Ex Vivo Expansion and Transplantation of Limbal Epithelial Stem Cells
Received 18 February 2008; received in revised form 31 March 2008; accepted 25 April 2008. published online 13 June 2008.
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.
Available online: June 10, 2008.
1Cells for Sight Transplantation and Research Programme, UCL Institute of Ophthalmology, London, United Kingdom
2Ocular Repair and Regeneration Biology Unit, UCL Institute of Ophthalmology, London, United Kingdom
3Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom
4Department of Pathology, UCL Institute of Ophthalmology, London, United Kingdom
Correspondence: Alex J. Shortt, MSc, MRCOphth, MRC, Clinical Research Fellow, Institute of Ophthalmology and Moorfields Eye Hospital, 11-43 Bath Street, London, EC1V 9EL, United Kingdom
Manuscript no. 2008-225.
Financial Disclosure(s): No author has a proprietary interest in this work, and there is no conflict of interest for any author.
Supported by the National Institutes of Health Biomedical Research Centre at Moorfields Eye Hospital and the UCL Institute of Ophthalmology. Funding was received in the form of a Clinical Research Training Fellowship awarded (AJS) by the UK Medical Research Council and from The Special Trustees of Moorfields Eye Hospital (AJS and JTD) and The Eranda Foundation (GAS). Funding for the Rostock adaptor and HRT-II used to perform in vivo confocal microscopy was provided by Moorfields Eye Hospital Special Trustees.
ABSTRACT