Nature Biotechnology: doi: /nbt Supplementary Figure 1. Generation of NSCs from hpscs in SDC medium.

Transcription

1 Supplementary Figure 1 Generation of NSCs from hpscs in SDC medium. (a) Q-PCR of pluripotent markers (OCT4, NANOG), neural markers (SOX1, PAX6, N-Cadherin), markers for the other germ layers (T, EOMES, SOX17) and Trophoblast marker CDX2 when cells were treated with different concentrations of CHIR (b) Cells stained for pluripotent marker OCT4 (Red) and neural stem cell marker SOX1 (Green) when treated with different concentrations of CHIR (c) FACS images of SOX2-expressing cells when treated with different concentrations of CHIR (d) Quantification of SOX2-expressing cells in c (n = 3). Data are represented as mean ± SEM. (e) Negative staining and positive staining of HOXA3- expressing cells (Red) when treated with 1.4μM CHIR99021 or 3.0μM CHIR99021 together with 0.1μM retinoic acid (RA) respectively. This antibody stained cells up to the r2 region in mouse embryos. Scale bar: 50μm; Hoechst staining is blue; CHIR: CHIR99021.

2 Supplementary Figure 2 The effects of SHH and FGF4 on the rostral hindbrain NSCs. (a) Q-PCR of regional neural markers along A-P axis before (1.4μM CHIR - SHH) or after (1.4μM CHIR + SHH) hindbrain NSCs treated with 1000ng/ml SHH. HFB: human fetal brain samples; Cells in 0μM CHIR SHH group were used as a positive control of markers in forebrain. (b) Cells stained with NKX2.2 and OLIG2 without treatment, treated with 1000ng/ml SHH or 1000ng/ml SHH together with 10ng/ml FGF4 during the second week of differentiation, respectively. (c) Quantification of NKX2.2- or OLIG2-expressing cells in b (n = 3). Data are represented as mean ± SEM. Scale bar: 50μm; Ho: Hoechst staining; CHIR: CHIR99021.

3 Supplementary Figure 3 The identity of neurons derived from rostral hindbrain NSCs not treated with SHH or FGF4. (a) Neurons (Tuj1+, Green) derived from progenitors without treatment of either SHH or FGF4 stained positively for GABA but not serotonin. (b) Quantification of GABA- or serotonin-expressing cells in a (n = 3). Data are represented as mean ± SEM. Scale bar: 50μm; Ho: Hoechst staining.

4 Supplementary Figure 4 Electrophysiological properties of the central serotonin neurons. (a) Inward Na+ and outward K+ currents were triggered upon - 50mV to + 50 mv voltage steps. The initial currents were enlarged in the insert panel. (b) Action potentials were induced from - 40 pa to pa injected current steps. (c) Plotting of action potential frequency to injected currents. (d) Spontaneous excitatory (downward currents) and inhibitory (upward currents) postsynaptic currents were recorded when neurons were held at 0 mv.

5 Supplementary Figure 5 Full western blot images of the selected portions shown in Figure 3h.

6 Supplementary Table 1. Summary of serotonin neuron differentiation from ESCs/iPSCs. Species Conditions Specification factors Mouse Not defined; FGF2; SHH; EBs; FCS; FGF8 KSR; Mouse Mouse Monkey Monkey Human Human Not defined; EBs; FCS; KSR; Not defined; stromal cells; KSR; Not defined; EBs; FBS; Not defined; EBs; FBS; Not defined; EBs; KSR; Chemically defined; Monolayer; SB; DMH1; CHIR FGF4; SHH; FGF8 FGF2; FGF4; SHH; FGF8 Systematic regional/ serotonergic markers En1; Pax2; Serotonin En1; Pax2; 5-HT Nkx6.1; Nkx2.2; Serotonin Electrophysiological 5-HT release assays References assays N/A N/A [24] N/A N/A [25] N/A N/A [23] FGF2 Serotonin; TPH N/A N/A [26] FGF2; FGF4; SHH RA; FGF2; 5- HT; FGF1; FSK Wnt (CHIR), SHH; FGF4 All the mouse work was focused on DA neurons. TPH2 Yes. N/A [27] Serotonin N/A Live serotonin imaging [28] HOXA2+;HOXA3-; HOXB1-; NKX2.2+;NKX6.1+;MASH1; FOXA2+; PHOX2B-; OLIG2-; Serotonin;TPH2; GATA2; GATA3 Yes. UPLC-ESI-MS/MS; Activity-dependent release; Response to drugs This study

