搜索结果: 'gmp t cell activator'

Aromatase inhibitors (AIs) are an effective therapy in treating estrogen receptor-positive breast cancer. Nonetheless,a significant percentage of patients either do not respond or become resistant to AIs. Decreased dependence on ER-signaling and increased dependence on growth factor receptor signaling pathways,particularly human epidermal growth factor receptor 2 (EGFR2/HER2),have been implicated in AI resistance. However,the role of growth factor signaling remains unclear. This current study investigates the possibility that signaling either through HER2 alone or through interplay between epidermal growth factor receptor 1 (EGFR/HER1) and HER2 mediates AI resistance by increasing the tumor initiating cell (TIC) subpopulation in AI-resistant cells via regulation of stem cell markers,such as breast cancer resistance protein (BCRP). TICs and BCRP are both known to be involved in drug resistance. Results from in vitro analyses of AI-resistant versus AI-sensitive cells and HER2-versus HER2+ cells,as well as from in vivo xenograft tumors,indicate that (1) AI-resistant cells overexpress both HER2 and BCRP and exhibit increased TIC characteristics compared to AI-sensitive cells; (2) inhibition of HER2 and/or BCRP decrease TIC characteristics in letrozole-resistant cells; and (3) HER2 and its dimerization partner EGFR/HER1 are involved in the regulation of BCRP. Overall,these results suggest that reducing or eliminating the TIC subpopulation with agents that target BCRP,HER2,EGFR/HER1,and/or their downstream kinase pathways could be effective in preventing and/or treating acquired AI resistance. View Publication

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA),known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as MELAS demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multi-lineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts,the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA,resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. Upon comparative differentiation of iPSC clones,improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared to isogenic clones with high heteroplasmy. Thus,mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny,and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases. View Publication

Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene,which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs,the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming,modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds,sodium butyrate (NaB) and Parnate,led to an increase in FXN expression and correction of repressive marks at the FXN locus,which persisted for several passages. However,prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore,we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together,these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications,thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA. View Publication

The polarization and migration of neural progenitor cells (NPCs) are critical for embryonic brain development and neurogenesis after brain injury. Although stromal-derived factor-1α (SDF-1α,CXCL12) and its receptor CXCR4 are well-known to mediate the migration of NPCs in the developing brain,the dynamic cellular processes and structure-related molecular events remain elusive. Transwell and microfluidic-based assays are classical assays to effectively study cellular migration. However,both of them have limitations in the analysis of a single cell. In this study,we modified the stripe assay and extended its applications in the study of NPC polarization and intracellular molecular events associated with CXCL12-mediated migration. In response to localized CXCL12,NPCs formed lamellipodia in the stripe assay. Furthermore,CXCR4 and Rac1 quickly re-distributed to the area of lamellipodia,indicating their roles in NPC polarization upon CXCL12 stimulation. Although the chemokine stripes in the assay provided concentration gradients that can be best used to study cellular polarization and migration through immunocytochemistry,they can also generate live imaging data with comparable quality. In conclusion,stripe assay is a visual,dynamic and economical tool to study cellular mobility and its related molecule mechanisms. View Publication

Craniosynostosis is a multigenic congenital condition in which one or more calvarial sutures have prematurely fused during the development of the fetus. Pathogenic variants in FGFR2 are associated with the development of syndromic craniosynostosis,such as Crouzon,Apert and Pfeifer syndromes. Investigation of FGFR2 -linked craniosynostosis is hindered by the lack of appropriate in vitro models. Patient-derived human induced pluripotent stem cell (hiPSC) in vitro disease models provide the opportunity to investigate the disease,identify molecular targets for pharmaceutical treatments,and enable the generation of autologous pluripotent stem cell catalogues. Here,we report three patient-derived hiPSC lines carrying the C342Y,S252W or E565G FGFR2 pathogenic variant. The patient hiPSC lines express characteristic pluripotency markers and display distinct phosphorylation profiles under unstimulated conditions. FGFR2 C342Y showed autophosphorylation in the absence of bFGF ligand,although downstream docking proteins PLCγ and FRS2α were not phosphorylated. FGFR2 S252W and FGFR2 E565G hiPSCs showed increased phosphorylation of docking proteins PLCγ and FRS2α,whereas FGFR2 was not phosphorylated. These patient hiPSC lines provide molecular and cellular options to investigate FGFR2 -linked craniosynostosis in the patient-specific genomic context and develop therapeutic modalities. View Publication

