American Academy of Ophthalmology Web Site: www.aao.org
Stem-Cell Research Takes Shape
Despite their microscopic size, stem cells are very big newsmakers. Their capacity to become any other cell type powers the miraculous engine of complex life. That capacity, in fact, may drive the future treatments of choice for many diseases.
In terms both literal and figurative, the eye provides a very good lens through which to assess stem-cell science. The visibility of the eye’s structures makes them far more open to this assessment than anatomical sites like the heart, gut or brain, where keeping tabs on transplanted cells and averting tragedies is no small matter.
And as a visible extension of the brain, the retina’s circuitry is already well-known and better understood than some other areas of the nervous system. “For stem-cell applications to central nervous system diseases, the eye will likely lead the way,” said David M. Gamm, MD, PhD, assistant professor of ophthalmology and visual sciences at the University of Wisconsin in Madison. “It’s accessible, it’s amenable to manipulation, and you can observe donor and host cells in real time. And the techniques and instrumentation needed to introduce cells into the eye are readily available.”
There are also advantages of focusing on eye diseases from a study safety standpoint, said Dr. Gamm. “You have two eyes and they are encapsulated organs, so if something were to go wrong, you could more easily contain the problem.”
Given the eye’s research strengths, some believe the fate of stem-cell medicine rests with ophthalmology. Here is a survey of this rapidly evolving field.
A TALE OF THREE CELL TYPES
The first stem cells to be studied as possible therapeutic weapons against human disease were the true originals—human embryonic stem (hES) cells. But stem-like cells can actually be obtained from differentiated tissue. These are induced pluripotent stem (iPS) cells and regeneration-capable adult (tissue-stem) cells.
Just when researchers thought they had mastered the intricacies of the hES cell, the iPS cell came along through a technique pioneered in 2006 by researchers at the University of Kyoto.1 This technique reprogrammed already differentiated adult mouse skin cells back to an undifferentiated state that mimicked many of the hES cells’ properties. Over time, it was found that reactivating just four dormant genes could induce pluripotency in these adult cells, said Deepak A. Lamba, MBBS, PhD, research assistant professor of ophthalmology at the University of Washington in Seattle. Many labs today are experimenting with both hES and iPS cells, and some have retrieved tissue-stem cells from the eye, as well.
But these three cell lines have epigenetic differences. And they can act differently in different settings, said Dr. Lamba. So exactly how useful each will become is up in the air. “It all comes down to the field maturing,” he said. Following are particulars of each cell line.
A GOAL WITH THREE STRATEGIES
With the overarching goal of exploiting stem cells to eliminate eye disease, ophthalmic researchers have generally chosen to work with three strategies:
TWO EYE SITES OF RESEARCH
Cornea out in front. Providing both easy access and well-defined stem cells at the narrow annulus circumscribing it, the cornea allowed stem-cell researchers a jump start in this field. These limbal stem cells are a focus of research for both Nick Di Girolamo, PhD, associate professor of pathology and director of ocular research at the University of New South Wales in Sydney, Australia, and Francisco C. Figueiredo, MD, PhD, of the North East England Stem Cell Institute in Newcastle upon Tyne in the United Kingdom.
After limbal stem-cell biopsy—with ex vivo expansion in the laboratory and subsequent transplantation—was pioneered in 1997 in Italy, Dr. Figueiredo and collaborators set a goal to improve on the technique for patients with total unilateral limbal stem-cell deficiency caused by chemical or thermal burns. “We wanted to do something that would pose less risk to the patient,” he said. “At the time, what was used was a technique for skin stem cells. For this technique, you had to use animal cells and products to expand them in vitro, putting them in a culture where they could grow but still retain their stem-cell properties.” The researchers’ goal was to emulate the technique but develop an animal-free method, one that used a human tissue substrate—amniotic membrane from the placenta—full of growth factors for facilitating stem-cell growth.3 Up to four years after transplantation, corneal stem cells are still functioning in all patients, producing greatly improved quality of life by reducing pain, glare and photophobia but also improving patients’ vision.
