culture and proliferation of human limbal stem cells in vitro

purpose: to evaluate the properties of cultured limbal stem cells for corneal surface reconstruction. methods: specimens of limbal explants were prepared from the eye bank of i.r. iran. the explants were cultured and expanded on acellular amniotic membrane for 21 days. the cultured cells were evaluated for expression of connexin 43 and keratinine k3 by immunocytochemistry and expression of k12 keratinine and p63 by rt-pcr. results: after 2 to 3 weeks, the limbal epithelial cells grew to form a sheet approximately 2×2 cm2 in size on the amniotic membrane. on histological examination the epithelial sheet was composed of 4 to 5 cell layers at the margin of the sheet and from 1 to 4 cell layers in the area between the margin and the original explant tissue. immunocytochemistry analysis showed keratinine k3 and connexin 43 were expressed in normal cornea. the markers were expressed weakly in cultured limbal cells. also, rt-pcr analysis revealed that p63 and k12 were expressed in cultured and normal limbal cells. the normal cornea expressed k3 and k12 but not p63. conclusions: these data support the notion that expansion of limbal stem cells on amniotic membrane can be used of tranplantation to patients with limbal stem cell deficiency.