搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Human-induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) have been shown to be useful for the development of cell-based regenerative strategies and for modelling drug discovery. However,stem cell-derived HLCs are not identical in nature to primary human hepatocytes (PHHs),which could affect the cell phenotype and,potentially,model reliability. Therefore,we employed the in-depth gene expression profiling of HLCs and other important and relevant cell types,which led to the identification of clear similarities and differences between them at the transcriptional level. Through gene set enrichment analysis,we identified that genes that are critical for immune signalling pathways become downregulated upon HLC differentiation. Our analysis also found that TAV.HLCs exhibit a mild gene signature characteristic of acute lymphoblastic leukaemia,but not other selected cancers. Importantly,HLCs present significant similarity to PHHs,making them genuinely valuable for modelling human liver biology in vitro and for the development of prototype cell-based therapies for pre-clinical testing. View Publication

The maturation of brain microvascular endothelial cells leads to the formation of a tightly sealed monolayer,known as the blood–brain barrier (BBB). The BBB damage is associated with the pathogenesis of age-related neurodegenerative diseases including vascular cognitive impairment and Alzheimer’s disease. Growing knowledge in the field of epigenetics can enhance the understanding of molecular profile of the BBB and has great potential for the development of novel therapeutic strategies or targets to repair a disrupted BBB. Histone deacetylases (HDACs) inhibitors are epigenetic regulators that can induce acetylation of histones and induce open chromatin conformation,promoting gene expression by enhancing the binding of DNA with transcription factors. We investigated how HDAC inhibition influences the barrier integrity using immortalized human endothelial cells (HCMEC/D3) and the human induced pluripotent stem cell (iPSC)-derived brain vascular endothelial cells. The endothelial cells were treated with or without a novel compound named W2A-16. W2A-16 not only activates Wnt/?-catenin signaling but also functions as a class I HDAC inhibitor. We demonstrated that the administration with W2A-16 sustained barrier properties of the monolayer of endothelial cells,as evidenced by increased trans-endothelial electrical resistance (TEER). The BBB-related genes and protein expression were also increased compared with non-treated controls. Analysis of transcript profiles through RNA-sequencing in hCMEC/D3 cells indicated that W2A-16 potentially enhances BBB integrity by influencing genes associated with the regulation of the extracellular microenvironment. These findings collectively propose that the HDAC inhibition by W2A-16 plays a facilitating role in the formation of the BBB. Pharmacological approaches to inhibit HDAC may be a potential therapeutic strategy to boost and/or restore BBB integrity. View Publication

BackgroundRNA-binding proteins (RBPs) are essential in cardiac development. However,a large of them have not been characterized during the process.MethodsWe applied the human embryonic stem cells (hESCs) differentiated into cardiomyocytes model and constructed SAMD4A-knockdown/overexpression hESCs to investigate the role of SAMD4A in cardiomyocyte lineage specification.ResultsSAMD4A,an RBP,exhibits increased expression during early heart development. Suppression of SAMD4A inhibits the proliferation of hESCs,impedes cardiac mesoderm differentiation,and impairs the function of hESC-derived cardiomyocytes. Correspondingly,forced expression of SAMD4A enhances proliferation and promotes cardiomyogenesis. Mechanistically,SAMD4A specifically binds to FGF2 via a specific CNGG/CNGGN motif,stabilizing its mRNA and enhancing translation,thereby upregulating FGF2 expression,which subsequently modulates the AKT signaling pathway and regulates cardiomyocyte lineage differentiation. Additionally,supplementation of FGF2 can rescue the proliferation defect of hESCs in the absence of SAMD4A.ConclusionsOur study demonstrates that SAMD4A orchestrates cardiomyocyte lineage commitment through the post-transcriptional regulation of FGF2 and modulation of AKT signaling. These findings not only underscore the essential role of SAMD4A in cardiac organogenesis,but also provide critical insights into the molecular mechanisms underlying heart development,thereby informing potential therapeutic strategies for congenital heart disease.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04269-7. View Publication

