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T. Kawakita, E.M. Espana, A.C. Romano, M. Di Pascuale, S.C. Tseng; Isolation and Differentiation of Intact Human and Rabbit Limbal Epithelial Sheets . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1347.
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© ARVO (1962-2015); The Authors (2016-present)
Objective: We would like to determine whether stemness of the limbal epithelium is influenced by preserving cell-cell junctions in a sheet, and by exposing it to different substrate and 3T3 fibroblasts (3T3). Methods: Fresh human and rabbit pigmented limbus were incubated in SHEM containing 50 mg/ ml Dispase II and 100 mM sorbitol for 18 h at 4 oC. Human limbal sheets (hL) were subjected to immunostaining with antibodies against keratin 3 (K3), connexin 43 (Cx43), p63 and integrins (In) ß1 and ß4, collagens (Coll) IV and VII and Laminin (Ln)-5. Rabbit limbal sheets (rL) were seeded directly or rendered into single cells by trypsin/EDTA on plastic with or without 3T3, or on epithelially denuded (dAM) or intact (iAM) AM. After confluence, epithelial sheets were removed by Dispase II at 37 oC for 5 min, reseeded on dAM for 3 days and air-lifted for 4 days. The resultant epithelial phenotype was determined by immunostaining to K3. Results: Isolated hL and rL retained pigmented palisades and exhibited large superficial squamous cells and small basal cuboidal cells. The basal layer of hL was negative to K3, Cx43, Ln-5 and Coll-VII, linearly positive to In ß4, scatteredly positive to p63 and Coll-IV. The remaining stroma was devoid of Ln-5 and Coll-IV but positive to Coll-VII. K3 staining of rL was full-thickness positive when seeded on dAM, but basally negative when seeded on iAM, suggesting stemness was preserved in iAM but lost in dAM. When rL was rendered single cells and cultured on plastic, K3 was full thickness positivity to K3 when reseeded on dAM. In contrast, rL on plastic without 3T3 fibroblasts showed sporadic positivity to K3, and with 3T3 fibroblasts retained basal negativity to K3 when reseeded on dAM. Conclusion: Both rabbit and human limbal sheets can be reproducibly isolated by cleaving the plane between lamina densa and lamina lucida of the basement membrane, and isolated sheets contain viable basal stem cells. The stemness of such isolated limbal sheets is adversely affected by disrupting cell-cell junctions and by plastic culturing, but preserved by coculturing with 3T3 or intact AM.
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