Quantification of dopaminergic neuron differentiation and neurotoxicity via a genetic reporter.
Human pluripotent stem cells provide a powerful human-genome based system for modeling human diseases in vitro and for potentially identifying novel treatments. Directed differentiation of pluripotent stem cells produces many specific cell types including dopaminergic neurons. Here, we generated a genetic reporter assay in pluripotent stem cells using newly-developed genome editing technologies in order to monitor differentiation efficiency and compare dopaminergic neuron survival under different conditions. We show that insertion of a luciferase reporter gene into the endogenous tyrosine hydroxylase (TH) locus enables rapid and easy quantification of dopaminergic neurons in cell culture throughout the entire differentiation process. Moreover, we demonstrate that the cellular assay is effective in assessing neuron response to different cytotoxic chemicals and is able to be scaled for high throughput applications. These results suggest that stem cell-derived terminal cell types can provide an alternative to traditional immortal cell lines or primary cells as a quantitative cellular model for toxin evaluation and drug discovery.
Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA.
Jun
J
Cui
Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA.
Megan
M
Rothstein
Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA.
Theo
T
Bennett
Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Pengbo
P
Zhang
Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA.
Ninuo
N
Xia
Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT 59717, USA.|Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA.
Renee A
RA
Reijo Pera
D002454:Cell Differentiation; D002478:Cells, Cultured; D003584:Cytological Techniques / Q000379:methods*; D003603:Cytotoxins / Q000378:metabolism*; D059290:Dopaminergic Neurons / Q000187:drug effects* / Q000502:physiology*; D000072669:Gene Editing; D017930:Genes, Reporter; D056426:Genetic Loci; D057166:High-Throughput Screening Assays; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000502:physiology*
D003603:Cytotoxins; D008156:Luciferases