Embryonic stem cell potency fluctuates with endogenous retrovirus activity

Journal name:
Nature
Volume:
487,
Pages:
57–63
Date published:
DOI:
doi:10.1038/nature11244
Received
Accepted
Published online

Abstract

Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.

At a glance

Figures

  1. The MuERV-L retrovirus and a reporter driven by its LTR marks the 2C state.
    Figure 1: The MuERV-L retrovirus and a reporter driven by its LTR marks the 2C state.

    a, Comparison of gene expression between oocytes and 2C embryos. Genes generating junctions to MuERV-L are shown in red and green, with those in red denoting significant change in expression. b, ORF status of predicted MuERV-L-linked chimaeric transcripts. c, Gene Ontology (GO) analysis of MuERV-L-linked protein-coding transcripts. The number of genes from the ten most enriched GO categories are shown. d, e, 2C (d) and blastocyst (e) embryos were mixed and immunostained with MuERV-L-Gag and Oct4 antibodies. Scale bars, 20μm. f, Zygotes were injected with the 2C::tdTomato transgene, and allowed to develop in vitro for 48h before imaging. DIC, differential interference contrast. Scale bar, 50μm. g, 2C::tdTomato+ ES cells express MuERV-L-Gag protein, as detected by immunofluorescence. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 50μm. h, Microarray analysis of 2C::tdTomato+ and 2C::tdTomato cells. Red indicates genes with a greater than fourfold change in expression. i, 2C::tdTomato+ MuERV-L-Gag+ ES and iPS cells lack Oct4 protein, as determined by immunofluorescence. Scale bars, 20μm.

  2. ES cells enter the 2C state regularly, but remain in the state transiently owing to cell intrinsic and extrinsic factors.
    Figure 2: ES cells enter the 2C state regularly, but remain in the state transiently owing to cell intrinsic and extrinsic factors.

    a, FACS analysis of 2C::ERT2-Cre-ERT2, ROSA::LSL-tdTomato ES cells at increasing passage (P) in the presence of 4HT. The percentage of tdTomato+ cells is indicated. b, 2C::ERT2-Cre-ERT2, ROSA::LSL-LacZ ES cells were cultured in the presence of 4HT, and at increasing passage, cells were fixed and immunostained with anti-β-galactosidase antibodies and counterstained with DAPI. Scale bars, 50μm. c, 2C::tdTomato+ and 2C::tdTomato cells were collected by FACS and plated before imaging 48h later. Scale bars, 50μm. d, 2C::tdTomato ES cells were cultured in 20% O2 (normoxia) or 5% O2 (hypoxia) for 48h, or 48h sequentially, and the percentage of tdTomato+ cells was determined by FACS. Error bars represent s.d., n = 3 e, 2C::tdTomato ES cells at the indicated passage were cultured in media containing 15% fetal calf serum (FCS), 20% knockout serum replacement (KOSR) or N2B27 media containing 3mM glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein-kinase kinase (MEK) inhibitors (2i) for 48h before counting the percentage of 2C::tdTomato+ cells by FACS. Error bars represent s.d., n = 3.

  3. The 2C state is associated with an active epigenetic signature and is antagonized by repressive chromatin-modifying enzymes.
    Figure 3: The 2C state is associated with an active epigenetic signature and is antagonized by repressive chromatin-modifying enzymes.

    a, 2C::tdTomato+ (+) and 2C::tdTomato (−) cells were collected by FACS and subjected to immunoblot analysis with indicated antibodies. H3K4me2, histone H3 dimethyl Lys4; AcH3, acetylated histone H3. b, Pairwise comparisons of the number of genes activated in Kap1, G9a and Kdm1a mutant ES cells compared with genes activated in 2C embryos. c, ES cell lines homozygous for mutant alleles of Kdm1a, Kap1 and G9a, and corresponding wild-type (WT) ES lines were immunostained with MuERV-L-Gag antibodies and counterstained with DAPI. GT, gene trap; KO, knockout. Scale bars, 50μm. d, 2C::tdTomato ES cells were treated with 40nM trichostatin A (TSA) for 24h before imaging. Scale bars, 50μm. e, Kdm1afl/fl; Cre-ERT ES cells containing a stably integrated 2C::tdTomato transgene were treated with vehicle or 4HT and subject to FACS analysis to determine the percentage of tdTomato+ cells. f, 2C::tdTomato; Kdm1afl/fl; Cre-ERT ES cells were treated with 4HT or vehicle for 24h, then passaged for 72h before collecting tdTomato cells by FACS. The percentage of tdTomato+ cells was plotted after increasing hours in culture. Error bars represent s.d., n = 3.

