Limbal stem cell transplantation shows high rate of success in long-term follow-up

Cells obtained from patients’ contralateral eyes were cultured and used to regenerate corneal epithelium after thermal and chemical ocular burns.

Issue: October 2010

Paolo Rama

Long-term success of limbal stem cell transplantation in the reconstruction of severe corneal damage from ocular burns was confirmed in a recently published study.

“The [New England Journal of Medicine] is known to be extremely selective, particularly with specialty studies. I believe that one of the reasons why our work has been accepted is because it contains a strong message for all translational medicine: Stem cells can be cultured under appropriate culture conditions, can be implanted and remain viable and functional in the long term,” Paolo Rama, MD, first author of the study, said.

Basic research on corneal stem cells dates back to the early 1990s, with the pioneering work of Graziella Pellegrini, PhD, and Michele De Luca, MD. They were the first researchers to find that cultured limbal cells include stem cells detectable as colonies, or holoclones. This discovery enabled the first therapeutic use of limbal cultures for the regeneration of corneal epithelium. These researchers also standardized a protocol for corneal limbal cell culture.

Study methods

Autologous limbal stem cells are obtained from a 1- to 2-mm biopsy specimen taken from the patient’s contralateral eye. These cells are then cultured on a feeder layer of a lethally irradiated 3T3-J2 clone of murine origin certified for clinical use. This allows 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 fibrin disc, ready to be implanted in the patient’s eye.

“The fibrin sheet, which is very similar to a soft contact lens, is placed on the eye and fitted under the dissected conjunctiva,” Dr. Rama said in an interview with Ocular Surgery News. “The eyelids are kept closed 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.”

A total of 112 patients were enrolled in the study between 1998 and 2006. The most common causes of stem cell deficiency were chemical and thermal burns, unilateral or bilateral. Permanent restoration of a transparent, renewing corneal epithelium was obtained in 76.6% of eyes. Restored eyes remained stable over time, with up to 10 years of follow-up.

“With this technique, all you need is a tiny portion of healthy limbus in one of the eyes to harvest your stem cell biopsy sample. Most of the eyes in our study had had previous keratoplasty surgery, which didn’t work, with recurrence of the fibrovascular pannus over the graft, because there were not enough stem cells to support the corneal epithelium turnover. Also, autologous limbal transplantation needs a fairly large portion of healthy limbus, basically a contralateral healthy eye. The advantage of this technique is that you can start from just a few survivor cells to reconstruct the surface of both eyes,” Dr. Rama said.

Validating results

The high number of patients, the high rate of success and the long-term follow-up confirm that this method of culture and transplantation of stem cells is reproducible, safe and effective. A further method of validation of results has been the use of the p63 marker, a marker of corneal epithelial stem cells, through which the presence and percentage of stem cells in successful implants can be assessed.

Corneal stem cell culture has been attempted by several other researchers, but the number of failures, usually within 1 year from transplantation, clearly demonstrated that those cultured cells were not stem cells, and could not, therefore, re-create a new, healthy limbus, Dr. Rama said.

“Our method of culturing cells has often been attacked because we use a feeder substrate of murine origin, which is FDA-approved, as well as calf serum in the culture medium. There is some doubt that the use of animal derivatives might carry the risk of transmitting pathogens or stimulate an immune reaction. However, we had no problems with our patients in 12 years, and the follow-up of cultured skin grafts is even longer, more than 20 years. No complications or side effects have been reported,” he said.

Other culture methods, however, have failed to prove effective in maintaining the “stemness” of stem cells, he said.

“We have a method that works, and we should start from here to see if, eventually, something else can be used in place of the feeder layer,” he said.

Now that the first goal has been achieved, new opportunities may arise in the future. Cultured stem cells could be used for gene therapy. Stem cells in which the genetic deficit has been corrected can be expanded in culture. Once reimplanted, these cells can repopulate the limbus. Theoretically, various forms of hereditary corneal dystrophies could be treated with this method, Dr. Rama said. – by Michela Cimberle

Reference:

Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med. 2010;363(2):147-155.

Paolo Rama, MD, can be reached at Ospedale San Raffaele, UO Oculistica – Malattie della Cornea e Superficie Oculare, Via Olgettina 60, 20132 Milan, Italy; +39-02-2643-3598; fax: +39-02-2643-4600; e-mail: rama.paolo@hsr.it.