Mezenkimal Kök Hücrelerin Kardiyomiyositlere Farklılaştırılmasında Kimyasal ve Biyolojik İndükleyicilerin Kombine Etkisinin Değerlendirilmesi

Abstract

Mezenkimal kök hücrelerden (MKH) elde edilen kardiyomiyositler, biyomedikal uygulamalar için önemli bir potansiyele sahiptir. Ancak, mevcut protokoller ile MKH'leriden yeterli sayıda olgun ve fonksiyonel kardiyomiyosit üretilememiştir. Bu çalışmada, sıçan kemik iliği kök hücrelerinin (rt-MKH'ler) kardiyomiyositlere trans-diferansiyasyonunu artırmada biyolojik ve kimyasal indükleyicilerin kombine etkisi araştırılmıştır. rt-MKH'ler, 5-Azasitidin (5-Aza), Dimetil sülfoksit (Me2SO) ve biyolojik molekül kokteyline (TGF-β1, BMP-2 ve FGF-2, topluca DM olarak adlandırılmıştır) tek başına veya kombinasyon halinde 14 gün boyunca maruz bırakılmıştır. Kardiyak transkripsiyon faktörleri ve kardiyak spesifik proteinler sırasıyla qPCR ve immünofloresan boyama yöntemleriyle analiz edilmiştir. Kardiyomiyosit oranı ise akış sitometri yöntemiyle değerlendirilmiştir. qPCR analizi, 5-Aza+Me2SO grubunda 7. günde GATA-4 ve NKX2.5 ekspresyon seviyelerinin sırasıyla 19 kat ve 8 kat arttığını göstermiştir (p<0,001). Kültür süresi 14 güne uzatıldığında, kardiyak transkripsiyon faktörlerinin ekspresyon seviyeleri 7. güne kıyasla azalmış olup, en yüksek GATA-4 ve NKX2.5 seviyeleri Me2SO grubunda gözlemlenmiştir. Özellikle, Me2SO grubundaki GATA-4 ekspresyonu, 5-Aza grubuna kıyasla 11 kat daha yüksek bulunmuştur (p<0,05). İmmünofloresan boyama, 5-Aza ve Me2SO ile muamele edilen rt-MKH'lerin, kontrol grubuna kıyasla yüksek seviyelerde cTnT, SAC, Cnx43 ve α-SMA eksprese ettiğini doğrulamıştır. Akış sitometri analizi ise, 5-Aza+Me2SO grubundaki indüklenmiş hücrelerin %42’sinin kardiyomiyosit olduğunu ortaya koymuştur. Sonuç olarak, kimyasal indükleyicilerin (5-Aza ve Me2SO) birlikte kullanımı, kimyasal ve biyolojik indükleyicilerin kombinasyonuna kıyasla rt-MKH’lerin kardiyomiyositlere farklılaşmasını teşvik etmede daha etkili olduğu tespit edilmiştir.

Keywords

• Mezenkimal Kök Hücre
• Kardiyomiyojenik Farklılaşma
• 5-Azasitidin
• Dimetil Sulfoksit

Assessment of Combine Effect of Chemical and Biological Inducers to Differentiate Mesenchymal Stem Cells into Cardiomyocyte

Abstract

Cardiomyocytes derived from mesenchymal stem cells (MSCs) hold significant potential for biomedical applications. However, existing protocols for differentiating MSCs into cardiomyocytes have not successfully generated a large number of mature, functional cells. This study aims to investigate the combined effects of biological and chemical inducers to enhance the trans-differentiation of rat bone marrow stem cells (rt-BMSCs) into cardiomyocytes. rt-BMSCs were treated with 5-Azacytidine (5-Aza), Dimethyl Sulfoxide (Me2SO), and biological molecule coctail (TGF-β1, BMP-2, and FGF-2, collectively referred to as DM) either individually or in combination for 14 days. Cardiac transcription factors and cardiac-specific proteins were analyzed using qPCR and immunofluorescence staining, respectively. The cardiomyocyte ratio was assessed by flow cytometry. qPCR analysis revealed that, after 7 days, GATA-4 and NKX2.5 expression levels increased 19-fold and 8-fold, respectively, in the 5-Aza+Me2SO group compared to the 5-Aza group (p<0.001). By day 14, the expression of cardiac transcription factors declined relative to day 7, with the highest GATA-4 and NKX2.5 levels observed in the Me2SO group. Notably, GATA-4 expression in the Me2SO group was 11 times higher than in the 5-Aza group (p<0.05). Immunofluorescence staining confirmed that rt-BMSCs treated with 5-Aza and Me2SO exhibited high expression of cTnT, SAC, Cnx43, and α-SMA compared to the control group. Flow cytometry analysis further revealed that 42% of the induced cells in the 5-Aza+Me2SO group were cardiomyocytes. In conclusion, the combination of chemical inducers (5-Aza and Me2SO) was more effective in promoting cardiomyocyte differentiation from rt-BMSCs than the combined use of chemical and biological inducers.

