Generation of Human Corneal Fibroblasts-Like Phenotype From Mesenchymal Stem Cells (msc) Differentiation in vitro

Abstract

Purpose: : Tissue engineering offers considerable promise in damaged tissues repair or replacement. In the cornea, fibroblasts result from mesodermal cell extension into optic cap. They participate in the genesis of corneal stroma during the fifth week of embryonic development. Here, we used MSC to generate corneal fibroblast phenotype cells for corneal tissue engineering and potential clinical applications.

Methods: : Genotype of corneal fibroblasts (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 or co-cultured with isolated corneal epithelial cells. 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 immunocytochemistry 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, vascular endothelial growth factor receptor (VEGFRs), Aquaporin 1, and α-SMA (α-smooth musculus actin). Finally, these phenotypes were compared to CF microarray analysis results.

Results: : Microarray analysis indicates corneal fibroblasts expression of SDF1, CXCR4, VEGF C, Flt1 and Flt4 mRNAs. Corresponding proteins were highlighted in corneal fibroblasts and DCFL cells by immunocytochemistry. 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⁺.

Conclusions: : 3G5 antigen is constitutively expressed by cultured human corneal fibroblast and is found in CD34⁺ bone marrow derived fibroblasts cells which suggests the potential for bone marrow to generate corneal fibroblast like cells. 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.

Keywords: cornea: stroma and keratocytes • differentiation