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

Paper ID Volume ID Publish Year Pages File Format Full-Text
139 11 2016 12 PDF Available
Title
Preservation of human limbal epithelial progenitor cells on carbodiimide cross-linked amniotic membrane via integrin-linked kinase-mediated Wnt activation
Abstract

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 significanceWe 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.

Graphical abstractSchematic illustration of the interaction of ILK and Wnt pathway with subsequent activation of p63 transcription to preserve limbo-corneal epithelial progenitor cells. Topographic cues on the basement membrane components of cross-linked amniotic membrane such as laminins are recognized by the integrins. Upon binding with the ligands, integrin β1 undergoes dimerization and clustering, and focal adhesion kinase (FAK) and integrin-linked kinase (ILK) which are connected to integrin β1 are phosphorylated and activated. ILK in turn inhibits the phosphorylation of APC–Axin–GSK3β complex and degradation of β-catenin. Free β-catenin can then translocate to the nucleus and together with TCF/LEF transcription factors to activate the transcription of delta Np63α, the master regulator of corneal epithelial stem cells.Figure optionsDownload full-size imageDownload high-quality image (240 K)Download as PowerPoint slide

Keywords
Limbal epithelial stem cells; Carbodiimide cross-linked amniotic membranes; Integrins; Wnt signaling pathways; Stem cell–microenvironment interactions
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Preservation of human limbal epithelial progenitor cells on carbodiimide cross-linked amniotic membrane via integrin-linked kinase-mediated Wnt activation
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Publisher
Database: Elsevier - ScienceDirect
Journal: Acta Biomaterialia - Volume 31, February 2016, Pages 144–155
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us