Feasibility Study Of Tbx3 Or Tbx18 Overexpression In The Biased Differentiation Of Human Induced Pluripotent Stem Cells Into Sinoatrial Node Cells

Objective: To explore the feasibility of the biased differentiation of human induced pluripotent stem cells（iPSCs）into sinoatrial node cells,the human Tbx3 or Tbx18 transcription factors were overexpressed by lentiviral tranduction during the directional differentiation of human iPSCs into cardiomyocytes.Methods: 1.Human iPS culture medium（Bio CISO,China）was used to culture iPSCs（LHpb-Yaab C3）.Subculture was done using a 1:4¹:7 ratio.Rho-associated,coiled-coil containing protein kinase（ROCK）inhibitor（10 μmol/ml）was added to reduce cell apoptosis and increase the survival rate of the clones.2.The pluripotent detection of iPSCs was ascertained by QRT-PCR analysis and immunofluorescent method.Pluripotent markers（Oct3/4,Sox2,Nanog）of iPSCs were detected by a Quantitative Real-time polymerase chain reaction（QRT-PCR）.The protein levels of iPSC specific markers,nuclear transcription factors（OCT4,NANOG）,and cell membrane proteins（SSEA4,TRA-1-60）of iPSCs were evaluated by immunofluorescence.3.The directional induction of the differentiation of iPSCs to cardiomyocytes commenced when the cells had achieved near-confluent growth.For induction,RPMI + B27（-）insulin-free medium containing the glycogen synthase kinase 3（GSK3）inhibitor CHIR（6 μM）was used at day 0 for the transient activation of the Wnt pathway,followed by the RPMI + B27（-）medium from day 1 to day 3.At day 3,the Wnt inhibitor IWP2（5 μM）was added to the medium.At day 5,culturing in the RPMI+B27（-）medium was resumed until day 7,when the medium was refreshed by RPMI+B27（+）containing insulin medium.The medium was changed every 3 days.4.Detection of the directional differentiation of iPSCs to cardiomyocytes.Recording the change in cell morphology and pulsation frequency.Cardiac precursor cell-specific genes（ISL1,NKX2₅）and cardiomyocyte-specific genes（ACTN1,TNNT2）were detected by QRT-PCR.Cardiac precursor cell-specific protein NKX2₅ and myocardial cell-specific contractile proteins（c Tn T,α-actinin）of differentiated cells were evaluated by immunofluorescence,as were markers of the atrial muscle（MLC-2A）and ventricular myocytes（MLC-2V）.Detection of the proportion of α-actinin-positive cells was done by flow cytometry.5.To induce the biased differentiation of human iPSCs into sinoatrial node cells,four groups were used: Tbx3 over-expression group,Tbx18 over-expression group,virus negative control group,and cell blank control group.Human Tbx3 and Tbx18 transcription factors were overexpressed by lentivirus transduction（multiplicity of infection of 50）during the cardiac mesoderm stage.6.To detect the biased differentiation of human iPSCs into sinoatrial node cells,sinoatrial node specific transcription factors（Tbx3,Tbx18,SHOX2）,ion channel markers（HCN4,HCN1）,and the cardiac precursor cell-specific marker NKX2₅ were detected by QRT-PCR.Results: 1.Under low magnification,human iPSCs displayed typical clonal growth.High magnification examination revealed the regular arrangement of iPSCs.The cells were smaller in size,with larger nuclei,less cytoplasm,and a higher nucleocytoplasmic ratio.Human iPSCs and human embryonic stem cells highly expressed pluripotent markers（Oct3/4,Sox2,Nanog）（all P>0.05）.The results suggested a similar pluripotency of iPSCs and embryonic stem cells.Immunofluorescence revealed iPSC expression of specific nuclear transcription factors（OCT4,NANOG）and cell membrane proteins（SSEA4,TRA-1-60）,and their localization in the nucleus or cell membrane.2.Differentiation of iPSCs to cardiomyocytes produced closely arranged cells,with growth in a fish-like or spiral shape.Pulsing of the cardiomyocytes began at day 9.The pulse frequency and proportion of pulsating cells gradually increased with time.After day 14,the pulse frequency was 40–90 beats per minute and the proportion of pulsating cells was approximately 70%.Cardiac precursor cell-specific genes（ISL1,NKX2₅）and cardiomyocyte-specific genes（ACTN1,TNNT2）were expressed at day 3.ISL1 and NKX2₅ gene expression was the highest at day 5 and day 7,respectively,and gradually declined thereafter.At day 28,the expression levels remained higher than the expression level of normal adult cardiomyocytes（P<0.05）,suggesting that the cells were less mature than adult cardiomyocytes.The expression levels of cardiomyocyte-specific genes（ACTN1 and TNNT2）increased with the culture time,and were only slightly lower than that of the normal adult cardiomyocytes at day 28（P>0.05）.Immunofluorescence assay performed on differentiated cells at day 21 revealed the expressions of NKX2₅,c Tn T,α-actinin,MLC-2A,and MLC-2V in cardiac precursor cells.NKX2₅ was localized in the nucleus and c Tn T and α-actinin were distributed in the cytoplasm and were visible in the striated structure of the sarcomere.The proportion of positive cells in the experimental group in a flow cytometry analysis was 78.5%.3.In the Tbx3 overexpression group,expression of Tbx18,SHOX2,and HCN1 were all higher than those in the control group.Only SHOX2 displayed a significant difference（P<0.05）.NKX2₅ expression was lower than that in the control group,but was not significantly different（P>0.05）.The results suggest that overexpression of Tbx3 can have positive effects in promoting the biased differentiation of iPSCs into sinoatrial node cells.However,more data are needed for a definitive conclusion.The expression levels of all markers in the Tbx18 overexpression group were significantly different from those in the control group（all P>0.05）,suggesting that overexpression of transcription factor Tbx18 in the mesoderm stage of cardiomyocytes failed to promote the biased differentiation of iPSCs into sinoatrial node cells.Conclusion: 1.Human iPSC culture and pluripotent maintenance techniques were established.Human iPSCs displayed similar pluripotent characteristics to human embryonic stem cells.2.Directed differentiation of human iPSCs into cardiomyocytes was achieved.3.Preliminary results suggested that overexpression of the transcription factors Tbx3 or Tbx18 in the mesoderm stage of cardiomyocytes failed to promote the biased differentiation of iPSCs into sinoatrial node cells.However,the overexpression of Tbx3 had at least some positive effects that were not statistically significant.