Figure 6. RA induces GABAergic differentiation of human embryonic stem cells.

Embryoid bodies derived from H9 human embryonic stem cells were treated for 3 d with vehicle, 1 µM RA, or 10 µM RA; then neural rosettes differentiating from these cultures were analyzed immunocytochemically 18 d after RA treatment ended. (A–F) RA treatment increased the number of neurons positive for Gad67 and GABA. (G–I) Tuj1 and DCX double-staining shows that RA treatment increases the number of Tuj1-positive neurons, and essentially all are also positive for DCX, which marks migrating neurons. (J–L) RA treatment decreased the number of neurons positive for the neural progenitor marker Pax6. (M) Quantitative analysis of various cell types in differentiating cultures showed that 1 µM RA or 10 µM RA significantly induced the GABAergic neuron phenotype (Gad67-positive) with a concomitant decrease of Pax6-expressing neural progenitors. Percentages were calculated by dividing the immunopositive cell number with the total number of DAPI-stained nuclei. Data are presented as mean ± SEM; * p<0.01 and ** p<0.001 (untreated versus treated).