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C. Jacobi, E. A. Blazejewska, E. Nkenke, T. Inatomi, S. Kinoshita, F. E. Kruse, U. Schlötzer-Schrehardt; Phenotypic Programming of Human Oral Mucosal Epithelial Stem Cells by Limbus-Specific Environmental Culture Conditions. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6067. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In order to improve the currently used culture technique for oral mucosal epithelial cells, we analyzed the effect of culture conditions mimicking the limbal microenvironment on the cellular phenotype and differentiation of oral mucosal epithelial cells in vitro.
Buccal mucosa biopsies were obtained from patients undergoing dental surgery after informed consent. Dissociated epithelial cells were enriched for stem and progenitor cell populations by clonal growth on 3T3 feeder cells and subcultivated on various extracellular matrices (collagen type IV, laminin-1, laminin-5, fibronectin) in serum-free DMEM/F12 calcium low-medium supplemented with different conditioned media which were harvested from cultured human fibroblasts derived from central cornea, peripheral cornea and limbal stroma as well as from 3T3 fibroblasts. Growth potential, proliferative activity and epithelial phenotype were evaluated by light and electron microscopy, BrdU labelling, and immunohistochemistry using antibodies to progenitor cell (ABCG2) and differentiation markers characteristic for corneal epithelium (K3/K12) or epidermis (K10).
Rapid cell adhesion, proliferation, and generation of confluent, regularly arranged epithelial cell sheets were obtained after seeding clonal cells on laminin-5, a major component of the corneal and limbal basement membrane zone, and on collagen type IV, whereas laminin-1 and fibronectin adversely affected cell adhesion and growth. Addition of conditioned media differentially influenced cellular phenotype and differentiation. Medium derived from limbal fibroblasts produced a corneal epithelium-like cellular phenotype and markedly increased the number of ABCG2 and K3/K12-immunopositive cells while decreasing expression of K10 in cultivated cell sheets as compared to the other conditioned media and controls.
The phenotypic manipulation of oral mucosal epithelial stem cells through modulation of limbus-specific environmental factors suggests that stromal-epithelial interactions are crucial for the development of corneal epithelial cell sheets ex vivo and may provide the basis for an improved tissue-engineering strategy for the purpose of ocular surface reconstruction.
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