Method Article

Generating Postganglionic Sympathetic Neurons from Human Pluripotent Stem Cells

July 8th, 2025

In This Article

Abstract

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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).

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.

Protocol

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1. Set-up for dish coating, media preparation, and hPSC maintenance

  1. Dish coating
    1. Vitronectin (VTN) coating
      1. Place vials of VTN in a 37 °C water bath until fully thawed, then mix thoroughly.
      2. For a 100 mm Petri dish, mix 7 mL of 1x phosphate buffered saline (PBS) with 0.5 mg/mL VTN, add VTN solution to the dish, and incubate at room temperature (RT) for 1 h.
    2. Basement membrane matrix coating​
      1. Thaw vials of basement membrane matrix (see Table of Materials) on ice at 4 °C overnight.
      2. For one....

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Results

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Neural crest induction diagram, cell differentiation micrographs, gene expression charts.
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 .......

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
100 mm cell culture dishesFalcon353003
15 mL conical tissue culture tubesVWR/Corning89039-664
24-well tissue culture platesFalcon353047
24-well ultra-low-attachment platesCorning07 200 601 and 07 200 602
5% CO2/20% O2 tissue culture incubatorThermo Fisher/Life TechnologiesHeracell VIOS 160i
50 ml conical tissue culture tubesVWR/Corning89039-656
6-well tissue culture platesCostar3516
AccutaseInnovation Cell TechnologiesAT104500Cell dissociation solution
Anti-AP2a antibodyAbcamab108311Host: Rabbit; 1:400 dilution
Anti-Ascl1 antibodyBD Pharmingen556604Host: Mouse IgG1; 1:200 dilution
Anti-CD49D antibodyBioLegend304313Host: Mouse IgG1; 5 μl/million cells in 100 μl volume
Anti-CD49D antibody (isotype)BioLegend400125Host: Mouse IgG1; 5 μl/million cells in 100 μl volume
Anti-DAPI antibodySigmaD95421:1000 dilution
Anti-DBH antibodyImmunostar22806Host: Rabbit; 1:500 dilution
Anti-GFP antibodyAbcamab13970Host: Chicken; 1:1000 dilution
Anti-HOXC9 antibodySanta Cruz Biotechnologysc-365692Host: Mouse IgG1; 1:100 dilution
Anti-NET1 antibodyMabNET17-1Host: Mouse; 1:1000 dilution
Anti-PRPH antibodySanta Cruz BiotechnologySC-377093/H0112Host: Mouse IgG2a; 1:200 dilution
Anti-SOX10 antibodyAbcamab50839Host: Mouse; 1:100 dilution
Anti-TH antibodyPel-FreezP40101- 150Host: Rabbit; 1:500 dilution
Ascorbic acidSigmaA8960-5GStock concentration: 100 mM
B27 supplementThermo Fisher/Life Technologies12587-010Stock concentration: 50x
BDNFR&D Systems248-BDStock concentration: 10 μg/mL
BMP4R&D Systems314-BPStock concentration: 6 mM
Cell counterThermo Fisher/Life TechnologiesCountess II
Cell counting chamber slidesInvitrogenC10312
CentrifugeEppendorf57021&5424R
CHIR99021R&D Systems4423Stock concentration: 6 mM
Cryo-vialThermo Fisher/Life Technologies375353
dbcAMPSigmaD0627Stock concentration: 100 mM
DMEMThermo Fisher/Life Technologies10829-018Stock concentration: 1x
DMEM/F12Thermo Fisher/Life Technologies11330-057Stock concentration: 1x
DMSOThermo Fisher/Life TechnologiesBP231-100
E6 mediumgibcoA15165-01
E8 mediumgibcoA15169-01Stock concentration: 1x
E8 supplementgibcoA15171-01Stock concentration: 50x
EDTASigmaED2SSStock concentration: 0.5 M
FACS machineBeckman CoulterCytoFLEX (for FACS)
FACS machineBeckman CoulterMoFlo Astrios EQ (for sorting)
FACS tubes (blue filter cap)Falcon352235
FACS tubes (white cap)Falcon352063
Fetal bovine serum (FBS)Atlanta BiologicalsS11150
GDNFPeproTech450Stock concentration: 10 μg/mL
GeltrexInvitrogenA1413202Basement membrane matrix; Stock concentration: 100x
hPSCsThomson et al., (1998)WA09
hPSCsOh et al. (2016)H9-PHOX2B::eGFP
Human fibronectin (FN)VWR/Corning47743-654Stock concentration: 1 mg/mL
L-glutamineThermo Fisher/Gibco25030-081Stock concentration: 200 mM
LN tankCustom Biogenic SystemsV-1500AB
Mouse laminin I (LM)R&D Systems3400-010-01Stock concentration: 1 mg/mL
N2 supplementThermo Fisher/Life Technologies17502-048Stock concentration: 100x
Neurobasal mediumgibco21103-049Stock concentration: 1x
NGFPeproTech450-01Stock concentration: 25 μg/mL
Phosphate-buffered saline (PBS)Gibco14190-136Stock concentration: 1x
Poly-L-ornithine hydrobromide (PO)SigmaP3655Stock concentration: 15 mg/mL
Primocin (antibiotics)InvivoGenANTPM1Stock concentration: 50 mg/mL
qPCR machineBio-Rad LaboratoriesC1000 Touch
qPCR platesBio-Rad LaboratoriesHSP9601
recombinant FGF2R&D Systems233-FB/CFStock concentration: 10 μg/mL
Retinoic acidSigmaR2625Stock concentration: 1 mM
SB431542Tocris/R&D Systems1614Stock concentration: 10 mM
Trypan blueCorningMT-25-900-CI
Vitronectin (VTN)Thermo Fisher/Life TechnologiesA14700Stock concentration: 0.5 mg/mL
Water bathVWR/Corning706308
Y27632R&D Systems1254Stock concentration: 10 mM

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Tags

Neural Crest CellsSympathetic Neuron DifferentiationBasement Membrane MatrixSpheroid FormationRetinoic Acid TreatmentNeural Progenitor CellsPeripheral Nervous SystemCell Culture IncubatorEDTA Dissociation

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