Abstract
Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. Specifically, ocular burns cause depletion of limbal stem cells, which leads to corneal opacification and visual loss. Corneal stem cells are segregated in the basal layer of the limbus, which is the transitional zone of the epithelium located between the cornea and the bulbar conjunctiva. Autologous cultured limbal epithelial cells can restore damaged corneas. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets. We outline some quality criteria, which assure the clinical performance of keratinocyte culture: evaluation of the number of holoclones within a cultured epithelial graft, proportion of aborting colonies, and percentage of cells expressing high levels of ΔNp63α.
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References
De Luca M, Pellegrini G, Green H (2006) Regeneration of squamous epithelia from stem cells of cultured grafts. Regen Med 1:45–57
Majo F, Rochat A, Nicolas M, Jaoudé GA, Barrandon Y (2008) Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature 456:250–4
Schermer A, Galvin S, Sun TT (1986) Differentiation-related expression of a major 64 K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 103:49–62
Pellegrini G, Golisano O, Paterna P, Lambiase A, Bonini S, Rama P, De Luca M (1999) Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 145:769–82
Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M (1997) Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 349:990–3
Rama P, Bonini S, Lambiase A, Golisano O, Paterna P, De Luca M, Pellegrini G (2001) Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation 72:1478–85
Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84:2302–6
Pellegrini G, Rama P, Mavilio F, De Luca M (2009) Epithelial stem cells in corneal regeneration and epidermal gene therapy. J Pathol 217:217–28
Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, Ponzin D, McKeon F, De Luca M (2001) p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98:3156–61
Di Iorio E, Barbaro V, Ruzza A, Ponzin D, Pellegrini G, De Luca M (2005) Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration. Proc Natl Acad Sci USA 102:9523–8
Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G (2010) Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 363:147–55
Pellegrini G, Ranno R, Stracuzzi G, Bondanza S, Guerra L, Zambruno G, Micali G, De Luca M (1999) The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. Transplantation 68:868–79
Di Iorio E, Barbaro V, Ferrari S, Ortolani C, De Luca M, Pellegrini G (2006) Q-FIHC: quantification of fluorescence immunohistochemistry to analyse p63 isoforms and cell cycle phases in human limbal stem cells. Microsc Res Tech 69:983–91
Acknowledgments
We thank Professor Howard Green, the father of Regenerative Medicine, for supplying 3T3-J2 cell line which were used in these years for the manipulation of corneal stem cells; Tung-Tien Sun who kindly gave us for years as a gift the antibody α-cytokeratin 3 for the selection of patients’ cells. We thank all the collaborators of previous laboratories who supported us in the epithelia characterization, in particular Sergio Bondanza and Patrizia Paterna (IDI, Rome); Dr. Osvaldo Golisano, Dr. Enzo Di Iorio, and Dr.ssa Vanessa Barbaro (FBOV, Venice); the ophthalmologists Dott. Paolo Rama, Prof. Carlo Traverso, Dott. Alessandro Lambiase, and Dott. Stefano Bonini who helped us in the definition of clinical protocols.
We thank MIUR, Italian Ministry of Health, and European Community for the research funding.
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Corradini, F., Venturi, B., Pellegrini, G., De Luca, M. (2012). Methods for Characterization/Manipulation of Human Corneal Stem Cells and their Applications in Regenerative Medicine. In: Mace, K., Braun, K. (eds) Progenitor Cells. Methods in Molecular Biology, vol 916. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-980-8_26
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DOI: https://doi.org/10.1007/978-1-61779-980-8_26
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Publisher Name: Humana Press, Totowa, NJ
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