技术资料

BACKGROUND: Despite increasing demand,current protocols for human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) remain time,labor,and cost intensive. Additionally,absence of robust methods for selective RPE purification and removal of non-RPE cell impurities prevents upscaling of clinical quality RPE production. We aimed to address these challenges by developing a simplified hPSC-derived RPE production and purification system that yields high-quality RPE monolayers within 90 days. METHODS: Human pluripotent stem cells were differentiated into RPE using an innovative time and cost-effective protocol relying entirely on 2D cultures and minimal use of cytokines. Once RPE identity was obtained,cells were transferred onto permeable membranes to acquire mature RPE morphology. RPE differentiation was verified by electron microscopy,polarized VEGF expression,establishment of high transepithelial electrical resistance and photoreceptor phagocytosis assay. After 4 weeks on permeable membranes,RPE cell cultures were incubated with Dil-AcLDL (DiI-conjugated acetylated low-density lipoproteins) and subjected to fluorescence-activated cell sorting (FACS) for purification and subculture. RESULTS: Using our 2D cytokine scarce protocol,hPSC-derived functional RPE cells can be obtained within 2 months. Nevertheless,at this stage,most samples contain a percentage of non-RPE/early RPE progenitor cells that make them unsuitable for clinical application. We demonstrate that functional RPE cells express high levels of lipoprotein receptors and that this correlates with their ability to uptake lipoproteins. Combining photoreceptor uptake assay with lipoprotein uptake assay further confirms that only functional RPE cells uptake AcLDL. Incubation of mixed RPE/non-RPE cell cultures with fluorophore conjugated AcLDL and subsequent FACS-based isolation of labeled cells allows selective purification of mature functional RPE. When subcultured,DiI-AcLDL-labeled cells rapidly form pure homogenous high-quality RPE monolayers. CONCLUSIONS: Pure functional RPE monolayers can be derived from hPSC within 90 days using simplified 2D cultures in conjunction with our RPE PLUS protocol (RPE Purification by Lipoprotein Uptake-based Sorting). The simplicity of this protocol makes it scalable,and the rapidity of production and purification allows for high-quality RPE to be produced in a short span of time making them ideally suited for downstream clinical and in vitro applications. View Publication

The network topology of a protein interactome is shaped by the function of each protein,making it a resource of functional knowledge in tissues and in single cells. Today,this resource is underused,as complete network topology characterization has proved difficult for large protein interactomes. We apply a matrix visualization and decoding approach to a physical protein interactome of a dendritic cell,thereby characterizing its topology with no prior assumptions of structure. We discover 294 proteins,each forming topological motifs called bow-ties" that tie together the majority of observed protein complexes. The central proteins of these bow-ties have unique network properties display multifunctional capabilities are enriched for essential proteins and are widely expressed in other cells and tissues. Collectively the bow-tie motifs are a pervasive and previously unnoted topological trend in cellular interactomes. As such these results provide fundamental knowledge on how intracellular protein connectivity is organized and operates." View Publication

Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore,the objective was to study the effects of SCFAs on biomarkers of ISC activity,differentiation,barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET),propionate (PROP),or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments,but increased with ACET. As a result of BUT and PROP treatments,there was an increase in differentiation markers for enterocyte,Paneth,goblet,and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3$\beta$,Reg3$\gamma$,and Defb1 were stimulated by BUT and PROP,but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures,although tight junctions were influenced by BUT and PROP,but not ACET in monolayer format. Furthermore,BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall,individual SCFAs are potent stimulators of cellular gene expression,however,PROP and especially BUT show great efficacy for driving cell differentiation and gene expression. View Publication

Cerebrospinal fluid (CSF) is a vital liquid,providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP),a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here,we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally,the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes. View Publication

The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here,we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days,inhibitory GABAergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that,although basal neuronal firing rate is unaffected,there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening. View Publication

