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A simple and robust method for establishing homogeneous mouse epiblast stem cell lines by wnt inhibition.

Sugimoto M, Kondo M, Koga Y, Shiura H, Ikeda R, Hirose M, Ogura A, Murakami A, Yoshiki A, Chuva de Sousa Lopes SM, Abe K - Stem Cell Reports (2015)

Bottom Line: Here, we devised a simple and robust technique to derive high-quality EpiSCs using an inhibitor of WNT secretion.Expression analyses revealed that these EpiSCs maintained a homogeneous, undifferentiated status, yet showed high potential for differentiation both in vitro and in teratomas.The homogeneous properties of this new version of EpiSCs should facilitate studies on the establishment and maintenance of a "primed" pluripotent state, and directed differentiation from the primed state.

View Article: PubMed Central - PubMed

Affiliation: Technology and Development Team for Mammalian Genome Dynamics, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.

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Cellular Pluripotency Marker Expression Levels in EpiSCs Derived by the IWP-2 Method(A–J) Immunofluorescence images for SSEA1 (A and B), PECAM1 (red) and OCT4 (green, C and D), NANOG (E and F), and histone H3K27 trimethylation (G and H) in mESCs (A, C, E, and G) and EpiSCs (B, D, F, and H). Nuclear staining is shown using TO-PRO3 (blue). Cytograms of EpiSCs and mESCs are shown using anti-SSEA1 (I) and anti-PECAM1 (J) antibodies.(K) Relative expression levels of marker genes in EpiSCs detected by qRT-PCR compared with J1 mESCs. For each gene, technical triplicate assays and two independent experiments were performed. Error bars represent the standard SEM.(L) Growth curves of EpiSCs. At the indicated time points, 2.0 × 104 cells were plated and counted. Averaged data from three independent experiments were plotted.Scale bars, 20 μm (A, B, C, D, G, and H) and 50 μm (E and F).
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fig2: Cellular Pluripotency Marker Expression Levels in EpiSCs Derived by the IWP-2 Method(A–J) Immunofluorescence images for SSEA1 (A and B), PECAM1 (red) and OCT4 (green, C and D), NANOG (E and F), and histone H3K27 trimethylation (G and H) in mESCs (A, C, E, and G) and EpiSCs (B, D, F, and H). Nuclear staining is shown using TO-PRO3 (blue). Cytograms of EpiSCs and mESCs are shown using anti-SSEA1 (I) and anti-PECAM1 (J) antibodies.(K) Relative expression levels of marker genes in EpiSCs detected by qRT-PCR compared with J1 mESCs. For each gene, technical triplicate assays and two independent experiments were performed. Error bars represent the standard SEM.(L) Growth curves of EpiSCs. At the indicated time points, 2.0 × 104 cells were plated and counted. Averaged data from three independent experiments were plotted.Scale bars, 20 μm (A, B, C, D, G, and H) and 50 μm (E and F).

Mentions: To characterize our newly established EpiSCs, we examined the expression of pluripotency cell markers. Immunofluorescence analysis allowed us to detect the expression of SSEA1 and OCT4 in EpiSCs as well as in mESCs, whereas PECAM1 was detected only in mESCs, as reported previously (Figures 2A–2D; Rugg-Gunn et al., 2012). The NANOG protein was detected in the nuclei of mESCs and EpiSCs, albeit at much lower levels in the EpiSCs (Figures 2E and 2F). It is known that one of two X chromosomes in female EpiSCs is inactivated (Bao et al., 2009). As shown in Figures 2G and 2H, a single, strong immunofluorescence signal of histone H3K27 trimethylation (H3K27me3) was observed in the nuclei of female EpiSCs, whereas no such signals for H3K27me3 were observed in male EpiSCs, indicating that X inactivation occurred in our female EpiSC lines. Flow-cytometry analysis also confirmed that our EpiSCs were SSEA1-positive and PECAM1-negative (Figures 2I and 2J). Furthermore, we carried out qRT-PCR analyses to measure the expression levels of mRNAs for the mESC and EpiSC lines. As shown in Figure 2K, the EpiSC lines expressed Oct4 and Sox2 at levels similar to those observed in the mESCs. Nanog expression was much lower in our EpiSCs than in the mESCs, as expected from the immunofluorescence data described above. Three genes—Rex1, Pecam1, and Dppa3 (which are known to be highly expressed in naive mESCs)—were not detected in our EpiSC lines, consistent with previous reports (Brons et al., 2007; Tesar et al., 2007). Marker genes for epiblast (Fgf5, Cldn6, and Otx2) were expressed at much higher levels in the EpiSCs than in mESCs. We also examined marker gene expression in one of the EpiSC lines established by the original protocol, using the 129C1 line (Brons et al., 2007) (a gift from Dr. S. Pauklin) as a control. The expression profile of the marker genes in this cell line was essentially the same as those of our EpiSCs, except for Dppa3. These results indicate that the EpiSCs produced by our method using the Wnt inhibitor IWP-2 possess a transcription profile characteristic of “bona fide” EpiSCs.


