技术资料

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis,temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility,a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound,pifithrin-alpha,protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus,inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction. View Publication

The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (200-fold) the efficiency of iPSC generation from human fibroblasts,within seven days of treatment. This will provide a basis for developing safer,more efficient,nonviral methods for reprogramming human somatic cells. View Publication

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy with one of the worst outcomes among all cancers. PDA often recurs after initial treatment to result in patient death despite the use of chemotherapy or radiation therapy. PDA contains a subset of tumor-initiating cells capable of extensive self-renewal known as cancer stem cells (CSC),which may contribute to therapeutic resistance and metastasis. At present,conventional chemotherapy and radiotherapy are largely ineffective in depleting CSC pool,suggesting the need for novel therapies that specifically target the cancer-sustaining stem cells for tumor eradication and to improve the poor prognosis of PDA patients. In this study,we report that death receptor 5 (DR5) is enriched in pancreatic CSCs compared with the bulk of the tumor cells. Treating a collection of freshly generated patient-derived PDA xenografts with gemcitabine,the first-line chemotherapeutic agent for PDA,is initially effective in reducing tumor size,but largely ineffective in diminishing the CSC populations,and eventually culminated in tumor relapse. However,a combination of tigatuzumab,a fully humanized DR5 agonist monoclonal antibody,with gemcitabine proved to be more efficacious by providing a double hit to kill both CSCs and bulk tumor cells. The combination therapy produced remarkable reduction in pancreatic CSCs,tumor remissions,and significant improvements in time to tumor progression in a model that is considered more difficult to treat. These data provide the rationale to explore the DR5-directed therapies in combination with chemotherapy as a therapeutic option to improve the current standard of care for pancreatic cancer patients. View Publication

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AIM OF THE STUDY Human breast milk reduces the risk and severity of necrotizing enterocolitis (NEC). Exosomes are extracellular vesicles (EVs) found in high concentrations in milk,and they mediate intercellular communication and immune responses. The aim of this study is to compare the protective effects of exosomes that are derived from different time periods of breast milk production against intestinal injury using an ex vivo intestinal organoid model. METHODS Colostrum,transitional and mature breast milk samples from healthy lactating mothers were collected. Exosomes were isolated using serial ultracentrifugation and filtration. Exosomes' presence was confirmed using transmission electron microscopy (TEM) and western blot. To form the intestinal organoids,terminal ileum was harvested from neonatal mice pups at postnatal day 9,crypts were isolated and organoids were cultured in matrigel. Organoids were either cultured with exposure to lipopolysaccharide (LPS),or in treatment groups where both LPS and exosomes were added in the culturing medium. Inflammatory markers and organoids viability were evaluated. MAIN RESULTS Human milk-derived exosomes were successfully isolated and characterized. LPS administration reduced the size of intestinal organoids,induced inflammation through increasing TNF$\alpha$ and TLR4 expression,and stimulated intestinal regeneration. Colostrum,transitional and mature human milk-derived exosome treatment all prevented inflammatory injury,while exosomes derived from colostrum were most effective at reducing inflammatory cytokine. CONCLUSIONS Human breast milk-derived exosomes were able to protect intestine organoids against epithelial injury induced by LPS. Colostrum exosomes offer the best protective effect among the breast-milk derived exosomes. Human milk exosomes can be protective against the development of intestinal injury such as that seen in NEC. View Publication

Background: Preclinical cell tracking is enhanced with a multimodal imaging approach. Bioluminescence imaging (BLI) is a highly sensitive optical modality that relies on engineering cells to constitutively express a luciferase gene. Magnetic particle imaging (MPI) is a newer imaging modality that directly detects superparamagnetic iron oxide (SPIO) particles used to label cells. Here,we compare BLI and MPI for imaging cells in vitro and in vivo. Methods: Mouse 4T1 breast carcinoma cells were transduced to express firefly luciferase,labeled with SPIO (ProMag),and imaged as cell samples after subcutaneous injection into mice. Results: For cell samples,the BLI and MPI signals were strongly correlated with cell number. Both modalities presented limitations for imaging cells in vivo. For BLI,weak signal penetration,signal attenuation,and scattering prevented the detection of cells for mice with hair and for cells far from the tissue surface. For MPI,background signals obscured the detection of low cell numbers due to the limited dynamic range,and cell numbers could not be accurately quantified from in vivo images. Conclusions: It is important to understand the shortcomings of these imaging modalities to develop strategies to improve cellular detection sensitivity. View Publication

The Wiskott-Aldrich syndrome protein (WASP) is a product of the gene defective in an Xid disorder,Wiskott-Aldrich syndrome. WASP expression is limited to hemopoietic cells,and WASP regulates the actin cytoskeleton. It has been reported that monocytes/macrophages from WASP-deficient Wiskott-Aldrich syndrome patients are severely defective in chemotaxis,resulting in recurrent infection. However,the molecular basis of such chemotactic defects is not understood. Recently,the WASP N-terminal region was found to bind to the three mammalian verprolin homologs: WASP interacting protein (WIP); WIP and CR16 homologous protein (WICH)/WIP-related protein (WIRE); and CR16. Verprolin was originally found to play an important role in the regulation of actin cytoskeleton in yeast. We have shown that WASP,WIP,and WICH/WIRE are expressed predominantly in the human monocyte cell line THP-1 and that WIP and WICH/WIRE are involved in monocyte chemotaxis. When WASP binding to verprolins was blocked,chemotactic migration of monocytes was impaired in both THP-1 cells and primary human monocytes. Increased expression of WASP and WIP enhanced monocyte chemotaxis. Blocking WASP binding to verprolins impaired cell polarization but not actin polymerization. These results indicate that a complex of WASP with mammalian verprolins plays an important role in chemotaxis of monocytes. Our results suggest that WASP and mammalian verprolins function as a unit in monocyte chemotaxis and that the activity of this unit is critical to establish cell polarization. In addition,our results also indicate that the WASP-verprolin complex is involved in other functions such as podosome formation and phagocytosis. View Publication