7 Supplementary Table 2. Primers used in the study. Related to Fig. 1-3, Supplementary Fig Name NCBI Reference Forward (5' to 3') Reverse (5' to 3') Sequence CDX2 NM_ TGGAGCTGGAGAAGGAGTTT CTGCTGCTGCTGTTGCTG DBX1 NM_ AAGACCTTCGCCTTTCCCTAC CTGCACGTCGGAGAAGACTG EMX1 NM_ CGAGACGCAGGTGAAGGTG CTGCCCTCGTGGGTTTGT EN1 NM_ GGACAATGACGTTGAAACGCAGCA AAGGTCGTAAGCGGTTTGGCTAGA EOMES NM_ ATCATTACGAAACAGGGCAGGC CGGGGTTGGTATTTGTGTAAGG FOXG1 NM_ GCAGCACTTTGAGTTACAACGGCA AGTTCTGAGTCAACACGGAGCTGT GAPDH NM_ GCACCGTCAAGGCTGAGAAC AGGGATCTCGCTCCTGGAA GBX2 NM_ CTCGCTGCTCGCCTTCTC GCCAGTCAGATTGTCATCCG HOXA1 NM_ GGGTGTCCTACTCCCACTCA GGACCATGGGAGATGAGAGA HOXA2 NM_ CGTCGCTCGCTGAGTGCCTG TGTCGAGTGTGAAAGCGTCGAGG HOXA3 NM_ CCAGAATCGCCGCATGAAGTACAA GGGCTCATACGGGACGCTGTTGA HOXA4 NM_ ACGCTCTGTTTGTCTGAGCGCC AGAGGCCGAGGCCGAATTGGA HOXB1 NM_ GAGCTTTGCACCGGCCTAT CTTCATCCAGTCGAAGGTCCG HOXB2 NM_ CCTAGCCTACAGGGTTCTCTC CACAGAGCGTACTGGTGAAAAA HOXB3 NM_ AACGCCTTACACTCCATGACC ATTCTGGTGGGCTTTACCGAA HOXB4 NM_ AAAGAGCCCGTCGTCTACC GTGTAGGCGGTCCGAGAG HOXB6 NM_ GAACTGAGGAGCGGACTCAC CTGGGATCAGGGAGTCTTCA HOXB8 NM_ AGCCTCCTTGTGCAATTG GTAACAATTGCCCACAGC HOXC5 NM_ TTGTGGGAACTATGGATCGGC CGGTGGGAAAGTGATGCTT HOXC6 NM_ / CACCGCCTATGATCCAGTGAGGCA GCTGGAACTGAACACGACATTCTC NM_ IRX3 NM_ CCTGGGACCCATTTCGGAC TCTAAGCCCTCAGAGCTATCTTC KROX20 NM_ TTGACCAGATGAACGGAGTG CAGAGACGGGAGCAAAGC LIM1 NM_ ATCCTGGACCGCTTTCTCTT GTACCGAAACACCGGAAGAA LMX1A NM_ AGGCCATCGCTCCCAATGAG TGGTTCTCGGACGTTTGGGG

