Directed differentiation of specific lineages has been a focal point in the field of human embryonic stem cell (hESC) research. Cell replacement therapy using hESCs have the potential for treating Parkinson's disease and other neurodegenerative disorders. This chapter describes the procedure for the derivation of dopaminergic (DA) neurons from hESCs.
Required Materials
Cells
- GIBCO Mouse Embryonic Fibroblasts (MEF), irradiated (Cat. No. S1520-100)
- Human embryonic stem cells (hESC)
Media and Reagents
- Dulbecco's Phosphate-Buffered Saline (D-PBS) without Ca2+ and Mg2+ (Cat. No. 14190)
- D-MEM/F-12 with GlutaMAX-I (Cat. No. 10565-018)
- Neurobasal Medium (Cat. No. 21103-049)
- Knockout Serum Replacement (Cat. No. 10828-028)
- 10% Bovine Serum Albumin (BSA) (Cat. No. P2489)
- Fetal Bovine Serum (FBS) (Cat. No. 16000-044)
- Dulbecco's Modified Eagle Medium (D-MEM) (Cat. No. 10569-010)
- Non-essential Amino Acids Solution (NEAA) (Cat. No. 11140)
- B-27 Supplement without Vitamin A (Cat. No. 12587-010)
- N-2 supplement (Cat. No. 17502-048)
- β-Mercaptoethanol (Cat. No. 21985-023)
- Attachment Factor (Cat. No. S-006-100)
- Natural Mouse Laminin (Cat no. 23017-015)
- StemPro Accutase Cell Dissociation Reagent (Cat. No. A11105-01)
- Recombinant Human FGF Basic (bFGF) (Cat. No. 13256-029)
- FGF-8b Recombinant Human (Cat. No. PHG0271)
- B-DNF Recombinant Human (Cat. No. PHC7074)
- G-DNF Recombinant Human (Cat. No. PHC7045)
- Trypan Blue Stain (Cat. No. 15250-061)
- Distilled water (Cat. No. 15230-162)
- Poly-L-Ornithine (Sigma, Cat. No. P3655)
- Heparin (Sigma, Cat. No. H3149)
- Ascorbic Acid (Sigma, Cat. No. A4403)
- Dibutyryl cyclic-AMP (dcAMP) (Sigma, Cat. No. D0627)
- Recombinant human sonic hedgehog (SHH) (R&D systems, Cat. No. 1314-SH-025)
Special Tools
- StemPro EZPassage Disposable Stem Cell Passaging Tool (Cat. No. 23181-010)
- Cell scraper (Fisher, Cat. No. 087711A)
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Stock solutions
Knockout Serum Replacement (KSR)
Thaw a bottle of KSR, prepare 50 mL aliquots, and store at –20°C. Use KSR within a week of thawing.
Recombinant human FGF, basic
Prepare a 10-μg/mL stock solution in D-PBS with 0.1% BSA, aliquot into sterilized tubes, and store at –20°C.
Heparin
Prepare a 2-mg/mL stock solution in D-PBS, aliquot 0.5 mL into sterilized tubes, and store at –80°C.
Ascorbic acid
Prepare a 200-mM stock solution in D-PBS, aliquot 0.5 mL into sterilized tubes, and store at –20°C.
Recombinant human sonic hedgehog
Prepare a 0.2-mg/mL stock solution in D-PBS with 0.1% BSA, aliquot into sterilized tubes, and store at –20°C.
Recombinant human FGF8b
Prepare a 0.1-mg/mL stock solution in D-PBS with 0.1% BSA, aliquot into sterilized tubes, and store at –20°C.
Recombinant human BDNF
Prepare a 25-μg/mL stock solution in D-PBS with 0.1% BSA, aliquot into sterilized tubes, and store at –20°C.
Recombinant human GDNF
Prepare a 20-μg/mL stock solution in D-PBS with 0.1% BSA, aliquot into sterilized tubes, and store at –20°C.
Dibutyryl cyclic-AMP (dcAMP)
Prepare a 1-mM stock solution in distilled water, aliquot 0.5 mL into sterilized tubes, and store at –20°C.
Poly-L-Ornithine
Prepare a 10-mg/mL stock solution in distilled water, aliquot 0.5 mL into sterilized tubes, and store at –20°C.
Mouse Embryonic Fibroblast (MEF) Medium
To prepare 100 mL of MEF medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally.
Component | Amount |
---|
D-MEM | 90 mL |
FBS | 10 mL |
Human Embryonic Stem Cell (hESC) Medium
To prepare 100 mL of hESC medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally. hESC medium lasts for up to 7 days at 4°C.
