The p63 gene generates transactivating and N-terminally truncated transcripts (⌬Np63) initiated by different promoters. Alternative splicing gives rise to three different C termini, designated ␣, ␤, and ␥. In the ocular epithelium, the corneal stem cells, which are segregated in the basal layer of the limbus, contain the ␣ isoform but not ␤ or ␥. Holoclones derived from the limbus are rich in ␣, meroclones contain little, and paraclones contain none. In normal resting corneal epithelium, p63 of all isoforms is absent. Upon corneal wounding, cells originating from the limbus and containing ␣ migrate progressively through the epithelium of the peripheral and central cornea. In the absence of an attached limbus, no ␣ isoform appears in the corneal epithelium. When migrating cells containing the ␣ isoform appear in the wounded corneal epithelium, they are confined to the basal layer, but the suprabasal cells, not only of the cornea but of the limbus as well, contain mRNA encoding ␤ and ␥. These data support the concept that the ␣ isoform of p63 is necessary for the maintenance of the proliferative potential of limbal stem cells and their ability to migrate over the cornea. The ␤ and ␥ isoforms, being suprabasal and virtually absent from the resting limbus, are not stem cell markers but are likely to play a role in epithelial differentiation specifically during the process of corneal regeneration. The corneal epithelium provides an ideal experimental system in which to distinguish keratinocyte stem and TA cells (8). Corneal stem cells are segregated in the basal layer of the limbus, which is the zone encircling the cornea and separating it from the bulbar conjunctiva. TA cells that migrate from the limbus form the corneal epithelium (8). That the limbus is the site of stem cell precursors of the corneal epithelium is clear for several reasons: (i) the basal layer of the limbus lacks keratin 3 (a marker for corneal differentiation), whereas limbal suprabasal layers and all layers of the corneal epithelium express keratin 3 (9); (ii) the limbus contains slow-cycling cells and holoclone-forming cells, but the corneal epithelium does not (7, 10); (iii) the corneal epithelial cells are not self-sustaining; they divide only a few times during their migration from the limbus to the central cornea (11); (iv) restoration of destroyed limbal͞corneal epithelium requires limbal transplantation (12) or grafts of autologous limbal cultures (13-15).The gene with the most striking effects on the development of stratified epithelia is p63 (16-19). Ablation of the p63 gene in mice results in the absence of these epithelia (17,18). In humans, mutations of the p63 gene cause disorders of the epithelia and of nonepithelial structures whose development depends on the epithelial functions (20). The p63 gene generates six isoforms (21). The transactivating isoforms are generated by the activity of an upstream promoter; the ⌬N isoforms are produced from a downstream intronic promoter and lack the transactivation domain. For both transcripts,...

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Descemet membrane endothelial keratoplasty tissues can be tri-folded (endo-in) with no significantly less cell loss as compared to endo-out. Spontaneous unfolding of endo-in may reduce overall time and surgical manipulation.

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Human corneal endothelial cells (HCEnCs) are responsible for maintaining the transparency of the cornea. Damaged or diseased HCEnCs may cause blindness. Replacement of the diseased cells with a healthy donor endothelium is the only currently available treatment. Tissue-engineering can serve as an alternative to conventional donor corneal transplantation. Due to the global shortage of donor corneas, a wide interest in the development of cultured graft substitutes and artificial corneas has increased. Availability of the old donor corneas is higher especially for research. Although it can be proposed as a valuable source for cell culture, its less proliferative capability emerges a challenge for the researchers. This article describes the use of hyaluronic acid (HA) in combination with Rho-kinase inhibitor (ROCK) Y-27632 for the cultivation of HCEnCs from older donor corneas (age > 60 years). Four conditions including and excluding HA + ROCK and its effect on early attachment rates and proliferation was studied on forty-eight corneas. It was observed that HCEnCs reach confluence within 10–15 days when cultured with HA + ROCK. This approach improves the efficiency of cell adhesion due to force attachment. HCEnCs from old donor corneas can be cultured using this method which may further lead to cell-based therapy for treating corneal endothelial dysfunction.