CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 () CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the on-target locus, as well as segmental loss and gain of chromosome 6, on which the gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.

The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here.

The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process.

Follicular helper T (T) cells control antibody responses by supporting antibody affinity maturation and memory formation. Inadequate T function has been found in individuals with ineffective responses to vaccines, but the mechanism underlying T regulation in vaccination is not understood. Here, we report that lower serum levels of the metabolic hormone leptin associate with reduced vaccine responses to influenza or hepatitis B virus vaccines in healthy populations. Leptin promotes mouse and human T differentiation and IL-21 production via STAT3 and mTOR pathways. Leptin receptor deficiency impairs T generation and antibody responses in immunisation and infection. Similarly, leptin deficiency induced by fasting reduces influenza vaccination-mediated protection for the subsequent infection challenge, which is mostly rescued by leptin replacement. Our results identify leptin as a regulator of T cell differentiation and function and indicate low levels of leptin as a risk factor for vaccine failure.

The enzyme-linked immunosorbent assay (ELISA) technique has been developed half a century ago, and yet its role in molecular biology remains significant. Among the most sensitive of immunoassays, it offers high throughput, combined with affordability and ease of use. This chapter provides the procedure of a highly reproducible indirect sandwich ELISA protocol, which can be applied to a variety of semi-quantitative assays for the investigation of the molecular biology of 5-hydroxymethylcytosine (5hmC) or TET enzymes. Three variations of this protocol are described: assessment and validation of 5hmC-binding proteins, screening and validation of anti-5hmC antibodies, or a readout of TET catalytic activity in in vitro experiments. The assay principle is based on the use of a high affinity avidin-biotin system for efficient immobilization of DNA fragments for further detection by high specificity antibodies. A colorimetric enzymatic reaction is ultimately developed with intensity correlating with the amount of attached antigen.

5-Hydroxymethylcytosine (5hmC) is an abundant DNA modification in human and mouse brain, as well as in embryonic stem cells, while severely depleted in multiple types of cancer. Assays for 5hmC detection and quantification, both on a locus-specific and global level, are limited in number and often resource-intensive. Immunodetection of 5hmC through antibodies remains a cost-effective and widely accessible approach. This chapter describes an ELISA-based protocol for 5hmC detection and quantification in genomic or in vitro modified DNA. It is based on the passive adsorption of DNA onto a solid polystyrene surface and the specific detection of 5hmC, which generates a measurable chemiluminescent signal, proportional to the amount of immobilized 5hmC. The assay utilizes a standard curve for interpolation of 5hmC percentage and a loading standard for monitoring loading precision.

Cryopreservation offers the potential to increase the availability of pancreatic islets for treatment of diabetic patients. However, current protocols, which use dimethyl sulfoxide (DMSO), lead to poor cryosurvival of islets. We demonstrate that equilibration of mouse islets with small molecules in aqueous solutions can be accelerated from > 24 to 6 h by increasing incubation temperature to 37 °C. We utilize this finding to demonstrate that current viability staining protocols are inaccurate and to develop a novel cryopreservation method combining DMSO with trehalose pre-incubation to achieve improved cryosurvival. This protocol resulted in improved ATP/ADP ratios and peptide secretion from β-cells, preserved cAMP response, and a gene expression profile consistent with improved cryoprotection. Our findings have potential to increase the availability of islets for transplantation and to inform the design of cryopreservation protocols for other multicellular aggregates, including organoids and bioengineered tissues.

Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. Even the advent of spectral cytometry cannot circumvent the spillover problem, with spectral unmixing an intrinsic part of such systems. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Here, we present AutoSpill, an alternative method for calculating spillover coefficients. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software. AutoSpill allows simpler and more robust workflows, while reducing the magnitude of compensation errors in high-parameter flow cytometry.

The annotation of small open reading frames (smORFs) of less than 100 codons (<300 nucleotides) is challenging due to the large number of such sequences in the genome.

Neutrophils, the most abundant circulating leukocytes in humans have key roles in host defense and in the inflammatory response. Agonist-activated phosphoinositide 3-kinases (PI3Ks) are important regulators of many facets of neutrophil biology. PIP3 is subject to dephosphorylation by several 5' phosphatases, including SHIP family phosphatases, which convert the PI3K product and lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) into PI(3,4)P2, a lipid second messenger in its own right. In addition to the leukocyte restricted SHIP1, neutrophils express the ubiquitous SHIP2. This study analyzed mice and isolated neutrophils carrying a catalytically inactive SHIP2, identifying an important regulatory function in neutrophil chemotaxis and directionality and in neutrophil recruitment to sites of sterile inflammation , in the absence of major defects of any other neutrophil functions analyzed, including, phagocytosis and the formation of reactive oxygen species. Mechanistically, this is explained by a subtle effect on global 3-phosphorylated phosphoinositide species. This work identifies a non-redundant role for the hitherto overlooked SHIP2 in the regulation of neutrophils, and specifically, neutrophil chemotaxis/trafficking. It completes an emerging wider understanding of the complexity of PI3K signaling in the neutrophil, and the roles played by individual kinases and phosphatases within.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

The development of single-cell multimodal assays provides a powerful tool for investigating multiple dimensions of cellular heterogeneity, enabling new insights into development, tissue homeostasis and disease. A key challenge in the analysis of single-cell multimodal data is to devise appropriate strategies for tying together data across different modalities. The term 'data integration' has been used to describe this task, encompassing a broad collection of approaches ranging from batch correction of individual omics datasets to association of chromatin accessibility and genetic variation with transcription. Although existing integration strategies exploit similar mathematical ideas, they typically have distinct goals and rely on different principles and assumptions. Consequently, new definitions and concepts are needed to contextualize existing methods and to enable development of new methods.

