技术资料

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast,lung,colon and prostate. Using CR,we have established matched normal and tumor cultures,GUMC-29 and GUMC-30 respectively,from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly,only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice,demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry,both normal and tumor CR cells expressed basal,luminal,and stem cell markers,with the majority of the normal and tumor CR cells expressing prostate basal cell markers,CD44 and Trop2,as well as luminal marker,CD13,suggesting a transit-amplifying phenotype. Consistent with this phenotype,real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5,KRT14 and p63),and low levels of luminal markers. When the CR tumor cells were injected into SCID mice,the expression of luminal markers (AR,NKX3.1) increased significantly,while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor,but undergo differentiation once injected into SCID mice. Genomic analyses,including SNP and INDEL,identified genes mutated in tumor cells,including components of apoptosis,cell attachment,and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer. View Publication

RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here,we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development,from early CD7-CD5- to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7-CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells,whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements,normalized T cell development,and corrected the NK cell-like phenotype. In conclusion,we succeeded in generating an iPSC-based RAG2-SCID model,which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks. View Publication

Human-induced Pluripotent Stem Cell (hiPSC)-derived models have advanced the study of neurodegenerative diseases,including Parkinson's disease (PD). While age is the strongest risk factor for these disorders,hiPSC-derived models represent rejuvenated neurons. We developed hiPSC-derived Aged dopaminergic and cholinergic neurons to model PD and related synucleinopathies. Our new method induces aging through a `semi-natural' process,by passaging multiple times at the Neural Precursor Cell stage,prior to final differentiation. Characterization of isogenic hiPSC-derived neurons using heterochromatin and nuclear envelope markers,as well as DNA damage and global DNA methylation,validated our age-inducing method. Next,we compared neurons derived from a patient with SNCA-triplication (SNCA-Tri) and a Control. The SNCA-Tri neurons displayed exacerbated nuclear aging,showing advanced aging signatures already at the Juvenile stage. Noteworthy,the Aged SNCA-Tri neurons showed more $\alpha$-synuclein aggregates per cell versus the Juvenile. We suggest a link between the effects of aging and SNCA overexpression on neuronal nuclear architecture. View Publication

Human embryonic stem cells (hESCs) hold great value for future clinical applications. However,standard culture conditions maintain hESCs in a primed state,which bears heterogeneity in pluripotency and a tendency for spontaneous differentiation. To counter these drawbacks,primed hESCs have been converted to a naive state,but this has restricted the efficiency of existing directed differentiation protocols. In mouse,WNT inhibition by inhibitor of WNT production-2,together with a higher dose of fibroblast growth factor 2 (12 ng/mL) in DMEM/F12 basal medium (DhiFI),markedly improved derivation and maintenance of primed mouse epiblast stem cells. In this study,we show that DhiFI conditions similarly improved primed hESC traits,such as conferring a primed transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. When triggered to differentiate to neuronal and cardiac lineages,DhiFI hESCs and isogenic primed hESCs progressed similarly. Moreover,DhiFI conditions supported the derivation of hESC lines from a post-inner cell mass intermediate (PICMI). DhiFI-derived hESCs showed less spontaneous differentiation and expressed significantly lower levels of lineage-specific markers,compared to primed-derived lines from the same PICMI. Overall,DhiFI hESCs retained advantages of both primed and naive pluripotency and may ultimately represent a more favorable starting point for differentiation toward clinically desired cell types. View Publication

Fibroblast-like synoviocytes (FLS) are joint-lining cells that promote rheumatoid arthritis (RA) pathology. Current disease-modifying antirheumatic agents (DMARDs) operate through systemic immunosuppression. FLS-targeted approaches could potentially be combined with DMARDs to improve control of RA without increasing immunosuppression. Here,we assessed the potential of immunoglobulin-like domains 1 and 2 (Ig1{\&}2),a decoy protein that activates the receptor tyrosine phosphatase sigma (PTPRS) on FLS,for RA therapy. We report that PTPRS expression is enriched in synovial lining RA FLS and that Ig1{\&}2 reduces migration of RA but not osteoarthritis FLS. Administration of an Fc-fusion Ig1{\&}2 attenuated arthritis in mice without affecting innate or adaptive immunity. Furthermore,PTPRS was down-regulated in FLS by tumor necrosis factor (TNF) via a phosphatidylinositol 3-kinase-mediated pathway,and TNF inhibition enhanced PTPRS expression in arthritic joints. Combination of ineffective doses of TNF inhibitor and Fc-Ig1{\&}2 reversed arthritis in mice,providing an example of synergy between FLS-targeted and immunosuppressive DMARD therapies. View Publication

