Cyclic adenosine 3',5'-monophosphate induces differentiation of mouse embryonic stem cells into cardiomyocytes.
Embryonic stem (ES) cells, derived from blastocyst-stage of early mammalian embryos, have the potential to differentiate into derivatives of all three embryonic germ layers. Here we reported the first evidence that murine pluripotent ES cells could be induced to differentiate into cardiomyocytes by cyclic adenosine 3',5'-monophosphate (cAMP) in vitro. Spontaneously beating of cardiac cell clusters began to be observed within the outgrowths of embryoid bodies (EBs) as early as 2 days after the onset of differentiation. By days 5-8 after induction, a maximum level of cardiomyocyte differentiation could be achieved. Incubation of EBs with cAMP at concentrations ranging from 0.01 mg/L to 1 mg/L resulted in a significant elevation in differentiation rate, reaching a maximum value of 44.0 +/- 1.3% at 0.03 mg/L of exposure. At 0.03 mg/L concentration point, an approximately 8.1-fold increase in cardiomyocyte differentiation was observed in comparison with 5.4 +/- 0.9% of untreated controls. The differentiation rate induced by cAMP was shown to be similar to that of RA/DMSO treated controls, indicating that cAMP has the same inducing effect as RA/DMSO. However, no significant co-inducing effects between cAMP and RA/DMSO were seen. Cardiomyocytes were evident as they expressed cardiac cell specific genes and protein markers including GATA4, Nkx2.5, beta-MHC, atrial natriuretic factor (ANF) and alpha-actin when analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining. The results from the present study suggested a novel role of cAMP in cardiomyocyte differentiation and provided a new research model for the study of cardiac cell biology.
Chen
Ye
Y
College of Life Sciences, Zhejiang University, Wenshan Road 232, Hangzhou, Zhejiang 310012, PR China.
Shao
Jian-Zhong
JZ
Xiang
Li-Xin
LX
Guo
Jian
J
Zhou
Qing-Jun
QJ
Yao
Xing
X
Dai
Li-Cheng
LC
Lu
Yong-Liang
YL
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology*; D005786:Gene Expression Regulation; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects*
D012333:RNA, Messenger; D000242:Cyclic AMP