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

Abstract

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.

Establishment of an in vitro model for analyzing mitochondrial ultrastructure in PRKN-mutated patient iPSC-derived dopaminergic neurons

“…In the present study, we first established TH-GFP iPSC lines from not only controls but also a patient with a PRKN mutation and first analyzed these cells using live cell imaging and CLEM to evaluate the structure and function of mitochondria specifically in dopaminergic neurons. Several studies have reported the establishment of TH reporter iPSC lines using gene editing technology for transcriptome profiling or the electrical properties of dopaminergic neurons [ 23 – 25 ]. Immunohistochemistry confirmed that the specificity of our TH-GFP iPSC lines was sufficient for the identification of dopaminergic neurons, similar to that of other knock-in reporters using genome editing technology.…”

“…It is therefore necessary to selectively label dopaminergic neurons among the mixture of dopaminergic and non-dopaminergic neurons that are derived from iPSCs. Recently, several studies have reported the successful purification of dopaminergic neurons carrying a tyrosine hydroxylase (TH; a marker for identifying dopaminergic neurons) knock-in reporter, either using the genome editing technology TALEN (transcription activator-like effector nuclease) or CRISPR/Cas9 systems [ 23 – 25 ]. However, the generation of TH reporter iPSC lines derived from PD patients, including those with PRKN mutations, has not been reported.…”

Current State-of-the-Art and Unresolved Problems in Using Human Induced Pluripotent Stem Cell-Derived Dopamine Neurons for Parkinson’s Disease Drug Development

“…The use of reporter gene that is selectively expressed in post-mitotic DA neurons is a convenient solution to detect these cells in heterogenous cultures. Thus, reporter iPS cell lines expressing GFP [ 87 , 88 ] or the brighter reporter protein mOrange [ 89 ] under the control of TH promoter were developed using the CRISPR Cas9 genome editing system. These genetic modifications facilitated the identification of DA neurons in mixed neuron cultures and xenotransplants, and even enabled their isolation in a viable state by FACS [ 89 ].…”

Profiling parvalbumin interneurons using iPSC: challenges and perspectives for Autism Spectrum Disorder (ASD)

“…Generation of tagged-parvalbumin neurons by a CRISPR/Cas9 approach as a tool to follow PVALB neuron cell fate The CRISPR/Cas9 genome-editing technology (reviewed in [53]) has been broadly used to modify iPSC and recently, many studies successfully demonstrated the generation of human iPSC lines expressing a fluorescent protein that faithfully recapitulates the endogenous expression of a given protein [54,55]. Through a CRISPR/Cas9 approach, one could insert a reporter cassette (i.e., eGFP, tdTomato), whose expression is driven by the endogenous PVALB promoter, allowing to follow the fate of these neurons once PV expression starts.…”