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

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

Crystalline Mg-Zinc (Zn)-Strontium (Sr) ternary alloys consist of elements naturally present in the human body and provide attractive mechanical and biodegradable properties for a variety of biomedical applications. The first objective of this study was to investigate the degradation and cytocompatibility of four Mg-4Zn-xSr alloys (x=0.15,0.5,1.0,1.5wt%; designated as ZSr41A,B,C,and D respectively) in the direct culture with human umbilical vein endothelial cells (HUVEC) in vitro. The second objective was to investigate,for the first time,the early-stage inflammatory response in cultured HUVECs as indicated by the induction of vascular cellular adhesion molecule-1 (VCAM-1). The results showed that the 24-h in vitro degradation of the ZSr41 alloys containing a β-phase with a Zn/Sr at% ratio ∼1.5 was significantly faster than the ZSr41 alloys with Zn/Sr at% ∼1. Additionally,the adhesion density of HUVECs in the direct culture but not in direct contact with the ZSr41 alloys for up to 24h was not adversely affected by the degradation of the alloys. Importantly,neither culture media supplemented with up to 27.6mM Mg(2+) ions nor media intentionally adjusted up to alkaline pH 9 induced any detectable adverse effects on HUVEC responses. In contrast,the significantly higher,yet non-cytotoxic,Zn(2+) ion concentration from the degradation of ZSr41D alloy was likely the cause for the initially higher VCAM-1 expression on cultured HUVECs. Lastly,analysis of the HUVEC-ZSr41 interface showed near-complete absence of cell adhesion directly on the sample surface,most likely caused by either a high local alkalinity,change in surface topography,and/or surface composition. The direct culture method used in this study was proposed as a valuable tool for studying the design aspects of Zn-containing Mg-based biomaterials in vitro,in order to engineer solutions to address current shortcomings of Mg alloys for vascular device applications. STATEMENT OF SIGNIFICANCE Magnesium (Mg) alloys specifically designed for biodegradable implant applications have been the focus of biomedical research since the early 2000s. Physicochemical properties of Mg alloys make these metallic biomaterials excellent candidates for temporary biodegradable implants in orthopedic and cardiovascular applications. As Mg alloys continue to be investigated for biomedical applications,it is necessary to understand whether Mg-based materials or the alloying elements have the intrinsic ability to direct an immune response to improve implant integration while avoiding cell-biomaterial interactions leading to chronic inflammation and/or foreign body reactions. The present study utilized the direct culture method to investigate for the first time the in vitro transient inflammatory activation of endothelial cells induced by the degradation products of Zn-containing Mg alloys. View Publication

BACKGROUND The robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research,including mechanistic studies of hematopoiesis,the development of cellular therapies for autoimmune diseases,induced transplant tolerance,anticancer immunotherapies,disease modeling,and drug/toxicity screening. Over the past years,significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal,endothelial,and hematopoietic specification. Thus,it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free,defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis. METHODS The hemogenic endothelium differentiation was accomplished in an adherent,serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial,myeloid,and lymphoid potential. RESULTS Monolayer induction based on GSK3 inhibition,described here,yielded a large number of CD31(+)CD34(+) hemogenic endothelium cells. When isolated and propagated in adherent conditions,these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells,these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid,T-lymphoid,and B-lymphoid cells. CONCLUSION The results of this study substantiate a method that significantly reduces the complexity of current protocols for hematopoietic induction,offers a defined system to study the factors that affect the early stages of hematopoiesis,and provides a new route of lymphoid and myeloid cell derivation from human pluripotent stem cells,thus enhancing their use in translational medicine. View Publication

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 microM. The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells,indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells,suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary,cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells,indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation. View Publication

BACKGROUND The increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects,including a reduction in cancer risk. METHODS We investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations. RESULTS In vitro studies showed that cerivastatin,pitavastatin,and fluvastatin were the most potent anti-proliferative,autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently,pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment,tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells. CONCLUSIONS Our study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies. 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

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

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

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification,recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines,mouse xenograft models and matched human primary and metastatic tissues,we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER(+)),HER2(-) luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts,administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts,as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore,this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-$$B (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore,these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635-46. textcopyright2012 AACR. View Publication

A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line,the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However,when the same methods were used to reprogram three different primary fibroblasts lines,two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer,we consistently observed higher reprogramming efficiencies with the episomal plasmid method,which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally,with the plasmid reprogramming protocol,recombinant vitronectin and synthemax® could be used together with commercially available,fully defined,xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol,we reprogrammed a further 2 primary patient cell lines,one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols,a prerequisite for generating clinically relevant patient specific iPS cells. View Publication

The challenge of expanding haematopoietic stem/progenitor cells (HSPCs) in vitro has limited their clinical application. Human hair follicle mesenchymal stem cells (hHFMSCs) can be reprogrammed to generate intermediate stem cells by transducing OCT4 (hHFMSCs OCT4 ) and pre-inducing with FLT3LG/SCF,and differentiated into erythrocytes. These intermediate cells exhibit gene expression patterns similar to pre-HSCs,making them promising for artificial haematopoiesis. However,further investigation is required to elucidate the in vitro proliferation ability and mechanism underlying the self-renewal of pre-HSCs derived from hHFMSCs. hHFMSCs OCT4 were pre-treated with FLT3LG and SCF cytokines,followed by characterization and isolation of the floating cell subsets for erythroid differentiation through stimulation with hematopoietic cytokines and nutritional factors. Cell adhesion was assessed through disassociation and adhesion assays. OCT4 expression levels were measured using immunofluorescence staining,RT-qPCR,and Western blotting. RNA sequencing and Gene Ontology (GO) enrichment analysis were then conducted to identify proliferation-related biological processes. Proliferative capacity was evaluated through CCK-8,colony formation assays,Ki67 index,and cell cycle analysis. Cytoskeleton was observed through Wright‒Giemsa,Coomassie brilliant blue,and phalloidin staining. Expression of adherens junction (AJ) core members was confirmed through RT‒qPCR,Western blotting,and immunofluorescence staining before and after ZO-1 knockdown. A regulatory network was constructed to determine relationships among cytoskeleton,proliferation,and the AJ pathway. Student’s t tests (GraphPad Prism 8.0.2) were used for group comparisons. The results were considered significant at P < 0.05. Pre-treatment of hHFMSCs OCT4 with FLT3LG and SCF leads to the emergence of floating cell subsets exhibiting small,globoid morphology,suspended above adherent cells,forming colonies,and displaying minimal expression of CD45. Excessive OCT4 expression weakens adhesion in floating hHFMSCs OCT4 . Floating cells moderately enhanced proliferation and undergo cytoskeleton remodelling,with increased contraction and aggregation of F-actin near the nucleus. The upregulation of ZO-1 could impact the expressions of F-actin,E-cadherin,and β-catenin genes,as well as the nuclear positioning of β-catenin,leading to variations in the cytoskeleton and cell cycle. Finally,a regulatory network revealed that the AJ pathway cored with ZO-1 critically bridges cytoskeletal remodelling and haematopoiesis-related proliferation in a β-catenin-dependent manner. ZO-1 improved the self-renewal of pre-HSCs from OCT4-overexpressing hHFMSCs by remodeling the cytoskeleton via the ZO-1-regulated AJ pathway,suggesting floating hHFMSCs OCT4 as the promising seed cells for artificial hematopoiesis. The online version contains supplementary material available at 10.1186/s13287-024-04080-w. View Publication