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

N6-methyladenosine (m6A) modification plays a pivotal role in cell fate determination. Previous studies show that eliminating m6A using Mb1-Cre dramatically impairs B cell development. However,whether disturbing m6A modification at later stages affects B cell development and function remains elusive. Here,we deleted m6A methyltransferase Mettl3 from the pro-B stage on using Cd19-Cre (Mettl3 cKO) and found that the frequency of total B cells in peripheral blood,peritoneal cavity,and liver is comparable between Mettl3 cKO mice and wild-type (WT) littermates,while the percentage of whole splenic B cells slightly increases in Mettl3 cKO individuals. The proportion of pre-pro-B,pro-B,pre-B,immature,and mature B cells in the bone marrow were minimally affected. Loss of Mettl3 resulted in increased apoptosis but barely affected B cells' proliferation and IgG production upon LPS,CD40L,anti-IgM,or TNF-$\alpha$ stimulation. Different stimuli had different effects on B cell activation. In addition,B cell-specific Mettl3 knockout had no influence on the pro-fibrogenic activity of B cells in liver fibrosis,evidenced by comparable fibrosis in carbon tetrachloride- (CCl4-) treated Mettl3 cKO mice and WT controls. In summary,our study demonstrated that deletion of Mettl3 from the pro-B stage on has minimal effects on B cell development and function,as well as profibrogenic activity of B cells in liver fibrosis,revealing a stage-specific dependence on Mettl3-mediated m6A of B cell development. View Publication

Metastatic breast cancer remains largely incurable,and the mechanisms driving the transition from primary to metastatic breast cancer remain elusive. We analyzed the complex landscape of estrogen receptor (ER)-positive breast cancer primary and metastatic tumors using scRNA-seq data from twenty-three female patients with either primary or metastatic disease. By employing single-cell transcriptional profiling of unpaired patient samples,we sought to elucidate the genetic and molecular mechanisms underlying changes in the metastatic tumor ecosystem. We identified specific subtypes of stromal and immune cells critical to forming a pro-tumor microenvironment in metastatic lesions,including CCL2+ macrophages,exhausted cytotoxic T cells,and FOXP3+ regulatory T cells. Analysis of cell-cell communication highlights a marked decrease in tumor-immune cell interactions in metastatic tissues,likely contributing to an immunosuppressive microenvironment. In contrast,primary breast cancer samples displayed increased activation of the TNF-α signaling pathway via NF-kB,indicating a potential therapeutic target. Our study comprehensively characterizes the transcriptional landscape encompassing primary and metastatic breast cancer. View Publication

We evaluated a cocktail of HLA-A2-specific peptides including heteroclitic XBP1 US184-192 (YISPWILAV),heteroclitic XBP1 SP367-375 (YLFPQLISV),native CD138260-268 (GLVGLIFAV) and native CS1239-247 (SLFVLGLFL),for their ability to elicit multipeptide-specific cytotoxic T lymphocytes (MP-CTLs) using T cells from smoldering multiple myeloma (SMM) patients. Our results demonstrate that MP-CTLs generated from SMM patients' T cells show effective anti-MM responses including CD137 (4-1BB) upregulation,CTL proliferation,interferon-γ production and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. Phenotypically,we observed increased total CD3(+)CD8(+) T cells (textgreater80%) and cellular activation (CD69(+)) within the memory SMM MP-CTL (CD45RO(+)/CD3(+)CD8(+)) subset after repeated multipeptide stimulation. Importantly,SMM patients could be categorized into distinct groups by their level of MP-CTL expansion and antitumor activity. In high responders,the effector memory (CCR7(-)CD45RO(+)/CD3(+)CD8(+)) T-cell subset was enriched,whereas the remaining responders' CTL contained a higher frequency of the terminal effector (CCR7(-)CD45RO(-)/CD3(+)CD8(+)) subset. These results suggest that this multipeptide cocktail has the potential to induce effective and durable memory MP-CTL in SMM patients. Therefore,our findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease. View Publication

BACKGROUND: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47,CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. DESIGN AND METHODS: We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. RESULTS: We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47,higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression,arguing against a direct reciprocal relationship. CONCLUSIONS: We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated,in part,by increased CD44-cytoskeleton binding. View Publication

