技术资料

The recent advances,offered by cell therapy in the regenerative medicine field,offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors,such as myoblasts or satellite cells,possess a marked regenerative capacity,but the exploitation of this potential still encounters significant challenges in clinical application,due to low rate of proliferation in vitro,as well as a reduced self-renewal capacity. In this scenario,induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches,but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs,generated from pericytes and fibroblasts,exploiting skeletal muscle-derived extracellular vesicles (EVs),in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological shuttles" to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches." View Publication

The incidence of Merkel cell carcinoma (MCC),a rare and highly metastatic skin malignancy,has sharply increased in the last decade. Clinical biomarkers are urgently needed for MCC prognosis,treatment response monitoring,and early diagnosis of relapse. The clinical interest of circulating tumors cells (CTCs) has been validated in many solid cancers. The aim of this study was to compare CTC detection and characterization in blood samples of patients with MCC using the CellSearch System and the RosetteSep -DEPArray workflow,an innovative procedure to enrich,detect and isolate single CTCs. In preliminary experiments (using spiked MCC cell lines) both methods allowed detecting very few MCC cells. In blood samples from 19 patients with MCC at different stages,CellSearch detected MCC CTCs in 26{\%} of patients,and the R-D workflow in 42{\%} of patients. The detection of CTC-positive patients increased to 52{\%} by the cumulative positivity rate of both methodologies. Moreover,Merkel cell polyomavirus DNA,involved in MCC oncogenesis,was detected in tumor biopsies,but not in all single CTCs from the same patient,reflecting the tumor heterogeneity. Our data demonstrate the possibility to detect,isolate and characterize CTCs in patients with MCC using two complementary approaches. View Publication

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age,but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further,we examined the combined and separate effects of an oncogene (c-MYC) and exposure to IL3,GM-CSF and SCF on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized,rapidly fatal and serially transplantable blast population,phenotypically and transcriptionally similar to human AML cells,but only in mice producing IL3,GM-CSF and SCF transgenically,or in regular mice in which the cells were exposed to IL3 or GM-CSF delivered using a co-transduction strategy. In their absence,the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months but,upon transfer to secondary mice producing the human cytokines,the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late (GMPs) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them. View Publication

BACKGROUND/OBJECTIVE A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. The vascular phenotype of the choroidal neovascular (CNV) lesions may contribute to the resistance. Animal studies of CNV lesions have shown that cells originating from bone marrow are capable of forming varying cell types in the lesions. This raised the possibility of a similar cell population in human nvAMD subjects. MATERIALS AND METHODS Blood draws were obtained from subjects with active nvAMD while patients were receiving standard of care anti-VEGF injections. Subjects were classified as refractory or non-refractory to anti-VEGF treatment based on previous number of injections in the preceding 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated and CD34-positive cells purified using magnetic bead sorting. The isolated cells were expanded in StemSpan SFEM media to increase cell numbers. After expansion,the cells were split and plated in either endothelial or mesenchymal promoting conditions. Phenotype analysis was performed via qPCR. RESULTS There was no significant difference in the number of PBMCs and CD34-positive cells between refractory and non-refractory nvAMD subjects. The growth pattern distribution between endothelial and mesenchymal media conditions were very similar between refractory and non-refractory subjects. qPCR and immunostaining demonstrated positive expression of endothelial markers in endothelial media,and markers such as NG2 and $\alpha$SMA in mesenchymal media. However,analysis of subsequent samples from AMD subjects demonstrated high variability in both the numbers and differentiation properties of this cell population. CONCLUSIONS CD34+ cells can be isolated from nvAMD subjects and show both endothelial and pericyte-like characteristics after differentiation in certain media conditions. However,nvAMD subjects show high variability in both numbers of cells and differentiation characteristics in repeat sampling. This variability highlights the importance of taking multiple samples from nvAMD subjects for any clinical trials focused on biomarkers for the disease. View Publication

