The process for human central serotonergic neuron generation is shown in Figure 1a. Human PSCs were seeded onto laminin (Life Technologies)-coated plastic plates or polyornithine (PO, from Sigma-Aldrich) and laminin-coated 12-mm glass coverslips. PSCs at approximately 20% confluence (1 d after passaging) were cultured for 1 week in a chemically defined medium modified from our previous work11. Briefly, the medium consists of DMEM/F12: neurobasal (1:1), 1 × N2, 1 × B27, 1 × nonessential amino acids (NEAA), 1% GlutaMAX (all from Life Technologies), 2 µM SB431542 (Stemgent) and 2 µM DMH1 (Tocris Bioscience). Different concentrations of CHIR99021 (0–3µM, Tocris Bioscience) were applied to identify a dose that specifies progenitors with the hindbrain fate. After 1 week of differentiation, cells were passed mechanically in the same chemically defined medium at the ratio of 1:3 onto precoated plates or coverslips and different concentrations of SHH C25II (0–1,000 ng/ml, R&D Systems) were applied to ventralize cells. After two weeks of differentiation, cells were passed as previously in the same CDM with 1,000 ng/ml SHH, and FGF4 (10 ng/ml) was used to help the