Keywords

• Mesenchymal Stem Cells
• Cardiomyogenic Differentiation
• 5-azacytidine
• Dimethyl Sulfoxide

References

1. Laranjo L, Lanas F, Sun MC, Chen DA, Hynes L, Imran TF, et al. World Heart Federation roadmap for secondary prevention of cardiovascular disease: 2023 update. Global Heart 2024;19. doi:10.1016/j.repc.2024.04.009.
2. Scafa Udriște A, Niculescu AG, Iliuță L, Bajeu T, Georgescu A, Grumezescu AM, et al. Progress in Biomaterials for Cardiac Tissue Engineering and Regeneration. Polymers 2023;15:1177. doi:10.3390/polym15051177.
3. Guo Y, Yu Y, Hu S, Chen Y, Shen Z. The therapeutic potential of mesenchymal stem cells for cardiovascular diseases. Cell Death and Disease 2020;11. doi:10.1038/s41419-020-2542-9.
4. Pecha S, Eschenhagen T, Reichenspurner H. Myocardial tissue engineering for cardiac repair. The Journal of Heart and Lung Transplantation 2016;35:294-298. doi:10.1016/j.healun.2015.12.007.
5. Jiang Y, Park P, Hong SM, Ban K. Maturation of cardiomyocytes derived from human pluripotent stem cells: current strategies and limitations. Molecules and Cells 2018;41:613. doi:10.14348/molcells.2018.0143.
6. Sulaiman N, Sainik NQAV, Sulaiman SB, Hafez P, Min N. The combination of bFGF and hydrocortisone is a better alternative compared to 5-azacytidine for cardiomyogenic differentiation of bone marrow and adipose stem cells. Sains Malaysiana 2021;50:1987-1996. doi:10.17576/jsm-2021-5007-13.
7. Iwatani M, Ikegami K, Kremenska Y, Hattori N, Tanaka S, Yagi S, et al. Dimethyl Sulfoxide Has an Impact on Epigenetic Profile in Mouse Embryoid Body. Stem Cells 2006;24:2549-2556. doi:10.1634/stemcells.2005-0427.
8. Kang MH, You SY, Hong K, Kim JH. DMSO impairs the transcriptional program for maternal-to-embryonic transition by altering histone acetylation. Biomaterials 2020;230:119604. doi:10.1016/j.biomaterials.2019.119604.

