技术资料

As autologous induced pluripotent stem cell (iPSC) therapy requires a custom-made small-lot cell production line,and the cell production method differs significantly from the existing processes for producing allogeneic iPSC stocks for clinical use. Specifically,mass culture to produce stock is no longer necessary; instead,a series of operations from iPSC production to induction of differentiation of therapeutic cells must be performed continuously. A three-dimensional (3D) culture method using small,closed-cell manufacturing devices is suitable for autologous iPSC therapy. The use of such devices avoids the need to handle many patient-derived specimens in a single clean room; handling of cell cultures in an open system in a cell processing facility increases the risk of infection. In this study,atelocollagen beads were evaluated as a 3D biomaterial to assist 3D culture in the establishment,expansion culture,and induction of differentiation of iPSCs. It was found that iPSCs can be handled in a closed-cell device with the same ease as use of a two-dimensional (2D) culture when laminin-511 is added to the medium. In conclusion,atelocollagen beads enable 3D culture of iPSCs,and the quality of the obtained cells is at the same level as those derived from 2D culture. View Publication

Pluripotent stem cells provide a scalable approach to analyse molecular regulation of cell differentiation across developmental lineages. Here,we engineer barcoded induced pluripotent stem cells to generate an atlas of multilineage differentiation from pluripotency,encompassing an eight-day time course with modulation of WNT,BMP,and VEGF signalling pathways. Annotation of in vitro cell types with reference to in vivo development reveals diverse mesendoderm lineage cell types including lateral plate and paraxial mesoderm,neural crest,and primitive gut. Interrogation of temporal and signalling-specific gene expression in this atlas,evaluated against cell type-specific gene expression in human complex trait data highlights the WNT-inhibitor gene TMEM88 as a regulator of mesendodermal lineages influencing cardiovascular and anthropometric traits. Genetic TMEM88 loss of function models show impaired differentiation of endodermal and mesodermal derivatives in vitro and dysregulated arterial blood pressure in vivo. Together,this study provides an atlas of multilineage stem cell differentiation and analysis pipelines to dissect genetic determinants of mammalian developmental physiology. Shen et al. report a method for multiplexing isogenic iPSCs for single-cell RNA-seq. With it,they created an atlas of in vitro differentiation and identified TMEM88 as a regulator of cardiovascular development,impacting blood pressure in adult mice. View Publication

Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are common in acute myeloid leukemia (AML) and drive leukemic cell growth and survival. Although FLT3 inhibitors have shown considerable promise for the treatment of AML,they ultimately fail to achieve long-term remissions as monotherapy. To identify genetic targets that can sensitize AML cells to killing by FLT3 inhibitors,we performed a genome-wide RNA interference (RNAi)-based screen that identified ATM (ataxia telangiectasia mutated) as being synthetic lethal with FLT3 inhibitor therapy. We found that inactivating ATM or its downstream effector glucose 6-phosphate dehydrogenase (G6PD) sensitizes AML cells to FLT3 inhibitor induced apoptosis. Examination of the cellular metabolome showed that FLT3 inhibition by itself causes profound alterations in central carbon metabolism,resulting in impaired production of the antioxidant factor glutathione,which was further impaired by ATM or G6PD inactivation. Moreover,FLT3 inhibition elicited severe mitochondrial oxidative stress that is causative in apoptosis and is exacerbated by ATM/G6PD inhibition. The use of an agent that intensifies mitochondrial oxidative stress in combination with a FLT3 inhibitor augmented elimination of AML cells in vitro and in vivo,revealing a therapeutic strategy for the improved treatment of FLT3 mutated AML. View Publication

We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent,stem cell-derived cardiomyocytes,including contraction force,rate,duration,and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy,and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive,inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents,or as a platform for studying cardiomyocyte biology. 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

ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial-mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness,we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers,we showed this to be the case in A549 cells,which harbor a Ras mutation,and in two other non-small-cell lung cancer cell lines,H1975 and H1650,driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover,treatment with hydroxycitrate phenocopied the effects of ACL KD,suggesting that the enzymatic activity of ACL was critical. Indeed,acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway,not the Ras-mitogen-activated protein kinase (MAPK) pathway,and that EMT can be reversed by PI3K inhibitors,we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling,and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells,ACL overexpression increased snail expression and stemness,both of which were reduced by ACL KD. Furthermore,ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally,ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function,namely its impact on cancer stemness in a broad range of genetically diverse cell types. View Publication

The ATR (ATM (ataxia telangiectasia mutated) and rad3-related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder,ATR-Seckel syndrome. To understand how ATR functions during neurogenesis,we conditionally deleted Atr broadly throughout the murine nervous system,or in a restricted manner in the dorsal telencephalon. Unexpectedly,in both scenarios,Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16(Ink4a/Arf))-independent proliferation arrest,other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs,in the nervous system,p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion,supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress,our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis. View Publication

Under specific conditions,cultured human embryonic stem cells (hESCs) corresponding to primed post-implantation epiblasts can be converted back to a ‘naïve pluripotency’ state that resembles the pre-implantation epiblasts. The core pluripotency factor OCT4 is known to be crucial in regulating different states of pluripotency,but its potential regulatory role in human naïve pluripotency remains unexplored. In this study,we systematically mapped out phosphorylation sites in OCT4 protein that are differentially phosphorylated between two states of pluripotency,and further identified ATR as a key kinase that phosphorylated OCT4 in naïve but not primed hESCs. The kinase activity levels of ATR in naïve hESCs were higher than those in primed hESCs. Ablating cellular ATR activity significantly halted the induction of naïve hESCs from their primed counterparts,and increased early apoptotic death of naïve hESCs upon UV and CPT treatment. Thus,our work reveals the importance of ATR activity in safeguarding human naïve pluripotency,and implicates a potential association of OCT4 phosphorylation,DNA damage sensing and repairing system in regulating different states of pluripotency during early development. View Publication

