搜索结果: 'gmp t cell activator'

Recombinant adeno-associated viruses (rAAV) are promising for applications in many genome editing techniques through their effectiveness as carriers of DNA homologous donors into primary hematopoietic stem and progenitor cells (HSPCs),but they have many outstanding concerns. Specifically,their biomanufacturing and the variety of factors that influence the quality and consistency of rAAV preps are in question. During the process of rAAV packaging,a cell line is transfected with several DNA plasmids that collectively encode all the necessary information to allow for viral packaging. Ideally,this process results in the packaging of complete viral particles only containing rAAV genomes; however,this is not the case. Through this study,we were able to leverage single-stranded virus (SSV) sequencing,a next-generation sequencing-based method to quantify all DNA species present within rAAV preps. From this,it was determined that much of the DNA within some rAAV preps is not vector-genome derived,and there is wide variability in the contamination by DNA across various preps. Furthermore,we demonstrate that transducing CD34 + HSPCs with preps with higher contaminating DNA resulted in decreased clonogenic potential,altered transcriptomic profiles,and decreased genomic editing. Collectively,this study characterized the effects of DNA contamination within rAAV preps on CD34 + HSPC cellular potential. View Publication

Tracheal replacement is a promising approach for treating tracheal defects that are caused by conditions such as stenosis,trauma,or tumors. However,slow postoperative epithelial regeneration often leads to complications,such as infection and granulation tissue formation. Ferroptosis,which is an iron-dependent form of regulated cell death,limits the proliferation of tracheal basal cells (TBCs),which are essential for the epithelialization of tissue-engineered tracheas (TETs). This study explored the potential of ferrostatin-1 (FER-1),which is a ferroptosis inhibitor,to increase TBC proliferation and accelerate the epithelialization of 3D-printed TETs. TBCs were isolated from rabbit bronchial mucosal tissues and cultured in vitro. Ferroptosis was induced in TBCs at passage 2,as shown by increased reactive oxygen species (ROS) levels,Fe 2 ⁺ accumulation,decreased ATP contents,and mitochondrial damage. TBCs were treated with FER-1 (1 μM) for 48 h to inhibit ferroptosis. The effects on ROS levels,Fe 2 ⁺ levels,ATP contents,and mitochondrial morphology were measured. For in vivo experiments,FER-1-treated TBCs were seeded onto 3D-printed polycaprolactone (PCL) scaffolds,which were implanted into rabbits with tracheal injury. Epithelial regeneration and granulation tissue formation were evaluated 6 months after surgery. FER-1 treatment significantly reduced ferroptosis marker levels in vitro; that is,FER-1 treatment decreased ROS and Fe 2 ⁺ accumulation,ameliorated mitochondrial structures,and increased ATP levels. TBC proliferation and viability were increased after ferroptosis inhibition. In vivo,the group that received 3D-printed scaffolds seeded with TBCs exhibited accelerated TET epithelialization and reduced granulation tissue formation compared with the control groups. These results suggest that inhibiting ferroptosis with FER-1 improves TBC function,leading to more efficient tracheal repair. Ferrostatin-1 effectively inhibits ferroptosis in tracheal basal cells,promoting their viability and proliferation. This results in faster epithelialization of tissue-engineered tracheas,offering a promising strategy for improving tracheal reconstruction outcomes and reducing complications such as infection and granulation tissue formation. Future studies are needed to further investigate the molecular mechanisms underlying ferroptosis in TBCs and its potential clinical applications. The online version contains supplementary material available at 10.1186/s13287-025-04263-z. View Publication

Long-term management for leukemia is challenging due to the painful and invasive procedure of bone marrow (BM) biopsy. At present,non-invasive liquid (blood) biopsy is not utilized for leukemia,due to lower counts of leukemia blast cells in the blood. Here,we described a robust system for the simultaneous detection and enrichment of rare blast cells. Enrichment of blast cells was achieved from blood with a one-step microfluidic blast cell biochip (BCB) sorting system,without specific targeting of proteins by antibodies. Non-target cells encountered a differential net force as compared to stiffer blast cells and were removed. The efficiency of the BCB promotes high detection sensitivity (1 in 106 cells) even from patients with minimal residual disease. The procedure was validated using actual blast cells from patients with various types of leukemia. Outcomes were compared to current evaluation standards,such as flow cytometry,using BM aspirates. Blast cell detection efficiency was higher in 55.6{\%} of the patients using the BCB as compared to flow cytometry,despite the lower concentrations of blast cells in liquid biopsy. These studies promote early-stage detection and routine monitoring for minimal residual disease in patients. 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

