Limbal stem cell transplantation achieves high standards in Europe and Asia

The concept that stem cells can renew, maintain and repair damaged or diseased tissues has raised a lot of interest in recent years. It is an idea that, for some organs, is progressively being translated from research into clinical practice.

In the realm of corneal stem cells, both the science and practice have made giant steps. Different techniques of allo-limbal and auto-limbal stem cell transplantation have been developed. Among them, the implantation of cultured limbal cells appears to be promising and has become the mission of a group of scientists and clinicians. Their efforts have already produced results that only a few years ago would have appeared as science fiction.

Most of these procedures have been standardized, although outcomes still maintain some degree of dishomogeneity due to the variability of cases, the learning curve of the procedure and, in particular, the complex issues related to quality criteria for graftability, compatibility, stem cell identification, culture methods and substrates.

Limbal stem cells

Several researchers have contributed to finding the specific location of corneal stem cells in the basal layers of the limbus, the transitional zone between the cornea and the bulbar conjunctiva.

Evidence was provided through a variety of immunohistochemical and morphogenetic procedures, which showed that cells in the basal layer of the limbus have a different morphology, biochemical composition and mitosis rate compared with adjacent corneal cells.

Recent research also found specific anatomical microenvironments, called “niches,” in which stem cells reside in the limbus.

“We termed these locations the limbal epithelial crypts. Compared to other parts of the limbus, they contain a much higher concentration of stem cells and are therefore likely to be the niches in which our limbal stem cells live and from which they migrate in response to injury,” Harminder Dua, MD, of Nottingham University, said.

Visual function requires an intact ocular surface, and corneal stem cells play a major role in maintaining this integrity. Congenital conditions such as aniridia, systemic diseases such as Stevens-Johnson syndrome, or, more frequently, infection or trauma can be the cause of various degrees of limbal stem cell deficiency, leading to a cascade of sight-threatening complications.

In more severe cases, without the regenerating support of healthy stem cells, the corneal epithelium can no longer survive, and its territory is invaded by conjunctival cells, which cover the cornea with an opaque membrane. Neovascularization, stromal scarring and chronic inflammation, as well as loss of vision, are the consequences of this conjunctivalization process. Corneal grafts are also unable to survive in these conditions and are soon taken over by the conjunctiva.

The only way to prevent this invasion is to restore the limbus. Depending on whether limbal damage is partial or total, bilateral or unilateral, due to an external insult or to systemic conditions, different techniques can be used and have been tried over the years. What they have in common is the principle that limbal functions can be restored by making fresh, healthy limbal cells repopulate the area.

Auto-limbal and allo-limbal grafting

Harminder Dua

“Limbal transplantation involves taking a lamellar strip of limbal tissue, usually with some adjacent peripheral cornea, and transplanting it to the host eye,” Dr. Dua said.

Before surgery, a suitable environment should be restored on the recipient bed by treating any signs of dry eye and inflammation.

Whenever possible, auto-limbal transplantation, is preferable to allo-limbal grafting. If the limbal graft is harvested from the contralateral healthy eye, there are no problems of compatibility and no immunosuppressive therapy is necessary.

However, auto-limbal transplantation can be performed when only one eye is affected and never in cases of unilateral manifestations of systemic diseases.

“Harvesting the tissue from an apparently normal eye is not recommended, as this is likely to present the same latent conditions of the affected eye,” Dr. Dua said.

Allo-limbal transplantation can be performed by harvesting the tissue from living related donor eyes or cadaver eyes. The use of this procedure is normally limited to extreme cases of bilateral total limbal deficiency caused by chemical and thermal injuries or systemic disorders that destroy the peripheral cornea, such as Stevens-Johnson syndrome and ocular pemphigoid.

Allo-limbal transplantation has numerous limitations. It requires complicated and not always successful HLA-matching tests, and the patient is exposed to the severe side effects of indefinite systemic immunosuppression. The rate of graft survival is low, and multiple surgical procedures are often required. In addition, visual outcome is usually limited.

Results of limbal allograft transplantations from living related donor have been published by several researchers. The rate of graft survival varied between approximately 75% and 92%, but the mean follow-up was relatively short, between 18 and 26 months. For better results, Sheraz Daya, MD, of the Moorfields Eye Hospital in London, suggested the systemic use of long-term, low-maintenance doses of cyclosporine in addition to systemic steroids to prolong graft survival.

