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

Studies have shown that neural stem/progenitor cell (NPC) transplantation is beneficial in experimental autoimmune encephalomyelitis (EAE),an established animal model of multiple sclerosis (MS). It is unclear whether NPCs have the ability to integrate into the host CNS to replace lost cells or if their main mechanism of action is via bystander immunomodulation. Understanding the mechanisms by which NPCs exert their beneficial effects as well as exploring methods to increase post-transplantation survival and differentiation is critical to advancing this treatment strategy. Using the EAE model and Fas-deficient (lpr) NPCs,we investigated the effects of altering the Fas system in NPC transplantation therapy. We show that transplantation of NPCs into EAE mice ameliorates clinical symptoms with greater efficacy than sham treatments regardless of cell type (wt or lpr). NPC transplantation via retro-orbital injections significantly decreased inflammatory infiltrates at the acute time point,with a similar trend at the chronic time point. Both wt and lpr NPCs injected into mice with EAE were able to home to sites of CNS inflammation in the periventricular brain and lumbar spinal cord. Both wt and lpr NPCs have the same capacity for inducing apoptosis of Th1 and Th17 cells,and minimal numbers of NPCs entered the CNS. These cells did not express terminal differentiation markers,suggesting that NPCs exert their effects mainly via bystander peripheral immunomodulation. View Publication

Objective Dendritic cells (DCs) are key orchestrators of immune function. To date,rheumatoid arthritis (RA) researchers have predominantly focused on a potential pathogenic role for CD1c+ DCs. In contrast,CD141+ DCs and plasmacytoid DCs (pDCs) have not been systematically examined,at least in early RA. In established RA,the role of pDCs is ambiguous and,since disease duration and treatment both impact RA pathophysiology,we examined pDCs,and CD1c+ and CD141+ conventional DCs (cDCs),in early,drug-na{\{i}}ve RA (eRA) patients. Methods We analyzed the frequency and phenotype of pDCs View Publication

Activated neutrophil-derived exosomes reportedly contribute to the proliferation of airway smooth muscle cells (ASMCs),thereby aggravating the airway wall remodeling during asthma; however,the specific mechanism remains unclear. Lipopolysaccharide (LPS)-EXO and si-CRNDE-EXO were extracted from the media of human neutrophils treated with LPS and LPS??+??si-CRNDE (a siRNA targets long non-coding RNA CRNDE),respectively. Human ASMCs were co-cultured with LPS-EXO or si-CRNDE-EXO,and cell viability,proliferation and migration were measured. The interplay of colorectal neoplasia differentially expressed (CRNDE),inhibitor of nuclear factor kappa B kinase subunit beta (IKK$\beta$) and nuclear receptor subfamily 2 group C member 2 (TAK1) was explored using RNA immunoprecipitation (RIP) and Co-IP assays. A mouse model of asthma was induced using ovalbumin. CRNDE was upregulated in LPS-EXO and successfully transferred from LPS-treated neutrophils to ASMCs through exosome. Mechanically,CRNDE loaded in LPS-EXO reinforced TAK1-mediated IKK$\beta$ phosphorylation,thereby activating the nuclear factor kappa B (NF-$\kappa$B) pathway. Functionally,silencing CRNDE in LPS-EXO,an IKK$\beta$ inhibitor,and an NF-$\kappa$B inhibitor all removed the upregulation of cell viability,proliferation and migration induced by LPS-EXO in ASMCs. In the end,the in vivo experiment demonstrated that CRNDE knockdown in neutrophils effectively reduced the thickness of bronchial smooth muscle in a mouse model for asthma. Activated neutrophils-derived CRNDE was transferred to ASMCs through exosomes and activated the NF-$\kappa$B pathway by enhancing IKK$\beta$ phosphorylation. The latter promoted the proliferation and migration of ASMCs and then contributed to airway remodeling in asthma. View Publication

Measles virus envelope pseudotyped LV (MV-LV) can achieve high B cell transduction rates (up to 50%),but suffers from low titers. To overcome current limitations,we developed an optimized MV-LV production protocol that achieved consistent B cell transduction efficiency up to 75%. We detail this protocol along with analytical assays to assess the results of MV-LV mediated B cell transduction,including flow cytometry for B cell phenotypic characterization and measurement of transduction efficiency,and ddPCR for VCN analysis. View Publication

