Journal: PLoS ONE

Article Title: Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network

doi: 10.1371/journal.pone.0024351

Figure Lengend Snippet: A. Retrovirus-based expression of OSKM was detected by immunofluorescence at four different time points (HFF1, 24 h, 48 h, 72 h). For reference, nuclear DNA is stained with DAPI (4′,6-diaminidino-2-phenylindole). B. Heat map of microarray hybridization values (log scale). ‘absent’ values were set to zero in this heatmap. Samples are HFF1 (-a and -b denote duplicates), 24, 48, 72 h, two HFF1-derived iPS cells (iPS2, iPS4) and two human ES cell lines (H1, H9). Illumina Ref-8 V3 microarrays detect the exogenous and endogenous forms of OCT4 ( POU5F1 ), since the microarray probe was located within the coding region of the OCT4 transcript. Illumina probes for SOX2, KLF4 and c-MYC were designed to the 3′UTR end, and therefore do not detect the virally expressed transcripts of these genes.

Article Snippet: Human neonatal foreskin fibroblast-HFF1 cells (ATCC) and Phoenix™ Ampho cells (Orbigen, Inc.) were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Invitrogen) and 0.5% penicillin and streptomycin (Invitrogen).

Techniques: Expressing, Immunofluorescence, Staining, Microarray, Hybridization, Derivative Assay

Journal: PLoS ONE

Article Title: Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network

doi: 10.1371/journal.pone.0024351

Figure Lengend Snippet: Expression profiles were obtained from donor cells (HFF1; human foreskin fibroblasts), donor cells transduced with OSKM at 24 h, 48 h and 72 h, HFF1-derived iPS cell lines (iPS2, iPS4) and ES cell lines (H1, H9). A. Principal component analysis shows the projection of the vectors of hybridization values (24526 probes) on the first two principal components. B. Differential regulation between HFF1 cells and each of the three timepoints (24, 48, and 72 h), iPS and ES cells was determined using the Bioconductor package limma (see Methods). The normalized expression values (z-score) of 6179 transcripts regulated at any timepoint or in iPS/ES cells with respect to HFF1 cells (p adj <0.05; fold change >1.5) are shown. C. Venn diagram depicting the overlap between regulated transcripts (1476) at each timepoint. D. Alterations in the number of pluripotency- and fibroblast-associated transcripts during the time-course towards an increasing pluripotent and decreasing somatic (HFF1) transcriptome. E. Increasing numbers of pluripotency-associated transcripts linked to the GO terms ‘integral to membrane’ or ‘cell surface’ are detected in the time series.

Article Snippet: Human neonatal foreskin fibroblast-HFF1 cells (ATCC) and Phoenix™ Ampho cells (Orbigen, Inc.) were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Invitrogen) and 0.5% penicillin and streptomycin (Invitrogen).

Techniques: Expressing, Transduction, Derivative Assay, Hybridization

Journal: PLoS ONE

Article Title: Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network

doi: 10.1371/journal.pone.0024351

Figure Lengend Snippet: A. For selected GO categories, the fraction of transcripts in each GO category compared to the number of regulated transcripts is depicted as a heatmap. Colours indicate no enrichment (white) or proportion of enriched transcripts (shades of black to white), as a percentage of regulated transcripts at each timepoint. B. Regulated transcripts (fold change >1.5; p adj <0.05) in selected GO categories are shown. Colours indicate the log2 fold change, either up-regulated (red), down-regulated (blue), or not regulated (white) with respect to HFF1 cells. Heatmaps shown are transcripts from stress-related GO categories (panel B) or groups of genes known to have an influence on reprogramming (panel C). Other categories are shown in .

Article Snippet: Human neonatal foreskin fibroblast-HFF1 cells (ATCC) and Phoenix™ Ampho cells (Orbigen, Inc.) were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Invitrogen) and 0.5% penicillin and streptomycin (Invitrogen).

