技术资料

The tumor microenvironment (TME) comprises complex interactions of multiple cell types that determines cell behavior and metabolism such as nutrient competition and immune suppression. We discuss the various types of heterogeneity that exist in solid tumors,and the complications this invokes for studies of TME. As human subjects and in vivo model systems are complex and difficult to manipulate,simpler 3D model systems that are compatible with flexible experimental control are necessary for studying metabolic regulation in TME. Stable Isotope Resolved Metabolomics (SIRM) is a valuable tool for tracing metabolic networks in complex systems,but at present does not directly address heterogeneous metabolism at the individual cell level. We compare the advantages and disadvantages of different model systems for SIRM experiments,with a focus on lung cancer cells,their interactions with macrophages and T cells,and their response to modulators in the immune microenvironment. We describe the experimental set up,illustrate results from 3D cultures and co-cultures of lung cancer cells with human macrophages,and outline strategies to address the heterogeneous TME. View Publication

Tissue resident intestinal macrophages are known to exhibit an anti-inflammatory phenotype and produce little pro-inflammatory cytokines upon TLR ligation,allowing symbiotic co-existence with the intestinal microbiota. However,upon acute events such as epithelial damage and concomitant influx of microbes,these macrophages must be able to quickly mount a pro-inflammatory response while more inflammatory macrophages are recruited from the blood stream simultaneously. Here,we show that dietary intake of vitamin A is required for the maintenance of the anti-inflammatory state of tissue resident intestinal macrophages. Interestingly,these anti-inflammatory macrophages were characterized by high levels of Dectin-1 expression. We show that Dectin-1 expression is enhanced by the vitamin A metabolite retinoic acid and our data suggests that Dectin-1 triggering might provide a switch to induce a rapid production of pro-inflammatory cytokines. In addition,Dectin-1 stimulation resulted in an altered metabolic profile which is linked to a pro-inflammatory response. Together,our data suggests that presence of vitamin A in the small intestine enhances an anti-inflammatory phenotype as well as Dectin-1 expression by macrophages and that this anti-inflammatory phenotype can rapidly convert toward a pro-inflammatory state upon Dectin-1 signaling. View Publication

Mast cells (MCs) arise from hematopoietic stem cells (HSCs) that mature within vascularized tissues. Fibroblasts and endothelial cells (ECs) play a role in the maturation of HSCs in the tissues. Due to difficulties in isolating MCs from tissues,large numbers of committed MC precursors can be generated in 2D culture systems with the use of differentiation factors. Since MCs are tissue-resident cells,the development of a 3D tissue-engineered model with ancillary cells that more closely mimics the 3D in vivo microenvironment has greater relevance for MC studies. The goals of this study were to show that MCs can be derived from HSCs within a 3D matrix and to determine a media to support MCs,fibroblasts,and ECs. The results show that HSCs within a collagen matrix cultured in StemSpan media with serum added at the last week yielded a greater number of c-kit+ cells and a greater amount of histamine granules compared to other media tested. Media supplemented with serum were necessary for EC survival,while fibroblasts survived irrespective of serum with higher cell yields in StemSpan. This work demonstrates the development of functional MCs within a 3D collagen matrix using a stem cell media that supports fibroblast and ECs. View Publication

Gelatin coatings are effective in increasing the retention of MSCs injected into the heart and minimizing the damage from acute myocardial infarction (AMI),but early studies suffered from low fractions of the MSCs coated with gelatin. Biotinylation of the MSC surface is a critical first step in the gelatin coating process,and in this study,we evaluated the use of biotinylated cholesterol lipid insertion" anchors as a substitute for the covalent NHS-biotin anchors to the cell surface. Streptavidin-eosin molecules where eosin is our photoinitiator can then be bound to the cell surface through biotin-streptavidin affinity. The use of cholesterol anchors increased streptavidin density on the surface of MSCs further driving polymerization and allowing for an increased fraction of MSCs coated with gelatin (83{\%}) when compared to NHS-biotin (52{\%}). Additionally the cholesterol anchors increased the uniformity of the coating on the MSC surface and supported greater numbers of coated MSCs even when the streptavidin density was slightly lower than that of an NHS-biotin anchoring strategy. Critically this improvement in gelatin coating efficiency did not impact cytokine secretion and other critical MSC functions. Proper selection of the cholesterol anchor and the biotinylation conditions supports cellular function and densities of streptavidin on the MSC surface of up to {\~{}}105 streptavidin molecules/$\mu$m2 . In all these cholesterol anchors offer an effective path towards the formation of conformal coatings on the majority of MSCs to improve the retention of MSCs in the heart following AMI." View Publication