The human bone marrow (BM) microenvironment involves hematopoietic and non-hematopoietic cell subsets organized in a complex architecture. Tremendous efforts have been made to model it in order to analyze normal or pathological hematopoiesis and its stromal counterpart. Herein,we report an original,fully-human in vitro 3D model of the BM microenvironment dedicated to study interactions taking place between mesenchymal stromal cells (MSC) and hematopoietic stem and progenitor cells (HSPC) during the hematopoietic differentiation. This fully-human Artificial Marrow Organoid (AMO) model is highly efficient to recapitulate MSC support to myeloid differentiation and NK cell development from the immature CD34 + HSPCs to the most terminally differentiated CD15 + polymorphonuclear neutrophils,CD64 + monocytes or NKG2A-KIR2D + CD57 + NK subset. Lastly,our model is suitable for evaluating anti-leukemic NK cell function in presence of therapeutic agents. Overall,the AMO is a versatile,low cost and simple model able to recapitulate normal hematopoiesis and allowing more physiological drug testing by taking into account both immune and non-immune BM microenvironment interactions.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-07717-9. View Publication

BackgroundChimeric antigen receptor T (CAR-T) cell therapy holds promise for cancer treatment,but its efficacy is often hindered by metabolic constraints in the tumor microenvironment. This study investigates the role of glutamine in enhancing CAR-T cell function against ovarian cancer.MethodsMetabolomic profiling of blood samples from ovarian cancer patients treated with MSLN-CAR-T cells was conducted to identify metabolic changes. In vitro,glutamine pretreatment was applied to CAR-T cells,and their proliferation,CAR expression,tumor lysis,and cytokine production (TNF-α,IFN-γ) were assessed. Mechanistic studies focused on the mTOR-SREBP2 pathway and its effect on HMGCS1 expression,membrane stability and immune synapse formation. In vivo,the antitumor effects and memory phenotype of glutamine-pretreated CAR-T cells were evaluated.ResultsElevated glutamine levels were observed in the blood of ovarian cancer patients who responded to MSLN-CAR-T cell treatment. Glutamine pretreatment enhanced CAR-T cell proliferation,CAR expression,tumor lysis,and cytokine production. Mechanistically,glutamine activated the mTOR-SREBP2 pathway,upregulating HMGCS1 and promoting membrane stability and immune synapse formation. In vivo,glutamine-pretreated CAR-T cells exhibited superior tumor infiltration,sustained antitumor activity,and preserved memory subsets.ConclusionsOur findings highlight glutamine-driven metabolic rewiring via the mTOR-SREBP2-HMGCS1 axis as a strategy to augment CAR-T cell efficacy in ovarian cancer.Trial registrationNCT05372692Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06853-0. View Publication

Purpose: Noninfectious uveitis is a sight-threatening autoimmune eye disease lacking effective targeted therapies. Dipyridamole (DIP),a phosphodiesterase (PDE) inhibitor,has demonstrated anti-inflammatory properties in inflammatory diseases. However,its application in uveitis remains unexplored. Methods: We used single-cell RNA sequencing (scRNA-seq) data from experimental autoimmune uveitis (EAU) mice and uveitis patients to assess the potential association of PDE gene expression with disease development. Subsequently,EAU mice received oral DIP (300 mg/kg/day),starting at different time points (preventative,early-therapeutic,or late-therapeutic),and treatment efficacy was assessed. To explore immune components and signaling changes,we profiled cervical draining lymph nodes (CDLNs) from control,EAU,and DIP-treated mice by scRNA-seq and validated key findings with additional experiments. Mechanistically,pharmacologic interventions (an adenylyl cyclase inhibitor and the STAT3 agonist) were used in vitro. Results: Expression of several PDE genes correlated with uveitis severity in both human and mouse. Preventative DIP treatment most effectively reduced fundus inflammation in EAU and modulated the Teff/Treg ratio in the CDLNs and spleens. In vitro,DIP suppressed CD4+ T cell proliferation,and inhibited pathogenic Teff. scRNA-seq analysis revealed that DIP partially reversed EAU-induced transcriptional alterations,with notable changes in immune cell composition and pathway activity. Mechanistically,DIP downregulated STAT3 activity and PIM1 expression in Th17 cells via cAMP,suggesting the involvement of the cAMP-STAT3-PIM1 axis in modulating immune homeostasis. Conclusions: DIP ameliorated intraocular inflammation,modulated Th17/Treg balance,and reduced Th17 pathogenicity in EAU,potentially via cAMP-STAT3-PIM1 signaling. These findings highlight DIP as a promising therapeutic candidate for autoimmune uveitis. View Publication

