Establishment of mouse mesenchymal stem cells overexpressing CXCR4 gene and evaluation of their functions

Abstract

This study was purposed to establish the mesenchymal stem cells (MSCs) stably overexpressing mouse CXC chemokine receptor type 4 (CXCR4) gene and to explore their function. The recombinant lentiviral vector LV-CXCR4-IRES-EGFP with packaging plasmid pSPAX2 and envelope plasmid pMD.2G were co-transfected into 293FT packaging cell line using lipofectamine 2000 to produce the recombinant lentiviral vectors. The recombinant viruses were harvested and concentrated by using ultracentrifugation. Mouse bone marrow MSC were infected with the viral supernatants. Variable methods were used to optimize the transduction condition. EGFP expression was visualized using fluorescence microscope and efficiency of infection was determined by flow cytometry (FCM). Proliferation and apoptosis were detected by proliferation curve and FCM, respectively. Migration capacity was assessed by a chemotaxis assay using transwell. Expression of EGFP were detected by fluorescence microscopy in MSCs after infection. The results showed that through optimization of infection condition, the recombination lentiviral vectors had higher infection efficacy; after infection for 72 h, the higher expression of EGFP could be observed under fluorescence microscope; the expression of CXCR4 protein on MSC surface in CXCR4-MSC group significantly increased compared with those in the control group. Meanwhile, over-expression of CXCR4 had no effect on their capacity of proliferation and did not induce apoptosis. Moreover, CXCR4 enhanced the migration of cells in the transwell induced by SDF-1 gradient compared with the EGFP control group. It is concluded that the lentiviral vector can not only infect mouse MSCs efficiently, but also can make CXCR4 express stably in MSC.