Refinement of a neuronal differentiation protocol predominantly yields human iPS cell-derived dopaminergic neurons for the investigation of neurodegenerative pathomechanisms in vitro

Abstract

Abstract. BACKGROUND:Major pathomechanisms underlying neurodegenerative diseases, such as Parkinson's Disease, are still not well understood. Induced human pluripotent and rodent embryonic stem cells provide powerful disease models to address neurodegeneration-inducing pathomechanisms on a molecular and cellular level. OBJECTIVE: Our aim is to establish a refined protocol to generate healthy and patient donor stem cell-derived dopaminergic neurons to investigate neurodegenerative events in vitro. METHODS: Human healthy donor-and patient-derived induced pluripotent stem cells were differentiated into stable dopaminergic progenitor cell lines and further differentiated into dopaminergic neurons. Induced pluripotent stem cells, neuronal progenitors and terminally differentiated neurons were characterized by confocal laser microscopy-based immunofluorescence analysis, live cell imaging demonstrating dopamine transporter-specific uptake of a fluorescent substrate and transcriptome analysis. RESULTS: Based on our immunofluorescence analysis, dopaminergic differentiation approaches predominantly yield dopaminergic neurons and GFAP-expressing glial cells. We detected a small partition of GABAergic neurons, yet neither serotonergic nor glutamatergic neurons. Dopaminergic neurons were successfully stained for pre-and postsynaptic and mitochondrial markers. Live cell imaging experiments verified dopamine transporter-dependent uptake of the fluorescent monoamine transporter substrate ASP+. CONCLUSION: Human stem cell-derived dopaminergic neurons are a suitable cellular system for fluorescence-based experimental approaches to address neurodegenerative events in vitro.