Hepatoblasts play a key role in liver organogenesis by differentiating into hepatocytes and cholangiocytes, the main functional cell types of the liver. Mouse studies have demonstrated an association of Wnt signalling with proliferation and differentiation of hepatoblasts. However, the exact function of this pathway in hepatic development has not been fully uncovered, especially in human. Here, we use hepatoblast organoids derived from human foetal livers to investigate the importance of Wnt signalling in self-renewal and cell fate decisions during liver development. We first showed that Wnt plays a key role in hepatoblast self-renewal capacity in vitro by maintaining their proliferative state. However, Wnt was not sufficient to block differentiation of hepatoblast organoids into hepatocytes or cholangiocytes, suggesting that other factors are necessary to maintain hepatoblast bipotency. Finally, single-cell transcriptomic analyses revealed that Wnt signalling activity correlates with hepatoblast proliferation in the human foetal liver, suggesting that the role for Wnt could be conserved in vivo. Taken together, our results support a model in which Wnt signalling acts to preserve the proliferative capacity of hepatoblasts without being sufficient to maintain their bipotent state.

Production of large quantities of hepatocytes remains a major challenge for a number of clinical applications in the biomedical field. Directed differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells (HLCs) provides an advantageous solution and a number of protocols have been developed for this purpose. However, these methods usually follow different steps of liver development in vitro, which is time consuming and requires complex culture conditions. In addition, HLCs lack the full repertoire of functionalities characterising primary hepatocytes. Here, we explore the interest of forward programming to generate hepatocytes from hPSCs and to bypass these limitations. This approach relies on the overexpression of three hepatocyte nuclear factors (HNF1A, HNF6, and FOXA3) in combination with different nuclear receptors expressed in the adult liver using the OPTi-OX platform. Forward programming allows for the rapid production of hepatocytes (FoP-Heps) with functional characteristics using a simplified process. We also uncovered that the overexpression of nuclear receptors such as RORc can enhance specific functionalities of FoP-Heps thereby validating its role in lipid/glucose metabolism. Together, our results show that forward programming could offer a versatile alternative to direct differentiation for generating hepatocytes in vitro.

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances, especially in regenerative medicine, are currently hampered by the lack of knowledge concerning human hepatic cell development. Here, we addressed this limitation by describing the developmental trajectories of different cell types comprising the human fetal liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing critical, supportive roles during organogenesis. We utilised this information to derive bipotential hepatoblast organoids and then exploited this novel model system to validate the importance of key signalling pathways and developmental cues. Furthermore, these insights into hepatic maturation enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a new platform to investigate the basic mechanisms of human liver development and to produce cell types for clinical applications.

BACKGROUND AND AIMS:Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. APPROACH AND RESULTS:Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. CONCLUSIONS:Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Clusters of enhancers, referred as to super-enhancers (SEs), control the expression of cell identity genes. The organisation of these clusters, and how they are remodelled upon developmental transitions remain poorly understood. Here, we report the existence of two types of enhancer units within SEs typified by distinctive CpG methylation dynamics in embryonic stem cells (ESCs). We find that these units are either prone for decommissioning or remain constitutively active in epiblast stem cells (EpiSCs), as further established in the peri-implantation epiblast in vivo. Mechanistically, we show a pivotal role for ESRRB in regulating the activity of ESC-specific enhancer units and propose that the developmentally regulated silencing of ESRRB triggers the selective inactivation of these units within SEs. Our study provides insights into the molecular events that follow the loss of ESRRB binding, and offers a mechanism by which the naive pluripotency transcriptional programme can be partially reset upon embryo implantation.

In Fig. 1b of this Article, owing to a production error, the time points on the x axis should read ‘0, 6, 12, 18’rather than ‘0, 4, 12, 24’. This error has been corrected online.

