Culture of hESCs and iPS Cells

Clinical-grade hESCs (H9) were purchased from the WiCell Research Institute (Madison, WI, http://www.wicell.org), and iPS cells were derived from skin fibroblast cells obtained by a needle punch biopsy with an approved institutional review board protocol from the University of California, Irvine.
Following the biopsy, the skin fibroblast cells were cultured and infected with retroviruses expressing the human transcription factors OCT4, SOX2, KLF4, and c-MYC to induce pluripotency, as described previously [31].
Multiple iPS cell colonies were generated and examined for the presence of iPS cell markers and the ability to form teratomas. The iPS cells and hESCs were maintained in human embryonic stem (hES) medium (Knockout Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 [DMEM/F-12] containing 20% knockout serum replacement, 1 mM GlutaMAX (Life Technologies, Grand Island, NY, http://www.lifetechnologies.com), 1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM penicillin, and 1 mM streptomycin) supplemented with 10 ng/ml basic fibroblast growth factor (bFGF; Millipore, Billerica, MA, http://www.millipore.com) and grown on a feeder layer of mitotic-inactivated primary mouse embryo fibroblasts (PMEFs).
After the iPS cells and hESCs reached confluence, the medium was removed, the cells were rinsed with phosphate-buffered saline (PBS), collagenase type IV (1 mg/ml) was added (1 ml per well of a six-well plate), and the cells were incubated at 37°C. After 15 minutes, the collagenase was removed, and hES medium was added (1 ml per well). The cells were scraped with a 5-ml pipette, centrifuged at 1,000g for 3 minutes, dissociated into small clumps, and plated on fresh PMEFs.