Cultivated Limbal Stem Cell Transplantation Research Articles

The role of eye banking with cell-based therapies.

Cell-based therapies are an exciting new frontier in managing corneal diseases. The introduction of these novel therapies may provide new alternatives to corneal transplantation and decrease the dependence on donor corneal tissue. These changes have the potential to significantly impact eye banking in the future. The current article reviews current research involving cell-based therapy for treating corneal disorders, including cultivated limbal stem cell transplantation, limbal mesenchymal stem cells for stromal regeneration, and the use of human-cultivated endothelial cells. We will look at barriers to the development and implementation of these therapies. As corneal surgery expands to include cell-based therapies; eye banks will need to redefine their role to support the everchanging landscape of corneal surgery and the decreased demand for corneal donor tissue.

Research progress on limbal stem cell transplantation

Limbal Stem Cell Deficiency (LSCD) is an ocular surface disease caused by the decrease of the quantity and dysfunction of limbal stem cell, which is characterized by conjunctivalization and other signs of epithelial dysfunction. For sever LSCD, surgery is the main treatment way. Recently, plenty of researches published the outcomes of different operation methods. This article summarized five major operations, including conjunctival limbal autograft (CLAU), simple limbal epithelial transplantation (SLET), limbal allograft, cultivated limbal stem cell transplantation (CLET) and cultivated oral mucosal epithelial transplantation (COMET). (Chin J Ophthalmol, 2020, 56:956-960).

Herpetic keratitis mimicking chronic limbal stem cell failure

To describe four cases of herpetic keratitis after cultivated limbal stem-cell transplantation (CLET). Four patients affected by limbal stem cell deficiency (LSCD) caused by alkali burns received CLET. During regular follow-up, epithelial defects were detected and corneal scraping was performed to identify active viral keratitis. Herpetic epithelial keratitis was successfully diagnosed by corneal scraping and polymerase chain reaction of HSV-1 DNA in all four patients affected by epithelial defects. The defects resolved after appropriate therapy with topical and/or systemic antiviral drugs. Epithelial defects due to herpetic keratitis after cultivated limbal stem-cell transplantation may mimic chronic stem-cell deficiency. A low-threshold for corneal scraping can yield rapid identification of herpetic viral DNA allowing for appropriate treatment.

Amniotic membrane transplantation with or without autologous cultivated limbal stem cell transplantation for the management of partial limbal stem cell deficiency.

PurposeTo compare the outcomes of amniotic membrane transplantation (AMT) vs cultivated limbal stem cell transplantation (LSCT) in eyes with partial limbal stem cell deficiency (LSCD) following chemical burns.MethodsEyes with unilateral partial LSCD (#180° involvement) were randomized in two groups to undergo either pannus resection combined with AMT or pannus resection combined with LSCT in a tertiary eye care hospital. Primary outcome measures were time to corneal epithelialization and absence of conjunctivalization of the cornea. Patients were followed up at 1 week, 1, 3, 6, and 12 months after the surgical procedure.ResultsThere was no difference between mean age (30.85±5.8 vs 28.64±6.4 years, P=0.40) and sex distribution of patients between the two groups at baseline. Mean time to corneal epithelialization was 10.45±5.8 days in the AMT group and 11±3.9 days in the LSCT group (P=0.43). At the end of 1 year, there was no significant difference between the degree of conjunctivalization of cornea, (P=0.06) corneal vascularization, (P=0.08), and clarity (P=0.07) in both groups.ConclusionOur study showed that AMT alone is a useful therapeutic modality in cases with partial LSCD due to ocular chemical injury. Stem cell transplantation may not be required in these cases.

Limbal Stem Cell Transplantation and Complications

ABSTRACT Corneal epithelial stem cells are adult somatic stem cells located at the limbus and represent the ultimate source of transparent corneal epithelium. When these limbal stem cells become dysfunctional or deficient, limbal stem cell deficiency (LSCD) develops. LSCD is a major cause of corneal scarring and is particularly prevalent in chemical and thermal burns of the ocular surface. LSCD leads to conjunctivalization of the corneal surface, neovascularization, recurrent or persistent epithelial defects, ocular surface inflammation, and scarring that, in turn, lead to decreased vision, pain, and impaired quality of life. Several techniques have been reported for limbal stem cell transplantation (LSCT). We introduce the surgical techniques, examine the success rate, and discuss the postoperative complications of conjunctival limbal autograft (CLAU), cultivated limbal stem cell transplantation (CLET), simple limbal epithelial transplantation (SLET), and limbal allograft, including keratolimbal allografts (KLAL) and living-related conjunctival allograft (LR-CLAL).

Allogenic cultivated limbal stem cell transplantation versus cadaveric keratolimbal allograft in ocular surface disorder: 1-year outcome.