B cells orchestrate autoimmune responses in patients with systemic lupus erythematosus (SLE),but broad-based B cell-directed therapies show only modest efficacy while blunting humoral immune responses to vaccines and inducing immunosuppression. Development of more effective therapies targeting pathogenic clones is a currently unmet need. Here,we demonstrate enhanced activation of the ATR/Chk1 pathway of the DNA damage response (DDR) in B cells of patients with active SLE disease. Treatment of B cells with type I IFN,a key driver of immunity in SLE,induced expression of ATR via binding of interferon regulatory factor 1 to its gene promoter. Pharmacologic targeting of ATR in B cells,via a specific inhibitor (VE-822),attenuated their immunogenic profile,including proinflammatory cytokine secretion,plasmablast formation,and antibody production. Together,these findings identify the ATR-mediated DDR axis as the orchestrator of the type I IFN-mediated B cell responses in SLE and as a potential novel therapeutic target. View Publication

New vaccines against pertussis are needed to evoke full protection and long-lasting immunological memory starting from the first administration in neonates--the major target of the life-threatening pertussis infection. A novel live attenuated Bordetella pertussis vaccine strain,BPZE1,has been developed by eliminating or detoxifying three important B. pertussis virulence factors: pertussis toxin,dermonecrotic toxin,and tracheal cytotoxin. We used a human preclinical ex vivo model based on monocyte-derived dendritic cells (MDDCs) to evaluate BPZE1 immunogenicity. We studied the effects of BPZE1 on MDDC functions,focusing on the impact of Bordetella-primed dendritic cells in the regulation of Th and suppressor T cells (Ts). BPZE1 is able to activate human MDDCs and to promote the production of a broad spectrum of proinflammatory and regulatory cytokines. Moreover,conversely to its parental wild-type counterpart BPSM,BPZE1-primed MDDCs very efficiently migrate in vitro in response to the lymphatic chemokine CCL21,due to the inactivation of pertussis toxin enzymatic activity. BPZE1-primed MDDCs drove a mixed Th1/Th17 polarization and also induced functional Ts. Experiments performed in a Transwell system showed that cell contact rather than the production of soluble factors was required for suppression activity. Overall,our findings support the potential of BPZE1 as a novel live attenuated pertussis vaccine,as BPZE1-challenged dendritic cells might migrate from the site of infection to the lymph nodes,prime Th cells,mount an adaptive immune response,and orchestrate Th1/Th17 and Ts responses. View Publication

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with a complex and multifactorial etiology,making it challenging to treat. While recent advances in immunomodulatory biologics,such as antitumor necrosis factor-α (TNF-α) antibodies,have shown moderate success,systemic administration of antibody therapeutics may lead to several adverse effects,including the risk of autoimmune disorders due to systemic cytokine depletion. Transient RNA interference using exogenous short interfering RNA (siRNA) to regulate target gene expression at the transcript level offers an alternative to systemic immunomodulation. However,siRNAs are susceptible to premature degradation and have poor cellular uptake. Graphene oxide (GO) nanoparticles have been shown to be effective nanocarriers for biologics due to their reduced cytotoxicity and enhanced bioavailability. In this study,we evaluate the therapeutic efficacy of GO mediated TNF-α_siRNA using in vitro models of chronic inflammation generated by treating murine small intestines (enteroids) and large intestines (colonoids) with inflammatory agents IL-1β,TNF-α,and LPS. The organotypic mouse enteroids and colonoids developed an inflammatory phenotype similar to that of IBD,characterized by impaired epithelial homeostasis and an increased production of inflammatory cytokines such as TNF-α,IL-1β,and IL-6. We assessed siRNA delivery to these inflamed organoids using three different GO formulations. Out of the three,small-sized GO with polymer and dendrimer modifications (smGO) demonstrated the highest transfection efficiency,which led to the downregulation of inflammatory cytokines,indicating an attenuation of the inflammatory phenotype. Moreover,the transfection efficiency and inflammation-ameliorating effects could be further enhanced by increasing the TNF-α_siRNA/smGO ratio from 1:1 to 3:1. Overall,the results of this study demonstrate that ex vivo organoids with disease-specific phenotypes are invaluable models for assessing the therapeutic potential of nanocarrier-mediated drug and biologic delivery systems. View Publication

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however,their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC),human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC),and compared their in-vitro tube formation,migration and cytokine expression profiles,and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless,BM-EC,hESC-EC and hiPSC-EC exhibited typical cobblestone morphology,had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein,and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (Ptextgreater0.05). While increased expression of major angiogenic factors including epidermal growth factor,hepatocyte growth factor,vascular endothelial growth factor,placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (Ptextless0.05),the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (Ptextless0.05). Compared with medium,transplanting BM-EC (n = 6),HUVEC (n = 6),hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion,functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases,and hESC-EC or iPSC-EC are readily available as off-the-shelf" format for the treatment of tissue ischemia." View Publication