The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C-ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.

The complex nature of folliculogenesis regulation accounts for its susceptibility to maternal physiological fitness. In obese mothers, progressive expansion of adipose tissue culminates with severe hyperestrogenism and hyperleptinemia with detrimental effects for ovarian performance. Indeed, maternal obesity is associated with the establishment of ovarian leptin resistance. This review summarizes current knowledge on potential effects of impaired leptin signaling throughout folliculogenesis and oocyte developmental competence in mice and women.

Dysregulation of glucose homeostasis leading to metabolic syndrome and type 2 diabetes is the cause of an increasing world health crisis. New intriguing roles have emerged for Rho family GTPases and their Rho guanine nucleotide exchange factor (GEF) activators in the regulation of glucose homeostasis. This review summates the current knowledge, focusing in particular on the roles of Rho GEFs in the processes of glucose-stimulated insulin secretion by pancreatic β cells and insulin-stimulated glucose uptake into skeletal muscle and adipose tissues. We discuss the ten Rho GEFs that are known so far to regulate glucose homeostasis, nine of which are in mammals, and one is in yeast. Among the mammalian Rho GEFs, P-Rex1, Vav2, Vav3, Tiam1, Kalirin and Plekhg4 were shown to mediate the insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane and/or insulin-stimulated glucose uptake in skeletal muscle or adipose tissue. The Rho GEFs P-Rex1, Vav2, Tiam1 and β-PIX were found to control the glucose-stimulated release of insulin by pancreatic β cells. In vivo studies demonstrated the involvement of the Rho GEFs P-Rex2, Vav2, Vav3 and PDZ-RhoGEF in glucose tolerance and/or insulin sensitivity, with deletion of these GEFs either contributing to the development of metabolic syndrome or protecting from it. This research is in its infancy. Considering that over 80 Rho GEFs exist, it is likely that future research will identify more roles for Rho GEFs in glucose homeostasis.

Genomic imprinting, an epigenetic phenomenon that causes the expression of a small set of genes in a parent-of-origin-specific manner, is thought to have co-evolved with placentation. Many imprinted genes are expressed in the placenta, where they play diverse roles related to development and nutrient supply function. However, only a small number of imprinted genes have been functionally tested for a role in nutrient transfer capacity in relation to the structural characteristics of the exchange labyrinthine zone. Here, we examine the transfer capacity in a mouse model deficient for the maternally expressed gene, which results in placental overgrowth and a transient reduction in fetal growth. Using stereology, we show that the morphology of the labyrinthine zone in mutants is normal at E16 and E19. In vivo placental transfer of radiolabeled solutes C-methyl-D-glucose and C-MeAIB remains unaffected at both gestational time points. However, placental passive permeability, as measured using two inert hydrophilic solutes (C-mannitol; C-inulin), is significantly higher in mutants. Importantly, this increase in passive permeability is associated with fetal catch-up growth. Our findings uncover a key role played by the imprinted gene in modifying placental passive permeability that may be important for determining fetal growth.

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.

The origin of the jaw is a long-standing problem in vertebrate evolutionary biology. Classical hypotheses of serial homology propose that the upper and lower jaw evolved through modifications of dorsal and ventral gill arch skeletal elements, respectively. If the jaw and gill arches are derived members of a primitive branchial series, we predict that they would share common developmental patterning mechanisms. Using candidate and RNAseq/differential gene expression analyses, we find broad conservation of dorsoventral patterning mechanisms within the developing mandibular, hyoid and gill arches of a cartilaginous fish, the skate (Leucoraja erinacea). Shared features include expression of genes encoding members of the ventralising BMP and endothelin signalling pathways and their effectors, the joint markers nkx3.2 and gdf5 and pro-chondrogenic transcription factor barx1, and the dorsal territory marker pou3f3. Additionally, we find that mesenchymal expression of eya1/six1 is an ancestral feature of the mandibular arch of jawed vertebrates, while differences in notch signalling distinguish the mandibular and gill arches in skate. Comparative transcriptomic analyses of mandibular and gill arch tissues reveal additional genes differentially expressed along the dorsoventral axis of the pharyngeal arches, including scamp5 as a novel marker of the dorsal mandibular arch, as well as distinct transcriptional features of mandibular and gill arch muscle progenitors and developing gill buds. Taken together, our findings reveal conserved patterning mechanisms in the pharyngeal arches of jawed vertebrates, consistent with serial homology of their skeletal derivatives, as well as unique transcriptional features that may underpin distinct jaw and gill arch morphologies.

