Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation
Authors
Khurram Hashmani, Matthew James Branch, Laura Elizabeth Sidney, Permesh Singh Dhillon, Megha Verma, Owen Douglas McIntosh, Andrew Hopkinson and Harminder Singh Dua
Institution
Uni Nottingham
Country
United Kingdom
Year
2013
Journal
Stem Cell Research & Therapy
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.
Keywords: Corneal stroma, Corneal epithelium, Mesenchymal stromal cells, Cell transdifferentiation, CD34,
Epithelial-mesenchymal transition