Comparison of functional limbal epithelial stem cell isolation methods

Marina López-Paniagua; Teresa Nieto-Miguel; Ana de la Mata; Marc Dziasko; Sara Galindo; Esther Rey; José M Herreras; Rosa M Corrales; Julie T Daniels; Margarita Calonge

doi:10.1016/j.exer.2015.12.002

Comparison of functional limbal epithelial stem cell isolation methods

Exp Eye Res. 2016 May:146:83-94. doi: 10.1016/j.exer.2015.12.002. Epub 2015 Dec 17.

Authors

Affiliations

¹ 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.
² 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.
³ 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.
⁴ 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.
⁵ 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.
⁶ 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.
⁷ 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.
⁸ 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.
⁹ 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.
¹⁰ 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.

PMID: 26704459
DOI: 10.1016/j.exer.2015.12.002

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.

Keywords: Cell culture; Cell suspensions; Explants; Limbal stem cells; Ocular surface.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

Adult
Aged
Aged, 80 and over
Biomarkers / metabolism
Cell Culture Techniques / methods*
Cell Separation
Cells, Cultured
Epithelium, Corneal / metabolism
Epithelium, Corneal / ultrastructure*
Female
Humans
Limbus Corneae / metabolism
Limbus Corneae / ultrastructure*
Male
Microscopy, Electron, Transmission
Middle Aged
Stem Cells / metabolism
Stem Cells / ultrastructure*
Tissue Donors

Substances

Biomarkers