The heterogeneous murine corneal stromal cell populations in vitro

Edgar M. Espana, Tetsuya Kawakita, Mario A. Di Pascuale, Wei Li, Lung Kun Yeh, Jean Marie Parel, Chia Yang Liu, Scheffer C G Tseng

Research output: Contribution to journalArticle

8 Citations (Scopus)

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)-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.

Original languageEnglish
Pages (from-to)4528-4535
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume46
Issue number12
DOIs
Publication statusPublished - 2005 Dec
Externally publishedYes

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Stromal Cells
Population
Nestin
Corneal Stroma
Transforming Growth Factors
In Vitro Techniques
Glial Fibrillary Acidic Protein
Tyrosine 3-Monooxygenase
Vimentin
Smooth Muscle
Actins
Neurofilament Proteins
Phase-Contrast Microscopy
Choline O-Acetyltransferase
Intermediate Filaments
Collagenases
Transferrin
Selenium

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Espana, E. M., Kawakita, T., Di Pascuale, M. A., Li, W., Yeh, L. K., Parel, J. M., ... Tseng, S. C. G. (2005). The heterogeneous murine corneal stromal cell populations in vitro. Investigative Ophthalmology and Visual Science, 46(12), 4528-4535. https://doi.org/10.1167/iovs.05-0117

The heterogeneous murine corneal stromal cell populations in vitro. / Espana, Edgar M.; Kawakita, Tetsuya; Di Pascuale, Mario A.; Li, Wei; Yeh, Lung Kun; Parel, Jean Marie; Liu, Chia Yang; Tseng, Scheffer C G.

In: Investigative Ophthalmology and Visual Science, Vol. 46, No. 12, 12.2005, p. 4528-4535.

Research output: Contribution to journalArticle

Espana, EM, Kawakita, T, Di Pascuale, MA, Li, W, Yeh, LK, Parel, JM, Liu, CY & Tseng, SCG 2005, 'The heterogeneous murine corneal stromal cell populations in vitro', Investigative Ophthalmology and Visual Science, vol. 46, no. 12, pp. 4528-4535. https://doi.org/10.1167/iovs.05-0117
Espana EM, Kawakita T, Di Pascuale MA, Li W, Yeh LK, Parel JM et al. The heterogeneous murine corneal stromal cell populations in vitro. Investigative Ophthalmology and Visual Science. 2005 Dec;46(12):4528-4535. https://doi.org/10.1167/iovs.05-0117
Espana, Edgar M. ; Kawakita, Tetsuya ; Di Pascuale, Mario A. ; Li, Wei ; Yeh, Lung Kun ; Parel, Jean Marie ; Liu, Chia Yang ; Tseng, Scheffer C G. / The heterogeneous murine corneal stromal cell populations in vitro. In: Investigative Ophthalmology and Visual Science. 2005 ; Vol. 46, No. 12. pp. 4528-4535.
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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)-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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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

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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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