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T. Mimura, S. Yamagami, T. Usui, S. Yokoo, K. Ono, M. Araie, S. Amano; Isolation of Adult Stem Cell From Corneal Limbal Epithelium and Autologous Transplantation for Limbal Stem Cell Deficiency in Rabbit . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2080.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To isolate adult stem cells of corneal limbal epithelial cells and investigate a novel strategy for total limbal stem cell deficiency. Methods: An ocular surface injury was created in right eye of each of thirty adult New Zealand white rabbits by a lamellar keratectomy. Autologous adult stem cells, isolated from rabbit corneal limbal epithelial cells by sphere–forming assay were cultivated for 2 weeks on a denuded amniotic membrane. The sphere colonies and their progeny were examined by immunocytochemistry and RT–PCR. At 3 to 4 weeks after the ocular surface injury, the conjunctivalized corneal surfaces were surgically removed and the autologous cultivated adult stem cells were transplanted (sphere group). The no transplantation, AM (rabbits with an amniotic membrane transplantation only), and limbs–2 and 4 weeks (rabbit with transplantation of corneal limbal epithelial cells cultured on amniotic membrane for 2 or 4 weeks) groups were the controls. Each group, observed for 8 weeks after surgery, was underwent external examination and immunhistochemical analysis. Results: Immunocytochemistry and/or RT–PCR shows the sphere colonies expressed BrdU, P63, P75NGFR and nestin and the progeny expressed cytokeratins 3, cytokeratin 12, vimentin, α–SMA, NSE, and MAP2. The cultivated epithelial stem cells for 2 weeks were completely stratified over ten layers on denuded amniotic membrane and expressed both P63 and P75NGFR throughout the epithelium, whereas cultured limbal epithelium on amniotic membrane for 4 weeks consisted of 4–5 layers and expressed these markers at only the basal layer 8 weeks after transplantation. In the limbs–4 weeks and the sphere groups, injured corneas showed no epithelial defects, no neovascularization into the cornea, and improved corneal clarity. Histologically corneal epithelium formed monolayer in the AM, double layers in the limbs–2 weeks, 4–5 layers in the limbs–4 weeks, and approximately 10–12 layers in the sphere group. The new stratified epithelium showed the highest expression levels of P63 and P75NGFR in the sphere group as compared with the other groups. Conclusions: We have isolated adult stem cells of rabbit corneal limbal epithelium by sphere–forming assay. Autologous adult stem cell transplantation can be an effective strategy to supply a large number of possible stem cell marker, P63–postive cells, for corneal epithelial cell deficiency.
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