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts1. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types2,3. However,whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis,we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E2 (PGE2). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic,autochthonous tumorigenesis. Mechanistically,single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE2 drives the expansion οf a population of Sca-1+ reserve-like stem cells. These express a strong regenerative/tumorigenic program,driven by the Hippo pathway effector Yap. In vivo,Yap is indispensable for Sca-1+ cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments,we identified a molecular mechanism whereby PGE2 promotes Yap dephosphorylation,nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1+ cell expansion and sporadic tumour initiation in mutant mice,thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE2-Ptger4. Analyses of patient-derived organoids established that PGE2-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE2-Ptger4-Yap signalling axis. View Publication

The cerebellum plays a critical role in all vertebrates,and many neurological disorders are associated with cerebellum dysfunction. A major limitation in cerebellar research has been the lack of adequate disease models. As an alternative to animal models,cerebellar neurons differentiated from pluripotent stem cells have been used. However,previous studies only produced limited amounts of Purkinje cells. Moreover,in vitro generation of Purkinje cells required co-culture systems,which may introduce unknown components to the system. Here we describe a novel differentiation strategy that uses defined medium to generate Purkinje cells,granule cells,interneurons,and deep cerebellar nuclei projection neurons,that self-formed and differentiated into electrically active cells. Using a defined basal medium optimized for neuronal cell culture,we successfully promoted the differentiation of cerebellar precursors without the need for co-culturing. We anticipate that our findings may help developing better models for the study of cerebellar dysfunctions,while providing an advance toward the development of autologous replacement strategies for treating cerebellar degenerative diseases. View Publication

NF-$\kappa$B is a key regulator of inflammation and cancer progression,with an important role in leukemogenesis. Despite its therapeutic potential,targeting NF-$\kappa$B using pharmacologic inhibitors has proven challenging. Here,we describe a myeloid cell-selective NF-$\kappa$B inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a,C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6),thereby blocking activation of NF-$\kappa$B in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-$\kappa$B activation in myeloid cells,and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly,C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions,miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set,we found an inverse correlation of miR-146a levels with NF-$\kappa$B-related genes and with patient survival. Correspondingly,C-miR146a induced cytotoxic effects in human MDSL,HL-60,and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-$\kappa$B target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders. View Publication

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here,we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1{\&}2),a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS,for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1{\&}2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1{\&}2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore,PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway,and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1{\&}2 reversed arthritis in mice,providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies. View Publication

RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here,we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development,from early CD7-CD5- to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7-CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells,whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements,normalized T cell development,and corrected the NK cell-like phenotype. In conclusion,we succeeded in generating an iPSC-based RAG2-SCID model,which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks. View Publication

Although the association between the microbiome and IBD and liver diseases is known,the cause and effect remain elusive. By connecting human microphysiological systems of the gut,liver,and circulating Treg and Th17 cells,we created a multi-organ model of ulcerative colitis (UC) ex vivo. The approach shows microbiome-derived short-chain fatty acids (SCFAs) to either improve or worsen UC severity,depending on the involvement of effector CD4 T cells. Using multiomics,we found SCFAs increased production of ketone bodies,glycolysis,and lipogenesis,while markedly reducing innate immune activation of the UC gut. However,during acute T cell-mediated inflammation,SCFAs exacerbated CD4+ T cell-effector function,partially through metabolic reprograming,leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity,metabolism,and tissue homeostasis. View Publication

Vaccine efficacy can be increased by arraying immunogens in multivalent form on virus-like nanoparticles to enhance B-cell activation. However,the effects of antigen copy number,spacing and affinity,as well as the dimensionality and rigidity of scaffold presentation on B-cell activation remain poorly understood. Here,we display the clinical vaccine immunogen eOD-GT8,an engineered outer domain of the HIV-1 glycoprotein-120,on DNA origami nanoparticles to systematically interrogate the impact of these nanoscale parameters on B-cell activation in vitro. We find that B-cell signalling is maximized by as few as five antigens maximally spaced on the surface of a 40-nm viral-like nanoparticle. Increasing antigen spacing up to {\~{}}25-30 nm monotonically increases B-cell receptor activation. Moreover,scaffold rigidity is essential for robust B-cell triggering. These results reveal molecular vaccine design principles that may be used to drive functional B-cell responses. View Publication