技术资料

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

Graphene quantum dots (GQDs) have gained significant attention in various biomedical applications. The physicochemical properties of these nanoparticles,including toxic effects,are largely determined by their surface modifications. Previous studies have demonstrated high in vitro cytotoxicity of the hydroxylated GQDs (OH-GQDs). The focus of this study was on the intestinal toxicity of OH-GQDs. Briefly,C57BL/6J mice were given daily oral gavage of 0.05,0.5 or 5 mg/kg OH-GQD for 7 days,and the indices of intestinal damage were evaluated. Higher doses of the OH-GQDs caused significant intestinal injuries,such as enhanced intestinal permeability,shortened villi and crypt loss. The number of Lgr5+ intestinal stem cells also decreased dramatically upon OH-GQDs exposure,which also inhibited the Ki67+ proliferative progenitor cells. In addition,an increased number of crypt cells harboring the oxidized DNA base 8-OHdG and $\gamma$H2AX foci were also detected in the intestines of OH-GQD-treated mice. Mechanistically,the OH-GQDs up-regulated both total and phosphorylated p53. Consistent with this,the average number of TUNEL+ and cleaved caspase-3+ apoptotic intestinal epithelial cells were significantly increased after OH-GQDs treatment. Finally,a 3-dimensional organoid culture was established using isolated crypts,and OH-GQDs treatment significantly reduced the size of the surviving intestinal organoids. Taken together,the intestinal toxicity of the OH-GQDs should be taken into account during biomedical applications. View Publication

Despite implementation of virtual crossmatches,flow cytometry crossmatches (FCXM) are still used by many transplant centers to determine immunological risk before kidney transplantation. To determine if common profiles of patients prone to false positive FCXM exist,we examined the demographics and native diseases of kidney patients tested with autologous FCXM (n = 480). Improvements to FCXM and cell isolation methods significantly reduced the positive rate from 15.1{\%} to 5.3{\%}. Patients with native diseases considered 'immunological' (vasculitis,lupus,IgA nephropathy) had more positive autologous FCXM (OR = 3.36,p = 0.003) vs. patients with all other diseases. Patients who were tested using our updated method (n = 321) still showed that these immunological diseases were a significant predictor for positive autologous FCXM (OR = 4.79,p = 0.006). Interestingly,patients on peritoneal dialysis (PD) also had significantly more positive autologous FCXM than patients on hemodialysis or waiting for pre-emptive kidney transplants (OR = 3.27,p = 0.02). These findings were confirmed in patients who had false positive allogeneic FCXM. Twenty of 24 (83.3{\%}) patients with false positive allogeneic FCXM tested with updated method either had immunological diseases originally or were on PD. Our findings are helpful when interpreting an unexpected positive FCXM,especially for transplantation from deceased donors. View Publication

The vascular wall is a source of progenitor cells that are able to induce skeletal repair,primarily by paracrine mechanisms. Here,the paracrine role of extracellular vesicles (EVs) in bone healing was investigated. First,purified human perivascular stem cells (PSCs) were observed to induce mitogenic,pro-migratory,and pro-osteogenic effects on osteoprogenitor cells while in non-contact co-culture via elaboration of EVs. PSC-derived EVs shared mitogenic,pro-migratory,and pro-osteogenic properties of their parent cell. PSC-EV effects were dependent on surface-associated tetraspanins,as demonstrated by EV trypsinization,or neutralizing antibodies for CD9 or CD81. Moreover,shRNA knockdown in recipient cells demonstrated requirement for the CD9/CD81 binding partners IGSF8 and PTGFRN for EV bioactivity. Finally,PSC-EVs stimulated bone repair,and did so via stimulation of skeletal cell proliferation,migration,and osteodifferentiation. In sum,PSC-EVs mediate the same tissue repair effects of perivascular stem cells,and represent an 'off-the-shelf' alternative for bone tissue regeneration. View Publication

Ethanol (EtOH)-induced alterations in intestinal homeostasis lead to multi-system pathologies,including liver injury. $\omega$-6 PUFAs exert pro-inflammatory activity,while $\omega$-3 PUFAs promote anti-inflammatory activity that is mediated,in part,through specialized pro-resolving mediators [e.g.,resolvin D1 (RvD1)]. We tested the hypothesis that a decrease in the $\omega$-6:$\omega$-3 PUFA ratio would attenuate EtOH-mediated alterations in the gut-liver axis. $\omega$-3 FA desaturase-1 (fat-1) mice,which endogenously increase $\omega$-3 PUFA levels,were protected against EtOH-mediated downregulation of intestinal tight junction proteins in organoid cultures and in vivo. EtOH- and lipopolysaccharide-induced expression of INF-$\gamma$,Il-6,and Cxcl1 was attenuated in fat-1 and WT RvD1-treated mice. RNA-seq of ileum tissue revealed upregulation of several genes involved in cell proliferation,stem cell renewal,and antimicrobial defense (including Alpi and Leap2) in fat-1 versus WT mice fed EtOH. fat-1 mice were also resistant to EtOH-mediated downregulation of genes important for xenobiotic/bile acid detoxification. Further,gut microbiome and plasma metabolomics revealed several changes in fat-1 versus WT mice that may contribute to a reduced inflammatory response. Finally,these data correlated with a significant reduction in liver injury. Our study suggests that $\omega$-3 PUFA enrichment or treatment with resolvins can attenuate the disruption in intestinal homeostasis caused by EtOH consumption and systemic inflammation with a concomitant reduction in liver injury. View Publication