Variability among pluripotent stem cell (PSC) lines is a prevailing issue that hampers not only experimental reproducibility but also large-scale applications and personalized cell-based therapy. This variability could result from epigenetic and genetic factors that influence stem cell behavior. Naive culture conditions minimize epigenetic fluctuation, potentially overcoming differences in PSC line differentiation potential. Here we derived PSCs from distinct mouse strains under naive conditions and show that lines from distinct genetic backgrounds have divergent differentiation capacity, confirming a major role for genetics in PSC phenotypic variability. This is explained in part through inconsistent activity of extra-cellular signaling, including the Wnt pathway, which is modulated by specific genetic variants. Overall, this study shows that genetic background plays a dominant role in driving phenotypic variability of PSCs.

Metformin, the world’s most prescribed anti-diabetic drug, is also effective in preventing Type 2 diabetes in people at high risk, by lowering body weight, fat mass and circulating insulin levels through mechanisms that are incompletely understood. Recent observational studies reporting the association of metformin use and circulating levels of GDF15 led us to hypothesize that GDF15, which signals through a specific receptor complex in the hindbrain to reduce body weight, might mediate these effects. We measured GDF15 in people without diabetes from a randomized placebo-controlled trial of metformin. Over 18 months, participants allocated metformin lost significant weight and levels of GDF15 were persistently elevated compared to placebo. The change in plasma GDF15 in this study correlated positively with weight loss. In wild-type mice, oral metformin increased circulating GDF15 with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to high fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GFRAL. In obese, high fat-fed mice, the effects of metformin to reduce body weight were reversed by a GFRAL antagonist antibody. Metformin had effects on both energy intake and energy expenditure that required GDF15. The insulin sensitising effects of metformin determined by insulin tolerance were abolished in mice lacking GDF15. Metformin significantly reduced fasting glucose and insulin levels in wild type but not in mice lacking GDF15. In summary, metformin increases the circulating levels of GDF15, which appears to be necessary for many of its actions as a metabolic chemopreventive agent.

Understanding the physiopathology of disease remains an essential step in developing novel therapeutics. Although animal models have certainly contributed to advancing this enterprise, their limitation in modeling all the aspects of complex human disorders is one of the major challenges faced by the biomedical research field. Human induced pluripotent stem cells (hiPSCs) derived from patients represent a great opportunity to overcome this deficiency because these cells cover the genetic diversity needed to fully model human diseases. Here, we provide an overview of the history of hiPSC technology and discuss common challenges and approaches that we and others have faced when using hiPSCs to model disease. Our emphasis is on liver disease, and consequently, we review the progress made using this technology to produce functional liver cells in vitro and how these systems are being used to recapitulate a diversity of developmental, metabolic, genetic, and infectious liver disorders.

Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk1,2. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner3. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to the shortage of donor organs. Developing renewable sources of human liver tissue is therefore attractive. Pluripotent stem cell-derived liver tissue represents a potential alternative to cadaver derived hepatocytes and whole organ transplant. At present, two-dimensional differentiation procedures deliver tissue lacking certain functions and long-term stability. Efforts to overcome these limiting factors have led to the building of three-dimensional (3D) cellular aggregates. Although enabling for the field, their widespread application is limited due to their reliance on variable biological components. Our studies focused on the development of 3D liver tissue under defined conditions. In vitro generated 3D tissues exhibited stable phenotype for over 1 year in culture, providing an attractive resource for long-term in vitro studies. Moreover, 3D derived tissue provided critical liver support in two animal models, including immunocompetent recipients. Therefore, we believe that our study provides stable human tissue to better model liver biology ‘in the dish’, and in the future may permit the support of compromised liver function in humans.

Cell cycle progression and cell fate decisions are closely linked in human pluripotent stem cells (hPSCs). However, the study of these interplays at the molecular level remains challenging due to the lack of efficient methods allowing cell cycle synchronization of large quantities of cells. Here, we screened inhibitors of cell cycle progression and identified nocodazole as the most efficient small molecule to synchronize hPSCs in the G2/M phase. Following nocodazole treatment, hPSCs remain pluripotent, retain a normal karyotype and can successfully differentiate into the three germ layers and functional cell types. Moreover, genome-wide transcriptomic analyses on single cells synchronized for their cell cycle and differentiated toward the endoderm lineage validated our findings and showed that nocodazole treatment has no effect on gene expression during the differentiation process. Thus, our synchronization method provides a robust approach to study cell cycle mechanisms in hPSCs.

