Growth, propagation and lentiviral transduction of hES cells
The NIH approved human ES H1 cell line was obtained from WiCell (Madison, Wisconsin). hES cell colonies were cultured on mouse embryonic fibroblasts (MEF) (Chemicon, Temecula, CA) in the presence of DMEM-F12 (Invitrogen, Carlsbad, CA) supplemented with 20% KNOCKOUT serum replacement with 1 mM L-glutamine, 1% Nonessential Amino Acids, 0.1 mM β-mercaptoethanol, 0.5% penicillin/streptomycin, and 4 ng/ml human basic fibroblast growth factor. Culture medium was replaced daily with fresh complete DMEM-F12. Mature colonies were subcultured weekly by digesting with collagenase IV as previously described [5]. A VSV-G pseudotyped lentiviral vector (SINF-EF1a-GFP) containing a GFP reporter gene (kindly supplied by R. Hawley, George Washington University) was used for hES cell transductions as previously described (30, 58). Generation of the pseudotyped vector in 293T cells and its concentration by ultracentrifugation were described previously [30,48]. For vector transduction, the undifferentiated hES cells were prepared into small clumps of 50–100 cells with enzyme digestion as done for routine passaging of cells. The cell clumps were incubated with the vector for 2 hrs in the presence of polybrene 6 ug/ml. A secondary cycle of transduction was done by adding fresh vector and incubating for another 2 hrs. The general vector titers were 1 × 107 and the multiplicity of infection was 10. The transduction efficiency was about 50%. The transduced colonies were cultured on MEF like above.