003 Génération de fibroblastes “like” cornéens issus de la différenciation de cellules souches mésenchymales humaines in vitro

Aim Tissue engineering (TE) offers considerable promise in damaged tissues repair or replacement. Corneal Fibroblasts (CF) result from mesodermal cell extension into optic cap. They participate in the genesis of corneal stroma during the 5th week of embryonic development. We used MSC to generate CF phenotype cells for corneal TE and clinical applications. Materials and Methods Genotype CF, obtained from 8 corneal grafts, were evaluated by genomic profiling of total RNA using microarray analysis (1300 genes). The MSC were isolated from 12 human bone marrows with the magnetic beads separation method. These cells were cultured in specific media. Three weeks later, capacity of CD34+ cells to differentiate into fibroblast like cells was analyzed. The resulting cells were renamed as mesenchymal stem cell “derivated corneal fibroblast-like” (DCFL) cells. Their phenotypes were analysed by imunocytochemistry (ICC) using specific antibodies against CD34, Von Willbrand factor (VWF), 3G5 (CF ganglioside being used as corneal keratocytes marker), stromal derived factor SDF-1 and its receptor CXCR4, VEGF receptors, Aqp 1, and α-SMA. These phenotypes were compared to CF microarray results. Results Microarray indicates corneal fibroblasts expression of SDF1, CXCR4, VEGF C, Flt1 and Flt4 mRNAs. Corresponding proteins were highlighted in corneal fibroblasts and DCFL cells by ICC. Indeed Aquaporin-1, SDF1alpha, CXCR4, VEGF and Flt1 and Flt4 were detected. 3G5 expression was detected on 0,2% DCFL. Both corneal fibroblasts and endothelial cells were stained with anti-alphaSMA antibody whereas only endothelial cells were positive for anti VWF staining. Interestingly, no corneal fibroblasts analysed by gene array (n = 2) and immunohistochemistry (n = 8) expressed CD34 + . Discussion 3G5 antigen is constitutively expressed by cultured human corneal fibroblast and is found in CD34 + BMDF cells which suggests the potential for bone marrow to generate CF like cells. Conclusion These cells, characterized by surface expression of CXCR4 + , SDF1- alpha + , Flt-4 + , VEGF-C + and 3G5 + should be considered as candidates for corneal tissue engineering.