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.
Highlights
The development of hiPSC-based strategies to treat or model Parkinson’s disease (PD), is hampered by the lack of efficient protocols for the directed differentiation of stem cells into dopaminergic neurons (DAn) with the appropriate characteristics of A9-subtype www.nature.com/scientificreports/
We first confirmed the exact colocalization of mOrange expression from the reporter gene with endogenous tyrosine hydroxylase (TH) expression in TH-mOrange hiPSC differentiated into DAn
Our results show that the generated TH-mOrange induced pluripotent stem cells (iPSCs) line faithfully reports endogenous TH expression and that the genetic modification does not impair mDA specification
Summary
CRISPR/Cas9-mediated knock-in of a P2A-mOrange cassette in-frame with TH gene. With the aim of identifying TH+ neurons among living cells, we generated a genetic reporter construct that could robustly and faithfully label DAn. To obtain a homogenous population of mOrange-labeled iPSC-derived DAn, edited-iPSC DAn were isolated using fluorescence-activated cell sorting (FACS) at 27 days of differentiation. For this specific analysis, we utilized an alternative DAn differentiation protocol using dual-SMAD inhibition to obtain neural progenitor cells (NPCs) (Fig. S2A, B). MOrange-negative sorted cells showed similar levels of mOrange+/ TH+ neurons as the unsorted cells (60.8% vs 67.5%), suggesting that the mOrange− population contains mitotically active undifferentiated progenitors or neurons that have not yet switched on TH expression (Fig. 4E) These results were confirmed by FACS analysis of the sorted neurons seven days after the initial isolation (Fig. 4B). These results demonstrate that post-mitotic fluorescent DAn can be purified from heterogeneous cultures and maintained in culture after sorting
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