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Involvement of stem cell factor and c-kit in corneal wound healing in mice.

ABSTRACT: To study the roles played by stem cell factor (SCF) and SCF receptor c-kit in wound healing of corneal epithelial cells.A 2 mm corneal epithelial wound was made in control (WBB6F1(+/+)), SCF (Sl/Sl(d))-, and c-kit (W/W(v)) mutant mice, and the speed of wound healing, 5-bromo-2'-deoxyuridine (BrdU) incorporation, and scanning electron microscopic (SEM) morphology of the corneas were examined. The incorporation of BrdU and the degree of cell attachment in cultured mouse corneal epithelial cells (MCECs) isolated from WBB6F1(+/+), Sl/Sl(d), and W/W(v) mice were examined. Cultured immortalized human corneal epithelial cells (HCECs) were examined by a cell attachment assay after their exposure to anti-SCF antibodies, tyrosine kinase inhibitor (genistein), and competitive Arg-Gly-Asp (RGD) peptide, as well as on cultures treated with extracellular matrix.The speed of corneal wound healing was slower in Sl/Sl(d) and W/W(v) mice than in controls (p<0.01) and the speed of healing in Sl/Sl(d) mice recovered after topical application of SCF (8 ng/ml). No significant difference was found in the BrdU incorporation assay either in vivo or in vitro. Loosened epithelial cells were detected at wound margins in W/W(v) mice by SEM. The cell attachment rate was increased by 157% in cells from WBB6F1(+/+) and 252% in Sl/Sl(d) MCECs by recombinant mouse SCF; however, no significant difference was found in W/W(v) MCECs. Anti-SCF antibodies (Ab), genistein, and RGD peptide reduced the percentage of attached HCECs. Anti-SCF Ab inhibited the attachment of HCECs on fibronectin, laminin, or type IV collagen coated dishes.These findings indicate that the SCF/c-kit system may play a role in corneal wound healing through epithelial cell attachment.

SUBMITTER: Miyamoto K

PROVIDER: S-EPMC3381705 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Involvement of stem cell factor and c-kit in corneal wound healing in mice.

Miyamoto Kazuhisa K Kobayashi Takeshi T Hayashi Yasuhito Y Zhang Yuan Y Hara Yuko Y Higashine Masakatsu M Shiraishi Atsushi A Ohashi Yuichi Y

Molecular vision 20120607

<h4>Purpose</h4>To study the roles played by stem cell factor (SCF) and SCF receptor c-kit in wound healing of corneal epithelial cells.<h4>Methods</h4>A 2 mm corneal epithelial wound was made in control (WBB6F1(+/+)), SCF (Sl/Sl(d))-, and c-kit (W/W(v)) mutant mice, and the speed of wound healing, 5-bromo-2'-deoxyuridine (BrdU) incorporation, and scanning electron microscopic (SEM) morphology of the corneas were examined. The incorporation of BrdU and the degree of cell attachment in cultured m ...[more]

PMID: 22736941

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Project description:BackgroundThe mouse corneal epithelium is a continuously renewing 5-6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing. Mosaic analysis can provide insights into LSC function, cell movement and cell mixing during tissue maintenance and repair. The present study investigates cell streaming during corneal maintenance and repair and changes in LSC function with age.ResultsThe initial pattern of corneal epithelial patches in XLacZ+/- X-inactivation mosaics was replaced after birth by radial stripes, indicating activation of LSCs. Stripe patterns (clockwise, anticlockwise or midline) were independent between paired eyes. Wound healing in organ culture was analysed by mosaic analysis of XLacZ+/- eyes or time-lapse imaging of GFP mosaics. Both central and peripheral wounds healed clonally, with cells moving in from all around the wound circumference without significant cell mixing, to reconstitute striping patterns. Mosaic analysis revealed that wounds can heal asymmetrically. Healing of peripheral wounds produced stripe patterns that mimicked some aberrant striping patterns observed in unwounded corneas. Quantitative analysis provided no evidence for an uneven distribution of LSC clones but showed that corrected corneal epithelial stripe numbers declined with age (implying declining LSC function) but stabilised after 39 weeks.ConclusionStriping patterns, produced by centripetal movement, are defined independently and stochastically in individual eyes. Little cell mixing occurs during the initial phase of wound healing and the direction of cell movement is determined by the position of the wound and not by population pressure from the limbus. LSC function declines with age and this may reflect reduced LSCs numbers, more quiescent LSCs or a reduced ability of older stem cells to maintain tissue homeostasis. The later plateau of LSC function might indicate the minimum LSC function that is sufficient for corneal epithelial maintenance. Quantitative and temporal mosaic analyses provide new possibilities for studying stem cell function, tissue maintenance and repair.

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Involvement of stem cell factor and c-kit in corneal wound healing in mice.

Publications

Involvement of stem cell factor and c-kit in corneal wound healing in mice.

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