9. Pal R, Mamidi MK, Das AK, Bhonde R. Diverse effects of dimethyl sulfoxide (DMSO) on the differentiation potential of human embryonic stem cells. Archives of Toxicology 2012;86:651-661. doi:10.1007/s00204-011-0782-2.
10. Budash HV, Bilko NM. Embryonic and Induced Pluripotent Stem Cells and Their Differentiation in the Cardiomyocyte Direction in the Presence of Dimethyl Sulfoxide. Cytology and Genetics 2019;53:34-41. doi:10.3103/s0095452719010055.
11. Schultheiss TM, Burch JB, Lassar AB. A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes & Development 1997;11:451-462. doi:10.1101/gad.11.4.451.
12. Shi S, Wu X, Wang X, Hao W, Miao H, Zhen L, et al. Differentiation of bone marrow mesenchymal stem cells to cardiomyocyte-like cells is regulated by the combined low dose treatment of transforming growth factor-β1 and 5-azacytidine. Stem Cells International 2016;2016:3816256. doi:10.1155/2016/3816256.
13. Hahn JY, Cho HJ, Kang HJ, Kim TS, Kim MH, Chung JH, et al. Pre-treatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes, and therapeutic efficacy for myocardial infarction. Journal of the American College of Cardiology 2008;51:933-943. doi:10.1016/j.jacc.2007.11.040.
14. Ramesh S, Govarthanan K, Ostrovidov S, Zhang H, Hu Q, Camci-Unal G, et al. Cardiac Differentiation of Mesenchymal Stem Cells: Impact of Biological and Chemical Inducers. Stem Cell Reviews and Reports 2021;17:1343-1361. doi:10.1007/s12015-021-10165-3.
15. Hasani S, Javeri A, Asadi A, Taha MF. Cardiac differentiation of adipose tissue-derived stem cells is driven by BMP4 and bFGF but counteracted by 5-azacytidine and valproic acid. Cell Journal (Yakhteh) 2020;22:273. doi:10.22074/cellj.2020.6582.
16. Ozturk S, Shahbazi R, Zeybek ND, Kurum B, Gultekinoglu M, Aksoy E, et al. Assessment of electromechanically stimulated bone marrow stem cells seeded acellular cardiac patch in a rat myocardial infarct model. Biomedical Materials 2021. doi:10.1088/1748-605X/ac199a.
17. Kakkar A, Nandy SB, Gupta S, Bharagava B, Airan B, Mohanty S. Adipose tissue derived mesenchymal stem cells are better respondents to TGFβ1 for in vitro generation of cardiomyocyte-like cells. Molecular and Cellular Biochemistry 2019;460:53-66. doi:10.1007/s11010-019-03570-3.
18. Wang XY, Tan YZ, Wang HJ, Zhang GT, He QZ. Effects of the Smad signaling pathway on differentiation of marrow-derived cardiac stem cells toward cardiomyocytes. Acta Anatomica Sinica 2006;37:525-529.
19. Neri T, Merico V, Fiordaliso F, Salio M, Rebuzzini P, Sacchi L, et al. The differentiation of cardiomyocytes from mouse embryonic stem cells is altered by dioxin. Toxicology Letters 2011;202:226-236. doi:10.1016/j.toxlet.2011.02.008.
20. Huang Y, Li YH, Dai ZQ, Yang F. Cardiomyogenic differentiation characteristics of adult stem cells derived from bone marrow in vivo and in vitro. Chinese Journal of Clinical Rehabilitation 2006;10:138-140. doi:10.1016/j.tsf.2005.11.062.
21. Kim HS, Yoon JW, Li H, Jeong GO, Park JJ, Shin SE, et al. Functional expression and pharmaceutical efficacy of cardiac-specific ion channels in human embryonic stem cell-derived cardiomyocytes. Scientific Report-Uk 2017;7. doi:10.1038/s41598-017-14198-y.
22. Hao W, Shi S, Zhou S, Wang X, Nie S. Aspirin inhibits growth and enhances cardiomyocyte differentiation of bone marrow mesenchymal stem cells. European Journal of Pharmacology 2018;827:198-207. doi:10.1016/j.ejphar.2018.03.016.
23. Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, et al. Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell 2010;142:375-386. doi:10.1016/j.cell.2010.07.002.
24. Kumar BM, Maeng GH, Lee YM, Kim TH, Lee JH, Jeon BG, et al. Neurogenic and cardiomyogenic differentiation of mesenchymal stem cells isolated from minipig bone marrow. Research in Veterinary Science 2012;93:749-757. doi:10.1016/j.rvsc.2011.09.012.
25. Zhang Y, Chu Y, Shen W, Dou Z. Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells. Interactive Cardiovascular and Thoracic Surgery 2009;9:943-946. doi:10.1510/icvts.2009.211490.
26. Schultheiss TM, Xydas S, Lassar AB. Induction of avian cardiac myogenesis by anterior endoderm. Development 1995;121:4203-4214. doi:10.1242/dev.121.12.4203.
27. Schwartz RJ, Olson EN. Building the heart piece by piece: modularity of cis-elements regulating Nkx2-5 transcription. Development 1999;126:4187-4192. doi:10.1242/dev.126.19.4187.
28. Shirinsky VP, Khapchaev AY, Stepanova OV. Molecular mechanisms of cardiomyogenesis and the prospects for cardiomyocyte regeneration in cardiac failure. Molecular Biology 2008;42:762-772. doi:10.1134/s0026893308050130.
