Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body development
Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body development
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DOI：
10.1152/ajpheart.01288.2008
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发表时间：
2009-06-01
影响因子：
4.8
通讯作者：
Sasaki, Katsunori
中科院分区：
文献类型：
--
作者：
Takei, Shunsuke;Ichikawa, Hinako;Sasaki, Katsunori
骨形态发生蛋白-4促进从人胚胎干细胞向心肌细胞诱导基于血清的类胚体发育。Am J Physiol心脏圈Physiol 296：H1793-H1803,2009。首次发表于2009年4月10日；来自人类胚胎干细胞的doi:10.1152/ajpheart.01288.2008.-Cardiomyocytes是基于细胞的心脏病治疗的潜在来源。研究了骨形态发生蛋白(BMP)-4在胎牛血清(FBS)存在下对人胚胎干细胞(H1ES细胞)在类胚体(EB)发育过程中心脏诱导分化的影响。含20%胎牛血清的悬浮培养4d对早期中胚层和心肌细胞的分化效果最好。加入Noggin后，EBS搏动发生率由23.6%降至5.3%，提示BMP信号参与心脏自发分化过程。在此条件下，在4d悬液中加入12.5~25 ng/mlBMP-4对心肌细胞的分化有促进作用。25 ng/mlBMP-4刺激EBS的发生率在扩增第6天达到95.8%，之后一直维持到第20天。实时荧光定量PCR分析显示，25 ng/mlBMP-4可上调EBS中心脏发育相关基因MESP1和NKX2.5的表达。Smad1/5/8的磷酸化增加以及Smad1/5/8和Smad4的核定位证实了BMP信号在EBS中的激活。加入150 ng/mlNoggin可显著减少EB突起中EBS和NKX2.5的表达，Noggin单独作用可增加EB突起中Nestin的表达和神经分化。25 ng/mlBMP-4诱导的心肌细胞在异丙肾上腺素作用、基因表达、蛋白检测、免疫反应性和亚细胞结构等方面均表现出良好的细胞生物学特性和分化过程。BMP-4对心肌细胞的凋亡和增殖程度无明显影响。这些结果表明，BMP-4在适当的时间和浓度联合FBS可显著促进人ES细胞向心肌细胞的诱导。
Takei S, Ichikawa H, Johkura K, Mogi A, No H, Yoshie S, Tomotsune D, Sasaki K. Bone morphogenetic protein-4 promotes induction of cardiomyocytes from human embryonic stem cells in serum-based embryoid body development. Am J Physiol Heart Circ Physiol 296: H1793-H1803, 2009. First published April 10, 2009; doi:10.1152/ajpheart.01288.2008.-Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for the differentiation of early mesoderm and cardiomyocytes. The addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5-25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on day 6 of expansion and then plateaued until day 20. In real-time PCR analysis, the cardiac development-related genes MESP1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. The activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smad1/5/8 and by the nuclear localization of phospho-Smad1/5/8 and Smad4. The addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. The cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and a course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promotes cardiomyocyte induction from human ES cells.