Elsevier

Ophthalmology

Volume 115, Issue 11, November 2008, Pages 1989-1997
Ophthalmology

Original article
Ex Vivo Expansion and Transplantation of Limbal Epithelial Stem Cells

https://doi.org/10.1016/j.ophtha.2008.04.039Get rights and content

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.

Section snippets

Patients

Ten eyes of 10 patients with corneal LESC deficiency were treated. Ethical approval was obtained from the institutional Medical Research Ethics Committee, and informed consent was obtained from all patients before treatment. A diagnosis of LESC deficiency was based on characteristic clinical changes confirmed by the presence of conjunctival epithelial cells on the cornea on impression cytology. The clinical signs included some or all of the following changes: loss of corneal epithelial

Clinical Outcome

There were 3 serious adverse clinical events. Patient 5 developed a presumed microbial keratitis that resolved after treatment with topical ceftazidime 5% without evident toxicity to the graft. In patient 8, the graft detached and was lost on the first postoperative day. Patient 3 developed tremor, hypertension, and increased serum creatinine at 8 weeks after treatment. This was attributed to her treatment with oral cyclosporine, which was therefore stopped. Shortly thereafter, her tremor

Discussion

This study reports the detailed clinical, impression cytologic, and confocal microscopic results of the use of ex vivo LESCs cultured in compliance with GMP standards without the use of a 3T3 fibroblast feeder layer. The majority of previous studies of LESC transplantation have used a mouse-derived 3T3 feeder layer in the culture protocol.1, 2, 3, 5, 6, 7, 10, 13, 14 The discovery that human embryonic stem cells cultured on such a feeder layer begin to express animal glycoproteins on their

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    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.

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