Figure 6.
Overexpression of Sox2 and Oct4 maintains ESC self-renewal and pluripotency in the absence of Rbbp4 in vitro and in vivo
(A) Schematic illustration of the embryoid body (EB) formation procedure for differentiation of ESCs.
(B) The diameter statistics of EB. Thirty 30 EB diameters of each genotype were measured at each time point.
(C) Phase-contrast images of floating EB derived from indicated ESCs at day 6 and day 12. Scale bar, 100 μm.
(D) qRT-PCR analysis of lineage-specific markers at day 0, day 6, and day 12 during EB formation from the indicated ESC lines. Fgf5, Nestin (ectoderm), Brachyury, Flk1 (mesoderm), and Gata4, Gata6 (endoderm). Data are normalized to the expression levels in ESCs. Error bars represent ± SD (n = 3).
(E) qRT-PCR mRNA analysis of pluripotency markers during time-course differentiation of Rbbp4F/F, and rescue of Rbbp4 in Rbbp4Δ/Δ EBs. Samples were collected at different days.
(F) In vivo differentiation potential of formation teratomas of indicated ESCs. Left: the morphological characteristics of teratomas are shown. Right: average teratoma size at 4 weeks after injection in the indicated groups.
(G) Histological analysis of teratomas. (A–F) Histological sections of hematoxylin and eosin-stained teratomas. (A, B, C, F) Each teratoma contained three embryonic germ layer tissues. (D and E) Immature germ layer differentiation is shown. Scale bar, 50 μm.
Data in (B and D–F) represent mean ± SD obtained from three independent experiments. ∗p < 0.05 and ∗∗p < 0.01 (Student's t test) compared with the control.