Embryonic stem cells represent a valuable germplasm resource with significant implications for breed conservation,development,and utilization. However,the scarcity of genetic resources in endangered species poses a fundamental constraint on obtaining gametes for embryonic stem cell derivation. Therefore,generating embryonic stem cells from somatic cell nuclear transfer blastocysts offers an optimal alternative for conservation cloning. In this study,we established ApèiJiaza somatic cell nuclear transfer ESCs (APNT-ESCs) from cloned embryos,using ApèiJiaza cattle ear fibroblasts as nuclear donors. APNT-ESCs could be passaged for over 30 generations in vitro,exhibiting high expression of key pluripotency markers,genomic stability,and the ability to form embryoid bodies and differentiate into cell types of all three germ layers. This research established an effective biotechnological framework for the genetic conservation of other endangered species lacking accessible gametes. View Publication

Background: Acute erythroid leukemia (AEL) or AML-M6 predominantly affects older adults and is rare in childhood. Compared with other AML subtypes,AEL remains relatively understudied because of its rarity. We established LS-CHM,a novel AEL cell line derived from the ascitic fluid of a patient with congenital leukemia. Interestingly,leukemic cells persisted in the ascitic fluid even after successful eradication from the bone marrow and extramedullary sites. Method: Leukemia cells from the ascites fluid exhibited robust proliferation in culture independent of cytokine requirement and were further characterized by flow cytometric immunophenotyping,cytogenetics,cell cycle and doubling time analysis,colony formation,genome and RNA sequencing,myeloid gene next generation sequencing,and cytotoxicity analysis. Results: LS-CHM displayed CD36,partial CD235a,CD31,CD43,and CD71 expression and demonstrated in vitro robust growth and high sensitivity to chemotherapeutic agents. A PDX mouse model showed development of leukemia. Genomic analysis revealed a frameshift BCOR mutation in the absence of additional mutations and downregulated TP53 expression with an exonic non-deleterious mutation. RNA sequencing of LS-CHM cells revealed upregulation of two cohesin complex genes,RAD21 and SMC3,whose high levels are associated with hematopoietic stem cell differentiation into erythroid lineage. Conclusions: LS-CHM represents the first congenital AEL-derived cell line,in contrast to the predominantly adult-origin and often secondary erythroid leukemia cell lines available currently. Thus,LS-CHM provides a unique pediatric and extramedullary AEL model,expanding the existing spectrum of AEL cell lines and offering valuable opportunities for biologic and therapeutic investigations. View Publication

Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However,responses triggered by programmable nucleases in HSPCs are poorly characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here,we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction,cell-cycle progression,and transcriptional responses in HSPC subpopulations,with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation,constraining proliferation,yield,and engraftment of edited HSPCs. However,functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs. View Publication

Transplantation-associated stress can compromise the hematopoietic potential of hematopoietic stem cells (HSCs). As a consequence,HSCs may undergo exhaustion" in serial transplant recipients� View Publication

Neural stem cells (NSCs) yield both neuronal and glial progeny,but their differentiation potential toward multiple region-specific neuron types remains remarkably poor. In contrast,embryonic stem cell (ESC) progeny readily yield region-specific neuronal fates in response to appropriate developmental signals. Here we demonstrate prospective and clonal isolation of neural rosette cells (termed R-NSCs),a novel NSC type with broad differentiation potential toward CNS and PNS fates and capable of in vivo engraftment. R-NSCs can be derived from human and mouse ESCs or from neural plate stage embryos. While R-NSCs express markers classically associated with NSC fate,we identified a set of genes that specifically mark the R-NSC state. Maintenance of R-NSCs is promoted by activation of SHH and Notch pathways. In the absence of these signals,R-NSCs rapidly lose rosette organization and progress to a more restricted NSC stage. We propose that R-NSCs represent the first characterized NSC stage capable of responding to patterning cues that direct differentiation toward region-specific neuronal fates. In addition,the R-NSC-specific genetic markers presented here offer new tools for harnessing the differentiation potential of human ESCs. View Publication