Thrombosis is a major cause of mortality in patients with myeloproliferative neoplasms (MPNs),though there is currently little to offer patients with MPN beyond aspirin and cytoreductive therapies such as hydroxyurea for primary prevention. Thrombogenesis in MPN involves multiple cellular mechanisms,including platelet activation and neutrophil-extracellular trap formation; therefore,an antithrombotic agent that targets one or more of these processes would be of therapeutic benefit in MPN. Here,we treated the JAK2V617F knockin mouse model of polycythemia vera with N-acetylcysteine (NAC),a sulfhydryl-containing compound with broad effects on glutathione replenishment,free radical scavenging,and reducing disulfide bonds,to investigate its antithrombotic effects in the context of MPN. Strikingly,NAC treatment extended the lifespan of JAK2V617F mice without impacting blood counts or splenomegaly. Using an acute pulmonary thrombosis model in vivo,we found that NAC reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin. In vitro analysis of platelet activation revealed that NAC reduced thrombin-induced platelet-leukocyte aggregate formation in JAK2V617F mice. Furthermore,NAC reduced neutrophil extracellular trap formation in primary human neutrophils from patients with MPN as well as healthy controls. These results provide evidence that N-acetylcysteine inhibits thrombosis in JAK2V617F mice and provide a pre-clinical rationale for investigating NAC as a therapeutic to reduce thrombotic risk in MPN. View Publication

Cancer stem cells are critical for cancer initiation,development,and treatment resistance. Our understanding of these processes,and how they relate to glioblastoma heterogeneity,is limited. To overcome these limitations,we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells,and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells,and,using RNA velocity,is often the originator of the other cell types. Finally,we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development,suggests a possible origin for glioblastoma hierarchy,and helps to identify cancer stem cell-specific targets. View Publication

Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis,binding and uptake of exosomes are well characterized4,5,delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology,we show that these two lipid receptors,which interact in the late endosome9,are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway,which Ebola virus exploits to infect cells9,mediates HAV infection by exo-HAV,which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen,but not the endosomal uncoating of HAV particles contained in the exosomes,is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV,infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses,and call for a reassessment of the role of envelope glycoproteins in infection. View Publication

Human-based body-on-a-chip" technology provides powerful platforms in developing models for drug evaluation and disease evaluations in phenotypic models. Induced pluripotent stem cells (iPSCs) are ideal cell sources for generating different cell types for these in vitro functional systems and recapitulation of the neuromuscular reflex arc would allow for the study of patient specific neuromuscular diseases. Regarding relevant afferent (intrafusal fibers sensory neurons) and efferent (extrafusal fibers motoneurons) cells in vitro differentiation of intrafusal fiber from human iPSCs has not been established. This work demonstrates a protocol for inducing an enrichment of intrafusal bag fibers from iPSCs using morphological analysis and immunocytochemistry. Phosphorylation of the ErbB2 receptors and S46 staining indicated a 3-fold increase of total intrafusal fibers further confirming the efficiency of the protocol. Integration of induced intrafusal fibers would enable more accurate reflex arc models and application of this protocol on patient iPSCs would allow for patient-specific disease modeling." View Publication

Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly,we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2,3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized),and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli. View Publication

BACKGROUND/OBJECTIVE A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects. MATERIALS AND METHODS Blood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion,the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR. RESULTS There was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media,and markers such as NG2 and $\alpha$SMA in mesenchymal media. However,analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population. CONCLUSIONS CD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However,nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease. View Publication

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here,we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes,14 potent neutralizing antibodies were identified,with the most potent one,BD-368-2,exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2,respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally,the 3.8 {\AA} cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover,we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether,we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases. View Publication

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age,but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further,we examined the combined and separate effects of an oncogene (c-MYC) and exposure to IL3,GM-CSF and SCF on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized,rapidly fatal and serially transplantable blast population,phenotypically and transcriptionally similar to human AML cells,but only in mice producing IL3,GM-CSF and SCF transgenically,or in regular mice in which the cells were exposed to IL3 or GM-CSF delivered using a co-transduction strategy. In their absence,the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months but,upon transfer to secondary mice producing the human cytokines,the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late (GMPs) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them. View Publication