{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,3]],"date-time":"2026-11-03T00:32:49Z","timestamp":1793665969878,"version":"4.5.1"},"reference-count":64,"publisher":"SAGE Publications","issue":"9","license":[{"start":{"date-parts":[[2015,9,1]],"date-time":"2015-09-01T00:00:00Z","timestamp":1441065600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/journals.sagepub.com\/page\/policies\/text-and-data-mining-license"}],"content-domain":{"domain":["journals.sagepub.com"],"crossmark-restriction":true},"short-container-title":["Cell Transplant"],"published-print":{"date-parts":[[2015,9]]},"abstract":"<jats:p>Keratoconus is a corneal disorder characterized by a thinning of stromal tissue, and the affected patients have induced astigmatism and visual impairment. It is associated with a loss of corneal stromal keratocytes (CSKs). Hence, reconstructing stromal tissue with autologous CSK replacement can be a viable alternative to corneal transplantation, which is restricted by the global donor material shortage and graft rejection. Human CSKs are normally quiescent and express unique markers, like aldehyde dehydrogenases and keratocan. In serum culture, they proliferate, but lose their characteristic phenotype and become stromal fibroblasts. Here we report a novel culture cocktail to ex vivo propagate and maintain CSKs. Primary human CSKs were obtained from adult donors and cultured with soluble human amnion stromal extract (ASE), rho-associated coiled-coil-forming protein serine\/threonine kinase inhibitor Y-27632, and insulin-like growth factor-1 (collectively named as ERI). Protein profiling using mass spectrometry followed by MetaCore\u201d pathway analysis predicted that ASE proteins might participate in transforming growth factor-\u03b2 (TGF-\u03b2) signaling and fibroblast development, cell adhesion, extracellular matrix remodeling, and immune response. In culture with 0.5% fetal bovine serum and ERI, the population of \u201cactivated keratocytes\u201d was expanded. They had much lowered expression of both keratocyte and fibroblast markers, suppressed TGF-\u03b2-mediated Smad2\/3 activation, and lacked fibroblast-mediated collagen contractibility. These \u201cactivated keratoctyes\u201d could be propagated for six to eight passages ex vivo, and they regained CSK-specific dendritic morphology and gene marker expression, including aldehyde dehydrogenases, lumican, and keratocan biosynthesis, expression, and secretion when returned to serum-depleted ERI condition. This novel cocktail maintained human CSKs in both adherent and suspension cultures with proper keratocyte features and without the transformation to stromal fibroblasts. Thus, human CSKs can be ex vivo propagated as transient \u201cactivated keratocytes.\u201d This could provide sufficient number of genuine CSKs for corneal tissue engineering.<\/jats:p>","DOI":"10.3727\/096368914x685069","type":"journal-article","created":{"date-parts":[[2015,8,28]],"date-time":"2015-08-28T22:04:55Z","timestamp":1440799495000},"page":"1845-1861","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":43,"title":["Ex Vivo Propagation of Human Corneal Stromal \u201cActivated Keratocytes\u201d for Tissue Engineering"],"prefix":"10.1177","volume":"24","author":[{"given":"Gary Hin-Fai","family":"Yam","sequence":"first","affiliation":[{"name":"Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore"},{"name":"Duke-NUS Graduate Medical School, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nur Zahirah Binte M.","family":"Yusoff","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aishwarya","family":"Kadaba","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dechao","family":"Tian","sequence":"additional","affiliation":[{"name":"Department of Statistics and Applied Probability, National University of Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Htoon Hla","family":"Myint","sequence":"additional","affiliation":[{"name":"Singapore National Eye Centre, Singapore"},{"name":"Duke-NUS Graduate Medical School, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roger W.","family":"Beuerman","sequence":"additional","affiliation":[{"name":"Duke-NUS Graduate Medical School, Singapore"},{"name":"Ocular Proteomics Group, Singapore Eye Research Institute, Singapore"},{"name":"Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Zhou","sequence":"additional","affiliation":[{"name":"Duke-NUS Graduate Medical School, Singapore"},{"name":"Ocular Proteomics Group, Singapore Eye Research Institute, Singapore"},{"name":"Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jodhbir S.","family":"Mehta","sequence":"additional","affiliation":[{"name":"Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore"},{"name":"Singapore National Eye Centre, Singapore"},{"name":"Duke-NUS Graduate Medical School, Singapore"},{"name":"Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"179","published-online":{"date-parts":[[2015,9,1]]},"reference":[{"key":"bibr1-096368914X685069","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0056831"},{"key":"bibr2-096368914X685069","doi-asserted-by":"publisher","DOI":"10.1097\/01.ico.0000160970.58330.09"},{"key":"bibr3-096368914X685069","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.08-3276"},{"key":"bibr4-096368914X685069","doi-asserted-by":"publisher","DOI":"10.1002\/stem.91"},{"key":"bibr5-096368914X685069","doi-asserted-by":"publisher","DOI":"10.1046\/j.1442-9071.2001.d01-16.x"},{"key":"bibr6-096368914X685069","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.03-0484"},{"key":"bibr7-096368914X685069","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.04-0201"},{"key":"bibr8-096368914X685069","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.08-3077"},{"key":"bibr9-096368914X685069","doi-asserted-by":"publisher","DOI":"10.1016\/j.matbio.2010.05.003"},{"key":"bibr10-096368914X685069","first-page":"2720","volume":"51","author":"Foty R.","year":"2011","journal-title":"J. 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