ES-MN differentiation Protocol

From Wichterle et al.

Wild-type (MM13 or W9.5) or HB9::GFP transgenic mouse-derived (HBG3) ES cells were grown on mouse embryonic fibroblasts in ES cell medium. ES cell colonies were partially dissociated after 2 days and cultured in DFK5 medium (see Supplemental Data at http://www.cell.com/cgi-/content/full/110/3/385/DC1). Medium was replaced at 2 days and supplemented with retinoic acid (100 nM to 2 µM) (Sigma), Sonic hedgehog (Shh-N; 300 nM) (Curis Inc.), hedgehog agonist Hh-Ag1.3 (1–1000 nM) (Curis Inc.), or hedgehog antibody (5E1, 30 µg/ml), and EBs were cultured for 2–5 days. For some experiments, ES cells were grown on a PA6 cell monolayer (Riken Cell Bank, Japan) (Kawasaki et al., 2000) in DFK5 medium alone, or supplemented with 2 µM RA. 

HBG3 ES cell-derived EBs were dissociated (Papain, Worthington) 4 days after induction, FACS-sorted by eGFP expression, plated on Petri dishes or Terasaki wells coated with matrigel (BD), and cultured in Supplemented F12 medium (see Supplemental Data). GDNF, NT3, CNTF, and BDNF (10 ng/ml, R&D Systems) were included in selected experiments. 

Todd’s notes

  1. Plate feeders on gelatin coated dishes at least 4 hours before splitting or thawing ES cells.
  2. Use ES-FCS media on day of splitting ES cells or when thawing cells, FCS stops trypsin reaction and helps cells to attach.
  3. The day after splitting, change media with ES-KOSR media, which is the media the cells were derived with.
  4. Split cells every 2 days approx. 1:5.
  5. When differentiating cells, split one confluent dish of good looking ES colonies 1:5 onto bacterial grade dishes with mDiff media:
    6. It may be beneficial to first plate onto regular cell culture treated dish for 1-2 hours so that feeders attach, then you can collect the ES cells by pipetting up and down, ES cells should only be loosely attached if attached at all while feeders should be attached well.
  6. Change mDiff media after two days with fresh mDiff media containing shh 200ng/ml-1000ng/ml, and 1 µM RA (stock concentration of 1mg/ml in EtOH good for couple months at -20°C):
    7. This is done by allowing the embryoid bodies to settle down in a 15 ml conical tube for a few minutes, aspirate media, add fresh media (don’t pipette vigorously or you will break up EB’s).
  7. Change media as described in 6. after two days
  8. At day 5, you should start to see green cells, even more at day 6. At this point the cells can be dissociated with papain, or plated onto laminin coated dishes as EBs (use high laminin for this purpose, similar to explants):
    9. In our hands the axons grow out nicely by two days, if cells are dissociated, MNs only survive for two days (maybe they need supporting cells for long term survival or perhaps just right cocktail of growth factors).
 

ES-FCS Media

80 ml ES qualified FCS (Chemicon)
5 ml 1 M HEPES (Invitrogen)
5 ml  Penn/strep/fungizone
5 µl BME
5 ml NEAA (Invitrogen)
5ml L-glutamine
500 µl LIF (ESGRO)
400 ml DMEM  (we have used many types, use high glucose, can use ES specific DMEM from Invitrogen)

ES-KOSR Media

Same as above except use 100 ml KOSR instead of FCS 

Todd’s mDiff Media

250 ml DMEM/F12
200 ml DMEM (high glucose)
5 ml L-glut
5 ml NEAA
5 ml Penn/strep with fungizone
5 ml N-2 supplement (Gibco)


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