title: Cultivation and characterization of limbal epithelial stem cells on contact lenses with a feeder layer: toward the treatment of limbal stem cell deficiency.
abstract: OBJECTIVE
  Limbal epithelial sheets are used to promote corneal surface reconstruction after the detection of limbal epithelial stem cell deficiency. The aim of this study was to evaluate a novel combination of limbal stem cells (LSCs) maintained on contact lenses (CLs) in the presence of a 3T3 feeder cell layer regarding preservation of stem cell phenotype and the potential use for future in vivo transplantation.
  
  
  METHODS
  Limbal epithelial cells were isolated from rabbit cornea and cultured with 3T3 cells on CLs. The preservation of LSC phenotype was determined using p63α and ABCG2 immunostaining, whereas epithelial differentiation was evaluated using CK3 and CK19. The colony-forming assay was used to determine the percentage of LSCs in cultures. Finally, CLs seeded with PKH26-labeled LSCs were transferred to rabbit eyes after performing a surgical keratectomy, and the transition and phenotype of labeled cells on the corneal surface were evaluated in whole-mount corneas.
  
  
  RESULTS
  Proliferation of individual limbal cells was observed on CLs with a 3T3 feeder cell layer, showing holoclone formation and retention of viable stem or progenitor cell phenotype. Finally, a higher transition of cultivated cells after a dual sequential CL transplantation to the ocular surface was observed, showing the preservation of the LSC phenotype in the corneal surface.
  
  
  CONCLUSIONS
  Limbal cells cultivated on a CL carrier overlaying a 3T3 feeder layer are mitotically active and retain the LSC phenotype. This novel technique of using CLs as a carrier offers an easily manipulable and nonimmunogenic method for transferring LSCs for ocular surface reconstruction in patients with limbal epithelial stem cell deficiency.
authors.affiliation: *Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, Israel; and †Cell and Molecular Biology, USAMRICD, Aberdeen Proving Ground, MD.
authors.forename: Ariel
authors.initials: A
authors.lastname: Gore
authors.forename: Vered
authors.initials: V
authors.lastname: Horwitz
authors.forename: Hila
authors.initials: H
authors.lastname: Gutman
authors.forename: Liat
authors.initials: L
authors.lastname: Tveria
authors.forename: Liat
authors.initials: L
authors.lastname: Cohen
authors.forename: Orit
authors.initials: O
authors.lastname: Cohen-Jacob
authors.forename: Joseph
authors.initials: J
authors.lastname: Turetz
authors.forename: Patrick Michael
authors.initials: PM
authors.lastname: McNutt
authors.forename: Shlomit
authors.initials: S
authors.lastname: Dachir
authors.forename: Tamar
authors.initials: T
authors.lastname: Kadar
mesh_terms: D016475:3T3 Cells / Q000166:cytology; D000704:Analysis of Variance; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003261:Contact Lenses; D016039:Corneal Transplantation / Q000379:methods; D004847:Epithelial Cells / Q000166:cytology*; D016850:Limbus Corneae / Q000166:cytology*; D051379:Mice; D023421:Models, Animal; D011817:Rabbits; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000166:cytology*