Explore the vast range of titles available.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1 −/− chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine. Single-cell profiling is used to create a molecular-level atlas of cell differentiation trajectories during gastrulation and early organogenesis in the mouse.

Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of ‘customized’ embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells—derived from mouse fibroblasts—can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro -reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells. Stem cells with potential The search for new ways of coaxing readily available cells to show the pluripotent activity of embryonic stem cells — the potential to differentiate into virtually any cell type — continues. The stakes are high, since if it can be achieved safely for human cells, cell transplantation therapy, even patient-specific therapy, will have come a step closer. Two groups now report an important advance in this direction: the creation of pluripotent stem cells from mouse fibroblasts. The epigenetic reprogramming requires the expression of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4. The resulting cells resemble embryonic stem cells in both biological potency and epigenetic state. Four transcription factors Oct4, Sox2, c-Myc and Klf4 are known to convert fibroblasts to pluripotent stem cells, if Fbx 15 expression is also selected. But the induced stem cells were shown to be distinct from normal embryonic stem cells. However, if cells expressing Nanog and Oct4 are selected, then the reprogrammed fibroblasts are similar to embryonic stem cells in both biological potency and epigenetic state.

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a \"cytokine storm\" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses.

Depression and anxiety disorders are associated with increased release of peripheral cytokines; however, their functional relevance remains unknown. Using a social stress model in mice, we find preexisting individual differences in the sensitivity of the peripheral immune system that predict and promote vulnerability to social stress. Cytokine profiles were obtained 20 min after the first social stress exposure. Of the cytokines regulated by stress, IL-6 was most highly up-regulated only in mice that ultimately developed a susceptible behavioral phenotype following a subsequent chronic stress, and levels remained elevated for at least 1 mo. We confirmed a similar elevation of serum IL-6 in two separate cohorts of patients with treatment-resistant major depressive disorder. Before any physical contact in mice, we observed individual differences in IL-6 levels from ex vivo stimulated leukocytes that predict susceptibility versus resilience to a subsequent stressor. To shift the sensitivity of the peripheral immune system to a pro- or antidepressant state, bone marrow (BM) chimeras were generated by transplanting hematopoietic progenitor cells from stress-susceptible mice releasing high IL-6 or from IL-6 knockout (IL-6 ⁻/⁻) mice. Stress-susceptible BM chimeras exhibited increased social avoidance behavior after exposure to either subthreshold repeated social defeat stress (RSDS) or a purely emotional stressor termed witness defeat. IL-6 ⁻/⁻ BM chimeric and IL-6 ⁻/⁻ mice, as well as those treated with a systemic IL-6 monoclonal antibody, were resilient to social stress. These data establish that preexisting differences in stress-responsive IL-6 release from BM-derived leukocytes functionally contribute to social stress-induced behavioral abnormalities. Significance Depression and anxiety have been linked to increased inflammation. However, we do not know if inflammatory status predates onset of disease or whether it contributes to depression symptomatology. We report preexisting individual differences in the peripheral immune system that predict and promote stress susceptibility. Replacing a stress-naive animal’s peripheral immune system with that of a stressed animal increases susceptibility to social stress including repeated social defeat stress (RSDS) and witness defeat (a purely emotional form of social stress). Depleting the cytokine IL-6 from the whole body or just from leukocytes promotes resilience, as does sequestering IL-6 outside of the brain. These studies demonstrate that the emotional response to stress can be generated or blocked in the periphery, and offer a potential new form of treatment for stress disorders.

Atherosclerosis is a major cause of death and disability in cardiovascular disease. Atherosclerosis associated with lipid accumulation and chronic inflammation leads to plaques formation in arterial walls and luminal stenosis in carotid arteries. Current approaches such as surgery or treatment with statins encounter big challenges in curing atherosclerosis plaque. The infiltration of proinflammatory M1 macrophages plays an essential role in the occurrence and development of atherosclerosis plaque. A recent study shows that TRIM24, an E3 ubiquitin ligase of a Trim family protein, acts as a valve to inhibit the polarization of anti-inflammatory M2 macrophages, and elimination of TRIM24 opens an avenue to achieve the M2 polarization. Proteolysis-targeting chimera (PROTAC) technology has emerged as a novel tool for the selective degradation of targeting proteins. But the low bioavailability and cell specificity of PROTAC reagents hinder their applications in treating atherosclerosis plaque. In this study we constructed a type of bioinspired PROTAC by coating the PROTAC degrader (dTRIM24)-loaded PLGA nanoparticles with M2 macrophage membrane (MELT) for atherosclerosis treatment. MELT was characterized by morphology, size, and stability. MELT displayed enhanced specificity to M1 macrophages as well as acidic-responsive release of dTRIM24. After intravenous administration, MELT showed significantly improved accumulation in atherosclerotic plaque of high fat and high cholesterol diet-fed atherosclerotic (ApoE −/− ) mice through binding to M1 macrophages and inducing effective and precise TRIM24 degradation, thus resulting in the polarization of M2 macrophages, which led to great reduction of plaque formation. These results suggest that MELT can be considered a potential therapeutic agent for targeting atherosclerotic plaque and alleviating atherosclerosis progression, providing an effective strategy for targeted atherosclerosis therapy.

