Volume 143, Issue 4, Pages 617-627 (November 2010) Epiblast Stem Cell Subpopulations Represent Mouse Embryos of Distinct Pregastrulation Stages  Dong Wook Han, Natalia Tapia, Jin Young Joo, Boris Greber, Marcos J. Araúzo-Bravo, Christof Bernemann, Kinarm Ko, Guangming Wu, Martin Stehling, Jeong Tae Do, Hans R. Schöler  Cell  Volume 143, Issue 4, Pages 617-627 (November 2010) DOI: 10.1016/j.cell.2010.10.015 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Characterization of GOF18 EpiSCs (A) Morphology and Oct4-GFP expression in the established GOF18 EpiSC line at early and late passages. (B and C) Scatter plots of global gene expression microarrays comparing GOF18 EpiSCs with T9 EpiSCs (B) and ESCs (C). The black lines delineate the boundaries of 2-fold difference in gene expression levels. Genes highly expressed in ordinate samples compared with abscissas samples are shown as green circles; those less expressed are shown as red. Positions of pluripotent cell (Pou5f1/Oct4, Sox2, Nanog, Klf2, Klf4, Klf5, Sall4, Dnmt3l, Esrrb, Fbxo15, and Zfp42/Rex1), germ cell (Stella [Dppa3], Dppa4, Dppa5, Iftm3 [Fragilis]), and epiblast (Fgf5, T) markers are indicated with orange dots. The color bar to the right indicates the scattering density; the higher the scattering density, the darker the blue color. Gene expression levels are depicted on log2 scale. (D) Protein levels of pluripotency markers in EpiSCs. Levels of Oct4, Nanog, and Sox2 were compared in ESCs and EpiSCs by western blot; 3T3 cells were used as a negative control. (E) Immunofluorescence analysis for Oct4, Nanog, and Sox2 in ESCs and EpiSCs. DAPI was used for nuclear staining. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Dynamic Expression of Oct4-GFP in GOF18 EpiSCs (A) Percentage of Oct4-GFP positive cells in EpiSCs measured by FACS sorting. (B) Oct4-GFP positive and Oct4-GFP negative EpiSCs were FACS sorted and cultured separately. An Oct4-GFP negative colony from Oct4-GFP positive cells and an Oct4-GFP positive colony from Oct4-GFP negative cells observed under fluorescence microscope after 1 week in culture are shown. (C) Percentage of Oct4-GFP positive cells from both Oct4-GFP positive and Oct4-GFP negative cells was measured by FACS 7 days after the initial sorting. (D) Summary of the clonal assay. FACS-sorted Oct4-GFP positive and negative single cells were plated on 96-well plates, and Oct4-GFP expression was monitored under fluorescence microscope during 1 week. The numbers of clonal cell lines containing only GFP positive cells, only GFP negative cells, or a mixture of GFP positive and negative cells are indicated. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Oct4-GFP Positive and Negative EpiSCs Have Distinct Gene Expression and Epigenetic Profiles (A) Scatter plots of global gene expression microarray comparing Oct4-GFP negative and positive EpiSCs with nonsorted EpiSCs. (B) Scatter plots of global gene expression microarrays comparing Oct4-GFP negative and positive EpiSCs with ESCs. The black lines delineate the boundaries of 2-fold difference in gene expression levels. Genes highly expressed in ordinate samples compared with abscissas samples are shown as green circles; those less expressed are shown as red. Positions of pluripotent cell (Pou5f1/Oct4, Sox2, Nanog, Klf2, Klf4, Klf5, Sall4, Dnmt3l, Esrrb, Fbxo15, and Zfp42/Rex1), germ cell (Stella [Dppa3], Dppa4, Dppa5, Iftm3 [Fragilis]), and epiblast (Fgf5, T) markers are indicated with orange dots. The color bar to the right indicates the scattering density; the higher the scattering density, the darker the blue color. Gene expression levels are depicted on log2 scale. (C) Heat map of global gene expression patterns in ESCs, Oct4-GFP positive and negative EpiSCs, T9 EpiSCs, and GOF18 EpiSCs. The color bar at top codifies the gene expression in log2 scale. Red and blue colors indicate high and low gene expression, respectively. (D) Hierarchical clustering shows that Oct4-GFP positive EpiSCs clustered close to the parental EpiSCs or T9 EpiSCs, but not to ESCs. (E) DNA methylation status of the promoter regions of both the endogenous Oct4 gene and the Oct4-GFP transgene was determined by bisulfite sequencing PCR. (F) Dppa5, Stella (Dppa3), Vasa, and Fragilis (Iftm3) promoter regions were analyzed by bisulfite sequencing PCR in ESCs, MEFs, and PGCs, as well as in Oct4-GFP positive and negative EpiSCs. See also Figure S1. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Oct4-GFP Positive and Negative EpiSCs Show Distinct ESC Conversion Efficiency (A) Conversion of both the Oct4-GFP positive and negative EpiSCs to an ES-like cell state using a conversion condition (2i+LIF). Morphology and alkaline phosphatase (AP) activity of EpiSCs after 4 days of treatment are shown. (B) Relative conversion efficiency of Oct4-GFP positive and negative EpiSCs was determined by counting the number of AP-positive colonies. Data represent mean ± SEM of triplicates; n = 3. (C) Dppa5, Stella, Vasa, and Fragilis promoter regions in the converted ES-like cells were analyzed by bisulfite sequencing PCR. See also Figure S2. