Effect of perturbation of key self-renewal signaling pathways in ESCs and iPSCs.

<p>ESCs and iPSCs were seeded in KO SR plus or minus LIF as indicated. After 24 hours 2 µM 1 m and 10% (v/v) Hyclone serum (HY) alone or together (<b>A</b>), 2 µM 1 m (<b>B</b>), 5 µM LY294002 and 10% (v/v) HY (<b>C</b>) or DMSO 1∶10000 (in all controls), were added. After a further 24 hours proteins were extracted and immunoblotting performed as indicated. Blots were stripped and re-probed with Gapdh, pan Akt or Shp2 antibodies to assess loading.</p

Expression and phosphorylation status of key signaling intermediates in ESCs and iPSCs.

<p><b>A</b> and <b>B</b> ESCs or iPSCs were seeded 8000 cells/cm² in KO SR plus LIF. After 24 hours, 10% (v/v) Hyclone serum (HY) was added where indicated (<b>A</b>), or cells were washed 3 times with PBS before LIF-containing or LIF-free KO SR was added to the cultures as indicated (<b>B</b>). After a further 24 hours incubation, protein extracts were prepared, separated through 10% acrylamide gels using SDS-PAGE and immunoblotted using the antibodies indicated. Blots in <b>A</b> and <b>B</b>(i) were all generated using cell extracts from one replicate and those in <b>B</b>(ii) from an independent experimental replicate. <b>C</b> ESCs or iPSCs were seeded at 8500 cells/cm² in GMEM 10% (v/v) Hyclone serum (HY) plus LIF, then 24 h later washed and deprived of LIF and serum for 4 h. 5000 U/ml of LIF or 10% (v/v) HY were added and proteins extracted following 0, 5, 30 min treatment with LIF or 30 min treatment with serum. The phospho-proteins indicated were detected by immunoblotting of the same membrane, which had been cut referring to the size of the protein marker, then stripped and re-probed with the corresponding pan antibody. Results generated from the same blots are grouped, with each series terminating with the respective Gapdh as loading control.</p

Culture conditions influence iPSC self-renewal and proliferation.

<p><b>A</b> ESCs or iPSCs (passage 21) were plated onto gelatin-coated dishes in KnockOut-DMEM (KO) supplemented with KO Serum Replacement (SR) and LIF in the presence or absence of 10%(v/v) Hyclone serum (HY) or GMEM supplemented with LIF and 10% (v/v) HY, as indicated. 48 hours after plating cultures were observed by light microscopy and photographs taken. Representative images are shown, scale bar = 200 µm. <b>B</b> The self-renewal capacity of ESCs and iPSCs were evaluated by alkaline phosphatase (AP) expression in clonal assays following 4 days culture in different conditions described in <b>A</b>. The average percentage of AP positive colonies ± SEM are shown from three independent experiments. Two-tailed paired t-tests indicate the following significance *** = p<0.005. <b>C</b> and <b>D</b> Cells where plated at day 0 in KO SR+LIF (<b>C</b>) or the same media supplemented with 10% (v/v) HY (<b>D</b>). Rapamycin (5 nM) or DMSO (1∶10000, as control) were added 24 hours after seeding, and kept in the medium for the duration of the experiment. Cells were harvested after 48 (D2) 72 (D3) and 96 (D4) hours and counted in triplicate. The average values ± SEM are shown from three independent experiments. <b>E</b> ESCs or iPSCs were seeded in KO SR+LIF at 8000 cells/cm². After 24 h DMSO 1∶10000 (ctrl), 5 nM Rapamycin (Rap), 10% (v/v) Hyclone serum (HY) or serum and rapamycin together (HY Rap) were added to the cultures. After 24 h treatment protein extracts were prepared, separated through 10% acrylamide gels using SDS PAGE and immunoblotted using the antibodies indicated.</p

High passage iPSCs exhibit altered morphology and response to stimuli.

