技术资料

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays,as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here,we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders,such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs,yielding electrophysiologically active neurons within just 3 wk. Using this platform,we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus,this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types. View Publication

We present a simple microfluidic system that generates water-in-water,aqueous two phase system (ATPS) droplets,by passive flow focusing. ATPS droplet formation is achieved by applying weak hydrostatic pressures,with liquid-filled pipette tips as fluid columns at the inlets,to introduce low speed flows to the flow focusing junction. To control the size of the droplets,we systematically vary the interfacial tension and viscosity of the ATPS fluids and adjust the fluid column height at the fluid inlets. The size of the droplets scales with a power law of the ratio of viscous stresses in the two ATPS phases. Overall,we find a drop size coefficient of variation (CV; i.e.,polydispersity) of about 10%. We also find that when drops form very close to the flow focusing junction,the drops have a CV of less than 1%. Our droplet generation method is easily scalable: we demonstrate a parallel system that generates droplets simultaneously and improves the droplet production rate by up to one order of magnitude. Finally,we show the potential application of our system for encapsulating cells in water-in-water emulsions by encapsulating microparticles and cells. To the best of our knowledge,our microfluidic technique is the first that forms low interfacial tension ATPS droplets without applying external perturbations. We anticipate that this simple approach will find utility in drug and cell delivery applications because of the all-biocompatible nature of the water-in-water ATPS environment. View Publication

Cockayne syndrome (CS) is a severe neurodevelopmental disorder characterized by growth abnormalities,premature aging,and photosensitivity. Mutation of Cockayne syndrome B (CSB) affects neuronal gene expression and differentiation,so we attempted to bypass its function by expressing downstream target genes. Intriguingly,ectopic expression of Synaptotagmin 9 (SYT9),a key component of the machinery controlling neurotrophin release,bypasses the need for CSB in neuritogenesis. Importantly,brain-derived neurotrophic factor (BDNF),a neurotrophin implicated in neuronal differentiation and synaptic modulation,and pharmacological mimics such as 7,8-dihydroxyflavone and amitriptyline can compensate for CSB deficiency in cell models of neuronal differentiation as well. SYT9 and BDNF are downregulated in CS patient brain tissue,further indicating that sub-optimal neurotrophin signaling underlies neurological defects in CS. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for pharmacological intervention,these data suggest an important role for SYT9 in neuronal differentiation. View Publication

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short,enzymatically biotinylated tag,compatible with SRI techniques including uPAINT,STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues,with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β,neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody,and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore,Nlg1 is dynamic,disperse and sensitive to synaptic stimulation,whereas LRRTM2 is organized in compact and stable nanodomains. Thus,mSA is a versatile tool to image membrane proteins at high resolution in complex live environments,providing novel information about the nano-organization of biological structures. View Publication

BACKGROUND Self-renewing,chemoresistant breast cancer stem cells are believed to contribute significantly to cancer invasion,migration and patient relapse. Therefore,the identification of signaling pathways that regulate the acquisition of stem-like qualities is an important step towards understanding why patients relapse and towards development of novel therapeutics that specifically target cancer stem cell vulnerabilities. Recent studies identified a role for the aryl hydrocarbon receptor (AHR),an environmental carcinogen receptor implicated in cancer initiation,in normal tissue-specific stem cell self-renewal. These studies inspired the hypothesis that the AHR plays a role in the acquisition of cancer stem cell-like qualities. RESULTS To test this hypothesis,AHR activity in Hs578T triple negative and SUM149 inflammatory breast cancer cells were modulated with AHR ligands,shRNA or AHR-specific inhibitors,and phenotypic,genomic and functional stem cell-associated characteristics were evaluated. The data demonstrate that (1) ALDH(high) cells express elevated levels of Ahr and Cyp1b1 and Cyp1a1,AHR-driven genes,(2) AHR knockdown reduces ALDH activity by 80%,(3) AHR hyper-activation with several ligands,including environmental ligands,significantly increases ALDH1 activity,expression of stem cell- and invasion/migration-associated genes,and accelerates cell migration,(4) a significant correlation between Ahr or Cyp1b1 expression (as a surrogate marker for AHR activity) and expression of stem cell- and invasion/migration-associated gene sets is seen with genomic data obtained from 79 human breast cancer cell lines and over 1,850 primary human breast cancers,(5) the AHR interacts directly with Sox2,a master regulator of self-renewal; AHR ligands increase this interaction and nuclear SOX2 translocation,(6) AHR knockdown inhibits tumorsphere formation in low adherence conditions,(7) AHR inhibition blocks the rapid migration of ALDH(high) cells and reduces ALDH(high) cell chemoresistance,(8) ALDH(high) cells are highly efficient at initiating tumors in orthotopic xenografts,and (9) AHR knockdown inhibits tumor initiation and reduces tumor Aldh1a1,Sox2,and Cyp1b1 expression in vivo. CONCLUSIONS These data suggest that the AHR plays an important role in development of cells with cancer stem cell-like qualities and that environmental AHR ligands may exacerbate breast cancer by enhancing expression of these properties. View Publication

Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here,memory B cells are activated and amplified using Epstein-Barr virus infection,co-cultured with CHO-muCD40L cells,and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells,and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly,our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family,influenza A neutralizing antibodies,contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. View Publication

Summary Human pluripotent stem cells (hPSCs) are uniquely dependent on aerobic glycolysis to generate ATP. However,the importance of oxidative phosphorylation (OXPHOS) has not been elucidated. Detailed amino acid profiling has revealed that glutamine is indispensable for the survival of hPSCs. Under glucose- and glutamine-depleted conditions,hPSCs quickly died due to the loss of ATP. Metabolome analyses showed that hPSCs oxidized pyruvate poorly and that glutamine was the main energy source for OXPHOS. hPSCs were unable to utilize pyruvate-derived citrate due to negligible expression of aconitase 2 (ACO2) and isocitrate dehydrogenase 2/3 (IDH2/3) and high expression of ATP-citrate lyase. Cardiomyocytes with mature mitochondria were not able to survive without glucose and glutamine,although they were able to use lactate to synthesize pyruvate and glutamate. This distinguishing feature of hPSC metabolism allows preparation of clinical-grade cell sources free of undifferentiated hPSCs,which prevents tumor formation during stem cell therapy. View Publication

Suppression of the cytotoxic T cell (CTL) immune response has been proposed as one mechanism for immune evasion in cancer. In this study,we have explored the underlying basis for CTL suppression in the context of B cell malignancies. We document that human B cells have an intrinsic ability to resist killing by freshly isolated cytotoxic T cells (CTLs),but are susceptible to lysis by IL-2 activated CTL blasts and CTLs isolated from immunotherapy-treated patients with chronic lymphocytic leukemia (CLL). Impaired killing was associated with the formation of dysfunctional non-lytic immune synapses characterized by the presence of defective linker for activation of T cells (LAT) signaling and non-polarized release of the lytic granules transported by ADP-ribosylation factor-like protein 8 (Arl8). We propose that non-lytic degranulation of CTLs are a key regulatory mechanism of evasion through which B cells may interfere with the formation of functional immune synapses by CTLs. View Publication

Surgical and medical androgen deprivation therapy (ADT) is a cornerstone for prostate cancer treatment,but relapse usually occurs. We herein show that orchiectomy synergizes with immunotherapy,whereas the more widely used treatment of medical ADT involving androgen receptor (AR) antagonists suppresses immunotherapy. Furthermore,we observed that the use of medical ADT could unexpectedly impair the adaptive immune responses through interference with initial T cell priming rather than in the reactivation or expansion phases. Mechanistically,we have revealed that inadvertent immunosuppression might be potentially mediated by a receptor shared with γ-aminobutyric acid. Our data demonstrate that the timing and dosing of antiandrogens are critical to maximizing the antitumor effects of combination therapy. This study highlights an underappreciated mechanism of AR antagonist-mediated immunosuppression and provides a new strategy to enhance immune response and prevent the relapse of advanced prostate cancer. View Publication

The establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism,genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs,the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells,which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation,metabolism,genetic network,and response to infection or other external stimuli. View Publication

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid,we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3,Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells,whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine. View Publication

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP),we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels,IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes,including a reduction in cell adhesion and increase in cell death. For cell adhesion,we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally,we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus,transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. View Publication