Objective To explore the role of over-expression of TBX3 and TBX18 in inducing human induced pluripotent stem cells (HiPS) to enrich and differentiate into sinoatrial node-like cells. Methods The expression of stemness markers OCT3/4, SOX2, and NANOG in HiPS was detected by real-time fluorescence quantitative PCR (qRT- PCR), and compared with human embryonic stem cells (hESCs). Immunofluorescence staining was used to observe the expression of HiPS stemness markers OCT3/4, NANOG, SSEA4, and TRA-1-60. The HiPS were directional differentiated into cardiomyocytes, the expressions of ISL1, NK2 homeobox 5 (NKX2-5), ACTN1, and TNNT2 were detected by qRT-PCR, and human adult cardiomyocytes (hACM) were used as positive control. Immunofluorescence staining was used to observe the expressions of NKX2-5, cardiac troponin (cTnT), α-actinin, atria myosin light chain 2A (MLC-2A), and ventricular myosin light chain 2V (MLC-2V). The positive rate of α-actinin was detected by flow cytometry. On the 3rd day after HiPS were differentiated into cardiomyocytes (mesodermal stage), lentiviral over-expressions of sinoatrial node-related genes TBX3 and TBX18 were carried out for 21 days. The relative expressions of specific markers TBX3, TBX18, SHOX2, NKX2-5, HCN4, and HCN1 in sinoatrial node cells were detected by qRT-PCR, and compared with enhanced green fluorescent protein blank virus. Results OCT3/4, SOX2, and NANOG were highly expressed in HiPS and ESCs, and there was no significant difference in the relative expression of each gene (P>0.05); OCT3/4 and NANOG were specifically distributed in the nucleus of HiPS, while SSEA4 and TRA-1-60 were distributed in the cell membrane. The relative expressions of ISL1 gene at 5, 7, 21, and 28 days and NKX2-5 gene at 7, 21, and 28 days of HiPS differentiation into cardiomyocytes were significantly higher than those of hACM (P<0.05), and the relative expressions of ACTN1 and TNNT2 genes at 3, 5, 7, and 21 days of HiPS differentiation into cardiomyocytes were significantly lower than those of hACM (P<0.05). NKX2-5 was expressed in most of the nuclei, cTnT and α-actinin, MLC-2A and MLC-2V signals were localized in the cytoplasm, presenting a texture-like structure of muscle nodules. Flow cytometry results showed that HiPS was successfully induced to differentiate into cardiomyocytes. The expressions of TBX18, SHOX2, HCN4, and HCN1 in the over-expression TBX3 group were up-regulated when compared with the control group, and difference in the relative expression of SHOX2 gene was significant (P<0.05); the relative expression of NKX2-5 gene was lower than that in the control group, but there was no significant difference (P>0.05). There was no significant difference in the relative expression of each gene between the over-expressed TBX18 group and the control group (P>0.05). Conclusion HiPS and hESCs have similar pluripotency, and we have established a stable method for maintaining and culturing the stemness of HiPS. A technological platform for the efficient differentiation of HiPS into cardiomyocytes has been successfully established. Although TBX3 and TBX18 do not play a significant role in promoting the enrichment and differentiation of HiPS into sinoatrial node-like cells, TBX3 shows a certain promoting trend, which can be further explored in the future.
ObjectiveTo 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.MethodsHiPSCs 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.ResultsThe 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).ConclusionJoint regulation of Wnt and BMP signaling pathways can improve the differentiation efficiency of hiPSCs into cardiomyocytes.