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CD34⁺ Corneal Stromal Cells Are Bone Marrow–Derived and Express Hemopoietic Stem Cell Markers

^a Department of Ophthalmology, University of Aberdeen, Scotland, United Kingdom;
^b Department of Neuroimmunology, Center for Brain Research, Medical University of Vienna, Austria

Key Words. Cornea • CD34 • Hemopoietic stem cell • Dendritic cell • Leukocytes

Correspondence: John V. Forrester, M.D., Department of Ophthalmology, University of Aberdeen, AB255ZD, Aberdeen, Scotland, United Kingdom. Telephone: 0044-122-455-3782; Fax: 0044-122-455-5955; e-mail: j.forrester{at}abdn.ac.uk

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 cytospins 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.

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