HuESC-12 (hESC Cell Facility, Harvard University)

Culture isolated ICMs on a feeder layer of mitotically inactivated mouse embryonic fibroblasts plated on gelatin-coated tissue culture plates. Culture medium: Knockout DMEM, supplemented with KO-Serum Replacement (8% or 10%), Plasmanate (8% or 10%), fetal calf serum (5%), Glutamax-I (2 mM), non-essential amino acids (1%), penicillin (50 U/ml) and streptomycin (50 ug/mL), beta-mercaptoethanol (0.055mM), recombinant hLIF (12ng/mL), and bFGF (5ng/mL). Mechanically dissociate ICM-like clumps 6 to 10 days after the initial plating, and replate on fresh feeder layers. Cells can be cryopreserved in freezing media consisting of FCS (90%) and DMSO (10%). Cryopreservation in hES culture media plus DMSO (10%) results in similar yield upon thawing.