Small Molecules Greatly Improve Conversion of Human-Induced Pluripotent Stem Cells to the Neuronal Lineage

Figure 6

Analysis of number of TH+ and TUJ+ neurons (A)–(C). Image under phase-contrast microscopy showed plating density of NPCs at day 1 in DA1 media (5x magnification) (A), cell morphology at day 12 (arrows indicate the formation of processes) (B) 10x magnification) and neurons at day 30 during dopaminergic maturation (C) 10x magnification). (D)–(F), Neurons treated with Pur (D), SAG (E) and Shh (F) were characterized by immunostaining with TUJ1 (green) and TH (red) and counterstained with DAPI (blue). Scale bar represents 200 μm. (G)–(J) Flow cytometry: No TUJ1 and TH immunoreactive NPCs were detected (H), which is comparable to unstained NPCs (G). In contrast to the population of unstained differentiated neurons (I), differentiated cells showed positive immunoreactivity to TUJ1 and TH (j). (k) Table illustrates percentage of TUJ1 relative to total cells, TH immunoreactive neurons relative to TUJ1 immunoreactive neurons, and also relative to total cells quantified using stereology and flow cytometry in control NPCs after dopaminergic maturation.