Researchers tackle engineering the eye&#8212from cornea to retina

May 2 - Fort Lauderdale, FL - Recent advances in biotechnology and tissue engineering are providing promise that in the future, tissue replacements or renewal techniques might be used to restore lost vision in eyes with genetic, malignant, or degenerative diseases.

James Zieske, PhD, of the Schepens Eye Research Institute at Harvard University, Boston, and collaborators have been focusing on cornea organogenesis. He reported at the Association for Research in Vision and Ophthalmology annual meeting that adult human corneal fibroblasts grown on top of a porous membrane with the addition of the vitamin C derivative L-ascorbic acid 2-phosphate stratify to form multiple cell layers and assemble an extracellular matrix containing type V and VI collagen.

Electron microscopy studies reveal the presence of collagen fibrils structurally aligned in a manner similar to the lamellar pattern of the in vivo cornea and that are requisite for that tissue's strength and clarity.

To further optimize the fibril alignment and produce a material with properties that closely mimic the cornea, ongoing studies are investigating the use of aligned type I collagen as a template for cell growth.

Dr. Zieske noted that another important take-home message from this research is that it demonstrates adult human corneal fibroblasts retain the ability to stratify in an orthogonal array. "This is an important point because there has been concern that generation of an artificial cornea would require developing fibroblasts," he added.

Scientists developing a chronically implanted retinal prosthesis need to consider the possibility that the device may fail, not on the basis of some engineering or structural feature, but rather because nothing has been done to arrest the progressive degeneration of the retina, said Raymond Iezzi, MD.

Together with colleagues at the Kresge Eye Institute at Wayne State University, Detroit, Dr. Iezzi has been undertaking research aimed to heal or maintain the retinal tissue with engineering improvements at the tissue interface so as to enhance its receptivity to an artificial stimulus.

"From studies by Rizzo and Humayun, we know that patients with more advanced retinal degeneration require more electrical current to perceive electrophosphenes," explained Dr. Iezzi. "Therefore, as a result of disease progression and because the prosthesis itself is a stressor, these devices could eventually become ineffective. Against that background, any measure we might take to improve the long-term health of already diseased tissue could enhance the ultimate functionality of an implanted sensory device."

He presented results from a study using Royal College of Surgeon (RCS) rats showing that intravitreal infusion of ciliary neurotrophic factor (CNTF) for 21 days had a dose-related neuroprotective effect for reducing the threshold for transcorneal electrically evoked potential relative to untreated control eyes. Histological studies have shown that benefit was accompanied by preservation of cells in the retinal outer nuclear layer (ONL).

In another study, intravitreal infusion of the steroid fluocinolone acetonide improved the biocompatibility of a subretinal implant as measured by ONL cell thickness.

"Encouragingly, electrical stimulation itself may have a neuroprotective effect, but we may need to approach neuroprotection using multiple strategies," Dr. Iezzi said.

Derek van der Kooy, PhD, and colleagues at the University of Toronto have been working to characterize adult mouse and human retinal stem cells in vivo and in vitro.

In their studies, they found that single pigmented cells isolated from the ciliary margin zone of the adult mouse peripheral retina proliferated in protein-free culture to form within 1-week spheres containing 10,000 cells. The clonally derived spheres demonstrated the two hallmarks of stem cells, i.e., multi-potentiality and self-renewal.

Using tissue dissected from the ciliary margin zone of eye bank eyes, the researchers also found that stem cells could be easily generated from the human retina. Surprisingly, the proliferation was as rapid as that which occurred using mouse cells.

Dr. van der Kooy also reported that by using gene transfection methods, it has been possible to influence progenitor cell development. For example, while photoreceptors normally account for 10% to 30% of the progeny of human retinal stem cells, it has been possible to increase that proportion to 70%.

In addition, while in vitro the photoreceptors that were differentiated from retinal stem cells did not assume the normal in vivo morphology, when human retinal stem cell clones were transplanted into the eyes of immunodeficient mice, the photoreceptor progeny appeared to make inner and outer segments resembling the morphology of photoreceptor in the native host eye.

"These results are encouraging because they suggest human retinal stem cells placed into the retinal environment will derive endogenous signals and differentiate into normal photoreceptors," Dr. van der Kooy said.

Ongoing studies are attempting to put the human retinal stem cells back into the eyes of blind mice to see if the progeny can functionally integrate into the host retina and even perhaps gain visual function.