Inducing Human Embryonic Stem Cells to Become Cardiomyocytes by Direct Adherence Method_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

MU Junsheng , LI Xianshuai , YUAN Shumin , ZHANG Jianqun , BOPing.

1. Department of Cardiac Surgery，Beijing Anzhen Hospital，Capital Medical University，Beijing 100029，P. R. China;;
2. State Key Laboratory of Biomembrane and MembraneBiotechnology，Institute of Zoology，Chinese Academy of Sciences，Beijing 100101，P. R. China;

Corresponding author：

Keywords：

Human embryonic stem cell; 　Differentiation; 　Cardiomyocyte; 　Adherence method

DOI：

10.7507/1007-4848.20130175

Video：

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Objective To use direct adherent method to induce human embryonic stem cells （hESCs） to become cardiomyocytes in vitro and examine their differentiation rate. Methods Undifferentiated hESCs were seeded onto Matrigel-coated plates at a density of 1×105 cells/cm2 and cultured in MEF-conditioned medium （MEF-CM） with 8 ng/ml basic fibroblast growth factor （bFGF） for 6 days. Then MEF-CM was replaced with RPMI 1640/B27 medium supplemented with 100 ng/ml human recombinant activin A for 24 hours in hESCs culture，followed by supplementation of 10 ng/ml human recombinant bone morphogenetic protein 4 （BMP4） for 4 days in hESCs culture. The medium was then replaced with RPMI 1640/B27 medium without supplementary cytokines，and hESCs were refed every 2-3 days for 2-3 additional weeks. Self-beating cardiomyocytes and the beating frequency were observed under the microscope，and the percentage of colonies showing beating cardiomyocytes was calculated. Cardiac troponin T （cTnT），a specific marker of cardiomyocytes，was examined by immunofluorescence. Spontaneous action potentials of cardiomyocytes were measured with patch clamp technique.Apoptotic rate of cardiomyocytes was detected with apoptosis-hoechst staining kit after beating cardiomyocytes were culturedunder hypoxia for 24 hours. Results A large number of spontaneous beating cardiomyocytes were observed 13 days after induction. The average time to show beating cardiomyocytes was 13.0±1.1 days after induction，the percentage of colonies showing beating cardiomyocytes was 66.7%，and the beating frequency of cardiomyocytes was 63.0±7.0 times/minutes. Beating cardiomyocytes were cTnT-positive. Spontaneous action potentials were detected in beating cardiomyocytes.Apoptotic rate of cardiomyocytes was 8.0%±0.5% after beating cardiomyocytes were cultured under hypoxia for 24 hours. Conclusion It’s the first time to use direct adherent method to induce hESCs to become cardiomyocytes in vitro in China with the differentiation rate of 66.7% and differentiation time of 13 days.

Citation： MU Junsheng,LI Xianshuai,YUAN Shumin,ZHANG Jianqun,BOPing.. Inducing Human Embryonic Stem Cells to Become Cardiomyocytes by Direct Adherence Method. Chinese Journal of Clinical Thoracic and Cardiovascular Surgery, 2013, 20(5): 564-569. doi: 10.7507/1007-4848.20130175 Copy