5-Ethynyl-2′-deoxyuridine (EdU) has revolutionized DNA replication and cell cycle analyses through fast,efficient click chemistry detection. However,commercial EdU kits suffer from high costs,proprietary formulations,limited antibody multiplexing capabilities,and difficulties with larger biological specimens. Here,we present OpenEMMU (Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics),an optimized,affordable,and user-friendly click chemistry platform utilizing off-the-shelf reagents. OpenEMMU enhances efficiency,brightness,and multiplexing capabilities of EdU staining with both non-conjugated and conjugated antibodies across diverse cell types,including T cell activation and proliferation assays. We validated its effectiveness for the fluorescent imaging of nascent DNA synthesis in developing embryos and organs,including embryonic heart,forelimbs,and 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled the deep-tissue 3D imaging of DNA synthesis in zebrafish larvae and under replication stress in embryos at high spatial resolution. This approach opens new avenues for understanding organismal development,cell proliferation,and DNA replication dynamics with unprecedented precision and flexibility. Subject areas: Biochemistry,Cell biology,Developmental biology,Computational bioinformatics View Publication

STAT3 is constitutively activated in many cancer types,including lung cancer,and can induce cancer cell proliferation and cancer stem cell (CSC) maintenance. STAT3 is activated by tyrosine kinases,such as JAK and SRC,but the mechanism by which STAT3 maintains its activated state in cancer cells remains unclear. Here,we show that PRMT5 directly methylates STAT3 and enhances its activated tyrosine phosphorylation in non-small cell lung cancer (NSCLC) cells. PRMT5 expression is also induced by STAT3,suggesting the presence of a positive feedback loop in cancer cells. Furthermore,methylation of STAT3 at arginine 609 by PRMT5 is important for its transcriptional activity and support of tumour growth and CSC maintenance. Indeed,NSCLC cells expressing the STAT3 mutant which R609 was replaced to alanine (R609K) show significantly impaired tumour growth in nude mice. Overall,our study reveals a mechanism by which STAT3 remains activated in NSCLC and provides a new target for cancer therapeutic approaches. Subject terms: Oncogenes,Non-small-cell lung cancer,Growth factor signalling View Publication

Cancer-associated fibroblasts (CAFs) play pivotal roles in solid tumor initiation,growth,and immune evasion. However,the optimal biomimetic modeling conditions remain elusive. In this study,we investigated the effects of 2D and 3D culturing conditions on human primary CAFs integrated into a modular tumor microenvironment (TME). Using single-nucleus RNA sequencing (snRNAseq) and Proteomics’ Proximity Extension Assays,we characterized CAF transcriptomic profiles and cytokine levels. Remarkably,when cultured in 2D,CAFs exhibited a myofibroblast (myCAF) subtype,whereas in 3D tumor spheroid cultures,CAFs displayed a more inflammatory (iCAF) pathological state. By integrating single-cell gene expression data with functional interrogations of critical TME-related processes [natural killer (NK)-mediated tumor killing,monocyte migration,and macrophage differentiation],we were able to reconcile form with function. In 3D TME spheroid models,CAFs enhance cancer cell growth and immunologically shield cells from NK cell-mediated cytotoxicity,in striking contrast with their 2D TME counterparts. Notably,3D CAF-secreted proteins manifest a more immunosuppressive profile by enhancing monocyte transendothelial migration and differentiation into M2-like tumor-associated macrophages (TAMs). Our findings reveal a more immunosuppressive and clinically relevant desmoplastic TME model that can be employed in industrial drug discovery campaigns to expand the cellular target range of chemotherapeutics. View Publication

