title: Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells.
abstract: The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.
authors.affiliation: Queensland Eye Institute, 140 Melbourne St, South Brisbane, QLD 4101, Australia. Electronic address: jennifer.young@qei.org.au.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Walshe
authors.affiliation: Queensland Eye Institute, 140 Melbourne St, South Brisbane, QLD 4101, Australia; School of Biomedical Sciences, Queensland University of Technology, 2 George Street, Brisbane, Queensland 4001, Australia; Institute of Health and Biomedical Innovation, 60 Musk Avenue, Kelvin Grove, Queensland 4059, Australia. Electronic address: d.harkin@qut.edu.au.
authors.forename: Damien G
authors.initials: DG
authors.lastname: Harkin
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D053673:Glypicans / Q000378:metabolism; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D064231:Nestin / Q000378:metabolism; D010641:Phenotype; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D053673:Glypicans; C577484:NES protein, human; D064231:Nestin
keywords: cornea; endothelium; explant; glypican-4; limbus; nestin; progenitor; telomerase