Cultivated corneal epithelium recreates healthy substratum

Corneal epithelial stem cells grow rapidly when cultured and restore the damaged mechanism of cell proliferation when transplanted.

ROME — An important condition for the success of keratoplasty is the presence of a healthy epithelium in the recipient’s eye. In the absence of healthy epithelium, keratoplasty causes corneal re-epithelialization by conjunctival cells. A key point in understanding this process is the alteration of the normal functions of corneal stem cells, which are the progenitors of the specialized population of corneal epithelial cells.

The transplantation of a corneal epithelial graft, obtained by in vitro cultivation of the corneal stem cells of the uninjured eye of the same patient, may offer a solution to the problem, because the transplant actually can recreate the healthy substratum for keratoplasty.

This was confirmed by the studies of Carlo Traverso, MD, of Genoa University Eye Clinic, Graziella Pellegrini, PhD, and Michele De Luca, MD, of the Laboratory of Tissue Engineering of I.D.I. Institute of Rome, published in The Lancet in 1997.

Further updates also were given by the clinical trials of Paolo Rama, MD, of Venice San Giovanni e Paolo Hospital Eye Clinic, and Stefano Bonini, MD, and Alessandro Lambiase, MD, of Rome Tor Vergata University Eye Clinic. The A.I.S.Co. meeting during the S.O.I Congress in Rome dedicated an entire session to this subject.

An idea is born

--- Prof. Traverso said the “idea was to take the corneal stem cells in the eye, cultivate them in vitro and transplant them in the hope that they could restore the natural mechanisms of cell proliferation in the damaged corneal epithelium, thus recreating the conditions for successful keratoplasty.”

Prof. Traverso said the “idea was to take the corneal stem cells in the eye, cultivate them in vitro and transplant them in the hope that they could restore the natural mechanisms of cell proliferation in the damaged corneal epithelium, thus recreating the conditions for successful keratoplasty.”

These studies were initiated with the Laboratory for Cell Differentiation of the IST in Genoa with Prof. Ranieri Cancedda, Dr. De Luca and Dr. Pellegrini, where studies on stem cell cultivation and transplantation already had been carried out in relation to the epidermis, the bladder and the arteries. “We started with a long series of experiments,” Prof. Traverso said. “My research colleagues at the IST Laboratory were very enthusiastic. Without their expertise, this project would have been impossible. At first, it was necessary to discuss some specific aspects of corneal stem cells and their function in the limbus. For limbal cells, animal experiments were carried out first.

“Compared with the autologous transplantation of limbal grafts, the advantage of this procedure was that only a very small biopsy sample was needed, with much lower risk and far less stress for the patient,” he said.

When Drs. Pellegrini and De Luca moved to the Laboratory of Tissue Engineering at I.D.I. in Rome, research collaboration continued.

Dr. Pellegrini said, “The limbus differs from the rest of the cornea by its characteristics of vascularization and cellular differentiation. Results of animal studies have shown the presence of a specific keratin in the corneal epithelium. This keratin seemed uniformly distributed in all the layers of the cornea, but it only was detected in the suprabasal layer in the limbus. This suggested the presence of stem cells in that area. Nevertheless, a more precise identification of stem cells in the limbus, based on their proliferative capacity, never had been made.”

High clonogenic potential

--- Dr. Pellegrini further explained that the stem cell differs from other cellular types because of its high clonogenic and proliferative potential. The stem cells cause tissue to regenerate continuously during the tissue’s life span.

Dr. Pellegrini further explained that the stem cell differs from other cellular types because of its high clonogenic and proliferative potential. The stem cells cause tissue to regenerate continuously during the tissue’s life span. Stem cells transplanted on patients with third-degree burns, such as those that encompass a large amount of the body’s surface, are able to regenerate the entire epidermis over a period of about 25 years.

Dr. Pellegrini said, “We investigated the presence of stem cells on the basis of these two parameters: clonogenic capacity and proliferative capacity. We explored the entire surface of the eye, taking from the same eye of the same donor, biopsy samples of the two fornices, of four quadrants of the bulbar conjunctiva, four quadrants of the limbus and of the central and paracentral cornea.”

She continued, “Clonogenic activity was registered in all areas of conjunctival epithelium. In the corneal epithelium, however, the clonogenic activity was strictly limited to the limbus and completely absent from the central and paracentral cornea. As far as the proliferative potential, the number of cellular duplications was equal in all districts of the conjunctiva, but only the limbus was active in the corneal area. The study then was extended to other donors to verify the validity of our findings. We then could conclude that the limbus was the seat of corneal stem cells. Once cultivated, the cells gave evidence of their extraordinary proliferative capacity: a single stem cell was able to generate a clone that in 12 days reached a diameter superior to that of the entire cornea.”

Autologous transplantation

Prof. Traverso said he established the transplantation procedure and performed the procedure on two patients with severe alkali burns in one eye.

A 1-mm full-thickness biopsy sample was taken from the limbus of the uninjured eye and was cultivated for about 2 weeks. The two limbal grafts were mounted on petrolatum gauze and on a soft contact lens, respectively.

The conjunctival epithelium covering the cornea and limbus of the injured eye was removed under topical anesthesia. For patient 1, the cultured epithelial graft was placed on the prepared eye mounted on a petrolatum gauze, which was gently removed under a microscope immediately after grafting. A soft hydrophilic contact lens was then placed over the graft. For patient 2, the cultured epithelium was placed already mounted on the hydrophilic contact lens.

