ISL1), NK2 homeobox 5 (NKX2-5)] and cardiomyocyte specific markers [myocyte enhancer factor 2C (MEF2C), myosin light chain 2 (MYL2), MYL7, and TNNT2] were detected by real-time fluorescent quantitative PCR. Cells were collected on the 28th day of differentiation, and the expression of cardiac troponin T (cTnT) was detected by flow cytometry and immunofluorescence staining.
The results of cell mophology and immunoflurescence staining showed that the OCT3/4, NANOG, and TRA-1-60 were highly expressed in hiPSCs, which suggested that hiPSCs had characteristics of pluripotency. The optimal concentration of IWP4 was 10.0 μmol/L ( <0.05) and the best effective period was the 3rd day ( <0.05) in inducing hiPSCs to differentiate into cardiomyocytes. The optimal concentration of BMP-4 was 20.0 ng/mL ( <0.05) and the best effective period was the 3rd day ( <0.05). The relative expressions of ISL1, NKX2-5, MEF2C, MYL2, MYL7, and TNNT2 mRNAs, the positive expression ratio of cTnT detected by flow cytometry, and sarcomere structure detected by immunofluorescence staining of Wnt+BMP group were superior to those of Wnt group ( <0.05).
Joint regulation of Wnt and BMP signaling pathways can improve the differentiation efficiency of hiPSCs into cardiomyocytes.
探讨Wnt和骨形态发生蛋白（BMP）信号通路的联合调控在人诱导多能干细胞（hiPSCs）向心肌细胞分化中的作用。
在倒置相差显微镜下培养并观察hiPSCs。采用免疫荧光染色观察hiPSCs多能性标志物（OCT3/4、NANOG和TRA-1-60）的表达。对hiPSCs进行传代，取第35代以内的细胞用于后续实验。当hiPSCs融合度接近100%时，在分化第0天添加CHIR99021（Wnt通路激活剂）。在分化第3天添加不同浓度的IWP4（Wnt产生抑制剂），并在不同时间点添加IWP4的最佳浓度。通过实时荧光定量PCR检测肌钙蛋白T（TNNT2）mRNA的表达，获得IWP4的最佳浓度和最佳作用时间。然后，在添加CHIR99021和IWP4的基础上，在分化第5天添加不同浓度的BMP-4，并在不同时间点添加BMP-4的最佳浓度。通过检测TNNT2 mRNA的表达，获得BMP-4的最佳浓度和最佳作用时间。最后，将hiPSCs分为三组：Wnt组、BMP组和Wnt+BMP组。在分化第0天添加CHIR99021的基础上，根据筛选结果分别向Wnt组、BMP组和Wnt+BMP组添加IWP4、BMP-4和IWP4+BMP-4。在分化第7天和第15天收集细胞。通过实时荧光定量PCR检测心肌前体细胞标志物[ISL LIM同源框1（ISL1）、NK2同源框5（NKX2-5）]和心肌细胞特异性标志物[肌细胞增强因子2C（MEF2C）、肌球蛋白轻链2（MYL2）、MYL7和TNNT2]的表达。在分化第28天收集细胞，通过流式细胞术和免疫荧光染色检测心肌肌钙蛋白T（cTnT）的表达。
细胞形态学和免疫荧光染色结果显示，OCT3/4、NANOG和TRA-1-60在hiPSCs中高表达，提示hiPSCs具有多能性特征。IWP4诱导hiPSCs分化为心肌细胞的最佳浓度为10.0 μmol/L（P＜０.０５），最佳作用时间为第3天（P＜０.０５）。BMP-4的最佳浓度为20.0 ng/mL（P＜０.０５），最佳作用时间为第3天（P＜０.０５）。Wnt+BMP组ISL1、NKX2-5、MEF2C、MYL2、MYL7和TNNT2 mRNA的相对表达量、流式细胞术检测的cTnT阳性表达率以及免疫荧光染色检测的肌节结构均优于Wnt组（P＜０.０５）。
Wnt和BMP信号通路的联合调控可提高hiPSCs向心肌细胞的分化效率。