Jmjd2 H3K9 demethylases cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. Here, we reveal that Jmjd2c facilitates this process by stabilising the assembly of mediator-cohesin complexes at lineage-specific enhancers. Functionally, we show that Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while Jmjd2c-knockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a (also known as Ehmt2), independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and further destabilises loading of the mediator and cohesin components Med1 and Smc1a at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c and G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with an impact on timely gene activation.

Jmjd2/Kdm4 H3K9-demethylases cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. Here, we uncover that Jmjd2c facilitates this process by stabilizing the assembly of Mediator-Cohesin complexes at lineage-specific enhancers. Functionally, we show that Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while Jmjd2cknockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a/Ehmt2, independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and furthermore destabilizes loading of the Mediator and Cohesin components Med1 and Smc1a at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c-G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with impact on timely gene activation. D ev el o pm en t • A dv an ce a rt ic le Introduction Pluripotency, the ability of a cell to generate all somatic lineages, is transiently acquired in vivo during mammalian pre-implantation development. Upon blastocyst formation, pluripotent cells develop within the inner cell mass (ICM), a mosaic of cells surrounded by an extra-embryonic layer the trophectoderm. By the time of implantation, a second extra-embryonic lineage, the primitive endoderm, emerges at the ICM surface. Concurrently, the ICM maintains its pluripotency as it matures into the epiblast but ultimately goes on to form the three primary germ layers and germ cells upon gastrulation (Boroviak and Nichols, 2014; Rossant, 2008). Pluripotent mouse embryonic stem cells (ESCs) are derived from ICM cells, and can self-renew and faithfully maintain an undifferentiated state in vitro in the presence of leukaemia inhibitory factor (LIF) and serum components while preserving their multi-lineage differentiation capacity (Evans and Kaufman, 1981; Martin, 1981; Niwa et al., 1998; Ying et al., 2003). Most recently, stem cell lines with similar lineage potential were established from other developmental stages (Chung et al., 2006; Tesar, 2005), including a number of post-implantation epiblast-derived stem cells (EpiSCs) (Brons et al., 2007; Osorno et al., 2012; Tesar et al., 2007). While ESCs are thought to represent an immature (pre-implantation) phase of pluripotency, EpiSCs exist in a more advanced state on the verge of differentiation (Nichols and Smith, 2009). Moreover, ESCs can stably transit into self-renewing EpiSCs, acquiring characteristics of post-implantation epiblast-like cells (Guo et al., 2009). ESC abilities depend on the potent expression of self-renewal genes and transcriptional priming of silent, lineage-affiliated genes – a critical balance of gene expression maintained through crosstalk between transcriptional factors and chromatin regulators (Azuara et al., 2006; Bernstein et al., 2006; Chen and Dent, 2014; Ng and Surani, 2011; Stock et al., 2007). Remarkably, both active (ESC-specific) and primed (lineagespecific) genes are expressed in a heterogeneous manner, a feature long considered a hallmark of ESC cultures to safeguard the swift response to differentiation cues (Efroni et al., 2008; Torres-Padilla and Chambers, 2014). Yet, it is now possible to derive and maintain ESCs with reduced D ev el o pm en t • A dv an ce a rt ic le heterogeneity and transcriptional gene priming through chemical inhibition of two differentiation-associated pathways Mek and Gsk3 (2i conditions), capturing a naïve pluripotent state in vitro (Marks et al., 2012; Ying et al., 2008). Gene promoter regions enriched in CpG islands and H3K4me3 function as genomic platforms for the recruitment of transcription factors and co-regulators as well as the basal transcriptional machinery (Deaton and Bird, 2011; Illingworth and Bird, 2009). Moreover, distal DNA elements such as enhancers play a significant role in potentiating gene expression being typically decorated by H3K4me1 and bound by pioneer transcription factors (Calo and Wysocka, 2013; Gibcus and Dekker, 2013; Spitz and Furlong, 2012). For example, the core pluripotency factor Oct4 was commonly shown to mark both active and poised enhancers in ESCs and EpiSCs (Buecker et al., 2014; Calo and Wysocka, 2013). Enhancer activity and robust ESCspecific gene expression entail long-range DNA interactions with the transcriptional apparatus at promoters, involving the cooperative action of Mediator-Cohesin complexes (Kagey et al., 2010). Yet, relatively little is