September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Peripheral corneal spheres facilitate ocular surface regeneration in healthy and diseased tissue
Author Affiliations & Notes
  • Salim Ismail
    Department of Ophthalmology, Faculty of Medical and Health Sciences,The University of Auckland, Auckland, New Zealand
  • Jeremy Mathan
    Department of Ophthalmology, Faculty of Medical and Health Sciences,The University of Auckland, Auckland, New Zealand
  • Himanshu Wadhwa
    Department of Ophthalmology, Faculty of Medical and Health Sciences,The University of Auckland, Auckland, New Zealand
  • Jane McGhee
    Department of Ophthalmology, Faculty of Medical and Health Sciences,The University of Auckland, Auckland, New Zealand
  • Trevor Sherwin
    Department of Ophthalmology, Faculty of Medical and Health Sciences,The University of Auckland, Auckland, New Zealand
  • Footnotes
    Commercial Relationships   Salim Ismail, None; Jeremy Mathan, None; Himanshu Wadhwa, None; Jane McGhee, None; Trevor Sherwin, None
  • Footnotes
    Support  Auckland Medical Research Foundation Grant 1111010; Save Sight Society Grant 3622588
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4903. doi:
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    • Get Citation

      Salim Ismail, Jeremy Mathan, Himanshu Wadhwa, Jane McGhee, Trevor Sherwin; Peripheral corneal spheres facilitate ocular surface regeneration in healthy and diseased tissue. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4903.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Cells isolated from human peripheral cornea and cultured using a sphere-forming assay generate stem cell-enriched spheres composed of a heterogenous mix of stromal, limbal and epithelial components. We hypothesise that these spheres are transplantable units that can be used to restore the ocular surface in patients with corneal dystrophies such as keratoconus and limbal stem cell deficiency.

Methods : Spheres from 5 donors (N=21) were labelled using the fluorescence ubiquitination cell cycle indicator (FUCCI) system, placed onto collagen-coated surfaces in-vitro and analysed by time lapse microscopy for cell migration and cell cycle progression. Subsequently, spheres (N=4 donors) were surgically implanted into decellularised human corneoscleral rims (from healthy donors, N=6) and diseased corneal buttons (N=2) in-situ and monitored over 10-14 days using a live/dead cytotoxicity assay. Implanted spheres were imaged for cell viability and evidence of cell migration by fluorescence microscopy at 1, 3 and 9 days post implantation. Sphere-implanted tissue was fixed and sectioned for immunohistochemical detection of stem cell and differentiation markers by confocal microscopy.

Results : Spheres responded to collagen stimulus with cell migration across the in-vitro surface. FUCCI labelling revealed both proliferative and non-proliferative cells with proliferating cells present both within the central sphere and amongst outwardly migrating cells. Implantation into corneoscleral rims resulted in preferential cell migration towards the cornea with complete repopulation of the available corneal surface within 10 days. Sphere-implantation into keratoconic and decompensated corneal buttons showed a similar pattern of cell migration outward from implanted spheres. Cross sectional analysis of implanted tissue showed markers for stem (ABCG2, ΔNp63α ), limbal (Notch 1) and differentiated cells (Laminin, Vimentin).

Conclusions : Our results support our hypothesis that peripheral corneal spheres are capable of repopulating the ocular surface in both normal and diseased tissue in-situ. These findings hold great promise for development of these spheres into transplantable units for in-vivo corneal repair in therapies targeting ocular surface regeneration and stem cell repopulation.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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