Brain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. We studied the in vitro response of neural cultures exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex,cerebellum and remaining areas. First,each brain region was evaluated with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second,the cerebral regions were subjected to the decellularization process and their respective characterization using molecular,histological,and ultrastructural techniques. Third,the levels of neurotrophic factors in the decellularized brain scaffold were analyzed. Fourth,we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells,as a unidirectional model of differentiation. Finally,in vitro studies were carried out to evaluate the cell recovery capacity of brain decellularized ECM under stroke-mimetic conditions. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and,in addition,it possesses a combination of structural and biochemical signals (e.g.,neurotrophic factors) that are capable of inducing cell phenotypic changes and promote viability and cell recovery in a stroke/ischemia model in vitro. The online version contains supplementary material available at 10.1038/s41598-025-95656-w. View Publication

Highlights•Preformation of aggregates tuned by cell density enable cultivation of hiPSCs in scale-down shear environments.•Scale-down systems utilizing preformation protocols achieve comparable fold expansion with commercial systems.•Expression of pluripotency markers and functional differentiation capacity is maintained following passage in scale-down culture.•Successful application of hiPSC protocols at < 20 mL scales enable rapid and cost-effective research into cell phenotype under dynamic conditions. Human induced pluripotent stem cell (hiPSC) derived therapeutics require clinically relevant quantities of high-quality cell populations for applications in regenerative medicine. The lack of efficacy exhibited across clinical trials suggests deeper understanding of the networks governing phenotype is needed. Further,costs limit study throughput in characterizing the artificial niche relative to outcomes. We present herein an optimized strategy to enable high-throughput hiPSC expansion at <20 mL research scale. We assessed viability of single cell inoculation and aggregate preformation to facilitate proliferation. We modeled aggregate characteristics against agitation rate. Our results demonstrate tunable control with fold expansion comparable to commercial systems. Marker quantification and teratoma assay confirm functional pluripotency. This approach constitutes a scalable protocol to accelerate hiPSC research,and a significant step in advancing the rate of progress in elucidating links to derivative functionality. This work will enable statistically rigorous studies targeting hiPSC and downstream phenotype for clinical manufacturing. Graphical abstractImplementation of adapted protocols enable scale-down systems as a tool for high-throughput iPSC biomanufacturing research,in platforms conducive to scale-up for clinical manufacturing.Image,graphical abstract View Publication

Immune dysregulation-induced inflammation serves as a driving force in the progression of metabolic dysfunction-associated steatohepatitis (MASH),while the underlying cellular and molecular mechanisms remain largely uncharted. A Western diet (WD) is employed to construct mouse models of metabolic dysfunction associated steatotic liver disease (MASLD) or MASH. Mass cytometry identifies a c-kit+ cDC1 subset whose frequency is reduced in the livers of mice and patients with MASH compared with healthy controls. Adoptive cell transfer of c-kit+ cDC1 protects the progression of MASH. Moreover,analysis of gut microbe sequence shows that WD-fed mice and MASLD/MASH patients exhibit gut microbiota dysbiosis,with an elevated abundance of H2S-producing Desulfovibrio_sp. Transplanting of MASH-derived fecal flora,Desulfovibrio_sp.,or injecting H2S intraperitoneally into MASLD mice decreases the c-kit+cDC1 population and exacerbates liver inflammation. Mechanistically,H2S induces autophagic cell death of cDC1 in a c-kit-dependent manner in cDC-specific c-kit-/- and Atg5-/- mice. We thus uncover that microbiota-derived H2S triggers the autophagic cell death of c-kit+ cDC1 and ignites the liver inflammatory cascade in MASH. The immune regulatory mechanism for metabolic dysfunction-associated steatohepatitis (MASH) remains elusive. Here,the authors identify a c-kit+ cDC1 subset,which can be depleted by Desulfovibrio_sp.-induced H2S via autophagic cell death and contributing to uncontrolled inflammation for MASH progression. View Publication

BackgroundHematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression,which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury,while if and how the niche is reshaped and regulates HSC regeneration are poorly understood.MethodsA mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number,distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry,immunofluorescence,colony assay and bone marrow transplantation,in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro,and was consolidated using megakaryocyte-specific knockout mice and transgenic mice.ResultsMegakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile,transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury,whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically,HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion,and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs,but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently,the delicate coordination between proliferation,mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly,punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury,representing a superior therapeutic approach for myelosuppression.ConclusionsOur study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01651-5. View Publication