Demyelination in the setting of oligodendrocytic loss is a significant contributor to neurological dysfunction in a variety of subcortical pathologies, including posthypoxic leukoencephalopathy, capsular stroke, and head trauma, as well as in the inflammatory, hereditary and degenerative leukoencephalopathies. We have found that a distinct and separate pool of mitotically-competent oligodendrocyte (oligo) progenitor cells resides in the adult human capsular white matter. This glial progenitor does not appear to be uncommon; it may comprise as many as 4 percent of the cells of the mature white matter. Although the existence of an analogous cell-type has been postulated in rats, its presence in humans had remained controversial, in part because of the difficulty in specifically identifying or obtaining these cells. We therefore developed a means of isolating these cells from adult human patients, by transfecting dissociates of resected white matter with plasmids that selectively identify oligo progenitors, by their expression of fluorescent transgenes controlled by early oligodendrocyte promoters. Specifically, we use the early promoter (P2) for cyclic nucleotide phosphodiesterase (CNP), which is preferentially active in mitotic oligo progenitors, to direct expression of green fluorescence protein (GFP) to these cells. This approach has allowed us not only to identify live oligo progenitors in vitro, but to enrich them by fluorescence-activated cell sorting (FACS). As a result, we can use FACS based upon CNP2:hGFP expression to purify oligodendrocyte progenitor cells from the adult human white matter, in high-yield, and with retained mitotic potential, differentiation competence and in vitro survival. In this application, I will capitalize upon our acquisition of these purified adult human oligodendrocyte progenitor cells, with experiments designed to assess their capacity for structural remyelination. To this end, I plan to define several therapeutically-relevant aspects of the biology of these cells, including 1) their lineage potential and capacity for myelination, and the humoral control thereof, and 2) the humoral regulation of their clonal expansion, and 3) their capacity for oligodendrocytic maturation and myelination upon engraftment. To these ends, I plan to transplant P/CNP2:hGFP-defined, FACS-purified populations of adult human oligo progenitors into a rodent model of focal demyelination. These latter studies would serve as a prelude to preclinical analysis of this cell type's response to implantation in nonhuman primates, in models of both chemical and radiation-induced demyelination. This application is intended to develop oligodendrocyte precursor implants, as well as induced oligoneogenesis from endogenous progenitors, as feasible therapeutic options for the structural repair of demyelinated brain.
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