Optimizing repair of damaged tissue such as demyelinated CNS lesions by precursor cell transplantation entails identification of precursor cell population with extensive self-renewal capacity and of means of expanding these populations in vitro to increase cell numbers available for transplantation. The applicant has discovered (a) multiple precursor phenotypes which all generate oligodendrocytes but which differ greatly in self renewal potential and (b) multiple means of enhancing precursor cell self renewal and expansion in vitro. These discoveries provide the foundation for deeper elucidation of principles underlying oligodendrocyte precursor cell division and differentiation and will also enable significant progress in optimizing strategies for remyelinating damaged tissue by precursor cell transplantation. The differing precursor cell populations will be immunopurified and grown in conditions which enhance precursor cell self renewal. Clonal and time-lapse microcinematographic analysis of division and differentiation, together with serial passaging studies will reveal precursor cell self renewal potential and mitotic lifespan. Studies on phenotypic changes occurring over time (in vivo and in vitro) will reveal stability of precursor cell phenotype. Analysis of the timing of differentiation of purified progenitors will provide information on the role of cell intrinsic mechanisms in promoting differentiation and limiting mitotic lifespan. Growth of cells in cytokine combinations that enhance precursor cell renewal will reveal environmental conditions that override cell intrinsic limitations to populations expansion. Finally in vitro analysis of myelination and transplantation of populations of cells to the CNS of dysmyelinating mouse mutants will reveal the capacity of the different populations for oligodendrocyte replacement and myelin creation. This research will enable us to make significant advances in (1) better characterizing the phenotypically distinct oligodendrocyte precursors cells that we have discovered (ii) determining their biological relationship with each other (iii) establishing contributions of intrinsic biological clocks to modulating differentiation and limiting self renewal (iv) identifying growth conditions that override theses cell-intrinsic limitations and which enable expansion of populations without compromising ability to carry out tissue repair and (v) establishing the capacity of these primary and expanded populations to replace oligodendrocyte in vitro and in vivo models of remyelination. The combination of fundamental biological analysis with specific attention to issues of tissue repair will provide new insights into both basic issues in precursor cell biology and into aspects of oligodendrocyte precursor cell biology relevant to enhancing repair of demyelinating damage.
