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The optimized method for mesenchymal stem cell differentiation into pacemaker-like cell

  • Faris Faris Basalamah ,
  • Ismail Hadisoebroto Dilogo ,
  • Sunu Budhi Raharjo ,
  • Muchtaruddin Mansyur ,
  • Nuryati Chairani Siregar ,
  • Nurhadi Ibrahim ,
  • Budi Yuli Setianto ,
  • Yoga Yuniadi ,


Abstract

Link of Video Abstract: https://www.youtube.com/watch?v=HIbeTAOCFcA


Background: Mesenchymal stem cells (MSCs) differentiate into pacemaker-like cells by activating TBX3 and downregulating Pitx2c by inhibiting the nodal signaling pathway. This optimization study aimed to determine the optimal conditions for differentiating MSCs into pacemaker-like cells.

Methods: The differentiation of MSCs into pacemaker-like cells was carried out in three ways: transfecting TBX3 pcDNA into MSCs, inhibiting the nodal signaling pathway using the small molecule SB431542, and a combination of both methods. This optimization study was conducted in two batches. The first batch was an experiment with transfection carried out on the 5th day of differentiation and addition of 20 μM/mL of the small molecule SB431542. The second batch was an experiment with transfection carried out on the 3rd day of differentiation and the addition of 2 μM/mL of the small molecule SB431542. From the two batches, the expression patterns of TBX3, Cx30.2, Cx40, Cx43, HCN4, HCN1, HCN3, and KCNN4 genes were analyzed using the qRT-PCR method.

Results: The results showed that the second batch had a better gene expression pattern in pacemaker-like cells than the first batch. These results were indicated by the increased expression of the TBX3, Cx30.2, HCN4, HCN1, HCN3, and KCNN4 genes. Furthermore, there was a decrease in Cx40 and Cx43 expression levels.

Conclusion: The results showed that TBX3 transfection on the third day and a smaller dose (2 μM/mL) of small molecules and combination treatment induced differentiation from the initial MSC, leading to the expression of pacemaker-like cells.

Keywords: Mesenchymal stem cells nodal inhibitor pacemaker-like cells TBX3 transfection