NF-$\kappa$B is a key regulator of inflammation and cancer progression,with an important role in leukemogenesis. Despite its therapeutic potential,targeting NF-$\kappa$B using pharmacologic inhibitors has proven challenging. Here,we describe a myeloid cell-selective NF-$\kappa$B inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a,C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6),thereby blocking activation of NF-$\kappa$B in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-$\kappa$B activation in myeloid cells,and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly,C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions,miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set,we found an inverse correlation of miR-146a levels with NF-$\kappa$B-related genes and with patient survival. Correspondingly,C-miR146a induced cytotoxic effects in human MDSL,HL-60,and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-$\kappa$B target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders. View Publication

R-2-hydroxyglutarate (R-2HG),produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes,was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically,R-2HG inhibits fat mass and obesity-associated protein (FTO) activity,thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells,which in turn decreases the stability of MYC/CEBPA transcripts,leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG,whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively,while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation,our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling. View Publication

Immune surveillance against pathogens and tumours in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. However,the characterization of the meningeal lymphatic network has shed light on previously unappreciated ways that an immune response can be elicited to antigens that are expressed in the brain1-3. Despite progress in our understanding of the development and structure of the meningeal lymphatic system,the contribution of this network in evoking a protective antigen-specific immune response in the brain remains unclear. Here,using a mouse model of glioblastoma,we show that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumour is confined to the central nervous system,resulting in uncontrolled tumour growth. However,ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes,migration of CD8 T cells into the tumour,rapid clearance of the glioblastoma and a long-lasting antitumour memory response. Furthermore,transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal the capacity of VEGF-C to promote immune surveillance of tumours,and suggest a new therapeutic approach to treat brain tumours. View Publication

Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging,however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS),DNA damage,senescence,and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC,as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically,CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress,as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC),improved their niche supporting activity,and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production,driving an HSC aging phenotype. Thus,modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function. View Publication

The diagnosis of Parkinson's disease (PD) is initiated after the occurrence of motor symptoms,such as resting tremors,rigidity,and bradykinesia. According to previous reports,non-motor symptoms,notably gastrointestinal dysfunction,could potentially be early biomarkers in PD patients as such symptoms occur earlier than motor symptoms. However,connecting PD to the intestine is methodologically challenging. Thus,we generated in vitro human intestinal organoids from PD patients and ex vivo mouse small intestinal organoids from aged transgenic mice. Both intestinal organoids (IOs) contained the human LRRK2 G2019S mutation,which is the most frequent genetic cause of familial and sporadic PD. By conducting comprehensive genomic comparisons with these two types of IOs,we determined that a particular gene,namely,Iroquois homeobox protein 2 (IRX2),showed PD-related expression patterns not only in human pluripotent stem cell (PSC)-derived neuroectodermal spheres but also in human PSC-derived neuronal cells containing dopaminergic neurons. We expected that our approach of using various cell types presented a novel technical method for studying the effects of multi-organs in PD pathophysiology as well as for the development of diagnostic markers for PD. View Publication

The cerebellum plays a critical role in all vertebrates,and many neurological disorders are associated with cerebellum dysfunction. A major limitation in cerebellar research has been the lack of adequate disease models. As an alternative to animal models,cerebellar neurons differentiated from pluripotent stem cells have been used. However,previous studies only produced limited amounts of Purkinje cells. Moreover,in vitro generation of Purkinje cells required co-culture systems,which may introduce unknown components to the system. Here we describe a novel differentiation strategy that uses defined medium to generate Purkinje cells,granule cells,interneurons,and deep cerebellar nuclei projection neurons,that self-formed and differentiated into electrically active cells. Using a defined basal medium optimized for neuronal cell culture,we successfully promoted the differentiation of cerebellar precursors without the need for co-culturing. We anticipate that our findings may help developing better models for the study of cerebellar dysfunctions,while providing an advance toward the development of autologous replacement strategies for treating cerebellar degenerative diseases. View Publication

Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here,we report a pivotal role of mitochondrial oxidative phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development,and this is necessary for metabolic reprogramming following T cell activation. Surprisingly,specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably,cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3,rather than Th17 genes,and become suppressive Treg cells. Mechanistically,OXPHOS promotes the Th17 pioneer transcription factor,BATF,and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly,overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together,our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment. View Publication