title: Regeneration of the corneal epithelium with conjunctival epithelial equivalents generated in serum- and feeder-cell-free media.
abstract: OBJECTIVE
  An alternative autologous tissue for ocular surface reconstruction is a potential treatment for the patients with bilateral limbal stem cell deficiency. For the purpose of regenerative procedures in patients, it is desirable to eliminate the involvement of xenogeneic components, such as nonhuman sera and feeder cells. In the present study, we examined the behavior and phenotypic features of cultured conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions when transplanted into the de-epithelialized limbal corneal surface.
  
  
  METHODS
  Epithelial cells from normal conjunctiva obtained by neutral protease digestion were expanded by culture in a serum-free low-calcium medium and set in an air-liquid interface culture for 14 days. The resulting multilayered epithelial sheets were grafted onto rabbit ocular surfaces made epithelial-free by alkali treatment. Pre-grafted and post-grafted epithelia were analyzed by electron microscopy and immunohistochemistry.
  
  
  RESULTS
  At graft time the cultured epithelial sheet consisted of 6-8 layers of properly stratified epithelium that displayed a CK19(+)/MUC5AC(+)/ CK3 (-)/CK12(-) phenotype, consistent with the conjunctival epithelial lineage. Two weeks after xeno-grafting the in vivo epithelium consisted of 5-6 well compacted layers expressing the precursor cell-related protein p63, the proliferation marker Ki67, desmosomes, hemidesmosomes and its integrin (β4), and the corneal specific cytokeratins CK3, and CK12. Conjunctival goblet cell mucin (MUC5AC) was not visible. The engrafted epithelium stained positively for the anti-human nuclei antibody, confirming that the epithelial cells on the rabbit corneas were of human origin.
  
  
  CONCLUSIONS
  Our results suggest that conjunctival epithelial sheets generated in serum- and 3T3-free culture conditions can acquire the corneal epithelial phenotype when transferred to the in vivo corneal stromal environment.
authors.affiliation: Catholic High Performance Cell Therapy Center, The Catholic University of Korea, Seoul, Korea ; Department of Medical Life Science, The Catholic University of Korea, Seoul, Korea.
authors.forename: Sohee
authors.initials: S
authors.lastname: Jeon
authors.affiliation: Catholic Institute of Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea.
authors.forename: Seong Hyun
authors.initials: SH
authors.lastname: Choi
authors.affiliation: Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY ; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY.
authors.forename: J Mario
authors.initials: JM
authors.lastname: Wolosin
authors.affiliation: Catholic Institute of Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea ; Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea.
authors.forename: So-Hyang
authors.initials: SH
authors.lastname: Chung
authors.affiliation: Catholic Institute of Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea ; Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea.
authors.forename: Choun-Ki
authors.initials: CK
authors.lastname: Joo
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D054338:Cell Transdifferentiation / Q000235:genetics* / Q000276:immunology; D003228:Conjunctiva / Q000166:cytology* / Q000276:immunology / Q000637:transplantation; D016895:Culture Media, Serum-Free; D004847:Epithelial Cells / Q000166:cytology* / Q000276:immunology / Q000637:transplantation; D019573:Epithelium, Corneal / Q000276:immunology* / Q000293:injuries / Q000473:pathology; D005260:Female; D015870:Gene Expression; D006085:Graft Survival / Q000276:immunology*; D006801:Humans; D007633:Keratins / Q000235:genetics / Q000276:immunology; D019394:Ki-67 Antigen / Q000235:genetics / Q000276:immunology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology; D008875:Middle Aged; D055271:Mucin 5AC / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D012038:Regeneration / Q000502:physiology*; D046509:Tissue Culture Techniques; D014183:Transplantation, Heterologous
chemical_list: D015415:Biomarkers; C081876:CKAP4 protein, human; D016895:Culture Media, Serum-Free; D019394:Ki-67 Antigen; C524678:MUC5AC protein, human; D008565:Membrane Proteins; D055271:Mucin 5AC; D007633:Keratins