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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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Effects of IWP-2 on Gene Expression in the 129C1 EpiSC LineImmunofluorescence images for GATA4 (red) and OCT4 (green) (A and B), SOX17 (C and D), T (E and F), and CER1 (G and H) in EpiSCs cultured without IWP-2 (A, C, E, and G) or with IWP-2 (B, D, F, and H). Nuclei were stained with TO-PRO3 (blue). Scale bar, 100 μm. See also Figures S5 and S6.
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fig5: Effects of IWP-2 on Gene Expression in the 129C1 EpiSC LineImmunofluorescence images for GATA4 (red) and OCT4 (green) (A and B), SOX17 (C and D), T (E and F), and CER1 (G and H) in EpiSCs cultured without IWP-2 (A, C, E, and G) or with IWP-2 (B, D, F, and H). Nuclei were stained with TO-PRO3 (blue). Scale bar, 100 μm. See also Figures S5 and S6.

Mentions: Next, we validated the suppression of several developmental regulators (GATA4, CER1, T, and SOX17) in 129C1 cells treated with IWP-2 by immunostaining (Figures 5 and S5). In 129C1 cells, GATA4- or SOX17-positive cells were clustered in OCT4-negative patches (Figures 5A–5D). However, such GATA4-positive cells or OCT4-negative regions were never observed in the EpiSCs cultured in the IWP-2 medium. Relatively large numbers of CER1-positive cells were found in the absence of IWP-2, whereas the IWP-2 treatment almost completely suppressed the expression of CER1. CER1-positive cells showed weaker OCT4 expression than the surrounding CER1-negative EpiSCs (Figure S5C). Small numbers of T-positive cells were found among the colonies in the absence of IWP-2 treatment, whereas T-positive cells were hardly seen in the IWP-2 cultures. T was expressed weakly in OCT4-positive cells, whereas cells weakly positive for OCT4 showed higher expression of this marker (Figure S5D). These results suggest that the EpiSC cultures were composed of subpopulations exhibiting different degrees of spontaneous differentiation: Cer1 was expressed in cells weakly positive for OCT4, while Gata4 and Sox17 were expressed in OCT4-negative cells. The IWP-2 treatment suppressed such developmental regulators by inhibiting WNT secretion, reducing the degree of spontaneous differentiation and thus producing a more homogeneous population of pluripotent EpiSCs. Whether or not “undifferentiated” EpiSCs cultured with IWP-2 differ significantly from EpiSCs cultured without IWP-2 remains to be clarified.


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)

Effects of IWP-2 on Gene Expression in the 129C1 EpiSC LineImmunofluorescence images for GATA4 (red) and OCT4 (green) (A and B), SOX17 (C and D), T (E and F), and CER1 (G and H) in EpiSCs cultured without IWP-2 (A, C, E, and G) or with IWP-2 (B, D, F, and H). Nuclei were stained with TO-PRO3 (blue). Scale bar, 100 μm. See also Figures S5 and S6.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400648&req=5

fig5: Effects of IWP-2 on Gene Expression in the 129C1 EpiSC LineImmunofluorescence images for GATA4 (red) and OCT4 (green) (A and B), SOX17 (C and D), T (E and F), and CER1 (G and H) in EpiSCs cultured without IWP-2 (A, C, E, and G) or with IWP-2 (B, D, F, and H). Nuclei were stained with TO-PRO3 (blue). Scale bar, 100 μm. See also Figures S5 and S6.
Mentions: Next, we validated the suppression of several developmental regulators (GATA4, CER1, T, and SOX17) in 129C1 cells treated with IWP-2 by immunostaining (Figures 5 and S5). In 129C1 cells, GATA4- or SOX17-positive cells were clustered in OCT4-negative patches (Figures 5A–5D). However, such GATA4-positive cells or OCT4-negative regions were never observed in the EpiSCs cultured in the IWP-2 medium. Relatively large numbers of CER1-positive cells were found in the absence of IWP-2, whereas the IWP-2 treatment almost completely suppressed the expression of CER1. CER1-positive cells showed weaker OCT4 expression than the surrounding CER1-negative EpiSCs (Figure S5C). Small numbers of T-positive cells were found among the colonies in the absence of IWP-2 treatment, whereas T-positive cells were hardly seen in the IWP-2 cultures. T was expressed weakly in OCT4-positive cells, whereas cells weakly positive for OCT4 showed higher expression of this marker (Figure S5D). These results suggest that the EpiSC cultures were composed of subpopulations exhibiting different degrees of spontaneous differentiation: Cer1 was expressed in cells weakly positive for OCT4, while Gata4 and Sox17 were expressed in OCT4-negative cells. The IWP-2 treatment suppressed such developmental regulators by inhibiting WNT secretion, reducing the degree of spontaneous differentiation and thus producing a more homogeneous population of pluripotent EpiSCs. Whether or not “undifferentiated” EpiSCs cultured with IWP-2 differ significantly from EpiSCs cultured without IWP-2 remains to be clarified.

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