Title |
Hematopoietic differentiation |
Date Submitted |
4/2012 |
Submitted by - |
Sunita D’Souza |
Adapted from - |
Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells |
Contributors - |
Kyung-Dal Choi, Maxim Vodyanik & Igor I Slukvin |
Affiliation(s) - |
|
Introduction
Flowchart
Materials and Reagent Preparation
Protocol
Acknowledgements
Contributors
Hematopoietic differentiation adapted from Igor Slukvin Lab (Nature Protocols 2011)
A protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin−CD34+CD43+CD45+ multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5–19 d of culture with cytokines, depending on the cell type.
Reagents |
Company |
Cat # |
---|---|---|
hESC WA01 and WA09 |
National stem cell bank |
- |
Lentivirally reprogrammed iPSCs iPS (Foreskin) |
National stem cell bank |
- |
Transgene-free iPSCs DF-19-9-7T and 4-3-7T |
WiscBank |
iPS-DF19-9-7T / iPS-DF4-3-7T.A |
OP9 mouse bone marrow stromal cell line | ATCC | CRL-2749 |
Mouse Embryonic Fibroblasts | WiCell Research Institute | - |
Dulbecco’s modified eagle medium (DMEM), powder | GIBCO-Invitrogen | 12100-046 |
KNOCKOUT SR (KO), serum replacement for ES cell | GIBCO-Invitrogen | 10828-023 |
Serum-free medium, Stemline Hematopoietic stem cell expansion medium | Sigma Aldrich | S-0189 |
Sodium azide, NaN3 | Fisher Scientific | BP922-500 |
Ascorbic acid | SIGMA-ALDRICH | A4544 |
DMEM/nutrient mixture F-12, powder | GIBCO-Invitrogen | 12400-024 |
α-MEM basal medium, powder | GIBCO-Invitrogen | 12000-022 |
Iscove’s modified Dulbecco’s medium (IMDM), powder | GIBCO-Invitrogen | 12200-036 |
L-glutamine | CellGro | 61-030-RM |
MEM non-essential amino acid, 100× solution | GIBCO-Invitrogen | 11140 |
2-mercaptoethanol | SIGMA-ALDRICH | M7522 |
Sodium Bicarbonate | Fisher Scientific | S233-500 |
Poly (2-hydroxyethyl methacrylate) (p-HEMA) | SIGMA-ALDRICH | P3932 |
NaOH | Fisher Scientific | S318 |
Percoll, solution | SIGMA-ALDRICH | P-1644 |
PBS powdered, without calcium and magnesium | GIBCO-Invitrogen | 21600-044 |
PBS, 10× | GIBCO-Invitrogen | 70011-044 |
Gelatin from porcine skin, Type A | GIBCO-Invitrogen | G-1890 |
EDTA 0.5M, pH 8.0 | GIBCO-Invitrogen | 15575-038 |
MTG, monothioglycerol | SIGMA-ALDRICH | M-6145 |
Fetal bovine serum defined | Hyclone | SH30070.03 |
Trypsin 0.05%/EDTA 0.5mM | Hyclone | SH30236.02 |
Cell dissociation buffer, enzyme free, PBS-based | GIBCO-Invitrogen | 13151-014 |
Collagenase Type IV | GIBCO-Invitrogen | 17104-019 |
Ex-Cyte | Celliance-Millipore | 81-129-1 |
Neupogen as a human G-CSF | Amgen | |
Leukine as a human GM-CSF | Berlex | |
Human M-CSF | Peprotech | 300-25 |
Human FGF-basic | Peprotech | 100-18B |
Human Flt3-Ligand | Peprotech | 100-18B |
Human IL-1β | Peprotech | 200-01B |
Human IL-3 | Peprotech | 200-03 |
Human IL-4 | Peprotech | 200-04 |
Human IL-5 | Peprotech | 200-05 |
Human sRANKL | Peprotech | 300-01 |
Human TGF-β1 | Peprotech | 100-21 |
Human TNF-α | Peprotech | 300-01A |
1α, 25-Dihydroxyvitamin D3, Biologically active form of vitamin D3 | SIGMA-ALDRICH | D-1530 |
Trypan blue solution, 0.4% | SIGMA-ALDRICH | T8154 |
Protocol Wright stain | Fisher Scientific | 23-264980 |
Protocol Phosphate buffer solution for Wright stain,pH 6.4 | Fisher Scientific | 23-262234 |
