Effects of over-expression of Notch1 intracellular domain on the differentiation of c-Kit+ bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2016.06.004

Abstract

Abstract:

BACKGROUND: Activation of Notch signaling plays a critical role in stem cell differentiation, and this effect seems to be cell-type dependent. Little is reported on the role of activation of Notch1 signaling in the differentiation of c-Kit+ bone marrow mesenchymal stem cells.
OBJECTIVE: To analyze the influence of activation of Notch1 signaling on the differentiation of c-Kit+ bone marrow mesenchymal stem cells.
METHODS: The Notch1 intracellular domain (N1-ICD) was obtained from the cDNA library by PCR and cloned into BamHI/AgeI digested adenoviral GV314 plasmid to construct N1-ICD overexpressing shuttle plasmid, and the positive clones were verified by sequencing. N1-ICD shuttle plasmid and helper plasmids pBHGloxΔE1,3 Cre were used to co-transfect HEK293T cells to obtain N1-ICD overexpressing adenoviral particles (N1-ICD-Ad). The c-Kit+ subpopulation were isolated from bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur via magnetic activated cell sorting. After transfection of the c-Kit+ BMSCs with N1-ICD-Ad adenovirus, we assessed the activation of Notch1 signaling and differentiation in c-Kit+ bone marrow mesenchymal stem cells by quantitative RT-PCR and immunofluorescent staining.
RESULTS AND CONCLUSION: N1-ICD coding sequence was successfully generated from the cDNA library, and then was cloned into the linearized adenoviral vectors GV314. The resistant clones were verified by sequencing. With the assistance of packaging plasmids, recombinant N1-ICD-Ad adenovirus plasmids were successful packaged in HEK293T cells, and its title was 2×1012 PFU/L. c-Kit+ bone marrow mesenchymal stem cells with the purity of 91.6% were successfully isolated from the bone marrow mesenchymal stem cells of the Sprague-Dawley rat femur. Compared with the blank and negative controls, N1-ICD-Ad infection in the c-Kit+ bone marrow mesenchymal stem cells led to substantial accumulation of N1-ICD in the cytoplasm and nuclei, significantly unregulated expressions of Hes1 (a downstream gene of Notch) and cardiomyocyte differentiation genes Nkx2.5 and cTnT, significantly increased the expression of von Willebrand factor, an endothelial cell differentiation gene, and mildly increased the expression of smooth muscle 22α, a smooth muscle cell differentiation gene. These experimental results indicate that the activation of Notch1 signaling contributes to multi-lineages differentiation of c-Kit+ bone marrow mesenchymal stem cells, and the construction of N1-ICD overexpressing adenoviral vector makes the foundation for further research on the role of Notch1 signaling in stem cell biology.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Ha Yan-ping, Wang Zhen-liang, Lei Hong, Ding Ran-ran, Jiang Xiao-fan, Wang Ke-ke, Shen Zhia-hua, Jie Wei. Effects of over-expression of Notch1 intracellular domain on the differentiation of c-Kit+ bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(6): 785-792.

Figures/Tables 5

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