Abstract
A major hallmark of Parkinson's disease is loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The pathophysiological mechanisms causing this relatively selective neurodegeneration are poorly understood, and thus experimental systems allowing to study dopaminergic neuron dysfunction are needed. Induced pluripotent stem cells (iPSCs) differentiated toward a dopaminergic neuronal phenotype offer a valuable source to generate human dopaminergic neurons. However, currently available protocols result in a highly variable yield of dopaminergic neurons depending on the source of hiPSCs. We have now developed a protocol based on HBA promoter-driven transient expression of transcription factors by means of adeno-associated viral (AAV) vectors, that allowed to generate very consistent numbers of dopaminergic neurons from four different human iPSC lines. We also demonstrate that AAV vectors expressing reporter genes from a neuron-specific hSyn1 promoter can serve as surrogate markers for maturation of hiPSC-derived dopaminergic neurons. Dopaminergic neurons differentiated by transcription factor expression showed aggravated neurodegeneration through α-synuclein overexpression, but were not sensitive to γ-synuclein overexpression, suggesting that these neurons are well suited to study neurodegeneration in the context of Parkinson’s disease.
Highlights
Parkinson’s disease is one of the most common neurological disorders
The highest numbers of differentiated dopaminergic neurons were detected in the CT-01 human induced pluripotent stem cells (hiPSCs) line (~65%), whereas the lowest numbers were detected in the Parkinson’s Disease (PD)-02 hiPSC line (~13%)
Statistical power (1−β error probability) >0.85 for all significant conditions. These results suggest that the efficiency of the patterning protocol to generate neurons is not consistent and varies substantially when different hiPSC lines are used
Summary
Parkinson’s disease is one of the most common neurological disorders. The disease is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. Reasoned that expression of transcription factors related to dopaminergic neuronal differentiation might be able to generate a better reproducible and more homogenous outcome concerning numbers and cell type from several different hiPSC lines. We report that the protocol using pharmacological compounds generated variable numbers of neurons in general, and percentages of dopaminergic neurons from four different hiPSC lines. These results suggest that these compounds trigger divergent cellular activation states in different cell lines and might not be able to pattern different hiPSC lines with sufficient stringency. We demonstrate that our transcription factordifferentiated neurons can be readily used to study neurotoxicity of α-synuclein
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
