Tbx18-dependent differentiation of brown adipose tissue-derived stem cells toward cardiac pacemaker cells

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A cell-sourced biological pacemaker is a promising therapeutic approach for sick sinus syndrome (SSS) or severe atrial ventricular block (AVB). Adipose tissue-derived stem cells (ATSCs), which are optimal candidate cells for possible use in regenerative therapy for acute or chronic myocardial injury, have the potential to differentiate into spontaneous beating cardiomyocytes. However, the pacemaker characteristics of the beating cells need to be confirmed, and little is known about the underlying differential mechanism. In this study, we found that brown adipose tissue-derived stem cells (BATSCs) in mice could differentiate into spontaneous beating cells in 15% FBS Dulbecco’s modified Eagle’s medium (DMEM) without additional treatment. Subsequently, we provide additional evidence, including data regarding ultrastructure, protein expression, electrophysiology, and pharmacology, to support the differentiation of BATSCs into a cardiac pacemaker phenotype during the course of early cultivation. Furthermore, we found that silencing Tbx18, a key transcription factor in the development of pacemaker cells, terminated the differentiation of BATSCs into a pacemaker phenotype, suggesting that Tbx18 is required to direct BATSCs toward a cardiac pacemaker fate. The expression of Tbx3 and shox2, the other two important transcription factors in the development of pacemaker cells, was decreased by silencing Tbx18, which suggests that Tbx18 mediates the differentiation of BATSCs into a pacemaker phenotype via these two downstream transcription factors.

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  1. 1.

    Rosen MR, Robinson RB, Brink PR, Cohen IS (2011) The road to biological pacing. Nat Rev Cardiol 8:656–666

  2. 2.

    Bakker ML, Boink GJ, Boukens BJ, Verkerk AO, van den Boogaard M, den Haan AD, Hoogaars WM, Buermans HP, de Bakker JM, Seppen J, Tan HL, Moorman AF, ‘t Hoen PA, Christoffels VM (2012) T-box transcription factor TBX3 reprogrammes mature cardiac myocytes into pacemaker-like cells. Cardiovasc Res 94:439–449

  3. 3.

    Kapoor N, Liang W, Marban E, Cho HC (2013) Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18. Nat Biotechnol 31:54–62

  4. 4.

    Scavone A, Capilupo D, Mazzocchi N, Crespi A, Zoia S, Campostrini G, Bucchi A, Milanesi R, Baruscotti M, Benedetti S, Antonini S, Messina G, DiFrancesco D, Barbuti A (2013) Embryonic stem cell-derived CD166 + precursors develop into fully functional sinoatrial-like cells. Circ Res 113:389–398

  5. 5.

    Kleger A, Seufferlein T, Malan D, Tischendorf M, Storch A, Wolheim A, Latz S, Protze S, Porzner M, Proepper C, Brunner C, Katz SF, Varma Pusapati G, Bullinger L, Franz WM, Koehntop R, Giehl K, Spyrantis A, Wittekindt O, Lin Q, Zenke M, Fleischmann BK, Wartenberg M, Wobus AM, Boeckers TM, Liebau S (2010) Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells. Circulation 122:1823–1836

  6. 6.

    Amin S, Banijamali SE, Tafazoli-Shadpour M, Shokrgozar MA, Dehghan MM, Haghighipour N, Mahdian R, Bayati V, Abbasnia P (2014) Comparing the effect of equiaxial cyclic mechanical stimulation on GATA4 expression in adipose-derived and bone marrow-derived mesenchymal stem cells. Cell Biol Int 38:219–227

  7. 7.

    Rangappa S, Fen C, Lee EH, Bongso A, Sim EK (2003) Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes. Ann Thorac Surg 75: 775–779

  8. 8.

    Planat-Bénard V, Menard C, André M, Puceat M, Perez A, Garcia-Verdugo JM, Pénicaud L, Casteilla L (2004) Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res 94:223–229

  9. 9.

    Yamada Y, Wang X, Yokoyama S, Fukuda N, Takakura N (2006) Cardiac progenitor cells in brown adipose tissue repaired damaged myocardium. Biochem Biophys Res Commun 342:662–670

  10. 10.

