Role of over-expression of TBX3 and TBX18 in the enrichment and differentiation of human induced pluripotent stem cells into sinoatrial node-like cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIU Feng ¹ , FANG Yibing ¹ , XIONG Qi ¹ , LUO Liangqin ² ,  ZHOU Rui ³ ,  LIAO Bin ¹

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;

Corresponding author：

ZHOU Rui, Email: zhouhuaxizhu@126.com; LIAO Bin, Email: 1875153677@qq.com

Keywords：

Human derived induced pluripotent stem cells; TBX3; TBX18; sinoatrial node-like cells

DOI：

10.7507/1002-1892.201812009

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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.

Citation： LIU Feng, FANG Yibing, XIONG Qi, LUO Liangqin, ZHOU Rui, LIAO Bin. Role of over-expression of TBX3 and TBX18 in the enrichment and differentiation of human induced pluripotent stem cells into sinoatrial node-like cells. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(4): 497-506. doi: 10.7507/1002-1892.201812009 Copy

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2.	Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663-676.
3.	Huangfu D, Osafune K, Maehr R, et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol, 2008, 26(11): 1269-1275.
4.	邹刚, 李豫皖, 金瑛, 等. TGF-β₁ 联合 VEGF 对人羊膜间充质干细胞向韧带成纤维细胞体外分化作用的研究. 中国修复重建外科杂志, 2017, 31(5): 582-593.
5.	周兰庭, 冯雁婷, 戴景兴, 等. miRNA 调控脂肪干细胞分化的研究进展. 中国修复重建外科杂志, 2017, 31(12): 1506-1511.
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7.	Nicaise AM, Banda E, Guzzo RM, et al. HiPS-derived neural progenitor cells from PPMS patients reveal defect in myelin injury response. Exp Neurol, 2017, 288: 114-121.
8.	Lian X, Zhang J, Azarin SM, et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat Protoc, 2013, 8(1): 162-175.
9.	Kapoor N, Liang W, Marbán E, et al. Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18. Nat Biotechnol, 2013, 31(1): 54-62.
10.	Wiese C, Grieskamp T, Airik R, et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res, 2009, 104(3): 388-397.
11.	Li Y, Zhang Q, Yin X, et al. Generation of HiPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Res, 2011, 21(1): 196-204.
12.	Wei X, Chen Y, Xu Y, et al. Small molecule compound induces chromatin de-condensation and facilitates induced pluripotent stem cell generation. J Mol Cell Biol, 2014, 6(5): 409-420.
13.	Stadtfeld M, Nagaya M, Utikal J, et al. Induced pluripotent stem cells generated without viral integration. Science, 2008, 322(5903): 945-949.
14.	Okita K, Nakagawa M, Hyenjong H, et al. Generation of mouse induced pluripotent stem cells without viral vectors. Science, 2008, 322(5903): 949-953.
15.	Warren L, Manos PD, Ahfeldt T, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 2010, 7(5): 618-630.
16.	Miyoshi N, Ishii H, Nagano H, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell, 2011, 8(6): 633-638.
17.	Lin T, Ambasudhan R, Yuan X, et al. A chemical platform for improved induction of human HiPSCs. Nat Methods, 2009, 6(11): 805-808.
18.	Esteban MA, Wang T, Qin B, et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 2010, 6(1): 71-79.
19.	Hoogaars WM, Engel A, Brons JF, et al. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev, 2007, 21(9): 1098-1112.
20.	Wiese C, Grieskamp T, Airik R, et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res, 2009, 104(3): 388-397.

1. Jung JJ, Husse B, Rimmbach C, et al. Programming and isolation of highly pure physiologically and pharmacologically functional sinus-nodal bodies from pluripotent stem cells. Stem Cell Reports, 2014, 2(5): 592-605.
2. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006, 126(4): 663-676.
3. Huangfu D, Osafune K, Maehr R, et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol, 2008, 26(11): 1269-1275.
4. 邹刚, 李豫皖, 金瑛, 等. TGF-β₁ 联合 VEGF 对人羊膜间充质干细胞向韧带成纤维细胞体外分化作用的研究. 中国修复重建外科杂志, 2017, 31(5): 582-593.
5. 周兰庭, 冯雁婷, 戴景兴, 等. miRNA 调控脂肪干细胞分化的研究进展. 中国修复重建外科杂志, 2017, 31(12): 1506-1511.
6. Zwi L, Caspi O, Arbel G, et al. Cardiomyocyte differentiation of human induced pluripotent stem cells. Circulation, 2009, 120(15): 1513-1523.
7. Nicaise AM, Banda E, Guzzo RM, et al. HiPS-derived neural progenitor cells from PPMS patients reveal defect in myelin injury response. Exp Neurol, 2017, 288: 114-121.
8. Lian X, Zhang J, Azarin SM, et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat Protoc, 2013, 8(1): 162-175.
9. Kapoor N, Liang W, Marbán E, et al. Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18. Nat Biotechnol, 2013, 31(1): 54-62.
10. Wiese C, Grieskamp T, Airik R, et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res, 2009, 104(3): 388-397.
11. Li Y, Zhang Q, Yin X, et al. Generation of HiPSCs from mouse fibroblasts with a single gene, Oct4, and small molecules. Cell Res, 2011, 21(1): 196-204.
12. Wei X, Chen Y, Xu Y, et al. Small molecule compound induces chromatin de-condensation and facilitates induced pluripotent stem cell generation. J Mol Cell Biol, 2014, 6(5): 409-420.
13. Stadtfeld M, Nagaya M, Utikal J, et al. Induced pluripotent stem cells generated without viral integration. Science, 2008, 322(5903): 945-949.
14. Okita K, Nakagawa M, Hyenjong H, et al. Generation of mouse induced pluripotent stem cells without viral vectors. Science, 2008, 322(5903): 949-953.
15. Warren L, Manos PD, Ahfeldt T, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 2010, 7(5): 618-630.
16. Miyoshi N, Ishii H, Nagano H, et al. Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell, 2011, 8(6): 633-638.
17. Lin T, Ambasudhan R, Yuan X, et al. A chemical platform for improved induction of human HiPSCs. Nat Methods, 2009, 6(11): 805-808.
18. Esteban MA, Wang T, Qin B, et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 2010, 6(1): 71-79.
19. Hoogaars WM, Engel A, Brons JF, et al. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev, 2007, 21(9): 1098-1112.
20. Wiese C, Grieskamp T, Airik R, et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res, 2009, 104(3): 388-397.

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Role of over-expression of TBX3 and TBX18 in the enrichment and differentiation of human induced pluripotent stem cells into sinoatrial node-like cells

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