title: One for all: A standardized protocol for ex vivo culture of limbal, conjunctival and oral mucosal epithelial cells into corneal lineage.
abstract: OBJECTIVE
  Autologous transplantation of ex vivo cultured cells the treatment of choice for patients with limbal stem cell deficiency. The most commonly used cell sources for transplantation limbal, conjunctival or oral mucosal tissue. Protocols vary for culturing each tissue type, and there are no comparative studies on transplantation outcomes using these different culture techniques. To overcome this limitation, we devised a simple protocol that can uniformly promote growth and differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage.
  
  
  METHODS
  Biopsies were cultured as explants on de-epithelialized human amniotic membrane in the presence of recombinant epidermal growth factor and insulin. Cultured cells were characterized using immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction for stem/progenitor markers (ABCG2 and P63α) and differentiation markers (CK3, CK12, CK4, CK13, CK15 and CONNEXIN 43). Fluorescence-activated cell sorter analysis was performed for ABCG2.
  
  
  RESULTS
  The results revealed that cells of all three biopsies differentiated into the corneal lineage. Positivity of CK3/12, CK4, CK12 and CONNEXIN 43 immunostaining and the relative mRNA expression of CK3, CK4, CK12, CK13, CK15 and CONNEXIN 43 could be detected in the cultured biopsies.
  
  
  CONCLUSIONS
  Unlike tissue-specific protocols, our protocol can unequivocally promote differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. This simple standardized protocol can be adapted for ocular surface reconstruction using stem cell transplantation.
authors.affiliation: Stem Cell Research Laboratory, GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, India; School of Biosciences and Technology, VIT University, Vellore, India.
authors.forename: Kamesh
authors.initials: K
authors.lastname: Dhamodaran
authors.affiliation: Stem Cell Research Laboratory, GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, India; Post Graduate Department of Biotechnology, Jamal Mohamed College (Autonomous), Tiruchirappalli, India.
authors.forename: Murali
authors.initials: M
authors.lastname: Subramani
authors.affiliation: Department of Cornea and Refractive Surgery, Bangalore, India.
authors.forename: Himanshu
authors.initials: H
authors.lastname: Matalia
authors.affiliation: Department of Vitreo-Retinal Services, Narayana Nethralaya Eye Hospital, Bangalore, India.
authors.forename: Chaitra
authors.initials: C
authors.lastname: Jayadev
authors.affiliation: Department of Cornea and Refractive Surgery, Bangalore, India.
authors.forename: Rohit
authors.initials: R
authors.lastname: Shetty
authors.affiliation: Stem Cell Research Laboratory, GROW Laboratory, Narayana Nethralaya Foundation, Bangalore, India. Electronic address: drdebashish@narayananethralaya.com.
authors.forename: Debashish
authors.initials: D
authors.lastname: Das
mesh_terms: D000650:Amnion / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods / Q000592:standards*; D002454:Cell Differentiation; D019070:Cell Lineage / Q000502:physiology*; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology*; D003315:Cornea / Q000166:cytology / Q000502:physiology*; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D005260:Female; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008297:Male; D009061:Mouth Mucosa / Q000166:cytology*; D012015:Reference Standards; D033581:Stem Cell Transplantation
keywords: cell therapy; conjunctival epithelial cells; human amniotic membrane; limbal epithelial cells; limbal stem cell deficiency; oral mucosal epithelial cells