<p>Cells were plated at a density of 8500 cells/cm² in KO SR plus LIF. <b>A</b> iPSCs changed morphology after several passages (iii), compared with ESC (i) or iPSC low passage (ii), but reinstate an ESC-like morphology after 24 h treatment with 2 µM 1 m (iv). iPSCs were considered as low passage (iPSC LP) with passages <25 and high passage (iPSC HP) with passages >40. <b>B</b> Chromosome spreads of ESCs and low/high passage iPSCs. The arrow indicates chromosome aberrations in iPSC HP. One representative karyotype out of 20 from each cell line is shown. <b>C</b> 4 days after seeding, ESC or iPSC proteins were extracted, separated through 10% acrylamide gels using SDS-PAGE and immunoblotted using phospho-S15 p53 antibody. β-actin antibody was used as loading control. As a positive control, protein extracted from irradiated T lymphocytes was used. <b>D</b> Cells were plated at day 0 and then harvested after 48 (D2), 72 (D3) and 96 (D4) hours. Cells were counted in triplicate. The average doubling times ± SEM are shown from two independent experiments. Doubling times were calculated using free software available from <a href="http://www.doubling-time.com" target="_blank">www.doubling-time.com</a>. Two-tailed paired t test: ** = p<0.01 * = p<0.05. <b>E</b> iPSCs low/high passage or ESCs were harvested 4 days after seeding and Nanog-GFP expression analyzed by flow cytometry. Contour plot graphs are shown and the missing Nanog-GFP low population in iPSC HP indicated by an arrow. <b>F</b> and <b>G</b> Alkaline phosphatase positive colonies were counted after growing in KO SR plus LIF supplemented with (<b>F</b>) or without 10% (v/v) Hyclone serum (HY, <b>G</b>). After 24 hours, 2 µM 1 m was added and kept in the medium until the cells were fixed and stained. The average values ±SEM are shown from three independent experiments.</p

iPSCs are less sensitive to LIF withdrawal than ESCs.

<p>ESCs or iPSCs were plated at a concentration of 8000 cells/cm², harvested after 4 days and analyzed for Nanog-GFP expression by flow cytometry. Cells were grown in KO SR (<b>A</b>), KO SR supplemented with 10% (v/v) Hyclone serum (HY) (<b>B</b>) or GMEM supplemented with HY (<b>C</b>) in presence or absence of LIF, as indicated. Numbers shown on the histograms are the mean of FL1 fluorescence intensity (MFI). One representative experiment out of four is shown. <b>D</b> ESCs or iPSCs were seeded at 8500 cells/cm² in KO SR in the presence or absence of LIF. Protein extracts were prepared after 4 days incubation and immunoblotting performed with the antibodies indicated. Following detection of pY705 Stat-3, blots were stripped and reprobed to detect total levels of Stat-3. <b>E</b> and <b>F</b> Cells were seeded at 8500 cells/cm² in KO SR plus 10% (v/v) HY (<b>E</b>) or GMEM supplemented with 10% (v/v) HY (<b>F</b>), in the presence or absence of LIF. After 48 h protein extracts were prepared and immunoblotting performed as indicated.</p

Metformin Targets the Metabolic Achilles Heel of Human Pancreatic Cancer Stem Cells

<div><p>Pancreatic ductal adenocarcinomas contain a subset of exclusively tumorigenic cancer stem cells (CSCs), which are capable of repopulating the entire heterogeneous cancer cell populations and are highly resistant to standard chemotherapy. Here we demonstrate that metformin selectively ablated pancreatic CSCs as evidenced by diminished expression of pluripotency-associated genes and CSC-associated surface markers. Subsequently, the ability of metformin-treated CSCs to clonally expand <i>in vitro</i> was irreversibly abrogated by inducing apoptosis. In contrast, non-CSCs preferentially responded by cell cycle arrest, but were not eliminated by metformin treatment. Mechanistically, metformin increased reactive oxygen species production in CSC and reduced their mitochondrial transmembrane potential. The subsequent induction of lethal energy crisis in CSCs was independent of AMPK/mTOR. Finally, in primary cancer tissue xenograft models metformin effectively reduced tumor burden and prevented disease progression; if combined with a stroma-targeting smoothened inhibitor for enhanced tissue penetration, while gemcitabine actually appeared dispensable.</p></div

Metformin specifically eliminates cancer stem cells.

