Effects of cardiotrophin-1 on differentiation and maturation of rat bone marrow mesenchymal stem cells induced with 5-azacytidine in vitro.
BACKGROUND
Cardiotrophin-1 (CT-1) is a cytokine involved in the growth and survival of cardiac cells that stimulates cardiomyogenesis in pluripotent murine embryonic stem (ES) cells. But it is not known whether CT-1 is responsible for the fate of differentiated bone marrow mesenchymal stem cells (BMMSCs).


METHODS
We investigated the effects of CT-1 on differentiation and maturation of BMMSCs in vitro induced with 5-azacytidine. BMMSCs isolated from femur of rats were induced by CT-1 only, by 5-azacytidine with or without CT-1,and an untreated control group was also set. After 4 weeks of induced culturing, we observed the levels of alpha-cardiac actin and troponin-I by immunohistochemical staining, the ultrastructure of induce-cultured BMMSCs, and the expression of GATA4, Nkx2.5, beta-myosin heavy chain (beta-MHC) and alpha-cardiac actin mRNA by real time RT-PCR analysis.


RESULTS
Differentiated BMMSCs treated by 5-azacytidine and CT-1 distinctly showed formations of myofilaments and myotube-like structures-morphological characteristics of myocyte like cells, and spontaneous contraction of a few cells was observed. The protein levels of alpha-cardiac actin and troponin-T were significantly higher than control. Furthermore, mRNA expression of GATA4, Nkx2.5, alpha-cardiac actin and beta-MHC was increased remarkably.


CONCLUSIONS
This study suggests that induced culturing of BMMSCs in the presence of 5-azacytidine combined with CT-1 can enhance cardiomyocytic characteristics. CT-1 upregulates the expression of GATA4, Nkx2-5, alpha-cardiac actin and beta-MHC mRNA, and rapidly promotes the differentiation and maturation of cardiomyocyte-like cells differentiated from BMMSCs induced with 5-azacytidine.
Xinyun
Chen
C
Department of Cardiology, West China Hospital, Sichuan University, Chengdu 610041, China.
Zhi
Zeng
Z
Bin
Zhou
Z
Li
Rao
R
Yucheng
Chen
C
Yafei
Yan
Y
Tingjie
Zhang
Z
Shengfu
Li
L
D000199:Actins / Q000235:genetics; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050991:GATA4 Transcription Factor / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics; D066298:In Vitro Techniques; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects*; D018995:Myosin Heavy Chains / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors / Q000235:genetics
D000199:Actins; D016207:Cytokines; D004791:Enzyme Inhibitors; D050991:GATA4 Transcription Factor; C084365:Gata4 protein, rat; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C000594638:MYH7 protein, rat; C524161:Nkx2-5 protein, rat; D014157:Transcription Factors; C092073:cardiotrophin 1; D018995:Myosin Heavy Chains; D001374:Azacitidine