Expansion of Undifferentiated Human Pluripotent Stem Cells

Low passage hESCs (H9, WiCell) were used. Similar results were obtained using HUES13 (Harvard) and hIPSCs derived in our laboratory. Undifferentiated hESCs were cultured as described [33] with slight modification. Briefly, cells were cultured in Knockout Dulbecco's modified Eagle's medium (KODMEM, Invitrogen, 10829-018) supplemented with 1 mM L-glutamine with 20% Knockout Serum Replacement medium (KOSR, Invitrogen), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids (NEAA, Invitrogen), 50 U/ml penicillin, 50 µg/ml streptomycin (Invitrogen), 0.1 mM beta-mercaptoethanol (Invitrogen) and 8 ng/ml basic fibroblast growth factor (bFGF, Sigma catalogue F0291-25UG). hESCs were grown on Matrigel (growth factor-reduced, BD Bioscience)-coated 6-well plates (Corning, Inc. catalogue 3506) on a feeder layer of primary MEFs from E13.5 CD-1 mice isolated as described [33] . Passage 3 to 4 MEFs were gamma-irradiated with 3,000 rads (30 Grays) and plated at 10⁴ cells per cm². All hESC lines were passaged following enzymatic digestion with either collagenase IV (Invitrogen, 17104-019) approximately every 7 days or Accutase (Chemicon) approximately every 10 days [34] (link), depending on cell condition and confluency. For collagenase treatment, cells were exposed to 1 mg/ml in KODMEM, sterile filtered, at room temperature. Once the edge of colonies were about to lift from the plate, the cells were rinsed twice with DPBS (Ca²⁺- and Mg²⁺-free), culture medium was added and cells were mechanically dispersed into 100–500-cell clusters by trituration using a 5 ml pipette and re-plated. For Accutase treatment, cells were washed twice with DPBS and then subsequently washed with a small amount of Accutase (1× concentration, Innovative Cell Technologies) and then exposed to Accutase at room temperature. After a few minutes, when MEFs and hESC-derived fibroblasts began to lift from the plate, accutase was removed and hESCs were washed twice with DPBS (Ca²⁺- and Mg²⁺-free) to remove MEFs and hESC-derived fibroblasts. A third of the volume of culture medium normally used was added and the stem cells were mechanically dispersed into 10–50-cell clusters by trituration as above. Each passage was a 1∶3 split ratio for collagenase IV-treated cells and 1∶4 to 1∶6 ratio for accutase-treated cells. Cells were routinely tested for mycoplasma (MycoAlert; Cambrex, Walkersville, MD).