Materials and Reagents

1. Human embryonic stem cells (hESCs) Undifferentiated hESCs were cultured on irradiated mouse embryonic fibroblast (MEF) feeders in DMEM/F12 medium supplemented with 20% KnockOut serum replacement, 0.1 mM nonessential amino acids (NEAA), 1 mM GlutaMAX^TM-1, 0.1 mM 2-mercaptoethanol (all from Invitrogen) and 8 ng/ml recombinant human FGF2 (Peprotech, catalog number: 100-18 B).
   
2. Matrigel (BD Biosciences, catalog number: 354230 )
   
3. Nocodazole (Sigma-Aldrich, catalog number: M1404 )
   
4. Thymidine (Sigma-Aldrich, catalog number: T1895 )
   
5. Aphidicholine (Sigma-Aldrich, catalog number: A0781 )
   
6. Phosphate buffered saline (PBS)
   
7. Dimethyl sulfoxide (DMSO)
   
8. Nocodazole stock solution (see Recipes)
   
9. Thymidine stock solution (see Recipes)
   
10. Aphidicholine stock solution (see Recipes)
    

Equipment

1. Centrifuges (Eppendorf 5415D centrifuge)
   
2. Fluorescence activated cell sorter (FACS, FACScan machine, Stanford FACS facility)
   

Procedure