Mapping systemic lupus erythematosus heterogeneity at the single-cell level.

Patients with systemic lupus erythematosus (SLE) display a complex blood transcriptome whose cellular origin is poorly resolved. Using single-cell RNA sequencing, we profiled ~276,000 peripheral blood mononuclear cells from 33 children with SLE with different degrees of disease activity and 11 matched controls. Increased expression of interferon-stimulated genes (ISGs) distinguished cells from children with SLE from healthy control cells. The high ISG expression signature (ISGhi) derived from a small number of transcriptionally defined subpopulations within major cell types, including monocytes, CD4+ and CD8+ T cells, natural killer cells, conventional and plasmacytoid dendritic cells, B cells and especially plasma cells. Expansion of unique subpopulations enriched in ISGs and/or in monogenic lupus-associated genes classified patients with the highest disease activity. Profiling of ~82,000 single peripheral blood mononuclear cells from adults with SLE confirmed the expansion of similar subpopulations in patients with the highest disease activity. This study lays the groundwork for resolving the origin of the SLE transcriptional signatures and the disease heterogeneity towards precision medicine applications.

Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow.

Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α(+) (CD103(+)) cDC1 lineage, and the CD11b(+) cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H(-)Ly6C(-) pre-DCs) or cDC2 lineage (Siglec-H(-)Ly6C(+) pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.

Glycine decarboxylase activity drives non-small cell lung cancer tumor-initiating cells and tumorigenesis.

Identification of the factors critical to the tumor-initiating cell (TIC) state may open new avenues in cancer therapy. Here we show that the metabolic enzyme glycine decarboxylase (GLDC) is critical for TICs in non-small cell lung cancer (NSCLC). TICs from primary NSCLC tumors express high levels of the oncogenic stem cell factor LIN28B and GLDC, which are both required for TIC growth and tumorigenesis. Overexpression of GLDC and other glycine/serine enzymes, but not catalytically inactive GLDC, promotes cellular transformation and tumorigenesis. We found that GLDC induces dramatic changes in glycolysis and glycine/serine metabolism, leading to changes in pyrimidine metabolism to regulate cancer cell proliferation. In the clinic, aberrant activation of GLDC correlates with poorer survival in lung cancer patients, and aberrant GLDC expression is observed in multiple cancer types. This link between glycine metabolism and tumorigenesis may provide novel targets for advancing anticancer therapy.

Simultaneous multifunctional transcriptome engineering by CRISPR RNA scaffold.

RNA processing and metabolism are subjected to precise regulation in the cell to ensure integrity and functions of RNA. Though targeted RNA engineering has become feasible with the discovery and engineering of the CRISPR-Cas13 system, simultaneous modulation of different RNA processing steps remains unavailable. In addition, off-target events resulting from effectors fused with dCas13 limit its application. Here we developed a novel platform, Combinatorial RNA Engineering via Scaffold Tagged gRNA (CREST), which can simultaneously execute multiple RNA modulation functions on different RNA targets. In CREST, RNA scaffolds are appended to the 3' end of Cas13 gRNA and their cognate RNA binding proteins are fused with enzymatic domains for manipulation. Taking RNA alternative splicing, A-to-G and C-to-U base editing as examples, we developed bifunctional and tri-functional CREST systems for simultaneously RNA manipulation. Furthermore, by fusing two split fragments of the deaminase domain of ADAR2 to dCas13 and/or PUFc respectively, we reconstituted its enzyme activity at target sites. This split design can reduce nearly 99% of off-target events otherwise induced by a full-length effector. The flexibility of the CREST framework will enrich the transcriptome engineering toolbox for the study of RNA biology.

Single-cell Transcriptome Landscape of DNA Methylome Regulators Associated with Orofacial Clefts in the Mouse Dental Pulp.