Techniques:

Journal: PLoS ONE

Article Title: Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network

doi: 10.1371/journal.pone.0024351

Figure Lengend Snippet: A. Quantitative analysis of reactive oxygen species (ROS) production in HFF1 cells, polybrene-treated HFF1 cells, OSKM (4 factor)-transduced HFF1 cells, and GFP-transduced HFF1 cells under retroviral transduction conditions. We measured ROS production 24 h after transduction by flow cytometry using 2′,7′-dichlorofluorescin diacetate (H2-DCFDA) in two independent experiments. Values are presented as relative changes compared to HFF1 cells without H2-DCFDA treatment. ROS levels from 4 factor-transduced HFF1 cells and GFP-transduced HFF1 cells are significantly up-regulated compared to HFF1 cells (significant changes: *, p<0.05). B. Quantification of ROS levels in HFF1 cells, mock cells, 4 factor-transduced HFF1 cells, and GFP-transduced HFF1 cells under nucleofection conditions. Measurements are as described in 5A. C. DNA damage in HFF1 fibroblasts. HFF1 cells were either left untreated or exposed to polybrene only, or to GFP- or OSKM-encoding virus. DNA damage was assayed by 8OHdG immuno-staining and monitored at three different time points (24 h, 48 h, and 72 h). Untreated fibroblasts or fibroblasts exposed to polybrene only, did not show any accumulation of DNA damage. In contrast, HFF1 transduced with GFP or 4 factors exhibited foci of nuclear and mitochondrial DNA damage (white arrows). HFF1 transduced with OSKM tended to cluster in cellular aggregates over time and showed a higher level of DNA damage. At 24 h, we observed the presence of small DAPI-positive dots in all transduced fibroblasts, which may correspond to viral DNA (green arrowheads). Magnification used was 63X, scale bar corresponds to 10 µm. D. Level of TP53 expression at 24, 48, and 72 h post-expression transduction of OSKM as measured by hybridization of the array of in real-time PCR confirmation. E. Western blot showing expression levels of phosphorylated p53 and non-phosphorylated p53 in untreated HFF1 cells, or HFF1 cells treated with polybrene, transduced with viruses expressing OSKM or GFP at 24 h post-transduction in two independent experiments. F. The ratio of expression values of phospho-p53 versus total p53 is presented as relative changes compared to untreated HFF1 cells for polybrene-treated cells, 4 factor-transduced HFF1 cells, and GFP-transduced HFF1 cells (significant changes: *, p<0.05).

Article Snippet: Human neonatal foreskin fibroblast-HFF1 cells (ATCC) and Phoenix™ Ampho cells (Orbigen, Inc.) were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Invitrogen) and 0.5% penicillin and streptomycin (Invitrogen).

Techniques: Transduction, Flow Cytometry, Immunostaining, Expressing, Hybridization, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Molecular Insights into Reprogramming-Initiation Events Mediated by the OSKM Gene Regulatory Network

doi: 10.1371/journal.pone.0024351

Figure Lengend Snippet: A. Bright-field images of HFF1 cells after staining for senescence beta-galactosidase activity (scale bar: 200 µm). B. Summary of the quantification of blue-stained senescent cells as a percentage of the total number of cells analyzed.

Article Snippet: Human neonatal foreskin fibroblast-HFF1 cells (ATCC) and Phoenix™ Ampho cells (Orbigen, Inc.) were maintained in Dulbecco's modified Eagle medium (DMEM, Gibco) containing 10% fetal bovine serum (Invitrogen) and 0.5% penicillin and streptomycin (Invitrogen).

Techniques: Staining, Activity Assay

Journal: PLoS ONE

Article Title: DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

doi: 10.1371/journal.pone.0030541

Figure Lengend Snippet: Quantification of global genome-nucleotide excision repair of UVC damage as percent of CPD repair in ( A ) ESCs and fibroblasts, and ( B ) iPSCs and their parental fibroblast lines. Values are mean±SEM (n = 3). The initial number of ESS/Mb following 10 J/m 2 UVC treatment in each cell line were: H9, 4.6±0.5; BG01, 6.3±0.1; iPSC1, 6.2±0.2; iPSC2, 3.2±0.2; human skin fibroblasts (CRL-2097), 25.5±1.1; human lung fibroblasts (IMR90), 14.5±0.3; and human foreskin fibroblasts (HF51), 13.9±0.4.

Article Snippet: Non-pluripotent IMR90 lung fibroblasts and CRL-2097 human foreskin fibroblasts were purchased from ATCC, GM03348E human foreskin diploid fibroblasts (HF02) were obtained from the Coriell Cell Repository and HF55 (HF01) and HF51 human neonatal foreskin fibroblasts were derived from discarded tissue provided by Arcadia Methodist Hospital from an approved protocol (City of Hope IRB# 92006).

Techniques: