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.
ObjectiveTo provide experimental data and theoretical support for further studying the maturity of cardiac patches in other in vitro experiments and the safety in other in vivo animal experiments, through standard chemically defined and small molecule-based induction protocol (CDM3) for promoting the differentiation of human induced pluripotent stem cells (hiPSCs) into myocardium, and preliminarily preparing cardiac patches. MethodsAfter resuscitation, culture and identification of hiPSCs, they were inoculated on the matrigel-coated polycaprolactone (PCL). After 24 hours, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and the stemness of hiPSCs was identified by OCT4 fluorescence. After fixation, electron microscope scanning was performed to observe the cell morphology on the surface of the patch. On the 1st, 3rd, 5th, and 7th days of culture, the cell viability was determined by CCK-8 method, and the growth curve was drawn to observe the cell growth and proliferation. After co-cultured with matrigel-coated PCL for 24 hours, hiPSCs were divided into a control group and a CDM3 group, and continued to culture for 6 days. On the 8th day, the cell growth was observed by DAPI fluorescence under a fluorescence microscope, and hiPSCs stemness was identified by OCT4 fluorescence, and cTnT and α-actin for cardiomyocyte marker identification. ResultsImmunofluorescence of hiPSCs co-cultured with matrigel-coated PCL for 24 hours showed that OCT4 emitted green fluorescence, and hiPSCs remained stemness on matrigel-coated PCL scaffolds. DAPI emitted blue fluorescence: cells grew clonally with uniform cell morphology. Scanning electron microscope showed that hiPSCs adhered and grew on matrigel-coated PCL, the cell outline was clearly visible, and the morphology was normal. The cell viability assay by CCK-8 method showed that hiPSCs proliferated and grew on PCL scaffolds coated with matrigel. After 6 days of culture in the control group and the CDM3 group, immunofluorescence showed that the hiPSCs in the control group highly expressed the stem cell stemness marker OCT4, but did not express the cardiac markers cTnT and α-actin. The CDM3 group obviously expressed the cardiac markers cTnT and α-actin, but did not express the stem cell stemness marker OCT4. ConclusionhiPSCs can proliferate and grow on matrigel-coated PCL. Under the influence of CDM3, hiPSCs can be differentiated into cardiomyocyte-like cells, and the preliminary preparation of cardiac patch can provide a better treatment method for further clinical treatment of cardiac infarction.