OBJECTIVE: Significant evidence links epigenetic processes governing the dynamics of DNA methylation and demethylation to an increased risk of syndromic and nonsyndromic cleft lip and/or cleft palate (CL/P). Previously, we characterized mesenchymal stem/stromal cells (MSCs) at different stages of osteogenic differentiation in the mouse incisor dental pulp. The main objective of this research was to characterize the transcriptional landscape of regulatory genes associated with DNA methylation and demethylation at a single-cell resolution. DESIGN: We used single-cell RNA sequencing (scRNA-seq) data to characterize transcriptome in individual subpopulations of MSCs in the mouse incisor dental pulp. SETTINGS: The biomedical research institution. PATIENTS/PARTICIPANTS: This study did not include patients. INTERVENTIONS: This study collected and analyzed data on the single-cell RNA expssion in the mouse incisor dental pulp. MAIN OUTCOME MEASURE(S): Molecular regulators of DNA methylation/demethylation exhibit differential transcriptional landscape in different subpopulations of osteogenic progenitor cells. RESULTS: scRNA-seq analysis revealed that genes encoding DNA methylation and demethylation enzymes (DNA methyltransferases and members of the ten-eleven translocation family of methylcytosine dioxygenases), methyl-DNA binding domain proteins, as well as transcription factors and chromatin remodeling proteins that cooperate with DNA methylation machinery are differentially expressed within distinct subpopulations of MSCs that undergo different stages of osteogenic differentiation. CONCLUSIONS: These findings suggest some mechanistic insights into a potential link between epigenetic alterations and multifactorial causes of CL/P phenotypes.

Social innovation, goal orientation, and openness: insights from social enterprise hybrids.

We empirically examine social innovation and openness through a survey of social enterprise hybrids in the United Kingdom (UK). Social innovation refers to new products, processes, and services that respond to grand challenges. Social enterprises pursue economic, social, and environmental goals but vary in their goal orientation, namely the relative importance ascribed to such goals. We first explore the relationships between commercial, social, and environmental goal orientation and social innovation performance. Next, we consider the moderating impact of openness to external knowledge and ideas on social innovation performance. Our analysis finds positive and significant relationships between commercial and social goal orientation and social innovation performance, but no relationship with environmental goal orientation. In addition, the use of external sources of knowledge and ideas positively strengthens these relationships for both commercial and social goal orientation but not for environmental goal orientation. Our results reveal some important influences on social innovation, openness, and hybrid organizing.

The hyperpolarization-activated, cyclic nucleotide-gated channel resides on myocytes in mouse bladders and contributes to adrenergic-induced detrusor relaxation.

Control of urinary continence is predicated on sensory signaling about bladder volume. Bladder sensory nerve activity is dependent on tension, implicating autonomic control over detrusor myocyte activity during bladder filling. Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are known contributors to bladder control, but their mechanism of action is not well understood. The lack of a definitive identification of cell type(s) expressing HCN in the bladder presents a significant knowledge gap. We recently reported a complete transcriptomic atlas of the C57BL/6 mouse bladder showing the dominant HCN paralog in mouse bladder, Hcn1, is limited to a subpopulation of detrusor smooth myocytes (DSMs). Here, we report details of these findings, along with results of patch-clamp experiments, immunohistochemistry, and functional myobath/tension experiments in bladder strips. With the use of a transgenic mouse expressing fluorescence-tagged α-smooth muscle actin, our data confirmed location and function of DSM HCN channels. Despite previous associations of HCN with postulated bladder interstitial cells, neither evidence of specific interstitial cell types nor an association of nonmyocytes with HCN was discovered. We confirm that HCN activation participates in reducing sustained (tonic) detrusor tension via cAMP, with no effect on intermittent (phasic) detrusor activity. In contrast, blockade of HCN increases phasic activity induced by a protein kinase A (PKA) blocker or a large-conductance Ca2+-activated K+ (BK) channel opener. Our findings, therefore, suggest a central role for detrusor myocyte HCN in regulating and constraining detrusor myocyte activity during bladder filling.

Cellular senescence in progenitor cells contributes to diminished remyelination potential in progressive multiple sclerosis.

Cellular senescence is a form of adaptive cellular physiology associated with aging. Cellular senescence causes a proinflammatory cellular phenotype that impairs tissue regeneration, has been linked to stress, and is implicated in several human neurodegenerative diseases. We had previously determined that neural progenitor cells (NPCs) derived from induced pluripotent stem cell (iPSC) lines from patients with primary progressive multiple sclerosis (PPMS) failed to promote oligodendrocyte progenitor cell (OPC) maturation, whereas NPCs from age-matched control cell lines did so efficiently. Herein, we report that expression of hallmarks of cellular senescence were identified in SOX2+ progenitor cells within white matter lesions of human progressive MS (PMS) autopsy brain tissues and iPS-derived NPCs from patients with PPMS. Expression of cellular senescence genes in PPMS NPCs was found to be reversible by treatment with rapamycin, which then enhanced PPMS NPC support for oligodendrocyte (OL) differentiation. A proteomic analysis of the PPMS NPC secretome identified high-mobility group box-1 (HMGB1), which was found to be a senescence-associated inhibitor of OL differentiation. Transcriptome analysis of OPCs revealed that senescent NPCs induced expression of epigenetic regulators mediated by extracellular HMGB1. Lastly, we determined that progenitor cells are a source of elevated HMGB1 in human white matter lesions. Based on these data, we conclude that cellular senescence contributes to altered progenitor cell functions in demyelinated lesions in MS. Moreover, these data implicate cellular aging and senescence as a process that contributes to remyelination failure in PMS, which may impact how this disease is modeled and inform development of future myelin regeneration strategies.