known about the identity of proteins that stabilize the formation of such assemblies. Histone demethylases have emerged as key players in the control of cell identity and development mainly through modulation of the chromatin environment of tissue-specific genes (Nottke et al., 2009). Recently, additional roles for these molecules independent of their enzymatic activity were reported (Shpargel et al., 2012; Wang et al., 2012; Yang et al., 2010), especially in regulating the recruitment of Polycomb repressive complexes (PRC) and poised RNA polymerase II to the promoter regions of developmental genes in ESCs (Farcas et al., 2012; Wu et al., 2013). Jmjd2c/Kdm4c is a member of the Jmjd2/Kdm4 gene family initially identified as H3K9me2/3 and/or H3K36me2/3 histone demethylases (Chen et al., 2006; Klose et al., 2006; Whetstine et al., 2006). Jmjd2c is highly expressed in the early embryo and in ESCs (Boroviak et al., 2015; Burton et al., 2013; Loh et al., 2007; Wang et al., 2010), and RNA interference-mediated depletion of the protein was shown to impair cleavage-stage development and ESC integrity as well as inhibiting somatic cell reprogramming (Das et al., 2014; Loh et al., D ev el o pm en t • A dv an ce a rt ic le 2007; Wang et al., 2010). Jmjd2c-null ESCs and mice could, however, be generated via gene trap approaches (Pedersen et al., 2014), in agreement with functional redundancy between Jmjd2 gene family members to support cell proliferation and survival (Pedersen et al., 2016). At the genomic level, Jmjd2c was shown to preferentially target H3K4me3-rich promoter regions of active and developmental-associated genes in ESCs via its Tudor domains (Das et al., 2014; Pedersen et al., 2014), where Jmjd2c was proposed to assist Jmjd2b-Nanog and PRC2 in transcriptional activation and repression, respectively (Das et al., 2014). In this study, we uncover a previously unrecognized link between Jmjd2c recruitment to lineage-specific enhancers and the establishment of a functionally primed state for differentiation in vitro. We show that, in the absence of Jmjd2c, ESC differentiation is severely impeded at an early postimplantation epiblast-like stage. While Jmjd2c-knockout ESCs can transit into self-renewing EpiSCs, these cells fail to form derivatives of the three primary germ layers, as revealed by their inability to initiate appropriate gene expression programs. In contrast, Jmjd2c-knockout cells remain capable of adopting extra-embryonic endoderm-like phenotypes under permissive conditions. Mechanistically, we show that Jmjd2c is re-distributed to lineagespecific enhancers in primed (serum/LIF) as opposed to naïve (2i/LIF) ESCs. Strikingly, Jmjd2c-bound enhancers are co-occupied by the antagonistic enzyme G9a/Ehmt2, independently of its silencing H3K9-modifying activity. We show that Jmjd2c-G9a co-occupancy coincides with the formation of activating, Med1-containing enhancer complexes. Loss of Jmjd2c abrogates G9a recruitment at Jmjd2c-bound distal sites, and this correlates with inefficient loading of Mediator-Cohesin complexes in ESC-derived EpiSCs with impact on gene activation upon lineage specification. Collectively, these data reveal that Jmjd2c is required for successful gene transcription and somatic differentiation in pluripotent stem cells, and propose a novel regulatory role for Jmjd2c in stabilizing the assembly of essential enhancerprotein complexes at the onset of ESC differentiation. D ev el o pm en t • A dv an ce a rt ic le Results Jmjd2c is required for embryonic stem cell differentiation towards somatic lineages Combining genetic deletion and functional assays, we tested whether Jmjd2c plays a role at the exit of pluripotency. Jmjd2c mutant and wild-type JM8-ESCs were obtained through the EUCOMM/IKMC repository (Bradley et al., 2012; Skarnes et al., 2011), and targeting of both Jmjd2c alleles in Jmjd2c-knockout (E2 and E3) ESC clones confirmed by long-range PCR and GFP expression (Fig. S1A-C). Western blotting validated that full-length Jmjd2c protein expression was completely abolished in Jmjd2c-knockout samples (Fig. 1A). This was accompanied by a notable increase in bulk H3K9me2 levels relative to wild-type (Fig. 1B, S1D); an effect attributed to Jmjd2c loss itself in the continued expression of other H3K9-demethylases and methyltransferases (Fig. S1D-E). Constitutively Jmjd2c-depleted E2 and E3 ESC clones grew normally in medium supplemented with serum plus LIF (Fig. S1F), in agreement with a similar conditional knockout model (Pedersen et al., 2014). When plated at low density these cells also displayed comparable proportion of undifferentiated colonies relative to their wild-type counterparts (Fig. 1C), despite reduced expression levels of pluripotencyassociated factors (Fig. S1G). Jmjd2c depletion did not, however, result in general de-repression of differentiation-associated genes (Fig. S1H), including those co-bound by Jmjd2c and PRC complexes at their promoter regions (Das et al., 2014). These