Component | Amount |
---|
D-MEM/F-12 | 79 mL |
Knockout Serum Replacement | 20 mL |
NEAA | 1 mL |
Basic FGF Solution | 40 μL |
β-Mercaptoethanol* | 182 μL |
*Add β-Mercaptoethanol (final 0.1 mM) at the time of medium change. |
Neural Induction Medium
To prepare 100 mL of neural induction medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally. Neural induction medium lasts for up to 7 days at 4°C.
Component | Amount |
---|
D-MEM/F-12 | 98 mL |
N-2 Supplement | 1 mL |
NEAA | 1 mL |
Basic FGF Solution | 200 μL |
Heparin Solution | 100 μL |
Neural Expansion Medium
To prepare 100 mL of neural expansion medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally. Neural expansion medium lasts for up to 7 days at 4°C.
Component | Amount |
---|
D-MEM/F-12 | 96 mL |
N-2 Supplement | 1 mL |
B-27 Supplement | 2 mL |
NEAA | 1 mL |
Basic FGF Solution | 200 μL |
Heparin Solution | 100 μL |
DA Neuronal Differentiation Medium
To prepare 100 mL of DA neural differentiation medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally. DA neural differentiation medium lasts for up to 7 days at 4°C.
Component | Amount |
---|
Neurobasal Medium | 96 mL |
L-Glutamine | 1 mL |
B-27 Supplement | 2 mL |
NEAA | 1 mL |
GDNF Solution* | 100 μL |
BDNF Solution* | 100 μL |
Ascorbic Acid Solution* | 100 μL |
dcAMP Solution* | 100 μM |
*Add GDNF, BDNF, ascorbic acid, and dcAMP at the time of medium change. |
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Gelatin Coating Culture Vessels
- Cover the whole surface of each culture vessel with Attachment Factor solution (1 mL for each well of a 6-well plate, 2 mL into each 60-mm dish, or 4 mL into each 100-mm dish) and incubate for 1 hour at room temperature. Wash once with distilled water before plating the MEF.
Note: AF is sterile 1X solution containing 0.1% gelatin, available from Invitrogen.
Thawing MEFs
- Wearing eye protection and ultra low-temperature cryo-gloves, remove the vials of irradiated MEF from the liquid nitrogen storage tank using metal forceps. Note: Transfer the vials into a container with a small amount of liquid nitrogen if the vials are exposed to ambient temperature for more than 15 seconds between removal and step 3.
- Briefly roll the vials containing MEF between your hands for about 10–15 seconds to remove frost and swirl them gently in a 37°C water bath. Do not submerge the vials completely.
- When only a small amount of ice remains in the vials, remove them from the water bath. Spray the outside of the vials with 70% ethanol before placing them in the cell culture hood.
- Pipet the thawed cells gently into a 15-mL conical tube using a 1-mL pipette.
- Rinse the cryovial with 1 mL of pre-warmed MEF medium. Transfer the medium to the same 15-mL tube containing the cells.
- Add 4 mL of pre-warmed MEF medium dropwise to the cells. Gently mix by pipetting up and down. Note: Adding the medium slowly helps cells to avoid osmotic shock.
- Centrifuge the cells at 200 × g for 5 minutes.
- Aspirate the supernatant and resuspend the cell pellet in 5 mL of pre-warmed MEF medium.
- Remove 10 μL of cell suspension and determine the viable cell count using your method of choice.
Note: We recommend using the Countess Automated Cell Counter for easy and accurate cell counting and viability measurements.
Plating MEFs
- Centrifuge the MEFs at 200 × g for 5 minutes and aspirate the supernatant.
- Resuspend the cell pellet in MEF medium to a concentration of 2.5 × 106 cells/mL.
- Aspirate the Attachment Factor solution from the coated culture vessels and wash the plates once with D-PBS.
- Add the appropriate amount of MEF medium into each culture vessel (2.5 mL into each well of 6-well plate, 5 mL into each 60-mm dish, or 10 mL into each 100-mm dish).
- Into each of these culture vessels, add the appropriate amount of MEF suspension (0.1 mL into each well of 6-well plate, 0.2 mL into each 60-mm dish, or 0.6 mL into each 100-mm dish). The recommended plating density for GIBCO Mouse Embryonic Fibroblasts (Irradiated) is 2.5 × 104 cells/cm2.
- Move the culture vessels in several quick back-and-forth and side-to-side motions to disperse cells across the surface of the wells and dishes. After plating the cells, place the vessels in a 37°C incubator with a humidified atmosphere of 5% CO2. Use the MEF plates and dishes within 3–4 days of plating.
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