Single-Cell RNA Sequencing Reveals Molecular Features of Heterogeneity in the Murine Retinal Pigment Epithelium.

Transcriptomic analysis of the mammalian retinal pigment epithelium (RPE) aims to identify cellular networks that influence ocular development, maintenance, function, and disease. However, available evidence points to RPE cell heterogeneity within native tissue, which adds complexity to global transcriptomic analysis. Here, to assess cell heterogeneity, we performed single-cell RNA sequencing of RPE cells from two young adult male C57BL/6J mice. Following quality control to ensure robust transcript identification limited to cell singlets, we detected 13,858 transcripts among 2667 and 2846 RPE cells. Dimensional reduction by principal component analysis and uniform manifold approximation and projection revealed six distinct cell populations. All clusters expressed transcripts typical of RPE cells; the smallest (C1, containing 1-2% of total cells) exhibited the hallmarks of stem and/or progenitor (SP) cells. Placing C1-6 along a pseudotime axis suggested a relative decrease in melanogenesis and SP gene expression and a corresponding increase in visual cycle gene expression upon RPE maturation. K-means clustering of all detected transcripts identified additional expression patterns that may advance the understanding of RPE SP cell maintenance and the evolution of cellular metabolic networks during development. This work provides new insights into the transcriptome of the mouse RPE and a baseline for identifying experimentally induced transcriptional changes in future studies of this tissue.

Corneal nonmyelinating Schwann cells illuminated by single-cell transcriptomics and visualized by protein biomarkers.

The cornea is the most innervated tissue in the human body. Myelinated axons upon inserting into the peripheral corneal stroma lose their myelin sheaths and continue into the central cornea wrapped by only nonmyelinating corneal Schwann cells (nm-cSCs). This anatomical organization is believed to be important for central vision. Here we employed single-cell RNA sequencing (scRNA-seq), microscopy, and transgenics to characterize these nm-cSCs of the central cornea. Using principal component analysis, uniform manifold approximation and projection, and unsupervised hierarchal cell clustering of scRNA-seq data derived from central corneal cells of male rabbits, we successfully identified several clusters representing different corneal cell types, including a unique cell cluster representing nm-cSCs. To confirm protein expression of cSC genes, we performed cross-species validation, employing corneal whole-mount immunostaining with confocal microscopy in mouse corneas. The expression of several representative proteins of nm-cSCs were validated. As the proteolipid protein 1 (PLP1) gene was also expressed in nm-cSCs, we explored the Plp1-eGFP transgenic reporter mouse line to visualize cSCs. Specific and efficient eGFP expression was observed in cSCs in adult mice of different ages. Of several putative cornea-specific SC genes identified, Dickkopf-related protein 1 was shown to be present in nm-cSCs. Taken together, our findings, for the first time, identify important insights and tools toward the study nm-cSCs in isolated tissue and adult animals. We expect that our results will advance the future study of nm-cSCs in applications of nerve repair, and provide a resource for the study of corneal sensory function.

A genetic and developmental pathway from STAT3 to the OCT4-NANOG circuit is essential for maintenance of ICM lineages in vivo.