SCOPE Necrotizing enterocolitis (NEC) is a devastating disease that is highly lethal in premature infants. Human milk oligosaccharides (HMOs) efficiently reduce the incidence of NEC. However,the protective mechanism of HMO treatment is unknown. It is hypothesized that HMOs protect against NEC by inhibiting the damage to intestinal epithelial cells. METHODS AND RESULTS C57BL/6 pups are challenged with hypoxia and cold stress to induce NEC. All pups are sacrificed after 72 h. It is found that HMO administration reduces the concentrations of IL-8 in the serum and ileum of all NEC mice. Ileum toll-like receptor 4 (TLR4) protein expression and nuclear factor kappa-B (NF$\kappa$B) pathway activation are inhibited. The proliferative ability of enterocytes in the ileum is restored as determined by labeling with proliferation markers (Ki67,SOX9). In a 3D culture intestinal crypt organoids study,HMO treatment improves the maturation of organoid cells and increases the ratio of proliferative cells under lipopolysaccharides (LPS) treatment. HMO treatment downregulates TLR4 expression in the organoid cells,thus reducing the effect of LPS. CONCLUSION HMOs protect intestinal epithelial cells from injury by accelerating the turnover of crypt cells by reducing the expression of TLR4 on intestinal epithelial cells. View Publication

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1),a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However,the regulatory network of SCA1 pathology,especially central regulators of the earliest developmental stages and inflammatory events,remains incompletely understood. Here,we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development,and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients. Molecular changes in neurodegeneration occur much earlier than previously expected. In this study,dynamic molecular network analysis of iPSC differentiation uncovers a temporal pathway from histone to ISG15 with the earliest molecular changes of SCA1. View Publication

Angiotensin (Ang)-(1–7) stimulates vasoprotective functions of diabetic (DB) CD34 + hematopoietic stem/progenitor cells partly by decreasing reactive oxygen species (ROS),increasing nitric oxide (NO) levels and decreasing TGFβ1 secretion. Telomerase reverse transcriptase (TERT) translocates to mitochondria and regulates ROS generation. Alternative splicing of TERT results in variants α-,β- and α-β-TERT,which may oppose functions of full-length (FL) TERT. This study tested if the protective functions of Ang-(1–7) or TGFβ1-silencing are mediated by mitoTERT and that diabetes decreases FL-TERT expression by inducing splicing. CD34 + cells were isolated from the peripheral blood mononuclear cells of nondiabetic (ND,n = 68) or DB (n = 74) subjects. NO and mitoROS levels were evaluated by flow cytometry. TERT splice variants and mitoDNA-lesions were characterized by qPCR. TRAP assay was used for telomerase activity. Decoy peptide was used to block mitochondrial translocation (mitoXTERT). TERT inhibitor or mitoXTERT prevented the effects of Ang-(1–7) on NO or mitoROS levels in DB-CD34 + cells. FL-TERT expression and telomerase activity were lower and mitoDNA-lesions were higher in DB cells compared to ND and were reversed by Ang-(1–7) or TGFβ1-silencing. The prevalence of TERT splice variants,with predominant β-TERT expression,was higher and the expression of FL-TERT was lower in DB cells (n = 25) compared to ND (n = 30). Ang-(1–7) or TGFβ1-silencing decreased TERT-splicing and increased FL-TERT. Blocking of β-splicing increased FL-TERT and protected mitoDNA in DB-cells. The findings suggest that diabetes induces TERT-splicing in CD34 + cells and that β-TERT splice variant largely contributes to the mitoDNA oxidative damage. View Publication

