Effects Of Limbal Niche Cells On The Culture Of Oral Mucosal Epithelial Cells In Vitro

Background: Autologous cultivated oral mucosal epithelial transplantation(COMET)is an important treatment for limbal stem cell deficiency.However,peripheral corneal neovascularization after surgery hinders its application.This study aims to employ a culture system using allogenic limbal niche cells(LNCs)instead of mouse-derived 3T3 cells as feeder layer that could relieve postoperative neovascularization.Methods: Rat oral mucosal epithelial cells(OMECs)were co-cultured with rat LNCs or 3T3 cells.Cultivated oral mucosal epithelial cells(COMECs)of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry.The expression levels of the angiogenesis-related factors were analyzed by RT-q PCR and Western Blotting/ELISA.Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells(HUVECs).Results: COMECs were obtained from both culture systems successfully.Immunocytochemistry showed approximately equal percentages of positive staining cells for p63?,ABCG2,Ki67,and CK3 in COMECs of different groups.RT-q PCR and Western Blotting/ELISA showed COMECs of the LNCs group expressed a significantly lower amount of b FGF but more PEDF and s Flt-1 than the COMECs of the 3T3 group.Furthermore,compared with COMECs of 3T3 group,COMECs of LNCs group could reduce the viability and tube formation of HUVECs.Conclusions: LNCs could substitute 3T3 cells for expanding OMECs in vitro,and the COMECs obtained in this system are less likely to induce postsurgical neovascularization,which provide an alternative option for ex vivo culture system and promote the application of COMET.Background: Autologous cultivated oral mucosal epithelial transplantation(COMET)is one of the ocular surface reconstruction strategies when severe bilateral limbal stem cell deficiency(LSCD)occurs,which is not limited by insufficient donor or long-term immunosuppressive therapy.We have previously used limbal niche cells(LNCs)as feeder cells to establish a culture system for oral mucosal epithelial cells(OMECs),which has the potential to inhibit peripheral corneal neovascularization.This study attempts to further validate this effect in animal models.Methods: The LSCD model was established using japanese white rabbits,and tested by slit lamp biomicroscope and H&E staining.The OMECs suspension was planted onto denuded amniotic membrane and co-cultured with LNCs or 3T3 cells.The cultured oral mucosa epithelial cells(COMECs)were transplanted into the model eyes(COMET-LNCs or COMET-3T3).The reconstruction of each group was compared by slit lamp biomicroscope examination and immunochemical staining.The expression of corneal angiogenesis-related factors in each group was compared by RT-q PCR and Western Blotting.Results: After modeling,the model eyes showed corneal opacity,conjunctivalization and neovascularization,and there was no tendency to recover.After COMET,corneas were relatively restored,with the exception of mild neovascularization in one cornea of the COMET-3T3 group.CD34 was detected in COMET-3T3 group corneas.Corneas of the COMET-LNCs group expressed high levels of PEDF and s Flt-1,but low levels of b FGF,compared with expression in COMET-3T3 corneas.Conclusions: In animal experiments,oral mucosal epithelial cells cultured with LNCs could be used for COMET surgery with a positive therapeutic effect,and could decrease the risk of neovascularization after COMET.