Parkinson's disease (PD) is a severe neurodegenerative disorder associated with the selective loss of midbrain dopaminergic (DA) neurons. There is proof of principal that DA neuronal replacement therapy can work in patients using fetal-derived tissue. However, this is afflicted with several technical and ethical problems. Muman embryonic stem cells (hESCs) represent a promising new cell source to generate functional DA neurons for cell replacement in PD. Current in vitro differentiation protocols have demonstrated that DA neurons can be produced from hESCs, however, the generation of a homogeneous DA neuronal cell population that is safe, stable and functional after transplantation into the brain has not yet been achieved. This proposal will focus on a novel idea for DA phenotype specification and selection. The transcriptional activator Nurrl plays a major role in DA cell specification during neuronal cell development. To enrich for the appropriate DA cellular phenotype, we will select Nurrl expressing cells by puromycin drug resistance using Lentiviral vectors engineered with a Nurrl-responsive promoter driving the expression of the puromycin resistance gene. Since Nurrl could potentially be expressed in non-neuronal cell populations, we will further select the DA neurons using Lentiviruses expressing the green fluorescence protein (GFP) from the neuronal-specific promoter synapsin by fluorescence activated cell sorting (FACS). The drug-selected and FACS-purified homogeneous DA neuronal cell population will be tested in a rat model of PD to determine its safety, stability and functionality. The use of this new technology in cell replacement paradigms for PD could be a major step towards hESC-based therapies for chronic neurodegenerative disorders. ? ?