Corneal myofibroblast generation from bone marrow-derived cells

Abstract

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha-smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha-smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP-, SMA-GFP+ and SMA-GFP- cells, as well as the number of DAPI+ cell nuclei, per 400x field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9x more SMA+GFP+ than SMA+GFP- myofibroblasts. This difference was significant (p < 0.01). There were significantly more (p < 0.01) SMA-GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP- cells, although SMA-GFP- cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60-95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.

Fig. 1

Double immunocytochemistry for SMA and GFP in chimeric mouse corneas with haze at 1 month after irregular PTK. The figure shows the staining results for 3 different corneas (A to C cornea 1, D to F cornea 2 and G to I cornea 3) that had PRK. The overlays are shown in panels A, D, and G. The red stain for SMA in are shown in B, E, and H. The green stain for GFP is shown in C, F, and I. The blue is DAPI staining of cell nuclei. E indicates the epithelium in several panels. In the first cornea (A to C) several myofibroblasts stain for both SMA and GFP (arrows). Several cells in the stroma do not stain for either SMA or GFP (arrowheads). In cornea 2, there are also several cells that stain for both SMA and GFP (arrows). In a third cornea (G to I), the arrowheads indicate the surface of the corneal epithelium. In panels G and H, arrows indicate stromal cells that stain for SMA, but not for GFP (compare to panel I). In panel I, the arrows indicate cells in the anterior stroma that stain for GFP, but not for SMA (compare to panel H). Panels J to L are control panels in which corneas that had irregular PTK are stained with the primary antibodies for SMA and GFP omitted. Magnification 300X.

Fig. 2

A high magnification view of a chimeric mouse cornea with haze at 1 month after irregular PTK. In the overlay, high concordance can be seen between the red stain for SMA and the green stain for GFP in several cells in the stroma (arrows). B shows red staining for SMA in these same cells (arrows). C shows green stain for GFP in the same cells (arrows). Blue is DAPI staining of nuclei. Magnification 800X.