Establishing cellular diversity is central to the development, structure and function of the brain. Underlying this diversity is a population of neural progenitors with stem cell properties that generates region-specific neurons and glia. Therefore understanding the molecular and cellular biology of neural progenitors holds the key to the mechanism(s) of development of specific regions of the brain including retina. Recapitulation of these developmental mechanisms is likely to open new avenues for treating the impairments of functions that arise due to death of specific neuronal populations as in the case of retinitis pigmentosa and macular degeneration. With these objectives in mind we have proposed to characterize neural progenitors isolated from derivatives of ocular neuroepithelium, the retina and the ciliary body, for their proliferative capacity, self-renewal, maintenance, and developmental potentials in vivo and in vitro under the following specific aims. First, proliferative and differentiation potentials of ocular progenitors will be characterized in the context of their responsiveness to the mitogens, FGF2 and EGF. We will use flow cytometry to examine their proliferation and survival, limiting dilution analysis to estimate their frequency to form clones, immunocytochemical and RT-PCR analyses of cell-type specific markers to test their multipotentiality, and clonal density culture to determine their self-renewal capacity. In addition, we will determine the distribution of mitogen receptors in different sub-populations of ocular progenitors in order to understand the basis of their responsiveness and relationships. Second, the role of Notch signaling in the maintenance of ocular progenitors will be evaluated. We will analyze proliferation and differentiation of these cells in response to gain-of-function and loss-of-function perturbations of Notch signaling. Third, the differentiation potentials of ocular progenitors will be determined in vitro in conditions that promote differentiation. We will use immunocytochemical and electrophysiological analyses to evaluate their ability to acquire specific phenotypes in response to growth factors and neurotrophins, and in co-culture conditions. Fourth, the potential of ocular progenitors to generate site-specific cells in vivo will be evaluated. We will use homotopic and heterotopic transplantation and in vivo activation of progenitors in response to injuries to achieve the aim. Accomplishing these aims will provide valuable information about the underlying mechanism(s) of retinal development and will allow the use of ocular progenitors in stem cell therapy to address degenerative changes in the retina whether inherited, age-related or due to injuries.
