技术资料

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study,we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg,HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV,thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection,we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients,we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions. Synopsis This study presents human pluripotent stem cell-derived hepatocyte-like cells (HLCs) as a culture system that expands the repertoire of research tools for studying hepatitis B and D viruses (HBV/HDV) and identifies CD63 as a potential HDV co-entry factor. Co-infection with HBV or virus-mediated delivery of HBV surface proteins enables HDV to complete its life cycle in HLCs.Extracellular HDV spread in HLCs enables the evaluation of anti-HDV therapies.HDV permissiveness along HLC differentiation reveals CD63 as a novel co-factor of HDV cell entry. This study presents human pluripotent stem cell-derived hepatocyte-like cells (HLCs) as a culture system that expands the repertoire of research tools for studying hepatitis B and D viruses (HBV/HDV) and identifies CD63 as a potential HDV co-entry factor. View Publication

Post-transcriptional modifications to RNA,which comprise the epitranscriptome,play important roles in RNA metabolism,gene regulation,and human disease,including viral pathogenesis. Modifications to the RNA viral genome and transcripts of human immunodeficiency virus 1 (HIV-1) have been reported and investigated in the context of virus and host biology. However,the diversity of experimental approaches used has made clear correlations across studies,as well as the significance of the HIV-1 epitranscriptome in biology and disease,difficult to assess. Therefore,we established a reference HIV-1 epitranscriptome. We sequenced the model NL4–3 HIV-1 genome from infected primary CD4+ T cells and the Jurkat cell line using the latest nanopore chemistry,optimized RNA preparation methods,and the most current and readily available base-calling algorithms. A highly reproducible sense and a preliminary antisense HIV-1 epitranscriptome were created,where N6-methyladenosine (m6A),5-methylcytosine (m5C),pseudouridine (psi),inosine,and 2’-O-methyl (Nm) modifications could be identified by rapid multiplexed base-calling. We observed that sequence and neighboring modification contexts induced modification miscalling,which could be corrected with synthetic HIV-1 RNA fragments. We validated m6A modification sites with STM2457,a small molecule inhibitor of methyltransferase-like 3 (METTL3). We find that modifications are quite stable under combination antiretroviral therapy (cART) treatment,in primary CD4+ T cells,and in HIV-1 virions. Sequencing samples from people living with HIV (PLWH) revealed conservation of m6A modifications. However,analysis of spliced transcript variants suggests transcript-dependent modification levels. Our approach and reference data offer a straightforward benchmark that can be adopted to help advance rigor,reproducibility,and uniformity across HIV-1 epitranscriptomics studies. They also provide a roadmap for the creation of reference epitranscriptomes for many other viruses or pathogens. View Publication

We have established several HLA-A2.1-transgenic rabbit lines to provide a host to study CD8(+) T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition,vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy. View Publication

Mechanisms that maintain cancer stem cells are crucial to tumour progression. The ID2 protein supports cancer hallmarks including the cancer stem cell state. HIFα transcription factors,most notably HIF2α (also known as EPAS1),are expressed in and required for maintenance of cancer stem cells (CSCs). However,the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. Here we report that DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Hypoxia downregulates this phosphorylation via inactivation of DYRK1A and DYRK1B. The activity of these kinases is stimulated in normoxia by the oxygen-sensing prolyl hydroxylase PHD1 (also known as EGLN2). ID2 binds to the VHL ubiquitin ligase complex,displaces VHL-associated Cullin 2,and impairs HIF2α ubiquitylation and degradation. Phosphorylation of Thr27 of ID2 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma,ID2 positively modulates HIF2α activity. Conversely,elevated expression of DYRK1 phosphorylates Thr27 of ID2,leading to HIF2α destabilization,loss of glioma stemness,inhibition of tumour growth,and a more favourable outcome for patients with glioblastoma. View Publication

With increasing worldwide demand for safe blood,there is much interest in generating red blood cells in vitro as an alternative clinical product. However,available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply,and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach,immortalizing early adult erythroblasts generating a stable line,which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature,functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level,and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture. View Publication

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species,the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest,followed by an immunodepletion step using microbeads coated with CD11b,CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion,a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1),CD90,CD105 and CD73 cell surface markers,but negative for the hematopoietic surface markers CD14,CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic,osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time. View Publication

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Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this,time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis,including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique,in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work,and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality,fast chromosomal analysis of human ES and iPS cells. View Publication

