Differentiation of dopaminergic neurons from human embyronic stem cell derived neural progenitors using glial cell-line derived neurotrophic factor
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Parkinson’s disease, the second most common neurodegenerative disease, causes the degeneration of the dopaminergic neurons projecting from the substantia nigra to the basal ganglia. Current treatments for Parkinson’s disease become progressively less effective or cause serious side effects. Human neural progenitor cells derived from human embryonic stem cells provide a source for modeling the development of dopaminergic neurons, a source for drug screens of potential Parkinson’s disease drugs or a potential source for a cell therapy for Parkinson’ disease. Glial cell-line derived neurotrophic factor is a known dopaminergic neurotrophic factor, which has been shown in rodent and non-human primate models to protect dopaminergic neurons from 6-OHDA and MPTP. In addition, glial cell-line derived neurotrophic factor has been used in human clinical trials; however, these trials were halted due to unexpected side effects. We previously derived a stable, adherent monolayer culture of human neural progenitor cells that can be maintained as proliferative cells or differentiated into neurons, astrocytes or oligodendrocytes. In this dissertation, we determined the human neural progenitors to have functional ion channels and ionotrophic receptors. These human neural progenitors also had a basal level of dopamine transporter expression that we were able to enhance using glial cell-line derived neurotrophic factor. The addition of 25ng/ml glial cell-line derived neurotrophic factor advanced the human neural progenitor cells from dopamine progenitors through a dopamine specification stage to a dopaminergic-like neuron as marked by NURR1, EN1, TH, PITX3, VMAT2 and DAT expression. The pathway through which glial cell-line derived neurotrophic factor enhanced dopaminergic-like neurons from human neural progenitor cells occurred through RET receptor activation of the ERK and p38MAPK pathways as well as the mTOR pathway. The results from these studies provide a novel one-step mechanism for obtaining dopaminergic-like neurons from human embryonic stem cell derived neural progenitor cells. These dopaminergic-like neurons progress developmentally through the stages of dopaminergic differentiation providing a model for dopaminergic differentiation. Additionally, these cells provide a human neural model to screen future drugs, which may treat Parkinson’s disease.