Cytoseal 60 mounting medium | Richard-Allan Scientific-ThermoFisher | 8310-4 |
Immersion oil | SIGMA-ALDRICH | 56822 |
7-AAD; 7-Aminoactinomycin D | SIGMA-ALDRICH | A9400 |
Bovine serum albumin Fraction V | Fisher Scientific | BP1605-100 |
Ethanol | SIGMA-ALDRICH | E7023 |
Cell strainer, 40μm | BD Bioscience | 352340 |
Cell strainer, 70μm | BD Bioscience | 352350 |
MACS separation columns, LD | Miltenyi Biotec | 130-042-303 |
MACS separation columns, LS | Miltenyi Biotec | 130-042-401 |
Midi MACS separation unit | Miltenyi Biotec | 130-042-302 |
MACS Multistand | Miltenyi Biotec | 130-042-303 |
MACSmix Tube Rotator | Miltenyi Biotec | 130-090-753 |
Pre-separation filters with 30μm nylon mesh | Miltenyi Biotec | 130-041-407 |
Nalgene Disposable bottle top filter, Polyethersulfone membrane with 0.2μm pore size | Fisher Scientific | 595-4520 |
T25 Tissue culture flask canted neck with 0.2μm vented plug seal cap, 50ml, Nonpyrogenic polystyrene | BD Bioscience | 353108 |
T75 Tissue culture flask canted neck with 0.2μm vented plug seal cap, 250ml, Nonpyrogenic polystyrene | BD Bioscience | 353136 |
Tissue culture dishes, polystyrene 100×20 mm | BD Bioscience | 353003 |
Tissue culture 6well plate, Polystyrene flat bottom | BD Bioscience | 353046 |
0.5ml microcentrifuge tube, autoclavable | Fisher Scientific | 05-408-120 |
5ml Polystyrene round-bottom tube, 12×75mm, non-sterile | BD Bioscience | 352008 |
5ml Polystyrene round-bottom tube with 35μm cell strainer cap, 12×75mm | BD Bioscience | 352235 |
15ml Polypropylene Conical tubes | BD Bioscience | 352097 |
50ml Polypropylene Conical tubes | BD Bioscience | 352098 |
50ml Vacuum filtration system with 0.22μm pore size membrane | Millipore | SCGP00525 |
Serological pipet, 1ml Nonpyrogenic | Fisher Scientific | 13-678-11B |
Serological pipet, 5ml Nonpyrogenic | Fisher Scientific | 357543 |
Serological pipet, 10ml Nonpyrogenic (. Cat. no. 357551) | BD Bioscience | 357551 |
Borosilicate glass pipets, 5ml | Corning | 7077-5N |
Object marker, Cell dotter for inverted microscope | Nikon | MBW10020 |
Hemocytometer, Reichert Bright-Line counting chamber | Fisher Scientific | 02-671-5 |
9″ Pasteur pipets, Flint glass | Fisher Scientific | 13-678-6B |
Sterling Nitrile-xtra powder-free exam gloves | 53139 |
Antibody list
Name |
Flurophore |
Clone |
Company |
Catalogue # |
---|---|---|---|---|
Anti-human CD41a |
PE |
HIP8 | BD Biosciences | 555467 |
Anti-human CD45 |
FITC |
HI30 | BD Biosciences | 555482 |
Anti-human CD235a |
PE |
GA-R2 (HIR2) | BD Biosciences | 555570 |
Anti-Mouse CD29 | PE | HM beta 1-1 | Serotec | MCA2298PE |
Anti-FITC microbeads | None | None | Miltenyi Biotec | 120-000-293 |
Anti-PE microbeads | None | None | Miltenyi Biotec | 120-000-294 |
Antibodies used to analyze differentiation of hematopoietic progenitors and myeloid lineages from human pluripotent stem cells
Name |
Fluorochrome |
Clone |
Company |
Cat. no. |
---|---|---|---|---|
Anti-human CD1a |
PE |
VIT6b | CALTAG-Invitrogen | MHCD1a04 |
Anti-human CD2 |
FITC |
RPA-2.10 | BD Biosciences | 555326 |
Anti-human CD3 |
PE |
SK7 | BD Biosciences | 349201 |
Anti-human CD7 | PE | M-T701 | BD Biosciences | 555361 |
