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  • Today many strategies have been developed to enhance CXCR

    2020-02-06

    Today, many strategies have been developed to enhance CXCR-4 receptor Ganetespib on the cell surface, either by modifying the cell culture medium or by modifying the CXCR-4 gene in BMSCs to stimulate stem cell recruitment (Jiang et al., 2012, Liu et al., 2014b). For instance, the pre-conditioning of sub-cultured BMSCs with hypoxia or cytokines (such as IL-6, HGF, etc.) before infusion has the potential to increase CXCR-4 expression and improve BMSCs\' migration in vitro (Ponte et al., 2007, Schioppa et al., 2003, Shi et al., 2007). However, this therapeutic method has little in vivo potential because the inflammatory environment would severely impair the BMSCs\' homing ability over the long term (Hung et al. 2007). Although CXCR-4 gene-modified BMSCs, which are modified by viral vectors (adenoviral and lentiviral vectors), proved effective for enhancing BMSC migration within the AKI micro-environment, their high toxicity and immunogenicity may cause some long-term safety issues and restrict clinical application (Clements et al., 2009, Liu et al., 2013). Unlike viral vectors, non-viral vectors provide a safe alternative for targeted gene delivery in BMSCs, but their transfection efficiency is not high (Liang et al. 2010). Thus, substantial improvement in the transfection efficiency is required for effective therapeutic application of BMSCs. Many reports have demonstrated that micro-bubble-mediated ultrasound irradiation could greatly enhance the delivery efficiency of extra-cellular genes and drugs by forming some transient pores in the plasma membrane (Deelman et al., 2010, Escoffre et al., 2013a, Zhou et al., 2012). Our group has previously proved the synergistic effect of combining ultrasound with micro-bubbles and PEI, which could greatly enhance hepatocyte growth factor (HGF) gene transfection efficiency in sub-cultured BMSCs, and the transfected BMSCs can still maintain their reproductive activity and multi-directional differentiation capability (Li et al. 2013). It would provide a novel non-viral transfection method for effective gene therapy in BMSCs that may be applied to clinical application. In our study, we tested CXCR-4 transfection efficiency in BMSCs by combining UTMD and PEI and examined the effect of CXCR-4 over-expression on the migratory capacity of experimental BMSCs both in vitro and in vivo. FCM assay showed the percentage of cells expressing CXCR-4 protein on the cell surface was significantly higher in the P+UTMD group compared to other groups at 24 and 48 h after CXCR-4 transfection. WB analysis also indicated that combining UTMD and PEI would increase functional CXCR-4 receptors on the cell surface at 24 and 48 h post-treatment. Transwell migration assay showed migration was most pronounced in the P+UTMD group, which expresses the highest level of cell surface CXCR-4. Thus, all these results suggest that combining UTMB and PEI could markedly increase CXCR-4 gene delivery in BMSCs, and this may enhance stem cells migration capacity in vitro. We also analyzed the homing ability of experimental BMSCs toward AKI kidney tissues. The number of DAPI-labeled BMSCs in AKI kidney tissues was much larger in the P+UTMD group than those of other groups at 24, 48 and 72 h post-treatment; this is consistent with the finding that this group expressed the highest levels of functional CXCR4 on the cell membrane. The goal of this study was to find an effective non-viral method of achieving high CXCR-4 gene transfection efficiency in sub-cultured BMSCs, which will be important for enhancing stem cell migration capacity both in vitro and in vivo. We demonstrated that over-expression of CXCR-4, via combining UTMD and PEI, could markedly augment BMSCs\' homing ability within the AKI micro-environment. The combination of UTMD and PEI has never previously been used to improve stem cell migration; thus, to the best of our knowledge, this is the first report to do so. The improvement of BMSC homing ability is critical for its clinical application, and much work needs to be done to evaluate the safety and efficacy of this method in vivo. To become a useful therapeutic option, certain parameters, like acoustic intensity, micro-bubble dosage and treatment time, must be optimized. In conclusion, we found that combining UTMD and PEI could enhance CXCR-4 gene expression in sub-cultured BMSCs. This, in turn, facilitates cell migration and cell retention at the targeted AKI kidney Ganetespib tissues. This finding may open a new approach in cell-based therapy of AKI.