Cui et al. (2016). Scientific Reports. doi: 10.1038/srep25181
…t 1:200, TH (Pel Freez) at 1:500, TUJ (Abcam) at 1:1000. Directed dopaminergic differentiation and quantification of TH using luciferase assay. ES cells were differentiated towards a dopamingeric neuron cell fate for up to 6 weeks following the floor-plate induction method23. Cell supernatants were collected every day for the first 20 days of neuronal differentiation and every other day afterwa
Abstract
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.