No abstract

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.

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 b) 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 genomewide 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.

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.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2280-2) contains supplementary material, which is available to authorized users.

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.

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.

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.

SummaryCell 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.