TY - JOUR
T1 - The heterogeneous murine corneal stromal cell populations in vitro
AU - Espana, Edgar M.
AU - Kawakita, Tetsuya
AU - Di Pascuale, Mario A.
AU - Li, Wei
AU - Yeh, Lung Kun
AU - Parel, Jean Marie
AU - Liu, Chia Yang
AU - Tseng, Scheffer C.G.
PY - 2005/12
Y1 - 2005/12
N2 - 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)-β1 for 5 days. Cell morphology and expression of α-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. α-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-β1. 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-β1 to the culture medium.
AB - 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)-β1 for 5 days. Cell morphology and expression of α-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. α-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-β1. 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-β1 to the culture medium.
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U2 - 10.1167/iovs.05-0117
DO - 10.1167/iovs.05-0117
M3 - Article
C2 - 16303944
AN - SCOPUS:33644797955
SN - 0146-0404
VL - 46
SP - 4528
EP - 4535
JO - Investigative Ophthalmology
JF - Investigative Ophthalmology
IS - 12
ER -