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

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 sta-ble 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 nei-ther 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.