The long-term goal of our research is to develop therapeutic applications for stem cell transplantation to treat hereditary neurodegenerative diseases. Our emphasis is on the neuronal ceroid-lipofuscinoses (NCLs). The NCLs are a group of inherited, progressive degenerative disorders of childhood characterized by loss of nerve cells in the retina and central nervous system (CNS). One NCL is commonly known as Batten Disease. The focus of this proposal is on degeneration in the retina, but our results will be applicable to degeneration throughout the CNS. We will transplant neuralized mouse embryonic stem (ES) cells that express enhanced green fluorescent protein (EGFP; for cell tracing) into the eyes of mouse models for NCLs. Our primary hypothesis is that transplantation of specifically engineered, neural-induced ES cells will retard or prevent retinal degeneration resulting in visual recovery. To test our hypothesis, the following specific aims will be performed.
Specific Aim #1 is to induce (neuralize) and transplant ES cells into host eyes and assess stem cell survival and integration within the retina. After induction, the ES cells will be injected into the eyes of mutant mice, and the fate of transplanted cells will be evaluated with antibodies specific for neural and glial markers.
Specific Aim #2 is to test in vitro for cross-correction of the cellular phenotype, due to factors secreted by donor ES cells. To promote survival of host neurons, ES cells will be engineered to express a growth factor (IGF-1) or the normal enzyme that is mutated in the Clnl mutant mouse. Use of a CMV promoter will ensure over-expression of the genes by neuralized ES cells.
Specific Aim #3 is to assess transplanted cell function and whether host retinal cells exhibit enhanced survival after stem cell transplantation. To determine if a normal ultrastructural phenotype is restored in host retinal cells and whether cellular connections are established between transplanted stem cells and host retinal cells immunoelectron microscopy (using an EGFP antibody), and electrophysiology will be performed. We will use electroretinograms to test for maintenance and/or recovery of visual function, intracellular recordings and dye-injections in retinal slice preparations will be used to determine the functional fates of donor cells as well as that of host cells. This research will provide essential new information about the use of stem cells in potential therapies for the NCLs as well as for other inherited neurodegenerative disorders of the CNS.
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