Plating Mouse embryonic fibroblasts (MEFs):
• Irradiated MEFs from commercial sources or home-made MEFs can be used
• MEF culture medium: DMEM-high glucose with 10% FBS
• Plate MEFs on 0.1% gelatin (autoclaved, made in PBS) coated plates (gelatin coating is done for 20 min at 37 0C)
• Plating density for 1 well of a 6-well plate is 0.2 x 106 MEFs per well, plating density for one 10 cm plate is 1 x 106 MEFs per plate
Human ES/iPS culture on irradiated MEFs
ES cell medium:
DMEM/F12 (# 11330-032, Gibco) containing 20% KO-SR (# 10828-010, Gibco), 2 mM L-Glutamine (# 35050-038), 0.1 mM non-essential amino acids (# 11140-035), 0.1 mM beta-mercaptoethanol (# 31350-010) (all from Life Technologies) and 10 ng/ml basic FGF (R&D systems).
Culturing and splitting human ES cells on MEFs:
• Mark differentiated and dark centered ES colonies, remove these colonies with aspirator, later remove all media
• Wash once with PBS
• Add Collagenase type IV (# 17104019, Life Technologies) at final concentration of 1 mg/ml prepared in DMEM/F12
• Incubate the cells with Collagenase at 37 0C, 5 min
• Aspirate collagenase, wash once with ES media
• Add 1 ml ES media, scrape cells with scraper, triturate gently thrice with a 1 ml pipette and distribute* equally on MEFs.
*- After splitting, ES cells become confluent usually in 6-7 days
Embryoid body formation and Hematopoietic Differentiation:
To obtain at least 8 wells of a 6-well plate during ES-to-blood differentiation, four 10cm plates of ESCs are a good start.
• Remove media from ESCs (10 cm dishes), wash once with PBS
• Add Dispase, 5 ml / plate (Dispase # 17105041, Life Technologies), at final concentration of 0.5 mg/ml in DMEM/F12
• Incubate cells at 37 0C for 30 min (tap the plate at every 10 min, 10 min, 5 min, 5 min)
• Collect cells in a 50 ml tube
• Add 15 ml DMEM/F12, let cells get washed and sink
• Wash one more time in the same way (this removes Dispase)
• Add 10 ml Mesototal to the cells
• Plate the detached ESCs in ultra-low attachment 10 cm dishes (Corning), (this is Day 0 of the blood differentiation protocol)
• Until Day 8, these EBs will be in suspension culture in Mesototal medium, for mesoderm commitment. On Day 1 and 2, there will be complete media change (100% Mesototal) to get rid of dead cells and debris. On day 4 and 6, there will be 50% Mesototal media change to supply fresh growth factors to the developing EBs.
• At day 8, EBs are plated on 0.08 μg/mm2 Matrigel (BD Biosciences) in 6-well tissue culture plates and 50% Mesototal media was exchanged of fresh media on day 10 and 12. Further differentiation toward hematopoietic cells was carried out until day 14 in MesoTotal medium.
Cyclic AMP induction:
Application of fresh medium and cAMP induction with 10 μM forskolin (Stemgent) and 500 μM IBMX (3-isobutyl-1-methylxanthine; Santa Cruz Biotechnology) was carried out at days 10 and 12 of differentiation. Forskolin specifically increases intracellular cAMP levels by activating the catalytic subunit of adenylyl cyclase 16. IBMX is a phosphodiesterase (PDE) inhibitor that specifically prevents PDE-mediated dephosphorylation of cAMP to AMP 17. Thus, combining forskolin with IBMX elevates intracellular cAMP by increasing cAMP production and preventing its dephosphorylation.
EB harvest at D14 for FACS or CFU assay:
• Collect media with the EBs in 15 ml tube, let the EBs sink and aspirate media
• Wash EBs twice with 1 ml PBS
• Add 1 ml Tryple (# 12604-013, Life Technologies), incubate 5 min, triturate 3 times
• Another 5-8 min incubation, triturate 10 times, collect cells
• Add DMEM+10%FCS, wash and collect rest of the EBs
• Add the collected cells/EBs slowly in a 5 ml syringe fitted with a 21G needle (BRAUN, Sterican, # 4665503; 0.80x50 mm, 21G x 2’’)
• Pass through the syringe 4 times (inclined to tube’s wall)
• Push cells through cell strainer (BD # 340632; 50 µm cup filcons) fitted on top of a new 15 ml tube
• Flush the cell strainer with 3 ml PBS+2%FCS to collect any remaining cells
• Spin down, resuspend in 1 ml PBS+2%FCS, count cells
• Proceed for FACS or CFU assay