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

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 β1, and integrin-linked kinase (ILK). Nuclear β-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 β-catenin, TCF-4 and deltaNp63α, 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 β1/ILK, which indirectly activated Wnt/β-catenin and subsequently deltaNp63α. Crosstalk between the integrin β1/ILK and Wnt/β-catenin pathways appears to play a crucial role in limbal progenitor cell survival on EBM. Statement of significance We demonstrated the superior capability of carbodiimide cross-linked denuded amniotic membrane (CLDAM) than natural DAM to preserve limbo-corneal epithelial progenitor cells in vitro, then we used CLDAM as a simulated epithelial basement membrane (EBM) to study how EBM maintains limbal epithelial stem cells (LESCs). We found that integrin-linked kinase (ILK) is an important mediator that transfers survival signals detected by integrin β1 to the Wnt/β-catenin pathway, which in turn up-regulates deltaNp63α, a master gene that regulates LESC function. The rougher surface of the limbal EBM suggests that the surface complexity of the LESC niche may be important in regulating LESC function, which is triggered by the recognition of topographic cues by integrin β1, followed by activation of the ILK/Wnt/β-catenin/p63 cascade.