title: Consecutive expansion of limbal epithelial stem cells from a single limbal biopsy.
abstract: OBJECTIVE
  Corneal epithelium is maintained by limbal epithelial stem cells (LESCs), the loss of which can be catastrophic for corneal transparency. Effective therapies include the transplantation of cultivated LESCs, requiring optimization of in vitro cultivation protocols. Unfortunately, optimization studies are hampered by the limited number of ocular tissue donors. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same 1-2 mm(2) limbal explant (LE).
  
  
  METHODS
  LEs were plated and maintained until outgrowth surrounded each, being removed at this point. LPCs were allowed to reach confluence (LPC0). The same removed LE was plated again, following the same procedure, obtaining LPC1. This procedure was repeated as often as possible up to six times. LPCs from each passage were analysed by real time reverse transcription-polymerase chain reaction and immunofluorescence-microscopy.
  
  
  RESULTS
  LPCs from LPC0 to LPC2 presented a heterogeneous cell population, with cells positive for LESC markers K14, K15, ABCG2 and p63, differentiated corneal epithelial cell-specific markers K3 and K12, and for the fibroblast marker S100A4. These cells had an epithelial-like morphology. In LPC3-LPC4, elongated cell morphology appeared, and the presence of LESC markers decreased, while the presence of differentiated corneal epithelial-cell and fibroblast markers increased.
  
  
  CONCLUSIONS
  One LE can be successfully cultivated up to three consecutive times while maintaining the LESC phenotype in the LPC cells. This protocol provides several homologous LPCs for basic research. Additionally, by using a cell-carrier, the resulting LPCs could serve reservoirs for potential autologous expanded LESC transplantations and/or for making correlations between laboratory and clinical outcomes.
authors: Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain. marina@ioba.med.uva.es
authors: Marina
authors: M
authors: López-Paniagua
authors: Teresa
authors: T
authors: Nieto-Miguel
authors: Ana
authors: A
authors: de la Mata
authors: Sara
authors: S
authors: Galindo
authors: José M
authors: JM
authors: Herreras
authors: Rosa M
authors: RM
authors: Corrales
authors: Margarita
authors: M
authors: Calonge
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001706:Biopsy; D002102:Cadaver; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D016039:Corneal Transplantation / Q000379:methods*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology*; D005125:Eye Banks; D005240:Feasibility Studies; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005819:Genetic Markers; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D015415:Biomarkers; D005819:Genetic Markers