Dopamine cell bodies exist in the midbrain, where they are mainly located in the substantia nigra, the ventral tegmental area, and the retrorubral field. These dopaminergic (DA) neurons give rise to extensive forebrain dopamine innervation ( e.g.projections to the caudate nucleus, putamen nucleus accumbens, olfactory tubercle, and several cortical regions and limbic regions, including the amygdala, lateral septum and ventral hippocampus). The basic organization of midbrain dopamine neurons and their projections is consistent across most mammalians. The midbrain dopamine projections to the striatum have been among the most extensively studied catecholamine neurons, in part because the degeneration of DA neurons in the substantia nigra results in Parkinsons disease. Both animal models and clinical trials have suggested that cell replacement therapies may be effective in treating Parkinsons disease. This approach is limited, however, by the availability of a rich, effective source of neural precursors for DA neuron generation.

Embryonic stem (ES) cells, derived from the inner cell mass of pre-implantation embryos, have been recognized as the most pluripotent stem cell population. ES cells give rise to a collection of differentiated cells and tissues, including neurons. Thus, they may serve as a potential source of replacement DA neurons in Parkinsons disease. Two groups have reported methods for generating DA neurons from ES cells, thus demonstrating the feasibility of identifying conditions required for functional DA neuron differentiation of ES cells. Experimental models established from these studies might facilitate the in vitroexamination of various aspects of neural development, in particular the underlying mechanisms controlling cell lineage and fate commitment.

DESIGN OF THE KIT

The Mouse Dopaminergic Neuron Differentiation Kit is a system designed for in vitroneural differentiation of mouse ES cells in a serum-free environment. This kit contains specially formulated ITS and N-2 Plus Media Supplements, which are used to select and enrich neural stem cell populations. Bovine Fibronectin is included to provide proper support for cell attachment and spreading. A growth factor panel, consisting of human fibroblast growth factor (FGF) basic, mouse FGF-8b, and mouse sonic hedgehog amino-terminal peptide (Shh-N), is included for effective DA differentiation. The quantity of each component provided in the kit is estimated to be sufficient for the induction of 3 x 10⁷ ES cells. This kit has been shown to generate an average of 15 plus/minus 5% DA neurons as estimated from double labeling various mouse ES cell lines with antibodies specific to tyrosine hydroxylase and neuron-specific β-III tubulin.