Quantification of dopaminergic neuron differentiation and neurotoxicity via a genetic reporter.

Jun Cui,Renee A Reijo Pera,Megan Rothstein,Theo Bennett,Pengbo Zhang,Ninuo Xia

doi:10.1038/srep25181

Jun Cui, Renee A Reijo Pera + Show 4 more

Open Access

https://doi.org/10.1038/srep25181

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Journal: Scientific Reports	Publication Date: Apr 28, 2016
Citations: 18	License type: CC BY 4.0

Affiliation: Montana State University, Stanford University

Abstract

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.

Highlights

Provides a robust and specific measurement of target cell types and is suitable to be used in large scale quantitative experiments and screening assays
The hybrid mRNA expression was detected in DA neurons from reporter clones 9 and 15 but not from H9 cells (Fig. 2B), which is consistent with the luciferase activity
In previous studies we found that DA neurons were especially vulnerable to oxidative stress induced by toxins such as H2O2 and 6-hydroxydopamine (6-OHDA)[12,26] and studying DA neuron response under oxidative stress could be a useful in vitro model for Parkinson’s Disease (PD)

Summary

Introduction

Provides a robust and specific measurement of target cell types and is suitable to be used in large scale quantitative experiments and screening assays. We differentiated the reporter cells to DA neurons using an established floor-plate induction protocol[13,23]. The above results combined indicate the reporter hESCs maintain pluripotency and the similar differentiation potential towards TH neurons to the parental H9 cells.

Results

Conclusion