Differentiation of Mouse ES Cells into Motor Neurons DFNB Medium

Day I
Thaw a vial of ES cells and culture in gelatinized T25 flask in ES medium
Day II
Change medium
Trypsinize ES cells (2.5 ml of 0.05% Trypsin/EDTA, Gibco) (~5 minutes in 37deg) and break the colonies into single cells. Add 5 ml of DFNB medium, transfer cells into 15 ml falcon tube and spin for 5 minutes at 800 rpm in table top Beckman. Remove SN, resuspend the pellet in 10 ml of DFNB medium. (optional: plate cells on gelatinized T75 in ES medium and let fibroblasts adhere for 30-60 minutes. Collect ES cells, spin them and resuspend in 10ml of DFNB medium). Repeat centrifugation to wash off remaining trypsin. Plate ES cells in 60mm bacteriological Petri dishes in 5 ml of DFNB medium (plate ~106 cells per dish).
Day IV
Transfer EBs into 15 ml falcon tube and let them settle down by gravity ~15-30 minutes (or pulse spin the tube). Resuspend EBs in fresh DFNB medium and replate them in a new 60 mm Petri dish.
Day V/Day 0
Swirl the Petri dish and transfer the EBs, which collect in the center of the dish into a 15 ml Falcon tube. Let EBs settle by gravity remove most of the medium and add 1ml of fresh DFNB medium. Distribute EBs using a wide orifice pipette tip into 3-5 60mm Petri dishes containing DFNB medium supplemented with RA (100nM-1µM; stock: 1mM in DMSO keep in dark at –20C, Sigma) and HhAg1.3 (100nM-1µM; stock: 10mM at –80C, compound 186 from Curis Inc.). Change medium whenever turns yellow (usually day 3-4).
Day 2
EBs contain many Sox1 expressing cells (Neural stem cell stage)
Day 3
Peak of spinal cord progenitor markers (Nkx6.1, Olig2) (Neural progenitor stage)
Day 5
Postmitotic pan- and motor neuron-specific markers (NeuN, HB9, Isl1/2, Lim3) (Postmitotic stage)
  ES Medium (500ml):
400 ml
DMEM for ES cells (Specialty Media, EmbryoMax)
75 ml
FCS (HyClone, Lot AHM9368)
5 ml
L-glutamine (Gibco)
5 ml
Pen-strep (Gibco)
5 ml
NEAA (Specialty media)
5 ml
Nucleosides (Specialty media)
5 ml
2-ME (diluted 25ul in 35 ml PBS) (Sigma)
50 ul
LIF (final 1000u/ml, Chemicon ESGRO)
  DFNB Medium (500ml):
125 ml
DMEM (Specialty Media, EmbryoMax)
125 ml
F12 (Gibco)
250 ml
Neurobasal medium (Gibco)
5 ml
Penicillin/Streptomycin (Gibco)
5 ml
B27 supplement (Gibco)
3.75 ml
200 mM L-Glutamine (Gibco)
3.6 ul
2-mercaptoethanol (final 0.1 mM, Sigma)
  Store in dark in cold room.