PAX6 expression identifies progenitor cells for corneal keratocytes

Abstract

Keratocytes of the corneal stroma produce a transparent extracellular matrix required for vision. During wound-healing and in vitro, keratocytes proliferate, becoming fibroblastic, and lose biosynthesis of unique corneal matrix components. This study sought identification of cells in the corneal stroma capable of assuming a keratocyte phenotype after extensive proliferation. About 3% of freshly isolated bovine stromal cells exhibited clonal growth. In low-mitogen media, selected clonal cultures displayed dendritic morphology and expressed high levels of keratan sulfate, aldehyde dehydrogenase 3A1, and keratocan, molecular markers of keratocyte phenotype. In protein-free media, both primary keratocytes and selected clonal cells aggregated to form attachment-independent spheroids expressing elevated levels of those marker molecules. The selected clonal cells exhibited normal karyotype and underwent replicative senescence after 65-70 population doublings; however, they continued expression of keratocyte phenotypic markers throughout their replicative life span. The progenitor cells expressed elevated mRNA for several genes characteristic of stem cells and also for genes expressed during ocular development PAX6, Six2, and Six3. PAX6 protein was detected in the cultured progenitor cells and a small number of stromal cells in intact tissue but was absent in cultured keratocytes and fibroblasts. Cytometry demonstrated PAX6 protein in 4% of freshly isolated stromal cells. These results demonstrate the presence of a previously unrecognized population of PAX6-positive cells in adult corneal stroma that maintain the potential to assume a keratocyte phenotype even after extensive replication. The presence of such progenitor cells has implications for corneal biology and for cell-based therapies targeting corneal scarring.

Figure 1

Properties of clonal cultures from corneal stroma. Phase contrast micrographs illustrate the range of cellular morphologies observed in cells growing clonally from bovine corneal stroma, fibroblastic (A) and dendritic (B). C) Keratan sulfate was detected by dot blot in proteoglycans isolated by 96 different clones 2 wk after a shift to low-mitogen media. The amount of keratan sulfate is calculated relative to cell abundance.

Figure 2. Phenotype of cloned stromal cells

A) Proteoglycans from culture media of 3 selected stromal clones (CPD 22) were assayed for keratan sulfate-containing proteoglycan using monoclonal antibody J19 with (+) or without (−) pretreatment with keratanase. B) mRNA pools for keratocan (shaded) and ALDH (open) were determined by quantitative RT-PCR in primary keratocytes, clonal stromal cells (CPD 24), and passage-four fibroblasts. Values are normalized to keratocytes = 100. C) Primary keratocytes were immunostained for f-actin (green) and vinculin (red). D) Clonal stromal cells (CDP 50) were stained similarly to C. E) Fibroblasts, passage four, were stained similarly to C and D.

Figure 3

Spheroid formation by stromal cells. Primary keratocytes in mitogen-free medium (A) attach and spread with a dendritic morphology. After 1 wk in medium containing 10 ng/ml FGF and ITS (B), cells form refractile aggregates. After 3 wk (C), aggregates become spherical and could be readily separated from individual cells (D). Live cells in spheres were stained with calcein AM (green) and propidium iodide (red) to show live and dead cells (E). Fixed spheres (F) were stained for keratan sulfate (green) and keratocan (red). Photos E and F are optical sections through the center of a sphere using confocal microscopy as described in Materials and Methods. RNA from spheres and from cells not aggregated into spheres from the same culture was subjected to analysis by quantitative RT-PCR for keratocan mRNA as described in Materials and Methods. mRNA pools were compared with those of noncultured stromal cells and with corneal fibroblasts in G. Bars in A–D = 100 μm and in E–F = 50 μm.

Figure 4

Replicative lifespan of cloned progenitor cells. After the initial 6 passages, estimated to require 22 CPD, 3 clonal progenitor lines were passaged at 7 day intervals by trypsinizing, counting, and replating at a fixed density of 1 × 10⁴ cells/cm². Points represent population doublings calculated from average cell counts of 2 cultures from each cell line.

Figure 5

Marker expression during expansion of progenitor cells. RNA was purified from primary keratocytes (CPD 0) and from clonal progenitor cells at early passage (27 CPD) and late passage (60 CPD). Cells were maintained in cloning medium (CM) (open bars) or after a week in differentiation medium (solid bars). Relative mRNA abundance was calculated as a percentage of that of primary keratocytes. A) ALDH mRNA. B) keratocan mRNA.

Figure 6

Stem cell gene expression by stromal progenitor cells. Relative abundance of mRNA for 11 genes reported in stem and ocular progenitor cells was examined using quantitative RT-PCR in clonal stromal (progenitor) cells compared with primary keratocytes as described in Materials and Methods. As controls, GAPDH, keratocan (KERA), and ALDH were determined in the same samples.

Figure 7

PAX6 expression by stromal progenitor cells. A) PAX6 protein in cloned progenitor cells was immunostained (green) and counterstained for cytoplasmic myosin (red). B) PAX6 protein was not detected in primary keratocytes cultured under identical conditions. C) PAX6 was detected only in rare cells in intact corneal stroma (arrowhead). Keratocyte cell membranes were counterstained with DiD (red). D) Freshly isolated stromal cells were fixed and stained for PAX6 protein and then analyzed by flow cytometry. Solid line shows results with a PAX6 primary antibody. Dashed line shows a nonimmune rabbit IgG primary. Fluorescence intensity is shown as a logarithmic scale. Arrow indicates a population of stained cells representing 4% of the total. White bars = 50 μm.