Effects of over-expression of Notch1 intracellular domain on the differentiation of c-Kit+ bone marrow mesenchymal stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Activation of Notch signaling plays a critical role in stem cel differentiation, and this effect seems to be cel-type dependent. Little is reported on the role of activation of Notch1 signaling in the differentiation of c-Kit+ bone marrow mesenchymal stem cels. OBJECTIVE:To analyze the influence of activation of Notch1 signaling on the differentiation of c-Kit+ bone marrow mesenchymal stem cels. METHODS:The Notch1 intracelular domain (N1-ICD) was obtained from the cDNA library by PCR and cloned intoBamHI/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 cels to obtain N1-ICD overexpressing adenoviral particles (N1-ICD-Ad). The c-Kit+ subpopulation were isolated from bone marrow mesenchymal stem cels of the Sprague-Dawley rat femurviamagnetic activated cel 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 cels by quantitative RT-PCR and immunofluorescent staining. RESULTS AND CONCLUSION:N1-ICD coding sequence was successfuly 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 cels, and its title was 2×1012 PFU/L. c-Kit+ bone marrow mesenchymal stem cels with the purity of 91.6% were successfuly isolated from the bone marrow mesenchymal stem cels 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 cels 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 Wilebrand factor, an endothelial cel differentiation gene, and mildly increased the expression of smooth muscle22α, a smooth muscle cel differentiation gene. These experimental results indicate that the activation of Notch1 signaling contributes to multi-lineages differentiation of c-Kit+ bone marrow mesenchymal stem cels, and the construction of N1-ICD overexpressing adenoviral vector makes the foundation for further research on the role of Notch1 signaling in stem cel biology.