Rat Limbal Niche Cells Can Induce Transdifferentiation Of Oral Mucosal Epithelial Cells Into Corneal Epithelial-Like Cells For Treatment Of Limbal Stem Cell Deficiency

Purpose: Cultivated oral mucosal epithelial cells are widely used in the treatment of limbal stem cell deficiency(LSCD)for their ocular reconstruction capability.As the most important component of the limbal microenvironment,limbal niche cells(LNCs)play a key role in the direction of stem cell differentiation.This study investigated whether LNCs can induce the transdifferentiation of rat oral mucosal epithelial cells(OMECs)to corneal epithelial-like cells.Methods: OMECs and LNCs were isolated from rats by dispase and collagenase,respectively,to establish a 3D or Transwell co-culturing system composed of them.3T3 and renewed LNCs were also used as feeder layers in the Transwell system to compare their ability to support the OMECs.The airlift method was used for the culture of OMECs to obtain a stratified epithelial sheet.Co-cultured OMECs were characterized by reverse transcriptionpolymerase chain reaction,western blotting,H-E staining and immunohistochemistry.Results: The co-cultured OMECs showed corneal epithelial-like morphology and expressed corneal epithelial markers CK12 and Pax6 in most co-cultured systems.Furthermore,it is observed that the expression level of CK12,Pax6,and proliferation marker Ki67 was upregulated when compared with that of other groups by renewing the LNCs in the Transwell system,suggesting that this might be a potential method for improving the efficiency of transdifferentiation.The obtained stratified epithelial sheet expressed CK3 and CK12.Conclusion: Through co-culturing OMECs and LNCs in vitro,corneal epithelial-liked OMECs were successfully obtained.This investigation is of great significance for the treatment of LSCD and ocular surface reconstruction.Purpose: Limbal stem cell deficiency is a serious threat to the visual acuity of patients.This investigation is to observe the ability of oral mucosal epithelial stem cells co-cultured with limbal niche cells to treat limbal stem cell deficiency.Methods: A rat model of limbal stem cell deficiency was established by alkali burns.After digesting the multilayer oral epithelial cells cultured in Transwell into a single cell,subconjunctival injection was performed.The corneal transparency,corneal fluorescein sodium staining and the length and area of neovascularization were compared between the threated group and the control group.CM-Dil was used to label the injected cells and observe their migration and survival after subconjunctival injection.Results: HE-staining,impression cytology and immunofluorescence staining confirmed that the rat model of limbal stem cell deficiency was successfully constructed by alkali burn.Subconjunctival injection of transdifferentiated oral mucosal epithelial cells had less neovascularization area score than PBS injection group,and had a certain role in preventing conjunctival invasion after alkali burn.14 days after injection,a few CM-Dil labeled cells entered the corneal limbus and still expressed the corneal specific marker CK12.Conclusion: Subconjunctival injection of transdifferentiated oral mucosal epithelial cells has certain effect on treating limbal stem cell deficiency,preventing conjunctival invasion and maintaining normal corneal phenotype.