| BACKGROUND: Limbal epithelial stem/progenitor cells(LESCs)are adult stem cells residing in corneal limbal epithelial basement,playing a vital role in restoring corneal transparency,wound healing,and epithelial integrity.Limbal niche cells(LNCs)have been proven to have a potent capacity in the maintenance of limbal epithelial stem/progenitor cells(LESCs).Four methods have been reported to isolate and expand LNCs: digestion by collagenase alone(C-LNC),collagenase following dispase removal of the limbal epithelium(DC-LNC),dissection of dispase-isolated limbal epithelial sheets(D-LNC),and explant cultures of limbal stromal tissues(Ex-LNC).This study aimed to isolate LNCs using those four methods and compare their capacity to maintain LESCs.METHODS: LNCs were isolated from the rat corneal limbus by the following methods:C-LNC,DC-LNC,D-LNC,and Ex-LNC.RT-q PCR and immunofluorescence staining analyzed the expression of embryonic stem cell(ESC)markers.The ability to maintain LESCs was assessed on the basis of colony-forming capacity and the expression of progenitor,proliferation,and differentiation markers in three-dimensional(3D)Matrigel and Transwell system.Notch signaling of LESCs supported by different LNCs in Tranwell inserts was analyzed by RT-q PCR.RESULTS: DC-LNCs exhibited lower expression of CK12 during isolation and expansion.Among P4-expanded LNCs,DC-LNCs expressed significantly higher levels of Sox2,Oct4,Nanog,and N-cadherin than C-LNCs,D-LNCs,and Ex-LNCs.Compared with other LNCs,DC-LNCs were more effective in maintaining LESCs with higher holoclone-forming efficiency,more expression of ΔNp63α and Ki67,and less expression of CK12.Meanwhile,DC-LNCs were more capable to downregulate Notch signaling of LESCs.CONCLUSIONS: DC-LNCs were more effective in expressing ESC markers and maintaining LESCs compared to other LNCs.This study identifies an optimal method for the isolation of LNCs in corneal epithelial tissue engineering. |
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