Sains Malaysiana 50(7)(2021): 1987-1996

http://doi.org/10.17576/jsm-2021-5007-13

 

The Combination of bFGF and Hydrocortisone is a Better Alternative Compared to 5-Azacytidine for Cardiomyogenic Differentiation of Bone Marrow and Adipose Stem Cells

(Gabungan bFGF dan Hidrokortison adalah Alternatif yang Lebih Baik Berbanding dengan 5-Azasitidin bagi Perbezaan Kardomiogen Sumsum Tulang dan Sel Stem Adipos)

 

NADIAH SULAIMAN1*, NUR QISYA AFIFAH VERONICA SAINIK1,2, SHAMSUL BIN SULAIMAN1, PEZHMAN HAFEZ1, NG MIN HWEI1 & RUSZYMAH BT HJ IDRUS1,2

 

1Tissue Engineering Centre, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

 

2Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

 

Diserahkan: 27 April 2020/Diterima: 13 November 2020

 

ABSTRACT

Stem cells can be differentiated into cardiomyocytes by induction with 5-azacytidine (5-aza) but its carcinogenicity is of concern for future translational application. Alternatively, growth factors and hormones such as basic fibroblast growth factor (bFGF) and hydrocortisone have been reported to act as a therapeutic inducer for cardiomyocytes differentiation. In this study, we aim to investigate the ability of bFGF and hydrocortisone in combination to stimulate the differentiation of mesenchymal stem cells (MSC) into cardiomyocytes lineage. Sheep adipose tissue stem cell (ATSC) and bone marrow stem cell (BMSC) were isolated, cultured and induced with the three groups of induction factors; 5-aza alone, the combination of hydrocortisone and bFGF and all three factors in combination for cardiomyogenic differentiation. Morphological, protein and functional ability of both ATSC and BMSC were observed and analysed to confirm cardiomyocyte differentiation. Viability of BMSC and ATSC in each treated group was significantly higher (P < 0.05) on both cells after treated with 10 nM of bFGF and 50 μM of hydrocortisone. Cardiomyocyte proteins; α-Sarcomeric actin (αSA) and Phospolamban (Plb) was detected in both ATSC and BMSC exposed to induction factors but not in the control negative group. Both ATSC and BMSC without induction factors showed only minute cell number possesses αSA and Plb. Calcium ion (Ca2+) spark was observed in primary heart cells. Similarly, Ca2+ spark was also detected in induced ATSC and BMSC, proving some functionality of induced cells. In conclusion, bFGF and hydrocortisone are safer induction factor compared to the currently used 5-aza as both showed higher viability after induction, therefore more cells are available for future use in cardiac tissue engineering.

 

Keywords: 5-Azacytidine; basic Fibroblast Growth Factor; cardiomyocytes differentiation; hydrocortisone; stem cells

 

