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Human/Mouse Dopaminergic Neuron Differentiation Kit

Key Product Details

Characterization of Dopaminergic Neurons Generated from Human Pluripotent Stem Cells.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, pluripotent stem cells are differentiated into dopaminergic neurons using the following procedure:

  • Generate embryoid bodies from pluripotent cells
  • Select for and expand Nestin positive cells
  • Differentiate Nestin positive cells into dopaminergic neurons
 

 

Reagents Provided

Differentiation of Pluripotent Cells into Dopaminergic Neurons using the Human/Mouse Dopaminergic Neuron Differentiation Kit (Catalog # SC001B)

  • ITS Supplement - 5 mL of a 100X concentrated solution, containing Bovine Insulin, Human Transferrin, and Sodium Selenite.
  • N-2 MAX Supplement - 5 mL of a 100X concentrated solution, containing Bovine Insulin, Human Transferrin, Sodium Selenite, Putrescine, and Progesterone.
  • Bovine Fibronectin Stock - 1 vial containing 2 mL of a 1000X (1 mg/mL) solution of purified Bovine Fibronectin.
  • Human FGF basic - 1 vial of lyophilized Recombinant Human FGF basic; enough to make 250 μL of a 1000X stock.
  • Mouse FGF-8b - 1 vial of lyophilized Recombinant Mouse FGF-8b; enough to make 250 μL of a 1000X stock.
  • Mouse Shh-N - 1 vial of lyophilized Recombinant Mouse Shh-N; enough to make 250 μL of a 1000X stock.

 

Other Supplies Required

Reagents

  • DMEM/F-12, no HEPES
  • Fetal Bovine Serum, ES Cell Qualified
  • Phosphate Buffered Saline (PBS)
  • 0.05% Trypsin/EDTA
  • Gelatin
  • ESGRO® (recombinant mouse LIF) (Millipore or equivalent)
  • Knock-out DMEM
  • MEM Non-essential AA Solution
  • Penicillin-Streptomycin-Glutamine, 100X
  • Penicillin-Streptomycin, 100X
  • 2-Mercaptoethanol, 1000X
  • Glucose
  • L-Glutamine
  • Sodium Bicarbonate (NaHCO3)
  • Poly-L-ornithine
  • Ascorbic Acid (Catalog # 4055/50)
  • Sterile, deionized water
  • BSA, very low endotoxin
  • Acetic acid
  • Anti-Nestin antibody (Catalog # AF2736 or IC1259)
  • Anti-Tyrosine Hydroxylase antibody (Catalog # MAB7566 or Catalog # AF7566)
  • Anti-Neuron-specific beta-III Tubulin antibody (Catalog # NL1195V or Catalog # MAB1195)

Materials

  • Mouse embryonic stem (ES) cells
  • Irradiated mouse embryonic fibroblast (iMEF) feeder cells (Catalog # PSC001)
  • 10 cm tissue culture dishes
  • 10 cm bacterial culture dishes
  • 12 mm cover slips
  • 24-well culture plates
  • 15 mL centrifuge tubes
  • 0.2 μm syringe filter
  • 0.2 μm, 500 mL filter units
  • Cryotubes
  • Serological pipettes
  • Pipettes and pipette tips
  • 10 mL syringes

Equipment

  • 37 °C and 5% CO2 incubator
  • 37 °C water bath
  • 60 °C hot plate
  • Centrifuge
  • Hemocytometer
  • Microscope
 
Procedure Overview

Protocol for the Differentiation of Pluripotent Cells into Dopaminergic Neurons using the Human/Mouse Dopaminergic Neuron Differentiation Kit (Catalog # SC001B)

Selection of Nestin-positive Cells

Generate embryoid bodies (EB) from pluripotent stem cells.

Transfer EB to a 10 cm culture dish containing KO-ES Media.

Culture the cells for 24 h at 37 °C and 5% CO2.

Replace the KO-ES Media with ITS/Fibronectin Media

Replace the KO-ES Media with ITS/Fibronectin Media.

Culture the cells for 6-8 days at 37 °C and 5% CO2.

Replace the media every 2 days.

Verify successful differentiation by staining cells for Nestin.

Replace the KO-ES Media with ITS/Fibronectin Media

Expansion of Nestin-Positive Cells

 

Wash the cells twice with sterile PBS.

Dissociate the cells with 0.05% Trypsin/EDTA.

Add 5 mL of KO-ES Media to neutralize the Trypsin

Wash the cells twice with sterile PBS

Transfer the cells to a 15 mL tube.

Remove the cell clumps by allowing the tube to stand for approximately 5 minutes and then transferring the suspended cells to a 15 mL tube.

Transfer the cells to a 15 mL tube

Centrifuge the samples at 220 x g for 5 minutes.

Resuspend the cell pellet in N-2 MAX/FGF basic/FGF-8b/Shh-N/Ascorbic Acid Media.

Centrifuge the samples at 220 x g for 5 minutes

Perform a cell count.

Perform a cell count

Plate the cells on Poly-L-ornithine/Fibronectin-coated plates at 3-5 x 105 cells/well in 500 μl of media.

Replace the media daily for 4-6 days with N-2 MAX/FGF basic/FGF-8b/Shh-N/Ascorbic Acid Media.

Plate the cells on Poly-L-ornithine/Fibronectin-coated plates at 3-5 x 105 cells/well in 500 µl of media

Differentiation of Nestin-positive Cells to Dopaminergic Neurons

 

Culture the cells in N-2 MAX/Ascorbic Acid Media without growth factors for 10-15 days.

Replace the media every 2 days.

Culture the cells in N-2 MAX/Ascorbic Acid Media without growth factors for 10-15 days

After 10-15 days, dopaminergic neurons can be identified by staining for expression of Tyrosine Hydroxylase and Neuron-specific beta-III Tubulin.

After 10-15 days, dopaminergic neurons can be identified by staining for expression of Tyrosine Hydroxylase and Neuron-specific beta-III Tubulin

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FAQs

  • Can the Human/Mouse Dopaminergic Neuron Differentiation Kit work with a starting population of neural progenitor cells instead of mouse pluripotent stem cells?

    The kit may work with a starting population of neural progenitor cells, but we have not tested this. If starting with neural progenitor cells we recommend starting at Stage 5 of the Dopaminergic Neuron Differentiation Kit protocol. The starting cell density would also have to be optimized.

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