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2.	Mummery C， Ward-van Oostwaard D， Doevendans P， et al. Differentiation of human embryonic stem cells to cardiomyocytes：role of coculture with visceral endoderm-like cells. Circulation， 2003， 107 （21）：2733-2740.
3.	Kehat I， Kenyagin-Karsenti D， Snir M， et al. Human embryonic stem cells can differentiate into myocytes with structural and functionalproperties of cardiomyocytes. J Clin Invest， 2001， 108 （3）：407-414.
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6.	Laflamme MA， Gold J， Xu C， et al. Formation of human myocardiumin the rat heart from human embryonic stem cells. Am J Pathol， 2005，167 （3）：663-671.
7.	Blau HM， Brazelton TR， Weimann JM. The evolving concept of a stem cell：entity or function ? Cell， 2001， 105 （7）：829-841.
8.	Orlic D， Hill JM， Arai E， et al. Stem cells for myocardial regeneration. Circ Res， 2002， 91 （12）：1092-1102.
9.	Roell W， Lewaher T， Sasse P， et al. Engraftment of connexin43expressing prevents post-infarct arrhythmia. Nature， 2007， 450 （7171）：819.
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13.	Nakajima Y， Yamagishi T， Ando K， et al. Significance of bone morphogenetic protein-4 function in the initial myofibrillogenesis of chick cardiogenesis. Dev Biol， 2002， 245 （2）：291-303.
14.	Ladd AN， Yatskievych TA， Antin PB. Regulation of avian cardiac myogenesis by activin/TGFbeta and bone morphogenetic proteins. Dev Biol， 1998， 204 （2）：407-419.
15.	Sugi Y， Lough J. Activin-A and FGF-2 mimic the inductive effects of anterior endoderm on terminal cardiac myogenesis in vitro. Dev Biol， 1995， 168 （2）：567-574.
16.	Lough J， Barron M， Brogley M， et al. Combined BMP-2 and FGF-4，but neither factor alone， induces cardiogenesis in non-precardiac embryonic mesoderm. Dev Biol， 1996， 178 （1）：198-202.
17.	Barron M， Gao M， Lough J. Requirement for BMP and FGF signalingduring cardiogenic induction in non-precardiac mesoderm is specific， transient， and cooperative. Dev Dyn， 2000， 218 （2）：383-393.
18.	Takahashi T， Lord B， Schulze PC， et al. Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes.Circulation， 2003， 107 （14）：1912-1916.
19.	Hsieh PC， Segers VF， Davis ME， et al. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med， 2007， 13 （8）：970-974.
20.	Kühn B， del Monte F， Hajjar RJ， et al. Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair. Nat Med， 2007， 13 （8）：962-969.
21.	Xu C， Police S， Rao N， et al. Characterization and Enrichment of Cardiomyocytes Derived From Human Embryonic. Stem Cells Circ Res， 2002， 91 （6）：501-508.
22.	Laflamme MA， Chen KY， Naumova AV， et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol， 2007， 25 （9）：1015-1024.
23.	Mandel Y， Weissman A， Schick R， et al. Human embryonic and induced pluripotent stem cell-derived cardiomyocytes exhibit beat rate variability and power-law behavior. Circulation， 2012， 125 （7）：883-893.
24.	Shiba Y， Fernandes S， Zhu WZ， et al. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature， 2012， 489 （7415）：322-325.
25.	Gepstein L， Ding C， Rahmutula D， et al. In vivo assessment of the electrophysiological integration and arrhythmogenic risk of myocardial cell transplantation strategies. Stem Cells， 2010， 28 （12）：2151-2161.