References

  1. Lam CW. Permanent cardiac pacemaker: an emergency perspective. Hong Kong j.emerg.med. 2001;8:169-175.
  2. Lopes JP, Fiarresga A, PS Cunha, Feliciano J, Ferreira RC. Mesenchymal stem cell therapy in heart disease. Rev Port Cardiol. 2013;32(1):43-47.
  3. Yokokawa M, Ohnishi S, Ishibashi-Ueda H, Obata H, Otani K, Miyahara Y, et al. Transplantation of mesenchymal stem cells improves atrioventricular conduction in a rat model of complete atrioventricular block. Cell Transplant. 2008;17(10-11):1145-1155.
  4. Schneider S, Unger M, van Griensven M, Balmayor ER. Adipose-derived mesenchymal stem cells from liposuction and resected fat are feasible sources for regenerative medicine. Eur J Med Res. 2017;22(1):17.
  5. Mueller P, Wolfien M, Ekat K, Lang CI, Koczan D, Wolkenhauer O, et al. RNA-based strategies for cardiac reprogramming of human mesenchymal stromal cells. Cells. 2020;9(2):504.
  6. Yechikov S, Kao HKJ, Chang CW, Pretto D, Zhang XD, Sun YH, et al. NODAL inhibition promotes differentiation of pacemaker-like cardiomyocytes from human induced pluripotent stem cells. Stem Cell Res. 2020;49:102043.
  7. Singh R, Hoogaars WM, Barnett P, Grieskamp T, Rana MS, Buermans H, et al. Tbx2 and Tbx3 induce atrioventricular myocardial development and endocardial cushion formation. Cell Mol Life Sci. 2012;69(8):1377-1389.
  8. Mommersteeg MT, Hoogaars WM, Prall OW, de Gier-de Vries C, Wiese C, Clout DE, et al. Molecular pathway for the localized formation of the sinoatrial node. Circ Res. 2007;100(3):354-362.
  9. Hoogaars WMH, Engel A, Brons JF, Verkerk AO, de Lange FJ, Wong LY, et al. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev. 2007;21(9):1098-1112.
  10. Khan SF, Damerell V, Omar R, Du Toit M, Khan M, Maranyane HM, et al. The roles and regulation of TBX3 in development and disease. Gene. 2020;726:144223.
  11. Yan Y, Liu F, Dang X, Zhou R, Liao B. TBX3 induces biased differentiation of human induced pluripotent stem cells into cardiac pacemaker-like cells. Gene Expr Patterns. 2021;40:119184.
  12. Bakker ML, Boukens BJ, Mommersteeg MTM, Brons JF, Wakker V, Moorman AF, et al. Transcription factor Tbx3 is required for the specification of the atrioventricular conduction system. Circ Res. 2008;102(11):1340-1349.
  13. Whitman M. Nodal signaling in early vertebrate embryos: themes and variations. Dev Cell. 2001;1(5):605-617.
  14. Shen MM. Nodal signaling: developmental roles and regulation. Development. 2007;134(6):1023-1034.
  15. Perea-Gomez A, Vella FD, Shawlot W, Oulad-Abdelghani M, Chazaud C, Meno C, et al. Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks. Dev Cell. 2002;3(5):745-756.
  16. Logan M, Pagán-Westphal SM, Smith DM, Paganessi L, Tabin CJ. The transcription factor pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. Cell. 1998;94(3):307-317.
  17. Liu C, Liu W, Palie J, Lu MF, Brown NA, Martin JF. Pitx2c patterns anterior myocardium and aortic arch vessels and is required for local cell movement into atrioventricular cushions. Development. 2002;129(21):5081-5091.
  18. Ammirabile G, Tessari A, Pignataro V, Szumska D, Sutera Sardo F, Benes J Jr, et al. Pitx2 confers left morphological, molecular, and functional identity to the sinus venosus myocardium. Cardiovasc Res. 2012;93(2):291-301.
  19. Hu W, Xin Y, Zhao Y, Hu J. Shox2: The role in differentiation and development of cardiac conduction system. Tohoku J Exp Med. 2018;244(3):177-186.
  20. Ye W, Wang J, Song Y, Yu D, Sun C, Liu C, et al. A common Shox2–nkx2-5 antagonistic mechanism primes the pacemaker cell fate in the pulmonary vein myocardium and sinoatrial node. Dev. 2015;142(14):2521-2532.
  21. Pawitan JA, Liem IK, Suryani D, Bustami A, Purwoko RY. Simple lipoaspirate washing using a coffee filter. Asian Biomed. 2013;7(3):333-338.
  22. Bakker ML, Boink GJ, Boukens BJ, Verkerk AO, van den Boogaard M, den Haan AD, et al. T-box transcription factor TBX3 reprogrammes mature cardiac myocytes into pacemaker-like cells. Cardiovascular Research. 2012;94:439-449.
  23. Raghunathan S, Islas JF, Mistretta B, Iyer D, Shi L, Gunaratne PH, et al. Conversion of human cardiac progenitor cells into cardiac pacemaker-like cells. Journal of Molecular and Cellular Cardiology. 2020;138:1222.
  24. Mangoni ME, Nargeot J. Genesis and regulation of the heart automaticity. Physiological Review. 2008;88:919-982.
  25. Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovascular Research. 2000;47:658-687.
  26. Zhao L, Yang G, Zhao X. Rho-associated protein kinases play an important role in the differentiation of rat adipose-derived stromal cells into cardiomyocytes in vitro. PLoS One. 2014;9(12):e115191.
  27. Vedantham, V. New approaches to biological pacemakers: links to sinoatrial node development. Trends in Molecular Medicine. 2015;21:749-761.
  28. Li Q, Kang C. Mechanisms of Action for small molecules revealed by structural biology in drug discovery. Int J Mol Sci. 2020;21:5262.
  29. Choudhury M, Boyett MR, Morris GM. Biology of the sinus node and its disease. Arrhythmia Electrophysiol Review. 2015;4:28-34.
  30. Clauss S, Kääb S. Is Pitx2 growing up?. Circ Cardiovasc Genet. 2011;4:105-107.
  31. Yin YQ, Zhong Y, Zhu Y, Tian L. Changes in gap junction proteins Connexin30.2 and Connexin40 expression in the sinoatrial node of rats with dexmedetomidine-induced sinus bradycardia. Braz J Anesthesiol. 2022;72(6):768-773.
  32. Liang X, Zhang Q, Cattaneo P, Zhuang S, Gong X, Spann NJ, et al. Transcription factor ISL1 is essential for pacemaker development and function. The Journal of Clinical Investigation. 2015;125:3256-3268.
  33. Zhang XD, Thai PN, Lieu DK, Chiamvimonvat N. Cardiac small-conductance calcium-activated potassium channels in health and disease. Pflugers Archiv: European Journal of Physiology. 2021;473:477-489.
  34. Kleger A, Seufferlein T, Malan D, Tischendorf M, Storch A, Wolheim A, et al. Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells. Circulation. 2010;122:1823-1836.

How to Cite

Faris Basalamah, F., Ismail Hadisoebroto Dilogo, Sunu Budhi Raharjo, Muchtaruddin Mansyur, Nuryati Chairani Siregar, Nurhadi Ibrahim, Budi Yuli Setianto, & Yoga Yuniadi. (2023). The optimized method for mesenchymal stem cell differentiation into pacemaker-like cell. Bali Medical Journal, 12(2), 2289–2297. https://doi.org/10.15562/bmj.v12i2.4475
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Faris Faris Basalamah
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Ismail Hadisoebroto Dilogo
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Sunu Budhi Raharjo
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Muchtaruddin Mansyur
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Nuryati Chairani Siregar
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Nurhadi Ibrahim
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Budi Yuli Setianto
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Yoga Yuniadi
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