Preservation of human limbal epithelial progenitor cells on carbodiimide cross-linked amniotic membrane via integrin-linked kinase-mediated Wnt activation

摘要

The Wnt pathway is a major signaling pathway that regulates corneal epithelial stem cells. However, little is known about how the ultrastructure of the limbal epithelial basement membrane (EBM) affects Wnt activity. Due to its enhanced matrix stability, the cross-linked amniotic membrane (AM) has gained increasing interest in the field of regenerative medicine. For the first time, we used EDC/NHS cross-linked denuded AM (CLDAM) as a simulated EBM substrate to investigate this mechanism. Human limbal epithelial (HLE) cells were cultured on dishes (HLE/dish), denuded AM (HLE/DAM) or CLDAM (HLE/CLDAM). Compared with HLE/dish or HLE/DAM cultures, HLE/CLDAM cultures showed greater BrdU retention and colony formation efficiency and expressed higher levels of p63, ABCG2, integrin beta 1, and integrin-linked kinase (ILK). Nuclear beta-catenin and TCF-4 levels were higher in HLE/CLDAM cultures compared with HLE cells cultured on collagen IV, laminin, Matrigel, or DAM. Silencing of ILK in HLE/CLDAM cultures resulted in decreased levels of nuclear beta-catenin, TCF-4 and deltaNp63 alpha, whereas cytokeratin 12 expression increased. Over-expression of ILK in HLE/dish cultures had the opposite effects. Accordingly, we proposed that the CLDAM matrix, with its higher rigidity and rougher ultrastructure, better preserved HLE progenitor cells in vitro, possibly by activating integrin beta 1/ILK, which indirectly activated Wnt/beta-catenin and subsequently deltaNp63 alpha. Crosstalk between the integrin beta 1/ILK and Wnt/beta-catenin pathways appears to play a crucial role in limbal progenitor cell survival on EBM.