    Chang W, Lim S, Song BW, Lee CY, Park MS, Chung YA, Yoon C, Lee SY, Ham O, Park JH, Choi E, Maeng LS, Hwang KC (2012) Phorbol myristate acetate differentiates human adipose-derived mesenchymal stem cells into functional cardiogenic cells. Biochem Biophys Res Commun 424:740–746

  11. 11.

    Jin MS, Shi S, Zhang Y, Yan Y, Sun XD, Liu W, Liu HW (2010) Icariin-mediated differentiation of mouse adipose-derived stem cells into cardiomyocytes. Mol Cell Biochem 344:1–9

  12. 12.

    Choi YS, Dusting GJ, Stubbs S, Arunothayaraj S, Han XL, Collas P, Morrison WA, Dilley RJ (2010) Differentiation of human adipose-derived stem cells into beating cardiomyocytes. J Cell Mol Med 14:878–889

  13. 13.

    Lee WC, Sepulveda JL, Rubin JP, Marra KG (2009) Cardiomyogenic differentiation potential of human adipose precursor cells. Int J Cardiol 133:399–401

  14. 14.

    Song YH, Gehmert S, Sadat S, Pinkernell K, Bai X, Matthias N, Alt E (2007) VEGF is critical for spontaneous differentiation of stem cells into cardiomyocytes. Biochem Biophys Res Commun 354:999–1003

  15. 15.

    Takahashi T, Nagai T, Kanda M, Liu ML, Kondo N, Naito AT, Ogura T, Nakaya H, Lee JK, Komuro I, Kobayashi Y (2015) Regeneration of the cardiac conduction system by adipose tissue-derived stem cells. Circ J 79:2703–2712

  16. 16.

    Bunnell BA, Flaat M, Gagliardi C, Patel B, Ripoll C (2008) Adipose-derived stem cells: isolation, expansion and differentiation. Methods 45:115–120

  17. 17.

    Savage J, Conley AJ, Blais A, Skerjanc IS (2009) SOX15 and SOX7 differentially regulate the myogenic program in P19 cells. Stem Cells 27:1231–1243

  18. 18.

    Cypess AM, Lehman S, Williams G, Tal I, Rodman D, Goldfine AB, Kuo FC, Palmer EL, Tseng YH, Doria A, Kolodny GM, Kahn CR (2009) Identification and importance of brown adipose tissue in adult humans. N Engl J Med 360:1509–1517

  19. 19.

    Virtanen KA, Lidell ME, Orava J, Heglind M, Westergren R, Niemi T, Taittonen M, Laine J, Savisto NJ, Enerbäck S, Nuutila P (2009) Functional brown adipose tissue in healthy adults. N Engl J Med 360:1518–1525

  20. 20.

    Cinti S (2011) Between brown and white: novel aspects of adipocyte differentiation. Ann Med 43:104–115

  21. 21.

    Jumabay M, Zhang R, Yao Y, Goldhaber JI, Bostrom KI (2010) Spontaneously beating cardiomyocytes derived from white mature adipocytes. Cardiovasc Res 85:17–27

  22. 22.

    Palpant NJ, Yasuda S, MacDougald O, Metzger JM (2007) Non-canonical Wnt signaling enhances differentiation of Sca1+/c-kit + adipose-derived murine stromal vascular cells into spontaneously beating cardiac myocytes. J Mol Cell Cardiol 43:362–370

  23. 23.

    Tholpady SS, Llull R, Ogle RC, Rubin JP, Futrell JW, Katz AJ (2006) Adipose tissue: stem cells and beyond. Clin Plast Surg 33:55–62

  24. 24.

    Dromard C, Barreau C, André M, Berger-Müller S, Casteilla L, Planat-Benard V (2014) Mouse adipose tissue stromal cells give rise to skeletal and cardiomyogenic cell sub-populations. Front Cell Dev Biol 2:42

  25. 25.

    Di Rocco G, Iachininoto MG, Tritarelli A, Straino S, Zacheo A, Germani A, Crea F, Capogrossi MC (2006) Myogenic potential of adipose-tissue-derived cells. J Cell Sci 119:2945–2952

  26. 26.

