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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
2. Seeding hPSCs to start the differentiation (day 0)
NOTE: hPSCs should be ready for differentiation after being stabilized (i.e., being split 2–3x after thawing). Be sure that the colonies are healthy, with smooth, shiny edges, and minimal differentiation (Figure 2B).
3. Neural crest cell induction (day 1 to day 10, Figure 1A)
4. Fluorescence activated cell sorting (FACS) for neural crest marker CD49D and aggregating NC cells in spheroids
NOTE: For FACS sorting, the samples should be kept on ice and not be exposed to light after staining until sorting.
5. Aggregating NC cells in spheroids
6. NC spheroid maintenance and sympathetic progenitor induction (day 10 to day 14, Figure 3A)
7. Sympathetic neuron (SymN) differentiation and maturation (Option 1: after day 14; Option 2: after day 28)
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 th...
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 |
This article has been published
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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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