8 LMX1B NM_ TTCCTGATGCGAGTCAACGAG GCAGTACAGTTTCCGATCCCG NANOG NM_ ACAACTGGCCGAAGAATAGCA GGTTCCCAGTCGGGTTCAC N-Cadherin NM_ TCCTGATATATGCCCAAGACAA TGACCCAGTCTCTCTTCTGC NKX2.1 NM_ CGCATCCAATCTCAAGGAAT CAGAGTGTGCCCAGAGTGAA NKX2.2 NM_ AAACCATGTCACGCGCTCA GGCGTTGTACTGCATGTGCT NKX6.1 NM_ ACACGAGACCCACTTTTTCCG GCCCCGCCAAGTATTTTGTT OCT3/4 NM_ AGTTTGTGCCAGGGTTTTTG ACTTCACCTTCCCTCCAACC OLIG2 NM_ GGTAAGTGCGCAATGCTAAGCTGT TACAAAGCCCAGTTTGCAACGCAG OTX2 NM_ GGGCTGAGTCTGACCACTTC CTGGGTGGAAAGAGAAGCTG PAX6 NM_ TCTTTGCTTGGGAAATCCG CTGCCCGTTCAACATCCTTAG PAX7 NM_ ACCCCTGCCTAACCACATC GCGGCAAAGAATCTTGGAGAC PET1 NM_ CACGGCGAGTTCAAGCTCA CTGGAAGTCGAAGCGGTAGG SERT NM_ ATGGAGACGACGCCCTTGA CACACGCTGATAGCTGCTTCT SIM1 NM_ TTGCCAACACTTCACCATGT TGGTCTCCTGCTGTCTGATG SIX3 NM_ CGAGCAGAAGACGCATTGCTTCAA AGTTGCCTACTTGTGTGGGAGTGA SOX1 NM_ GTTTTTTGTAGTTGTTACCGC GCATTTACAAGAAATAATAC SOX17 NM_ GGCGCAGCAGAATCCAGA CCACGACTTGCCCAGCAT T NM_ TGCTTCCCTGAGACCCAGTT GATCACTTCTTTCCTTTGCATCAAG TPH2 NM_ ACGGAGAGGGTTTTCCCTG TGCCAAGTAGCTGATGCTCTT

9 Supplementary Table 3. List of antibodies used in this study. Name Company Catalog# Host Dilution 5-HT Abcam ab16007 Mouse 1:100 AADC Chemicon AB1569 Rabbit 1:500 EN1 DSHB 4G11c Mouse 1:800 FOXA2 Santa Cruz sc-6554 Goat 1:1000 FOXG1 Abcam ab18259 Rabbit 1:100 GABA Sigma A2052 Rabbit 1:1000 GAPDH Thermo Scientific PIMA Mouse 1:4000 GATA2 Sigma HPA Rabbit 1:500 GATA3 R&D MAB6330 Mouse 1:1000 GFAP Dako Z0344 Rabbit 1:5000 HOXA2 Sigma HPA Rabbit 1:200 HOXA3 Sigma HPA Rabbit 1:1000 HOXB1 R&D AF6318 Sheep 1:500 HOXB4 DSHB I12 anti-hoxb4 Rat 1:50 HTR1A Millipore MAB11041 Mouse 1:400 Ki67 ZYMED Z Rabbit 1:200 NESTIN Santa Cruz sc Goat 1:1000 NKX2.2 DSHB 74.5A5 Mouse 1:50 NKX6.1 Sigma HPA Rabbit 1:500 OCT3/4 Santa Cruz sc5279 Mouse 1:500 OLIG2 Chemicon AB9610 Rabbit 1:500 OTX2 R&D AF1979 Goat 1:2000 PAX3/7 Santa Cruz sc-7748 Goat 1:100 PAX6 DSHB PAX6 Mouse 1:5000

10 PET1 Novus Biologicals H A01 Mouse 1:500 PHOX2B R&D AF4940 Goat 1:2000 SERT ImmunoStar Rabbit 1:5000 SOX1 R&D AF3369 Goat 1:1000 SOX2 R&D AF2018 Goat 1:1000 TH Pel-Freez P40101 Rabbit 1:1000 TPH2 Novus Biologicals NB Rabbit 1:2000 Tuj1 Sigma T8660 Mouse 1:10000 Tuj1 Covance PBR-435P Rabbit 1:10000 VMAT2 Abcam ab Mouse 1:50