The ovary is one of the first organs to exhibit signs of aging,characterized by reduced tissue function,chronic inflammation,and fibrosis. Multinucleated giant cells (MNGCs),formed by macrophage fusion,typically occur in chronic immune pathologies,including infectious and non-infectious granulomas and the foreign body response,but are also observed in the aging ovary. The function and consequence of ovarian MNGCs remain unknown as their biological activity is highly context-dependent,and their large size has limited their isolation and analysis through technologies such as single-cell RNA sequencing. In this study,we define ovarian MNGCs through a deep analysis of their presence across age and species using advanced imaging technologies as well as their unique transcriptome using laser capture microdissection. MNGCs form complex interconnected networks that increase with age in both mouse and nonhuman primate ovaries. MNGCs are characterized by high Gpnmb expression,a putative marker of ovarian and non-ovarian MNGCs. Pathway analysis highlighted functions in apoptotic cell clearance,lipid metabolism,proteolysis,immune processes,and increased oxidative phosphorylation and antioxidant activity. Thus,MNGCs have signatures related to degradative processes,immune function,and high metabolic activity. These processes were enriched in MNGCs compared to primary ovarian macrophages,suggesting discrete functionality. MNGCs express CD4 and colocalize with T-cells,which were enriched in regions of MNGCs,indicative of a close interaction between these immune cell types. These findings implicate MNGCs in modulation of the ovarian immune landscape during aging given their high penetrance and unique molecular signature that supports degradative and immune functions. Ovarian multinucleated giant cells are a unique macrophage population that arise within the aging mammalian ovary. This study characterizes their transcriptome in mice,uncovering a potential role in degradation of cellular debris and immune signaling,suggesting a potential contribution to ovarian inflammation during aging. View Publication

High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics,yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput,low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation,allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities,demonstrating efficient delivery of various cargo,with high rates of transfection,gene knockout via non-homologous end joining,and precise knock-in through homology-directed repair. To showcase its utility in functional genomics,we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4⁺ T cells,identifying novel regulators of exhaustion,including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-13532-z. View Publication

The transition of naive T lymphocytes into antigenically activated effector cells is associated with a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This shift facilitates production of the key anti-tumor cytokine interferon (IFN)-γ; however,an associated loss of mitochondrial efficiency in effector T cells ultimately limits anti-tumor immunity. Memory phenotype (MP) T cells are a newly recognized subset that arises through homeostatic activation signals following hematopoietic transplantation. We show here that human CD4 + MP cell differentiation is associated with increased glycolytic and oxidative metabolic activity,but MP cells retain less compromised mitochondria compared to effector CD4 + T cells,and their IFN-γ response is less dependent on glucose and more reliant on glutamine. MP cells also produced IFN-γ more efficiently in response to weak T cell receptor (TCR) agonism than effectors and mediated stronger responses to transformed B cells. MP cells may thus be particularly well suited to carry out sustained immunosurveillance against neoplastic cells. Subject areas: immunity,cell biology View Publication

Of all gynecologic cancers,epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts,90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa,we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover,treatment with LIFR inhibitor,EC359 significantly reduced OCa cell viability and cell survival with an IC 50 ranging from 5-50 nM. Furthermore,EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro,ex vivo and in vivo models including cell-based xenografts,patient-derived explants,organoids,and xenograft tumors,we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally,EC359 therapy considerably improved tumor immunogenicity by robust CD45 + leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-,B-,and other immune cells. Collectively,our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa. Subject terms: Molecular medicine,Ovarian cancer View Publication

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this,the molecular mechanisms that allow H . influenzae to establish persistent infections of human epithelia are not well understood. Here,we have investigated how H . influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H . influenzae infection with an initial,‘unproductive’ inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently,an apparent tolerance to large extracellular and intraepithelial burdens of H . influenzae developed,with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations,and appears to involve interruption of NFκB signalling. This is the first time that large-scale,persistence-promoting immunomodulatory effects of H . influenzae during infection have been observed,and we were able to demonstrate that only infections with live,but not heat-killed H . influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β,IL-36γ and TNFα. Interestingly,NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection,resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H . influenzae in the host,our data further indicate the presence of infection stage-specific gene expression modules,highlighting fundamental similarities between immune responses in NHNE and canonical immune cells,which merit further investigation. View Publication

Liver cancer stem cells (LCSCs) are thought to drive the metastasis and recurrence,however,the heterogeneity of molecular markers of LCSCs has hindered the development of effective methods to isolate them. This study introduced an effective approach to isolate and culture LCSCs from human primary liver cancer (HPLC),leveraging mouse embryonic fibroblasts (MEFs) as feeder cells in conjunction with using defined medium. Isolated LCSCs were further characterized by multiple approaches. Transcriptome sequencing data analysis was conducted to identify highly expressed genes in LCSCs and classify different subtypes of liver cancers. Total sixteen cell strains were directly isolated from 24 tissues of three types of HPLC without sorting,seven of which could be maintained long-term culture as colony growth on MEFs,which is unique characteristics of stem cells. Even 10 of cloned cells formed the tumors in immunodeficient mice,indicating that those cloned cells were tumorgenic. The histologies and gene expression pattern of human xenografts were very similar to those of HPLC where these cloned cells were isolated. Moreover,putative markers of LCSCs were further verified to all express in cloned cells,confirming that these cells were LCSCs. These cloned LCSCs could be cryopreserved,and still maintained the feature of colony growth on MEFs after the recovery. Compared to suspension culture as conventional approach to culture LCSCs,our approach much better maintained stemness of LCSCs for a long time. To date,these cloned cells could be cultured on MEFs over 12 passages. Moreover,bioinformatics analysis of sequencing data revealed the gene expression profiles in LCSCs,and liver cancers were classified into two subtypes C1 and C2 based on genes associated with the prognosis of LCSCs. Patients of the C2 subtype,which is closely related to the extracellular matrix,were found to be sensitive to treatments such as Cisplatin,Axitinib,JAK1 inhibitors,WNT-c59,Sorafenib,and RO-3306. In summary,this effective approach offers new insights into the molecular landscape of human liver cancers,and the identification of the C2 subtype and its unique response to the treatment pave the way for the creation of more effective,personalized therapeutic strategies. The online version contains supplementary material available at 10.1186/s12967-024-05870-9. View Publication

