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기사 소개

  • Overview
  • 프로토콜
  • 결과
  • 공개
  • 자료
  • 참고문헌

Overview

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.

프로토콜

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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결과

figure-results-58
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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공개

No conflicts of interest declared.

자료

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

참고문헌

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