Amyotrophic lateral sclerosis is a progressive disease characterized by the loss of upper and lower motor neurons,leading to paralysis of voluntary muscles. About 10% of all ALS cases are familial (fALS),among which 15-20% are linked to Cu/Zn superoxide dismutase (SOD1) mutations,usually inherited in an autosomal dominant manner. To date only one FDA approved drug is available which increases survival moderately. Our understanding of ALS disease mechanisms is largely derived from rodent model studies,however due to the differences between rodents and humans,it is necessary to have humanized models for studies of disease pathogenesis as well as drug development. Therefore,we generated a comprehensive library of a total 22 of fALS patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized before being deposited into the library. The library of cells includes a variety of C9orf72 mutations,sod1 mutations,FUS,ANG and FIG4 mutations. Certain mutations are represented with more than one line,which allows for studies of variable genetic backgrounds. In addition,these iPSCs can be successfully differentiated to astroglia,a cell type known to play a critical role in ALS disease progression. This library represents a comprehensive resource that can be used for ALS disease modeling and the development of novel therapeutics. View Publication

Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells,whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3-/- pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues,whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability,DNA binding,and gene transcription. Moreover,NEUROG3 was highly unstable in the intestinal epithelium,explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships. View Publication

Sickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium,we generated a diverse,comprehensive,and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities,β-globin gene (HBB) haplotypes,and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development,model disease,and perhaps eventually,treat patients. Here,we describe this unique resource for the study of sickle cell disease,including novel haplotype-specific polymorphisms that affect disease severity,as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library,and as proof of principle for future cell- and gene-based therapies,we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation. View Publication

The Y-linked SRY gene initiates mammalian testis-determination. However,how the expression of SRY is regulated remains elusive. Here,we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5’ regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination,including a large multigenerational family,identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing,in a novel in vitro cellular model recapitulating human Sertoli cell formation,resulted in a significant reduction in expression of SRY. Therefore,human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression,a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare,the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process. Disease-causing variants define a conserved and unique NR5A1 responsive enhancer for SRY expression to initiate testis-determination in humans. Modelling regulatory variants causing sex-reversal provides a tool to understand global enhancer activity. View Publication

SummaryCryosectioning remains the gold standard for antibody and transcriptomic/in situ hybridization tissue analysis. However,tissue processing is time-consuming and costly,limiting routine and diagnostic use. Currently,no commercially available protocols or products exist for multiplexing this process. Here,we introduce multiplexed tissue molds (MTMs) that enable high-throughput cryoprocessing—cutting costs and workload by up to 96% while permitting the processing of tissues of various sizes and origins. We demonstrate compatibility with heterogeneous tissues by processing 19 different adult mouse tissues in parallel. Furthermore,we process up to ?110 neural organoids of different ages and sizes simultaneously and assess their neural differentiation marker expression. MTMs allow sectioning-based tissue analysis when labor,time,and cost are limiting factors. MTMs could be used to compare high specimen numbers in histopathological settings,organism-wide antigen and antibody targeting studies,high-throughput tissue screens,and defined tissue section positioning for,e.g.,spatial transcriptomics experiments. Graphical abstract Highlights•Multiplexed tissue molds (MTMs) drastically upscale cryosectioning procedures•MTMs can simultaneously accommodate up to 19 mouse organs and ?110 cerebral organoids•MTMs reduce analysis costs and processing times of tissues by up to 96%•MTMs could be used to reduce diagnostic costs and for spatial transcriptomics MotivationEfficient cryosectioning remains a critical yet labor- and cost-intensive step for immunohistochemistry and in situ hybridization,limiting routine diagnostic and research applications. The increasing demand for high-throughput tissue analysis—driven by advances in organoid and three-dimensional (3D) culture systems and tissue analysis for diagnostics—necessitates methods capable of processing numerous heterogeneous samples simultaneously. Current protocols lack multiplexing capabilities,leading to variability and extended processing times. Our work introduces multiplexed tissue molds (MTMs),a scalable solution that drastically reduces costs and labor by up to 96% while maintaining tissue integrity and consistency,thereby enabling large-scale (>100 tissues) comparative analyses and enhanced experimental reproducibility as well as access to tissue analysis,where cost is a restrictive factor. Reumann et al. develop multiplexed tissue molds (MTMs),which allow upscaling of tissue processing (up to 19 mouse organs or ?110 cerebral organoids simultaneously) while reducing workload and associated analysis costs by up to 96%. MTMs allow cryosection-based tissue analysis when labor,time,and cost are limiting factors and could be used for patient sample analysis as well as spatial transcriptomics approaches. View Publication