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First published online 18 February 2009
doi: 10.1242/dev.030957

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Klf4 reverts developmentally programmed restriction of ground state pluripotency

Ge Guo^1,2, Jian Yang^1,2, Jennifer Nichols^1,3, John Simon Hall^1,2,*, Isobel Eyres¹, William Mansfield¹ and Austin Smith^1,2,

¹ Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.
² Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.
³ Department of Physiology, Development and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.

View larger version (54K): [in this window] [in a new window]   Fig. 1. EpiSCs are distinct from, and do not spontaneously convert to, ES cells. (A) Phase contrast and fluorescence images of established EpiSC line. (B) qRT-PCR analysis of marker gene expression in ES cells and EpiSCs. ES, ES cells in 2i/Lif. Epi6 and Epi7 are two independent EpiSC lines. y-axis, relative expression normalised to Gapdh. (C) Immunostaining of male and female EpiSCs for me3H3K27 and Oct4. White arrow indicates focus of staining diagnostic of an inactive X chromosome. (D) EpiSCs lose Oct4 expression and differentiate or die in 2i/Lif. AF, EpiSC cultured in activin A plus Fgf2. (E) qRT-PCR analysis of ES cell differentiation into EpiSCs upon culture in Fgf2 and activin. Epi3 and Epi10 indicate cells cultured in Fgf2 and activin for three and ten passages, respectively. y-axis, relative expression normalised to Gapdh. (F) Oct4 and me3H3K27 immunostaining of female ES cell-derived EpiSCs. EpiSCs both express Oct4 and exhibit a nuclear body indicative of the inactive X (white arrow). Blue arrow indicates a dividing cell.

View larger version (48K): [in this window] [in a new window]   Fig. 2. Klf4 neither prevents ES cell differentiation into EpiSCs nor converts an EpiSC population into ES cells in the presence of activin and FGF. (A) qRT-PCR analysis of Lif induction of Klf4 in ES cells but not in EpiSCs. Cells were stimulated with Lif (+LIF) for 1 hour. (B) ES cells constitutively expressing Klf4 acquire an EpiSC marker profile in Fgf2 plus activin A. MT, empty vector transfectants. P0, P2 and P10 indicate passage numbers in Fgf2/activin. (C) Constitutive Klf4 expression permits continued recovery of ES cell colonies after culture in activin and Fgf2. One thousand cells were plated for each sample in triplicate at the indicated passage (P) number. MT, empty vector transfectants; K4, Klf4 transfectants. (D) PiggyBac vector for expression of Klf4 (pGG137Klf4), and control PiggyBac vector (pGG131). Arrows (P) indicate PCR primers used to amplify the PB LTR fragment after Cre-mediated recombination. (E) Hygromycin-selected Klf4 and control vector-transfected EpiSCs. (F) qRT-PCR analysis showing that forced Klf4 expression does not induce ES cell marker gene expression in EpiSC culture. ES, ES cells; Epi, EpiSCs; Vec, EpiSC transfected with control vector pGG131; Klf4, EpiSCs transfected with pGG137Klf4. y-axis, relative expression normalised to Gapdh.

View larger version (68K): [in this window] [in a new window]   Fig. 3. EpiSCs transfected with Klf4 can convert to ground state pluripotency. (A) Oct4-positive colonies obtained by transfection with Klf4 and transfer to 2i/Lif after 72 hours. Images were taken after 9 days in 2i/Lif. (B) qRT-PCR analysis of marker gene expression in ES cells, EpiSCs and derivative Epi-iPS cells isolated in 2i/Lif. y-axis, relative expression normalised to Gapdh. (C) me3H3K27 staining of female EpiSCs and derivative Epi-iPS cells. (D) Images of Epi-iPS colonies after 10 days in 2i/Lif, showing mutually exclusive expression of DsRed and Oct4-GFP. (E) Flow cytometry analysis of four expanded Epi-iPS clones. Two clones retain weak but detectable red fluorescence. (F) qRT-PCR analysis of Klf4 transgene and DsRed expression in Epi-iPS cell clones and parental EpiSC line. y-axis, relative expression normalised to Gapdh. (G) Chimeric mouse produced from the K4C12 Epi-iPS clone and agouti germline offspring.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Retention of ground state pluripotency after transgene excision. (A) Splinkerette-PCR reveals 1-3 PB insertions in each iPS clone. (B) Flow cytometry showing the DsRed-negative population in the K4C3 line before and after Cre transfection. (C) Genomic PCR showing loss of the Klf4 transgene and gain of the PB-LTR fragment in two revertant clones. (D) RT-PCR analysis showing the lack of Klf4 transgene and DsRed expression in expanded Cre-reverted cells. (E) Marker gene expression in Cre-reverted Epi-iPS cells (iPS-Cre) compared with ES cells and EpiSCs. Data are for two independent Cre-revertant clones derived from each of Epi-iPS cell lines K4C3 and K4C5. y-axis, relative expression normalised to Gapdh. (F) Maintained morphology and Oct4-GFP expression in a Cre-reverted Epi-iPS cell line. (G) me3H3K27 staining of Klf4 transgene-deleted iPS cells as compared with parental EpiSCs. (H) Chimeric mouse made with revertant K4C3-A3 cells, and agouti offspring denoting germline transmission.

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