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Three Lineage Differentiation

The following protocols are for verification of pluripotency only and do not claim to be highly efficient or to generate pure populations for subsequent study. For all protocols, pluripotent cells should be grown as colonies on Matrigel (1.25%) in mTeSR. One plate should suffice—2 wells per differentiation protocol. High quality cultures with little spontaneous differentiation are essential for success.

Independent verification of these protocols is important so we would appreciate feedback if you have tried them and found them to work or not in your hands. Please contact us at with your comments.

Mesoderm—Beating Cardiomyocytes; James Thomson (modified)

  1. Wash colonies once with D-PBS and add 1ml dispase (1-2mg/ml) per well of a 6-well plate
  2. Incubate at 37oC until colonies detach intact
  3. Remove colonies and transfer to a 15ml tube, wash each well with 1-2ml DMEM:F12 and add wash to the 15ml tube
  4. Allow to sediment for 3 minutes, aspirate down to the pellet and add 5ml DMEM:F12
  5. Repeat the sediments twice more
  6. Remove wash, resuspend (without trituration) in Alpha-MEM (Invitrogen #12571–063) containing 20% FBS and transfer to a Corning low-attachment dish (we use 1 well of a 6–well plate per well of starting material)–Day 0
  7. Change the medium by sedimenting on Day 2
  8. Pre-coat tissue culture plasticware with 0.1% gelatin in water for at least 2 hours, preferably overnight
  9. On Day 4 change the medium by sedimenting and transfer to the gelatin-coated dish. May need to consolidate into 1 well of a 6-well plate
  10. Change the medium every 2 days. 'Beaters' should appear as early as Day 8 but may not appear until Day 14.

Endoderm—Liver; Si-Tayeb (2010) Hepatology 51 (1) 297–305

  1. Change TeSR1 medium to RPMI containing B27 supplement, glutamine and 100ng/ml Activin A—Day 0
  2. Change the medium after 2-3 days
  3. On day 5 change the medium to RPMI containing B27 supplement, glutamine and 20ng/ml BMP4 + 10ng/ml bFGF. This should be done at low oxygen (5%) but will work with lower efficiency at regular oxygen levels.
  4. Change the medium after 2-3 days
  5. On day 10 change the medium to RPMI containing B27 supplement, glutamine and 20ng/ml HGF. This should also be done at low oxygen (5%) but will work with lower efficiency at regular oxygen levels.
  6. On day 15, change the medium to Lonza Hepatocyte Culture Medium containing 20ng/ml oncostatin M
  7. Change medium after 2-3 days.
  8. Fix and stain with HNF4a (Cell Signaling Technology 1:200) and Albumin (Cedarlane 1:5000) overnight.

Ectoderm—Generation of Neural Progenitor Cells from hPSCs Using STEMdiff Neural Induction Medium; Stem Cell Technologies. Neuronal Differentiation; Cohen (2007) Current Protocols in Cell Biology
23.7.1–23.7.20, (modified O. Kozhich, B. Mallon)

Manufacturer's instructions should be followed—please refer to the user manual. A brief description and subsequent differentiation protocol is provided.

  1. Prepare an AggreWell 800 plate with Neural Induction Medium (NIM) containing 10uM Rock inhibitor (Y27632, Calbiochem) according to instructions.
  2. Wash colonies once with D-PBS and incubate for 10 mins with Accutase at 37oC. Remove with 5ml/well of DMEM/F12 and transfer to a 15ml tube.
  3. Spin at 200g for 5 mins, resuspend in NIM containing Rock inhibitor and count. Adjust the cell density to between 1.5 and 6x106/ml.
  4. Add 1ml of cell suspension to the appropriate number of wells of the AggreWell 800 plate and spin to generate EBs of between 5000 and 20000 cells.
  5. Feed daily by removing 1.5ml of medium carefully using a pipet (not aspirating) for 4 days.
  6. Precoat plates with 20ug/ml poly-D-ornithine in water (overnight @ 37oC) and 10ug/ml laminin (Sigma L-2020) in PBS (overnight @ 37oC)
  7. On day 5, harvest the EBs according to the instructions. Alternatively, EBs may be flushed and wells washed using 2ml pipets; combine the washes and allow the EBs to sediment for 3-5 minutes.
  8. Transfer all EBs from one well of the AggreWell plate to one well of a coated 6-well plate in NIM. Feed daily.
  9. When rosettes have formed, either use Stem Cell Technologies Rosette Selection Solution to remove (see manufacturer's instructions) or incubate with Accutase for 10 mins and replate in NIM.
  10. For propagation, resuspend in NPM (DMEM #11965044 Invitrogen containing B27 supplement minus Vitamin A and glutamine) containing 20ng/ml each of bFGF and EGF and plate on polyO/laminin coated plates at high density—1.5–2x106/well. Cells may be passaged 1:2 or 1:3 with Accutase for expansion or 1:12 for differentiation.
  11. For differentiation, plate cells in mTeSR (Stem Cell Technologies) on polyO/laminin or Matrigel (1.25%) at low density (1:12 split ratio). When confluent, usually after 5-7days, pass the cells 1:3 with Accutase and replate. This generates immature neurons which appear to retain propagation potential. Further differentiation to mature neurons requires the use of other growth factors and media such as Neurobasal medium or Millipore's Neural Differentiation Medium.
  12. Fix and stain with Nestin (Chemicon 1:100) and TuJ1 (Covance 1:500). Note this particular combination requires the use of IgG subtype-specific secondary antibodies. Can also triple stain with TH (PelFreeze 1:500).