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Phenotype of human corneal stroma‐derived cells obtained by different isolation techniques from various corneal regions
Author(s) -
Nagymihaly R.,
Veréb Z.,
Facskó A.,
Moe M.,
Petrovski G.
Publication year - 2017
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2017.01371
Subject(s) - vimentin , stroma , ex vivo , microbiology and biotechnology , nestin , immunostaining , stromal cell , biology , pathology , chemistry , immunohistochemistry , stem cell , immunology , in vitro , cancer research , medicine , biochemistry , neural stem cell
Purpose Investigating the effect of isolation technique and location upon the phenotype of human corneal stroma‐derived cells ( CSC s). Methods Cells were obtained from the center and periphery of the corneal stroma using the explant and enzymatic digestion methods. The native tissue was stained for markers of functionality, stemness, proliferation, adhesion and matrix proteins. Surface immunophenotyping was performed on ex vivo cultured CSC s obtained by the different methods by three‐colour fluorescence‐activated cell sorting ( FACS ) analysis. Finally, RT qPCR was used to determine the expression of corneal stroma‐specific genes and to verify the results of immunostaining. Results Cultures of human CSC s were established by explant and enzymatic techniques from the central and peripheral corneal stroma regions. The corneal stroma, in situ was positive for a‐actinin, ALDH 1A1, CD 31, CD 34, Collagen I and Vimentin, while ABCG 2, ABCG 5, fibroblast marker, CD 73, CD 90, CD 105, Collagen IV , Fibronectin, Ki‐67, Nestin and VE ‐Cadherin were not detected. Ex vivo cultured CSC s expressed CD 73, CD 90, CD 105, CD 51, Nestin, CD 49a, CD 49d, ABCG 2, CD 47, while CD 34 and CD 31 were absent. RT qPCR analysis revealed a significant downregulation of ALDH 1A1, AQP 1, ITGB 4, ALDH 1A1 and CD 34 in cultured versus native cells, with the upregulation of ABCG 2, ITGAV , Nestin, CD 73, CD 90, CD 105 and Vimentin. KLF 4, GPC 4 and CD 31 were not significantly different. Conclusions The present study finds no significant difference between the phenotype of the CSC s generated by the explant or enzymatic digestion technique from the central or peripheral part of the stroma. For research purposes, any type of corneal stroma tissue may be used, however the change in cultivated cells’ surface marker and genotype prolife during ex vivo expansion needs to be considered.