29. Mohan G, Shamsuddin AA, Hasan Adli D, Moghadamtousi SZ, Khanabdali R, Saadat A, et al. Promoting effect of small molecules in cardiomyogenic and neurogenic differentiation of rat bone marrow-derived mesenchymal stem cells. Drug Design, Development and Therapy 2015:81. doi:10.2147/dddt.s89658.
30. Akavia UD, Veinblat O, Benayahu D. Comparing the transcriptional profile of mesenchymal cells to cardiac and skeletal muscle cells. Journal of Cellular Physiology 2008;216:663-672. doi:10.1002/jcp.21442.
31. McKinsey TA, Zhang CL, Olson EN. MEF2: a calcium-dependent regulator of cell division, differentiation and death. Trends in Biochemical Sciences 2002;27:40-47. doi:10.1016/s0968-0004(01)02031-x.
32. Olson EN. Sizing up the heart: development redux in disease. Genes & Development 2003;17:1937-1956. doi:10.1101/gad.1110103.
33. Skerjanc IS, Petropoulos H, Ridgeway AG, Wilton S. Myocyte enhancer factor 2C and Nkx2–5 up-regulate each other's expression and initiate cardiomyogenesis in P19 cells. Journal of Biological Chemistry 1998;273:34904-34910. doi:10.1074/jbc.273.52.34904.
34. Ventura C, Maioli M. Opioid peptide gene expression primes cardiogenesis in embryonal pluripotent stem cells. Circulation Research 2000;87:189-194. doi:10.1161/01.RES.87.3.189.
35. Ohtsu Y, Johkura K, Ito Ki, Akashima T, Asanuma K, Ogiwara N, et al. Stimulation of P19CL6 with multiple reagents induces pulsating particles in vivo. Current Medical Research and Opinion 2005;21:795-803. doi:10.1185/030079905X41499.
36. Okura H, Matsuyama A, Lee CM, Saga A, Kakuta-Yamamoto A, Nagao A, et al. Cardiomyoblast-like cells differentiated from human adipose tissue-derived mesenchymal stem cells improve left ventricular dysfunction and survival in a rat myocardial infarction model. Tissue Engineering Part C: Methods 2010;16:417-425. doi:10.1089/ten.tec.2009.0362.
37. Somekawa S, Fukuhara S, Nakaoka Y, Fujita H, Saito Y, Mochizuki N. Enhanced Functional Gap Junction Neoformation by Protein Kinase A–Dependent and Epac-Dependent Signals Downstream of cAMP in Cardiac Myocytes. Circulation Research 2005;97:655-662. doi:10.1161/01.res.0000183880.49270.f9.
38. Gasiūnienė M, Valatkaitė E, Navakauskaitė A, Navakauskienė R. The Effect of Angiotensin II, Retinoic Acid, EGCG, and Vitamin C on the Cardiomyogenic Differentiation Induction of Human Amniotic Fluid-Derived Mesenchymal Stem Cells. International Journal of Molecular Sciences 2020;21:8752. doi:10.3390/ijms21228752.
39. Vreeker A, Van Stuijvenberg L, Hund TJ, Mohler PJ, Nikkels PGJ, Van Veen TAB. Assembly of the Cardiac Intercalated Disk during Pre- and Postnatal Development of the Human Heart. PLoS ONE 2014;9:e94722. doi:10.1371/journal.pone.0094722.
40. Woodcock-Mitchell J, Mitchell JJ, Low RB, Kieny M, Sengel P, Rubbia L, et al. α-Smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles. Differentiation 1988;39:161-166. doi:10.1111/j.1432-0436.1988.tb00091.x.
41. Potta SP, Liang H, Winkler J, Doss MX, Chen S, Wagh V, et al. Isolation and Functional Characterization of α-Smooth Muscle Actin Expressing Cardiomyocytes from Embryonic Stem Cells. Cellular Physiology and Biochemistry 2010;25:595-604. doi:10.1159/000315078.
42. Kobayashi T, Jin L, De Tombe PP. Cardiac thin filament regulation. Pflügers Archiv - European Journal of Physiology 2008;457:37-46. doi:10.1007/s00424-008-0511-8.
43. Markmee R, Aungsuchawan S, Tancharoen W, Narakornsak S, Pothacharoen P. Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine. Heliyon 2020;6:e04844. doi:10.1016/j.heliyon.2020.e04844.
44. Sjöblom B, Salmazo A, Djinović-Carugo K. α-Actinin structure and regulation. Cellular and Molecular Life Sciences 2008;65:2688-2701. doi:10.1007/s00018-008-8080-8.
45. Balana B, Nicoletti C, Zahanich I, Graf EM, Christ T, Boxberger S, et al. 5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells. Cell Research 2006;16:949-960. doi:10.1038/sj.cr.7310116.
46. Liu Y, Song J, Liu W, Wan Y, Chen X, Hu C. Growth and differentiation of rat bone marrow stromal cells: does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovascular Research 2003;58:460-468. doi:10.1016/S0008-6363(03)00265-7.
47. Siegel G, Krause P, Wohrle S, Nowak P, Ayturan M, Kluba T, et al. Bone marrow-derived human mesenchymal stem cells express cardiomyogenic proteins but do not exhibit functional cardiomyogenic differentiation potential. Stem Cells and Development 2012;21:2457-2470. doi:10.1089/scd.2011.0626.