Klf4 reverts developmentally programmed restriction of ground state pluripotency

Abstract

Mouse embryonic stem (ES) cells derived from pluripotent early epiblast contribute functionally differentiated progeny to all foetal lineages of chimaeras. By contrast, epistem cell (EpiSC) lines from post-implantation epithelialised epiblast are unable to colonise the embryo even though they express the core pluripotency genes Oct4, Sox2 and Nanog. We examined interconversion between these two cell types. ES cells can readily become EpiSCs in response to growth factor cues. By contrast, EpiSCs do not change into ES cells. We exploited PiggyBac transposition to introduce a single reprogramming factor, Klf4, into EpiSCs. No effect was apparent in EpiSC culture conditions, but in ground state ES cell conditions a fraction of cells formed undifferentiated colonies. These EpiSC-derived induced pluripotent stem (Epi-iPS) cells activated expression of ES cell-specific transcripts including endogenous Klf4, and downregulated markers of lineage specification. X chromosome silencing in female cells, a feature of the EpiSC state, was erased in Epi-iPS cells. They produced high-contribution chimaeras that yielded germline transmission. These properties were maintained after Cre-mediated deletion of the Klf4 transgene, formally demonstrating complete and stable reprogramming of developmental phenotype. Thus, re-expression of Klf4 in an appropriate environment can regenerate the naïve ground state from EpiSCs. Reprogramming is dependent on suppression of extrinsic growth factor stimuli and proceeds to completion in less than 1% of cells. This substantiates the argument that EpiSCs are developmentally, epigenetically and functionally differentiated from ES cells. However, because a single transgene is the minimum requirement to attain the ground state, EpiSCs offer an attractive opportunity for screening for unknown components of the reprogramming process.

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.

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.

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.

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.