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1.
Figure 2.

Figure 2. From: Robust activation of the human but not mouse telomerase gene during the induction of pluripotency.

Isolation of fully reprogrammed clones (class III) in D-MEFs. A) D-MEFs infected with pBMN-NIG viruses and selected by neomycin for 7 d. B) D-MEFs at 6 d after transduction by the 4 viruses. Rapid cell proliferation and formation of patches of apparently transformed cells are visible. C) Formation of colonies that were initially all GFP-positive. D) Appearance of a GFP-silenced colony, as indicated by arrows. E) GFP-negative colony, III-10, after initial isolation. F) Morphology of clone III-10 grown on MEF feeder after 3 passages. Top panels: phase-contrast fields. Bottom panels: green fluorescence fields. Scale bars = 100 μm.

Renjith Mathew, et al. FASEB J. 2010 August;24(8):2702-2715.
2.
Figure 4.

Figure 4. From: Robust activation of the human but not mouse telomerase gene during the induction of pluripotency.

Expression of the endogenous mTERT gene and transgenic hTERT reporter in reprogrammed cells. A, B) Levels of mTERT (A) and Rluc (B) mRNAs in reprogrammed cells, determined by qRT-PCR (Taqman assay). Data are normalized to 18S ribosomal RNA and presented as relative values to those of MEFs. C) Rluc activities in reprogrammed cells, as determined by luciferase assays and presented for every 1000 cells. All experiments were performed at least twice and in triplicate. Data are averages of triplicates from one representative experiment. *P < 0.05, **P < 0.01, ***P < 0.001 vs. MEFs; Student’s t test.

Renjith Mathew, et al. FASEB J. 2010 August;24(8):2702-2715.
3.
Figure 1.

Figure 1. From: Robust activation of the human but not mouse telomerase gene during the induction of pluripotency.

Characterization of reprogrammed cells from D-MEFs. A) Morphological analysis of reprogrammed clones. a–c) I-3 (a), II-25 (b), and III-10 (c) are representative clones from classes I, II, and III, respectively. Top panel: initial appearance of colonies. Middle panel: passaged culture of each clone. Bottom panel: AP staining. d, e) MEFs (d) and HM1 mouse ESCs (e, top panel) and their AP staining (e, bottom panel) are shown as controls. Initial colonies were imaged using an ×40 objective, the rest with an ×10 objective. Scale bars = 100 μm. B, C) Expression levels of pluripotency markers (B) and retroviral transgenes (C) in representative clones of each class of reprogrammed cells, as determined by RT-PCR. Nat1 was used as an internal control. NTC, no template control.

Renjith Mathew, et al. FASEB J. 2010 August;24(8):2702-2715.
4.
Figure 5.

Figure 5. From: Robust activation of the human but not mouse telomerase gene during the induction of pluripotency.

Epigenetic analyses of Nanog and TERT promoters in reprogrammed cells. A) Analysis of CpG methylation of endogenous Nanog and mTERT promoters and transgenic hTERT promoter. Genomic DNAs were subjected to bisulfite conversion, PCR cloning, and sequencing. Six CpG sites immediately upstream of the Nanog ATG codon, and 13 and 43 CpGs immediately upstream of the mTERT and hTERT TSSs, respectively, are shown. Open circles indicate unmethylated CpG sites; solid circles denote methylated sites. Coordinates of Cs listed at bottom are relative to the Nanog ATG codon, and mTERT and hTERT TSSs. B) ChIP analysis of hTERT promoter. H3K4Me2 and H3K9Me3 are antibodies against dimethylated lysine 4 and trimethylated lysine 9 residues of histone H3, respectively. Precipitated DNA fragments were analyzed by quantitative PCR using primers and a probe specific for the transgenic hTERT promoter and normalized to input DNA. *P < 0.01 vs. D-MEFs; Student’s t test.

Renjith Mathew, et al. FASEB J. 2010 August;24(8):2702-2715.
5.
Figure 7.

Figure 7. From: Robust activation of the human but not mouse telomerase gene during the induction of pluripotency.

