Phenotype of human corneal stroma‐derived cells obtained by different isolation techniques from various corneal regions

Abstract

PurposeInvestigating the effect of isolation technique and location upon the phenotype of human corneal stroma‐derived cells (CSCs).MethodsCells 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 CSCs 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.ResultsCultures of human CSCs 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, ALDH1A1, CD31, CD34, Collagen I and Vimentin, while ABCG2, ABCG5, fibroblast marker, CD73, CD90, CD105, Collagen IV, Fibronectin, Ki‐67, Nestin and VE‐Cadherin were not detected. Ex vivo cultured CSCs expressed CD73, CD90, CD105, CD51, Nestin, CD49a, CD49d, ABCG2, CD47, while CD34 and CD31 were absent. RT qPCR analysis revealed a significant downregulation of ALDH1A1, AQP1, ITGB4, ALDH1A1 and CD34 in cultured versus native cells, with the upregulation of ABCG2, ITGAV, Nestin, CD73, CD90, CD105 and Vimentin. KLF4, GPC4 and CD31 were not significantly different.ConclusionsThe present study finds no significant difference between the phenotype of the CSCs 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.