Abstract
The aims of this study were to determine whether human limbal explant cultures without feeder cells result in expansion of epithelial progenitors and to estimate the optimal expansion time for progenitor cells. Limbal explants from ten human corneas were cultured for 7, 9, 11, 14, 18, and 21 days. Limbal explants from two corneas were enzymatically dissociated or directly cultured for 14 days. Progenitor cells were characterized by their ability to form colonies, by immunocytochemistry, and by quantitative real-time polymerase chain reaction. Colonies were identified after 9, 11, 14, and 18 days of culture, but not after 21 days. The number of colonies per explant was significantly higher after 14 days than after 9 and 21 days. The mean percentage of seeded cells giving rise to clones was 4.03% after 14 days of culture and 0.36% for non-cultured dissociated limbal epithelial cells. The number of cells giving rise to clones per cornea significantly increased from an average of 2275 for non-cultured cells to 24266 for cells cultured for 14 days. Immunocytochemical analysis detected positive staining for cytokeratin (CK) 3, CK5/6/8/10/13/18, CK19, vimentin, p63, and p63α, in both cultures and clones. CK3 expression increased significantly with culture time. Transcript expression was observed for CK3, CK19, vimentin, and Delta N p63α at each culture time point, both in cultures and clones. The optimal culture time for limbal explants in cholera toxin-free Green medium without feeder cells was 14 days leading to the expansion of progenitors.
Highlights
The ocular surface is covered with three non-keratinizing epithelia: the transparent corneal epithelium, the conjunctival epithelium, and the limbal epithelium overlying the limbal region which lies between the cornea and the sclera [1]
The stem cell niche of the limbal epithelial cells can be affected by the culture conditions, including murine 3T3 feeder cells, human amniotic membrane (AM), fibrin, and tissue-culture treated plastic
Whereas the presence of progenitors in limbal epithelial cell cultures has been demonstrated through expression of several markers and colony formation assays, little is known about the rate and kinetics of progenitor cell expansion [30,31,32]
Summary
The ocular surface is covered with three non-keratinizing epithelia: the transparent corneal epithelium, the conjunctival epithelium, and the limbal epithelium overlying the limbal region which lies between the cornea and the sclera [1]. In patients with limbal stem cell (SC) deficiency, one of the emerging surgical strategies for restoring the corneal epithelial surface is the transplantation of ex vivo expanded limbal epithelial SCs [3,4,5,6]. This therapeutic approach involves harvesting of small limbal biopsies from either the patient’s contralateral healthy eye or a donor eye, followed by cellexpansion to produce an epithelial sheet on a transplantable carrier such as fibrin or human amniotic membrane [6,7,8,9,10,11,12]. Whereas the presence of progenitors in limbal epithelial cell cultures has been demonstrated through expression of several markers and colony formation assays, little is known about the rate and kinetics of progenitor cell expansion [30,31,32]
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