Embryoid body (EB) medium:
To prepare 600 mL of EB medium:
488 mL Iscove’s DMEM, 1X (Corning Cellgro)
90 mL (15%) FBS (Hyclone, or Omega Lot #101331)
6 mL (1%) Pen/Strep (Invitrogen)
6 mL (1%) Glutamax-1 (Invitrogen)
9 μL (0.0015%) 2-mercaptoethanol (Sigma-Aldrich)
As outlined in the EB Generation stepwise protocol below, for days 0-4, use EB medium without cytokines.
At day 4 and 7 of EB differentiation, supplement EB medium with 300 ng/mL of SCF (Invitrogen), 50 ng/mL of FLT3-l (Peprotech), and 10 ng/mL BMP4 (Invitrogen)
At day 10 of EB differentiation, supplement EB medium with 300 ng/mL of SCF (Invitrogen) and 50 ng/mL of FLT3-l (Peprotech)
EB Media without cytokines can be stored refrigerated at 4C for 1 months, and 2 weeks with cytokines added.
hESC Collagenase Solution:
To prepare a 1X solution of collagenase:
For every mL of DMDM/F12 1X (Corning Cellgro)
Dissolve 1 mg of Collagenase Type IV (Invitrogen)
EB Dissociation Medium:
To prepare 10 mL of a 2X solution of EB dissociation media:
PBS 1X to volume
2 mL (10%) FBS (Omega)
20 mg Collagenase Type A (Worthington)
5 mg Dispase (Gibco)
83.3 ug DNase (Sigma)
Media can be stored refrigerated at 4C for 1 week.
Human Hematopoietic Stem Cell (HSC) Medium:
To prepare 50 mL of HSC medium:
39 mL MEM alpha 1X (Gibco)
10 mL (20%) FBS (Omega)
500 μL (1%) Pen/Strep (Invitrogen)
500 μL (1%) Glutamine (Invitrogen)
25 ng/mL each of hSCF (Invitrogen), hTPO (Peprotech), and hFLT3-l (Peprotech)
HSC medium can be stored refrigerated at 4C for 2 weeks.
Stock Dilutions of AM580:
To prepare 100X dilutions:
AM580 (Tocris) was dissolved first in DMSO (Corning Cellgro) and diluted 1:500 in PBS 1X (Corning Cellgro) to make a 100X dilution and applied to wells for a final concentration of 0.2 uM. Addition of DMSO to control wells was at a final dilution of 1:25000.
- Grow 60 10 cm plates of H1 ESCs to ~80% confluency
- Aspirate media from plates.
- Wash once with 5 ml of PBS 1X (Corning Cellgro) per plate
- Incubate each plate for 10 minutes with 3 mL of hESC collagenase solution in a 37C incubator
- Following incubation, run the StemPro EZ Passage Disposable Stem Cell Passaging tool once gently, but firmly, around the entire surface areas of the plate to detach cells.
- Collect the cell clumps by washing the plates three times each with 3 mL of 5% FBS (Omega) in PBS 1X (Corning Cellgro), scraping gently to help cells detach further.
- Collect washes with cells in 50 mL conical tubes and spin at 1300 rpm for 5 min. at 10C.
- Aspirate supernatant after the spin and resuspend pellet in 15 mL 5% FBS in PBS 1X per conical collection tube.
- Pool the pellets into as many conical tubes as necessary and spin again to pellet at 1300 rpm for 5 min. at 10C.
- Aspirate supernatant and resuspend the pellet in EB medium.
- Allocate cells from up to 8 of the 10 cm H1 ESC plates per Ultralow attachment flask (Corning) or 10 plates per Ultralow 6-well plate (Corning) and add to volume of 40-50 mL of EB medium per flask or 4 mL per well of 6-well plate
- On day 4, lift cells into 50 mL conical tubes and allow them to settle by gravity for 30 min. at room temperature.
- Carefully aspirate as much of the supernatant from the loose pellet as possible.
- Resuspend in fresh EB media supplemented with 300 ng/mL of SCF (Invitrogen), 50 ng/mL of FLT3 (Peprotech), and 10 ng/mL BMP4 (Invitrogen) and redistribute with appropriate volumes (as in Step 11) to each flask or well of 6-well plate.
- On day 7, repeat steps 12-14.
- On day 10, repeat steps 12-14, with the exception that BMP4 is not added to the fresh EB media.
- Leave in incubator until day 14.
EB Processing and CD34+ cell Isolation
- On day 14, lift cells into 50 mL conical tubes.
- Wash with 10 mL/flask or 1 mL/well of PBS 1X and collect wash in same tubes.
- Spin at 1300 rpm for 5 min. at 10C.
- Carefully aspirate supernatant and resuspend pellet in a 1:1 ration by volume in PBS 1X with the EB Dissociation Media (2X).
- Shake at 200 rpm at 37C for 15 min.
- Filter cells through a 70 μm nylon mesh sterile cell strainer (Fisher Scientific) into a 50 mL conical tube and count.
- Place the mesh strainer in a sterile 6-well plate and add 6 mL of accutase to cover. Incubate for 5 min. in the 37C incubator and wash the filter into the same 50 mL conical tube with the accutase followed by 10 mL PBS 1X to further dissociate any other cell clumps.
- Pass the strained cells through the Dead Cell Removal Kit (Miltenyi Biotec) to collect live cells.
- Isolate CD34+ cells obtained from the live cell fraction of the Dead Cell Removal Kit (Miltenyi Biotec) following instructions of the CD34 MicroBead Kit UltraPure, human (Miltenyi Biotec) and count.
AM580 treatment of EB derived CD34+ cells on stroma co-culture
- Irradiate OP9-M2 cells (2000 rad)
- Plate irradiated OP9-M2 at a density of 50,000 cells per well of 24-well tissue-culture treated plate in 500 uL of HSC media.
- Allow stromal cells to settle for at least 4 hours and use irradiated plates within 3 days of irradiation.
- Following at least 4 hours post-stromal cell plating, add 50,000 to 100,000 EB derived CD34+ cells in 500 uL of HSC media to each stroma-layered well.
- To control wells, add 10 uL of 100X DMSO dilution. To AM580 wells, add 10 uL of 100X AM580 dilution.
- Every 2-3 days, carefully remove 500 uL of the media from each well without disturbing cell layer on the bottom, and replace with 500 uL of fresh HSC media containing DMSO at a final dilution of 1:25000 or AM580 at 1:500000 per well.
- On day 6, carefully remove 900 uL of media from each well without disturbing cell layer on the bottom of the wells. Replace with 900 uL of fresh HSC media.
- On day 7, carefully remove 900 uL of media from each well without disturbing cell layer on the bottom of the wells. Replace with 900 uL of fresh HSC media.
- Continue with half media changes (removing 500 uL of media from each well and replacing with 500 uL fresh HSC media) every 2-3 days.
- To lift cells at any time for analysis, carefully remove all media from each well and strain through 70 uM cell strainer into 15 mL conical collection tubes. Incubate 5 min. at 37C with 200 uL/well of accutase. Add 500 uL of PBS 1X and strain into collection tubes. Wash with 1 mL/well of PBS 2X and strain washes through cell strainer into collection tubes.
- Spin at 1300 rpm for 5 min. at 10C.
- Aspirate supernatant and continue with tissue culture in same conditions described above but without the AM580 treatment, to assess HSPC expansion, or proceed immediately to FACS analysis or further experiments to assess HSC molecular and functional properties.