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Gary Hin-Fai Yam, Nur Zahirah Binte M Yusoff, Lei Zhou, Jodhbir S Mehta; Amnion Stromal Extract to Ex Vivo Propagate "Activated Keratocytes" from Human Corneal Stroma. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5135.
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© ARVO (1962-2015); The Authors (2016-present)
Human corneal stromal keratocytes (CSK) are quiescent but become proliferative fibroblasts under serum and cytokine culture with a total loss of keratocyte features. Although these proliferating cells play a crucial role in corneal wound repair, development of corneal haze and opacity is a problem. CSK expansion without fibroblast transition is therefore critical for corneal tissue engineering and ocular clarity. Here, we demonstrate the utility of stromal niche protein factors in the ex vivo maintenance of human CSK.
Primary human CSK were cultivated with soluble human amnion stromal extract (ASE), ROCK inhibitor Y-27632 and Insulin-like growth factor (collectively named as ERI) under serum-free or serum-added conditions. The cells were characterized for CSK markers (including ALDH1A1, lumican and keratocan) and fibroblast markers (F-actin stress fiber, α-smooth muscle actin αSMA) by immunofluorescence and quantitative PCR as well as collagen gel contraction assay. The ASE effect on fibroblast suppression was further examined by TGFβ-mediated Smad2 signaling in CSK cultured with various ASE fractions (with respect to molecular weight MW range) obtained by differential centrifugal filtration. The protein composition of the optimal fraction was profiled by nanoLC-MS/MS and subjected to MetaCoreTM for significant pathway analysis (P<0.001).
ERI supplement maintained human CSK with typical dendritic morphology even under serum culture. The condition expanded a population of "activated keratocytes" with loss of both keratocyte and fibroblast marker expression, and lack of fibroblast-mediated collagen contractibility. These cells could propagate for 10 culture passages and regained CSK marker expression, including keratocan biosynthesis, expression and secretion, when returned to serum-free ERI condition. ASE fraction without low MW proteins showed the highest efficiency to inhibit TGFβ-mediated Smad2 signaling to CSK nuclei. A total of 335 proteins were mapped by peptide homology ≥95% and 12 significant pathways (P<10-4 and false discovery rate <10-3) were predicted, which included TGFβ signaling, cytoskeleton and extracellular matrix modeling and immune response.
ERI supplement allowed the ex vivo propagation of "activated keratoctyes", which could revert to bona fide CSK for stromal tissue engineering without the risk of fibroblast development.
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