We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3′ and/or 5′ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5′ differences and in support of this we report that a 5′ isomiR-9–1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5′ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.

SUMMARYPluripotent cells develop within the inner cell mass of blastocysts, a mosaic of cells surrounded by an extra-embryonic layer, the trophectoderm. We show that a set of somatic lineage regulators (including Hox, Gata and Sox factors) that carry bivalent chromatin enriched in H3K27me3 and H3K4me2 are selectively targeted by Suv39h1-mediated H3K9me3 and de novo DNA methylation in extra-embryonic versus embryonic (pluripotent) lineages, as assessed both in blastocyst-derived stem cells and in vivo. This stably repressed state is linked with a loss of gene priming for transcription through the exclusion of PRC1 (Ring1B) and RNA polymerase II complexes at bivalent, lineage-inappropriate genes upon trophoblast lineage commitment. Collectively, our results suggest a mutually exclusive role for Ring1B and Suv39h1 in regulating distinct chromatin states at key developmental genes and propose a novel mechanism by which lineage specification can be reinforced during early development.

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.

Liver transplantation represents the standard treatment for people with an end-stage liver disease and some liver-based metabolic disorders; however, shortage of liver donor tissues limits its availability. Furthermore, whole liver replacement eliminates the possibility of using native liver as a possible target for future gene therapy in case of liver-based metabolic defects. Cell therapy has emerged as a potential alternative, as cells can provide the hepatic functions and engraft in the liver parenchyma. Various options have been proposed, including human or other species hepatocytes, hepatocyte-like cells derived from stem cells or more futuristic alternatives, such as combination therapies with different cell types, organoids and cell-biomaterial combinations. In this review, we aim to give an overview of the cell therapies developed so far, highlighting preclinical and/or clinical achievements as well as the limitations that need to be overcome to make them fully effective and safe for clinical applications.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

The high rate of glucose uptake to fuel the bioenergetic and anabolic demands of proliferating cancer cells is well recognized, and exploited with 18F-2-fluoro-2-deoxyglucose positron emission tomography (18F-FDG-PET) to image tumors clinically. In contrast, enhanced glucose storage as glycogen (glycogenesis) in cancer is less well understood and the availability of a non-invasive method to image glycogen in vivo could provide important biologic insights. Here, we demonstrate that 18F-N-(methyl-(2-fluoroethyl)-1H-[1,2,3]triazole-4-yl)glucosamine (18F-NFTG) annotates glycogenesis in cancer cells and tumors in vivo, measured by PET. Specificity of glycogen labeling was demonstrated by isolating 18F-NFTG-associated glycogen and with stable knockdown of glycogen synthase 1, which inhibited 18F-NFTG uptake, while oncogene (Rab25) activation-associated glycogen synthesis led to increased uptake. We further show that the rate of glycogenesis is cell cycle-regulated, enhanced during the non-proliferative state of cancer cells. We demonstrate that glycogen levels, 18F-NFTG, but not 18F-FDG uptake, increase proportionally with cell density and G1/G0 arrest with potential application in the assessment of activation of oncogenic pathways related to glycogenesis and the detection of post treatment tumor quiescence.