Publisher Correction: GDF15 mediates the effects of metformin on body weight and energy balance.

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

GDF15 mediates the effects of metformin on body weight and energy balance.

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.

Regional Differences in Human Biliary Tissues and Corresponding In Vitro-Derived Organoids.

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.

Jmjd2c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation.

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.

Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development.

The transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner.

Ncoa3 functions as an essential Esrrb coactivator to sustain embryonic stem cell self-renewal and reprogramming.

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 ß) 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.

3D human liver tissue from pluripotent stem cells displays stable phenotype in vitro and supports compromised liver function in vivo.

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.

FOXE1 polymorphisms are associated with familial and sporadic nonmedullary thyroid cancer susceptibility.

OBJECTIVE: FOXE1 is a transcription factor required for thyroid differentiation and function. FOXE1 locus polymorphisms (chromosome 9q22.33) were recently associated with increased sporadic thyroid cancer risk. In this study, we aimed to investigate the association of FOXE1 variants with nonmedullary thyroid cancer (NMTC), in both sporadic and familial (FNMTC) cases from the Portuguese population. DESIGN AND METHODS: Nine variants located at the FOXE1 locus were sequenced in genomic DNA from 60 FNMTC probands and 80 patients with sporadic NMTC. Alleles were tested for association with thyroid cancer, against 130 healthy matched Portuguese controls. RESULTS: All variants were significantly associated with increased thyroid cancer risk when combining familial and sporadic cases (OR range = 1·62-2·58). In particular, two reported risk variants were associated with the disease: rs965513 (allele A) with familial (OR = 2·30, 95% CI = 1·48-3·59, P = 0·0002) and sporadic (OR = 2·81, 95% CI = 1·87-4·22, P < 0·0001) NMTC and rs1867277 (allele A) with the sporadic (OR = 1·76, 95% CI = 1·18-2·62, P = 0·0052) and combined NMTC cases (OR = 1·70, 95% CI = 1·21-2·40, P = 0·0022). Interestingly, we also identified association of FOXE1 polyalanine tract expansions (>14 alanines) with thyroid cancer risk, in both familial (OR = 2·56, 95% CI = 1·64-4·01, P < 0·0001) and sporadic (OR = 2·44, 95% CI = 1·61-3·68, P < 0·0001) cases. CONCLUSIONS: We found compelling evidence of association between FOXE1 variants and thyroid cancer risk in the Portuguese population. To our knowledge, this is the first study supporting the association of this locus with both sporadic and familial NMTC susceptibility.

Generation of functional hepatocytes by forward programming with nuclear receptors.

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.

Single-cell atlas of human liver development reveals pathways directing hepatic cell fates.

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human 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 essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also 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 platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.

Dynamic CpG methylation delineates subregions within super-enhancers selectively decommissioned at the exit from naive pluripotency.

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.

Naive Pluripotent Stem Cells Exhibit Phenotypic Variability that Is Driven by Genetic Variation.

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.

Modeling Disease with Human Inducible Pluripotent Stem Cells.

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.

Method to Synchronize Cell Cycle of Human Pluripotent Stem Cells without Affecting Their Fundamental Characteristics.

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.

Differential methylation as a cause of allele dropout at the imprinted GNAS locus.

INTRODUCTION: We detected false homozygosity at the NESP55 differentially methylated region of the imprinted GNAS locus while analyzing the segregation of single-nucleotide polymorphisms (SNPs) in families with pseudohypoparathyroidism type Ib (PHP-Ib). We hypothesized that differential methylation of NESP55 could affect polymerase chain reaction (PCR) amplification, resulting in allele dropout. METHODS: We genotyped 10 normal controls for four SNPs in NESP55 differentially methylated region. SNPs were amplified by standard PCR conditions and with the addition of dimethyl sulfoxide. The methylated allele was identified by HpaII analysis, and haplotypes were confirmed using subcloning strategies. All SNPs were also genotyped in a PHP-Ib patient (P1), carrying methylation at both NESP55 alleles, and in an in vitro methylated control DNA (SSSI-N4). RESULTS: In the control samples, we identified allele dropout of the methylated allele in 85% of the amplifications, using standard PCR conditions. Addition of dimethyl sulfoxide to the PCR successfully prevented dropout in all cases. No amplification bias was observed for P1 and SSSI-N4 samples. CONCLUSIONS: For the first time, we report that differential methylation of imprinted regions can lead to preferential amplification of unmethylated alleles. Addition of coadjuvants to the PCR may facilitate amplification of both alleles, providing an accurate genotyping in cases with methylation-related diseases.