In another variation of this autologous approach, Dr. Di Girolamo and colleagues have harvested limbal stem cells from patients’ healthy eyes and placed them on an FDA-approved hydrogel contact lens, where the cells are nurtured in the patients’ own serum. These are patients with unilateral limbal stem-cell deficiency due to chemical or thermal burns, multiple surgeries to the ocular surface, or aniridia.4
Although a small percentage of the cells that migrate from the tissue biopsy are stem cells, said Dr. Di Girolamo, the rest are daughter cells with a high proliferative capacity, and they rapidly cover the contact lens surface over two to three weeks. In a cellular version of “Red Rover, Red Rover,” the cells “jump” from the contact lens polymer to the human ocular surface. “We’re not quite sure how this happens,” he said. But with this procedure his team has been able to create a stable, transparent corneal epithelium, partially restoring vision and correcting problems such as large painful ulcers and conjunctival pannus.
Momentum for the corneal stem-cell therapy is growing. A recent Italian study involving 112 patients with corneal burns reported “permanent restoration” of clear epithelium in 76.6 percent of eyes by treating them with autologous limbal stem cells that had been cultivated on fibrin.5
Retina brings up the rear. Dr. Gamm’s lab is focusing on the forks in the road that determine early cell fate decisions for the retina: “What makes an undifferentiated pluripotent human stem cell become virtually any cell type in the body? What makes it decide to be a neural cell, then a neural cell in the anterior portion of the brain? And later on, a cell belonging to the retina, and, finally, a photoreceptor or RPE cell? Right now, it’s still a black box.”
Every step in cell differentiation builds off preceding steps, so understanding early stages is essential, Dr. Gamm said. “What we’ve done is to develop a protocol whereby we can derive retinal cells in a step-by-step manner from hES and iPS cells.” This system allows his lab to follow the development of these cells from an undifferentiated state all the way through the production of cells that express markers of mature photoreceptors, revealing a pathway that closely mimics what’s expected in the normal course of human retinal development. Other labs have published similar protocols, he said, but with the like-minded goal of working forward from the most basic scientific principles to achieve efficient production of cell types with therapeutic value.
Dr. Lamba’s group was among the first in the United States to take undifferentiated hES and iPS cells and very efficiently transform them into retinal cells.6 “We have been characterizing these cells since 2006 to figure out if we have any kind of contamination.” In 2009, his team was able to show that these light-sensing cells could restore some vision in a mouse model of Leber congenital amaurosis.7
Remarkably, the visual improvement was comparable to restoring enough vision for a completely blind person to navigate across a room, said Dr. Lamba. “We feel it’s efficacious but want to make sure that it will not be deleterious to the patient in any way,” he said, explaining that the present goal is to thoroughly characterize the cells, ensuring that they won’t form tumors. “We’ve never seen it, and I think it is because our protocol is a very efficient way of making the cells; but we need to make sure we know everything we have in our dish before we put them in people.”
Referring to the 2009 paper, Dr. Kalil lauded the work of Dr. Lamba and his collaborators. “The transplanted cells integrated with the photoreceptor layer and behaved like photoreceptors,” he said, noting that the cells displayed electrical activity reminiscent of an eye’s normal response. “It’s one of the best papers in the field showing that the cells have some potential, at least in respect to photoreceptor degeneration, to do something productive after they’re transplanted into adults. And, that’s the $64,000 question: You can produce cells that replace cells, but are they going to integrate with the circuitry?”
In 2006, Dr. Coffey’s lab announced the audacious goal of taking stem-cell therapy into the clinic for macular degeneration within five years. “There is good clinical evidence to suggest that as long as you can get RPE back under the macula, you can stop someone from going blind,” he said, emphasizing that timing is also key. “We developed a human embryonic stem-cell line that gave good differentiation of retinal pigment epithelium and could be produced in quantity. And we’ve manufactured membranes of cells so we can grow these on patches and hope to place those patches surgically.” Dr. Coffey’s team is in preclinical stages. He said they are now 18 months away from a phase 1 clinical trial using patches of RPE in 10 patients. Dr. Coffey is optimistic this approach will be successful, in part because his team has not yet seen an immunological response to the cells in animals.
Also moving to clinical trials is the company Advanced Cell Technology, which hopes to use stem-cell therapy to replace RPE cells in patients with Stargardt macular dystrophy.
THE PLURIPOTENT FUTURE
Although the hope is that these studies will shed light on efficacy, they may only show safety, said Dr. Coffey. And, said Dr. Kalil, a single gold standard may remain elusive since different approaches will likely be used for different problems, providing something more akin to a stem-cell tool kit. “One of the beauties of this field is that it proceeds like an old motor vehicle, chugging along and then suddenly jumping forward. And who can predict when that will happen? There are extraordinarily talented people working on this, and we’ll likely see this come to fruition within the next 10 years.”