To compare the outcome of allogenic ex vivo cultivated limbal stem cell transplantation (LSCT) versus cadaveric keratolimbal allograft in patients with limbal stem cell deficiency (LSCD). In this prospective interventional study from Army Hospital Research and Referral in New Delhi, 50 eyes of 40 patients with ocular surface disorder having LSCD underwent either cultivated LSCT (group 1: 25 eyes of 20 patients) or keratolimbal allograft transplantation (group 2: 25 eyes of 20 patients). The outcome measures were gain of two or more lines of best-corrected visual acuity (BCVA), absence of conjunctival vascularization over cornea, corneal neovascularization regression, regression of visually significant corneal opacity (iris details poorly visible), Schirmer's 1 ≥10s, tear break-up time (TBUT)≥10s, and absence of fluorescein staining. At 1year of follow-up, both groups experienced significant improvement in all outcome measures, but there was no significant difference between both groups in improvement of two or more lines of BCVA (P=0.841), absence of conjunctival vascularization over cornea (P=0.812), corneal neovascularization (P=0.074), visually significant corneal opacity (P=0.075), Schirmer's 1 ≥10s (P=0.814), TBUT≥10s (P=0.382), or absence of fluorescein stain (P=1.00). Both allogenic ex vivo cultivated LSCT and cadaveric keratolimbal allograft transplantation are comparable in terms of visual gain and ocular surface restoration.

Long‐term results of the phase I/II clinical trial: standardized, non‐xenogenic, cultivated limbal stem cell transplantation

PurposeTo evaluate the long‐term success rate of transplanted non‐xenogenic, bio‐engineered, composite grafts of cultured limbal epithelial stem cells on standardized amniotic membranes in patients with limbal stem cell deficiency.MethodsWe report the extended outcome of standardized cultivated limbal stem cell transplantations of our previous phase I/II trial. Between July 2008 and May 2012, 18 patients with partial or total limbal stem cell deficiency received either autologous (n = 15) or allogenic (n = 3) stem cell explants, using a protocol free from xenogenic products and a reduced manipulation surgical technique.ResultsSix of the 18 patients were lost to follow‐up. The remaining 12 patients had a mean follow‐up of 46.6 months. Two of 10 patients experienced an evisceration, not related to the stem cell transplantation. 7 of the remaining 8 (87.5%) patients retained a persistent intact epithelium. Although we did see a significant reduction in corneal neovascularization of the corneas post transplantation, we did not see a significant improvement in visual acuity of the total cohort, although one patient gained a visual acuity from finger counting to 0.5 post limbal stem cell transplantation and post penetrating keratoplasty.ConclusionsThis standardized culture system and surgical approach is safe and effective in restoring a functional epithelial cell layer, with satisfying long‐term results. Due to the limited number of patients and the comorbidities, we did not see a significant improvement in visual acuity of the total cohort. We maintain that in selected cases this technique, followed by a penetrating keratoplasty can significantly improve visual acuity.

Results of a phase I/II clinical trial: standardized, non-xenogenic, cultivated limbal stem cell transplantation

BackgroundTo determine if a standardized, non-xenogenic, reduced manipulation cultivation and surgical transplantation of limbal stem cell grafts is a safe and effective treatment option for patients with total and partial limbal stem cell deficiency.MethodsIn vitro cellular outgrowth and phenotype of the limbal epithelial cell and composite grafts were validated using a new protocol. Patients received either autologous (n = 15) or allogenic (n = 3) explants cultured using a standardized protocol free from xenogenic products. The resulting grafts were transplanted using a reduced manipulation surgical technique.ResultsThe majority of cells (>50%) displayed a progenitor phenotype typified by positive immunofluorescence for ∆Np63, CK14 and ABCG2 and low immunofluorescence for CK3/12 and desmoglein 3 proteins. The surgical protocol was designed to minimize manipulation and the graft itself was secured without sutures. The transplant recipients were followed for a mean of 24 months. Twelve of the 18 transplant recipients were graded as anatomically successful (67%), based on the defined success parameters. There was a significant reduction in corneal neovascularization, which was accompanied by an improvement in pain though not photophobia or central corneal opacity post transplant. The transplantation protocol showed no measureable effect on visual acuity.ConclusionWe conclude that this standardized culture system and surgical approach is safe and effective in reducing corneal neovascularization. The technique is free from animal contaminants and maintains a large proportion of progenitor cells. Although this technique did not improve visual function, restoring a functional epithelial cell layer and reducing corneal neovascularization provides an improved platform for a penetrating keratoplasty to ultimately improve visual function.