    Huang SJ, Fu RH, Shyu WC, Liu SP, Jong GP, Chiu YW, Wu HS, Tsou YA, Cheng CW, Lin SZ (2013) Adipose-derived stem cells: isolation, characterization, and differentiation potential. Cell Transplant 22:701–709

  27. 27.

    Wiese C, Nikolova T, Zahanich I, Sulzbacher S, Fuchs J, Yamanaka S, Graf E, Ravens U, Boheler KR, Wobus AM (2011) Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin. Int J Cardiol 147:95–111

  28. 28.

    Christoffels VM, Smits GJ, Kispert A, Moorman AF (2010) Development of the pacemaker tissues of the heart. Circ Res 106:240–254

  29. 29.

    Hoogaars WM, Barnett P, Moorman AF, Christoffels VM (2007) T-box factors determine cardiac design. Cell Mol Life Sci 64:646–660

  30. 30.

    Wiese C, Grieskamp T, Airik R, Mommersteeg MT, Gardiwal A, de Gier-de Vries C, Schuster-Gossler K, Moorman AF, Kispert A, Christoffels VM (2009) Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3. Circ Res 104:388–397

  31. 31.

    Yang M, Zhang GG, Wang T, Wang X, Tang YH, Huang H, Barajas-Martinez H, Hu D, Huang CX (2016) TBX18 gene induces adipose-derived stem cells to differentiate into pacemaker-like cells in the myocardial microenvironment. Int J Mol Med 38:1403–1410

  32. 32.

    Hoogaars WM, Engel A, Brons JF, Verkerk AO, de Lange FJ, Wong LY, Bakker ML, Clout DE, Wakker V, Barnett P, Ravesloot JH, Moorman AF, Verheijck EE, Christoffels VM (2007) Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev 21:1098–1112

  33. 33.

    Blaschke RJ, Hahurij ND, Kuijper S, Just S, Wisse LJ, Deissler K, Maxelon T, Anastassiadis K, Spitzer J, Hardt SE, Schöler H, Feitsma H, Rottbauer W, Blum M, Meijlink F, Rappold G, Gittenberger-de Groot AC (2007) Targeted mutation reveals essential functions of the homeodomain transcription factor Shox2 in sinoatrial and pacemaking development. Circulation 115:1830–1838

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This work was supported by grants from the Natural Science Foundation of China (31170934, 31271050, and 81271717).

Author information

Correspondence to Yu-Quan Li or Xiang-Qun Yang.

Additional information

Lei Chen and Zi-Jun Deng have contributed equally to this work.

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Supplement Fig. 1 The expression of markers at transcriptional levels in ATSCs, qRT-PCR was used to detected the gene expression of LPL, Leptin, PPAR γ2(a); Collagen II, Aggrecan, Sox9(b); OP, OC, Runx2(c) after 2-week induced. Expression levels were normalized to α-actin. Error bars represent by ± SD. One-tailed Student’s t-tests were used as appropriate to evaluate the statistical significance of differences between two groups.*P <0.05 vs WATSCs, #P <0.05 vs BATSCs. (TIF 4034 KB)

Supplementary Fig. 2 Western blot analysis of Tbx18, Tbx3, sarcomeric α-actin (Sr) and HCN4 expression in shTbx18-treated cells transfected with Tbx18. (a) Western blot analysis of Tbx18, Tbx3, Sr and HCN4 protein expression; (b) quantitative assessment of Tbx18, Tbx3, Sr and HCN4 protein levels using integrated optical density analyses. *P <0.01 compared to the corresponding values for the shTbx18 groups. #P <0.01 compared to the corresponding values for the untreated groups. (TIF 6712 KB)

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Chen, L., Deng, Z., Zhou, J. et al. Tbx18-dependent differentiation of brown adipose tissue-derived stem cells toward cardiac pacemaker cells. Mol Cell Biochem 433, 61–77 (2017).

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  • Adipose tissue-derived stem cells
  • Brown adipose tissue
  • Pacemaker phenotype
  • Differentiation
  • Tbx18