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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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Differentiation Potential of EpiSCs Isolated by the IWP-2 Method(A and B) Expression levels of Brachyury (T) (A) and GATA4 (B) were detected in B129a4 EpiSCs isolated by the IWP-2 method when they were cultured in medium without IWP-2 for 1 week.(C) Bright-field image of EBs formed from 129Ba2 EpiSCs isolated with IWP-2 treatment.(D) Immunofluorescence images for TUBB3 (red) and NESTIN (green), detecting neural differentiation in 129 Ba2 EBs.(E–J) Hematoxylin and eosin-stained sections of teratomas from 129Ba1 EpiSCs: (E) cartilage, (F) skeletal muscle, (G) adipocytes, (H) gastrointestinal epithelium, (I) melanocytes, and (J) neural tissue.Scale bars, 50 μm (A, B, and D–J) and 0.5 mm (C). See also Figures S2 and S3.
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fig3: Differentiation Potential of EpiSCs Isolated by the IWP-2 Method(A and B) Expression levels of Brachyury (T) (A) and GATA4 (B) were detected in B129a4 EpiSCs isolated by the IWP-2 method when they were cultured in medium without IWP-2 for 1 week.(C) Bright-field image of EBs formed from 129Ba2 EpiSCs isolated with IWP-2 treatment.(D) Immunofluorescence images for TUBB3 (red) and NESTIN (green), detecting neural differentiation in 129 Ba2 EBs.(E–J) Hematoxylin and eosin-stained sections of teratomas from 129Ba1 EpiSCs: (E) cartilage, (F) skeletal muscle, (G) adipocytes, (H) gastrointestinal epithelium, (I) melanocytes, and (J) neural tissue.Scale bars, 50 μm (A, B, and D–J) and 0.5 mm (C). See also Figures S2 and S3.

Mentions: When IWP-2 was withdrawn from the EpiSC culture medium, our EpiSCs changed their morphologies and started to differentiate into cells expressing the mesoderm marker T or the endoderm marker GATA4 (Figures 3A, 3B, and S2). By contrast, 129C1, the EpiSC line established by the original protocol, could be maintained as an undifferentiated form in the absence of IWP-2. Flow-cytometry analysis of SSEA1-positive cells indicated that our EpiSC line downregulated SSEA1 expression, whereas a higher percentage of 129C1 cells continued to express SSEA1 4 days after IWP-2 removal (Figure S2).


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)

Differentiation Potential of EpiSCs Isolated by the IWP-2 Method(A and B) Expression levels of Brachyury (T) (A) and GATA4 (B) were detected in B129a4 EpiSCs isolated by the IWP-2 method when they were cultured in medium without IWP-2 for 1 week.(C) Bright-field image of EBs formed from 129Ba2 EpiSCs isolated with IWP-2 treatment.(D) Immunofluorescence images for TUBB3 (red) and NESTIN (green), detecting neural differentiation in 129 Ba2 EBs.(E–J) Hematoxylin and eosin-stained sections of teratomas from 129Ba1 EpiSCs: (E) cartilage, (F) skeletal muscle, (G) adipocytes, (H) gastrointestinal epithelium, (I) melanocytes, and (J) neural tissue.Scale bars, 50 μm (A, B, and D–J) and 0.5 mm (C). See also Figures S2 and S3.
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Related In: Results  -  Collection

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fig3: Differentiation Potential of EpiSCs Isolated by the IWP-2 Method(A and B) Expression levels of Brachyury (T) (A) and GATA4 (B) were detected in B129a4 EpiSCs isolated by the IWP-2 method when they were cultured in medium without IWP-2 for 1 week.(C) Bright-field image of EBs formed from 129Ba2 EpiSCs isolated with IWP-2 treatment.(D) Immunofluorescence images for TUBB3 (red) and NESTIN (green), detecting neural differentiation in 129 Ba2 EBs.(E–J) Hematoxylin and eosin-stained sections of teratomas from 129Ba1 EpiSCs: (E) cartilage, (F) skeletal muscle, (G) adipocytes, (H) gastrointestinal epithelium, (I) melanocytes, and (J) neural tissue.Scale bars, 50 μm (A, B, and D–J) and 0.5 mm (C). See also Figures S2 and S3.
Mentions: When IWP-2 was withdrawn from the EpiSC culture medium, our EpiSCs changed their morphologies and started to differentiate into cells expressing the mesoderm marker T or the endoderm marker GATA4 (Figures 3A, 3B, and S2). By contrast, 129C1, the EpiSC line established by the original protocol, could be maintained as an undifferentiated form in the absence of IWP-2. Flow-cytometry analysis of SSEA1-positive cells indicated that our EpiSC line downregulated SSEA1 expression, whereas a higher percentage of 129C1 cells continued to express SSEA1 4 days after IWP-2 removal (Figure S2).

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