Delineation of the domains of Klf2 required for the self-renewal of mouse ES cells.

<p>(A) Schematic illustration of Klf2 deletion (Δ) mutants. Klf2 is highly homologous to Klf4 and belongs to the same subclass. A FLAG-HA tandem epitope tag was fused to the N-terminus of each of the deletion mutants. The zinc finger domain is shown as green boxes. (B) Western blot analyses of Klf2 and Klf2 deletion mutants with an anti-HA antibody. Arrowheads indicate HA-Klf proteins. (C) Percentage of AP-positive colonies in ES cells overexpressing full-length Klf2 or Klf2 deletion mutants, grown in the presence or absence of LIF. (D) Expression of the pluripotency-related marker Rex1. Rex1-GFP expression is seen in full-length Klf2, and N-terminal deletion mutants (N1, N2) in the absence of LIF. Scale bar: 100 μm. Asterisks and hash symbols indicate statistical significance. *<i>P</i> < 0.05 vs. empty vector; # <i>P</i> < 0.05 vs. full-length Klf; * <i>P</i> < 0.01; n.s.; not significant; n.d.; not detected.</p

Comprehensive identification of <i>Krüppel-like factor</i> (<i>Klf</i>) family members whose overexpression achieves leukemia inhibitory factor (LIF)-independent self-renewal of mouse embryonic stem (ES) cells.

<p>(A) A schematic representation of Klfs. These proteins have highly homologous C-terminal DNA binding domains characterized by three C₂H₂ zinc finger motifs, shown in green boxes. The number of amino acids is shown to the left. (B) Western blot of proteins from mouse ES cells overexpressing FLAG-HA epitope-tagged Klf. Anti-HA and anti-β-actin antibodies were used to detect HA-tagged Klf and endogenous β-actin, respectively. Arrowheads indicate HA-tagged Klf proteins. (C) A schematic illustration of the experimental outline to assay the ability of a Klf protein to maintain self-renewal. (D) Generation of colonies from ES cells carrying either the empty vector or a Klf expression vector in the presence or absence of LIF. Scale bar: 2 mm. (E) The percentage of AP-positive colonies. Asterisks indicate statistical significance. *<i>P</i> < 0.05; **<i>P</i> < 0.01.</p

Expression of pluripotency-related markers in Klf-overexpressing mouse ES cells.

<p>(A) Immunostaining for Nanog in mouse ES cells overexpressing (OE) a Klf protein, cultured in the presence or absence of LIF. (B) Rex1-GFP expression in Klf-overexpressing ES cells grown in the presence or absence of LIF. Scale bar: 100 μm.</p

Chimera formation with iPSCs reprogrammed from EpiSCs.

<p>(A) Chimeras at E9.5 generated from two independent iPSC lines (#87 and 89). (B) Chimera and its genital ridge at E13.5 generated from iPSC line #89. Scale bar: 1 mm.</p

Reprogramming of <i>Fgf5-P2A-Venus</i> BAC Tg mEpiSCs into miPSCs.

<p>(A) Experimental scheme for reprogramming of the Tg mEpiSCs into miPSCs. The Tg mEpiSCs stably expressing <i>Klf5</i> or <i>Nanog</i> were cultured in NDiff227 medium supplemented with the Mek inhibitor (PD0325901), Gsk3 inhibitor (CHIR99021) and LIF. After 7 days, miPSC colonies were subjected to immunostaining. (B) Immunostaining for Venus (anti-GFP, green), CD31 (purple) and Nanog (red) in untransfected, vector control and miPSCs. OE: overexpression. Scale bar: 100 μm. (C) RT-qPCR analysis of Tg mEpiSCs and miPSCs. The mean and SD of two independent experiments are shown. *<i>P</i> < 0.05.</p

Identification of the ability of Klf family members to reprogram EpiSCs into iPSCs.

<p>(A) Experimental outline for evaluating the reprogramming activity of a Klf protein. EpiSCs carrying Oct3/4-∆PE-GFP were cultured in N2B27 medium supplemented with LIF, the Mek inhibitor PD0325901, and the Gsk3 inhibitor CHIRON (CHIR99021) for 5–7 days and were then subjected to AP assays. (B) Western blot analysis of EpiSCs expressing epitope-tagged Klf. (C) Percentage of AP-positive colonies generated from EpiSCs overexpressing a Klf protein. (D) RT-qPCR analysis of iPSCs. (E) Immunostaining for Nanog in iPSCs generated by overexpression of Klf2, Klf4 and Klf5. (F) GFP fluorescence in EpiSCs generated by Klf2, Klf4 and Klf5 overexpression. Scale bar: 100 μm. Asterisks indicate statistical significance. *<i>P</i> < 0.05; **<i>P</i> < 0.01; n.s.; not significant.</p

ChIP analysis of the binding of epitope-tagged Klf to <i>Nanog</i> regulatory regions in mouse ES cells.

<p>(A) Schematic representation of the <i>Nanog</i> proximal promoter and distal enhancer. Two key regulatory regions (the enhancer and proximal promoter) of <i>Nanog</i> are shown as red and blue bars, respectively. Horizontal arrows indicate the primers used for PCR in site-specific ChIP assays. (B) ChIP analysis of the binding of Klf to the promoter and enhancer of <i>Nanog</i>. Asterisks indicate statistical significance. *<i>P</i> < 0.05; **<i>P</i> < 0.01; n.s.; not significant.</p

Visualization of the Epiblast and Visceral Endodermal Cells Using <i>Fgf5-P2A-Venus</i> BAC Transgenic Mice and Epiblast Stem Cells

<div><p><i>Fibroblast growth factor 5</i> (<i>Fgf5</i>) has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs) in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells <i>in vivo</i> and <i>in vitro</i>. Here, we report for the first time the generation of <i>Fgf5-P2A-Venus</i> BAC transgenic (Tg) mice and show that the BAC Tg can recapitulate endogenous <i>Fgf5</i> expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that <i>Fgf5-P2A-Venus</i> BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even <i>Fgf5</i> expression shows dynamic heterogeneity in mEpiSCs. Taken together, <i>Fgf5-P2A-Venus</i> BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research.</p></div

Derivation and characterization of <i>Fgf5-P2A-Venus</i> BAC Tg mEpiSCs.

<p>(A) Immunofluorescence analysis of the Tg mEpiSCs for Venus (anti-GFP, green), Oct3/4 (purple), Nanog (red) and Nuclei (Hoechst33342, blue). Scale bar: 20 μm. All images were captured by a Leica TCS-SP8 confocal microscope using a 63x/1.4 oil objective lens. (B) RT-qPCR analysis of genes associated with pluripotency and lineage commitment in the Tg mEpiSCs and mESCs. <i>β-actin</i> was used as endogenous control for normalization. The mean and SD of three independent experiments are shown. *<i>P</i> < 0.05. (C) Venus expression in control and the Tg mEpiSCs was analyzed by flow cytometry.</p

Delineation of Klf2 domains required for reprogramming EpiSCs to iPSCs.

<p>(A) Western blot analysis of EpiSCs expressing epitope-tagged Klf2 and deletion (Δ) mutants. Closed arrowheads indicate epitope-tagged Klf2 proteins. (B) Fluorescence images of a reprogrammed Oct3/4-ΔPE-GFP EpiSC colony with a Klf2 deletion domain on day 7. Open arrowheads indicate GFP fluorescence. OE, overexpression. Scale bar: 100 μm.</p