Objective: 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.
Methods: 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.
Results: 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 ( P<0.05) and the best effective period was the 3rd day ( P<0.05) in inducing hiPSCs to differentiate into cardiomyocytes. The optimal concentration of BMP-4 was 20.0 ng/mL ( P<0.05) and the best effective period was the 3rd day ( P<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 ( P<0.05).
Conclusion: Joint regulation of Wnt and BMP signaling pathways can improve the differentiation efficiency of hiPSCs into cardiomyocytes.
目的: 探讨联合调控 Wnt 和 BMP 信号通路在人源诱导多能干细胞（human induced pluripotent stem cells，hiPSCs）分化为心肌细胞中的作用。.
方法: 复苏 hiPSCs 培养，倒置相差显微镜下观察细胞形态，免疫荧光染色鉴定多能性标记物 OCT3/4、NANOG 和 TRA-1-60 表达；传代培养 hiPSCs，取 35 代以内细胞进行后续实验。待细胞融合接近 100% 时开始分化（记为分化第 0 天），加入 Wnt 通路激活剂 CHIR99021。于分化第 3 天加入不同浓度的 IWP4（Wnt 通路抑制剂），实时荧光定量 PCR 检测心肌细胞特异性标记物肌钙蛋白 T（troponin T，TNNT2）mRNA 表达，筛选 IWP4 最佳浓度；在不同时间点加入最佳浓度 IWP4，通过比较 TNNT2 mRNA 表达筛选最佳作用时间。然后，在加入 CHIR99021 和 IWP4 基础上于分化第 5 天加入不同浓度 BMP-4 以及不同时间点加入 BMP-4，通过检测 TNNT2 mRNA 表达筛选 BMP-4 最佳浓度及最佳作用时间。最后，将 hiPSCs 分为 3 组，Wnt 调控组、BMP 调控组和 Wnt+BMP 联合调控组，在分化第 0 天加入 CHIR99021 基础上，分别按照筛选结果加入 IWP4、BMP-4、IWP4+BMP-4。收集分化第 7、15 天细胞，实时荧光定量 PCR 检测心肌前体细胞标记物 ISL1（ISL LIM homeobox 1）、NKX2-5（NK2 homeobox 5）以及心肌细胞特异性标记肌细胞增强因子 2C（myocyte enhancer factor 2C，MEF2C）、肌球蛋白轻链 2（myosin light chain 2，MYL2）、MYL7 及 TNNT2 mRNA 表达；收集分化第 28 天细胞，流式细胞术及免疫荧光染色检测心肌细胞特异性蛋白心肌肌钙蛋白 T（cardiac troponin T，cTnT）表达。.
结果: 细胞形态及免疫荧光染色观察显示 hiPSCs 表达多能性标记物 OCT3/4、NANOG 和 TRA-1-60，提示其具有良好的多能性特点。IWP4 促 hiPSCs 向心肌细胞分化的最佳浓度为 10.0 μmol/L（ P<0.05），最佳作用时间是分化第 3 天（ P<0.05）。BMP-4 促 hiPSCs 向心肌细胞分化的最佳浓度为 20.0 ng/mL（ P<0.05），最佳作用时间是分化第 3 天（ P<0.05）。Wnt+BMP 联合调控组 ISL1、NKX2-5 以及 MEF2C、MYL2、MYL7、TNNT2 mRNA 相对表达量，cTnT 阳性表达率，以及免疫荧光染色 cTnT 阳性表达和肌节结构，均优于 Wnt 调控组，相关定量指标差异均有统计学意义（ P<0.05）。.
结论: 联合调控 Wnt 和 BMP 信号通路可以提高 hiPSCs 诱导分化为心肌细胞的效率。.
Keywords: Human induced pluripotent stem cells; Wnt signaling pathway; bone morphogenetic protein signaling pathway; cardiomyocytes.