<p>(<b>A</b>) Number of cells grown in the presence of the indicated concentrations of metformin for 24 h. (n = 3). (<b>B</b>) Quantification for Ki67 and DAPI in adherent cells after 7 d of treatment with metformin or control (n = 3). (<b>C</b>) qPCR analysis for CyclinD1 in adherent and sphere-derived cells after 7 d of treatment with metformin or control. Data are normalized to the housekeeping gene and are presented as fold change in gene expression relative to untreated cells (n = 6). (<b>D</b>) Cell cycle analysis determined by Propidium Iodide staining in adherent cells and spheres after 7 d of treatment with metformin or control (n = 3). (<b>E</b>) Cytometry analysis of apoptotic cells by double staining for Annexin V/DAPI after treatment with metformin or control for adherent versus sphere-derived cells (n = 3).</p

Glycogen Synthase Kinase-3 Inhibition Enhances Translation of Pluripotency-Associated Transcription Factors to Contribute to Maintenance of Mouse Embryonic Stem Cell Self-Renewal

Maintenance of embryonic stem cell (ESC) self-renewal and pluripotency are controlled by extrinsic factors, molecular signaling pathways and transcriptional regulators. While many of the key players have been studied in depth, how the molecular signals interact with transcription factors of the pluripotency network to regulate their action remains less well understood. Inhibition of glycogen synthase kinase 3 (Gsk-3) has been implicated in the maintenance of mouse ESC pluripotency, although there is contradictory data on its role, with enhancement of cell survival and metabolism, stabilisation of c-Myc and activation of Wnt signalling proposed as potential mechanisms. We have discovered that suppression of Gsk-3 activity leads to enhanced protein levels of key transcriptional regulators of the pluripotency network, notably Nanog, Tbx3 and c-Myc. Protein stability was unchanged following Gsk-3 inhibition, although interestingly, Nanog and Tbx3 proteins were found to have half-lives of 1–3 h, while that of Oct4 protein was longer, at 6 h. We demonstrate that the effects on protein levels seen following inhibition of Gsk-3 are due to both enhanced de novo synthesis of Nanog protein and increases in the proportion of Nanog and Tbx3 RNAs bound to polysomes, findings consistent with Gsk-3 regulating translation of these factors. These effects were not due to changes in regulators of general translation initiation machinery nor mediated via the 5′ or 3′ UTR sequences of Nanog alone. The data we present provide both new conceptual insight into the mechanisms regulated by Gsk-3 that may contribute to ESC self-renewal and, importantly, establish control of protein translation as an additional mechanism involved in modulation of ESC pluripotency

The ability of Gsk-3 inhibition to promote ESC proliferation is dependent upon the culture environment.

<p>ESCs were grown in (<b>A</b>) GMEM supplemented with Serum and LIF; (<b>B</b>) N2B27 supplemented with BMP4 and LIF or <b>(C</b>) N2B27 without extrinsic stimuli, in the presence of Gsk3 inhibitor CHIR (CHIR99201) or MEK inhibitor PD (PD0325901) alone or in combination (2i) for 3 days and their growth measured. Control ESCs had DMSO vehicle added instead of inhibitors (CON). The data are the average and S.E.M of triplicate experiments. **, p<0.01, ***, p<i><</i>0.005 (2-way ANOVA with Bonferroni post-hoc test).</p

Association of Nanog and Tbx3 RNAs with polysomes increases upon inhibition of Gsk3.

<p>WT ESCs grown in the absence (WT) or presence of 2 µM 1 m (1 m) and Gsk-3 DKO ESCs (DKO) were cultured in serum supplemented with LIF for 4 and 8 hours before extracting cell lysates. Sedimentation through sucrose gradients was used to separate the polysomal-enriched fractions from the monosomal fractions. The levels of RNA bound to polysomal or monosomal fractions were measured using quantitative RT-PCR, with the primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060148#pone.0060148.s006" target="_blank">Table S1</a>. In each case gene expression was normalized relative to ß-actin levels. <b>A.</b> Expression of (i) Nanog, (ii) Tbx3 and (iii) Oct4 in individual fractions across the sedimentation gradient. A representative experiment is shown in each case (4 h time points for (i) and (ii) and 8 h time point for (iii)). Values show expression relative to ß-actin. <b>B.</b> Gene expression in pooled monosomal and polysomal fractions. Values show the proportion of RNA bound to the polysome fraction (Bound/Total RNA). The data are the average and S.E.M of three independent experiments run in duplicate for the 8-hour time point and the average and S.E.M of two independent experiments run in duplicate for the 4-hour time point. *, p<0.05; **, p<0.01, ***, p<0.005 (Student’s t-test).</p