Mammalian focal adhesion proteins Pinch1 and Pinch2 regulate integrin activation and cell-extracellular matrix adhesion and migration. Here,we show that deleting Pinch1 in osteocytes and mature osteoblasts using the 10-kb mouse Dmp1-Cre and Pinch2 globally (double KO; dKO) results in severe osteopenia throughout life,while ablating either gene does not cause bone loss,suggesting a functional redundancy of both factors in bone. Pinch deletion in osteocytes and mature osteoblasts generates signals that inhibit osteoblast and bone formation. Pinch-deficient osteocytes and conditioned media from dKO bone slice cultures contain abundant sclerostin protein and potently suppress osteoblast differentiation in primary BM stromal cells (BMSC) and calvarial cultures. Pinch deletion increases adiposity in the BM cavity. Primary dKO BMSC cultures display decreased osteoblastic but enhanced adipogenic,differentiation capacity. Pinch loss decreases expression of integrin $\beta$3,integrin-linked kinase (ILK),and $\alpha$-parvin and increases that of active caspase-3 and -8 in osteocytes. Pinch loss increases osteocyte apoptosis in vitro and in bone. Pinch loss upregulates expression of both Rankl and Opg in the cortical bone and does not increase osteoclast formation and bone resorption. Finally,Pinch ablation exacerbates hindlimb unloading-induced bone loss and impairs active ulna loading-stimulated bone formation. Thus,we establish a critical role of Pinch in control of bone homeostasis. View Publication

The perivascular niche within adipose tissue is known to house multipotent cells,including osteoblast precursors. However,the identity of perivascular subpopulations that may mineralize or ossify most readily is not known. Here,we utilize inducible PDGFR$\alpha$ (platelet-derived growth factor alpha) reporter animals to identify subpopulations of perivascular progenitor cells. Results showed that PDGFR$\alpha$-expressing cells are present in four histologic niches within inguinal fat,including two perivascular locations. PDGFR$\alpha$+ cells are most frequent within the tunica adventitia of arteries and veins,where PDGFR$\alpha$+ cells populate the inner aspects of the adventitial layer. Although both PDGFR$\alpha$+ and PDGFR$\alpha$- fractions are multipotent progenitor cells,adipose tissue-derived PDGFR$\alpha$+ stromal cells proliferate faster and mineralize to a greater degree than their PDGFR$\alpha$- counterparts. Likewise,PDGFR$\alpha$+ ectopic implants reconstitute the perivascular niche and ossify to a greater degree than PDGFR$\alpha$- cell fractions. Adventicytes can be further grouped into three distinct groups based on expression of PDGFR$\alpha$ and/or CD34. When further partitioned,adventicytes co-expressing PDGFR$\alpha$ and CD34 represented a cell fraction with the highest mineralization potential. Long-term tracing studies showed that PDGFR$\alpha$-expressing adventicytes give rise to adipocytes,but not to other cells within the vessel wall under homeostatic conditions. However,upon bone morphogenetic protein 2 (BMP2)-induced ossicle formation,descendants of PDGFR$\alpha$+ cells gave rise to osteoblasts,adipocytes,and pericyte-like" cells within the ossicle. In sum PDGFR$\alpha$ marks distinct perivascular osteoprogenitor cell subpopulations within adipose tissue. The identification of perivascular osteoprogenitors may contribute to our improved understanding of pathologic mineralization/ossification. Stem Cells 2019." View Publication

Molecular- and cellular-based therapies have the potential to reduce obesity-associated disease. In response to cold,beige adipocytes form in subcutaneous white adipose tissue and convert energy stored in metabolic substrates to heat,making them an attractive therapeutic target. We developed a robust method to generate a renewable source of human beige adipocytes from induced pluripotent stem cells (iPSCs). Developmentally,these cells are derived from FOXF1+ mesoderm and progress through an expandable mural-like mesenchymal stem cell (MSC) to form mature beige adipocytes that display a thermogenically active profile. This includes expression of uncoupling protein 1 (UCP1) concomitant with increased uncoupled respiration. With this method,dysfunctional adipogenic precursors can be reprogrammed and differentiated into beige adipocytes with increased thermogenic function and anti-diabetic secretion potential. This resource can be used to (1) elucidate mechanisms that underlie the control of beige adipogenesis and (2) generate material for cellular-based therapies that target metabolic syndrome in humans. View Publication

TFF1,a secreted protein,plays an essential role in keeping the integrity of gastric mucosa and its barrier function. Loss of TFF1 expression in the TFF1-knockout (KO) mouse leads to a pro-inflammatory phenotype with a cascade of gastric lesions that include low-grade dysplasia,high-grade dysplasia,and adenocarcinomas. In this study,we demonstrate nuclear localization of p-STATY705,with significant overexpression of several STAT3 target genes in gastric glands from the TFF1-KO mice. We also show frequent loss of TFF1 with nuclear localization of STAT3 in human gastric cancers. The reconstitution of TFF1 protein in human gastric cancer cells and 3D gastric glands organoids from TFF1-KO mice abrogates IL6-induced nuclear p-STAT3Y705 expression. Reconstitution of TFF1 inhibits IL6-induced STAT3 transcription activity,suppressing expression of its target genes. TFF1 blocks IL6R$\alpha$-GP130 complex formation through interfering with binding of IL6 to its receptor IL6R$\alpha$. These findings demonstrate a functional role of TFF1 in suppressing gastric tumorigenesis by impeding the IL6-STAT3 pro-inflammatory signaling axis. View Publication

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes,including the release of proteases,phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation,or NETosis,is a specific and highly efficient process,which is induced by a variety of stimuli leading to expulsion of DNA,proteases and antimicrobial peptides to the extracellular space. However,uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here,we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly,neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF),a Panx1 channel inhibitor,decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus,we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target. View Publication