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BACKGROUND CD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here,we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy. METHODS Exosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry,also in the presence of selective inhibitors of CD73. Interferon (IFN)-$\gamma$ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control. RESULTS Isolated exosomes carried CD73 on their surface,which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-$\gamma$ release,which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment,expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders,while exosomal CD73 resulted significantly increased in non-responders. CONCLUSIONS CD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment,elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy. View Publication

BACKGROUND Alcohol abuse produces an enormous impact on health,society,and the economy. Currently,there are very limited therapies available,largely due to the poor understanding of mechanisms underlying alcohol use disorders (AUDs) in humans. Oxidative damage of mitochondria and cellular proteins aggravates the progression of neuroinflammation and neurological disorders initiated by alcohol abuse. RESULTS Here we show that ethanol exposure causes neuroinflammation in both human induced pluripotent stem (iPS) cells and human neural progenitor cells (NPCs). Ethanol exposure for 24 hours or 7 days does not affect the proliferation of iPS cells and NPCs,but primes an innate immune-like response by activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. This leads to an increase of microtubule-associated protein 1A/1B-light chain 3(+) (LC3B(+)) autophagic puncta and impairment of the mitochondrial and lysosomal distribution. In addition,a decrease of mature neurons derived from differentiating NPCs is evident in ethanol pre-exposed compared to control NPCs. Moreover,a second insult of a pro-inflammatory factor in addition to ethanol preexposure enhances innate cellular inflammation in human iPS cells. CONCLUSIONS This study provides strong evidence that neuronal inflammation contributes to the pathophysiology of AUDs through the activation of the inflammasome pathway in human cellular models. View Publication

INTRODUCTION Despite attempts to prevent brain injury during the hyperacute phase of stroke,most sufferers end up with significant neuronal loss and functional deficits. The use of cell-based therapies to recover the injured brain offers new hope. In the current study,we employed human neural stem cells (hNSCs) isolated from subventricular zone (SVZ),and directed their differentiation into GABAergic neurons followed by transplantation to ischemic brain. METHODS Pre-differentiated GABAergic neurons,undifferentiated SVZ-hNSCs or media alone were stereotaxically transplanted into the rat brain (n=7/group) 7 days after endothelin-1 induced stroke. Neurological outcome was assessed by neurological deficit scores and the cylinder test. Transplanted cell survival,cellular phenotype and maturation were assessed using immunohistochemistry and confocal microscopy. RESULTS Behavioral assessments revealed accelerated improvements in motor function 7 days post-transplant in rats treated with pre-differentiated GABAergic cells in comparison to media alone and undifferentiated hNSC treated groups. Histopathology 28 days-post transplant indicated that pre-differentiated cells maintained their GABAergic neuronal phenotype,showed evidence of synaptogenesis and up-regulated expression of both GABA and calcium signaling proteins associated with neurotransmission. Rats treated with pre-differentiated cells also showed increased neurogenic activity within the SVZ at 28 days,suggesting an additional trophic role of these GABAergic cells. In contrast,undifferentiated SVZ-hNSCs predominantly differentiated into GFAP-positive astrocytes and appeared to be incorporated into the glial scar. CONCLUSION Our study is the first to show enhanced exogenous repopulation of a neuronal phenotype after stroke using techniques aimed at GABAergic cell induction prior to delivery that resulted in accelerated and improved functional recovery. View Publication

Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors,their interaction with the host immune system,and their host range. We have investigated the capacity of a panel of GPs of both retroviral (amphotropic murine leukemia virus [MLV-A]; gibbon ape leukemia virus [GALV]; RD114,feline endogenous virus) and nonretroviral (fowl plague virus [FPV]; Ebola virus [EboV]; vesicular stomatitis virus [VSV]; lymphocytic choriomeningitis virus [LCMV]) origins to pseudotype lentiviral vectors derived from simian immunodeficiency virus (SIVmac251). SIV vectors were efficiently pseudotyped with the FPV hemagglutinin,VSV-G,LCMV,and MLV-A GPs. In contrast,the GALV and RD114 GPs conferred much lower infectivity to the vectors. Capitalizing on the conservation of some structural features in the transmembrane domains and cytoplasmic tails of the incorporation-competent MLV-A GP and in RD114 and GALV GPs,we generated chimeric GPs encoding the extracellular and transmembrane domains of GALV or RD114 GPs fused to the cytoplasmic tail (designated TR) of MLV-A GP. Importantly,SIV-derived vectors pseudotyped with these GALV/TR and RD114/TR GP chimeras had significantly higher titers than vectors coated with the parental GPs. Additionally,RD114/TR-pseudotyped vectors were efficiently concentrated and were resistant to inactivation induced by the complement of both human and macaque sera,indicating that modified RD114 GP-pseudotyped lentiviral vectors may be of particular interest for in vivo gene transfer applications. Furthermore,as compared to vectors pseudotyped with other retroviral GPs or with VSV-G,RD114/TR-pseudotyped vectors showed augmented transduction of human and macaque primary blood lymphocytes and CD34+ cells. View Publication

The Kit receptor functions in hematopoiesis,lymphocyte development,gastrointestinal tract motility,melanogenesis,and gametogenesis. To investigate the roles of different Kit signaling pathways in vivo,we have generated knock-in mice in which docking sites for PI 3-kinase (KitY719) or Src kinase (KitY567) have been mutated. Whereas steady-state hematopoiesis is normal in KitY719F/Y719F and KitY567F/Y567F mice,lymphopoiesis is affected differentially. The KitY567F mutation,but not the KitY719F mutation,blocks pro T cell and pro B cell development in an age-dependent manner. Thus,the Src family kinase,but not the PI 3-kinase docking site in Kit,mediates a critical signal for lymphocyte development. In agreement with these results,treatment of normal mice with the Kit tyrosine kinase inhibitor imatinib (Gleevec) leads to deficits in pro T and pro B cell development,similar to those seen in KitY567F/Y567F and KitW/W mice. The two mutations do not affect embryonic gametogenesis but the KitY719F mutation blocks spermatogenesis at the spermatogonial stages and in contrast the KitY567F mutation does not affect this process. Therefore,Kit-mediated PI 3-kinase signaling and Src kinase family signaling is highly specific for different cellular contexts in vivo. View Publication

Because video data are complex and are comprised of many images,mining information from video material is difficult to do without the aid of computer software. Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article,we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments,hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT. To determine the rate of growth of these colonies,recipes were developed using CL-Quant software which enables users to extract various types of data from video images. To accurately evaluate colony growth,three recipes were created. The first segmented the image into the colony and background,the second enhanced the image to define colonies throughout the video sequence accurately,and the third measured the number of pixels in the colony over time. The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes,the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared,results were virtually identical,indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition,other video bioinformatics recipes can be developed in the future for other cell processes such as migration,apoptosis,and cell adhesion. View Publication

SUMMARY Resident cardiac macrophages are critical mediators of cardiac function. Despite their known importance to cardiac electrophysiology and tissue maintenance,there are currently no stem-cell-derived models of human engineered cardiac tissues (hECTs) that include resident macrophages. In this study,we made an induced pluripotent stem cell (iPSC)-derived hECT model with a resident population of macrophages (iM0) to better recapitulate the native myocardium and characterized their impact on tissue function. Macrophage retention within the hECTs was confirmed via immunofluorescence after 28 days of cultivation. The inclusion of iM0s significantly impacted hECT function,increasing contractile force production. A potential mechanism underlying these changes was revealed by the interrogation of calcium signaling,which demonstrated the modulation of ?-adrenergic signaling in +iM0 hECTs. Collectively,these findings demonstrate that macrophages significantly enhance cardiac function in iPSC-derived hECT models,emphasizing the need to further explore their contributions not only in healthy hECT models but also in the contexts of disease and injury. In brief Lock and Graney et al. develop a human engineered cardiac tissue with an incorporated iPSC-derived macrophage population to better mimic the complex cell landscape of the native myocardium. Macrophage inclusion leads to increased contractile function of the tissue,which is attributed to macrophage stimulation of the cardiomyocyte ?-adrenergic signaling pathway. Graphical Abstract View Publication

