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S. Schrader, M. Notara, S. J. Tuft, M. Beaconsfield, G. Geerling, J. T. Daniels; Simulation of an in vitro Niche Environment That Preserves Conjunctival Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1907.
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Maintenance and expansion of an epithelial stem cell population in vitro is crucial when patient cells have to be expanded for the purpose of transplantation. Aim of this study was to evaluate a serum free system where mitotically active subconjunctival fibroblasts were co-cultured with conjunctival epithelial cells to mimic a niche environment for conjunctival progenitor cells.
Human conjunctival epithelial cells were expanded from bulbar biopsies on a growth arrested 3T3 feeder layer and evaluated for their colony forming efficiency and clonal ability. The cells were then transferred to a serum free co-culture system, where they were cultured in the presence of mitotically active subconjunctival fibroblasts (HCEC-HCF). Cell proliferation dynamics were evaluated by Ki67 staining and total colony forming efficiency (CFE), the number of colonies with a surface area > 10 mm2, the expression of putative progenitor cell markers p63α, ABCG2, CK15 and the presence of MUC5AC and PAS positive cells was compared to standard culture conditions (HCEC-3T3).
Conjunctival epithelial cells cultured under HCEC-HCF and HCEC-3T3 conditions showed strong immunoreactivity to p63α and ABCG2. Co-localisation of CK15 and p63α revealed a subpopulation of CK15 positive cells under HCEC-3T3 conditions, whereas under HCEC-HCF conditions only few CK15 positive cells were found. MUC5AC and PAS positive cells were regularly observed under HCEC-3T3 conditions, whereas under HCEC-HCF conditions these cells were only found very rarely. These results were confirmed by RT-PCR. Cells in HCEC-HCF conditions showed a significantly higher total CFE and also a significantly higher percentage of colonies with holoclone-like morphology compared to HCEC-3T3 conditions.
The simulation of an in vitro niche environment by co-culturing mitotically active subconjunctival fibroblasts with conjunctival epithelial cells supports the maintenance of conjunctival cells with progenitor cell characteristics and therefore might be a useful tool to expand conjunctival epithelial cells with progenitor cell characteristics in vitro for clinical use.
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