Pretreatment of Mesenchymal Stem Cells and Stromal-derived Factor-1α Delivery from Chitosan-based Injectable Hydrogels for Better Cell Guidance and Retention

Abstract

Clinical applications of mesenchymal stem cells (MSCs) rely on their capacity to home and engraft in the appropriate target tissues for a long time. Homing and engraftment capacity of these stem cells depend on the expression of Chemokines and their receptors. Ex vivo expanded MSCs exhibit homing potential when grafted to injury tissue but their homing efficiency has been observed very poor because of modifications in homing receptor expression and/or functions during culture and/or preparation steps. Hence, this study was designed to investigate the expression of surface CXCR4 by flow cytometric analysis (FACS) and in vitro modified Boyden chamber assay in adipose-derive MSCs (ASCs) stimulated with a hypoxia mimicking agents such as desferrioxamine mesilate (DFX), cobalt chloride (CoCl2), lithium chloride (LiCl), valproic acid (VPA) and hypoxia. Intracellular CXCR4 were also evaluated by conventional and real-time PCR. Then we evaluated the homing ability of DFX-pretreated human DiI-labeled ASCs in vivo, 2 weeks after intravenous (IV), local infusion towards subcutaneously implanted chitosan-glycerophophate-hydroxyethyl cellulose (CH-GP-HEC) injectable hydrogels releasing SDF1 in dorsum of Wistar Rats. Presence of human ASCs in the CH-GP-HEC injectable, spleen, and lung were analyzed histologically by fluorescent microscope, and also quantified by PCR for human specific CXCR4 gene, 2 weeks after transplantation in recipients' Rats. Results showed that short-term (24 hours) pretreatment to ASCs with the hypoxia mimicking agents up-regulate the CXCR4, increase in vitro migration capacity toward 100ng/ml SDF-1 (P<0.001) and in vivo homing capacity to the implanted CH-GP-HEC injectable hydrogel releasing SDF1. Fluorescence microscopic examination disclosed enhanced local accumulation of fluorescence-labeled ASCs in CH-GP-HEC in the DFX-pretreated group at 16th post-transplantation day. These results suggest that the SDF-1/CXCR4 axis plays an important role in the regulation of motility of ASCs, and increased expression of CXCR4 might be a potential strategy to improve homing and engraftment of ASCs towards SDF1 released by injectable hydrogels in different lesions. Keywords: Chemotactic recruitment, Guided homing, Stem cells therapy.