Mounting evidence indicates that exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exosomes) combine the advantages of hucMSC pluripotency with their nanoscale dimensions,enhancing their clinical potential through prolonged circulation half-life. Despite these promising characteristics,research on their immunological toxicity remains insufficient. This study focuses on the impact of hucMSC-exosomes on the general toxicity and immunopathological indicators. When mice received tail vein injections of 6 × 10 10 hucMSC-exosomes particles,we observed no significant changes in body weight,feed intake,blood composition,organ indices,or histopathological findings throughout the 14 days observation period. Similarly,blood levels of immunoglobulins,cytokines,and lymphocyte subpopulations remained stable. The hucMSC-exosomes produced no detectable negative effects on immune organs including the thymus,spleen,and bone marrow. These findings indicate that intravenous administration of 6 × 10 10 particles of hucMSC-exosomes appears relatively safe at the murine level. This assessment of safety and immunological impact following intravenous hucMSC-exosomes infusion offers experimental support for potential clinical applications and future analyses in this field. View Publication

Targeted therapy interventions using tyrosine kinase inhibitors (TKIs) provide encouraging treatment responses in patients with ALK -rearranged lung adenocarcinomas,yet resistance occurs almost inevitably. In addition to tumor cell-intrinsic resistance mechanisms,accumulating evidence suggests that cancer-associated fibroblasts (CAFs) within the tumor microenvironment contribute to therapy resistance. This study aimed to investigate CAF-driven molecular networks that shape the therapeutic susceptibility of ALK -driven lung adenocarcinoma cells. Three-dimensional (3D) spheroid co-cultures comprising ALK -rearranged lung adenocarcinoma cells and CAFs were utilized to model the tumor microenvironment. Single-cell RNA sequencing was performed to uncover transcriptional differences between TKI-treated homotypic and heterotypic spheroids. Functional assays assessed the effects of CAF-conditioned medium and CAF-secreted factors on tumor cell survival,proliferation,lipid metabolism,and downstream AKT signaling. The therapeutic potential of targeting metabolic vulnerabilities was evaluated using pharmacological inhibition of lipid metabolism and by ferroptosis induction. CAFs significantly diminished the apoptotic response of lung tumor cells to ALK inhibitors while simultaneously enhancing their proliferative capacity. Single-cell RNA sequencing identified lipogenesis-associated genes as a key transcriptional difference between TKI-treated homotypic and heterotypic lung tumor spheroids. CAF-conditioned medium and the CAF-secreted factors HGF and NRG1 activated AKT signaling in 3D-cultured ALK-rearranged lung tumor cells,leading to increased de novo lipogenesis and suppression of lipid peroxidation. These metabolic adaptations were critical for promoting tumor cell survival and fostering therapy resistance. Notably,both dual inhibition of ALK and the lipid-regulatory factor SREBP-1,as well as co-treatment with ferroptosis inducers such as erastin or RSL3,effectively disrupted the CAF-driven metabolic-supportive niche and restored sensitivity of resistant lung tumor spheroids to ALK inhibition. This study highlights a critical role for CAFs in mediating resistance to ALK-TKIs by reprogramming lipid metabolism in ALK-rearranged lung cancer cells. It suggests that targeting these metabolic vulnerabilities,particularly through inhibition of lipid metabolism or induction of ferroptosis,could provide a novel therapeutic approach to overcome resistance and improve patient outcomes. The online version contains supplementary material available at 10.1186/s40170-025-00400-7. View Publication

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here,we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes,14 potent neutralizing antibodies were identified,with the most potent one,BD-368-2,exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2,respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally,the 3.8 {\AA} cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover,we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether,we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases. View Publication