Feng Liu1, Yibing Fang1, Qi Xiong1, Liangqin Luo2, Rui Zhou3, Bin Liao4. 1. Department of Cardiac Macrovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou Sichuan, 646000, P.R.China. 2. Department of General Thoracic Surgery, Affiliated Hospital of Southwest Medical University, Luzhou Sichuan, 646000, P.R.China. 3. Institute of Cardiovascular Medicine, Southwest Medical University, Luzhou Sichuan, 646000, P.R.China.zhouhuaxizhu@126.com. 4. Department of Cardiac Macrovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou Sichuan, 646000, P.R.China.1875153677@qq.com.
Abstract
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.
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.
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