Hypoxic culture in the isolation of corneal stem cells

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We succeeded in the isolation of stem cells from the mouse corneal stroma. We named these cells as cornea-derived progenitors (COPs) that we published in Stem Cells. COPs are neural crest derived, multipotent stem cells that have the ability to differentiate into corneal keratocytes, fibroblasts, neural cells as well as glial cells and adipocytes. COPs were propagated for over 10 passages in serum-free medium. We have also started the isolation of human COPs, and plan to find a protocol that allows the long-term culture of human COPs.We also devised a culture method to efficiently culture corneal epithelial progenitor cells under hypoxia. Under 2% 02, cytokeratin 12 (K12) negative progenitor cells proliferated more efficiently than under normoxia. Colony forming efficiency (CFE) was also higher under hypoxia indicating that immature cells are selectively cultivated. The efficient culture of corneal epithelial progenitors and stem cells will have impact on regenerative medicine. We already have the technique to engineer stratified epithelial sheets using progenitor cells. We also found from the current investigation that using 2 layers of feeder cells produces epithelial sheets that resemble the limbal epithelial phenotype. We also reported that K15 could be used as a marker of limbal progenitors cells, which can be used to characterize epithelial cells in stratified cells sheets. There are still many points that require clarification in elucidating the limbal epithelial stem cell niche, and we plan to pursue this further in upcoming projects.