Magnesium (Mg) is a promising biodegradable metallic material for applications in cellular/tissue engineering and biomedical implants/devices. To advance clinical translation of Mg-based biomaterials,we investigated the effects and mechanisms of Mg degradation on the proliferation and pluripotency of human embryonic stem cells (hESCs). We used hESCs as the in vitro model system to study cellular responses to Mg degradation because they are sensitive to toxicants and capable of differentiating into any cell types of interest for regenerative medicine. In a previous study when hESCs were cultured in vitro with either polished metallic Mg (99.9% purity) or pre-degraded Mg,cell death was observed within the first 30 hours of culture. Excess Mg ions and hydroxide ions induced by Mg degradation may have been the causes for the observed cell death; hence,their respective effects on hESCs were investigated for the first time to reveal the potential mechanisms. For this purpose,the mTeSR®1 hESC culture media was either modified to an alkaline pH of 8.1 or supplemented with 0.4-40 mM of Mg ions. We showed that the initial increase of media pH to 8.1 had no adverse effect on hESC proliferation. At all tested Mg ion dosages,the hESCs grew to confluency and retained pluripotency as indicated by the expression of OCT4,SSEA3,and SOX2. When the supplemental Mg ion dosages increased to greater than 10 mM,however,hESC colony morphology changed and cell counts decreased. These results suggest that Mg-based implants or scaffolds are promising in combination with hESCs for regenerative medicine applications,providing their degradation rate is moderate. Additionally,the hESC culture system could serve as a standard model for cytocompatibility studies of Mg in vitro,and an identified 10 mM critical dosage of Mg ions could serve as a design guideline for safe degradation of Mg-based implants/scaffolds. View Publication

Hematotoxicity is a significant issue for drug safety and can result from direct cytotoxicity toward circulating mature blood cell types as well as targeting of immature blood-forming stem cells/progenitor cells in the bone marrow. In vitro models for understanding and investigating the hematotoxicity potential of new test items/drugs are critical in early preclinical drug development. The traditional method,colony forming unit (CFU) assay,is commonly used and has been validated as a method for hematotoxicity screening. The CFU assay has multiple limitations for its application in investigative work. In this paper,we describe a detailed protocol for a liquid-culture,microplate-based in vitro hematotoxicity assay to evaluate lineage-specific (myeloid,erythroid,and megakaryocytic) hematotoxicity at different stages of differentiation. This assay has multiple advantages over the traditional CFU assay,including being suitable for high-throughput screening and flexible enough to allow inclusion of additional endpoints for mechanistic studies. Therefore,it is an extremely useful tool for scientists in pharmaceutical discovery and development. {\textcopyright} 2018 by John Wiley & Sons,Inc. View Publication

The inner ear organs responsible for hearing (cochlea) and balance (vestibular system) are susceptible to oxidative stress due to the high metabolic demands of their sensorineural cells. Oxidative stress-induced damage to these cells can cause hearing loss or vestibular dysfunction,yet the precise mechanisms remain unclear due to the limitations of animal models and challenges of obtaining living human inner ear tissue. Therefore,we developed an in vitro oxidative stress model of the pre-natal human inner ear using otic progenitor cells (OPCs) derived from human-induced pluripotent stem cells (hiPSCs). OPCs,hiPSCs,and HeLa cells were exposed to hydrogen peroxide or ototoxic drugs (gentamicin and cisplatin) that induce oxidative stress to evaluate subsequent cell viability,cell death,reactive oxygen species (ROS) production,mitochondrial activity,and apoptosis (caspase 3/7 activity). Dose-dependent reductions in OPC cell viability were observed post-exposure,demonstrating their vulnerability to oxidative stress. Notably,gentamicin exposure induced ROS production and cell death in OPCs,but not hiPSCs or HeLa cells. This OPC-based human model effectively simulates oxidative stress conditions in the human inner ear and may be useful for modeling the impact of ototoxicity during early pregnancy or evaluating therapies to prevent cytotoxicity. View Publication

Mutations in RB and PTEN are linked to castration resistance and poor prognosis in prostate cancer. Identification of genes that are regulated by these tumor suppressors in a context that recapitulates cancer progression may be beneficial for discovering novel therapeutic targets. Although various genetically engineered mice thus far provided tumor models with various pathological stages,they are not ideal for detecting dynamic changes in gene transcription. Additionally,it is difficult to achieve an effect specific to tumor progression via gain of functions of these genes. In this study,we developed an in vitro model to help identify RB- and PTEN-loss signatures during the malignant progression of prostate cancers. Trp53(-/-) ; Rb(f/f),Trp53(-/-) ; Pten(f/f),and Trp53(-/-) ; Rb(f/f) ; Pten(f/f) prostate epithelial cells were infected with AD-LacZ or AD-Cre. We found that deletion of Rb,Pten or both stimulated prostasphere formation and tumor development in immune-compromised mice. The GO analysis of genes affected by the deletion of Rb or Pten in Trp53(-/-) prostate epithelial cells identified a number of genes encoding cytokines,chemokines and extracellular matrix remodeling factors,but only few genes related to cell cycle progression. Two genes (Il-6 and Lox) were further analyzed. Blockade of Il-6 signaling and depletion of Lox significantly attenuated prostasphere formation in 3D culture,and in the case of IL-6,strongly suppressed tumor growth in vivo. These findings suggest that our in vitro model may be instrumental in identifying novel therapeutic targets of prostate cancer progression,and further underscore IL-6 and LOX as promising therapeutic targets. textcopyright 2015 Wiley Periodicals,Inc. View Publication