Human/Mouse Dopaminergic Neuron Differentiation Kit Summary
A kit optimized to efficiently differentiate pluripotent cells into dopaminergic neurons under serum-free conditions.
- Pre-optimized kit reduces experimental variation
- Defined conditions decreases variation
- Includes a panel of premium quality growth factors
Why Use Pre-optimized Reagents to Differentiate Pluripotent Stem Cells into Dopaminergic Neurons?
The loss of dopaminergic neurons in the substantia nigra is a characteristic of Parkinson’s disease (PD), a neurodegenerative disorder that affects motor function.
Both animal models and clinical trials have suggested that cell replacement therapies may be effective in treating PD. However, this approach is limited by the availability of a rich, effective source of neural precursors for dopaminergic neuron generation. While embryonic and induced pluripotent stem cells can be differentiated into dopaminergic neurons, successful differentiation is dependent on high quality media and defined differentiation supplements. R&D Systems offers the Human/Mouse Dopaminergic Neuron Differentiation Kit to drive successful and efficient differentiation of embryonic and induced pluripotent stem cells into dopaminergic neurons.
R&D Systems Human/Mouse Dopaminergic Neuron Differentiation Kit:
- Includes high quality reagents to generate dopaminergic neurons in 27 days.
- Includes pre-optimized components with low lot-to-lot variation.
- Includes sufficient reagents for differentiation of 3 x 107 pluripotent stem cells.
The Human/Mouse Dopaminergic Neuron Differentiation Kit contains ITS and N-2 MAX Media Supplements, which are used to select and enrich neural stem cell populations. Bovine Fibronectin is included to support cell attachment and spreading. A growth factor panel, consisting of Recombinant Human Fibroblast Growth Factor Basic (FGF basic), Recombinant Mouse Fibroblast Growth Factor 8b (FGF-8b) and Recombinant Mouse Sonic Hedgehog Amino-terminal Peptide (Shh-N), is included for effective dopaminergic differentiation.
The quantity of each component provided in the kit is estimated to be sufficient for differentiation of 3 x 107 embryonic stem cells.
- Recombinant Mouse FGF-8b
- Recombinant Human FGF basic
- Recombinant Mouse Shh-N
- Purified Bovine Fibronectin
- ITS Media Supplement
- N-2 MAX Media Supplement
Characterization of Dopaminergic Neurons Generated from Human Pluripotent Stem Cells. Dopaminergic neurons were generated from human pluripotent stem cells using the Dopaminergic Neuron Differentiation Kit (Catalog # SC001B). Tyrosine Hydroxylase was detected using a Mouse Anti-Human Tyrosine Hydroxylase Monoclonal Antibody (Catalog # MAB7566). The cells were stained with the NorthernLights™(NL) 557-conjugated Donkey Anti-Mouse Antigen Affinity-purified Secondary Antibody (Catalog # NL007, red). Neuron-specific beta-III Tubulin was detected using a Mouse Anti-Neuron-specific beta-III Tubulin (Clone Tuj-1) Monoclonal Antibody (Catalog # MAB1195) followed by the NL493-flourochrome-conjugated Donkey Anti-Mouse Antigen Affinity-purified Secondary Antibody (Catalog # NL009, green). View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips.
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
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.
- DMEM/F-12, no HEPES
- Fetal Bovine Serum, ES Cell Qualified
- Phosphate Buffered Saline (PBS)
- 0.05% Trypsin/EDTA
- 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
- Sodium Bicarbonate (NaHCO3)
- 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)
- 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
- Serological pipettes
- Pipettes and pipette tips
- 10 mL syringes
- 37 °C and 5% CO2 incubator
- 37 °C water bath
- 60 °C hot plate
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.
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.
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
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.
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.
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.
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.
After 10-15 days, dopaminergic neurons can be identified by staining for expression of Tyrosine Hydroxylase and Neuron-specific beta-III Tubulin.
Citations for Human/Mouse Dopaminergic Neuron Differentiation Kit
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 2
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Caveolin-1 downregulation promotes the dopaminergic neuron-like differentiation of human adipose-derived mesenchymal stem cells
Authors: C Han, YJ Wang, YC Wang, X Guan, L Wang, LM Shen, W Zou, J Liu
Neural Regen Res, 2021;16(4):714-720. 2021
Physiological transgene regulation and functional complementation of a neurological disease gene deficiency in neurons.
Authors: Peruzzi PP, Lawler SE, Senior SL, Dmitrieva N, Edser PA, Gianni D, Chiocca EA, Wade-Martins R
Mol. Ther., 2009;17(9):1517-26. 2009
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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