Anti-human CD10 | None | HI10a | BD Biosciences | 555375 |
Anti-human CD11b | None | VIM12 | CALTAG-Invitrogen | CD11b01 |
Anti-human CD13 | PE | TüK1 | CALTAG-Invitrogen | MHCD1304 |
Anti-human CD14 | FITC | M5E2 | BD Biosciences | 555397 |
Anti-human CD15 | FITC | VIMC6 | Miltenyi Biotec | 130-081-101 |
Anti-human CD16 | PE | 3G8 | CALTAG-Invitrogen | MHCD1604 |
Anti-human CD19 | PE | HIB19 | BD Biosciences | 555413 |
Anti-human CD34 | FITC | 581 | BD Biosciences | 555824 |
Anti-human CD41a | PE | HIP8 | BD Biosciences | 555467 |
Anti-human CD43 | FITC | 1G10 | BD Biosciences | 555475 |
Anti-human CD45 | APC | HI30 | BD Biosciences | 555485 |
Anti-human CD64 | FITC | 10.1 | CALTAG-Invitrogen | CD6401 |
Anti-human CD66b | FITC | G10F5 | BD Biosciences | 555724 |
Anti-human CD90 (Thy-1) | APC | 5E10 | BD Biosciences | 559869 |
Anti-human CD115 | PE | 61708 | R&D Systems | FAB329P |
Anti-human CD117 | APC | YB5.B8 | BD Biosciences | 550412 |
Anti-human CD123 | FITC | AC145 | Miltenyi Biotec | 130-090-897 |
Anti-human CD163 | PE | 215927 | R&D Systems | FAB1607P |
Anti-human CD235a | PE | GA-R2(HIR2) | BD Biosciences | 555570 |
DC-SIGN | FITC | DCN46 | BD Biosciences | 551264 |
HLA-DR | PE | Tü36 | CALTAG-Invitrogen | MHLDR04 |
Lactoferrin-* | PE | 3C5 | CALTAG-Invitrogen | GIC206 |
Langerin-* | PE | 343828 | R&D Systems | FAB2088P |
Major basic protein (MBP)-* | None | AHE-2 | BD Biosciences | 550843 |
Myeloperoxidase (MPO)-* | FITC | H-43-5 | CALTAG-Invirogen | GIC205 |
Anti-human TRA-1-85 | APC | TRA-1-85 | R&D Systems | FAB3195A |
Collagenase solution (1mg/ml): Add 50 mg of collagenase to 50 ml of DMEM/F-12 basal medium, and sterilize the solution by filtration using a 0.22 µm membrane filter. Keep solution at 2–8°C and use for up to one week.
0.1% Gelatin solution (w/v): Add 500 mg of gelatin to 500 ml of endotoxin-free reagent grade distilled water. Solubilize and sterilize by autoclaving for 20 min at 121°C. Store the solution at 4°C for up to 6 months. Keep sterile.
Magnetic cells sorting (MACS) buffer: MACS buffer contains 5% FBS (v/v) and 2 mM EDTA in PBS (Ca2+ and Mg2+ free). For 500 ml, add 25 ml of FBS and 2 ml of 0.5M EDTA (pH 8.0) into Ca2+ and Mg2+ free-PBS. Sterilize MACS buffer by filtration using a 0.22 µm membrane filter and keep at 2–8°C for up to 6 months. Optional: After filtration, close lid of filter unit and keep MACS buffer under vacuum for about 10–15min for degassing.
Flow cytometry buffer: Flow cytometry buffer contains 2% FBS (v/v), 0.05% sodium azide (NaN³, w/v) and 2 mM EDTA in PBS (Ca2+ and Mg2+ free). For 500 ml, add 10 ml of FBS, 0.25 g of NaN 3 and 2 ml of 0.5 M EDTA (pH 8.0) into Ca2+ and Mg2+ free-PBS. Filtrate the buffer using a 0.22 µm membrane filter and store at 2–8°C for up to 6 months.
10% pHEMA coating solution (w/v): Add 4 g of pHEMA to 40 ml of 95% ethanol containing 10 mM NaOH. Dissolve completely by continuously rotating at room temperature or 37°C overnight. Store at room temperature until needed for use. CAUTION: It is very hard to dissolve pHEMA crystals completely when they aggregate. Thus, shake the solution immediately after addition of pHEMA to prevent precipitation.
