1. Volume 11, Issue 6, Pages 769-782 (December 2012)
A Late Transition in Somatic Cell Reprogramming Requires Regulators Distinct from the Pluripotency Network 
Azadeh Golipour, Laurent David, Yu Liu, Gowtham Jayakumaran, Calley L. Hirsch, Dan Trcka, Jeffrey L. Wrana 
Cell Stem Cell 
Volume 11, Issue 6, Pages (December 2012)
DOI: /j.stem
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2. Cell Stem Cell 2012 11, 769-782DOI: (10.1016/j.stem.2012.11.008)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 OKMS Transgenes Suppress the Stabilization Phase
(A) Expression of stabilization-phase genes (SPGs) is inversely correlated with transgene expression. mRNA from R1 ESCs, secondary (2°) iPSCs, and secondary MEFs treated with Dox as indicated was isolated, and expression of the selected maturation-phase genes and SPGs as well as the OKMS transgenes was assessed by RT-qPCR. Secondary iPSCs were obtained after 6 days of Dox withdrawal from cells cultured 24 days prior in the presence of Dox. qPCR data are presented as a color log scale from blue (0%) to yellow (100% of maximal expression), as shown in the legend.
(B) Transgenes suppress expression of pluripotency-associated SPGs. mRNA from secondary MEFs cultured as indicated (top panel) was analyzed for the indicated SPGs and piggyBac transgene (-PB) expression. Expression levels at d20 (top panel) and d25 (bottom panel) are shown for mESC (triangle) and reprogramming cells cultured in the absence (open circle) or presence (filled circle) of Dox. Note that SPGs continue to display reduced expression after prolonged culture in Dox (green dotted line), but are upregulated by removal of Dox (red dotted line) to levels comparable to R1 ESCs (black dotted line).
(C) Appearance of Dppa4-positive cells in reprogramming cells upon loss of transgene expression. Secondary MEFs were cultured in Dox for 17 days and then split into parallel cultures in the presence or absence of Dox for an additional 3 or 8 days. These cells and control iPSCs were then stained for Dppa4 and analyzed by flow cytometry. Two-dimensional plots of side-scatter (SSC) versus Dppa4 intensity are shown with a color scale displaying the density of events from 0% to 100% (legend). The percentage of Dppa4-positive cells is indicated.
(D) Loss of transgene expression leads to the appearance of Dppa4-positive colonies. Secondary MEFs cultured for 20 days in Dox were split into parallel cultures and cultures were continued in the presence or absence of Dox for 2 more days. Control iPSCs cultures were stained for Nanog and Dppa4, and their nuclei were counterstained, as indicated. Note that colonies are Nanog+/Dppa4− in the presence of Dox but Nanog+/Dppa4+ in the absence of Dox. The scale bar represents 27μm. See also Figure S1.
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4. Figure 2
Successful Transition of Reprogramming Cells to Stabilization Requires Late Maturation Phase
(A and B) Increased time of transgene expression enhances transition upon transgene repression. Secondary MEFs cultured as indicated in the schematics were stained for AP and DAPI and then imaged by fluorescence microscopy (A) or scanned with a Celigo high-content microscope to quantify AP-positive areas (B). Fix, fixation.
(C–E) Schematic and quantification of clonal reprogramming. As indicated in the schematic (C), secondary MEFs were seeded as single cells in individual wells of a 96-well plate and cultured clonally in the presence of Dox for 14 or 21 days. Dox withdrawal at d14 resulted in no Dox-independent colonies. Certain clones then acquired competency to respond to Dox withdrawal (SC clones), whereas others remained incompetent (SI). In (D), AP-positive colony formation in response to Dox withdrawal (i.e., transgene repression) was quantified for four SC and four SI clones using the Celigo system, and the p value was calculated using a t test. Representative AP- and DAPI-stained images of the SC and SI clones in the presence and absence of Dox are presented in (E). Scale bars represent 200 μm.
(F) Average expression levels of Nanog, endogenous Oct4, and Sall4 in the four SC and four SI clones. mRNA from SC and SI clones cultured in the indicated conditions was isolated at the specified times. Expression of the indicated genes was quantified by RT-qPCR and is plotted as log2 of relative units. Feeders and R1 ESC are shown as controls. Error bars represent the SD; a t test was performed for d21 and d23; ns represents nonsignificant (p > 0.05) and ∗ represents p < 0.05. See also Figures S2 and S3.
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5. Figure 3 SC Clones Display an Altered Transcriptome
(A) Schematic of mRNA that was isolated for RNA-seq analysis.
