Aim: 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.
Methods: Human conjunctival epithelial cells were expanded in vitro and evaluated for their colony-forming efficiency and clonal ability. The cells were then transferred to a serum-free co-culture system and cultured in the presence of mitotically active subconjunctival fibroblasts (human conjunctival epithelial cells and human bulbar subconjunctival fibroblasts [HCEC-HCF]). Cells were evaluated by Ki67 staining, total colony-forming efficiency and the number of colonies with a surface area of more than 10 mm(2). The expression of putative progenitor cell markers p63α, ABCG2 and CK15, and the presence of MUC5AC- and periodic acid-Schiff-positive cells was compared with standard culture conditions (HCEC-3T3).
Results: Conjunctival epithelial cells cultured under HCEC-HCF and HCEC-3T3 conditions demonstrated strong immunoreactivity to p63α and ABCG2. Co-localization of CK15 and p63α revealed a subpopulation of CK15-positive cells under HCEC-3T3 conditions compared with only a few CK15-positive cells found under HCEC-HCF conditions. MUC5AC- and periodic acid-Schiff-positive cells were much more common under HCEC-3T3 conditions than under HCEC-HCF conditions. These results were confirmed by reverse transcription-PCR. Cells in HCEC-HCF conditions demonstrated a significantly higher total colony-forming efficiency and a significantly higher percentage of colonies with holoclone-like morphology.
Conclusions: The simulation of a niche environment in vitro 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 progenitor cells in vitro for clinical use.