A simple and robust method for establishing homogeneous mouse epiblast stem cell lines by wnt inhibition.

Sugimoto M, Kondo M, Koga Y, Shiura H, Ikeda R, Hirose M, Ogura A, Murakami A, Yoshiki A, Chuva de Sousa Lopes SM, Abe K - Stem Cell Reports (2015)

Cellular Pluripotency Marker Expression Levels in EpiSCs Derived by the IWP-2 Method(A–J) Immunofluorescence images for SSEA1 (A and B), PECAM1 (red) and OCT4 (green, C and D), NANOG (E and F), and histone H3K27 trimethylation (G and H) in mESCs (A, C, E, and G) and EpiSCs (B, D, F, and H). Nuclear staining is shown using TO-PRO3 (blue). Cytograms of EpiSCs and mESCs are shown using anti-SSEA1 (I) and anti-PECAM1 (J) antibodies.(K) Relative expression levels of marker genes in EpiSCs detected by qRT-PCR compared with J1 mESCs. For each gene, technical triplicate assays and two independent experiments were performed. Error bars represent the standard SEM.(L) Growth curves of EpiSCs. At the indicated time points, 2.0 × 104 cells were plated and counted. Averaged data from three independent experiments were plotted.Scale bars, 20 μm (A, B, C, D, G, and H) and 50 μm (E and F).
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fig2: Cellular Pluripotency Marker Expression Levels in EpiSCs Derived by the IWP-2 Method(A–J) Immunofluorescence images for SSEA1 (A and B), PECAM1 (red) and OCT4 (green, C and D), NANOG (E and F), and histone H3K27 trimethylation (G and H) in mESCs (A, C, E, and G) and EpiSCs (B, D, F, and H). Nuclear staining is shown using TO-PRO3 (blue). Cytograms of EpiSCs and mESCs are shown using anti-SSEA1 (I) and anti-PECAM1 (J) antibodies.(K) Relative expression levels of marker genes in EpiSCs detected by qRT-PCR compared with J1 mESCs. For each gene, technical triplicate assays and two independent experiments were performed. Error bars represent the standard SEM.(L) Growth curves of EpiSCs. At the indicated time points, 2.0 × 104 cells were plated and counted. Averaged data from three independent experiments were plotted.Scale bars, 20 μm (A, B, C, D, G, and H) and 50 μm (E and F).
Mentions: To characterize our newly established EpiSCs, we examined the expression of pluripotency cell markers. Immunofluorescence analysis allowed us to detect the expression of SSEA1 and OCT4 in EpiSCs as well as in mESCs, whereas PECAM1 was detected only in mESCs, as reported previously (Figures 2A–2D; Rugg-Gunn et al., 2012). The NANOG protein was detected in the nuclei of mESCs and EpiSCs, albeit at much lower levels in the EpiSCs (Figures 2E and 2F). It is known that one of two X chromosomes in female EpiSCs is inactivated (Bao et al., 2009). As shown in Figures 2G and 2H, a single, strong immunofluorescence signal of histone H3K27 trimethylation (H3K27me3) was observed in the nuclei of female EpiSCs, whereas no such signals for H3K27me3 were observed in male EpiSCs, indicating that X inactivation occurred in our female EpiSC lines. Flow-cytometry analysis also confirmed that our EpiSCs were SSEA1-positive and PECAM1-negative (Figures 2I and 2J). Furthermore, we carried out qRT-PCR analyses to measure the expression levels of mRNAs for the mESC and EpiSC lines. As shown in Figure 2K, the EpiSC lines expressed Oct4 and Sox2 at levels similar to those observed in the mESCs. Nanog expression was much lower in our EpiSCs than in the mESCs, as expected from the immunofluorescence data described above. Three genes—Rex1, Pecam1, and Dppa3 (which are known to be highly expressed in naive mESCs)—were not detected in our EpiSC lines, consistent with previous reports (Brons et al., 2007; Tesar et al., 2007). Marker genes for epiblast (Fgf5, Cldn6, and Otx2) were expressed at much higher levels in the EpiSCs than in mESCs. We also examined marker gene expression in one of the EpiSC lines established by the original protocol, using the 129C1 line (Brons et al., 2007) (a gift from Dr. S. Pauklin) as a control. The expression profile of the marker genes in this cell line was essentially the same as those of our EpiSCs, except for Dppa3. These results indicate that the EpiSCs produced by our method using the Wnt inhibitor IWP-2 possess a transcription profile characteristic of “bona fide” EpiSCs.

Bottom Line: Here, we devised a simple and robust technique to derive high-quality EpiSCs using an inhibitor of WNT secretion.Expression analyses revealed that these EpiSCs maintained a homogeneous, undifferentiated status, yet showed high potential for differentiation both in vitro and in teratomas.The homogeneous properties of this new version of EpiSCs should facilitate studies on the establishment and maintenance of a "primed" pluripotent state, and directed differentiation from the primed state.

View Article: PubMed Central - PubMed

Affiliation: Technology and Development Team for Mammalian Genome Dynamics, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.

Show MeSH
Related in: MedlinePlus