Bone marrow (BM) chimeric mice are a valuable tool in the field of immunology, with the genetic manipulation of donor cells widely used to study gene function under physiological and pathological settings. To date, however, BM chimera protocols require myeloablative conditioning of recipient mice, which dramatically alters steady-state hematopoiesis. Additionally, most protocols use fluorescence-activated cell sorting (FACS) of hematopoietic stem/progenitor cells (HSPCs) for ex vivo genetic manipulation. Here, we describe our development of cell culture techniques for the enrichment of functional HSPCs from mouse BM without the use of FACS purification. Furthermore, the large number of HSPCs derived from these cultures generate BM chimeric mice without irradiation. These HSPC cultures can also be genetically manipulated by viral transduction, to allow for doxycycline-inducible transgene expression in donor-derived immune cells within non-conditioned immunocompetent recipients. This technique is therefore expected to overcome current limitations in mouse transplantation models. Bone marrow chimaeric mice are a valuable tool in research, but require myeloablative conditioning. Here the authors demonstrate efficient FACS-free enrichment of haematopoietic stem and progenitor cells for transplantation into unconditioned recipient mice, as well as for genetic engineering using polyvinyl alcohol based media.

An orthogonal aminoacyl-tRNA synthetase/tRNA pair is a crucial prerequisite for site-specific incorporation of unnatural amino acids. Due to its high codon suppression efficiency and full orthogonality, the pyrrolysyl-tRNA synthetase/pyrrolysyl-tRNA pair is currently the ideal system for genetic code expansion in both eukaryotes and prokaryotes. There is a pressing need to discover or engineer other fully orthogonal translation systems. Here, through rational chimera design by transplanting the key orthogonal components from the pyrrolysine system, we create multiple chimeric tRNA synthetase/chimeric tRNA pairs, including chimera histidine, phenylalanine, and alanine systems. We further show that these engineered chimeric systems are orthogonal and highly efficient with comparable flexibility to the pyrrolysine system. Besides, the chimera phenylalanine system can incorporate a group of phenylalanine, tyrosine, and tryptophan analogues efficiently in both E. coli and mammalian cells. These aromatic amino acids analogous exhibit unique properties and characteristics, including fluorescence, post-translation modification. Orthogonal aminoacyl-tRNA synthetase/tRNA pairs are crucial for the incorporation of unnatural amino acids in a site-specific manner. Here the authors use rational chimera design to create multiple efficient pairs that function in bacterial and mammalian systems for genetic code expansion.

The derivation of induced pluripotent stem cells (iPSCs) over a decade ago sparked widespread enthusiasm for the development of new models of human disease, enhanced platforms for drug discovery and more widespread use of autologous cell-based therapy. Early studies using directed differentiation of iPSCs frequently uncovered cell-level phenotypes in monogenic diseases, but translation to tissue-level and organ-level diseases has required development of more complex, 3D, multicellular systems. Organoids and human–rodent chimaeras more accurately mirror the diverse cellular ecosystems of complex tissues and are being applied to iPSC disease models to recapitulate the pathobiology of a broad spectrum of human maladies, including infectious diseases, genetic disorders and cancer.Enthusiasm for patient-specific therapies based on induced pluripotent stem cells (iPSCs) has risen in parallel with rapid advances in genome editing. This Review summarizes the progress in iPSC-based disease modelling over the past decade, with a focus on 3D organoid systems and chimeric models being exploited for new therapeutic approaches.

Idiopathic pulmonary fibrosis is a devastating disease with poor prognosis. The pathogenic role of the lysophospholipid mediator sphingosine-1-phosphate and its receptor S1PR2 in lung fibrosis is unknown. We show here that genetic deletion of S1pr2 strikingly attenuated lung fibrosis induced by repeated injections of bleomycin in mice. We observed by using S1pr2LacZ/+ mice that S1PR2 was expressed in alveolar macrophages, vascular endothelial cells and alveolar epithelial cells in the lung and that S1PR2-expressing cells accumulated in the fibrotic legions. Bone marrow chimera experiments suggested that S1PR2 in bone marrow-derived cells contributes to the development of lung fibrosis. Depletion of macrophages greatly attenuated lung fibrosis. Bleomycin administration stimulated the mRNA expression of the profibrotic cytokines IL-13 and IL-4 and the M2 markers including arginase 1, Fizz1/Retnla, Ccl17 and Ccl24 in cells collected from broncho-alveolar lavage fluids (BALF), and S1pr2 deletion markedly diminished the stimulated expression of these genes. BALF cells from bleomycin-administered wild-type mice showed a marked increase in phosphorylation of STAT6, a transcription factor which is activated downstream of IL-13, compared with saline-administered wild-type mice. Interestingly, in bleomycin-administered S1pr2-/- mice, STAT6 phosphorylation in BALF cells was substantially diminished compared with wild-type mice. Finally, pharmacological S1PR2 blockade in S1pr2+/+ mice alleviated bleomycin-induced lung fibrosis. Thus, S1PR2 facilitates lung fibrosis through the mechanisms involving augmentation of IL-13 expression and its signaling in BALF cells, and represents a novel target for treating lung fibrosis.