CD34⁺ corneal stromal cells are bone marrow-derived and express hemopoietic stem cell markers

Previous studies have suggested that corneal stromal keratocytes express the CD34 antigen. We wished to investigate CD34 antigen expression in normal mouse cornea using dual and triple-staining techniques. Whole-mount preparations of mouse and rat corneas were examined with confocal microscopy using single, dual, or triple immunostaining to study their morphology, phenotype, and distribution. Single-cell suspensions from normal mouse corneas were also prepared and analyzed by flow cytometry. After short-term culture of corneal stromal explants, nonadherent cells were harvested and cyto-spins were prepared and stained for different markers. Combined staining for F-actin and leukocyte differentiation markers clearly showed that the corneal stroma contains a population of CD45(+) resident bone marrow-derived cells, whereas most cells were CD45-F-actin(+) keratocytes. A significant proportion (two thirds) of CD45(+) cells in the normal corneal stroma expressed CD34(+), whereas no CD45(-) cells (i.e., keratocytes) coexpressed CD34. In addition, CD34(+) cells were CD11c(-) and CD11b(+). Fewer than 10 % of the CD34(+) cells also coexpressed Sca-1(+), but no CD34(+) cells coexpressed major histocompatibility complex (MHC) class II+. In contrast, the remaining population of CD45(+)CD34(-) cells in the corneal stroma expressed CD11b, MHC class II+ but not CD11c and were found mostly in the anterior and peripheral part of stroma. These cells are in intimate contact with corneal keratocytes, which stained only for F-actin and were negative for all leukocyte markers. Very few CD45+ cells expressed the B220 marker, suggesting a plasmacytoid dendritic cell phenotype. Flow cytometry analyses confirmed the morphometric data showing that 68% of CD45(+) cells coexpress CD34 and CD11b, whereas 22% are CD11b(+)CD34(-). We conclude that the normal mouse cornea contains two populations of bone marrow-derived leukocytes, both of which are distinct from stromal keratocytes. The larger population resembles CD34(+) hemopoietic stem cells, whereas the smaller population are CD34(-)CD11b(+) MHC class II+ macrophages. A very small percentage comprises plasmacytoid dendritic cells.