title: Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface.
abstract: BACKGROUND
  The limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. Injuries to the limbus, through disease or accidents such as chemical injuries or burns, may lead to significant visual impairment due to depletion of the native stem cells of the tissue.
  
  
  METHODS
  Sphere-forming cells were isolated from peripheral cornea for potential use as transplantable elements for limbal stem cell repopulation and limbal reconstruction. Immunocytochemistry, live cell imaging and quantitative PCR were used to characterize spheres and elucidate activity post implantation into human cadaveric corneal tissue.
  
  
  RESULTS
  Spheres stained positively for stem cell markers ∆NP63α, ABCG2 and ABCB5 as well as the basal limbal marker and putative niche marker, notch 1. In addition, spheres also stained positively for markers of corneal cells, vimentin, keratin 3, keratocan and laminin, indicating a heterogeneous mix of stromal and epithelial-origin cells. Upon implantation into decellularized corneoscleral tissue, 3D, polarized and radially orientated cell migration with cell proliferation was observed. Cells migrated out from the spheres and repopulated the entire corneal surface over 14 days. Post-implantation analysis revealed qualitative evidence of stem, stromal and epithelial cell markers while quantitative PCR showed a quantitative reduction in keratocan and laminin expression indicative of an enhanced progenitor cell response. Proliferation, quantified by PCNA expression, significantly increased at 4 days subsequently followed by a decrease at day 7 post implantation.
  
  
  CONCLUSIONS
  These observations suggest great promise for the potential of peripheral corneal spheres as transplantable units for corneal repair, targeting ocular surface regeneration and stem cell repopulation.
authors.affiliation: Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand.
authors.forename: Jeremy John
authors.initials: JJ
authors.lastname: Mathan
authors.affiliation: Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand.
authors.forename: Salim
authors.initials: S
authors.lastname: Ismail
authors.affiliation: Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand.
authors.forename: Jennifer Jane
authors.initials: JJ
authors.lastname: McGhee
authors.affiliation: Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand.
authors.forename: Charles Ninian John
authors.initials: CN
authors.lastname: McGhee
authors.affiliation: Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand. t.sherwin@auckland.ac.nz.
authors.forename: Trevor
authors.initials: T
authors.lastname: Sherwin
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2 / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002102:Cadaver; D002454:Cell Differentiation; D002465:Cell Movement; D049109:Cell Proliferation; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D053558:Keratin-3 / Q000235:genetics / Q000378:metabolism; D007797:Laminin / Q000235:genetics / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D046509:Tissue Culture Techniques; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism
chemical_list: C480744:ABCB5 protein, human; C116614:ABCG2 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D015415:Biomarkers; C490948:KERA protein, human; C479360:KRT3 protein, human; D053558:Keratin-3; D007797:Laminin; C497761:NOTCH1 protein, human; D009363:Neoplasm Proteins; D018809:Proliferating Cell Nuclear Antigen; D011509:Proteoglycans; D051881:Receptor, Notch1; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D014746:Vimentin
keywords: Cell culture; Cornea; Holoclone; Immunocytochemistry; Quantitative PCR; Spheroid