title: Culture of corneal limbal epithelial stem cells: experience from benchtop to bedside in a tertiary care hospital in India.
abstract: OBJECTIVE
  To standardize the technique of ex vivo culture of limbal epithelial stem cells (LESCs) using locally available adapted methods and evaluate the outcome of transplantation in patients with limbal stem cell deficiency.
  
  
  METHODS
  Limbal tissue specimens were isolated from cadaveric corneoscleral rims, living related donors, or contralateral eye of the patients. Harvested tissue was cultured on denuded human amniotic membrane (dHAM) using various techniques to stabilize dHAM. The optimization of in vitro culture conditions was achieved by modifications in culture media (culture media 1 and 2). The LESCs were cultured in both types of media for 2 weeks, and growth patterns were observed. Expanded cells were further characterized by immunocytochemistry (K3/12, K19, and ABCG2) and reverse transcriptase polymerase chain reaction (K12, Cx43, Pax6, ABCG2, p63, and K19). The cultivated epithelium was transplanted in 50 patients with total and partial limbal stem cell deficiencies.
  
  
  RESULTS
  Stabilization of dHAM was successfully achieved using coverslips. The outgrowth was observed within 1-3 days of culture using both types of culture media (P = 0.20), but cultures in culture medium 1 reached confluency faster than cultures in culture medium 2 (P = 0.0004). Histopathological analysis showed multilayer formation and immunostaining, and reverse transcriptase polymerase chain reaction data confirmed the expression of both stem cell markers (K19, p63, and ABCG2) and differentiation markers (K3, K12, and Cx43). Patients who had undergone limbal stem cell transplantation showed a stable ocular surface with improved visual acuity over a long-term follow-up period.
  
  
  CONCLUSIONS
  LESCs were successfully cultured using locally available adapted methods, and their clinical benefits verified by transplantation.
authors.affiliation: Department of Ophthalmology, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Shweta
authors.initials: S
authors.lastname: Sharma
authors.forename: Radhika
authors.initials: R
authors.lastname: Tandon
authors.forename: Sujata
authors.initials: S
authors.lastname: Mohanty
authors.forename: Namrata
authors.initials: N
authors.lastname: Sharma
authors.forename: Vanathi
authors.initials: V
authors.lastname: M
authors.forename: Seema
authors.initials: S
authors.lastname: Sen
authors.forename: Seema
authors.initials: S
authors.lastname: Kashyap
authors.forename: Neeta
authors.initials: N
authors.lastname: Singh
mesh_terms: D000293:Adolescent; D000328:Adult; D000650:Amnion / Q000166:cytology; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002648:Child; D002675:Child, Preschool; D003316:Corneal Diseases / Q000378:metabolism / Q000601:surgery*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006761:Hospitals; D006801:Humans; D007124:Immunoenzyme Techniques; D007194:India; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014019:Tissue Donors; D014792:Visual Acuity / Q000502:physiology; D055815:Young Adult
chemical_list: D015415:Biomarkers