Cargando…

Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media

Purpose: An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it is desirable to eliminate the involvement of xenogeneic components, such as non...

Descripción completa

Detalles Bibliográficos
Autores principales: Jeon, Sohee, Choi, Seong Hyun, Wolosin, J. Mario, Chung, So-Hyang, Joo, Choun-Ki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Molecular Vision 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867160/
https://www.ncbi.nlm.nih.gov/pubmed/24357922
_version_ 1782296260655448064
author Jeon, Sohee
Choi, Seong Hyun
Wolosin, J. Mario
Chung, So-Hyang
Joo, Choun-Ki
author_facet Jeon, Sohee
Choi, Seong Hyun
Wolosin, J. Mario
Chung, So-Hyang
Joo, Choun-Ki
author_sort Jeon, Sohee
collection PubMed
description Purpose: An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it is desirable to eliminate the involvement of xenogeneic components, such as nonhuman sera and feeder cells. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions when transplanted into the de-epithelialized limbal corneal surface. Methods: Epithelial cells from normal conjunctiva obtained by neutral protease digestion were expanded by culture in a serum-free low-calcium medium and set in an air-liquid interface culture for 14 days. The resulting multilayered epithelial sheets were grafted onto rabbit ocular surfaces made epithelial-free by alkali treatment. Pre-grafted and post-grafted epithelia were analyzed by electron microscopy and immunohistochemistry. Results: At graft time the cultured epithelial sheet consisted of 6–8 layers of properly stratified epithelium that displayed a CK19(+)/MUC5AC(+)/ CK3 (-)/CK12(-) phenotype, consistent with the conjunctival epithelial lineage. Two weeks after xeno-grafting the in vivo epithelium consisted of 5-6 well compacted layers expressing the precursor cell-related protein p63, the proliferation marker Ki67, desmosomes, hemidesmosomes and its integrin (β4), and the corneal specific cytokeratins CK3, and CK12. Conjunctival goblet cell mucin (MUC5AC) was not visible. The engrafted epithelium stained positively for the anti-human nuclei antibody, confirming that the epithelial cells on the rabbit corneas were of human origin. Conclusions: Our results suggest that conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions can acquire the corneal epithelial phenotype when transferred to the in vivo corneal stromal environment.
format Online
Article
Text
id pubmed-3867160
institution National Center for Biotechnology Information
language English
publishDate 2013
publisher Molecular Vision
record_format MEDLINE/PubMed
spelling pubmed-38671602013-12-19 Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media Jeon, Sohee Choi, Seong Hyun Wolosin, J. Mario Chung, So-Hyang Joo, Choun-Ki Mol Vis Research Article Purpose: An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it is desirable to eliminate the involvement of xenogeneic components, such as nonhuman sera and feeder cells. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions when transplanted into the de-epithelialized limbal corneal surface. Methods: Epithelial cells from normal conjunctiva obtained by neutral protease digestion were expanded by culture in a serum-free low-calcium medium and set in an air-liquid interface culture for 14 days. The resulting multilayered epithelial sheets were grafted onto rabbit ocular surfaces made epithelial-free by alkali treatment. Pre-grafted and post-grafted epithelia were analyzed by electron microscopy and immunohistochemistry. Results: At graft time the cultured epithelial sheet consisted of 6–8 layers of properly stratified epithelium that displayed a CK19(+)/MUC5AC(+)/ CK3 (-)/CK12(-) phenotype, consistent with the conjunctival epithelial lineage. Two weeks after xeno-grafting the in vivo epithelium consisted of 5-6 well compacted layers expressing the precursor cell-related protein p63, the proliferation marker Ki67, desmosomes, hemidesmosomes and its integrin (β4), and the corneal specific cytokeratins CK3, and CK12. Conjunctival goblet cell mucin (MUC5AC) was not visible. The engrafted epithelium stained positively for the anti-human nuclei antibody, confirming that the epithelial cells on the rabbit corneas were of human origin. Conclusions: Our results suggest that conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions can acquire the corneal epithelial phenotype when transferred to the in vivo corneal stromal environment. Molecular Vision 2013-12-16 /pmc/articles/PMC3867160/ /pubmed/24357922 Text en Copyright © 2013 Molecular Vision. http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited, used for non-commercial purposes, and is not altered or transformed.
spellingShingle Research Article
Jeon, Sohee
Choi, Seong Hyun
Wolosin, J. Mario
Chung, So-Hyang
Joo, Choun-Ki
Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media
title Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media
title_full Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media
title_fullStr Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media
title_full_unstemmed Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media
title_short Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media
title_sort regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell–free media
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867160/
https://www.ncbi.nlm.nih.gov/pubmed/24357922
work_keys_str_mv AT jeonsohee regenerationofthecornealepitheliumwithconjunctivalepithelialequivalentsgeneratedinserumandfeedercellfreemedia
AT choiseonghyun regenerationofthecornealepitheliumwithconjunctivalepithelialequivalentsgeneratedinserumandfeedercellfreemedia
AT wolosinjmario regenerationofthecornealepitheliumwithconjunctivalepithelialequivalentsgeneratedinserumandfeedercellfreemedia
AT chungsohyang regenerationofthecornealepitheliumwithconjunctivalepithelialequivalentsgeneratedinserumandfeedercellfreemedia
AT joochounki regenerationofthecornealepitheliumwithconjunctivalepithelialequivalentsgeneratedinserumandfeedercellfreemedia