The long term goal of these experiments is to understand the molecular mechanisms regulating production of diverse cell types in the mammalian CNS. From studying fates of single cells in culture, we have obtained evidence for self-renewing, multipotential stem cells in the embryonic cerebral cortex. By analogy to hemopoiesis and neural crest formation, we suggest that stem cells are ancestor cells in the cerebral cortex, that they self-renew to produce germinal tissue and generate restricted precursors for different classes of cortical cells. In this proposal, we aim to test and expand this model. The development of cortical stem cells grown in a standardized environment will be followed using time-lapse microscopy. This will test the model by revealing whether stem cells self-renew and undergo asymmetric divisions yielding restricted precursors for neurons, astrocytes or oligodendrocyte, as predicted. It will also reveal whether there are patterns in stem cell lineage trees, in terms of cell divisions, differentiation or cell death, which might indicate how stem cell development is regulated. For example, stem cells might generate neurons first, then astrocytes and then oligodendrocyte, mirroring the time-course of generation of these cell types in vivo. The time-lapse study will also provide information on stem cell heterogeneity, indicating how the germinal tissue might be organized. To investigate environmental regulation, single stem cells from the Rosa 26 mouse (which contain a B-galactosidase marker gene) will be cultured in contact with clusters of unlabeled cells modeling the maturing cerebral cortex. The numbers of neurons, astrocytes and oligodendrocyte produced will be counted. This will test whether the environment of the maturing cortex influences stem cell output, and orchestrates the normal schedule of differentiation of these fundamental cell types. Stem cells from the Rosa 26 mouse will also be grown in contact with germinal tissue from other neural regions: olfactory epithelium, optic nerve, cerebral cortex, retina and spinal cord, and their output examined with regional markers. This will investigate whether cortical stem cells are committed to giving cortical cells, or whether they can respond to their environment by producing appropriate cell types. These experiments will lead us towards a molecular study of stem cell regulation. Increased knowledge about factors influencing CNS stem cells may contribute towards the development of therapeutic tools for patients who suffer from disorders of the nervous system, such as neural tumors or neurodegenerative disorders.
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