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Stem Cell Biology

Hematopoietic Stem Cells Protocols

Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Cell Layer
Culture of hES Cells
Induction of hES Cell Differentiation
Expansion of hES-blast Colony (BC) Cells

Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Cell Layer

  • Eviscerate 12.5 dpc ICR or CD-1 mouse embryos leaving the heads on; rinse with PBS (P7059) and mince with scissors for approximately 3–5 minutes in a dry 60 mm Petri dish.
  • Add 5 ml of 37 °C trypsin (T5266) per mouse, constantly pipetting with a 5 ml tissue culture pipette for 5 minutes; transfer to a 50 ml tube with 20 ml of MEF growth medium (DMEM medium (D5796) supplemented with 10% FBS (F6178), GlutaMax, Penicillin and streptomycin (G6784) and centrifuge at approximately 1000 rpm for 5 minutes.
  • Remove the supernatant and resuspend the pellet in MEF growth medium.
  • Plate the suspension onto gelatin-coated plates at a density of 1.5 embryos per 150 mm plate (P0).
  • Expand MEFs once (1:5 split) usually within 1–2 days and freeze (P1).
  • Add 10 µg/ml Mitomycin C (M4287) to the media of a confluent plate of MEFs and incubate at 37 °C for 3 hours.
  • Harvest by trypsinization and plate in MEF growth medium at a density of 50–60 thousand cells/cm2.
  • Pre-treat plate with 0.1% gelatin (G1393) for at least 4 hours, then plate 7.5 X 105 cells per well of six-well plate. Incubate at 37 °C with 5% CO2 for at least 24 hours. The MEF feeder layer is now ready for plating hES cells.

 


Culture of hES Cells

  • Add 0.5 ml 0.05% trypsin-0.53 mM EDTA (T4049) to one well (hES cells) of six-well plate, incubate at 37 °C for 3–4 min, then pipette to produce smaller cell clumps (2–5 cells).
  • Collect cells by adding 3 ml of hES cell growth medium (KO-DMEM medium with 10% Plasmanate, 10% Knockout Serum Replacement (S0638), 10 ng/ml of human LIF (L5283), 8 ng/ml of human bFGF (F0291). 1X non-essential amino acid (M7145), 1X Glutamax-I, and
    1X β-mercaptoethanol (M7522) and spin down for 4 min at 1000 rpm.
  • Resuspend cells in 9 ml of hES cell growth medium and re-plate in 3 wells of six-well plate with pre-formed MEF feeder.
  • Change half (1.5 ml) of the medium every 24 hours until the cells reach 70–80% confluence.

 


Induction of hES Cell Differentiation

  • Collect hES cells as above, and count cell numbers. Usually one well of 80% confluent, undifferentiated hES cells will generate approximately 2 million cells.
  • Resuspend cells from one well in 3 ml Stemline II medium (S0192) supplemented with 50 ng/ml of VEGF (V4512) and 50 ng/ml of BMP-4 (B2680), plate in one well of six-well ultra low plate, and incubate at 37 °C with 5% CO2. EBs will be formed in the first 24 hours.
  • Replace half the medium (1.5 ml) with fresh Stemline II medium (S0192) supplemented with 50 ng/ml of VEGF (V4512), 50ng/ml of BMP-4 (B2680), 40ng/ml of SCF (S7901), 40ng/ml of Tpo (T4184) and 40ng/ml of FLT3 (F3422) ligand, and continue incubation.
  • Collect EBs after 84 hours and the dissociate EBs by 0.05% trypsin-0.53 mM EDTA for 2–5 min. Prepare single cell suspension by passing through 22G needle 3–5 times and a 40 µm strainer.
  • Count cell numbers and resuspend cells in Stemline II medium at 2–5 X 106 cells/ml.

 


Expansion of hES-blast Colony (BC) Cells

  • Mix single cell suspension (0.1 ml, 2 to 5 X 105 cells) with 2–3 ml of BC growth medium (BGM, 1.0% methylcellulose in Isocve’s MDM (I6529), 1–2% bovine serumalbumin (A9418), 0.1 mM
    2-mercaptoethanol (M7522), 10 µg/ml rh-insulin, 200 µg/ml iron saturated human transferring (T0665) 20 ng/ml rh-GM-CSF (G0282), 20ng/ml rh-IL-3 (I1646), 20 ng/ml rh-IL-6 (I3268), 20 ng/ml rh-G-CSF (G8160), 3 to 6 units/ml rh-Epo (E5627), 50ng/ml rh-SCF (S7901), 50 ng/ml rh-VEGF (V7259), 50 ng/ml rh-BMP-4 (B2680), 50 ng/ml of Tpo (T1568) and FL) with brief vortex, and stand for 5 min.
  • Transfer the BGM-cell mixture to one well of six-well ultra low plate by using a syringe (3 ml) attached with a 16G needle, and incubate at 37 °C with 5% CO2.
  • Usually BCs are visible at 3 days, and after 4–6 days, grape-like hES-BCs can be easily identified under microscopy. After 6–7 days, BCs can be picked up using a mouth-glass capillary.

 

Protocols taken in part from:

  • Lu, S. J. et al. Generation of functional hemangioblasts from human embryonic stem cells. Nature Methods 2007, 418, 501–509.

 

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