| Limbal epithelial stem cells are completely lost or functional incapacitation in patients who have severe eye disease or trauma,leading to corneal vascularization,opacification and severe visual loss.Corneal reconstruction with limbal epithelial stem cells can be facilitated by transplantation of autologous limbal epithelial stem cells.But the treating bilateral limbal stem cell deficiencies are very limited.In recent years,there are some attempts on the application of stem cells that are not from eyes in reconstructing corneal epithelium,such as embryonic stem cells,oral mucosal epithelial progenitor cells,bone marrow mesenchymal stem cells and epidermal stem cells.At present,it is well established that many adult stem cells reside in the bulge area of hair follicle,which are easy acquired and plentiful.Hair follicle stem cells are multipotent as seed cells.The purpose of study approached for hair follicle stem cells thansdifferentiated into corneal epithelial cells,as a cell resource of reconstructing corneal epithelium.Differentiation and transdifferentiation of stem cells are regulated by many factors.Pax6 is a master control gene of cornea development,and is important in function of corneal epithelium.Wnt signal process has emerged as a critical regulator of stem cells,including hair follicle stem cells.In present investigation,the potential of hair follicle stem cells tansdifferentiation into corneal epithelial cells in vitro was discussed,and the expression changing of Pax6 andβ-catenin/Lef-1 were measured.The effects of Pax6 were studied.The reconstruction of hair follicle stem cells into corneal epithelium in vivo was observed.The main results are the following:Culture and characteristics of hair follicle stem cellsHair follicle stem cells were enriched and cultivated byⅣcollagen,and were characterized.The results indicated that hair follicle stem cells could be enriched by selective adhesion to typeⅣcollagen.Cultured cells had homogeneous shape and a large nuclear/cytoplasmic ratio,which suggest a primitive morphologic feature.The high colony-forming efficiency indicated cells proliferated rapidly.The expression of CD34 andα6-integrin were positive.2.transdifferentiation of hair follicle stem cells into corneal epithelial-like cells in vitroHair follicle stem cells were induced in the condition medium prepared by limbal tissue homogenate.The results indicated that hair follicle stem cells transdifferentiated into corneal epithelial-like cells in the condition medium,and the expression of keratin 12 is positive.In the induced process,the Pax6 was up regulated,and promoted the transdifferentiation.Wnt signal process was inhibited,appearanced as down-regulation of Lef-1 and translocation ofβ-catenin from nuclear to cytoplasmic.Induced cells formed barrier function,similar to normal corneal epithelum.3.Effect of Pax6 in the transdifferentiationOur results indicated that Pax6 also expressed in rat whisker hair follicle,similar to corneal epithelium.However,the expression of Pax6 is majored in Pax6-5a,which is majored in another transcript of Pax6.The cells transfected Pax6 expressed K12,which indicated that Pax6 is important in the tansdifferentiation of hair follicle stem cells and promoting the k12 expression.The inhibition ofβ-catenin and Lef-1 in trandifferentiation contributed to the condition medium,not to Pax6.The inhibition ofβ-catenin and Lef-1 is related to the expression of DKK2 in cornea.4.Effects of hair follicle stem cells on corneal epithelium reconstructionThe animal models of limbal stem cells deficiency were prepared by alkali burn.The carrier-free hair follicle stem cells sheets prepared by temperature-responsive cell culture surface transplanted onto injured corneal surface.The results indicated that hair follicle stem cells could reconstruct injured corneal epithelium,improve corneal clarity and inhibit new vessels.Hair follicle stem cells formed the epithelium expressing K12.The expression of MMP2 and MMP9 were decreased,contributed to inhibition of inflammation.But the mechanism was not clear.The main conclusion:The investigation indicated that hair follicle stem cells have the potential of tansdifferentiation into corneal epithelial-like cells.In the induced process,Pax6 was up-regulated,whileβ-catenin and Lef-1 were down-regulated.Pax6 was a key factor for regulating K12 expression.Hair follicle stem cells tansdifferentiation into corneal epithelial-like cells in vivo,and reconstructed injured corneal surface.The study opened a new path to the resource of seed cells in reconstructing corneal epithelium,and provided a new strategy.
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