FACS analysis of AP, SSEA1 and GFP reactivation was performed on Nanog-GFP/M2rtTA and Oct4-GFP/M2rtTA MEFs at different times after the induction of reprogramming. Cells were harvested at various time points after the addition of dox to the ES cell medium and incubated with an APC labeled anti-SSEA1 antibody and a fluorescent substrate that is activated in the presence of AP activity. The data from three independent experiments are summarized. Columns display average percentages of cells expressing AP (black), SSEA1 (white) and Nanog-GFP (diagonal) or Oct4-GFP (vertical). AP and SSEA1 values represent averages of Nanog-GFP and Oct4-GFP experiments. Error bars indicate standard deviations.

Oct4GFP/M2rTTA MEFs undergoing reprogramming were FACS sorted for SSEA1 expression after 9 days of transgene expression (A). The cells were cultured either in the presence or absence of dox for 20 days after the initial sorting and then re-analyzed for SSEA1 and GFP expression. Plates that were treated with dox contained SSEA1+/GFP+ and SSEA1+/GFP− cells whereas cells on untreated plates had lost their SSEA1 expression (A). Culture in the presence of dox yielded ES-like colonies, but in the absence of dox the SSEA1+/GFP− cells returned to a MEF like morphology (B).

Differentiation of iPS cells in teratomas was ablated by continuous expression of transgenes in iPS cells. Tumors derived from iPS cells expressing the four transgenes under the control of the constitutive Ubiquitin C promoter stained homogeneously positive for Oct4 and did not show normal cell differentiation (A). IPS cells harboring the inducible viral backbones differentiated readily after downregulation of the transgenes in the absence of dox (B). The tumors derived from these cells were similar in size, fraction of cells staining positive for Oct4 and differentiation to tumors derived from wildtype control ES cell lines (C) or ES cells harboring the R26-M2rtTA allele (D).

Somatic cells harboring a GFP reporter driven by the endogenous Oct4 or Nanog promoters were infected with tet-inducible lentiviral vectors carrying the cDNAs of Oct4, Sox2, Klf4 and c-Myc (A). The addition of doxycycline after infection induced lentiviral expression and subsequently led to reprogrammed, GFP positive iPS cell colonies (B). The iPS cells were pluripotent and contributed to viable chimeras after injection into BALB/C host blastocysts as indicated by coat color (C, D). White mice are non-chimeric BALB/C animals, whereas mixed coat color mice are chimeric.

SSEA1+/GFP− cells were sorted with an APC conjugated SSEA1-antibody after 21 days of transgene expression and seeded on feeder MEFs (A). GFP positive ES-like cell colonies were observed ten days after sorting (B). Supplementation of the media with dox for an additional three days after sorting increased the percentage of SSEA1+/GFP−cells that gave rise to GFP positive iPS cell colonies (C). Numbers from two independent experiments for Oct4-GFP and Nanog-GFP were averaged. Error bars indicate standard deviations. Confirmation of GFP-positive cells in the sorted SSEA1+/GFP− populations eleven days after initial sorting and seeding on feeder MEFs (D).

Infected MEFs were split into 6 dishes and cultured in the presence of dox for a different number of days as indicated by numbers next to the red lines (A). Dox was withdrawn at different time points as indicated by red bars and the cells were subsequently cultured in the absence of the drug until day 35. All plates were stained for AP at day 35 of reprogramming to determine how many transgene-independent cells existed at the time point of dox withdrawal. No AP positive colonies were observed when dox was withdrawn before day 12 and the number of AP positive colonies increased with the time the cells were exposed to the drug (A). Similarly, infected MEFs were grown in the presence of dox and dox was removed at different time points from the culture medium. GFP expressing iPS colonies were counted on day 35. Data for Nanog-GFP and Oct4-GFP experiments were averaged and the number of colonies detected in six wells (3 for Oct4-GFP and 3 for Nanog-GFP cells) for each time point is shown in the bar graph (B). Error bars indicate standard deviation.

The black arrow represents the time starting from MLV vector infection (above (Maherali et al., 2007; Meissner et al., 2007; Okita et al., 2007; Wernig et al., 2007)) or dox-induction (below) in days. For MLV infection, the earliest time points for two antibiotic selection strategies are indicated by blue bars and the expression of Nanog-GFP and Oct4-GFP is indicated by green bars. For dox-inducible transgenes, the minimum time of dox-induced transgene expression for successful iPS cell derivation is displayed as a black bar. The expression timing of four pluripotency marker genes is indicated by red (AP and SSEA1) and green bars (Nanog-GFP and Oct4-GFP)