Although it is known that OCT4-NANOG are required for maintenance of pluripotent cells in vitro, the upstream signals that regulate this circuit during early development in vivo have not been identified. Here we demonstrate, for the first time, signal transducers and activators of transcription 3 (STAT3)-dependent regulation of the OCT4-NANOG circuitry necessary to maintain the pluripotent inner cell mass (ICM), the source of in vitro-derived embryonic stem cells (ESCs). We show that STAT3 is highly expressed in mouse oocytes and becomes phosphorylated and translocates to the nucleus in the four-cell and later stage embryos. Using leukemia inhibitory factor (Lif)-null embryos, we found that STAT3 phosphorylation is dependent on LIF in four-cell stage embryos. In blastocysts, interleukin 6 (IL-6) acts in an autocrine fashion to ensure STAT3 phosphorylation, mediated by janus kinase 1 (JAK1), a LIF- and IL-6-dependent kinase. Using genetically engineered mouse strains to eliminate Stat3 in oocytes and embryos, we firmly establish that STAT3 is essential for maintenance of ICM lineages but not for ICM and trophectoderm formation. Indeed, STAT3 directly binds to the Oct4 and Nanog distal enhancers, modulating their expression to maintain pluripotency of mouse embryonic and induced pluripotent stem cells. These results provide a novel genetic model of cell fate determination operating through STAT3 in the preimplantation embryo and pluripotent stem cells in vivo.

Controlled comparisons between soil and hydroponic systems reveal increased water use efficiency and higher lycopene and ß-carotene contents in hydroponically grown tomatoes.

There are many different types of systems used to grow food that are distinguished by ideology or the technology used. It is often difficult to directly compare yield and quality in different growth systems due to the complicated interactions between genotype, physiology and environment. Many published comparisons do not identify and acknowledge confounding factors. However, there is urgency to undertake controlled comparisons to identify the most efficient and effective food production systems, because the world faces considerable challenges to food supply with population rise, ongoing environmental degradation and the threat of climatic change. Here we compared soil with two hydroponic growth systems, drip irrigation and deep-water culture (DWC). It is often claimed that such systems differ in water use, yield and crop quality; however, such comparisons are often confounded by assessing plant and system parameters in different growth environments or where factors that are difficult to standardise between systems, such as nutrient status, are not controlled. We grew tomato (Solanum lycopersicum L.) in the three growth systems in two replicated experiments, in either a polytunnel or glasshouse. We controlled and monitored water use and nutrient levels across all systems as different fertilizer applications can influence the nutritional values of produce. Plants in the two hydroponic systems transpired less water and were more water-efficient with a lower product water use than plants grown in soil. Fruit yield was similar and total soluble solids and sugar levels were not significantly different between the three growing systems. However, levels of lycopene and ß-carotene were either similar or significantly higher in DWC compared to growth systems using soil or drip irrigation. Our results identify hydroponic systems as more water use efficient with DWC also capable of producing higher quality produce.

Single-cell transcriptomes identify human islet cell signatures and reveal cell-type-specific expression changes in type 2 diabetes.

Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type-specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis.

Single-cell multimodal profiling reveals cellular epigenetic heterogeneity.

Sample heterogeneity often masks DNA methylation signatures in subpopulations of cells. Here, we present a method to genotype single cells while simultaneously interrogating gene expression and DNA methylation at multiple loci. We used this targeted multimodal approach, implemented on an automated, high-throughput microfluidic platform, to assess primary lung adenocarcinomas and human fibroblasts undergoing reprogramming by profiling epigenetic variation among cell types identified through genotyping and transcriptional analysis.

Novel Miscanthus genotypes selected for different drought tolerance phenotypes show enhanced tolerance across combinations of salinity and drought treatments.

BACKGROUND AND AIMS: Water deficit and salinity stresses are often experienced by plants concurrently; however, knowledge is limited about the effects of combined salinity and water deficit stress in plants, and especially in C4 bioenergy crops. Here we aim to understand how diverse drought tolerance traits may deliver tolerance to combinations of drought and salinity in C4 crops, and identify key traits that influence the productivity and biomass composition of novel Miscanthus genotypes under such conditions. METHODS: Novel genotypes used included M. sinensis and M. floridulus species, pre-screened for different drought responses, plus the commercial accession Miscanthus × giganteus (M×g.). Plants were grown under control treatments, single stress or combinations of water deficit and moderate salinity stress. Morphophysiological responses, including growth, yield, gas exchange and leaf water relations and contents of proline, soluble sugars, ash and lignin were tested for significant genotypic and treatment effects. KEY RESULTS: The results indicated that plants subjected to combined stresses showed more severe responses compared with single stresses. All novel drought-tolerant genotypes and M×g. were tolerant to moderate salinity stress. Biomass production in M. sinensis genotypes was more resilient to co-occurring stresses than that in M×g. and M. floridulus, which, despite the yield penalty produced more biomass overall. A stay-green M. sinensis genotype adopted a conservative growth strategy with few significant treatment effects. Proline biosynthesis was species-specific and was triggered by salinity and co-occurring stress treatments, mainly in M. floridulus. The ash content was compartmentalized differently in leaves and stems in the novel genotypes, indicating different mechanisms of ion accumulation. CONCLUSIONS: This study highlights the potential to select novel drought-tolerant Miscanthus genotypes that are resilient to combinations of stress and is expected to contribute to a deeper fundamental knowledge of different mechanistic responses identified for further exploitation in developing resilient Miscanthus crops.

