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Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation

Hashmani, Khurram; Branch, Matthew James; Sidney, Laura E.; Dhillon, Permesh Singh; Verma, Megha; McIntosh, Owen Douglas; Hopkinson, Andrew; Dua, Harminder Singh

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Authors

Khurram Hashmani

Matthew James Branch

LAURA SIDNEY LAURA.SIDNEY@NOTTINGHAM.AC.UK
Senior Research Fellow

Permesh Singh Dhillon

Megha Verma

Owen Douglas McIntosh

Andrew Hopkinson

Harminder Singh Dua



Abstract

Introduction: The corneal stroma is being increasingly recognized as a repository for stem cells. Like the limbal and endothelial niches, stromal stem cells often reside in the peripheral cornea and limbus. These peripheral and limbal corneal stromal cells (PLCSCs) are known to produce mesenchymal stem cells in vitro. Recently, a common corneal stromal and epithelial progenitor was hinted at. This study aims to examine the stem cell potential of corneal stromal cells and to investigate their epithelial transdifferentiation ability.

Methods: PLCSCs were grown in traditional Dulbecco modified Eagle medium (DMEM)-based keratocyte culture medium and an M199-based medium and analyzed for a profile of cell-surface markers by using flow cytometry and differentiated into mesenchymal phenotypes analyzed with quantitative polymerase chain reaction (qPCR) and histologic staining. PLCSCs in M199 were subsequently divided into subpopulations based on CD34 and CD105 expression by using fluorescence- activated cell sorting (FACS). Subpopulations were characterized by marker profile and mesenchymal differentiation ability. Both whole PLCSCs and subpopulations were also cultured for epithelial transdifferentiation.

Results: Cells cultured in M199 demonstrated a more stem-like cell-surface marker profile, and the keratocyte marker CD34 was retained for several passages but absent in cells cultured in DMEM. Cells cultured in M199 also exhibited a greater mesenchymal differentiation potential, compared with DMEM. PLCSCs could be divided into CD34+CD105+, CD34-CD105+, and CD34-CD105- subpopulations, of which CD34+CD105+ cells were the most stemlike with regard to marker expression and mesenchymal differentiation potential. Subpopulations of PLCSCs exhibited differing abilities to transdifferentiate into epithelial phenotypes. Cells that were initially CD34+CD105+ showed the greatest differentiation potential, producing CK3+ and CK19+ cells, and expressed a range of both epithelial progenitor (HES1, FRZB1, DCT, SOD2, ABCG2, CDH1, KRT19) and terminally differentiated (DSG3, KRT3, KRT12, KRT24) genes.

Conclusions: Culture medium has a significant effect on the phenotype and differentiation capacity of PLCSCs. The stroma contains a heterogeneous cell population in which we have identified CD34+ cells as a stem cell population with a capacity for mesenchymal and epithelial differentiation.

Citation

Hashmani, K., Branch, M. J., Sidney, L. E., Dhillon, P. S., Verma, M., McIntosh, O. D., …Dua, H. S. (2013). Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation. Stem Cell Research and Therapy, 4(3), Article 75. https://doi.org/10.1186/scrt226

Journal Article Type Article
Publication Date Jun 24, 2013
Deposit Date Apr 14, 2014
Publicly Available Date Apr 14, 2014
Journal Stem Cell Research & Therapy
Electronic ISSN 1757-6512
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
Volume 4
Issue 3
Article Number 75
DOI https://doi.org/10.1186/scrt226
Public URL https://nottingham-repository.worktribe.com/output/715552
Publisher URL http://stemcellres.com/content/4/3/75
Contract Date Apr 14, 2014

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