Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.

A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2 × 2 × 0.25 mm) were harvested from 77 cadavers and expanded e...

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Autores principales:	Réka Albert, Zoltán Veréb, Krisztián Csomós, Morten C Moe, Erik O Johnsen, Ole Kristoffer Olstad, Bjørn Nicolaissen, Eva Rajnavölgyi, László Fésüs, András Berta, Goran Petrovski
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c724980dc8784c869d5e105ab87d26dc
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spelling	oai:doaj.org-article:c724980dc8784c869d5e105ab87d26dc2021-11-18T08:12:40ZCultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.1932-620310.1371/journal.pone.0047187https://doaj.org/article/c724980dc8784c869d5e105ab87d26dc2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23056608/?tool=EBIhttps://doaj.org/toc/1932-6203A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2 × 2 × 0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.Réka AlbertZoltán VerébKrisztián CsomósMorten C MoeErik O JohnsenOle Kristoffer OlstadBjørn NicolaissenEva RajnavölgyiLászló FésüsAndrás BertaGoran PetrovskiPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 10, p e47187 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Réka Albert Zoltán Veréb Krisztián Csomós Morten C Moe Erik O Johnsen Ole Kristoffer Olstad Bjørn Nicolaissen Eva Rajnavölgyi László Fésüs András Berta Goran Petrovski Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.
description	A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2 × 2 × 0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.
format	article
author	Réka Albert Zoltán Veréb Krisztián Csomós Morten C Moe Erik O Johnsen Ole Kristoffer Olstad Bjørn Nicolaissen Eva Rajnavölgyi László Fésüs András Berta Goran Petrovski
author_facet	Réka Albert Zoltán Veréb Krisztián Csomós Morten C Moe Erik O Johnsen Ole Kristoffer Olstad Bjørn Nicolaissen Eva Rajnavölgyi László Fésüs András Berta Goran Petrovski
author_sort	Réka Albert
title	Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.
title_short	Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.
title_full	Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.
title_fullStr	Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.
title_full_unstemmed	Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.
title_sort	cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/c724980dc8784c869d5e105ab87d26dc
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Cultivation and characterization of cornea limbal epithelial stem cells on lens capsule in animal material-free medium.

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