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The heterogeneous murine corneal stromal cell populations in vitro
Edgar M Espana1, Tetsuya Kawakita, Mario A Di Pascuale
1TissueTech, Inc. and Ocular Surface Center, Miami, Florida 33173, USA.
Insights
Murine corneal stroma contains diverse cells, including progenitor cells expressing nestin. Transforming growth factor-beta1 (TGF-β1) inhibits cluster formation and nestin expression in these corneal stromal cells.
Area of Science:
- Ophthalmology
- Stem Cell Biology
- Cell Biology
Background:
- The corneal stroma, a key component of the eye, plays a vital role in maintaining corneal transparency and structure.
- Understanding the cellular composition of the corneal stroma is crucial for regenerative medicine and treating corneal diseases.
Purpose of the Study:
- To investigate the heterogeneity of cell populations within the murine corneal stroma.
- To identify cells expressing nestin, a potential progenitor cell marker, in the corneal stroma.
Main Methods:
- Corneal stromal cells were isolated from mice at different postnatal ages and cultured under various densities.
- Cells were analyzed for morphology and expression of specific markers including vimentin, keratocan, alpha-smooth muscle actin, neurofilament, GFAP, and nestin.
- The effect of transforming growth factor-beta1 (TGF-β1) on cell behavior and marker expression was examined.
Main Results:
- Freshly isolated corneal stromal cells exhibited morphological heterogeneity, including dendritic, stellate, and neuronal cells.
- Vimentin and keratocan were highly expressed in primary cultures, while alpha-smooth muscle actin and neurofilament expression was low.
- High-density cultures and TGF-β1 treatment revealed distinct cellular responses and marker expression patterns, with nestin expression observed in cell clusters, which was abolished by TGF-β1.
Conclusions:
- The murine corneal stroma harbors heterogeneous cell populations with progenitor potential, indicated by nestin expression and cluster formation.
- TGF-β1 disrupts the progenitor-like characteristics of these corneal stromal cells, suggesting a regulatory role in their differentiation or behavior.
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.
