Inherited and acquired eye diseases that target photoreceptors and/or retinal pigment epithelium (RPE) are a significant cause of visual morbidity in humans. The existence of regionally specific stem cell populations in human tissue, including brain, has raised interest in the therapeutic applications of stem cell biology. Innovative methods of propagating precursor cells from ocular tissue may produce populations of transplantable cells capable of preventing or repairing photoreceptor or RPE degeneration. This proposal outlines a five-year plan to 1) use a novel tissue culture technique to isolate and expand precursor cells obtained from retina and/or pigmented epithelial regions of fetal and adult human eyes, 2) characterize their growth and differentiation potential in response to different culture treatments and 3) examine their capacity to repopulate or rescue photoreceptors in animal models of retinal degeneration. Using a culture method developed in the host laboratory, we demonstrate that free-floating spheres of pigmented and nonpigmented cells can be successfully propagated from the posterior segment of human fetal eyes. Cells prompted to migrate from these spheres undergo differentiation and adopt distinct morphologies and patterns of pigmentation and cell marker expression. In particular, non-pigmented spheres from retina and pigmented spheres from the ciliary body produced neurons, while pigmented spheres from RPE produced regularly arranged sheets of pigmented epithelium. Building on these preliminary experiments, we will focus immediate efforts on dissecting additional specimens from both fetal and postnatal enucleated human eyes and monitoring their growth and differentiation potential over time. These studies will be carried over to the second and third years of the grant period, when we will also begin to examine the effects of specific growth factors and conditioned media on sphere behavior. During the final years of the project, strategies are proposed to examine the therapeutic potential of the different sphere populations in two distinct rat models of retinal degeneration. Together, these experiments are designed to build on recent advances in the burgeoning field of stem cell biology and apply them to the basic and clinical science of ophthalmology.