New perspective of cultivated stem cells

A new perspective on the treatment of ocular disorders characterized by stem cell deficiency was given by the pioneering work of two Italian biologists, Graziella Pellegrini, PhD, and Michele De Luca, MD. They were the first to find that cultured limbal cells include stem cells detectable as colonies, or holoclones, and this made possible the first therapeutic use of limbal cultures for the regeneration of corneal epithelium.

In 1997, The Lancet published the results of the first two procedures performed by Carlo Traverso, MD, at the University Eye Clinic of Genoa, on patients with severe alkali burns in one eye. The limbal cells were cultivated from a 1 mm² biopsy sample taken from the limbus of a healthy eye. The cultured cells were transferred in the operating room on the inner surface of a contact lens, which was applied to the patient’s eye.

Since then, new and more sophisticated methods of stem cell identification and culture have been developed, leading to a better, safer and more reproducible transplantation procedure.

The protocol standardized by Prof. Pellegrini and Prof. De Luca for corneal limbal cell culture now entails the use of a feeder layer of lethally irradiated 3T3-J2 clone of murine origin certified for clinical use, which allow colonies of stem cells to grow and maintain their integrity as holoclones in an immunologically safe environment. A portion of these cells is then transferred on a 3-cm diameter fibrin disc, ready to be implanted to the patient’s eye.

Large study shows high rate of success

The surgeon most closely involved in this research is Paolo Rama, MD, of San Raffaele Hospital in Milan. With Prof. Pellegrini, Prof. De Luca and other researchers, he published the results of the first 116 patients with ocular burns grafted with stem cells on a fibrin sheet substrate.

“The fibrin sheet, which is very similar to a soft contact lens, is placed on the eye and fitted under the dissected conjunctiva. The eyelids are sutured to be reopened approximately 2 days later. Fibrin is a quickly degradable, natural substrate. It dissolves within 24 to 48 hours, and the stem cells move away to search their niches, from where they will repopulate the surface,” Dr. Rama explained.

During the last 9 years, a large study involving 26 Italian ophthalmology departments has been conducted. A total of 246 transplantations, including bilateral treatments and re-treatments, in 212 patients with limbal stem cell deficiency, resulting from chemical burns and from abuse of contact lenses and ensuing bacterial infection, were treated with limbal cultures.

The mean success rate was 70%, ranging between 67% and 83%, Dr. Pellegrini said. The follow-up has confirmed the continuing integrity of the epithelium and the final recovery of almost normal visual acuity. Regrafting was required in a minority of patients, and this second intervention led to successful re-creation of a normal surface and recovery of vision.

Continued progress in India

Virender S. Sangwan, MS, of the L.V. Prasad Eye Institute in Hyderabad, India, is a pioneer of ocular surface disease through corneal regeneration using limbal stem cell transplantation in India.

Virender S. Sangwan

When he came upon the articles of the Italian group of scientists and clinicians who performed the first successful transplants of cultured limbal grafts, he was inspired and started on his own project with practically no money, but one enthusiastic partner, Geeta K. Vemuganti, MD.

“Within about a year, we had learned how to grow stem cells and discovered our own way of doing it,” he said. They applied for approval to conduct a clinical trial and managed to raise funds to establish the Sudhakar and Sreekanth Ravi Stem Cell Biology Laboratory.

“We have done more than 500 transplants,” Dr. Sangwan said. “In my office, I have two to four patients every day. Many centers have done 10 to 12 cases in a year or 2 years time. I do that in less than a week.”

Recently, Dr. Sangwan started a collaboration with Australian and Canadian groups doing research on new nonbiological substrates to grow stem cells. Similar research is being carried out by a group of Indian and Japanese ophthalmologists and biologists.

“The use of biological material as a substrate is associated with the risk of disease transmission. We started a study to evaluate the use of a synthetic polymer Mebiol gel as a substrate for the growth of limbal phenotype cells and cornea phenotype cells for limbal explants,” said H.N. Madhavan, MD, PhD, FAMS, FICPath, of the Vision Research Foundation.

The new substrate has so far been tested in vitro and on rabbit eyes. A clinical study on humans will soon be started.

