Figure 1.
E-cadherin-Overexpressing Mouse EpiSCs Show Highly Efficient Conversion to Naive-like PSCs in the Presence of LIF
(A) Schematic diagram of doxycycline-inducible system for the expression of E-cadherin transgene.
(B) Western blotting analyses for E-CADHERIN in mouse EB3DR ESCs (ESC), mouse EB3DR epiblast stem cells (EpiSC), and transgenic mouse EB3DR EpiSCs cultured with or without doxycycline (2 μg/ml) for 2 days (Dox−, Dox+). β-ACTIN was used as a loading control.
(C) FACS analysis of E-CADHERIN expression levels in transgenic mouse EB3DR EpiSCs cultured with (blue line) or without (red line) doxycycline for 2 days.
(D) Time schedule of all experiments with or without doxycycline in mouse EB3DR EpiSCs. Culture conditions were defined as primed (ESM + bFGF) and naive (N2B27 + LIF).
(E) FACS analysis of CD31-positive cells in mouse EB3DR EpiSC (DsRed) under various conditions. Shown are the percentages of CD31-expressing cells after culturing with or without doxycycline for 7 days under primed (ESM + bFGF) and naive (N2B27 + LIF) conditions.
(F) Average of PECAM1-expressing cells associated with (E). The frequency of PECAM1-expressing cells is significantly increased in E-cadherin upregulated mouse EpiSCs in the presence of LIF (mean ± SEM of three independent experiments, ∗p < 0.05).
(G) Timing and efficiency of PECAM1 expression. In the presence of LIF, shifts of E-cadherin upregulated mouse EpiSCs to PECAM1-expressing status differ significantly from those seen under other conditions (mean ± SEM of three independent experiments, ∗p < 0.05, and ∗∗p < 0.01).
(H) Photomicrograph of PECAM1-expressing cells obtained from E-cadherin upregulated mouse EpiSCs in the presence of LIF. Almost all PECAM1-expressing cells occurred as dome-like compact colonies. Scale bar, 50 um.
(I) Live-born chimeric mice obtained from DsRed-marked mouse Ecad-rESCs.
(J) Chimeric mice obtained by injection of mouse E-cad-rESCs (agouti) into BDF1 × C57BL/6 blastocysts (black) show coat-color contribution from mouse E-cad-rESCs.