4.6 Article

Ex Vivo Expansion and Transplantation of Limbal Epithelial Stem Cells

Journal

OPHTHALMOLOGY
Volume 115, Issue 11, Pages 1989-1997

Publisher

ELSEVIER SCIENCE INC
DOI: 10.1016/j.ophtha.2008.04.039

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Funding

  1. National Institutes of Health Biomedical Research Centre
  2. UCL Institute of Ophthalmology
  3. UK Medical Research Council
  4. The Special Trustees of Moorfields Eye Hospital
  5. The Eranda Foundation
  6. MRC [G84/6715] Funding Source: UKRI
  7. Medical Research Council [G84/6715] Funding Source: researchfish

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Objective: To determine, using objective measures, the outcome of ex vivo cultured limbal epithelial stem cell (LESC) transplantation performed in compliance with good manufacturing practice using a novel culture system without 3T3 feeder cells. Design: Prospective, noncomparative, interventional case series. Participants: Ten eyes of 10 patients with profound LESC deficiency arising from chemical injury (4 eyes), aniridia (3 eyes), ectodermal dysplasia (1 eye), Reiger's anomaly with Pax6 haploinsufficiency (1 eye), and unknown cause (1 eye). Methods: Allogeneic (7 eyes) or autologous (3 eyes) corneal LESCs were cultured on human amniotic membrane. Tissue was transplanted to the recipient eye after superficial keratectomy. Impression cytology and confocal microscopy were performed 6 months after surgery with clinical follow-up to 13 months. Success was defined as an improvement in the defined clinical parameters of LESC deficiency, an improvement in visual acuity, the restoration of a more normal corneal phenotype on impression cytology, and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy. Main Outcome Measures: Clinical parameters of LESC deficiency (loss of epithelial transparency, superficial corneal vascularization, epithelial irregularity, and epithelial breakdown), visual acuity, impression cytology and cytokeratin expression profiles, and in vivo confocal corneal confocal microscopy. Results: The success rate using this technique was 60% (autografts 33%, allografts 71%). All patients with a successful outcome experienced an improvement in visual acuity of >= 2 lines Snellen acuity. Preoperatively, CK3+ and CK19+ cells accounted for 12 +/- 2.4% (mean standard error of the mean) and 80 +/- 2.15% of cells, respectively, whereas postoperatively these accounted for 69 +/- 6.43% (P<0.0001) and 30 +/- 6.34% (P<0.0001) of cells, respectively. Goblet cells accounted for 8 +/- 1.19% of cells preoperatively and 1 +/- 0.35% of cells postoperatively (P<0.0001). Conclusions: These data demonstrate that it is possible to culture LESCs ex vivo in compliance with good manufacturing practice regulations. A set of objective outcome measures that confirm the efficiency of this technique in treating LESC deficiency is described. The widespread use of such standardized and objective outcome measures would facilitate a comparison between the different culture methods in use. Financial Disclosure(s): The authors have no proprietary or commercial interest in any materials discussed in this article. Ophthalmology 2008;115:1989-1997 (C) 2008 by the American Academy of Ophthalmology.

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