Ge Guo

Nanog Is the Gateway to the Pluripotent Ground State

Summary Pluripotency is generated naturally during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Pluripotency can be recreated by somatic cell reprogramming. Here we present evidence that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency. Production of pluripotent hybrids by cell fusion is promoted by and dependent on Nanog. In transcription factor-induced molecular reprogramming, Nanog is initially dispensable but becomes essential for dedifferentiated intermediates to transit to ground state pluripotency. In the embryo, Nanog specifically demarcates the nascent epiblast, coincident with the domain of X chromosome reprogramming. Without Nanog, pluripotency does not develop, and the inner cell mass is trapped in a pre-pluripotent, indeterminate state that is ultimately nonviable. These findings suggest that Nanog choreographs synthesis of the naive epiblast ground state in the embryo and that this function is recapitulated in the culmination of somatic cell reprogramming.

Klf4 reverts developmentally programmed restriction of ground state pluripotency

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.

Resetting Transcription Factor Control Circuitry toward Ground-State Pluripotency in Human

Summary Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.

Oct4 and LIF/Stat3 Additively Induce Krüppel Factors to Sustain Embryonic Stem Cell Self-Renewal

Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.

Stat3 Activation Is Limiting for Reprogramming to Ground State Pluripotency

Summary The cytokine leukemia inhibitory factor (Lif) sustains self-renewal of mouse embryonic and induced pluripotent stem cells by activating Jak kinase and the transcription factor Stat3. Here we investigate whether Jak/Stat3 may also contribute to induction of pluripotency. EpiSCs derived from postimplantation embryos express low levels of Lif receptor and Stat3. We introduced into EpiSCs a Jak/Stat3 activating receptor (GY118F) responsive to granulocyte colony stimulating factor (Gcsf). On transfer to ground state culture, in which MAPK signaling and glycogen synthase kinase are inhibited, Gcsf induced transcriptional resetting and functional reprogramming. Activation of a tamoxifen-regulatable fusion, Stat3ERT2, also converted EpiSCs into chimera-competent iPSCs. We exploited GY118F to increase Jak/Stat3 activity during somatic cell reprogramming. Incompletely reprogrammed cells derived from neural stem cells or fibroblasts responded to Gcsf with elevated frequencies of progression to ground state pluripotency. These findings indicate that Jak/Stat3 participate directly in molecular reprogramming and that activation of this pathway is a limiting component.

A genome-wide screen in EpiSCs identifies Nr5a nuclear receptors as potent inducers of ground state pluripotency

In rodents, the naïve early epiblast undergoes profound morphogenetic, transcriptional and epigenetic changes after implantation. These differences are maintained between blastocyst-derived embryonic stem (ES) cells and egg cylinder-derived epiblast stem cells (EpiSCs). Notably, ES cells robustly colonise chimaeras, whereas EpiSCs show little or no contribution. ES cells self-renew independently of mitogenic growth factors, whereas EpiSCs require fibroblast growth factor. However, EpiSCs retain the core pluripotency factors Oct4 and Sox2 and the developmental barrier dividing them from unrestricted pluripotency can be surmounted by a single reprogramming factor. This provides an opportunity to identify molecules that can reset the naïve state. We undertook a forward genetic screen for effectors of EpiSC reprogramming, employing piggyBac transposition to activate endogenous gene expression at random and selecting for undifferentiated colonies in the absence of growth factor signalling. Three recovered clones harboured integrations that activate the closely related orphan nuclear receptor genes Nr5a1 and Nr5a2. Activity of Nr5a1 and Nr5a2 was confirmed by direct transfection. Reprogrammed colonies were obtained without transgene integration and at 10-fold higher frequency than with other single factors. Converted cells exhibited the diagnostic self-renewal characteristics, gene expression profile and X chromosome activation signature of ground state pluripotency. They efficiently produced adult chimaeras and gave germline transmission. Nr5a receptors regulate Oct4 transcription but this is insufficient for reprogramming. Intriguingly, unlike previously identified reprogramming molecules, Nr5a receptors play no evident role in ES cell self-renewal. This implies a different foundation for their capacity to reset pluripotency and suggests that further factors remain to be identified.

Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass

Summary Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.

A PiggyBac-Based Recessive Screening Method to Identify Pluripotency Regulators

Phenotype driven genetic screens allow unbiased exploration of the genome to discover new biological regulators. Bloom syndrome gene (Blm) deficient embryonic stem (ES) cells provide an opportunity for recessive screening due to frequent loss of heterozygosity. We describe a strategy for isolating regulators of mammalian pluripotency based on conversion to homozygosity of PiggyBac gene trap insertions combined with stringent selection for differentiation resistance. From a screen of 2000 mutants we obtained a disruptive integration in the Tcf3 gene. Homozygous Tcf3 mutants showed impaired differentiation and enhanced self-renewal. This phenotype was reverted in a dosage sensitive manner by excision of one or both copies of the gene trap. These results provide new evidence confirming that Tcf3 is a potent negative regulator of pluripotency and validate a forward screening methodology to identify modulators of pluripotent stem cell biology.

Epigenetic resetting of human pluripotency

Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells. Highlighted article: A simple, transgene-free method is described for resetting human ESCs or iPSCs to a stable naïve status via transient histone deacetylase inhibition.

Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.

ABSTRACT Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies have provided transcriptome data from human pre-implantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors, such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here, we address these issues to assemble a complete gene expression time course spanning human pre-implantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post-hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naïve state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology. Highlighted Article: A comprehensive analysis of single-cell RNA-seq data from human pre-implantation embryos resolves cell-type ambiguities and defines consensus transcriptomes for emergent embryonic lineages.