1. 　Xu C， Police S， Rao N， et al. Characterization and enrichment of cardiomyocytesderived from human embryonic stem cells. Circ Res， 2002， 91 （6）：501-508.
2. 　Mummery C， Ward-van Oostwaard D， Doevendans P， et al. Differentiation of human embryonic stem cells to cardiomyocytes：role of coculture with visceral endoderm-like cells. Circulation， 2003， 107 （21）：2733-2740.
3. 　Kehat I， Kenyagin-Karsenti D， Snir M， et al. Human embryonic stem cells can differentiate into myocytes with structural and functionalproperties of cardiomyocytes. J Clin Invest， 2001， 108 （3）：407-414.
4. 　McDevitt TC， Laflamme MA， Murry CE. Proliferation of cardiomyocytes derived from human embryonic stem cells is mediated via the IGF/PI 3-kinase/Akt signaling pathway. J Mol Cell Cardiol， 2005， 39 （6）：865-873.
5. 　Snir M， Kehat I， Gepstein A， et al. Assessment of the ultrastructuraland proliferative properties of human embryonic stem cell-derived cardiomyocytes. Am J Physiol Heart Circ Physiol， 2003， 285 （6）：2355-2363.
6. 　Laflamme MA， Gold J， Xu C， et al. Formation of human myocardiumin the rat heart from human embryonic stem cells. Am J Pathol， 2005，167 （3）：663-671.
7. 　Blau HM， Brazelton TR， Weimann JM. The evolving concept of a stem cell：entity or function ? Cell， 2001， 105 （7）：829-841.
8. 　Orlic D， Hill JM， Arai E， et al. Stem cells for myocardial regeneration. Circ Res， 2002， 91 （12）：1092-1102.
9. 　Roell W， Lewaher T， Sasse P， et al. Engraftment of connexin43expressing prevents post-infarct arrhythmia. Nature， 2007， 450 （7171）：819.
10. 　Dove A. Cell-based therapies go live. Nat Biotechnol， 2002， 20 （4）：339-343.
11. 　Pal R， Mamidi MK， Das AK， et al. Comparative analysis of cardiomyocyte differentiation from human embryonic stem cells under 3-D and 2-D culture conditions. J Biosci Bioeng， 2013， 115 （2）：200-206.
12. 　Smith JC， Price BM， Van Nimmen K， et al. Identification of a potentXenopus mesoderm-inducing factor as a homologue of activin A. Nature， 1990， 345 （6277）：729-731.
13. 　Nakajima Y， Yamagishi T， Ando K， et al. Significance of bone morphogenetic protein-4 function in the initial myofibrillogenesis of chick cardiogenesis. Dev Biol， 2002， 245 （2）：291-303.
14. 　Ladd AN， Yatskievych TA， Antin PB. Regulation of avian cardiac myogenesis by activin/TGFbeta and bone morphogenetic proteins. Dev Biol， 1998， 204 （2）：407-419.
15. 　Sugi Y， Lough J. Activin-A and FGF-2 mimic the inductive effects of anterior endoderm on terminal cardiac myogenesis in vitro. Dev Biol， 1995， 168 （2）：567-574.
16. 　Lough J， Barron M， Brogley M， et al. Combined BMP-2 and FGF-4，but neither factor alone， induces cardiogenesis in non-precardiac embryonic mesoderm. Dev Biol， 1996， 178 （1）：198-202.
17. 　Barron M， Gao M， Lough J. Requirement for BMP and FGF signalingduring cardiogenic induction in non-precardiac mesoderm is specific， transient， and cooperative. Dev Dyn， 2000， 218 （2）：383-393.
18. 　Takahashi T， Lord B， Schulze PC， et al. Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes.Circulation， 2003， 107 （14）：1912-1916.
19. 　Hsieh PC， Segers VF， Davis ME， et al. Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med， 2007， 13 （8）：970-974.
20. 　Kühn B， del Monte F， Hajjar RJ， et al. Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair. Nat Med， 2007， 13 （8）：962-969.
21. 　Xu C， Police S， Rao N， et al. Characterization and Enrichment of Cardiomyocytes Derived From Human Embryonic. Stem Cells Circ Res， 2002， 91 （6）：501-508.
22. 　Laflamme MA， Chen KY， Naumova AV， et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol， 2007， 25 （9）：1015-1024.
23. 　Mandel Y， Weissman A， Schick R， et al. Human embryonic and induced pluripotent stem cell-derived cardiomyocytes exhibit beat rate variability and power-law behavior. Circulation， 2012， 125 （7）：883-893.
24. 　Shiba Y， Fernandes S， Zhu WZ， et al. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature， 2012， 489 （7415）：322-325.
25. 　Gepstein L， Ding C， Rahmutula D， et al. In vivo assessment of the electrophysiological integration and arrhythmogenic risk of myocardial cell transplantation strategies. Stem Cells， 2010， 28 （12）：2151-2161.

Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Inducing Human Embryonic Stem Cells to Become Cardiomyocytes by Direct Adherence Method

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