Generation and isolation of human embryonic stem cell (hESC)-derived serotonergic neurons in vitro. (a) Tryptophan hydroxylase (TPH) staining in fixed mouse brain coronal sections reveals TPH-immunopositive (green) cells in the midbrain dorsal raphe nuclei near the aqueduct (AQ). (b) TPH stain of 3-week-old differentiated hESC-derived neurons in the FGF-2 (top panel) and FGF-8+Shh (lower panel) treated groups. (c) Quantification of percentage of TPH+ cells (green) over MAP2ab+ (red) neurons shows increase in the FGF-8+Shh condition as compared with the FGF-2 condition. (d) 5-HT+ (red) neurons formed a subset of all TPH+ (green) neurons at three as shown by quantification in (e). (d, lower panel) At 8 weeks post differentiation, 5-HT+ (green) neurons formed a subset of all TPH+ (red) neurons as shown by quantification in (f). (g) TPH2::GFP reporter lentivirus staining showed that a majority of GFP+ cells (green) were also TPH+ (red) and vice versa, where a majority of TPH+ neurons were virally labeled and were GFP+. (h) Quantification of TPH2::GFP reporter lentivirus accuracy shows that a majority of GFP+ neurons were TPH+ and a majority of TPH+ neurons were also labeled with the TPH2::GFP lentiviral reporter. (i) QPCR analysis of sorted TPH2::GFP+/Syn::DsRed+ double positive neurons over TPH2::GFP−/Syn::DsRed+ neurons shows increased levels of mRNA of serotonergic transcription factors NKX2.2, FEV, GATA2 and LMX1B and TPH2, and no change in LHX6 (log scale). Data are represented as mean±s.e.m., *P-value<0.05. n=3 independent experiments. Arrowheads point to double positive cells. Scale bars 50 μm; in (d), lower panel, 30 μm. Syn::DsRed, Synapsin::DsRed; TPH::GFP, TPH2::GFP.