July 2017




Presentation spotlight
Fast and uncomplicated corneal regeneration

by Stefanie Petrou Binder, MD, EyeWorld Contributing Writer


The limbal mesenchymal stem cells are prepared for application on damaged corneas.

Dr. Basu applies the limbal mesenchymal stem cells in a patient with severe corneal burns in the ongoing clinical trial.
Source: Sayan Basu, MD

Clinical pilot study used autologous, limbal-derived mesenchymal stem cells to reduce corneal scars

Conventional corneal allograph therapy is a widely used and successful therapy for corneal scars, but donor tissue is not universally available, and grafts can sometimes be rejected. The use of stem cells could reduce the need for corneal grafts and at the same time be a more readily available therapy to patients with corneal scars and burns worldwide. A new pilot study is using human limbal-derived mesenchymal stem cells (MSC) to resolve corneal scars. The process is uncomplicated and effective, according to Sayan Basu, MD, and Vivek Singh, PhD, Tej Kohli Cornea Institute, LV Prasad Eye Institute, Hyderabad, India, who helped pioneer this treatment.
“The hypothesis that we started this work with was that in patients who have corneal stromal diseases, we could take a limbal biopsy from the same eye, the fellow eye of the patient, or from a donor, and we could grow the cells in a xeno-free culture system, have them differentiate into keratocytes that would help in modulation of the stromal problem, and restore the opaque cornea into transparent tissue,” Dr. Basu said at the 2016 European Society of Cataract and Refractive Surgeons (ESCRS) Congress in Copenhagen, Denmark. “This is basically regeneration instead of replacement. We use the same scaffold of the normal cornea but make the tissue transparent again by changing what is being produced in the tissue. The process involves embedding MSCs into debrided corneas, regenerating human corneal stroma, remodeling active corneal wounds, and preventing/reversing corneal scarring. The procedure is exciting and when we started seemed very audacious.”
The pilot trial included patients with corneal scars, ulcers, and burns up to 250 microns in depth, excluding the epithelium, who received limbal MSC therapy. A control group was given a fibrinogen/thrombin glue mixture without MSC. The study will evaluate the effect of MSCs on reduction in corneal haziness, improvement in uncorrected and best corrected vision, and an improvement in cosmesis as compared to controls.

How it’s done

Dr. Basu performed limbal biopsies, which yielded mesenchymal cells, by removing a small snip from the limbus of about 3 mm in diameter, at the 1 o’clock hour. The limbus healed without complications. He grew the sample in the lab, which generated both epithelial cells, which were easily isolated and removed, and mesenchymal cells. The cells expressed stem cell factors, and he identified normal human stromal tissue of type 1 collagen. The keratocytes aligned and changed orientation as the construct got thicker, reaching a thickness of about 150 microns in 6–8 weeks.
Once the cells were grown, the patients were prepared for the procedure by marking the area on the eye where the cells were to be placed and gently debriding it. The MSCs were mixed into a commercially available fibrin glue, which has two components: fibrinogen and thrombin. By mixing the MSC into the fibrinogen component of the glue, which is thicker and does not dissipate when placed on the cornea, Dr. Basu was able to retain control of the mixture once placed on the cornea. He placed one drop of the fibrinogen/MSC mixture and one drop of the thrombin component, and mixed it on the debrided area with a spatula to form a paste-like matrix that is perfectly suitable for mesenchymal cells to grow. The gel formed instantly with the cells trapped inside it. Once the epithelium grows over the gel mass, the cells become a part of the stroma and they do their work at restructuring it and reversing the corneal scarring. The patients received topical anesthesia and no peribulbar block, and the eyes were simply bandaged after the short procedure was completed.
“In our earlier work that preceded this, in the wound model, if we put the stem cells in at the acute stage, they would prevent scarring, and by putting them in at the chronic stage, once a scar formed, they could reverse the scarring and render the cornea much clearer,” Dr. Basu said. “This is what we are expecting to find in the present study. In the pre-clinical study, when we looked at the transmission electromicroscopic images, those corneas that were treated with the stem cells looked like anatomically normal corneas with a regular orientation of collagen fibers, while those that were not treated remained scarred and lost the normal orientation of the collagen fibers. In the control group we did the same thing but applied gel without MSCs, and we saw that the differences were clinically and visually significant.”

The no-bank alternative

In India, 1.8 million people have corneal blindness, of which 30% are children, and which accounts for 14% of all cases of blindness. Fifty-five percent of corneal blindness results from infection and scars, which is 30% from endothelial disease and 11% from keratoconus and dystrophies. Each year, the number of people with corneal blindness increases by 30,000.
More than 50% of the indications for transplants in India have a fair to poor prognosis, largely due to the fact that corneal transplants do not do well in eyes that have had inflammation, that are likely to have inflammation, or that are vascularized. In addition, the budding network of corneal banks in India has a deficient corneal collection of 30,000 per year along with an inadequate number of corneal specialists.
“Early on, MSCs were only known to exist in bone marrow, but we now know that stem cells can be found in adipose tissue, dental pulp, and corneal stroma as well, just below where the limbal epithelial stem cells are located,” Dr. Basu said. “They are an important component of the niche. Our idea, initially, was to have a tissue source with less risk of rejection and not have to depend on donor tissue as much because in the developing world there is an extreme scarcity of corneal tissue. We also thought we should have a system of delivery that is simple and does not require a complicated procedure, which in this case is a 5-minute procedure under topical anesthesia. Also, one vital aspect of this whole procedure is the fact that in diseases involving corneal opacities and scars, the limbus is not always affected. In the most common forms of corneal stromal diseases, the limbus is actually spared. And if one eye is affected, you always have the other eye that you can harvest the MSCs from.”
Drs. Basu and Singh are using both autologous and allogeneic limbal-derived MSCs in patients with corneal scars, ulcers, and burns. The MSCs can also be used in the acute inflammatory stage, without the application of topical or systemic steroids, which are normally used intensively in burn or ulcer patients. They anticipate rapid corneal healing. The clinical outcomes in these patients are pending.

Editors’ note: Dr. Basu has no financial interests related to his comments.

Contact information

Basu: sayanbasu@lvpei.org

Fast and uncomplicated corneal regeneration Fast and uncomplicated corneal regeneration
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