Cultivated corneal epithelial stem cell transplantation in ocular surface disorders

doi:10.1016/S0161-6420(01)00694-7

Ophthalmology

Volume 108, Issue 9, September 2001, Pages 1569-1574

Presented in part at the annual meeting of the American Academy of Ophthalmology, Dallas, Texas, October 2000.

https://doi.org/10.1016/S0161-6420(01)00694-7 Get rights and content

Abstract

Purpose

To investigate the outcome of cultivated corneal epithelial transplantation for severe stem cell deficiencies using denuded amniotic membrane (AM) as a carrier.

Design

Retrospective, noncomparative case series.

Participants

Thirteen eyes of 11 patients were studied. These consisted of five eyes with acute Stevens-Johnson syndrome (SJS), two with chronic SJS, one with an acute chemical injury, two with chronic chemical injuries, two with ocular cicatricial pemphigoid, and one with drug-induced pseudopemphigoid. All of these eyes had total stem cell deficiencies.

Main outcome measures

Adaptation of the cultivated corneal epithelium onto the host corneal surface was confirmed 48 hours after surgery. The reconstruction of the ocular surface and visual acuity were measured.

Methods

Corneal limbal epithelium from donor corneas was cultivated for 4 weeks on a denuded AM carrier, with 3T3 fibroblast coculture and air lifting. The cultivated corneal epithelium showed four to five layers of stratification and was well differentiated. After conjunctival tissue removal from the cornea up to 3 mm outside the limbus and subconjunctival tissue treatment with 0.04% mitomycin C, cultivated allocorneal epithelium, including the AM carrier, was transplanted onto the corneal surface up to the limbus. Lamellar keratoplasty, using preserved donor graft without epithelium, was performed simultaneously for five chronic-phase patients showing corneal stromal scarring. Systemic immunosuppression was used to prevent allograft rejection.

Results

In all 13 eyes, the entire corneal surface, on which cultivated allocorneal epithelium had been placed, was free from epithelial defects 48 hours after surgery, indicating complete survival of the transplanted corneal epithelium. Visual acuity improved in all eyes after surgery, and 10 of the 13 eyes were restored to good vision (postoperative visual acuity improved two or more lines) 6 months after the operation. During the follow-up period (mean ± standard deviation, 11.2 ± 1.3 months), the corneal surfaces were clear, although three eyes experienced epithelial rejection.

Conclusions

Cultivated corneal epithelial transplantation using denuded AM as a carrier can be used for severe stem cell deficiencies.

Section snippets

Subjects

The study consisted of 13 eyes of 11 patients who underwent cultivated allocorneal epithelial transplantation in our university hospital from July 1999 through November 1999. They consisted of six males and five females with a mean age (± standard deviation) of 42.5 years (± 24.5 years). The patients had been clinically diagnosed as being total stem cell deficient. This diagnosis was based on the complete disappearance of the palisades of Vogt and the presence of a total corneal epithelial

Results

As shown in Table 1 and Figure 2, 48 hours after transplantation the corneal surfaces of all 13 eyes were clear and smooth. We observed that the entire corneal surfaces were perfectly covered with transplanted allocorneal epithelium that did not stain with fluorescein. Although there was no inherent staining of the recipient epithelium, the transplanted corneal epithelium was surrounded by a conjunctival epithelial defect at 360°, suggesting there was no contamination of host conjunctival

Discussion

Cultivated corneal epithelial transplantation has been one of the dreams of corneal transplantation surgeons. Cultivated skin transplantation was established more than 10 years ago among dermatologists, and autocultivated epithelial transplantation has been widely performed for the treatment of severely burned skin. However, in vitro cultivation of corneal epithelium is much more difficult. In the past, many attempts have been made,18, 19, 20, 21 but they were never successful enough for

Acknowledgements

The authors thank Nigel Fullwood, PhD, for reviewing the manuscript.

References (25)

A Schermer et al.
Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells
J Cell Biol
(1986)
M.S Shapiro et al.
Corneal re-epithelialization from the conjunctiva
Invest Ophthalmol Vis Sci
(1981)
H.S Dua et al.
The corneoscleral limbus in human corneal epithelial wound healing
Am J Ophthalmol
(1990)
K Tsubota et al.
Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens-Johnson syndrome
Am J Ophthalmol
(1996)
J Shimazaki et al.
Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns
Ophthalmology
(1997)
S.C.G Tseng et al.
Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency
Arch Ophthalmol
(1998)
K Fukuda et al.
Differential distribution of subchains of the basement membrane components type IV collagen and laminin among the amniotic membrane, cornea, and conjunctiva
Cornea
(1999)
N Koizumi et al.
Growth factor mRNA and protein in preserved human amniotic membrane
Curr Eye Res
(2000)
G Pellegrini et al.
Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium
Lancet
(1997)
R.J.F Tsai et al.
Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells
N Engl J Med
(2000)

I.R Schwab et al.

Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease

Cornea

(2000)

N Koizumi et al.

Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits

Cornea

(2000)

Cited by (450)

Comparative analysis of long-term results of three epithelial cell transplantation procedures for treating limbal stem cell deficiency
2024, Ocular Surface
This study compared the long-term outcome of different epithelial transplantation techniques to treat limbal stem cell deficiency (LSCD). We conducted a retrospective 15-year comparative systematic cohort study of patients with LSCD who underwent either cultivated limbal epithelial transplantation (CLET), simple limbal epithelial transplantation (SLET), or cultivated oral mucosal epithelial transplantation (COMET). We reviewed the demographic data, etiology, LSCD severity, best-corrected visual acuity, surgical outcomes, and complications. A total of 103 eyes of 94 patients (mean age, 45.0 ± 16.4 years) with LSCD were enrolled. The most common cause of LSCD was chemical injury (42.7 %). The median follow-up time was 75 months. The success rates of CLET, SLET, and COMET were 45.5 %, 77.8 %, and 57.8 %, respectively. The 7-year survival rates after CLET, SLET, and COMET were 50.0 %, 72.2 %, and 53.2 %, respectively. Steven-Johnson syndrome (SJS) had a significantly lower survival rate than other causes (p < 0.001), but SLET had a significantly higher survival rate than CLET (p = 0.018) and COMET (p = 0.047). Visual improvement of more than four Snellen lines was achieved in 53.1 % of successful cases and 28.2 % of failed cases. SJS, Schirmer I test <5 mm, and the presence of postoperative recurrent epithelial defects were significant risk factors for a failed surgery. All epithelial transplantation techniques had favorable long-term surgical outcomes. More than half of the patients achieved a stable ocular surface and visual acuity improvement up to 7 years postoperatively. SLET tends to have a better surgical outcome than CLET and COMET, especially in patients with SJS.
Corneal regeneration strategies: From stem cell therapy to tissue engineered stem cell scaffolds
2023, Biomedicine and Pharmacotherapy
Corneal epithelial defects and excessive wound healing might lead to severe complications. As stem cells can self-renew infinitely, they are a promising solution for regenerating the corneal epithelium and treating severe corneal epithelial injury. The chemical and biophysical properties of biological scaffolds, such as the amniotic membrane, fibrin, and hydrogels, can provide the necessary signals for stem cell proliferation and differentiation. Multiple researchers have conducted investigations on these scaffolds and evaluated them as potential therapeutic interventions for corneal disorders. These studies have identified various inherent benefits and drawbacks associated with these scaffolds. In this study, we provided a comprehensive overview of the history and use of various stem cells in corneal repair. We mainly discussed biological scaffolds that are used in stem cell transplantation and innovative materials that are under investigation.
Insights into the clinical development of regenerative medical products through a comparison of three cell-based products recently approved for limbal stem cell deficiency
2023, Ocular Surface
Three regenerative medical products for limbal stem cell deficiency (LSCD), a rare and intractable ocular surface disease, have recently been approved in Japan. To our knowledge, this is the first time multiple stem-cell-based medical products have been approved for the same ocular disease. Development plans and study designs for each product differ, resulting in differences in indications. Since cell-based products have a heterogeneous formulation and often target rare diseases, they require a flexible approach to development. This review article describes the status and prospects of the clinical development of regenerative medical products by summarizing the issues of the three products from the Pharmaceuticals and Medical Devices Agency (PMDA) standpoint. Implementing stem cell-based products is challenging, requiring scientific and flexible review by regulatory authorities. To overcome these issues in the development process, developers and regulatory authorities need to communicate and fully discuss study protocols from the early stage of development.
Keratin films for ocular surface reconstruction: Wound healing in an in-vivo model
2023, Experimental Eye Research
The most commonly used tissue substitute for ocular surface reconstruction is human amniotic membrane (AM). Because of its low biomechanical strength and intransparency there is a need to search for alternatives of consistent quality. This study, further explored the biocompatibility of Keratin Film (KF) and its ability to sustain corneal epithelial wound healing.
In three equal groups of 5 New Zeeland white rabbits a 4 mm superficial keratectomy was created in the right eye. Five eyes received a KF, five a human AM graft and the remaining five no implant. All eyes were treated with ofloxacin and dexamethasone eye drops and followed up for 10 days. Corneal fluorescein staining, vascularization, and transparency were assessed using slit lamp biomicroscopy according to a standardized grading score during and at the end of follow-up. The corneal-scleral-button was excised and processed for histology.
After 10 days all eyes which had received a KF showed complete epithelial healing and no signs of neovascularization. In the AM group 1 eye showed a persistent epithelial defect at day 10 and 2 eyes showed neovascularization at day 7 resolving at day 10. Transparency improved progressively both in the KF group as well as in the AM group towards the end of the follow. Histology showed a multilayer epithelium firmly adherent to the KF with no evidence of keratocyte migration or inflammatory reaction in the corneal stroma.
In this study on rabbit eyes KF better supported corneal epithelial wound healing than amniotic membrane.
Stem cells and genetic engineering empower therapeutic development for blinding eye diseases
2023, The Eye: Volume 4
The major external information sensed by a human is visual perception. However, this perception is compromised and eventually abolished in a broad spectrum of blinding eye diseases, including corneal disease, cataract, glaucoma, inherited retinal degeneration, and age-related macular degeneration, and optic neuropathy, in an anatomically anterior to posterior order. Currently, traditional treatments remain incompetent to fully recover the histological structures and restore visual function. Anatomically, the eye holds natural vantages of easy accession and operation, immune isolation, and optical transparency, which prioritize ocular diseases for new technology trials, such as stem cell treatments, gene augmentation, and gene correction therapies. In the past two decades, genetic engineering and stem cell technologies have revolutionized the treatment of a wide range of blinding diseases. We present a comprehensive review of the current status of gene and stem cell-based translational and clinical research for the treatment of blinding eye diseases, with a focus on key ocular tissues from the cornea to the retina, and highlight the most promising preclinical research efforts.
Cultivated Autologous Limbal Epithelial Cell Transplantation: New Frontier in the Treatment of Limbal Stem Cell Deficiency: Cultivated Autologous Limbal Epithelial Cell Transplantation
2022, American Journal of Ophthalmology
Taking into consideration prior human experience with treating limbal stem cell deficiency (LSCD) with cultivated limbal epithelial cells (CLEC) from other countries, we have set a goal to optimize and standardize the techniques of CLEC preparation (called CALEC by our group) for the clinical trial in the United States.
We performed an extensive literature review of all human trials, case series, and reports involving autologous cultivated limbal epithelial cell transplantation. Allogeneic cultivated limbal epithelial cell transplantations were reported only when combined with autologous studies. We also searched prior animal data aiding in detailing regulatory toxicology requirements.
Between 1997 and 2020, the analysis of human trials revealed 21 studies on autologous grafts, and 13 studies analyzing both autologous grafts and allogeneic grafts. Of a total of 34 studies, 6 studies used good manufacturing process (GMP) facilities, and 11 studies had no animal-derived products or murine feeder layers, whereas only 1 study had both. Overall, the treatment with autologous CLEC grafts was 68.9% successful. In total there were 6 preclinical studies using rabbits, serving as surrogate studies to assess the safety and toxicity of cultivated limbal epithelial cells for human trials. Based on prior human experience, we further optimized the manufacturing conditions with GMP-grade and serum and animal-free reagents, and developed cell characterization assays for the CALEC product release.
These data were used to develop a novel and consistent manufacturing process using only qualified and validated reagents for performing the first clinical trial on CALEC transplantation to treat LSCD in the United States.