Genomic index selection provides a pragmatic framework for setting and refining multi-objective breeding targets in Miscanthus.

BACKGROUND: Miscanthus has potential as a biomass crop but the development of varieties that are consistently superior to the natural hybrid M. × giganteus has been challenging, presumably because of strong G × E interactions and poor knowledge of the complex genetic architectures of traits underlying biomass productivity and climatic adaptation. While linkage and association mapping studies are starting to generate long lists of candidate regions and even individual genes, it seems unlikely that this information can be translated into effective marker-assisted selection for the needs of breeding programmes. Genomic selection has emerged as a viable alternative, and prediction accuracies are moderate across a range of phenological and morphometric traits in Miscanthus, though relatively low for biomass yield per se. METHODS: We have previously proposed a combination of index selection and genomic prediction as a way of overcoming the limitations imposed by the inherent complexity of biomass yield. Here we extend this approach and illustrate its potential to achieve multiple breeding targets simultaneously, in the absence of a priori knowledge about their relative economic importance, while also monitoring correlated selection responses for non-target traits. We evaluate two hypothetical scenarios of increasing biomass yield by 20 % within a single round of selection. In the first scenario, this is achieved in combination with delaying flowering by 44 d (roughly 20 %), whereas, in the second, increased yield is targeted jointly with reduced lignin (-5 %) and increased cellulose (+5 %) content, relative to current average levels in the breeding population. KEY RESULTS: In both scenarios, the objectives were achieved efficiently (selection intensities corresponding to keeping the best 20 and 4 % of genotypes, respectively). However, the outcomes were strikingly different in terms of correlated responses, and the relative economic values (i.e. value per unit of change in each trait compared with that for biomass yield) of secondary traits included in selection indices varied considerably. CONCLUSIONS: Although these calculations rely on multiple assumptions, they highlight the need to evaluate breeding objectives and explicitly consider correlated responses in silico, prior to committing extensive resources. The proposed approach is broadly applicable for this purpose and can readily incorporate high-throughput phenotyping data as part of integrated breeding platforms.

Dynamic changes in Sox2 spatio-temporal expression promote the second cell fate decision through Fgf4/Fgfr2 signaling in preimplantation mouse embryos.

Oct4 and Sox2 regulate the expression of target genes such as Nanog, Fgf4, and Utf1, by binding to their respective regulatory motifs. Their functional cooperation is reflected in their ability to heterodimerize on adjacent cis regulatory motifs, the composite Sox/Oct motif. Given that Oct4 and Sox2 regulate many developmental genes, a quantitative analysis of their synergistic action on different Sox/Oct motifs would yield valuable insights into the mechanisms of early embryonic development. In the present study, we measured binding affinities of Oct4 and Sox2 to different Sox/Oct motifs using fluorescence correlation spectroscopy. We found that the synergistic binding interaction is driven mainly by the level of Sox2 in the case of the Fgf4 Sox/Oct motif. Taking into account Sox2 expression levels fluctuate more than Oct4, our finding provides an explanation on how Sox2 controls the segregation of the epiblast and primitive endoderm populations within the inner cell mass of the developing rodent blastocyst.

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal.

The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterized. Knowledge of the central transcription factors (TFs), the noncoding gene regulatory regions that they bind to, and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq, we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX, and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence.

Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.

There were errors published in Development 142, 3151-3165.In the issue published online on 22 September 2015, Fig. 3 was mislabelled: panels A, B, C and D should have been B, C, D and A, respectively. In the legend, the text prior to '(A) Cytoscape enrichment map…' should not have been included. The correct version of the figure and legend now appear online and in print.We apologise to the authors and readers for this mistake.

Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.

Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-ß signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-ß signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells.