技术资料

BACKGROUND Adult intestinal organoids have been used to study ex vivo intestinal injury in adulthood. However,the neonatal intestinal epithelium has many unique features that are different from adult mature intestine. Establishing a neonatal ex vivo organoid model is essential to study the epithelial physiology in early postnatal development and to investigate derangements associated with disease processes during the neonatal period like necrotizing enterocolitis (NEC). METHODS Fresh and frozen terminal ileum was harvested from mice pups on postnatal day 9. Crypts were isolated and organoids were cultured. Organoids were exposed to hypoxia and lipopolysaccharide (LPS) for 48 h to induce epithelial injury. Inflammatory cytokines and tight junction proteins were evaluated. RESULTS Robust intestinal organoids can be formed from both fresh and frozen intestinal tissue of neonatal mice pups. Hypoxia and LPS administration induced intestinal inflammation and disrupted tight junctions in these neonatal intestinal organoids. CONCLUSIONS We have established a novel method to grow organoids from neonatal intestine. We demonstrated that these organoids respond to the injury occurring during neonatal intestinal diseases such as NEC by increasing the organoid inflammation and by disrupting the organoid barrier function. Organoids provide an ex vivo platform to study intestinal physiology and pathology during the neonatal period. View Publication

Cystic Fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mutations cause dysregulation of channel function with intracellular accumulation of misfolded proteins and endoplasmic reticulum (ER) stress,with activation of the IRE1alpha-XBP1 pathway that regulates a subset of unfolded protein response (UPR) genes. This pathway regulates a group of genes that control proinflammatory and metabolic responses in different immune cells; however,the metabolic state of immune cells and the role of this pathway in CF remain elusive. Our results indicate that only innate immune cells from CF patients present increased levels of ER stress,mainly affecting neutrophils,monocytes,and macrophages. An overactive IRE1alpha-XBP1 pathway reprograms CF M1 macrophages toward an increased metabolic state,with increased glycolytic rates and mitochondrial function,associated with exaggerated production of TNF and IL-6. This hyper-metabolic state,seen in CF macrophages,is reversed by inhibiting the RNase domain of IRE1alpha,thereby decreasing the increased glycolic rates,mitochondrial function and inflammation. Altogether,our results indicate that innate immune cells from CF patients are primarily affected by ER stress. Moreover,the IRE1alpha-XBP1 pathway of the UPR is responsible for the hyper-metabolic state seen in CF macrophages,which is associated with the exaggerated inflammatory response. Modulating ER stress,metabolism and inflammation,by targeting IRE1alpha,may improve the metabolic fitness of macrophages,and other immune cells in CF and other immune-related disorders. View Publication

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM),a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation,we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast,in ML-DS,clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model,we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically,genetically,and transcriptionally mirrored ML-DS. View Publication

Intestinal fibrosis is a serious complication in inflammatory bowel disease (IBD). Despite the remarkable success of recent anti-inflammatory therapies for IBD,incidence of intestinal fibrosis and need for bowel resection have not significantly changed. To clarify the contribution of haematopoietic-derived cells in intestinal fibrosis,we prepared bone marrow (BM) chimeric mice (chimeras),which were reconstituted with BM cells derived from enhanced green fluorescent protein (EGFP)-transgenic mice or CC chemokine receptor 2 (CCR2)-deficient mice. After 2 months of transplantation,BM chimeras were treated with azoxymethane/dextran sodium sulphate. During chronic inflammation,CCR2+ BM-derived monocyte and fibrocyte infiltration into the colon and CC chemokine ligand 2 production increased,leading to colon fibrosis in EGFP BM chimeras. In CCR2-deficient BM chimeras,monocyte and fibrocyte numbers in the colonic lamina propria significantly decreased,and colon fibrosis was attenuated. In colon tissue,mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 but not of collagen I,transforming growth factor-beta1 or matrix metalloproteinases was significantly different between the two chimeras. CCR2+ monocytes and fibrocytes showed high Timp1 mRNA expression. Our results suggest that infiltrating CCR2+ monocytes and their progenies,fibrocytes,promote colon fibrosis by inhibiting collagen degradation through TIMP-1 production. View Publication

Rapidly proliferating cells are highly sensitive to ionizing radiation and can undergo apoptosis if the oxidative and genotoxic injury exceed the defensive and regenerative capacity of the cell. Our earlier work has established the antiapoptotic action of the growth factor-like lipid mediator lysophosphatidic acid (LPA). Activation of the LPA2 GPCR has been hypothesized to elicit antiapoptotic and regenerative actions of LPA. Based on this hypothesis we developed a novel nonlipid agonist of LPA2,which we designated Radioprotectin-1 (RP-1). We tested RP-1 at the six murine LPA GPCR subtypes using the transforming growth factor alpha shedding assay and found that it had a 25 nM EC50 that is similar to that of LPA18:1 at 32 nM. RP-1 effectively reduced apoptosis induced by gamma-irradiation and the radiomimetic drug Adriamycin only in cells that expressed LPA2 either endogenously or after transfection. RP-1 reduced gamma-H2AX levels in irradiated mouse embryonic fibroblasts transduced with the human LPA2 GPCR but was ineffective in vector transduced MEF control cells and significantly increased clonogenic survival after gamma-irradiation. gamma-Irradiation induced the expression of lpar2 transcripts that was further enhanced by RP-1 exposure within 30 min after irradiation. RP-1 decreased the mortality of C57BL/6 mice in models of the hematopoietic and gastrointestinal acute radiation syndromes. Using Lgr5-EGFP-CreER;Tdtomatoflox transgenic mice,we found that RP-1 increased the survival and growth of intestinal enteroids via the enhanced survival of Lgr5+ intestinal stem cells. Taken together,our results suggest that the LPA2-specific agonist RP-1 exerts its radioprotective and radiomitigative action through specific activation of the upregulated LPA2 GPCR in Lgr5+ stem cells. View Publication

Type 1 diabetes (T1D) is an autoimmune disease in which a strong inflammatory response causes the death of insulin-producing pancreatic beta-cells,while inefficient regulatory mechanisms allow that response to become chronic. Ethyl pyruvate (EP),a stable pyruvate derivate and certified inhibitor of an alarmin-high mobility group box 1 (HMGB1),exerts anti-oxidant and anti-inflammatory properties in animal models of rheumatoid arthritis and encephalomyelitis. To test its therapeutic potential in T1D,EP was administered intraperitoneally to C57BL/6 mice with multiple low-dose streptozotocin (MLDS)-induced T1D. EP treatment decreased T1D incidence,reduced the infiltration of cells into the pancreatic islets and preserved beta-cell function. Apart from reducing HMGB1 expression,EP treatment successfully interfered with the inflammatory response within the local pancreatic lymph nodes and in the pancreas. Its effect was restricted to boosting the regulatory arm of the immune response through up-regulation of tolerogenic dendritic cells (CD11c+CD11b-CD103+) within the pancreatic infiltrates and through the enhancement of regulatory T cell (Treg) levels (CD4+CD25highFoxP3+). These EP-stimulated Treg displayed enhanced suppressive capacity reflected in increased levels of CTLA-4,secreted TGF-beta,and IL-10 and in the more efficient inhibition of effector T cell proliferation compared to Treg from diabetic animals. Higher levels of Treg were a result of increased differentiation and proliferation (Ki67+ cells),but also of the heightened potency for migration due to increased expression of adhesion molecules (CD11a and CD62L) and CXCR3 chemokine receptor. Treg isolated from EP-treated mice had the activated phenotype and T-bet expression more frequently,suggesting that they readily suppressed IFN-gamma-producing cells. The effect of EP on Treg was also reproduced in vitro. Overall,our results show that EP treatment reduced T1D incidence in C57BL/6 mice predominantly by enhancing Treg differentiation,proliferation,their suppressive capacity,and recruitment into the pancreas. View Publication

The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens,but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells,we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs,40{\%} of antigen-specific SED B cells bind antigen within 2 h,whereas unspecifc cells do not,indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice,but is unperturbed in mice depleted of classical dendritic cells (DC). Thus,we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses,and should be taken into account when developing mucosal vaccines. View Publication

Deregulation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is found in cancer with STAT5A/B controlling leukemic cell survival and disease progression. As mutations in STAT5B,but not STAT5A,have been frequently described in hematopoietic tumors,we used BCR/ABL as model systems to investigate the contribution of STAT5A or STAT5B for leukemogenesis. The absence of STAT5A decreased cell survival and colony formation. Even more drastic effects were observed in the absence of STAT5B. STAT5B-deficient cells formed BCR/ABL+ colonies or stable cell lines at low frequency. The rarely evolving Stat5b-/- cell lines expressed enhanced levels of BCR/ABL oncoprotein compared to wild-type cells. In line,Stat5b-/- leukemic cells induced leukemia with a significantly prolonged disease onset,whereas Stat5a-/- cells rapidly caused a fatal disease superimposable to wild-type cells. RNA-sequencing (RNA-seq) profiling revealed a marked enhancement of interferon (IFN)-alpha and IFN-gamma signatures in Stat5b-/- cells. Inhibition of IFN responses rescued BCR/ABL+ colony formation of Stat5b-/--deficient cells. A downregulated IFN response was also observed in patients suffering from leukemia carrying STAT5B mutations. Our data define STAT5B as major STAT5 isoform driving BCR/ABL+ leukemia. STAT5B enables transformation by suppressing IFN-alpha/gamma,thereby facilitating leukemogenesis. Our findings might help explain the high frequency of STAT5B mutations in hematopoietic tumors. View Publication

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation,we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range,enabling targeting of many previously inaccessible PAMs. On average,enAsCas12a exhibits a twofold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a,and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing,endogenous gene activation and C-to-T base editing,and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively,enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing. View Publication

Development of targeted cancer therapy requires a thorough understanding of mechanisms of tumorigenesis as well as mechanisms of action of therapeutics. This is challenging because by the time patients are diagnosed with cancer,early events of tumorigenesis have already taken place. Similarly,development of cancer immunotherapies is hampered by a lack of appropriate small animal models with autologous human tumor and immune system. In this article,we report the development of a mouse model of human acute myeloid leukemia (AML) with autologous immune system for studying early events of human leukemogenesis and testing the efficacy of immunotherapeutics. To develop such a model,human hematopoietic stem/progenitor cells (HSPC) are transduced with lentiviruses expressing a mutated form of nucleophosmin (NPM1),referred to as NPM1c. Following engraftment into immunodeficient mice,transduced HSPCs give rise to human myeloid leukemia,whereas untransduced HSPCs give rise to human immune cells in the same mice. The de novo AML,with CD123+ leukemic stem or initiating cells (LSC),resembles NPM1c+ AML from patients. Transcriptional analysis of LSC and leukemic cells confirms similarity of the de novo leukemia generated in mice with patient leukemia and suggests Myc as a co-operating factor in NPM1c-driven leukemogenesis. We show that a bispecific conjugate that binds both CD3 and CD123 eliminates CD123+ LSCs in a T cell-dependent manner both in vivo and in vitro. These results demonstrate the utility of the NPM1c+ AML model with an autologous immune system for studying early events of human leukemogenesis and for evaluating efficacy and mechanism of immunotherapeutics. View Publication

Acute inflammatory bowel disease (AIBD) is a wide clinical entity including severe gastrointestinal pathologies with common histopathological basis. Epidemiologically increasing diseases,such as necrotizing enterocolitis (NEC),gastrointestinal graft versus host disease (GVHD),and the primary acute phase of chronic inflammatory bowel disease (CIBD),exhibit a high necessity for new therapeutic strategies. Mesenchymal stem cell (MSC) cellular therapy represents a promising option for the treatment of these diseases. In our study,we comparatively assess the efficacy of human MSCs derived from bone marrow (BM),umbilical cord blood (UCB),human embryonic stem cells (ESCs),or human-induced pluripotent stem cells (iPSCs) in a mouse model of chemically induced acute enterocolitis. The laboratory animals were provided ad libitum potable dextrane sulfate sodium solution (DSS) in order to reproduce an AIBD model and then individually exposed intraperitoneally to MSCs derived from BM (BM-MSCs),UCB (UCB-MSCs),ESCs (ESC-MSCs),or iPSCs (iPSC-MSCs). The parameters used to evaluate the cellular treatment efficacy were the animal survival prolongation and the histopathological-macroscopic picture of bowel sections. Although all categories of mesenchymal stem cells led to statistically significant survival prolongation compared to the control group,significant clinical and histopathological improvement was observed only in mice receiving BM-MSCs and UCB-MSCs. Our results demonstrated that the in vivo anti-inflammatory effect of ESC-MSCs and iPSC-MSCs was inferior to that of UCB-MSCs and BM-MSCs. Further investigation will clarify the potential of ESCs and iPSC-derived MSCs in AIBD treatment. View Publication

Human pluripotent stem cell (hPSC)-derived intestinal organoids (hIOs) form 3D structures organized into crypt and villus domains,making them an excellent in vitro model system for studying human intestinal development and disease. However,hPSC-derived hIOs still require in vivo maturation to fully recapitulate adult intestine,with the mechanism of maturation remaining elusive. Here,we show that the co-culture with human T lymphocytes induce the in vitro maturation of hIOs,and identify STAT3-activating interleukin-2 (IL-2) as the major factor inducing maturation. hIOs exposed to IL-2 closely mimic the adult intestinal epithelium and have comparable expression levels of mature intestinal markers,as well as increased intestine-specific functional activities. Even after in vivo engraftment,in vitro-matured hIOs retain their maturation status. The results of our study demonstrate that STAT3 signaling can induce the maturation of hIOs in vitro,thereby circumventing the need for animal models and in vivo maturation. View Publication