Neurons derived from induced pluripotent stem cells (h-iPSC-Ns) provide an invaluable model for studying the physiological aspects of human neuronal development under healthy and pathological conditions. However,multiple studies have demonstrated that h-iPSC-Ns exhibit a high degree of functional and epigenetic diversity. Due to the imprecise characterization and significant variation among the currently available maturation protocols,it is essential to establish a set of criteria to standardize models and accurately characterize and define the developmental properties of human neurons derived from iPSCs. In this study,we conducted comprehensive cellular and network level analysis of the functional development of human neurons,generated from iPSCs obtained from healthy young female peripheral blood mononuclear cells by BDNF and GDNF treatment. We provide a thorough description of the maturation process of h-iPSC-Ns over a 10-week in vitro period using conventional whole-cell patch clamp and dynamic clamp techniques,alongside with morphometry and immunocytochemistry. Additionally,we utilized calcium imaging to monitor the progression of synaptic activity and network communication. At the single cell level,human neurons exhibited gradually decreasing membrane resistance in parallel with improved excitability. By the fifth week of maturation,firing profiles were consistent with those of mature regular firing type of neurons. At the network level,fast glutamatergic and depolarizing GABAergic synaptic connections were abundant together with synchronized network activity from the sixth week of maturation. Alterations in the expression of GABA A receptor subunits were also observed during the process of maturation. The sequence of differentiation events was consistent,providing a robust temporal framework to execute experiments at defined stages of neuronal maturation as well as to use a specific set of experiments to assess a culture’s maturation. The uncovered progression of differentiation events provides a powerful tool to aid the planning and designing of targeted experiments during defined stages of neuronal maturation. View Publication

Colorectal cancer (CRC) is a prevalent malignant tumor globally,ranking third in incidence and second in mortality. Metastasis is the main cause of death in patients with CRC. Solanum nigrum L. (SNL),a traditional Chinese medicinal herb endowed with detoxification,blood circulation enhancement,and anti-swelling properties,has been widely used in folk prescriptions for cancer treatment in China. Solamargine (SM) is the major steroidal alkaloid glycoside purified from SNL. However,its role and mechanism against metastatic CRC are not yet clear. The purpose of this study was to evaluate the inhibitory effect of SM on human hepatic metastatic CRC and investigate its underlying mechanism. CCK-8 assay,colony-formation assay,transwell assay,flow cytometry,tumoursphere formation assay,reverse-transcription quantitative PCR (RT-qPCR),Western blotting,transcriptomic sequencing and ferroptosis analysis were performed to reveal the efficacy and the underlying mechanism of SM in CRC cell lines. In vivo,allograft model,patient-derived xenograft (PDX) model,and liver metastatic model were performed to verify the effect of SM on the growth and metastasis of CRC. SM was found to suppress hepatic metastasis in CRC by effectively targeting key cellular processes,including proliferation,survival,and stemness. RNA sequencing showed that SM could induce ferroptosis,which was confirmed by elevated lipid reactive oxygen species (ROS) and downregulated glutathione peroxidase 4 (GPX4) and glutathione synthetase (GSS) in CRC cells and xenografts. Induction of ferroptosis by SM was regulated by nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore,downregulation of β-catenin was found to be fundamental for the SM-enabled cancer stem cells (CSCs) elimination and metastasis blockage in CRC. Our results indicated that SM is a promising therapeutic drug to inhibit hepatic metastasis in CRC by inducing ferroptosis and impeding CSCs. The online version contains supplementary material available at 10.1186/s13020-025-01171-5. View Publication

Induced pluripotent stem cells (iPSCs) are widely used to model human genetic diseases. The most common strategy involves collecting cells from relevant individuals and then reprogramming them into iPSCs. This strategy is very powerful,but finding enough individuals with a specific genetic disease can be challenging,especially since most are rare. In addition,making numerous iPSC lines is time-consuming and expensive. As a result,most studies have included relatively small numbers of iPSC lines,sometimes from the same individual. Considering the experimental variability obtained using different iPSC lines,there has been great interest in delineating the most efficient number of lines needed to achieve a robust and reproducible result. Several recommendations have been published,although most conclusions have been based on methods where experimental variance from individual cases is difficult to separate from technical issues related to the preparation of iPSCs. The current study used gene expression profiles determined by RNA sequencing (RNAseq) to empirically evaluate the impact of the number of unique individuals and the number of replicate iPSC lines from each individual for modeling Lesch-Nyhan disease (LND). This disease is caused by mutations in the HPRT1 gene,which encodes the enzyme hypoxanthine-guanine phosphoribosyltransferase. Results for detecting disease-relevant changes in gene expression depended on the analytical method employed,and whether or not statistical procedures were used to address multiple iPSC lines from the same individual. In keeping with prior studies,the best results were obtained with iPSC lines from 3-4 unique individuals per group. In contrast to prior studies,results were improved with 2 lines per individual,without statistical corrections for duplicate lines from the same individual. In the current study where all lines were produced in parallel using the same methods,most variance in gene expression came from technical factors unrelated to the individual from whom the iPSC lines were prepared. View Publication