Cancer stem cells are critical for cancer initiation,development,and treatment resistance. Our understanding of these processes,and how they relate to glioblastoma heterogeneity,is limited. To overcome these limitations,we performed single-cell RNA sequencing on 53586 adult glioblastoma cells and 22637 normal human fetal brain cells,and compared the lineage hierarchy of the developing human brain to the transcriptome of cancer cells. We find a conserved neural tri-lineage cancer hierarchy centered around glial progenitor-like cells. We also find that this progenitor population contains the majority of the cancer's cycling cells,and,using RNA velocity,is often the originator of the other cell types. Finally,we show that this hierarchal map can be used to identify therapeutic targets specific to progenitor cancer stem cells. Our analyses show that normal brain development reconciles glioblastoma development,suggests a possible origin for glioblastoma hierarchy,and helps to identify cancer stem cell-specific targets. View Publication

Mast cells (MCs) arise from hematopoietic stem cells (HSCs) that mature within vascularized tissues. Fibroblasts and endothelial cells (ECs) play a role in the maturation of HSCs in the tissues. Due to difficulties in isolating MCs from tissues,large numbers of committed MC precursors can be generated in 2D culture systems with the use of differentiation factors. Since MCs are tissue-resident cells,the development of a 3D tissue-engineered model with ancillary cells that more closely mimics the 3D in vivo microenvironment has greater relevance for MC studies. The goals of this study were to show that MCs can be derived from HSCs within a 3D matrix and to determine a media to support MCs,fibroblasts,and ECs. The results show that HSCs within a collagen matrix cultured in StemSpan media with serum added at the last week yielded a greater number of c-kit+ cells and a greater amount of histamine granules compared to other media tested. Media supplemented with serum were necessary for EC survival,while fibroblasts survived irrespective of serum with higher cell yields in StemSpan. This work demonstrates the development of functional MCs within a 3D collagen matrix using a stem cell media that supports fibroblast and ECs. View Publication

Tissue resident intestinal macrophages are known to exhibit an anti-inflammatory phenotype and produce little pro-inflammatory cytokines upon TLR ligation,allowing symbiotic co-existence with the intestinal microbiota. However,upon acute events such as epithelial damage and concomitant influx of microbes,these macrophages must be able to quickly mount a pro-inflammatory response while more inflammatory macrophages are recruited from the blood stream simultaneously. Here,we show that dietary intake of vitamin A is required for the maintenance of the anti-inflammatory state of tissue resident intestinal macrophages. Interestingly,these anti-inflammatory macrophages were characterized by high levels of Dectin-1 expression. We show that Dectin-1 expression is enhanced by the vitamin A metabolite retinoic acid and our data suggests that Dectin-1 triggering might provide a switch to induce a rapid production of pro-inflammatory cytokines. In addition,Dectin-1 stimulation resulted in an altered metabolic profile which is linked to a pro-inflammatory response. Together,our data suggests that presence of vitamin A in the small intestine enhances an anti-inflammatory phenotype as well as Dectin-1 expression by macrophages and that this anti-inflammatory phenotype can rapidly convert toward a pro-inflammatory state upon Dectin-1 signaling. View Publication

The tumor microenvironment (TME) comprises complex interactions of multiple cell types that determines cell behavior and metabolism such as nutrient competition and immune suppression. We discuss the various types of heterogeneity that exist in solid tumors,and the complications this invokes for studies of TME. As human subjects and in vivo model systems are complex and difficult to manipulate,simpler 3D model systems that are compatible with flexible experimental control are necessary for studying metabolic regulation in TME. Stable Isotope Resolved Metabolomics (SIRM) is a valuable tool for tracing metabolic networks in complex systems,but at present does not directly address heterogeneous metabolism at the individual cell level. We compare the advantages and disadvantages of different model systems for SIRM experiments,with a focus on lung cancer cells,their interactions with macrophages and T cells,and their response to modulators in the immune microenvironment. We describe the experimental set up,illustrate results from 3D cultures and co-cultures of lung cancer cells with human macrophages,and outline strategies to address the heterogeneous TME. View Publication