Results of a phase I/II clinical trial: standardized, non‐xenogenic, cultivated limbal stem cell transplantation

AbstractWe describe the results of a phase I/II clinical trial for standardized, non‐xenogenic, cultivation and “no touch” surgical transplantation of limbal stem cell grafts. 18 eyes of 18 patients were transplanted with either autologous (n=15) or allogenic (n=3) limbo‐amnion composite grafts that were generated using a standardized culture protocol free of xenogenic culture products and transplanted using a standardized “no touch” surgical technique. In vitro cellular outgrowth and phenotype of the cultivated graft was assessed prior to transplantation. Corneal neovascularization, central corneal opacity, pain, photophobia and visual acuity were investigated pre and post transplantation. The limbal epithelial cells showed an average outgrowth of 14.2mm by day 14. The majority of the cells displayed a progenitor phenotype: p63, CK14, desmoglein, ABCG2 bright and CK3/12 dim protein expression. The transplant recipients were followed up for a mean of 22 months. 12 out of the 18 transplant recipients were graded successful, giving an overall success rate of 67%. The ocular surface photographs for pre‐ and post stem cell transplantation, showed a significant (p=0.007) reduction in the percentage area of corneal neovascularization.

Limbal Side Population Cells: A Future Treatment for Limbal Stem Cell Deficiency

Corneal blindness carries a morbidity that affects quality of life and is often associated with an increased economic burden. In this review, we focus on the severe and painful condition of limbal stem cell deficiency, an important cause of corneal blindness. Conventional corneal transplantation usually results in graft failure and is contraindicated in this condition. Ex vivo-expanded limbal epithelial transplantation has been used as a cellular-based therapy to regenerate and reconstruct the ocular surface as a mode of treatment. Enrichment methods for stem cells are a strategy to improve the outcome of limbal stem cell transplantation. Here we discuss the side population assay as a functional assay to enrich for stem cells as an important source of limbal stem cells. The challenges in ex vivo-expanded limbal stem cell transplantation are wide and varied and will be addressed in this review with regard to improving the clinical outcomes of cultivated limbal stem cell transplantation.

Cultivated Limbal Stem Cell Transplantation—The Surgical Technique

Limbal stem cell deficiency (LSCD) is characterized by loss of limbal palisades of Vogt, ingrowth of conjunctiva on to the cornea, and recurrent or persistent epithelial defects of the cornea. This results in pain, watering, decrease in vision, and light sensitivity. LSCD is most commonly caused by ocular injury, immune-mediated ocular surface diseases, and aniridia. A variety of surgical techniques have been described to treat LSCD, but cultivated limbal stem cell transplantation seems the most promising. In this technique a small biopsy is harvested from healthy limbus, either of the same eye or the other eye. The limbal tissue is shredded into small pieces and seeded on to de-epithelialized human amniotic membrane. The human corneal epithelial medium is used to grow the cells. Twelve to 14 days of culture gives enough cells for clinical transplantation.

Midterm Outcomes of Autologous Cultivated Limbal Stem Cell Transplantation With or Without Penetrating Keratoplasty

To report the midterm outcomes of autologous limbal stem cell transplantation cultivated on amniotic membrane (AM) with or without subsequent penetrating keratoplasty (PKP) in patients with total unilateral limbal stem cell deficiency (LSCD). Eight eyes of 8 consecutive patients with unilateral total LSCD underwent autologous limbal stem cell transplantation cultivated on AM. Four eyes underwent subsequent optical PKP. Main outcome measures were corneal vascularization and transparency. The patients were followed for 34.0 +/- 13.5 months (6-48 months). Seven cases had a stable corneal epithelium with marked decrease in opacification and vascularization. Progressive sectorial conjunctivalization was evident in all cases with subsequent PKP at the last follow-up. Primary failure was observed in one case because of exposure. Transplantation of autologous stem cells cultivated on AM with or without subsequent PKP seems to be an effective way for visual rehabilitation in total LSCD. More work with more cases and longer follow-up are needed to optimize this procedure to provide and maintain an adequate supply of limbal stem cells in these patients.

A Comparison Between Cultivated and Conventional Limbal Stem Cell Transplantation for Stevens-Johnson Syndrome

To compare the resolution of inflammation and long-term results of cultivated and conventional limbal stem cell transplantation (LSCT) in a patient with Stevens-Johnson syndrome (SJS). Interventional case report. A 32-year-old man with SJS and bilateral total limbal stem cell deficiency underwent cultivated LSCT in the right eye, followed by conventional LSCT in the left eye three weeks later. The postoperative medication included dexamethasone 0.1% and ofloxacin 0.3% eyedrops and a tapering dose of systemic corticosteroid, cyclosporine, and cyclophosphamide. Tear samples were collected and analyzed for interleukin (IL) 8 levels. Complete corneal epithelialization was achieved 48 hours after cultivated LSCT, compared with three weeks after conventional LSCT. Ocular inflammation and IL-8 levels decreased more rapidly in the eye with cultivated LSCT. Four years after surgery, more severe corneal scarring and opacification were noted in the conventional LSCT eye. Cultivated LSCT resulted in a better clinical result and vision, with less stromal scarring compared with conventional LSCT.