title: Comparison of functional limbal epithelial stem cell isolation methods.
abstract: The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either of the isolation techniques from the donor limbal tissue, then the cell suspension is probably the best option as long as the cells are expanded following our culture conditions.
authors.affiliation: IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Campus Miguel Delibes, Paseo de Belén 17, E-47011, Valladolid, Spain; CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials and Nanomedicine) Valladolid, Spain. Electronic address: marina@ioba.med.uva.es.
authors.forename: Marina
authors.initials: M
authors.lastname: López-Paniagua
authors.affiliation: CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials and Nanomedicine) Valladolid, Spain; IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Campus Miguel Delibes, Paseo de Belén 17, E-47011, Valladolid, Spain. Electronic address: tnietom@ioba.med.uva.es.
authors.forename: Teresa
authors.initials: T
authors.lastname: Nieto-Miguel
authors.affiliation: IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Campus Miguel Delibes, Paseo de Belén 17, E-47011, Valladolid, Spain; CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials and Nanomedicine) Valladolid, Spain. Electronic address: anus1981@hotmail.com.
authors.forename: Ana
authors.initials: A
authors.lastname: de la Mata
authors.affiliation: Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, 11-43 Bath Street, Greater London EC1V 9EL, London, UK. Electronic address: marc.dziasko.11@ucl.ac.uk.
authors.forename: Marc
authors.initials: M
authors.lastname: Dziasko
authors.affiliation: IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Campus Miguel Delibes, Paseo de Belén 17, E-47011, Valladolid, Spain; CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials and Nanomedicine) Valladolid, Spain. Electronic address: sgalindor@ioba.med.uva.es.
authors.forename: Sara
authors.initials: S
authors.lastname: Galindo
authors.affiliation: CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials and Nanomedicine) Valladolid, Spain; IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Campus Miguel Delibes, Paseo de Belén 17, E-47011, Valladolid, Spain. Electronic address: esther_fdz_rey@hotmail.com.
authors.forename: Esther
authors.initials: E
authors.lastname: Rey
authors.affiliation: IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Campus Miguel Delibes, Paseo de Belén 17, E-47011, Valladolid, Spain; CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials and Nanomedicine) Valladolid, Spain. Electronic address: herreras@ioba.med.uva.es.
authors.forename: José M
authors.initials: JM
authors.lastname: Herreras
authors.affiliation: CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials and Nanomedicine) Valladolid, Spain; IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Campus Miguel Delibes, Paseo de Belén 17, E-47011, Valladolid, Spain. Electronic address: rcorrales@pointguardllc.com.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Corrales
authors.affiliation: Department of Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, 11-43 Bath Street, Greater London EC1V 9EL, London, UK. Electronic address: j.daniels@ucl.ac.uk.
authors.forename: Julie T
authors.initials: JT
authors.lastname: Daniels
authors.affiliation: IOBA (Institute of Applied Ophthalmobiology), University of Valladolid, Campus Miguel Delibes, Paseo de Belén 17, E-47011, Valladolid, Spain; CIBER-BBN (Networking Research Center on Bioengineering, Biomaterials and Nanomedicine) Valladolid, Spain. Electronic address: calonge@ioba.med.uva.es.
authors.forename: Margarita
authors.initials: M
authors.lastname: Calonge
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000378:metabolism / Q000648:ultrastructure*; D005260:Female; D006801:Humans; D016850:Limbus Corneae / Q000378:metabolism / Q000648:ultrastructure*; D008297:Male; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D013234:Stem Cells / Q000378:metabolism / Q000648:ultrastructure*; D014019:Tissue Donors
chemical_list: D015415:Biomarkers
keywords: Cell culture; Cell suspensions; Explants; Limbal stem cells; Ocular surface