Mannose is a glucose-associated serum metabolite mainly released by the liver. Recent studies have shown several unexpected pleiotropic effects of mannose including increased regulatory T cells (Tregs),prevention of auto-immune disease and ability to reduce growth of human cancer cells. We have previously shown in large cohorts that elevated serum mannose levels are associated with future development of type 2 diabetes (T2D) and cardiovascular disease. However,potential direct effects of mannose on insulin sensitivity in vivo or in vitro are unknown. We here show that administration of mannose (0.1 g/kg BW twice daily) for one week in man did not elicit negative effects on meal-modified glucose tolerance,markers of inflammation or insulin levels. Tregs number and insulin signaling in human liver cells were unchanged. These data suggest that mannose is a marker,and not a mediator,of insulin resistance. To verify this,we examined serum mannose levels during long-term euglycemic hyperinsulinemic clamps in non-diabetic and T2D individuals. Mannose was reduced by insulin infusion in proportion to whole-body insulin sensitivity. Thus,mannose is a biomarker of insulin resistance which may be useful for the early identification of diabetic individuals with insulin resistance and increased risk of its complications. View Publication

The tumor immune microenvironment (TIME) plays a pivotal role in tumor development,progression,and prognosis. However,the characteristics of the TIME in diffuse astrocytoma (DA) are still unclear. Leveraging mass cytometry with a panel of 33 markers,we analyzed the infiltrating immune cells from 10 DA and 4 oligodendroglioma (OG) tissues and provided a single cell-resolution landscape of the intricate immune microenvironment. Our study profiled the composition of the TIME in DA and confirmed the presence of immune cells,such as glioma-associated microglia/macrophages (GAMs),CD8+ T cells,CD4+ T cells,regulatory T cells (Tregs),and natural killer cells. Increased percentages of PD-1+ CD8+ T cells,TIM-3+ CD4+ T cell subpopulations,Tregs and pro-tumor phenotype GAMs substantially contribute to the local immunosuppressive microenvironment in DA. DAs and OGs share similar compositions in terms of immune cells,while GAMs in DA exhibit more inhibitory characteristics than those in OG. View Publication

Activation-induced cytidine deaminase (AID) mediates immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM),critical processes for maturation of the antibody response. Epigenetic factors,such as histone deacetylases (HDACs),would underpin B cell differentiation stage-specific AID expression. Here,we showed that NAD+-dependent class III HDAC sirtuin 1 (Sirt1) is highly expressed in resting B cells and down-regulated by stimuli inducing AID. B cell Sirt1 down-regulation,deprivation of NAD+ cofactor,or genetic Sirt1 deletion reduced deacetylation of Aicda promoter histones,Dnmt1,and nuclear factor-$\kappa$B (NF-$\kappa$B) p65 and increased AID expression. This promoted class-switched and hypermutated T-dependent and T-independent antibody responses or led to generation of autoantibodies. Genetic Sirt1 overexpression,Sirt1 boost by NAD+,or allosteric Sirt1 enhancement by SRT1720 repressed AID expression and CSR/SHM. By deacetylating histone and nonhistone proteins (Dnmt1 and NF-$\kappa$B p65),Sirt1 transduces metabolic cues into epigenetic changes to play an important B cell-intrinsic role in modulating antibody and autoantibody responses. View Publication

Transcriptional status determines the HIV replicative state in infected patients. However,the transcriptional mechanisms for proviral replication control remain unclear. In this study,we show that,apart from its function in HIV integration,LEDGF/p75 differentially regulates HIV transcription in latency and proviral reactivation. During latency,LEDGF/p75 suppresses proviral transcription via promoter-proximal pausing of RNA polymerase II (Pol II) by recruiting PAF1 complex to the provirus. Following latency reversal,MLL1 complex competitively displaces PAF1 from the provirus through casein kinase II (CKII)-dependent association with LEDGF/p75. Depleting or pharmacologically inhibiting CKII prevents PAF1 dissociation and abrogates the recruitment of both MLL1 and Super Elongation Complex (SEC) to the provirus,thereby impairing transcriptional reactivation for latency reversal. These findings,therefore,provide a mechanistic understanding of how LEDGF/p75 coordinates its distinct regulatory functions at different stages of the post-integrated HIV life cycles. Targeting these mechanisms may have a therapeutic potential to eradicate HIV infection. View Publication