View all citing articles on Scopus

^☆: Supported by the Japanese Ministry of Health and Welfare and the Japanese Ministry of Education (grant no.: 12557149), Tokyo, Japan; the Kyoto Foundation for the Promotion of Medical Science, Kyoto, Japan; and the Intramural Research Fund of the Kyoto Prefectural University of Medicine, Kyoto, Japan.

¹: The authors have no financial interest in this work.

View full text

Cultivated corneal epithelial stem cell transplantation in ocular surface disorders1☆,

Abstract

Purpose

Design

Participants

Main outcome measures

Methods

Results

Conclusions

Section snippets

Subjects

Results

Discussion

Acknowledgements

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells

J Cell Biol

Corneal re-epithelialization from the conjunctiva

Invest Ophthalmol Vis Sci

The corneoscleral limbus in human corneal epithelial wound healing

Am J Ophthalmol

Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens-Johnson syndrome

Am J Ophthalmol

Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns

Ophthalmology

Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency

Arch Ophthalmol

Differential distribution of subchains of the basement membrane components type IV collagen and laminin among the amniotic membrane, cornea, and conjunctiva

Cornea

Growth factor mRNA and protein in preserved human amniotic membrane

Curr Eye Res

Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium

Lancet

Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells

N Engl J Med

Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease

Cornea

Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits

Cornea

Cultivated corneal epithelial stem cell transplantation in ocular surface disorders¹☆,