Abstract
Tau pathology is a major hallmark of many neurodegenerative diseases summarized under the term tauopathies. In most of these disorders, such as Alzheimer’s disease, the neuronal axonal microtubule-binding Tau protein becomes mislocalized to the somatodendritic compartment. In human disease, this missorting of Tau is accompanied by an abnormally high phosphorylation state of the Tau protein, and several downstream pathological consequences (e.g., loss of microtubules, degradation of postsynaptic spines, impaired synaptic transmission, neuronal death). While some mechanisms of Tau sorting, missorting, and associated pathologies have been addressed in rodent models, few studies have addressed human Tau in physiological disease-relevant human neurons. Thus, suitable human-derived in vitro models are necessary. This protocol provides a simple step-by-step protocol for generating homogeneous cultures of cortical glutamatergic neurons using an engineered Ngn2 transgene-carrying WTC11 iPSC line. We further demonstrate strategies to improve neuronal maturity, that is, synapse formation, Tau isoform expression, and neuronal activity by co-culturing hiPSC-derived glutamatergic neurons with mouse-derived astrocytes. Finally, we describe a simple protocol for high-efficiency lentiviral transduction of hiPSC-derived neurons at almost all stages of differentiation.
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Acknowledgments
We thank Dr. Magdalena Laugsch and Dr. Marlen Lauffer for their basic guidance and support in iPSC-related cell culture work. We thank Prof. Dr. Florian Klein (Institute of Virology, University Hospital Cologne) for providing lentiviral vectors and Lena Kluge and Helen Breuer for supporting the lentivirus production. We thank Prof. Dr. Brunhilde Wirth (Institute of Human Genetics, University Hospital Cologne) and Dr. Markus Chmielewski (Department I of Internal Medicine, University Hospital Cologne) for providing the HEK293T cell line. We thank Mhd. Aghyad Al-Kabbani for the methodological advice and critical manuscript proofreading. Our work is supported by the Koeln Fortune Program (Faculty of Medicine, University of Cologne), by the Else-Kröner-Fresenius-Stiftung, by a stipend from the Studienstiftung des deutschen Volkes, and by the Jürgen-Manchot-Stiftung.
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Buchholz, S., Bell-Simons, M., Cakmak, C., Klimek, J., Gan, L., Zempel, H. (2024). Cultivation, Differentiation, and Lentiviral Transduction of Human-Induced Pluripotent Stem Cell (hiPSC)-Derived Glutamatergic Neurons for Studying Human Tau. In: Smet-Nocca, C. (eds) Tau Protein. Methods in Molecular Biology, vol 2754. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3629-9_31
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DOI: https://doi.org/10.1007/978-1-0716-3629-9_31
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