5× Percoll solution: Add 5 ml of 10× PBS (Ca2+ and Mg2+ free) to 45 ml of Percoll solution (90% Percoll and 10% 10× PBS). Store at 4°C for 6 months. For 1× Percoll working solution, dilute 10 ml of 5× Percoll solution in 40 ml of 1× PBS (1/5 dilution). Use fresh.
100× (100 mM) L-glutamine/2-mercaptoethanol solution. Add 146 mg of L-glutamine and 7µl of 2-mercaptoethanol to 10 ml of PBS (Ca2+ and Mg2+ free). Sterilize the solution by filtration using a 0.22µm membrane filter and store up to 2 weeks at 2–8°C.
1000× (100 mM) MTG solution: Add 87 µl of MTG to 10 ml of endotoxin-free reagent grade distilled water. Mix well and divide into 500 µl aliquots. Store up to 6 months at -20°C. CAUTION: MTG has high viscosity, thus pipet slowly to dispense MTG accurately.
1000× (50 mg/ml) ascorbic acid solution: Add 500 mg of ascorbic acid to 10 ml of endotoxin-free reagent grade distilled water. Dissolve completely, divide into 500 µl aliquots, and store up to 6 months at -20°C.
1mM 1a,25-Dihydroxyvitamin D3 stock solution: Dissolve 10 µg of 1a,25-Dihydroxyvitamin D3 in 24 µl of 95% ethanol (Final concentration is 0.42 µg/µl in 95% EtOH). Store the stock solution at -20°C for up to 6 months.
50× 7-AAD solution: Dissolve 1 mg of 7-AAD in 50µl of absolute methanol, then add 950 µl of 1× PBS. Final concentration is 1 mg/ml. Store the solution in an amber glass bottle or tube at 4°C protected from light. Solution can be stored for at least up to 6 months. For working solution, the stock solution (1 mg/ml) is diluted with flow cytometry buffer to 20 µg/ml concentration. Use 10 µl for staining.
0.1% BSA/PBS solution: Dissolve 25mg of Bovine serum albumin Fraction V in 25ml of PBS (Ca2+ and Mg2+ free). Sterilize the solution by filtration using a 0.22µm membrane filter and store for up to 6 months at 2–8°C.
Reconstitution of cytokines: Centrifuge vials at maximum speed for 1 min to precipitate lyophilized pellet prior to opening vials. Reconstitute cytokines according to the product information provided by manufacturer. Dilute with 0.1% BSA/PBS solution for working concentration and store at -80°C until needed for use.
Gelatin-coated 10 cm culture dish and 6 well tissue-culture plate: Add 7–8 ml of autoclaved gelatin solution to a 10 cm culture dish or 2 ml to each well of a 6 well tissue-culture plate. Allow the gelatin solution to cover the entire plastic surface and incubate for at least 3 hrs at 37°C in an incubator. Dishes and 6-well plates containing gelatin solution can be stored for up to several days at 37°C in CO2 incubator. Do not allow the wells to dry. Before use, aspirate the gelatin solution from the dish.
Reconstitution of cytokines
Cytokines |
Product state |
Reconstitution |
Working dilution |
Storage |
---|---|---|---|---|
Lyophilized pellet |
Concentrated aqueous solution |
- | 100 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Leukine as a human GM-CSF |
Concentrated aqueous solution |
- | 100 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human FGF-basic |
Lyophilized pellet |
1 mg/ml with 5mM Tris (pH7.6) |
100 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human Flt3-Ligand | Lyophilized pellet | 1 mg/ml with distilled water |
100 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human IL-1β | Lyophilized pellet | 100 μg /ml with distilled water |
10 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human IL-3 | Lyophilized pellet | 100 μg /ml with distilled water |
10 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human IL-4 | Lyophilized pellet | 1mg/ml with distilled water |
100 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human IL-5 | Lyophilized pellet | 100 μg /ml with distilled water |
10 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human M-CSF | Lyophilized pellet | 100 μg /ml with distilled water |
10 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human sRANKL | Lyophilized pellet | 100 μg /ml with distilled water |
10 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human TGF-β1 | Lyophilized pellet | 50 μg/ml with 10 mM citric acid solution (pH 3.0) |
5 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
Human TNF-α | Lyophilized pellet | 1mg/ml with distilled water |
50 μg/ml in 0.1% BSA/PBS solution |
Aliquot 100 μl into autoclaved 0.5 ml tubes and store at −80°C until needed for use. |
CRITICAL: Perform entire procedure in a sterile biosafety cabinet. Read the product information sheet carefully before preparation of working aliquots of all cytokines.
