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This video demonstrates an in vitro method to generate postganglionic sympathetic neurons from human pluripotent stem cells (hPSCs). hPSCs are differentiated into neural crest cells (NCCs) on a basement membrane matrix support using a differentiation medium. The NCCs are then dissociated and formed into spheroids using another differentiation medium. Finally, the spheroids are transformed into postganglionic sympathetic neurons using a sympathetic neuron medium.
1. Set-up for dish coating, media preparation, and hPSC maintenance
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Figure 1: NC induction. (A) Timeline and treatments for NC induction from day 0 to day 10. (B) The morphology and formation of NC ridges were monitored every 2 days by bright field microscopy. Cells at each time point after day 2 were co-stained for AP2A (red) and SOX10 (green). All immunofluorescence pictures were counterstained with DAPI. Red arrows indicate the structures of ridges. (C) qRT-PCR analysis for the expression profile of NC markers from day 2&#....
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Name | Company | Catalog Number | Comments |
100 mm cell culture dishes | Falcon | 353003 | |
15 mL conical tissue culture tubes | VWR/Corning | 89039-664 | |
24-well tissue culture plates | Falcon | 353047 | |
24-well ultra-low-attachment plates | Corning | 07 200 601 and 07 200 602 | |
5% CO2/20% O2 tissue culture incubator | Thermo Fisher/Life Technologies | Heracell VIOS 160i | |
50 ml conical tissue culture tubes | VWR/Corning | 89039-656 | |
6-well tissue culture plates | Costar | 3516 | |
Accutase | Innovation Cell Technologies | AT104500 | Cell dissociation solution |
Anti-AP2a antibody | Abcam | ab108311 | Host: Rabbit; 1:400 dilution |
Anti-Ascl1 antibody | BD Pharmingen | 556604 | Host: Mouse IgG1; 1:200 dilution |
Anti-CD49D antibody | BioLegend | 304313 | Host: Mouse IgG1; 5 μl/million cells in 100 μl volume |
Anti-CD49D antibody (isotype) | BioLegend | 400125 | Host: Mouse IgG1; 5 μl/million cells in 100 μl volume |
Anti-DAPI antibody | Sigma | D9542 | 1:1000 dilution |
Anti-DBH antibody | Immunostar | 22806 | Host: Rabbit; 1:500 dilution |
Anti-GFP antibody | Abcam | ab13970 | Host: Chicken; 1:1000 dilution |
Anti-HOXC9 antibody | Santa Cruz Biotechnology | sc-365692 | Host: Mouse IgG1; 1:100 dilution |
Anti-NET1 antibody | Mab | NET17-1 | Host: Mouse; 1:1000 dilution |
Anti-PRPH antibody | Santa Cruz Biotechnology | SC-377093/H0112 | Host: Mouse IgG2a; 1:200 dilution |
Anti-SOX10 antibody | Abcam | ab50839 | Host: Mouse; 1:100 dilution |
Anti-TH antibody | Pel-Freez | P40101- 150 | Host: Rabbit; 1:500 dilution |
Ascorbic acid | Sigma | A8960-5G | Stock concentration: 100 mM |
B27 supplement | Thermo Fisher/Life Technologies | 12587-010 | Stock concentration: 50x |
BDNF | R&D Systems | 248-BD | Stock concentration: 10 μg/mL |
BMP4 | R&D Systems | 314-BP | Stock concentration: 6 mM |
Cell counter | Thermo Fisher/Life Technologies | Countess II | |
Cell counting chamber slides | Invitrogen | C10312 | |
Centrifuge | Eppendorf | 57021&5424R | |
CHIR99021 | R&D Systems | 4423 | Stock concentration: 6 mM |
Cryo-vial | Thermo Fisher/Life Technologies | 375353 | |
dbcAMP | Sigma | D0627 | Stock concentration: 100 mM |
DMEM | Thermo Fisher/Life Technologies | 10829-018 | Stock concentration: 1x |
DMEM/F12 | Thermo Fisher/Life Technologies | 11330-057 | Stock concentration: 1x |
DMSO | Thermo Fisher/Life Technologies | BP231-100 | |
E6 medium | gibco | A15165-01 | |
E8 medium | gibco | A15169-01 | Stock concentration: 1x |
E8 supplement | gibco | A15171-01 | Stock concentration: 50x |
EDTA | Sigma | ED2SS | Stock concentration: 0.5 M |
FACS machine | Beckman Coulter | CytoFLEX (for FACS) | |
FACS machine | Beckman Coulter | MoFlo Astrios EQ (for sorting) | |
FACS tubes (blue filter cap) | Falcon | 352235 | |
FACS tubes (white cap) | Falcon | 352063 | |
Fetal bovine serum (FBS) | Atlanta Biologicals | S11150 | |
GDNF | PeproTech | 450 | Stock concentration: 10 μg/mL |
Geltrex | Invitrogen | A1413202 | Basement membrane matrix; Stock concentration: 100x |
hPSCs | Thomson et al., (1998) | WA09 | |
hPSCs | Oh et al. (2016) | H9-PHOX2B::eGFP | |
Human fibronectin (FN) | VWR/Corning | 47743-654 | Stock concentration: 1 mg/mL |
L-glutamine | Thermo Fisher/Gibco | 25030-081 | Stock concentration: 200 mM |
LN tank | Custom Biogenic Systems | V-1500AB | |
Mouse laminin I (LM) | R&D Systems | 3400-010-01 | Stock concentration: 1 mg/mL |
N2 supplement | Thermo Fisher/Life Technologies | 17502-048 | Stock concentration: 100x |
Neurobasal medium | gibco | 21103-049 | Stock concentration: 1x |
NGF | PeproTech | 450-01 | Stock concentration: 25 μg/mL |
Phosphate-buffered saline (PBS) | Gibco | 14190-136 | Stock concentration: 1x |
Poly-L-ornithine hydrobromide (PO) | Sigma | P3655 | Stock concentration: 15 mg/mL |
Primocin (antibiotics) | InvivoGen | ANTPM1 | Stock concentration: 50 mg/mL |
qPCR machine | Bio-Rad Laboratories | C1000 Touch | |
qPCR plates | Bio-Rad Laboratories | HSP9601 | |
recombinant FGF2 | R&D Systems | 233-FB/CF | Stock concentration: 10 μg/mL |
Retinoic acid | Sigma | R2625 | Stock concentration: 1 mM |
SB431542 | Tocris/R&D Systems | 1614 | Stock concentration: 10 mM |
Trypan blue | Corning | MT-25-900-CI | |
Vitronectin (VTN) | Thermo Fisher/Life Technologies | A14700 | Stock concentration: 0.5 mg/mL |
Water bath | VWR/Corning | 706308 | |
Y27632 | R&D Systems | 1254 | Stock concentration: 10 mM |
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Source: Wu, H. F., et al. Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions. J. Vis. Exp. (2020).
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