1. Model of differentiation from pluripotent stem cells to terminal dopaminergic neurons
Model of differentiation from pluripotent stem cells to terminal dopaminergic neurons Schematic representation of the differentiation system used and the temporal expression dynamics of differentiation stage markers.RNA levels of differentiation stage markers were measured by qRT–PCR. Relative levels are normalized to Actb internal control, and values are plotted relative to the highest expressed time point. Mean and standard deviation (SD) are from three biological replicates.Indirect immunofluorescence confirms expression of stage‐specific markers at the single cell level. OCT4 is a marker of pluripotent ESCs. Tuj1 is an antibody that detects neuronal marker TUBB3 at day 16 and day 30 neurons. The cycling activity of ESCs, day 16, and day 30 neurons was assessed by BrdU incorporation (24 h) into replicating BrDNA. Nuclei are counterstained with DAPI. Scale bar, 100 μm.Tyrosine hydroxylase (TH; in red) is a marker of dopaminergic neurons. It is not expressed in ESCs and detected weakly in day 16 and broadly in day 30 neurons. Nuclei are counterstained with DAPI. Scale bar, 100 μm.Gene expression dynamics across the differentiation time line for genes whose expression peaks in a single time point (z‐score > 1.75; from mRNA‐seq). Representative enriched GO terms were calculated using as background all genes expressed (> 1 TPM) in at least one time point. n, number of genes per group. Permute P‐value (GO‐Elite) is shown.
Carmelo Ferrai et al. Mol Syst Biol 2017;13:946
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