Medium composition
All basal medium, α-MEM, DMEM, DMEM/F-12, and IMDM should be prepared freshly from powder according to the manufacturer’s instructions, sterilized by filtration using a 0.22μm membrane filter and stored for up to 2 months at 2–8°C.
Figures:
MEF growth medium |
Volume |
Final concentration |
---|---|---|
DMEM |
445 ml |
|
FBS |
50 ml |
10% |
100× NEAA solution (10 mM) |
5 ml |
100 μM |
Sterilize the medium by filtration using a 0.22 μm membrane filter and store for up to 3 weeks at 2–8°C |
hESC culture medium (250 ml) |
Volume |
Final concentration |
---|---|---|
DMEM/F-12 |
195 ml |
78% |
KO serum replacement |
50 ml |
20% |
100× NEAA solution (10 mM) |
2.5 ml |
100 μM |
L-glutamine/2-mercaptoethanol solution (100 mM) | 2.5 ml | 1 mM |
Basic FGF (100 μg/ml) for hESCs culture | 10 μl | 4 ng/ml |
Sterilize the medium by filtration using a 0.22 μm membrane filter and store for up to 2 weeks at 2–8°C. |
hiPSC culture medium |
Volume |
Final concentration |
---|---|---|
DMEM/F-12 |
195 ml |
78% |
KO serum replacement |
50 ml |
20% |
100× NEAA solution (10 mM) |
2.5 ml |
100 μM |
L-glutamine/2-mercaptoethanol solution (100 mM) | 2.5 ml | 1 mM |
Basic FGF (100 μg/ml) | 10 μl | 10 ng/ml |
Sterilize the medium by filtration using a 0.22 μm membrane filter and store for up to 2 weeks at 2–8°C. CRITICAL: Note that the basic FGF concentration for hiPSC maintenance should be 2.5 times higher than for hESCs to lessen the effect of variations in MEF quality on culture of hiPSCs. |
Mouse OP9 bone marrow stromal cells culture medium |
Volume |
Final concentration |
---|---|---|
α-MEM |
200 ml |
80% |
FBS |
50 ml |
20% |
Sterilize the medium by filtration using a 0.22 μm membrane filter and store for up to 2 weeks at 2–8°C. |
Differentiation (hPSC/OP9 coculture) medium |
Volume |
Final concentration |
---|---|---|
α-MEM |
450 ml |
90% |
FBS |
50 ml |
10% |
1000× MTG solution (100 mM) | 500 μl | 100 μM |
Sterilize the medium by filtration using a 0.22 μm membrane filter and store for up to 2 weeks at 2–8°C. Optional: Ascorbic acid solution (50 mg/ml) can be added to achieve 50 μg/ml final concentration. |
Multipotent myeloid progenitor expansion medium |
Volume |
Final concentration |
---|---|---|
Differentiation (hPSC/OP9 coculture) medium |
- |
|
GM-CSF (100 μg/ml) |
1/500 dilution |
200 ng/ml |
Volume of the medium will vary depending on input cell number. In this protocol, we use 1 ml of medium for ~1×106 cocultured cells. The complete medium is prepared freshly and GM-CSF is added right before use. |
Neutrophil differentiation medium |
Volume |
Final concentration |
---|---|---|
IMDM with 20% FBS medium |
- |
|
G-CSF (100 μg/ml) |
1/1000 dilution |
100 ng/ml |
Volume of the medium will vary depending on input cell number. In this protocol, we use 1ml of medium for 2×104 cells. The complete medium is prepared freshly and all cytokines and supplements are added right before use. Eosinophil differentiation medium |
Eosinophil differentiation medium |
Volume |
Final concentration |
---|---|---|
IMDM with 20% FBS medium |
- |
|
IL-3 (10 μg/ml) |
1/1000 dilution |
10 ng/ml |
IL-5 (10 μg/ml) | 1/2000 dilution | 5ng/ml |
Volume of the medium will vary depending on input cell number. In this protocol, we use 1ml of medium for 2×104 cells. The complete medium is prepared freshly and all cytokines and supplements are added right before use. |
DC differentiation medium |