CRISPR/Cas9 screens are a powerful approach to identify key regulators of biological processes. By combining pooled CRISPR/Cas9 screening with single-cell RNA-sequencing readout, individual perturbations can be assessed in parallel both comprehensively and at scale. Importantly, this allows gene function and regulation to be interrogated at a cellular level in an unbiased manner. Here, we present a protocol to perform pooled CRISPR-activation screens in mouse embryonic stem cells using 10× Genomics scRNA-seq as a readout. For complete information on the generation and use of this protocol, please refer to Alda-Catalinas et al. (2020).

Transient receptor potential (TRP) channels play prominent roles in ion homeostasis by their ability to control cation influx. Mouse placentation is governed by the processes of trophoblast proliferation, invasion, differentiation, and fusion, all of which require calcium signaling. Although certain TRP channels have been shown to contribute to maternal-fetal transport of magnesium and calcium, a role for TRP channels in specific trophoblast functions has been disregarded. Using qRT-PCR and in situ hybridisation, the spatio-temporal expression pattern of TRP channels in the mouse placenta across gestation (E10.5-E18.5) was assessed. Prominent expression was observed for Trpv2, Trpm6, and Trpm7. Calcium microfluorimetry in primary trophoblast cells isolated at E14.5 of gestation further revealed the functional activity of TRPV2 and TRPM7. Finally, comparing TRP channels expression in mouse trophoblast stem cells (mTSCs) and mouse embryonic stem cells (mESC) confirmed the specific expression of TRPV2 during placental development. Moreover, TRP channel expression was similar in mTSCs compared to primary trophoblasts and validate mTSC as a model to study TRP channels in placental development. Collectivity, our results identify a specific spatio-temporal TRP channel expression pattern in trophoblasts, suggesting a possible involvement in regulating the process of placentation.

Neutrophils play fundamental roles in innate immune response, shape adaptive immunity, and are a potentially causal cell type underpinning genetic associations with immune system traits and diseases. Here, we profile the binding of myeloid master regulator PU.1 in primary neutrophils across nearly a hundred volunteers. We show that variants associated with differential PU.1 binding underlie genetically-driven differences in cell count and susceptibility to autoimmune and inflammatory diseases. We integrate these results with other multi-individual genomic readouts, revealing coordinated effects of PU.1 binding variants on the local chromatin state, enhancer-promoter contacts and downstream gene expression, and providing a functional interpretation for 27 genes underlying immune traits. Collectively, these results demonstrate the functional role of PU.1 and its target enhancers in neutrophil transcriptional control and immune disease susceptibility.

CD4 regulatory T (Treg) cells, dependent upon the transcription factor Foxp3, contribute to tumour immunosuppression but are also required for immune homeostasis. There is interest in developing therapies that selectively target the immunosuppressive function of Treg cells within tumours without disrupting their systemic anti-inflammatory function. High levels of expression of Chemokine (C-C motif) receptor 8 (CCR8) discriminate Treg cells within tumours from those found in systemic lymphoid tissues. It has recently been proposed that disruption of CCR8 function using blocking anti-CCR8 antibodies results in reduced accumulation of Treg cells within tumours and disruption of their immunosuppressive function. Here, using Ccr8 mice, we show that CCR8 function is not required for Treg cell accumulation or immunosuppression in the context of syngeneic MC38 colorectal adenocarcinoma and B16 melanoma tumours. We observed high levels of CCR8 expression on tumour-infiltrating Treg cells which was abolished in Ccr8 mice. High levels of CCR8 marked cells with high levels of suppressive function. However, whereas systemic ablation of Treg cells resulted in strikingly diminished tumour burden, growth of subcutaneously implanted tumours was unaffected by systemic CCR8 loss. Consistently, we observed minimal impact of systemic CCR8 ablation on the frequency, phenotype and function of tumour-infiltrating Treg cells and conventional T (Tconv) function. These findings suggest that CCR8 is not required for Treg cell accumulation and immunosuppressive function within tumours and that depletion of CCR8 Treg cells rather than blockade of CCR8 function is a more promising avenue for selective immunotherapy.

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of HO By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of HO maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.

The transition from naive to primed pluripotency is accompanied by an extensive reorganisation of transcriptional and epigenetic programmes. However, the role of transcriptional enhancers and three-dimensional chromatin organisation in coordinating these developmental programmes remains incompletely understood. Here, we generate a high-resolution atlas of gene regulatory interactions, chromatin profiles and transcription factor occupancy in naive and primed human pluripotent stem cells, and develop a network-graph approach to examine the atlas at multiple spatial scales. We uncover highly connected promoter hubs that change substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merge to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. We identify widespread state-specific differences in enhancer activity and interactivity that correspond with an extensive reconfiguration of OCT4, SOX2 and NANOG binding and target gene expression. These findings provide multilayered insights into the chromatin-based gene regulatory control of human pluripotent states.