Establishing stable lines of human nigral dopaminergic progenitor cells

We intend to develop lines of clonal, stable and self-renewing human dopamine-producing neuronal progenitor cells. These will be derived from the part of the midbrain that gives rise to dopamine neurons in human fetal development. For this purpose, we will use fluorescence-activated cell sorting (FACS) of human fetal midbrain samples, treated with synthetic genes that specifically express a fluorescent protein in those precursor cells destined to give rise to neurons of the ventral midbrain. These neurons include those of the substantia nigra, the cell population lost in Parkinson's disease. We will use two different propagation modalities-primary neurosphere culture and immortalization-to establish stable lines of neuronal precursors committed to dopamine production. From the resultant lines, representative cells of each will be given genetic tags that will allow single lines to be isolated as stable clones. Each of the resultant clones will be assessed for expression of both dopamine synthetic enzymes and for genes typical of midbrain position. The most promising lines will then be assessed with regards to their efficiency of transplantation and dopamine replacement in rats that have been rendered Parkinsonian by chemical destruction of their own dopamine neurons.