Sensitivity to Oxidative Stress in DJ-1-Deficient Dopamine Neurons: An ES- Derived Cell Model of Primary Parkinsonism
Figure 3
DJ-1-Deficient ES Cell Cultures Display Reduced DN Production
(A) The SDIA coculture method. DJ-1 knockout or control heterozygous ES cells are cocultured with mouse stromal cells (MS5) in the absence of serum and leukemia inhibitory factor for 18 DIV.
(B) DN production was quantified at 18 DIV by 3H-dopamine uptake assay. DJ-1-deficient ES cell cultures were defective relative to heterozygous control cultures.
(C–D) Neuron production was quantified by immunohistochemical analysis as a percent of TuJ1-positive colonies that express TH (C) or GABA (D). Quantification of TH and GABA immunostaining was performed on all colonies in each of three independent wells. Colonies were scored as positive if any immunostained cells were present. * p ≤ 0.05.
(E) The absolute number of TuJ1-positive colonies was not significantly different between the two genotypes.
(F) Kinetic analysis of DN differentiation in DJ-1-deficient cultures (clone 32, solid square) and heterozygous controls (open circle) as quantified by 3H-dopamine uptake assay. * p ≤ 0.05.
(G) DJ-1-deficient (open bar) and heterozygous control (closed bar) cultures differentiated for 9 DIV and then exposed to 6-OHDA at the indicated dose for 72 h. DNs were quantified by 3H-dopamine uptake assay. Data represent the means ± SEM and were analyzed by ANOVA followed by Fisher's post-hoc test. * p ≤ 0.05.