ABSTRAK

Sel induk boleh dibezakan menjadi kardiomiosit dengan aruhan 5-azasitidin (5-aza) tetapi sifat karsinogeniknya menimbulkan kerisauan bagi kegunaan klinikal pada masa hadapan. Sebagai alternatif, faktor pertumbuhan dan pelbagai jenis hormon seperti faktor pertumbuhan fibroblas asas (bFGF) dan hidrokortison dilaporkan boleh bertindak sebagai pemacu terapi untuk pembezaan kardiomiosit. Kajian ini bertujuan untuk mengkaji kemampuan bFGF dan hidrokortison secara gabungan untuk merangsang pembezaan MSC kepada leluhur kardiomiosit. Sel dasar lemak (ATSC) dan tulang sum-sum kambing (BMSC) diasingkan, dikultur dan diaruh dengan tiga kumpulan faktor aruhan; 5-aza sahaja, gabungan hidrokortison dan bFGF dan ketiga-tiga faktor gabungan untuk pembezaan kardiomogenik. Perubahan morfologi, protein dan fungsi kedua-dua ATSC dan BMSC dikaji dan dianalisis untuk mengesahkan pembezaan leluhur kardiomiosit. Perkembangan ATSC dan BMSC pada setiap kumpulan yang dirawat jauh lebih tinggi (P <0.05) pada kedua-dua sel setelah dirawat dengan 10 nM bFGF dan 50 μM hidrokortison. Protein kardiomiosit, α-Sarcomeric actin (αSA) dan Phospolamban (Plb) dikesan pada kedua-dua ATSC dan BMSC yang terdedah kepada faktor aruhan tetapi tidak dalam kawalan negatif. Kedua-dua sel, ATSC dan BMSC tanpa faktor aruhan menunjukkan hanya sebilangan kecil sel mempunyai αSA dan Plb. Percikan ion kalsium (Ca2 +) diperhatikan pada sel jantung primer, yang turut dikesan pada ATSC dan BMSC yang diinduksi. Maka, sel yang diinduksi sedikit sebanyak berfungsi seperti kardiomiosit. Kesimpulannya, bFGF dan hidrokortison adalah faktor aruhan yang lebih selamat berbanding dengan 5-Aza yang digunakan sekarang. Hal ini demikian adalah kerana kedua-duanya menunjukkan perkembangan yang lebih tinggi selepas aruhan, oleh itu lebih banyak sel tersedia untuk kegunaan pada peringkat klinikal.

 

Kata kunci: 5-azasitidin; faktor tumbesaran fibroblas asas; hidrokortison; pembezaan kardiomiosit; sel dasar

 

RUJUKAN

Akin, B.L., Hurley, T.D., Chen, Z. & Jones, L.R. 2013. The structural basis for phospholamban inhibition of the calcium pump in sarcoplasmic reticulum. Journal of Biological Chemistry 288(42): 30181-30191.

Bird, S.D., Doevendans, P. A., van Rooijen, M.A., de la Riviere, A.B., Hassink, R.J., Passier, R. & Mummery, C.L. 2003. The human adult cardiomyocyte phenotype. Cardiovascular Research 58(2): 423-434.

Brade, T., Pane, L.S., Moretti, A., Chien, K.R. & Laugwitz, K.L. 2013. Embryonic heart progenitors and cardiogenesis. Cold Spring Harbor Perspectives in Medicine 3(10): a013847. 

Choi, Y.S., Dusting, G.J., Stubbs, S., Arunothayaraj, S., Han, X.L., Collas, P., Morrison, W.A. & Dilley, R.J. 2010. Differentiation of human adipose-derived stem cells into beating cardiomyocytes. Journal of Cellular and Molecular Medicine 14(4): 878-889.

Clifford, D.M., Fisher, S.A., Brunskill, S.J., Doree, C., Mathur, A., Watt, S. & Martin-Rendon, E. 2012. Stem cell treatment for acute myocardial infarction. Cochrane Database of Systematic Reviews (2): CD006536.

Colenci, R., da Silva Assunção, L.R., Bomfim, S.R.M., de Assis Golim, M., Deffune, E. & Oliveira, S.H.P. 2014. Bone marrow mesenchymal stem cells stimulated by bFGF up-regulated protein expression in comparison with periodontal fibroblasts in vitro. Archives of Oral Biology 59(3): 268-276. 

Giugliano, G.R., Giugliano, R.P., Michael Gibson, C. & Kuntz, R.E. 2003. Meta-analysis of corticosteroid treatment in acute myocardial infarction. American Journal of Cardiology 91(9): 1055-1059.

Guo, X., Bai, Y., Zhang, L., Zhang, B., Zagidullin, N., Carvalho, K., Du, Z. & Cai, B. 2018. Cardiomyocyte differentiation of mesenchymal stem cells from bone marrow: New regulators and its implications. Stem Cell Research and Therapy 9(1): 44.

