Feeder Free Culturing of hESCs

1. Fibronectin (human): human foreskins fibroblast cellular fibronectin (Sigma-Aldrich Inc.; cat. no. F6277); human plasma fibronectin (Sigma-Aldrich Inc.; cat. no. F2006 or Chemicon; cat. no. FC010-10) (see Note 6). Dilute 1 mg human fibronectin in 10 mL of sterile water (Sigma-Aldrich Inc.; cat. no. W1503). Coat plates (50 ^g/10-cm dish) at room temperature for at least 1 h before plating of hESCs (see Note 7).

2. Feeder-free culture medium: 85% ko-DMEM, 15% SR, 1% nonessential amino acids, 2 mM L-glutamine, 0.1 mM ß-mercaptoethanol, 4 ng/mL bFGF, 0.12 ng/mL TGFpj (R&D Systems, Minneapolis, MN; cat. no. 240-B). For the preparation of 500 mL of culture medium: add 75 mL SR, 5 mL nonessential amino acids, 2.5 mL L-glutamine, 1 mL ß-mercaptoethanol, 2000 ng bFGF, 60 ng TGFp1, and 416.5 mL ko-DMEM into a filter unit and filter (see Notes 3 and 8).

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