  4. Activation of the 2C state is associated with expanded potency in chimaeric embryos towards extraembryonic lineages.
    Figure 4: Activation of the 2C state is associated with expanded potency in chimaeric embryos towards extraembryonic lineages.

    a, 2C::tdTomato+ or 2C::tdTomato cytomegalovirus (CMV)–GFP ES cells were injected into morula-stage embryos, which were then grown in vitro. The resulting blastocysts were imaged to visualize the position of injected cells in either the trophectoderm (TE) or ICM. Scale bars, 20μm. b, 2C::tdTomato+ or 2C::tdTomato, Ef1a::GFP+ cells were injected into blastocysts that were then implanted into pseudopregnant females to generate chimaeric embryos. Arrows indicate 2C::tdTomato+, GFP+ cells contributing to the yolk sac and placenta. Bright denotes bright-field microscopy. c, 2C::tdTomato+, Ef1a::GFP+ cells contribute to embryonic endoderm, mesoderm, ectoderm, yolk sac, placental tissues (including giant trophoblast cells, white arrows) and primordial germ cells (PGCs, colabelled with anti-Ddx4 antibody in red, blue arrows). Scale bars, 500μM (endoderm, mesoderm, ectoderm and yolk sac) and 50μm (placenta and PGCs). d, Heterozygous (+/GT) or homozygous (GT/GT) Kdm1a-β-geo gene-trap ES cells were injected into wild-type blastocysts that were implanted into pseudopregnant females. (β-geo is a fusion of β-galactosidase and neomycin-resistance genes.) Embryonic (E) and extraembryonic (X) tissues were separated from chimaeric embryos, and subject to semiquantitative PCR with β-geo primers to determine the relative contribution of the injected cells to these lineages. Error bars represent s.e.m. e, A 1:1 mixture of Kdm1afl/fl control and Kdm1a knockout (KO) ES cells were co-injected into wild-type blastocysts. At embryonic day 12.5, chimaeric embryonic (E) tissue was separated from placenta (P), yolk sac (Y) and amnion (A) and subject to PCR to detect the floxed (fl) and knockout alleles of the injected cells relative to wild-type alleles of the resident injected embryo. M denotes PCR marker. f, Kdm1aGT/GT, Ef1a::GFP+ cells contribute to embryonic endoderm, mesoderm, ectoderm, yolk sac, placental tissues (including giant trophoblast cells, white arrow) and primordial germ cells (PGCs, colabelled with anti-Ddx4 antibody in red, blue arrow). Scale bars, 500μM (endoderm, mesoderm, ectoderm and yolk sac) and 50μm (placenta and PGCs).

  5. Model of the role of the MuERV-L-LTR-linked 2C gene network in regulating embryonic potency.
    Figure 5: Model of the role of the MuERV-L-LTR-linked 2C gene network in regulating embryonic potency.

    a, During zygote genome activation, a network of genes that use MuERV-L-LTRs as promoters is activated. This stage correlates with a period in which blastomeres are totipotent. As development progresses, the MuERV-L-LTR-linked 2C gene network is silenced by chromatin repressors, as the ICM segregates from the trophectoderm and primitive endoderm (PrE). HDACs, histone deacetylases. b, During the derivation of ES cells from blastocysts, a rare transient population of cells marked by the 2C::tdTomato reporter expresses high levels of 2C genes and low levels of pluripotency markers. In mouse chimaera assays, these cells contribute to embryonic and extraembryonic tissues (shown in green). Increasing the oxidative tension of ES cell cultures or deletion/inhibition of repressive histone-modifying enzymes alters the equilibrium between the 2C and ES states.

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Primary accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Howard Hughes Medical Institute, Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines, La Jolla, California 92037, USA

    • Todd S. Macfarlan,
    • Wesley D. Gifford,
    • Shawn Driscoll,
    • Karen Lettieri,
    • Dario Bonanomi &
    • Samuel L. Pfaff
  2. School of Life Sciences, Ecole Polytechnique Federale de Lausanne (EPFL), 1015 Lausanne, Switzerland

    • Helen M. Rowe &
    • Didier Trono
  3. Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines, La Jolla, California 92037, USA

    • Amy Firth &
    • Oded Singer
  4. Present address: Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

    • Todd S. Macfarlan

Contributions

T.S.M. designed and performed all experiments with assistance from W.D.G., S.D., D.B. and K.L. under the supervision of S.L.P. D.T. generated Kap1 mutant ES cells and H.M.R. and D.T. provided mRNA-seq data from these cells. A.F. and O.S. generated and provided iPS cell lines and lentivirus constructs. T.S.M., W.D.G. and S.L.P. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Microarray and RNA-seq files have been submitted to the NCBI Gene Expression Omnibus database under accession GSE33923.

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Supplementary information

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  1. Supplementary Information (4M)

    This file contains Supplementary Figures 1-11 and legends for Supplementary Movies 1-3.

Zip files

  1. Supplementary Tables (1.6M)

    This zipped file contains Supplementary Tables 1-7.

Movies

  1. Supplementary Movie 1 (543K)

    The 2C::tomato reporter is restricted to the zygote and 2C/4C stage - see Supplementary Information fie for full legend.

  2. Supplementary Movie 2 (2.1M)

    2C::tomato is transiently expressed in ES cultures - see Supplementary Information fie for full legend.

  3. Supplementary Movie 3 (2.1M)

    Entrance into the 2C::tomato (+) state is more rapid in Kdm1a mutant ES cells - see Supplementary Information fie for full legend.

Additional data