(B) Pairwise comparison of the transcriptome of SC and SI clones. The samples from (A), as well as three samples of feeder mRNA, as indicated in the legend, were analyzed by RNA-seq using Spearman's rank correlation coefficient and are plotted as a heatmap matrix after unsupervised hierarchical clustering with complete linkage. The dendrogram of the similarity relationship is shown beside the heatmap with the three major groups highlighted in black, red, and green. The color scale of correlation coefficients is depicted on right side.
(C) SC clones at d14 cocluster with SI clones at d14 and d21. A detailed view of the heatmap cluster of SI clones at d14 and d21 shows interspersed SC clones at d14. See also Figure S4 and Table S1.
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6. Figure 4 The SC Signature
(A) The SC signature. To identify genes that are specifically expressed before Dox withdrawal and mark competency to transition from maturation to stabilization, SC-d21 RNA-seq samples were compared to SC-d14, SI-d14, and SI-d21 RNA-seq samples by gene and sample two-dimensional hierarchical clustering with relative mean-centered gene expression presented as a green-black-red-colored heatmap (minimum to maximum, respectively). The dendrogram of the similarity relationship of the samples is shown above the heatmap, and for each gene the fold change was calculated as the average of SC-d21 over the averages of SC-d14, SI-d14, and SI-d21 samples and is presented as a bar graph next to the heatmap. This highlights four clusters (a, b, c, and d) with higher expression in SC-d21 samples.
(B) Schematic of gene-expression classes. Class 1 genes are upregulated more than 2-fold in SC versus SI clones and further upregulated by at least 2-fold in iPSCs. Class 2 genes are upregulated more than 2-fold in SC clones and stay relatively constant (less than a 2-fold change) upon Dox withdrawal.
(C and D) Examples of the two expression classes. Expression profiles of selected genes in SC and their iPSCs derivatives versus SI clones and feeders is shown at the indicated times and culture conditions. Gene expression is presented as in (A).
Cell Stem Cell  , DOI: ( /j.stem )
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7. Figure 5
Genes Regulating Transition to the Stabilization Phase and Maintenance of Pluripotency
(A) Schematic of the functional siRNA screen used to identify regulators of the transition between the maturation and stabilization phases versus regulators of pluripotency maintenance. See Supplemental Information for screen details.
(B and C) Representative Celigo images are shown of Dppa4- and DAPI-stained colonies identified by automated image analysis from mock-, siNT-, and siRNA-Oct4-treated cultures, for the transition screen (B) and the pluripotency-maintenance screen (C).
(D and E) Results of the siRNA screen. The siRNA screens were performed in quadruplicate (two biological replicates, done in duplicate), and the Dppa4 area measurements from all four individual wells were averaged and plotted as a rank-order plot of log2-transformed values. Values for screen controls such as mock, NT, and Oct4 siRNAs are shown as gray dots, whereas enhancers of transition (D) such as Mmrn2 and Morc1 or pluripotency maintenance (E) such as Dppa2 and Klf5 are shown as green dots.
(F and G) Expression profiles of representative regulators of the transition to stabilization (F) or pluripotency maintenance (G) are plotted as in Figures 4C and 4D. See also Figure S5 and Tables S2 and S3.
Cell Stem Cell  , DOI: ( /j.stem )
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8. Figure 6
Distinct Regulatory Networks Control Transition versus Maintenance of Pluripotency
(A) Interaction of genes that regulate formation of Dppa4-positive colonies. A Venn diagram summarizes unique and overlapping genes identified in the two screens. A color gradient (yellow-orange-red) depicts the three phases of reprogramming, with the maturation-to-stabilization transition upon transgene withdrawal marked as a dotted line. Genes that enhance and suppress transition only (yellow), maintenance only (brown), or both processes (orange) are displayed as nodes. Associations (gray edges) were obtained using GeneMANIA, with edge weight (thickness and darkness) reflecting confidence, as indicated.
(B) Distribution of gene-expression classes in regulators of transition versus maintenance. The number of class 1 and 2 genes (white and gray, respectively) that are present in the regulators of transition, maintenance, or both processes is plotted as a bar graph.
(C) Functional classifications in regulators of transition versus maintenance. Regulators of transition versus pluripotency maintenance are classified according to their function. The bar graph represents the number of unique (yellow for transitioning cells and brown for iPSCs) or common (orange) regulators. Note that some categories are only found in transitional regulators.
Cell Stem Cell  , DOI: ( /j.stem )
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