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. 2018 Feb 15;10(3):1102–1114. doi: 10.1016/j.stemcr.2018.01.014

Figure 4.

Figure 4

T-UCstem1 Sustains ESC Self-Renewal In Vitro and In Vivo

(A) Representative pictures of crystal violet-stained and colony-type frequency (∼100 colonies scored/condition) of colonies generated from Control (NT) and T-UCstem1 KD ESCs, at day 6 after plating. Data are mean ± SEM (n = 3 independent experiments); ∗∗p < 0.005. Scale bar, 100 μm.

(B) Representative immunofluorescence of NANOG/OCT4 and SOX2/OCT4 of Control (NT) and KD ESC colonies at day 6 after plating. Nuclei were stained with DAPI. Scale bars, 75 μm.

(C) qRT-PCR analysis of pluripotency (Nanog, Sox2, and Oct4), mesodermal (Fgf5 and Brachyury [Bra]), neural (Sox1 and Nestin), and endodermal-associated genes (Foxa2 and Sox17) in Control (NT) and two independent T-UCstem1 KD ESC clones. Relative RNA level was normalized to Gapdh expression. Data are mean ± SEM (n = 3 independent experiments); ∗p < 0.01, ∗∗p < 0.005, ∗∗∗p < 0.001.

(D) Representative pictures of NESTIN, BRA, and SOX17 immunostaining in KD ESC colonies. Nuclei were stained with DAPI. Scale bars, 75 μm.

(E) Representative pictures of alkaline phosphatase (AP)-stained colonies generated from Control (NT), KD, and KD + 2i (CHIR99021 + PD0325901) ESCs, at day 6 after plating. Scale bar, 100 μm.

(F) Western blot analysis of PARP full-length form and cleaved fragment, p21, NANOG, and SOX2 in Control (NT), KD, and KD + 2i (CHIR99021 + PD0325901) ESCs, at day 6 after plating. GAPDH was used as a loading control.

(G) Representative photomicrograph by Axio Zoom.V16 Zeiss microscopy (original magnification ×10) of chimeric embryos from EGFP-labelled T-UCstem1 KD ESCs injected into morula and dissected at embryonic day 9.5 (E9.5).

(H) Effect of T-UCstem1 depletion on teratoma formation. Representative picture of an immunodeficient mouse injected with Control (NT) and T-UCstem1KD ESCs, and the dissected ectopic tissues (left panel). Quantitative analysis of tissue weights (right panel). Data are mean ± SEM (seven mice/group).

(I) Immunohistochemistry analysis showing tissues deriving from ectoderm (nestin; a, a'), mesoderm (MF-20; b, b'). Scale bars, 75 μm., H&E staining displaying endoderm derivatives (glandular epithelial structures; c, c'). Scale bar, 150 μm. See also Figures S4 and S5.