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1.
Figure 2

Figure 2. Gene expression changes in nonisogenic ES and iPS cells undergoing directed differentiation to DE over 5 days.. From: Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes.

(A) Flow cytometry assessment of expression of GFP reporters targeted to each indicated locus in ST8 iPS cells (Sox2-GFP), Oct4 iPS cells (Oct4-GFP), 2D4 iPS cells (Nanog-GFP), and 129/Ola ES cells (T-GFP). (B) qRT-PCR assessment of the levels of gene expression of each indicated marker or transcription factor on day 0 versus day 5 of directed differentiation (mean fold-change expression ± SEM). ESC, ES cell; T-GFP/Foxa2-hCD4, 129/Ola ES cell line; iPS2D4, Nanog-GFP iPS cell line; iPSST5, Sox2-GFP clone 5 iPS cell line; iPSST8, Sox2-GFP clone 8 iPS cell line; iPSOct4, Oct4-GFP iPS cell line.

Constantina Christodoulou, et al. J Clin Invest. 2011 June 1;121(6):2313-2325.
2.
Figure 4

Figure 4. Methodology for purification of ES/iPS cell–derived endoderm.. From: Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes.

(A) Representative flow cytometry analysis of ckit and Sox2 expression levels in iPS cells after 5 days of directed differentiation and expression of CXCR4 and ENDM1 cell surface markers within each indicated subgate. (B) Comparison of gene expression profiles (qRT-PCR) of Sox2-GFPdim/ckit+ and Sox2-GFPbright/ckit sorted cell populations. Sox2-GFPdim/ckit+ fractions preferentially express endodermal gene markers, while Sox2-GFPbright/ckit fraction expresses residual Rex1 and the neuroectodermal maker Pax6. D0, day 0 undifferentiated cells; D5, cells differentiated for 5 days. Error bars represent mean fold change in expression ± SEM.

Constantina Christodoulou, et al. J Clin Invest. 2011 June 1;121(6):2313-2325.
3.
Figure 6

Figure 6. Analysis of cell-type effects between ES and iPS cell samples, regardless of differentiation stage, reveals aberrant silencing of genes encoded by the Dlk1-Dio3–imprinted gene cluster on chromosome 12qF1. . From: Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes.

(A) Unsupervised clustering analysis of the top 111 transcripts differentially expressed due to cell-type effects, based on 2-way ANOVA with FDR< 0.001. Yellow highlighting indicates the 36 transcripts that localize to chromosome 12qF1 in the region of the Dlk1-Dio3 gene cluster. (B) Schematic representation of the mouse Dlk1-Dio3–imprinted gene cluster. (C) Validation of microarray analysis through qRT-PCR quantification of each indicated gene, normalized to 18S rRNA (n = 3; data shown as average ± SEM). (D) qRT-PCR analysis of Gtl2 expression kinetics during 15 days of endoderm differentiation, followed by hepatic lineage specification. During differentiation, Gtl2 expression is upregulated in ES cells, while remaining silenced in ST5 and ST8 iPS clones. (E) Mouse Gtl2 (AJ320506) pyrosequencing indicates aberrant DNA methylation of the Dlk1-Dio3 gene cluster in ST5 and ST8 iPS cell clones at day 0 as well as day 5, in contrast to that of ES cells and parental tail-tip fibroblasts prior to reprogramming. The graph indicates the global percentage of methylation of each of 29 CpG islands, spanning the Gtl2 IG-DMR region (Nt 81262-81567). TTFs, tail-tip fibroblasts.

Constantina Christodoulou, et al. J Clin Invest. 2011 June 1;121(6):2313-2325.
4.
Figure 1

Figure 1. Kinetics of differentiation of ES cells into DE.. From: Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes.

