Effects of explant size on epithelial outgrowth, thickness, stratification, ultrastructure and phenotype of cultured limbal epithelial cells

Abstract

PurposeTransplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient’s eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function.MethodsHuman limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed.ResultsEpithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = –0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin β1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups.ConclusionFor donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient.

Current Perspectives of Limbal-Derived Stem Cells and its Application in Ocular Surface Regeneration and Limbal Stem Cell Transplantation

“…Moreover, the epithelial cell sheets developed from the limbal explants ex vivo showed greater integrity, as evident from the rich development of desmosomes, hemidesmosomes, and E-cadherins. 20,21 Furthermore, a study reported by Ekpo et al had cultured the cadaver tissues adjacent to the limbus, that is, toward corneal (L.cor) and conjunctival (L.conj) sides. Interestingly, they have noted that the cells cultured from the L.conj had more growth potential and stemness than L.cor, as evident from p63α expression.…”

“…Moreover, the epithelial cell sheets developed from the limbal explants ex vivo showed greater integrity, as evident from the rich development of desmosomes, hemidesmosomes, and E‐cadherins. 20 , 21 …”

Characterization of limbal explant sites: Optimization of stem cell outgrowth in in vitro culture

“…Utheim et al reported that larger (3 mm) limbal explants yield larger outgrowths and are preferable than small (1 mm) explants, although small explant exhibited higher growth rates. No differences between small and large explants were detected when considering other parameters such as explant thickness, strength, and expression of stem cell markers [19]. The explant must be harvested from an area of the limbus that has a sufficient…”

“…Several groups have described the ideal size of a limbal explant that will yield the greatest degree of stemness in cell cultures [16][17][18][19]. In a recent study, stemness of explants from four different locations within the limbus, including superior, inferior, nasal, and temporal, were compared; the results indicated that the superior and inferior limbal areas exhibited greater growth potential [20].…”

Ex vivo cultivated oral mucosal epithelial cell transplantation for limbal stem cell deficiency: a review

“…Immunohistochemistry and RT-PCR have shown that cultured OMECs are positive for keratin (K)3, K4, and K13 [ 13 , 14 , 17 , 21 , 26 , 31 – 33 ], the latter is not expressed in the corneal epithelium [ 51 ]. OMECs also express markers of corneal differentiation connexin 43, laminin 5 [ 52 , 53 ], and putative stem cell markers ß1-integrin, p75, p63, ABCG2, C/EBPδ [ 52 , 54 ]. They do not express corneal-specific K12 and transcription factor PAX6 [ 22 , 31 ].…”