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2019-05-02

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cc-by (c) Calatayud Aristoy, Carles et al., 2019
Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/148117

CRISPR/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons

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Recurs relacionat

https://doi.org/10.1038/s41598-019-43080-2

Resum

Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson's disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus. After differentiation of the TH-mOrange reporter iPSC line, we confirmed that mOrange expression faithfully mimicked endogenous TH expression in iPSC-derived DAn. We also employed calcium imaging techniques to determine the intrinsic functional differences between dopaminergic and non-dopaminergic ventral midbrain neurons. Crucially, the brightness of mOrange allowed direct visualization of TH-expressing cells in heterogeneous cultures, and enabled us to isolate live mOrange-positive cells through fluorescence-activated cell sorting, for further differentiation. This technique, coupled to refined imaging and data processing tools, could advance the investigation of PD pathogenesis and might offer a platform to test potential new therapeutics for PD and other neurodegenerative diseases.

Matèries

Malaltia de Parkinson, Ciències de la salut, Enginyeria genètica

Matèries (anglès)

Parkinson's disease, Medical sciences, Genetic engineering

Citació

Col·leccions

Articles publicats en revistes (Patologia i Terapèutica Experimental)
Articles publicats en revistes (Física de la Matèria Condensada)
Articles publicats en revistes (Institut d'lnvestigació Biomèdica de Bellvitge (IDIBELL))

Citació

CALATAYUD ARISTOY, Carles, et al. CRISPR/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons. Scientific Reports. 2019. Vol. 9, num. 6811. ISSN 2045-2322. [consulted: 20 of August of 2026]. Available at: https://hdl.handle.net/2445/148117

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