Kaptoge, S., Pennells, L., De Bacquer, D., Cooney, M.T., Kavousi, M., Stevens, G.,  Riley, L.M., Savin, S., Khan, T.,  Altay, S., Amouyel, P., Assmann, G., Bell, S.,  Ben-Shlomo, Y., Berkman, L., Beulens, J.W., Björkelund, C., Blaha, M., Blazer, D.G.,  Bolton, T., Beaglehole, R.B., Brenner, H., Brunner, E.J., Casiglia, E., Chamnan, P., Choi, Y-H., Chowdry, R., Coady, S., Crespo, C.J., Cushman, M., Dagenais, G.R., D'Agostino Sr., R.B., Daimon, M., Davidson, K.W., Engström, G., Ford, I.,  Gallacher, J., Gansevoort, R.T., Gaziano, T.A., Giampaoli, S., Grandits, G., Grimsgaard, S., Grobbee, D.E., Gudnason, V.,  Guo, Q.,  Tolonen, H., Humphries, S., Iso, H., Jukema, J.W., Kauhanen, J., Kengne, A.P., Khalili, D., Koenig, W., Kromhout, D., Krumholz, H., Lam, T.H., Laughlin, G., Ibañez, A.M., Meade, T.W., Moons, K.G.M., Nietert, P.J., Ninomiya, T., Nordestgaard, B.G., O'Donnell, C., Palmieri, L., Patel, A., Perel, P., Price, J.F., Providencia, R., Ridker, P.M.,  Rodriguez, B., Rosengren, A., Roussel, R., Sakurai, M., Salomaa, V., Sato, S.,  Schöttker, B., Shara, N., Shaw, J.E., Shin, H-C., Simons, L.A., Sofianopoulou, E., Sundström, J., Völzke, H., Wallace, R.B., Wareham, N.J., Willeit, P., Wood, D., Wood, A., Zhao, D., Woodward, M., Danaei, G., Roth, G., Mendis, S., Onuma, O., Varghese, C., Ezzati, M.,  Graham, I., Jackson, R., Danesh, J., Angelantonio, E.D. 2019. World Health Organization cardiovascular disease risk charts: Revised models to estimate risk in 21 global regions. The Lancet Global Health 7(10): e1332-e1345.

Kawai, T., Takahashi, T., Esaki, M., Ushikoshi, H., Nagano, S., Fujiwara, H. & Kosai, K.I. 2004. Efficient cardiomyogenic differentiation of embryonic stem cell by fibroblast growth factor 2 and bone morphogenetic protein 2. Circulation Journal 68(7): 691-702.

Kelecsényi, Z., Spencer, D.L. & Caspary, W.J. 2000. Molecular analysis of 5-azacytidine-induced variants in mammalian cells. Mutagenesis 15(1): 25-31.

Knot, H.J., Laher, I., Sobie, E.A., Guatimosim, S., Gomez-Viquez, L., Hartmann, H., Song, L.S., Lederer, W.J., Graier, W.F., Malli, R., Frieden, M. & Petersen, O.H. 2005. Twenty years of calcium imaging: Cell physiology to dye for. Molecular Interventions 5(2): 112-127.

Kuhn, N.Z. & Tuan, R.S. 2010. Regulation of stemness and stem cell niche of mesenchymal stem cells: Implications in tumorigenesis and metastasis. Journal of Cellular Physiology 222(2): 268-277. 

Leon, B.M. 2015. Diabetes and cardiovascular disease: Epidemiology, biological mechanisms, treatment recommendations and future research. World Journal of Diabetes 6(13): 1246-1258.

Li, K., Li, S.Z., Zhang, Y.L. & Wang, X.Z. 2011. The effects of dan-shen root on cardiomyogenic differentiation of human placenta-derived mesenchymal stem cells. Biochemical and Biophysical Research Communications 415(1): 147-151.

Li, W., Wu, D., Niu, Z., Jiang, D., Ma, H., He, H., Zuo, X., Xie, X. & He, Y. 2016. 5-azacytidine suppresses EC9706 cell proliferation and metastasis by upregulating the expression of SOX17 and CDH1. International Journal of Molecular Medicine 38(4): 1047-1054.

