Abstract
Purpose:
Mesenchymal cells from limbal stroma exhibit stem cell properties and can regenerate stroma in vivo and vitro. Recent publications showed stromal cells associating with limbal epithelial cells to express the chemokine receptor CXCR4. The study examined the hypothesis that stromal CXCR4 expression allows selection of stromal stem cells with a high potential for corneal regeneration.
Methods:
Limbal stromal cells were recovered from human corneal rims by collagenase digestion and expanded in culture at clonal density in a low-serum medium containing cholera toxin (PMID:22078813). Gene expression patterns were determined by qPCR. Protein expression was assayed by flow cytometry. CXCR4+ cells were isolated with MACS immunoadsorption or by FACS. Keratocyte phenotype was determined by gene and extracellular matrix expression after culture on collagen or aligned nanofiber substrata. Immunostaining was carried out on tissue wholemounts and sections using immunofluorescent detection with confocal microscopy.
Results:
Strong staining for CXCR4 protein was limited to small populations of limbal cells subjacent to the epithelial basement membrane. In uncultured digests and primary cultures, CXCR4 positive cells amounted to one third of limbal cells in flow cytometry, but in central stroma less than 1% total cells were CXCR4+. After passage, the proportion of CXCR4+ in cultured limbal cells decreased, particularly in confluent cultures. Separated CXCR4+ cells expressed pluripotent markers Oct4, SOX2, NANOG, and neural crest marker p75NTR. Under differentiation conditions, CXCR4+ cells exhibited enhanced expression of keratocyte genes and matrix compared to CXCR4- cells.
Conclusions:
CXCR4 is a cell surface protein the abundance of which correlates well with the potential of cells to differentiation to keratocytes in vitro. This correlation differs from other widely known mesenchymal stem cell markers and thereby provides a novel and useful tool in isolating and enriching populations of stromal stem cells for use in corneal bioengineering and direct-cell based corneal therapy.
Keywords: 484 cornea: stroma and keratocytes •
721 stem cells •
687 regeneration