Volume |
Final concentration |
---|---|---|
Serum free medium (StemLine, SIGMA-Aldrich) |
- |
|
GM-CSF (100 μg/ml) |
1/5000 dilution |
20 ng/ml |
IL-4 (100 μg/ml) | 1/5000 dilution | 10 ng/ml20 ng/ml |
TNF-α (50 μg/ml) | 1/20000 dilution | 2.5 ng/ml |
Ex-Cyte | 1/500 dilution | 2 μl/ml |
Volume of the medium will vary depending on input cell number. In this protocol, we use 1 ml of medium for 105 cells. The complete medium is prepared freshly and all cytokines and supplements are added right before use. |
LC differentiation medium |
Volume |
Final concentration |
---|---|---|
Serum free medium (StemLine, SIGMA-Aldrich) |
- |
|
GM-CSF (100 μg/ml) |
1/5000 dilution |
20 ng/ml |
TGF-β1 (5μg/ml) | 1/2000 dilution | 2.5ng/ml |
TNF-α (50μg/ml) | 1/50000 dilution | 1ng/ml |
Ex-Cyte | 1/500 dilution | 2 μl/ml |
Volume of the medium will vary depending on input cell number. In this protocol, we use 1ml of medium for 105 cells. The complete medium is prepared freshly and all cytokines and supplements are added right before use. |
IMDM medium containing 10% FBS |
Volume |
Final concentration |
---|---|---|
IMDM |
225ml |
90% |
FBS |
25ml |
10% |
Sterilize the medium by filtration with 0.22 μm membrane filter and keep sterile at 2–8°C for up to 2 weeks. |
Macrophage differentiation medium |
Volume |
Final concentration |
---|---|---|
IMDM with 10% FBS medium |
- |
|
M-CSF (10 μg/ml) |
1/500 dilution |
20 ng/ml |
IL-1β (10 μg/ml) | 1/1000 dilution | 10 ng/ml |
Volume of the medium will vary depending on input cell number. In this protocol, we use 1ml of medium for 105 cells. The complete medium is prepared freshly and all cytokines and supplements are added right before use. |
Osteoclast progenitor expansion medium |
Volume |
Final concentration |
---|---|---|
Differentiation (hPSC/OP9 coculture) medium |
- |
|
GM-CSF (100 μg/ml) |
1/2000 dilution |
50 ng/ml |
1α, 25-Dihydroxyvitamin D3 (1mM) | 1/5000 dilution | 200nM |
Volume of the medium will vary depending on input cell number. In this protocol, we use 1 ml of medium for 2×104 cells. The complete medium is prepared freshly and all cytokines and supplements are added right before use. |
Osteoclast maturation medium |
Volume |
Final concentration |
---|---|---|
Differentiation (hPSC/OP9 coculture) medium |
- | |
GM-CSF (100 μg/ml) |
1/2000 dilution |
50 ng/ml |
1α, 25-Dihydroxyvitamin D3 (1mM) | 1/5000 dilution | 200nM |
RANKL (10 μg/ml) | 1/1000 dilution | 10 ng/ml |
Volume of the medium will vary depending on input cell number. In this protocol, we use 1ml of medium for 5×104 cells. The complete medium is prepared freshly and all cytokines and supplements are added right before use. |
IMDM medium containing 20% FBS |
Volume |
Final concentration |
---|---|---|
α-MEM |
200 ml |
80% |
FBS |
50 ml |
20% |
Sterilize the medium by filtration using a 0.22μm membrane filter and keep sterile at 2–8°C for up to 2 weeks. Diluted Buffer for Wright Stain Procedure Mix 30 ml of Protocol phosphate buffer pH 6.4 with 100 ml of deionized water. Store at room temperature for up to 6 months. |
MEFs preparation for Human ES/iPSC culture TIMING 24 hours
Images
Hematopoietic differentiation and production of mature myeloid cells from human pluripotent stem cells
Kyung-Dal Choi, Maxim Vodyanik & Igor I Slukvin
Nature Protocols 6, 296–313 (2011) doi:10.1038/nprot.2010.18, Published online 17 February 2011
This page was last modified on October 18, 2012