The heterogeneous murine corneal stromal cell populations in vitro

Abstract

Purpose: To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin.

Methods: Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined.

Results: Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1.

Conclusions: Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.

Figure 1

Heterogeneous morphologies of corneal stroma cells seeded at different densities. At 5 cells/mm², cell morphologies included dendritic (arrows), stellate, small polyhedral, and neuronlike cells (A). A similar finding of neuronlike cells was seen at 50 cells/mm² (B, arrows). At 500 cells/mm², cells were polyhedral and formed a confluent monolayer with neuronlike cells (C, arrows). The observed heterogeneity was also seen among neuronlike cells. Cell groups had similar morphologies with multiple dendritic processes that suggested a common origin (D) or different morphologies containing both multiple and bipolar dendritic processes (E). Most of the neuronlike cells were found as single cells (F). Bar, 25 μm.

Figure 2

Corneal stroma phenotype in vivo. Noninflamed murine corneas expressed keratocan in the entire stroma (A). Scarce CD45-expressing cells were detected in the stroma (B, arrows). No α-SMA expression was observed (C). NF-150-positive corneal nerve stems entered the posterior sclera and peripheral cornea (D; inset: optic nerve as a positive control). Occasional, nucleated cells expressed NF (arrow) in the 2- (E) and 12- (F) week-old mice. Bar, 25 μm.

Figure 3

Freshly isolated corneal stroma cells obtained from 2- and 12-week-old mice were heterogeneous in phenotype after 24 hours of culture. Keratin 12–expressing epithelial cells were absent (A). All cells expressed vimentin (B). Most cells expressed keratocan (C). In contrast, few cells expressed CD45 (D; inset: murine macrophages as positive control). α-SMA was observed at intermediate (E) and high densities (F). Elongated and nucleated NF-150-expressing cells were consistently observed (G). The expression of α-SMA (H) and NF-150 (I) expression persisted in 12-week-old mice. Bar, 25 μm.

Figure 4

Cell clusters were present in corneal stromal cells isolated from 2-week-old mice. Freshly isolated stromal cells seeded at a 50 cells/mm² aggregated formed cell clusters, from which many bipolar neuronlike cells emigrated (Fig. 4A, inset). These cells grew on top of confluent stromal cells (Fig. 4B, coming from the inset of Fig. 4A). Immunostaining of these clusters was negative to NF (C) and GFAP (D). In contrast, strong immunoreactivity against nestin was noted in different cell clusters (E, F). Cells migrating out of the cluster also reacted against nestin (G). Bar, 25 μm.

Figure 5

Adult corneal stroma cells also formed nestin-expressing cell clusters. Stromal cells freshly isolated from 12-week-old mice formed similar cell clusters (A). DAPI expression (B). A cell cluster expressing nestin (C). Merged image showing the expression of nestin in the cluster and neuronlike elongated cells. Nestin was not expressed by the stromal cells (D). Cell clusters and migrating cells reacted against pgp 9.5 (E). Migrating cells but not the cell clusters reacted against tyrosine hydroxylase (F). Bar, 25 μm.

Figure 6

Fetal bovine serum abolished nestin-positive cell clusters. Already formed cell clusters (A) or neuronlike cells (B) in DMEM/F12/ITS were challenged by adding 10% FBS. In 2 days, the cluster began to disintegrate (C), and nestin expression was downregulated (D). Hoechst 33342 staining demonstrated highly fluorescent and fragmented nuclei (E, inset shows the higher magnification). Cells continued to express vimentin (F). Bar, 25 μm.

Figure 7

TGF-β1 abolishes cell cluster formation. Corneal keratocyte seeded with corneal stromal cells at 500 cells/mm² in DMEM/F12 containing ITS and supplemented with 10 ng/mL TGF-β1 formed a confluent monolayer but no cluster nor single neuronlike cells (A). After 5 days in culture, this monolayer contracted and formed a mass of cells in the center of the dish (B). Immunostaining showed that cells in this structure were strongly positive to α-SMA (C). Bar, 25 μm.