After grafting, the eye was patched tightly for 3 days. The lens was removed after 2 weeks.

“The cornea of both patients was covered by a transparent, normal looking epithelium. There was no sign of vascularization, and fluorescein staining was negative. Both patients felt comfortable and satisfied with the results,” Prof. Traverso said.

Keratoplasty was successfully performed on patient 1 approximately 4 months later. Recalling the poor outcome of two previous keratoplasties, patient 2 refused to undergo additional surgery. More than 2 years after grafting, both patients still were clinically stable.

Further developments

“The article published in The Lancet was a great spur for us,” said Paolo Rama, MD, of San Giovanni e Paolo Hospital in Venice. “We understood that a whole new chapter of our story could be started.”

In connection with the I.D.I. Laboratory and with the Tor Vergata University Eye Clinic of Rome, a new study group was formed to carry out further research on Prof. Traverso’s application of the discovery.

“We had three goals,” Dr. Rama said. “First, we wanted to confirm with further evidence that stem cells are located in the limbus. Second, we wanted to simplify some methodological aspects. The procedure, as published in The Lancet, only could be applied in the few centers in the world that have laboratory facilities close to the clinic. We wanted to make sure that the cultured grafts could be easily and safely delivered from the few specialized laboratories of tissue engineering to the many eye clinics that are potentially able to offer this type of surgery. Third, we wanted to increase the number of patients in the study.”

The results of the investigation for the location of stem cells in the limbus were published in the Journal of Cell Biology.

He added that a key point in simplifying the procedure was that of finding a substrate for cell culture that also could be a safe support for the corneal graft, thus making it less fragile, more transportable and easier to be applied on the eye.

Dr. Rama explained, “After many experiments, we opted for modified fibrin. This substance does not alter the differentiation and proliferation characteristics of the cells. It has the transparency that is necessary to monitor their growth, and it is elastic and easy to manipulate during surgery. Once the graft is applied to the eye of the patient, the fibrin dissolves within 3 days, leaving the cells adherent to the cornea,” Dr. Rama explained.

Once the procedure was standardized, the first clinical study was initiated, with the collaboration of Dr. Rama in Venice and Prof. Bonini and Dr. Lambiase in Rome.

Fourteen patients were treated, all of them with a diagnosis of unilateral limbal deficiency and re-epithelialization of the cornea by bulbar conjunctival cells. The uninjured eye was used as a cell do-nor. The biopsy was made under topical anesthesia.

Continued research needed

Dr. Rama explained, “The biopsy sample is refrigerated and sent immediately to the laboratory, where the culture is initiated. Fourteen to 29 days are necessary for the formation of a corneal epithelium, which is delivered to us directly from the laboratory. We then call the patient. The conjunctival epithelium covering the cornea is removed under local retrobulbar anesthesia, and the limbal graft is implanted.”

He continued, “Histological examinations of the first two patients’ corneal biopsies showed a normal corneal epithelium, which confirmed the presence and activity of corneal stem cells. More significant information will be available when all patients achieve 1-year follow-up. For now, we consider these as preliminary results, and the beginning of a new approach, which may become routine not only in ophthalmology, but in many branches of medicine. Personally, I strongly believe that the future of medicine is in research biology, in cell culture and in tissue engineering.”

Dr. Pellegrini said, “In the future, we will be perfecting what we have done so far, and we are, in fact, starting a similar study for the conjunctiva. Looking even further into the future, we can foresee the possibility of genetic therapy. In our study, Dr. DeLuca and I have found genetic pathologies of the corneal epithelium. So far, we have no evidence that they can be treated, but we tried to set up the conditions for genetic transduction, to verify if our cultured stem cells could be transduced with a transgene. The genetic transduction has worked. This is only the first, small step down a long path, but we are hopeful.”

Prof. Traverso said, “I am happy to see that other people are making progress on this discovery. It is the first time that cell stem therapy is applied to the eye.” In 1999, Prof. Traverso won the A.I.R.C.M.O. prize, an Italian award for scientific research in ophthalmology.

For Your Information:

• Carlo Enrico Traverso, MD, can be reached at e-mail: mc8620@mclink.it. Dr. Traverso has no direct financial interest in any of the products mentioned in this article, nor is he a paid consultant for any companies mentioned.
• Graziella Pellegrini, PhD, can be reached at IDI, Istituto Dermopatico dell’Immacolata, Via dei Castelli Romani 83/85, 00040 Pomezia, Italy; (39) 06-9112193; fax: (39) 06-9106765. Dr. Pellegrini has no direct financial interest in any of the products mentioned in this article, nor is she a paid consultant for any companies mentioned.
• Paolo Rama, MD, can be reached at Via Ospedale 9, 30174 Mestre – Venezia, Italy; (39) 041-962644; fax: (39) 041-5058405. Dr. Rama has no direct financial interest in any of the products mentioned in this article, nor is he a paid consultant for any companies mentioned.
• Michele DeLuca, MD, is head of the laboratory of tissue engineering at IDI.

Reference:

• Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Concedda R, de Luca M. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet. 1997;349(9057):990-993.
• Pellegrini G, Golisano O, Paterna P, et al. Location and clonal analysis of stem cells and their differentiated progreny in the human ocular surface. J Cell Biol. 1999;145:769-782.