(A) Schematic of the mouse E8.25–E9.0 developing embryo, indicating transcription factors and marker genes induced as the foregut endoderm is patterned into prospective organ domains of thyroid, lung, liver, and dorsal/ventral pancreas. Hrt, heart; vp, ventral pancreas; Li, liver; Lu, lung; Th, Thyroid; dp, dorsal pancreas. (B) Flow cytometry quantification of the kinetics of endodermal differentiation of the 129/Ola ES cell line containing GFP and hCD4 reporters knocked-in to the brachyury (T) and Foxa2 loci, respectively. Numbers in each quadrant indicate the percentage of cells in that quadrant. (C) Summary of endodermal differentiation kinetics of ES cells, displayed as the percentage of cells at each time point, displaying the flow cytometry profile of anterior primitive streak–like (APS-like) cells (T+/Foxa2+) or DE-like cells (T/Foxa2+), or coexpressing ckit+/CXCR4+ cells, which are considered surrogate markers of endoderm differentiation. Error bars represent average ± SEM. (D) Day 0 versus day 5 expression of transcription factors during endodermal differentiation of ES cells, as assessed by qRT-PCR. Error bars represent average ± SEM. (E) qRT-PCR assessment of the kinetics of gene expression of ES cells in a 2-step protocol designed to accomplish DE differentiation (stage 1), followed by lineage specification (stage 2; day 6–18). T+/Foxa2+/ckit+ APS-like cells were sorted on day 4 (left panel).

Constantina Christodoulou, et al. J Clin Invest. 2011 June 1;121(6):2313-2325.
5.
Figure 3

Figure 3. Comparison of strain-matched ES and iPS cell capacity to undergo directed differentiation to DE, followed by hepatic lineage specification.. From: Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes.

(A) iPS cell clones (ST5 and ST8) and their parental syngeneic ES cells (Sox2-GFP) were differentiated in parallel to endoderm. The kinetics of expression of ckit, CXCR4, and the DE marker ENDM1 were measured by flow cytometry. PE indicates autofluorescence. Numbers in each quadrant indicate the percentage of cells in that quadrant. (B) Summary of kinetics and cell counts from 3 repeated experiments. (C and D) Gene expression kinetics (qRT-PCR; n = 3) during hepatic lineage specification. Note sequential decrement of pluripotent markers and induction of α-fetoprotein (Afp), followed by α-1 antitrypsin (Aat), followed by expression of albumin (Alb). (E) Glycogen storage capacity of undifferentiated (day 0) cells versus day 19 hepatocyte-like cells derived from each ES and iPS cell clone. *P < 0.05, comparing the difference in glycogen storage capacity between ST8-derived and ES-derived cells (2-tailed t test). (F) Albumin (red) immunostaining in day 18 iPSST5-derived hepatocytes. Nuclei were stained with DAPI (blue). Original magnification, ×10. Graphs represent 3 biological replicates; error bars represent mean ± SEM.

Constantina Christodoulou, et al. J Clin Invest. 2011 June 1;121(6):2313-2325.
6.
Figure 5

Figure 5. Microarray analysis of global gene expression in ES and iPS cells before (day 0) and after (day 5) endodermal differentiation.. From: Mouse ES and iPS cells can form similar definitive endoderm despite differences in imprinted genes.

(A) Principal components analysis (PCA) of 18 samples reveals tight grouping of iPS cell clones in the undifferentiated state. Time effect (differentiation) is responsible for the majority of the variability in global gene expression. PC1, first principal component; PC2, second principal component. (B) Supervised heat map of samples across the top 1,000 genes differentially expressed with differentiation (time effect) in ES and iPS cell samples. Two-way ANOVA was used to calculate the top 1,000 probe sets, ranked by FDR-adjusted P value. (C) Venn diagram of the overlap between the genetic programs of in vivo DE from the E8.25 embryonic DE and putative DE derived from ES and iPS cells. The top 2,715 genes differentially expressed (FDR < 0.001) between undifferentiated stem cells reminiscent of the blastocyst inner cell mass and E8.25 embryonic DE are shown compared with the top 1,000 genes representing in vitro ES/iPS cell–derived DE (time effect) shown in B. The schematic (top) demonstrates the comparison algorithm used for each statistical analysis to calculate the 2 indicated gene kinetic signatures. (D) Unsupervised clustering of the 18 in vitro samples shown in A and B across the 2,715 embryonic DE gene signature list from the E8.25 embryo. Unsupervised clustering indicates similar transcriptome changes in ES and iPS cells with in vitro differentiation.

Constantina Christodoulou, et al. J Clin Invest. 2011 June 1;121(6):2313-2325.

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