1×105 human fibroblasts were plated in DMEM with 10%FBS on a gelatin-coated 35mm plastic tissue culture dish. The next day polybrene was added to the media (5 ug/mL) and the cells were infected with hSTEMCCA-loxP lentiviruses at a multiplicity of infection (MOI)=0.1, 1, or 10 where indicated in the text. On day 2, the media was changed to serum-free ‘iPSC media’ (see below), and on day 6 the entire well was trypsinized and passed at a 1:16 split by plating onto two 10cm gelatin-coated culture dishes which had been pre-seeded the day before with mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder cells. iPSC colonies were mechanically isolated 30 days post-infection with the 4 factor hSTEMCCA-loxP or 45 days post-infection with the 3 factor hSTEMCCA-RedLight-loxP based on morphology and expanded on MEF feeders in iPSC media. For 3 factor reprogramming, where indicated, GSK3 inhibitor (Bio) (EMD Biosciences, 361550; 10μM) was added to the culture media on days 7–30 of reprogramming. Reprogramming efficiency was calculated by dividing the number of total colonies obtained on day 30 by the number of starting input fibroblasts. To avoid miscalculating efficiency as can occur when counting the progeny of passaged cells, efficiency was determined from separate experiments in which the input fibroblasts were not passaged onto feeders prior to colony counting.
Candidate iPSC clones were characterized based on staining for expression of alkaline phosphatase (Alkaline Phophatase Substrate Kit I, Vector Laboratories, SK – 5100) or immunostaining of 4% paraformaldehyde-fixed cell colonies with antibodies against SSEA-4, TRA1-60, and TRA1-81 (ES Cell Characterization Kit, Millipore, SCR001). Primary antibodies were detected with secondary Alexa Fluor 488-conjugated, goat anti-mouse IgG or IgM (Invitrogen, A10680). In addition, the number of hSTEMCCA lentiviral integrations was determined by Southern blot of gDNA digested with BamHI and probed for the WPRE element as previously published21 (link). RT-PCR was performed as previously described21 (link).
In order to evaluate the degree of DNA methylation of the human NANOG promoter, gDNA extracts of each indicated sample underwent bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen). Quantitative methylation analyses of 6 CpG islands in the proximal NANOG promoter were performed via pyrosequencing by EpigenDx Inc (Worcester, MA) using the ADS502/Human NANOG promoter assay, spanning positions −565 to −431 relative to the NANOG ATG start site.
For teratoma formation assays, 6 wells of a 6 well plate of iPSC colonies were harvested with Collagenase IV and resuspended in 140μl of DMEM/F12. Immediately prior to injection, 60μl of Matrigel (BD Biosciences) was added to the cell suspension at 4°C, and the resulting mixture was injected sub-dermally between the scapulae of each anesthetized SCID–Beige mouse (Charles River, strain 250). Resulting tumors were harvested at 6–8 weeks after injection, fixed in 4% paraformaldehyde, and paraffin tissue sections were prepared and stained with hematoxylin and eosin according to standard methods.
Candidate iPSC clones were characterized based on staining for expression of alkaline phosphatase (Alkaline Phophatase Substrate Kit I, Vector Laboratories, SK – 5100) or immunostaining of 4% paraformaldehyde-fixed cell colonies with antibodies against SSEA-4, TRA1-60, and TRA1-81 (ES Cell Characterization Kit, Millipore, SCR001). Primary antibodies were detected with secondary Alexa Fluor 488-conjugated, goat anti-mouse IgG or IgM (Invitrogen, A10680). In addition, the number of hSTEMCCA lentiviral integrations was determined by Southern blot of gDNA digested with BamHI and probed for the WPRE element as previously published21 (link). RT-PCR was performed as previously described21 (link).
In order to evaluate the degree of DNA methylation of the human NANOG promoter, gDNA extracts of each indicated sample underwent bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen). Quantitative methylation analyses of 6 CpG islands in the proximal NANOG promoter were performed via pyrosequencing by EpigenDx Inc (Worcester, MA) using the ADS502/Human NANOG promoter assay, spanning positions −565 to −431 relative to the NANOG ATG start site.
For teratoma formation assays, 6 wells of a 6 well plate of iPSC colonies were harvested with Collagenase IV and resuspended in 140μl of DMEM/F12. Immediately prior to injection, 60μl of Matrigel (BD Biosciences) was added to the cell suspension at 4°C, and the resulting mixture was injected sub-dermally between the scapulae of each anesthetized SCID–Beige mouse (Charles River, strain 250). Resulting tumors were harvested at 6–8 weeks after injection, fixed in 4% paraformaldehyde, and paraffin tissue sections were prepared and stained with hematoxylin and eosin according to standard methods.