Our understanding of the signalling pathways regulating early human development is limited,despite their fundamental biological importance. Here,we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors,along with IGF1 ligand. Consequently,we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions,we derive several pluripotent stem cell lines that express pluripotency-associated genes,retain high viability and a normal karyotype,and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos,and in both primed and na{\{i}}ve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche." View Publication

BackgroundLung cancer has become one of the most fatal cancers at present. Traditional treatments showed limited therapeutic effects on lung cancer. The phototherapy has emerged as a powerful approach for lung cancer treatment. Aggregation-induced emission luminogens (AIEgens) exhibit excellent optical performance such as strong fluorescence,enhanced reactive oxygen species (ROS) generation,and effective thermal effect after aggregation,which show great potential in phototherapy. However,the disadvantages including hydrophobicity,low specificity,and short circulation lifetime limited their efficacy on cancer therapy.MethodsWe developed a biomimetic AIEgens constructed using CD8+ T cells membrane to camouflage the AIEgen C41H37N2O3S2 (named BITT) nanoparticles (termed TB). The prepared TB improved the tumor accumulation of AIEgen by PD-1/PD-L1 recognition on the CD8+ T and LLC cell membranes,respectively.ResultsThe prepared TB showed improved binding efficiency,photothermal effects,and ROS generation ability to kill the lung cancer cells. TB also showed improved circulation lifetime and excellent tumor targeting ability,leading to effective phototherapy and immunotherapy in vivo based on BITT and the CD8+ T cell-derived membranes. Based on the AIE and immune checkpoint blockade (ICB) strategies,TB enhanced the antitumor activities of lung cancer by phototherapy and immunotherapy.ConclusionThe present work developed a type of biomimetic AIEgens,which overcame the inherent limitations of conventional AIEgens and leveraged immune recognition for targeted tumor accumulation. Furthermore,the integration of AIE-driven phototherapy with immune checkpoint blockade demonstrated potent synergistic antitumor efficacy,establishing a promising combinatorial strategy against aggressive lung malignancies. View Publication

IntroductionNeuroinflammation is a key contributor to the pathogenesis of Alzheimer's disease (AD),and impaired clearance of amyloid-? (A?) by microglia is closely associated with disease progression. Oxytocin (OXT),a hypothalamic neuropeptide,has recently been reported to exert anti-inflammatory effects on microglia; however,its therapeutic potential in the human brain remains unclear.MethodsWe generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) to model early AD-like pathology. A? toxicity was induced by applying 3 ?M A?1–42 for 48 h. The protective effects of OXT were evaluated through immunohistochemistry,RT-qPCR,calcium imaging,and multielectrode array (MEA) recordings. The involvement of microglia in A? clearance was assessed by immunostaining and gene expression analysis of TREM2.ResultsA? exposure led to significant deposition of A? in the outer layers of hCOs,accompanied by suppressed neural activity and increased apoptotic signaling. Pretreatment with OXT attenuated A? deposition and caspase-3-mediated apoptosis in a concentration-dependent manner. OXT also restored calcium oscillations and neuronal network activity as measured by MEA. Notably,OXT enhanced the recruitment of microglia to A? deposits and upregulated the expression of TREM2,a key regulator of microglial phagocytosis. Co-expression of oxytocin receptors (OXTR) on Iba1-positive microglia suggests that OXT directly modulates microglial activation and A? clearance.ConclusionsOXT has neuroprotective effects on human cortical organoids by preserving their neuronal activity and promoting microglial-mediated A? clearance. This study provides novel insights into the therapeutic potential of OXT for targeting neuroinflammation and A? pathology in patients with AD. Graphical abstractImage 1 Highlights•Oxytocin reduces A? deposition and apoptosis in human cerebral organoids.•A? impairs neuronal activity,rescued by oxytocin preconditioning.•Oxytocin enhances microglial phagocytosis via OXTR and TREM2 upregulation.•Human iPSC-derived organoids model early A? pathology and oxytocin response. View Publication