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Expression changes of Notch--related genes during the differentiation of human mesenchymal stem cells into neurons

Xing Ying, Bai Ruiying, Yan Wenhai, Han Xuefei, Duan Ping, Xu Yan, Fan Zhigang

The Stem Cells Research Center,College of Basic Medical Sciences, Zhengzhou University.Zhengzhou 450052,Henan

Abstract

The Notch signaling pathway has been implicated in the regulation of cell-fate decisions such as differentiation of embryo stem cells and neural stem cells into neurons. We cultured human mesenchymal stem cells （hMSCs） in vitro and induced hMSCs to differentiate into neural cells by #beta#-mercaptoethanol （#beta#-ME）, DMSO and 3-tert-butyl-4-hydroxyanisole （BHA）. Immunocytochemistry was utilized to detect neuron-specific enolase （NSE） and Nissl body, and flow cytometry was used to determine cell growth phases. The expressions of signal molecules involved in the Notch pathway such as Notchl, Jagged 1 （JAG 1）, presenilin 1 （PS 1） and hairy and enhancer of split 1 filES 1） were observed by RT-PCR and immunofluorescent techniques. The results were as follows： （1） Before induction, the percentage of hMSCs at G_(0)/G_(1) was 58.5%, and the percentage at S+G_(2)/M was 41.5%. After induction, the percentage of hMSCs at G_(0)/G_(1) increased to 73.1%, 76.2% and 78.1%, respectively on days 2, 4 and 6, and the percentage at S+G_(2)/M decreased to 26.8%, 24.8% and 21.9%, respectively; The percentage of NSE-positive cells reached （77±0.35）%; Nissl's staining was positive in cytoplasm. （2） Notchl and JAG1 were both expressed in hMSCs before and after induction, but the mRNA expressions of both Notchl and JAG1, detected by RT-PCR, decreased obviously after induction（P<0.05）. Notchl mRNA/#beta#-actin was 1.157, 0.815, 0.756 and 0.570, and JAG1 mRNA/#beta#-actin was 0.437, 0.350, 0.314 and 0.362, respectively, on days 0, 2, 4 and 6 after induction. The Notch pathway activation participant PS1 mRNA and Notch pathway target gene HES1 mRNA also decreased apparently after induction （P<0.05）, and their mRNA/#beta#-actin was 0.990, 0.449, 0.441, 0.454 and 0.370, 0.256, 0.266, 0.240 on days 0, 2, 4 and 6, respectively. These observations indicate that the expressions of Notch signal molecules were suppressed when hMSCs were induced to differentiate into neural cells. Based on these findings, we propose that low level of Notch signalling activation may contribute to neural cell differentiation.

Key words: mesenchymal stem cell;Notch1;JAG1;PS1;HES1;Differentiation;Neuron

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Citing This Article：

Xing Ying, Bai Ruiying, Yan Wenhai, Han Xuefei, Duan Ping, Xu Yan, Fan Zhigang. Expression changes of Notch--related genes during the differentiation of human mesenchymal stem cells into neurons. Acta Physiol Sin 2007; 59 (3): 267-272 (in Chinese with English abstract).