Embryonic stem cell potency fluctuates with endogenous retrovirus activity

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.

Figure 1. The MERVL 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 MERVL are shown in red/green, with those in red showing significant change in expression. b, ORF status of predicted MERVL-linked chimeric transcripts. c, GO analysis of MERVL-linked protein coding transcripts. The number of genes from the 10 most enriched GO categories are shown. d-e, 2C (d) and blastocyst embryos (e) were mixed and immunostained with MERVL-Gag and Oct4 antibodies. Scale bar 20 μm. f, Zygotes were injected with the 2C::tomato transgene, and allowed to develop in vitro for 48 hours before imaging. Scale bar 50μm. g, 2C::tomato ⁺ ES cells express MERVL-Gag protein detected by immunofluorescence. Scale bar 50μm. h, Microarray analysis of 2C::tomato⁺ vs. ⁻ cells. Red indicates genes with greater than 4-fold change in expression. i, 2C::tomato⁺/MERVL-Gag⁺ ES cells and iPS cells lack Oct4 protein as determined by immuno-fluorescence. Scale bar 20μm

Figure 2. The 2C epigenetic state is transient, is activated within the majority of cells in ES cultures at increasing passages, and is controlled by cell intrinsic and extrinsic factors

a,, FACS analysis of 2C::ERT2-Cre-ERT2, ROSA LSL::Tomato ES cells at increasing passage in the presence of 4OHT. The percentage of tomato⁺ cells is indicated. b, 2C::ERT2-Cre-ERT2, LSL::LacZ ES cells were cultured in the presence of 4OHT, and at increasing passage, cells were fixed and immunostained with anti Beta-galactosidase antibodies and counterstained with DAPI. Scale bar 50μm. c, 2C::tomato⁺ and ⁻ cells were collected by FACS and plated before imaging 48 hours later. Scale bar 50μm. d, 2C::tomato ES cells were cultured in 20% O₂ (normoxia) or 5% O₂ (hypoxia) for the indicated number of hours, and the percentage of tomato⁺ cells was determined by FACS. Error bars represent s.d., n=3 e, 2C::tomato 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 GSK3β and MEK inhibitors (2i) for 48 hours before counting the percentage of 2C::tomato⁺ cells by FACS. Error bars represent s.d., n=3.

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

a, 2C::tomato⁺ and ⁻ cells were collected by FACS and subjected to immunoblot analysis with indicated antibodies. 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 ES lines were immunostained with MERVL Gag antibodies and counterstained with DAPI. Scale bar 50 μm. d, 2C::tomato ES cells were treated with 40nM TSA for 24 hours before imaging. Scale bar 50μm. e, Kdm1a Fl/Fl, Cre-ERT ES cells containing a stably integrated 2C::tomato transgene were treated with vehicle or 4OHT and subject to FACS to count the percentage of tomato⁺ cells. f, 2C::tomato; Kdm1a Fl/Fl, Cre:ERT ES cells were treated with 4OHT or vehicle for 24 hours, then passaged for 72 hours before collecting tomato negative cells by FACS. The percentage of tomato⁺ cells was plotted after increasing hours in culture. Error bars represent s.d., n=3.

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

a, 2C::tomato⁺ or 2C::tomato⁻ ,CMV::GFP ES cells were injected into morula-stage embryos, which were then grown in vitro. The resulting blastocysts were imaged to visualize position of injected cells in either the trophectoderm (TE) or ICM. Scale bar 20μm. b, 2C::tomato⁺ or 2C::tomato⁻ , Ef1a::GFP⁺ cells were injected into blastocysts which were then implanted into pseudopregnant females to generate chimeric embryos. Arrows indicate 2C::tomato⁺, GFP⁺ cells contributing to the yolk sac and placenta. c, 2C::tomato⁺, Ef1a::GFP⁺ cells contribute to embryonic endoderm, mesoderm, ectoderm, yolk sac, placental tissues (including trophoblast giant cells-white arrows) and primordial germ cells (PGCs, colabeled with anti-Ddx4 antibody in red, blue arrows). Scale bars represent 500uM (Endoderm, mesoderm, ectoderm, yolk sac) or 50um (placenta and PGCs). d, Heterozygous (+/GT) or homozygous (GT/GT )Kdm1a-ßgeo genetrap ES cells were injected into wild type blastocysts which were implanted into pseudopregnant females. Embryonic (E) and extraembryonic (X) tissues were separated from chimeric embryos, and subject to semi-quantitative PCR with ßgeo 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 Kdm1a Fl/Fl and KO/KO ES cells were co-injected into wild type blastocysts. At E12.5, chimeric embryonic tissue (E) was separated from placenta (P), yolk sac (Y) and amnion (A) and subject to PCR to detect the FL and KO alleles of the injected cells relative to WT alleles of the resident injected embryo. F, Kdm1a GT/GT, Ef1a::GFP⁺ cells contribute to embryonic endoderm, mesoderm, ectoderm, yolk sac, placental tissues (including trophoblast giant cells-white arrows) and primordial germ cells (PGCs, colabeled with anti Ddx4 antibody in red, blue arrows). Scale bars represent 500uM (Endoderm, mesoderm, ectoderm, yolk sac) or 50um (placenta and PGCs).

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

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