Causes of failure

Taking into account the severity of the conditions that are treated with this procedure, the percentage of failures is relatively low and is decreasing with the progressive improvements of the cell culture and surgical techniques, as well as with the increasingly clear criteria for patient selection.

“The most frequent cause of failure is persistent inflammation. Proper preparation of the receiving bed and control of inflammation with anti-inflammatory drugs, before and after surgery, is crucial to the success of the engraftment,” Dr. Rama said.

Another cause of failure might be the unsatisfactory quality of the limbal cell in some cases.

“We have progressively perfected our methods of clonal analysis and cell marking. By analyzing the cryopreserved cells of patients who needed a second procedure, we found they had a reduced p63 expression,” Prof. Pellegrini said

p63, in its ÄNp63á isoform, was recognized as a specific marker of human corneal cells. The low expression of p63 therefore indicated that what was thought to be a holoclone had in fact evolved into a transient-amplifying cell progeny, or paraclone, with a much lower proliferative capacity. Even more complex and accurate marking methods were subsequently developed and used to define the “stemness” of stem cells before transplantation.

“Grafting ‘true’ stem cells in the right amount is of course a prerequisite for successful grafting, and both the poor quality of the limbal biopsy or some inaccuracy in the cultivation could be responsible for subsequent failures,” Prof. Pellegrini said.

A precise quantification of the minimum number of implanted stem cells that are needed to effectively repopulate and re-create a functional limbus was also achieved.

“We know now that if we remain within a certain numeric range, the success rate is 85%. Below that range, it drops to 20%,” she noted.

A standardization of these quality control procedures is mandatory to optimize results and make these results comparable.

Alternatives for bilateral damage

The advantage of this procedure is that even bilaterally injured eyes can be treated, provided that a minimal portion of the limbus has been spared in one of the patient’s eyes.

In cases of total bilateral damage, two alternative methods based on the same concept of cell culture have been explored.

The first consists of the use of allo-limbal epithelial cells cultivated on denuded amniotic membrane. This procedure has been conducted since 1999 at Kyoto University in Japan by Noriko Koizumi, MD.

The amniotic membrane appears to be a healthy substrate to cultivate cells and to cover a damaged corneal stroma. However, Dr. Koizumi observed that in eyes that needed re-treatment after rejection, large patches of amniotic membrane were found to cover the cornea after removal of the failed graft. In addition, allo-limbal cultured cell transplantation requires immunosuppressive therapy.

However, the role of immunosuppression in prolonging graft survival is still unclear. DNA analysis conducted by Dr. Daya on seven eyes in which this method was used showed no ex vivo donor stem cell DNA beyond 9 months.

“The absence of donor DNA beyond 9 months suggests that ongoing immunosuppression may be unnecessary and raises questions regarding the origin of the host corneal epithelium,” he said.

The second option consists of using autologous cells taken from the patient’s own oral mucosa.

“We harvest a 3-mm-by-3-mm specimen of oral mucosal tissue and cultivate it for 2 weeks on a temperature-responsive cell-culture surface together with 3T3 feeder cells previously treated with mitomycin C,” said Kohji Nishida, MD, who developed this technique at Osaka University in Japan.

The epithelial cell sheet is then detached by lowering the temperature of the substrate and implanted on to the denuded cornea without sutures.

This technique appears to be promising. In the patients treated by Dr. Nishida, corneal transparency and restored visual acuity was achieved. However, a better understanding of how transplanted oral mucosa cells improve the ocular surface is needed. – by Michela Cimberle

How does cultured limbal stem cell transplantation play into the current practical use of stem cells for corneal regeneration?

For more information:

• Daya SM, Ilari FA. Living related conjunctival limbal allograft for the treatment of stem cell deficiency. Ophthalmology. 2001;108:126-133.
• De Luca M, Pellegrini G, Green H. Regeneration of squamous epithelia from stem cells of cultured grafts. Regen Med. 2006 Jan;1(1):45-57.
• Pellegrini G, Traverso CE, Franzi AT, et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet. 1997;349:990-993.
• Rama P, Bonini S, Lambiase A, et al. Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation. 2001;72:1478-1485.
• Tsubota K, Satake Y, Kaido M, et al. Treatment of severe ocular-surface disorders with corneal epithelial stem-cell transplantation. N Engl J Med. 1999;340:1697-1703.