Diagnostic and Surgical TechniquesTransplantation of Ex Vivo Cultured Limbal Epithelial Stem Cells: A Review of Techniques and Clinical Results
Section snippets
Limbal Epithelial Stem Cells and Corneal Epithelial Homeostasis
The corneal epithelium is essential in maintaining the clarity and the regular refractive surface of the cornea. As with other epithelial surfaces there is a continuous loss of cells from the surface of the corneal epithelium.52, 103 Desquamated cells are replenished by a small population of LESCs located in the basal layer of the limbal epithelium, which also play a central role in corneal epithelial regeneration and repair following injury.13, 14, 24, 26, 57, 81, 103 Clinically LSCD is
Etiology
A deficiency of LESCs occurs in a variety of disorders. Some, such as aniridia, are the result of a genetic disorder and are heritable.41, 70 More commonly, however, LSCD results from acquired factors such as chemical or thermal injury, ultraviolet and ionizing radiation, Stevens Johnson syndrome, advanced ocular cicatricial pemphigoid, contact lens wear, multiple surgeries, radiation, antimetabolites, or extensive microbial infection.9, 24, 27, 29, 33, 35, 40, 42, 92 If LESCs are depleted by
Characteristics of Reported Studies
We identified 17 peer-reviewed publications reporting the use of ex vivo cultured LECs18, 37, 55, 56, 63, 64, 67, 76, 80, 87, 89, 90, 91, 94, 95, 96, 102 and four publications reporting the transplantation of ex vivo cultured oral mucosal epithelial cells.46, 47, 62, 71 The main characteristics of these studies are listed in Table 1.
Methods Employed by Studies
The methods used to diagnose LSCD and produce ex vivo cultured LEC grafts varied widely.
Evidence for Presence of Stem Cells in Cultures and Grafts
The evidence for the presence of stem cells in the transplanted cell population is based upon the following: functional assays of the colony forming ability of transplanted cells (only stem cells can give rise to large colonies in culture4, 75), immunohistological analysis of grafts, and evidence of donor cell survival.
Three studies provided colony forming efficiency data that strongly support the presence of stem cells in these transplanted cell populations.76, 80, 95 Schwab et al estimated
Surgical Transplantation of Ex Vivo Cultured Epithelial Sheets
The surgical technique for transplanting ex vivo cultured LECs was similar in all the studies. Following 360° conjunctival peritomy, the fibrovascular pannus and ingrowing conjunctival tissue was dissected from the cornea and limbus. Hemostasis was achieved using cautery with or without topical 10% phenylephrine. In an attempt to prevent conjunctival ingrowth in the postoperative period some authors applied mitomycin C (0.04% for 5 minutes) to the subconjunctival space followed by vigorous
Postoperative Management
The basic principles are immediate control of inflammation, prophylaxis against infection, mechanical protection of the graft, and prevention of allograft rejection. In all studies topical unpreserved steroid and a broad spectrum antibiotic (ofloxacin37, 55, 56, 64, 67 or chloramphenicol18, 76, 80) were administered four times daily starting immediately after transplantation and continuing for 1 to 3 months. A 1-month course of oral corticosteroids (e.g., betamethasone or prednisolone 1
Donor Screening
Ex vivo LEC culture carries a risk of transfer of bacteria, viruses, and prions, both to the patient but also to the laboratory staff processing the tissue. For these reasons consideration must be given to the screening of tissue donors and the risk of cross-contamination of cultures. Screening should include HIV (human immunodeficiency virus), hepatitis B and C, syphilis and HTLV (human T lymphotrophic virus), and prion-related disease. This can be achieved through a combination of
Improvement in Ocular Surface
The outcome measures used to define successful treatment were poorly described by most studies. Rama et al devised a scoring system to grade the severity of stem cell deficiency.80 This was based on defined clinical and impression cytology findings. These were assessed and scored before and after treatment and statistical analysis of the difference was performed. This provided a reliable objective assessment of treatment success. Koizumi et al55 and Daya et al18 defined a set of clinical
Regulatory Considerations
Until recently, groups providing this treatment only required a local ethics committee's approval. However, groups providing cellular therapies in Europe will soon be required to comply with new mandatory European Union (EU) regulations. The Department of Health in the UK introduced a voluntary “Code of Practice for Tissue Banks Providing Tissues of Human Origin for Therapeutic Purposes” in 2001 (ISBN 1 84182 329 5). This code of practice applies to all non-commercial tissue banks. Currently
Discussion and Conclusions
Ex vivo expansion and transplantation of limbal epithelium has been performed by several unrelated groups in a number of countries. It has been used to treat a variety of ocular surface disorders that are thought to be the result of limbal stem cell failure.
Despite a substantial number of experimental models of this technique, and an ever-growing body of laboratory data on limbal epithelial stem cell biology, the scientific understanding of this procedure is poor. Some key questions still need
Method of Literature Search
This article was prepared by searching the National Library of Medicine database 1975–October 2006 using the following search words: tissue engineering, cultured cells, stem cell transplantation, deficiency, stem cell deficiency, cornea, amnion, corneal limbus, limbal stem cell, ocular surface. Additional sources included the textbooks referenced herein. The search was restricted to publications in English and other-language publications with English abstracts. The reference lists of published
References (113)
- et al.
Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting
Exp Eye Res
(2003) - et al.
Impression cytology of the ocular surface: a review
Exp Eye Res
(2004) - et al.
Treatment of contact lens-related ocular surface disorders with autologous conjunctival transplantation
Ophthalmology
(1992) - et al.
Suprabasal marker proteins distinguishing keratinizing squamous epithelia: cytokeratin 2 polypeptides of oral masticatory epithelium and epidermis are different
Differentiation
(1992) - et al.
Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells
Cell
(1989) - et al.
Living related conjunctival limbal allograft for the treatment of stem cell deficiency
Ophthalmology
(2001) - et al.
Outcomes and DNA analysis of ex vivo expanded stem cell allograft for ocular surface reconstruction
Ophthalmology
(2005) - et al.
Limbal stem cells of the corneal epithelium
Surv Ophthalmol
(2000) - et al.
Phenotypic study of a case with successful transplantation of ex vivo expanded human limbal epithelium for unilateral total limbal stem cell deficiency
Ophthalmology
(2002) - et al.
Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche
Surv Ophthalmol
(2003)
Management of aniridic keratopathy with keratolimbal allograft: a limbal stem cell transplantation technique
Ophthalmology
Long-term outcomes of keratolimbal allograft for the treatment of severe ocular surface disorders
Ophthalmology
Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures
Exp Eye Res
Characteristics of the human ocular surface epithelium
Prog Retin Eye Res
Cultivated corneal epithelial stem cell transplantation in ocular surface disorders
Ophthalmology
Transplantation of EGF-responsive neurospheres from GFP transgenic mice into the eyes of rd mice
Brain Res
Transplantation of autologous serum-derived cultivated corneal epithelial equivalents for the treatment of severe ocular surface disease
Ophthalmology
Ocular surface abnormalities in aniridia
Am J Ophthalmol
Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium
Lancet
Cytologic evidence of corneal diseases with limbal stem cell deficiency
Ophthalmology
Long-term results of allogeneic penetrating limbo-keratoplasty in total limbal stem cell deficiency
Ophthalmology
Serial cultivation of normal human epidermal keratinocytes
Methods Cell Biol
Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders
Ophthalmology
Long-term outcome of keratolimbal allograft with or without penetrating keratoplasty for total limbal stem cell deficiency
Ophthalmology
Penetrating limbo-keratoplasty for granular and lattice corneal dystrophy: survival of donor limbal stem cells and intermediate-term clinical results
Ophthalmology
Inferior conjunctival autograft for primary pterygia
Ophthalmology
Pterygium excision with conjunctival autografting: an effective and safe technique
Br J Ophthalmol
Amniotic membrane transplantation for partial limbal stem cell deficiency
Br J Ophthalmol
Three clonal types of keratinocyte with different capacities for multiplication
Proc Natl Acad Sci USA
Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells
J Cell Sci
Limbal stem cells: the search for a marker
Clin Experiment Ophthalmol
Characterization of putative stem cell phenotype in human limbal epithelia
Stem Cells
Facile isolation and the characterization of human retinal stem cells
Proc Natl Acad Sci USA
An investigation of removed cultivated epithelial transplants in patients after allocultivated corneal epithelial transplantation
Cornea
Corneal stem cells in review
Wound Repair Regen
Impression cytology of the ocular surface—research tool or routine clinical investigation?
Br J Ophthalmol
Stem cell therapy for retinal degeneration: retinal neurons from heterologous sources
Semin Ophthalmol
ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells
Stem Cells
Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration
Proc Natl Acad Sci USA
The conjunctiva in corneal epithelial wound healing
Br J Ophthalmol
Corneal epithelial wound healing
Br J Ophthalmol
Stem cell differentiation and the effects of deficiency
Eye
Limbal stem cell deficiency: concept, aetiology, clinical presentation, diagnosis and management
Indian J Ophthalmol
Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche
Br J Ophthalmol
Autologous limbal transplantation in patients with unilateral corneal stem cell deficiency
Br J Ophthalmol
Mucous membrane pemphigoid: an update
Curr Opin Ophthalmol
Characterization of corneal pannus removed from patients with total limbal stem cell deficiency
Invest Ophthalmol Vis Sci
Keratolimbal allograft in corneal reconstruction
Eye
Outcome of pterygium surgery: analysis over 14 years
Eye
Culture of animal cells: a manual of basic techniques
Cited by (295)
Update on stem/progenitor cell-based clinical trials for eye disease
2023, The Eye: Volume 4Surgical approaches to autologous limbal stem cell transplantation (LSCT) following severe corneal chemical burns
2022, Journal Francais d'Ophtalmologie
The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article. Supported by the National Institute of Health Biomedical Research Centre at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Medical Research Council UK (AJS) and Moorfields Eye Hospital Special Trustees (AJS, JTD).