The familial forms of non-medullary thyroid carcinoma (FNMTC) represent approximately 5 % of thyroid neoplasms. Nine FNMTC susceptibility loci have been mapped; however, only the DICER1 and SRGAP1 susceptibility genes have been identified. The transcription factors NKX2-1, FOXE1, PAX8, and HHEX are involved in the morphogenesis and differentiation of the thyroid. Recent studies have identified NKX2 - 1 germline mutations in FNMTC families. However, the role of high-penetrant FOXE1 variants in FNMTC etiology remains unclear. The aim of this study was to investigate the role of FOXE1 germline mutations in the pathogenesis of FNMTC. We searched for molecular changes in the FOXE1 gene in the probands from 60 Portuguese families with FNMTC. In this series, we identified nine polymorphisms and one variant (c.743C>G, p.A248G) which was not previously described. This variant, which involved an amino acid residue conserved in evolution, segregated with disease in one family, and was also detected in an apparently unrelated case of sporadic NMTC. Functional studies were performed using rat normal thyroid cells (PCCL3) clones and human papillary thyroid carcinoma cell line (TPC-1) pools, expressing the wild type and mutant (p.A248G) forms of FOXE1 . In these experiments, we observed that the p.A248G variant promoted cell proliferation and migration, suggesting that it may be involved in thyroid tumorigenesis. Additionally, somatic p.V600E BRAF mutations were also detected in the thyroid tumors of two members of the family carrying the p.A248G variant. This study represents the first evidence of involvement of a germline FOXE1 rare variant in FNMTC etiology and suggests that mutations in MAPK pathway-related genes may contribute to tumor development in these familial cases.

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Embryonic stem cell (ESC) pluripotency depends on a well-characterized gene regulatory network centered on Oct4, Sox2, and Nanog. In contrast, little is known about the identity of the key coregulators and the mechanisms by which they may potentiate transcription in ESCs. Alongside core transcription factors, the orphan nuclear receptor Esrrb (estrogen-related receptor β) is vital for the maintenance of ESC identity and furthermore is uniquely associated with the basal transcription machinery. Here, we show that Ncoa3, an essential coactivator, is required to mediate Esrrb function in ESCs. Ncoa3 interacts with Esrrb via its ligand-binding domain and bridges Esrrb to RNA polymerase II complexes. Functionally, Ncoa3 is critical for both the induction and maintenance of pluripotency. Through chromatin immunoprecipitation (ChIP) sequencing and microarray experiments, we further demonstrate that Ncoa3 shares overlapping gene regulatory functions with Esrrb and cooperates genome-wide with the Oct4-Sox2-Nanog circuitry at active enhancers to up-regulate genes involved in self-renewal and pluripotency. We propose an integrated model of transcriptional and coactivator control, mediated by Ncoa3, for the maintenance of ESC self-renewal and somatic cell reprogramming.