Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice

Nada Sagga, Lucia Kuffova, Neil Vargesson, Lynda Erskine, J. Martin Collinson (Corresponding Author)

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Abstract

Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2’-deoxyuridine (IdU), 5-chloro-2’-deoxyuridine (CldU) and 5-ethynyl-2’- deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ± 0.2 days) compared to 10 week old mice (4.97 ± 0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ± 1.4% of LESCs were shown to enter S phase in a 24 hour period and were estimated to divide every 2-3 weeks. Within 24 hours of corneal injury this rose significantly to 32.8 ± 10.0 % of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24-48 hour period after wounding in young adults, LESC activation continued to increase (86.5 ± 8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ± 9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.
Original languageEnglish
Pages (from-to)185-198
Number of pages14
JournalStem Cell Research
Volume33
Issue number12
Early online date5 Nov 2018
DOIs
Publication statusPublished - Dec 2018

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Cell Cycle
Stem Cells
Epithelial Cells
Deoxyuridine
S Phase
Corneal Epithelium
Young Adult
Wounds and Injuries
Inbred C57BL Mouse
Wound Healing
Thymidine
Maintenance

Keywords

  • cornea
  • wounding
  • limbal stem cells
  • proliferation
  • label-retaining cells

Cite this

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title = "Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice",
abstract = "Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2’-deoxyuridine (IdU), 5-chloro-2’-deoxyuridine (CldU) and 5-ethynyl-2’- deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ± 0.2 days) compared to 10 week old mice (4.97 ± 0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ± 1.4{\%} of LESCs were shown to enter S phase in a 24 hour period and were estimated to divide every 2-3 weeks. Within 24 hours of corneal injury this rose significantly to 32.8 ± 10.0 {\%} of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24-48 hour period after wounding in young adults, LESC activation continued to increase (86.5 ± 8.2{\%} of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ± 9.4{\%} of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.",
keywords = "cornea, wounding, limbal stem cells, proliferation, label-retaining cells",
author = "Nada Sagga and Lucia Kuffova and Neil Vargesson and Lynda Erskine and Collinson, {J. Martin}",
note = "NS was funded by a Ministry of Education in Saudi Arabia PhD studentship. LK was funded by Saving Sight in Grampian, University of Aberdeen Development Trust. This work was performed under University of Aberdeen Development Trust Funding (‘Research into Corneal Blindness’) to JMC, LE and NV and BBSRC Research Grant BB/J015237/1 to JMC.",
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T1 - Limbal epithelial stem cell activity and corneal epithelial cell cycle parameters in adult and aging mice

AU - Sagga, Nada

AU - Kuffova, Lucia

AU - Vargesson, Neil

AU - Erskine, Lynda

AU - Collinson, J. Martin

N1 - NS was funded by a Ministry of Education in Saudi Arabia PhD studentship. LK was funded by Saving Sight in Grampian, University of Aberdeen Development Trust. This work was performed under University of Aberdeen Development Trust Funding (‘Research into Corneal Blindness’) to JMC, LE and NV and BBSRC Research Grant BB/J015237/1 to JMC.

PY - 2018/12

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N2 - Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2’-deoxyuridine (IdU), 5-chloro-2’-deoxyuridine (CldU) and 5-ethynyl-2’- deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ± 0.2 days) compared to 10 week old mice (4.97 ± 0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ± 1.4% of LESCs were shown to enter S phase in a 24 hour period and were estimated to divide every 2-3 weeks. Within 24 hours of corneal injury this rose significantly to 32.8 ± 10.0 % of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24-48 hour period after wounding in young adults, LESC activation continued to increase (86.5 ± 8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ± 9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.

AB - Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2’-deoxyuridine (IdU), 5-chloro-2’-deoxyuridine (CldU) and 5-ethynyl-2’- deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12 months) and young adult (8 week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24 ± 0.2 days) compared to 10 week old mice (4.97 ± 0.5 days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52 ± 1.4% of LESCs were shown to enter S phase in a 24 hour period and were estimated to divide every 2-3 weeks. Within 24 hours of corneal injury this rose significantly to 32.8 ± 10.0 % of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24-48 hour period after wounding in young adults, LESC activation continued to increase (86.5 ± 8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0 ± 9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.

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KW - wounding

KW - limbal stem cells

KW - proliferation

KW - label-retaining cells

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DO - 10.1016/j.scr.2018.11.001

M3 - Article

VL - 33

SP - 185

EP - 198

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JF - Stem Cell Research

SN - 1873-5061

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ER -