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Oct4-GFP Positive and Negative EpiSCs Represent In Vivo Epiblasts of Different Developmental Stages (A and B) Comparison of gene expression patterns in in vivo samples (E5.5 versus E6.5 epiblasts) (A) and in vitro samples (Oct4-GFP positive versus negative EpiSCs) (B). Expression levels are normalized to those of E6.5 epiblast (A) and Oct4-GFP negative EpiSCs (B), respectively. (C) Heat map of the RT-PCR values. RT-PCR RQ ratios were used to compare in vivo with in vitro samples. The in vivo ratios were calculated by dividing the RT-PCR RQ value of the E6.5 epiblast by that of the E5.5 epiblast sample. The in vitro ratios were calculated by dividing the RT-PCR RQ value of the GFP negative EpiSC sample by that of the GFP positive EpiSC sample. The color bar at top codifies the ratio values; red and blue colors indicate high and low ratios, respectively. (D) Evaluation of the Oct4 enhancer activity in Oct4-GFP positive and negative EpiSCs. Relative luciferase activity was normalized to the activity of the empty vector. Data represent mean ± SEM of triplicates; n = 3. (E) Dynamics of the Oct4-GFP reporter expression in in vivo epiblasts. In vivo epiblasts were isolated from both GOF18 and GOF18ΔPE mice at E5.5, 6.5, and 7.5. Oct4-GFP expression was measured under fluorescence microscope. To show specific Oct4-GFP expression from GOF18ΔPE mice, all images were taken with the same overexposure, but the images from GOF18 mice were taken with normal exposure. The extra panel under the E7.5 GOF18ΔPE epiblast shows residual Oct4-GFP expression in PGCs. See also Figure S3. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Oct4-GFP Positive EpiSCs Can Contribute to Chimera Formation (A) Integration of Oct4-GFP positive EpiSCs into the ICM following blastocyst injection. (B) Germline contribution of Oct4-GFP positive EpiSCs, as shown by the expression of Oct4-GFP in the gonad of a 13.5 days postcoitum (dpc) embryo. (C) Coat-color chimera from Oct4-GFP positive EpiSCs. (D) Morphology of Epi-Red cells and expression of Td-tomato lentivirus. (E) Epi-Red cells failed to integrate into the ICM following blastocyst injection. See also Figure S4. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Dppa5 Overexpression Increases the Early-Stage Epiblast Fraction (A) The Oct4-GFP positive fraction was increased by overexpressing Dppa5. The numbers of Oct4-GFP positive cells from both control and Dppa5-overexpressing EpiSCs were compared by FACS. (B) Dynamics of Oct4-GFP expression in FACS-sorted GFP positive cells from both control and Dppa5-overexpressing EpiSCs. FACS-sorted Oct4-GFP positive cells were cultured for another 10 passages, and the number of Oct4-GFP positive cells was measured after every two passages. (C) A dynamic interchange exists between ESCs positive and negative for Stella (Dppa3), Nanog, and Rex1 (Zfp42). When all three genes are expressed, ESCs represent the well-known ground state of pluripotency. However, when the expression of one of these genes is reduced, the pluripotent capability is also considerably reduced. EpiSCs are also heterogeneous, with two different populations distinguished by Oct4-GFP transgene expression. Oct4-GFP positive and negative EpiSCs exist in a state of dynamic equilibrium and correspond to in vivo epiblasts of early and late stages of development, respectively. The endogenous Oct4 locus is unmethylated and expressed in both populations, whereas the Oct4-GFP transgene is differentially methylated, allowing to distinguish the early- and late-stage epiblast subpopulations. See also Figure S2. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S1 Distinct Gene Expression Patterns in Oct4-GFP Positive and Negative EpiSCs, Related to Figure 3 Real-time PCR of genes reveals a distinct expression of Oct4-GFP positive and negative EpiSCs. All data are normalized to β-actin expression and calibrated on ESCs. Data represent mean ± SEM of triplicates; n = 3. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S2 DNA Methylation Status of Rex1 Promoter Region in Oct4-GFP Positive and Negative EpiSCs, Related to Figures 4 and 7 DNA methylation pattern of Rex1 promoter region was analyzed by bisulfite sequencing PCR in Oct4-GFP positive and negative EpiSCs, reverted ES-like cells, and Oct4-GFP positive cells from Dppa5-overexpressing EpiSCs. Open and closed circles indicate unmethylated and methylated CpGs, respectively. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S3 Hierarchical Clustering Generated from Real-Time PCR RQ Data, Related to Figure 5 Blue lines connect GFP positive EpiSCs with E5.5 epiblast, and red lines connect GFP negative EpiSCs with E6.5 epiblast. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure S4 Time Course Analysis of ICM Incorporation of Epi-Red Cells, Related to Figure 6 Integration of Epi-Red cells into the ICM of recipient blastocyst was monitored at 1 and 8 hr after injection. Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions

Cell 2010 143, 617-627DOI: (10.1016/j.cell.2010.10.015) Copyright © 2010 Elsevier Inc. Terms and Conditions