Loughrey, C.M., MacEachern, K.E., Neary, P. & Smith, G.L. 2002. The relationship between intracellular [Ca(2+)] and Ca(2+) wave characteristics in permeabilised cardiomyocytes from the rabbit. The Journal of Physiology 543(3): 859-870.

Ludman, P.F. 2018. Percutaneous coronary intervention. Medicine (United Kingdom) 46(9): 547-554. 

MacLennan, D.H. & Kranias, E.G. 2003. Phospholamban: A crucial regulator of cardiac contractility. Nature Reviews Molecular Cell Biology 4: 566-577.

Marques, S.R., Lee, Y., Poss, K.D. & Yelon, D. 2008. Reiterative roles for FGF signaling in the establishment of size and proportion of the zebrafish heart. Developmental Biology  321(2): 397-406.

Ogawa, R., Akita, S., Akaishi, S., Aramaki-Hattori, N., Dohi, T., Hayashi, T., Kishi, K., Kono, T., Matsumura, H., Muneuchi, G., Murao, N., Nagao, M., Okabe, K., Shimizu, F., Tosa, M., Tosa, Y., Yamawaki, S., Ansai, S., Inazu, N., Kamo, T., Kazki, R. & Kuribayashi, S. 2019. Diagnosis and treatment of keloids and hypertrophic scars - Japan scar workshop consensus document 2018. Burns and Trauma 7: 39.

Olesen, C., Picard, M., Winther, A.M.L., Gyrup, C., Preben Morth, J., Oxvig, C., Vuust Møller, J. & Nissen, P. 2007. The structural basis of calcium transport by the calcium pump. Nature 450: 1036-1042.

Peter, A.K., Bjerke, M.A. & Leinwand, L.A. 2016. Biology of the cardiac myocyte in heart disease. Molecular Biology of the Cell 27(14): 2149-2160.

Pittenger, M.F. & Martin, B.J. 2004. Mesenchymal stem cells and their potential as cardiac therapeutics. Circulation Research 95(1): 9-20.

Segers, V.F.M. & Lee, R.T. 2008. Stem-cell therapy for cardiac disease. Nature 451: 937-942.

Shipunova, N.N., Petinati, N.A. & Drize, N.I. 2013. Effect of hydrocortisone on multipotent human mesenchymal stromal cells. Bulletin of Experimental Biology and Medicine 155(1): 159-163.

Steinhauser, M.L. & Lee, R.T. 2011. Regeneration of the heart. EMBO Molecular Medicine 3(12): 701-712.

Stern-Straeter, J., Bonaterra, G.A., Juritz, S., Birk, R., Goessler, U.R., Bieback, K., Bugert, P., Schultz, J., Hörmann, K., Kinscherf, R. & Faber, A. 2014. Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells. International Journal of Molecular Medicine 33(1): 160-170.

Yin, Z., Ren, J. & Guo, W. 2015. Sarcomeric protein isoform transitions in cardiac muscle: A journey to heart failure. Biochimica et Biophysica Acta - Molecular Basis of Disease 1852(1): 47-52.

Yoon, Y.S., Park, J.S., Tkebuchava, T., Luedeman, C. & Losordo, D.W. 2004. Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction. Circulation 109(25): 3154-3157.

Zhang, Y., Mignone, J. & Robb Maclellan, W. 2015. Cardiac regeneration and stem cells. Physiological Reviews 95(4): 1189-1204,

Zhao, Y., Li, T., Wei, X., Bianchi, G., Hu, J., Sanchez, P.G., Xu, K., Zhang, P., Pittenger, M.F., Wu, Z.J. & Griffith, B.P. 2012. Mesenchymal stem cell transplantation improves regional cardiac remodeling following ovine infarction. STEM CELLS Translational Medicine 1(9): 685-695.

 

*Pengarang untuk surat-menyurat; email: nadiahsulaiman@ukm.edu.my

 

 

sebelumnya