技术资料

The prostate transmembrane protein,androgen-induced 1 (PMEPA1) has been previously shown to promote solid malignancies in a variety of cancers,but the role and mechanisms of PMEPA1 in breast cancer has not been fully addressed. Here,we found that PMEPA1 was upregulated in breast cancer cell lines as well as in a set of clinical invasive breast ductal carcinomas. Interestingly,depletion of PMEPA1 decreased breast cancer stem cell (CSC)-enriched populations,while ectopic overexpression of PMEPA1 increased breast CSC-enriched populations. Furthermore,transforming growth factor-$$ (TGF-$$) treatment was also found to upregulate PMEPA1 expression and the CSC-enriched populations in triple-negative breast cancer cell lines. TGF-$$-induced PMEPA1 expression partially contributed to TGF-$$-induced breast CSC maintenance. These findings suggest that TGF-$$-PMEPA1 axis might provide new diagnosis and therapeutic targets for breast cancer treatment. View Publication

Recently,we reported that human neutrophils produce biologically active amounts of IL-6 when incubated with agonists activating TLR8,a receptor recognizing viral single strand RNA. In this study,we demonstrate that IFNα,a cytokine that modulates the early innate immune responses toward viral and bacterial infections,potently enhances the production of IL-6 in neutrophils stimulated with R848,a TLR8 agonist. We also show that such an effect is not caused by an IFNα-dependent induction of TLR7 and its consequent co-activation with TLR8 in response to R848,but,rather,it is substantially mediated by an increased production and release of endogenous TNFα. The latter cytokine,in an autocrine manner,leads to an augmented synthesis of the IkBζ co-activator and an enhanced recruitment of the C/EBPβ transcription factor to the IL-6 promoter. Moreover,we show that neutrophils from SLE patients with active disease state,hence displaying an IFN-induced gene expression signature,produce increased amounts of both IL-6 and TNFα in response to R848 as compared to healthy donors. Altogether,data uncover novel effects that type I IFN exerts in TLR8-activated neutrophils,which therefore enlarge our knowledge on the various biological actions which type I IFN orchestrates during infectious and autoimmune diseases. View Publication

There is little insight into or agreement about the signals that control differentiation of memory B cells (MBCs) and long-lived plasma cells (LLPCs). By performing BrdU pulse-labeling studies,we found that MBC formation preceded the formation of LLPCs in an adoptive transfer immunization system,which allowed for a synchronized Ag-specific response with homogeneous Ag-receptor,yet at natural precursor frequencies. We confirmed these observations in wild-type (WT) mice and extended them with germinal center (GC) disruption experiments and variable region gene sequencing. We thus show that the GC response undergoes a temporal switch in its output as it matures,revealing that the reaction engenders both MBC subsets with different immune effector function and,ultimately,LLPCs at largely separate points in time. These data demonstrate the kinetics of the formation of the cells that provide stable humoral immunity and therefore have implications for autoimmunity,for vaccine development,and for understanding long-term pathogen resistance. View Publication

The transplantation of glucose-responsive,insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically,but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-$\$),which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However,clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic,immunocompetent animal model using human SC-$\$ SC-$\$ were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin,which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells. View Publication

Germinal centers (GCs) are microanatomical structures critical for the development of high-affinity Abs and B cell memory. They are organized into two zones,light and dark,with coordinated roles,controlled by local signaling. The innate lectin-like transcript 1 (LLT1) is known to be expressed on B cells,but its functional role in the GC reaction has not been explored. In this study,we report high expression of LLT1 on GC-associated B cells,early plasmablasts,and GC-derived lymphomas. LLT1 expression was readily induced via BCR,CD40,and CpG stimulation on B cells. Unexpectedly,we found high expression of the LLT1 ligand,CD161,on follicular dendritic cells. Triggering of LLT1 supported B cell activation,CD83 upregulation,and CXCR4 downregulation. Overall,these data suggest that LLT1-CD161 interactions play a novel and important role in B cell maturation within the GC in humans. View Publication

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach,however,is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here,we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT." View Publication

BACKGROUND Protection of cerebral endothelial cells (ECs) from hypoxia/reoxygenation (H/R)-induced injury is an important strategy for treating ischemic stroke. In this study,we investigated whether co-culture with endothelial progenitor cells (EPCs) and neural progenitor cells (NPCs) synergistically protects cerebral ECs against H/R injury and the underlying mechanism. RESULTS EPCs and NPCs were respectively generated from inducible pluripotent stem cells. Human brain ECs were used to produce an in vitro H/R-injury model. Data showed: 1) Co-culture with EPCs and NPCs synergistically inhibited H/R-induced reactive oxygen species (ROS) over-production,apoptosis,and improved the angiogenic and barrier functions (tube formation and permeability) in H/R-injured ECs. 2) Co-culture with NPCs up-regulated the expression of vascular endothelial growth factor receptor 2 (VEGFR2). 3) Co-culture with EPCs and NPCs complementarily increased vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) levels in conditioned medium,and synergistically up-regulated the expression of p-Akt/Akt and p-Flk1/VEGFR2 in H/R-injured ECs. 4) Those effects could be decreased or abolished by inhibition of both VEGFR2 and tyrosine kinase B (TrkB) or phosphatidylinositol-3-kinase (PI3K). CONCLUSIONS Our data demonstrate that EPCs and NPCs synergistically protect cerebral ECs from H/R-injury,via activating the PI3K/Akt pathway which mainly depends on VEGF and BDNF paracrine. View Publication

Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy,reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative,but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC,comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated,through enrichment analysis,their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally,we report an unprecedented coverage of MSC CD markers,as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC. View Publication

The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation. View Publication

The use of induced pluripotent stem cells (iPSCs) as a source of cells for cell-based therapy in regenerative medicine is hampered by the limited efficiency and safety of the reprogramming procedure and the low efficiency of iPSC differentiation to specialized cell types. Evidence suggests that iPSCs retain an epigenetic memory of their parental cells with a possible influence on their differentiation capacity in vitro. We reprogramme three cell types,namely human umbilical cord vein endothelial cells (HUVECs),endothelial progenitor cells (EPCs) and human dermal fibroblasts (HDFs),to iPSCs and compare their hematoendothelial differentiation capacity. HUVECs and EPCs were at least two-fold more efficient in iPSC reprogramming than HDFs. Both HUVEC- and EPC-derived iPSCs exhibited high potentiality toward endothelial cell differentiation compared with HDF-derived iPSCs. However,only HUVEC-derived iPSCs showed efficient differentiation to hematopoietic stem/progenitor cells. Examination of DNA methylation at promoters of hematopoietic and endothelial genes revealed evidence for the existence of epigenetic memory at the endothelial genes but not the hematopoietic genes in iPSCs derived from HUVECs and EPCs indicating that epigenetic memory involves an endothelial differentiation bias. Our findings suggest that endothelial cells and EPCs are better sources for iPSC derivation regarding their reprogramming efficiency and that the somatic cell type used for iPSC generation toward specific cell lineage differentiation is of importance. View Publication

Bone is the most common site for metastasis in patients with solid tumours. Bisphosphonates are an effective treatment for preventing skeletal related events and preserving quality of life in these patients. Zoledronic acid (ZA) is the most potent osteoclast inhibitor and is licensed for the treatment of bone metastases. Clodronate and pamidronate are also licensed for this indication. In addition,ZA has been demonstrated to exhibit antitumour effect. Direct and indirect mechanisms of anti-tumour effect have been postulated and at many times proven. Evidence exists that ZA antitumour effect is mediated through inhibition of tumour cells proliferation,induction of apoptosis,synergistic/additive to inhibitory effect of cytotoxic agents,inhibition of angiogenesis,decrease tumour cells adhesion to bone,decrease tumour cells invasion and migration,disorganization of cell cytoskeleton and activation of specific cellular antitumour immune response. There is also clinical evidence from clinical trials that ZA improved long term survival outcome in cancer patients with and without bone metastases. In this review we highlight the preclinical and clinical studies investigating the antitumour effect of bisphosphonates with particular reference to ZA. View Publication

Limb girdle muscular dystrophies types 2B (LGMD2B) and 2D (LGMD2D) are degenerative muscle diseases caused by mutations in the dysferlin and alpha-sarcoglycan genes,respectively. Using patient-derived induced pluripotent stem cells (iPSC),we corrected the dysferlin nonsense mutation c.5713CtextgreaterT; p.R1905X and the most common alpha-sarcoglycan mutation,missense c.229CtextgreaterT; p.R77C,by single-stranded oligonucleotide-mediated gene editing,using the CRISPR/Cas9 gene editing system to enhance the frequency of homology-directed repair. We demonstrated seamless,allele-specific correction at efficiencies of 0.7-1.5%. As an alternative,we also carried out precise gene addition strategies for correction of the LGMD2B iPSC by integration of wild-type dysferlin cDNA into the H11 safe harbor locus on chromosome 22,using dual integrase cassette exchange (DICE) or TALEN-assisted homologous recombination for insertion precise (THRIP). These methods employed TALENs and homologous recombination,and DICE also utilized site-specific recombinases. With DICE and THRIP,we obtained targeting efficiencies after selection of ˜20%. We purified iPSC corrected by all methods and verified rescue of appropriate levels of dysferlin and alpha-sarcoglycan protein expression and correct localization,as shown by immunoblot and immunocytochemistry. In summary,we demonstrate for the first time precise correction of LGMD iPSC and validation of expression,opening the possibility of cell therapy utilizing these corrected iPSC.Molecular Therapy (2016); doi:10.1038/mt.2016.40. View Publication