Telomerase expression and telomere maintenance in reprogrammed human cells. A) Expression of endogenous pluripotency markers, as determined by RT- PCR analysis. B) qRT-PCR analysis of the hTERT mRNA, normalized to those of 18S ribosomal RNA and plotted relative to hTERT expression level in NHFs. Data are averages of triplicates; assays were repeated at least twice. *P < 0.0001 vs. all fibroblasts; Student’s t test. C) Telomerase activities as determined by TRAP assay; 10, 100, and 1000 ng of cell extracts were used in each set of assays. IC, internal control. D) Assessment of telomere lengths by Southern blot analysis of telomeric restriction fragments. DNA size markers (kb) are shown at left. Mouse telomere signals in reprogrammed human cells and hESCs are from feeder layers. Feeder, irradiated MEFs used as feeder layers for culturing hESCs and reprogrammed human cells.

Renjith Mathew, et al. FASEB J. 2010 August;24(8):2702-2715.
6.
Figure 6.

Figure 6. From: Robust activation of the human but not mouse telomerase gene during the induction of pluripotency.

Conversion of partially reprogrammed class II cells to fully reprogrammed cells by chemical inhibitors. Class II-25 cells were infected with pBMN-NIG retroviruses and selected with neomycin. These cells were then cultured for 4 wk in the presence of both MEK inhibitor U0126 (10 μM) and GSK3 inhibitor CHIR99021 (3 μM), or U0126 alone. Cells treated with both inhibitors (II-25+2i) were cultured for another 26 d in the absence of chemicals. A) Luciferase activities from the transgenic hTERT reporter, determined at each passage. B) Expression of retroviral GFP in II-25 cells. Day 0, pretreated cells; day 35, post-treated cells. Left panel: green fluorescence field. Right panel: phase-contrast field. C) Expression of pluripotency markers induced by chemical inhibitors in II-25 cells, as determined by RT-PCR. Klf4-Tg, transgenic retroviral Klf4; +2i, cells treated with both U0126 and CHIR99021; −2i, cells were cultured in medium without the two inhibitors; NTC, no template control. D) Pyrosequencing analysis of Nanog promoter. Representative pyrograms from pretreated II-25 (top panel) and II-25+2i (lower panel) cells are shown. Numbers in boxes indicate percentage of methylated residue at each CpG site. Table at bottom presents average methylation percentage at each CpG site in II-25 and II-25+2i cells, calculated from 3 independent pyrosequencing reactions. Positions A–D correspond to nucleotides −412, −434, −491, and −541 with respect to the Nanog TSS, respectively.

Renjith Mathew, et al. FASEB J. 2010 August;24(8):2702-2715.
7.
Figure 3.

Figure 3. From: Robust activation of the human but not mouse telomerase gene during the induction of pluripotency.

Characterization of differentiation potentials of reprogrammed cells. A) Kinetics of teratoma development following subcutaneous injection of cells into nude mice. Each clone was injected at 2 subcutaneous sites (106 cells/site). Once palpable, the volume of each tumor (length × breadth × thickness) was measured using calipers every 3 or 4 d. Data are from 1 injection site of each cell type. The other site differed in absolute tumor volume, but kinetics was the same. MEFs were injected as control, which did not develop any tumors (data not shown). B) Histological analysis of teratomas derived from reprogrammed cells. Tumors were removed about 1 mo postinjection and subjected to H&E staining. Respiratory epithelium (top panels), immature cartilage (middle panels), and neuroectoderm (bottom panels) represent differentiated cells that belong to the 3 germ layers, endoderm, mesoderm, and ectoderm, respectively. All images were photographed at ×400. Arrows indicate respective tissues as labeled at left. Scale bars = 100 μm. C) Osteogenic differentiation of reprogrammed cells. Differentiated bone colonies formed under osteogenic culture conditions were stained dark brown with silver nitrate in 6-cm plates, whereas nonbone colonies were counterstained with methylene blue. II-25+2i, II-25 cells treated with CHIR99021 and U0126 for 4 wk (see Fig. 6). D) Expression of osteogenic markers, as analyzed by RT-PCR. U, undifferentiated cells; B, bone colony; COL1, type I collagen.

Renjith Mathew, et al. FASEB J. 2010 August;24(8):2702-2715.

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