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5,were cross resistant to other small-molecule CCR5 antagonists,and were isolated from the patient's pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4(+) T cells. The V3 loop contained residues essential for viral resistance to APL,while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However,these mutations were context dependent,being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N' terminus of CCR5 in the presence of APL. In addition,the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However,recognition of drug-bound CCR5 was less efficient,resulting in a tropism shift toward effector memory cells upon infection of primary CD4(+) T cells in the presence of APL,with relative sparing of the central memory CD4(+) T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses,then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T(CM) subset of CD4(+) T cells and result in improved T cell homeostasis and immune function. View Publication

Alzheimer's disease (AD) involves the progressive degeneration of neurons critical for learning and memory. In addition,patients with AD typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD,we determined whether inefficient BER due to reduced DNA polymerase-β (Polβ) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild-type and 3xTgAD mice with normal or reduced Polβ levels. Compared to wild-type control mice,Polβ heterozygous (Polβ+/- ),and 3xTgAD mice,3xTgAD/Polβ+/- mice exhibited impaired performance in a buried food test of olfaction. Polβ deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However,numbers of newly generated neurons were reduced by approximately 25% in Polβ+/- and 3xTgAD mice,and by over 60% in the 3xTgAD/Polβ+/- mice compared to wild-type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Polβ+/- mice compared to 3xTgAD mice. Levels of amyloid β-peptide (Aβ) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Polβ+/- mice,and cultured Polβ-deficient neurons exhibited increased vulnerability to Aβ-induced death. Olfactory deficit is an early sign in human AD,but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons,and suggest a mechanism underlying early olfactory impairment in AD. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication

Insight into the normal function of PrP(C),and how it can be subverted to produce neurotoxic effects,is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants,Δ105-125 (called ΔCR),produces a spontaneous neurodegenerative illness when expressed in transgenic mice,and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous,or can be exerted on neighboring cells,is unknown. To investigate this question,we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice,which are marked by the presence or absence of GFP,are differentiated together to yield neurons,astrocytes,and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity,we assayed sensitivity of the cells to the cationic antibiotic,Zeocin. In a previous study,we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics,an effect that is suppressed by co-expression of wild type PrP,similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system,we find that while ΔCR-dependent toxicity is cell-autonomous,the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrP(C),and the cellular mechanisms underlying the rescuing process. View Publication

OBJECTIVE As an inhibitor of the AhR signalling pathway,StemRegenin 1 (SR1) not only promotes the expansion of CD34+ cells but also increases CD34- cell numbers. These CD34- cells influenced the ex vivo expansion of CD34+ cells. In this work,the effects of periodically removing CD34- cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated. MATERIALS AND METHODS CD34- cells were removed periodically with SR1 addition to investigate cell subpopulations,cell expansion,biological functions,expanded cell division mode and supernatant TGF-$\beta$1 contents. RESULTS After 10-day culture,the expansion of CD34+ cells in the CD34- cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover,periodically removing CD34- cells with SR1 addition improved the biological function of expanded CD34+ cells and significantly increased the percentage of self-renewal symmetric division of CD34+ cells. In addition,the concentration of total TGF-$\beta$1 and activated TGF-$\beta$1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT-qPCR results showed that the periodic removal of CD34- cells with cooperation from SR1 further reduced the expression of AhR-related genes. CONCLUSIONS Periodic removal of CD34- cells plus cooperation with SR1 improved the expansion of CD34+ cells,maintained better biological function of expanded CD34+ cells and reduced the TGF-$\beta$1 contents by downregulating AhR signalling. View Publication

Spinal cord injury (SCI) is one of the most debilitating injuries,and transplantation of stem cells in a scaffold is a promising strategy for treatment. However,stem cell treatment of SCI has been severely impaired by the increased generation of reactive oxygen species in the lesion microenvironment,which can lead to a high level of stem cell death and dysfunction. Herein,a MnO2 nanoparticle (NP)-dotted hydrogel is prepared through dispersion of MnO2 NPs in a PPFLMLLKGSTR peptide modified hyaluronic acid hydrogel. The peptide-modified hydrogel enables the adhesive growth of mesenchymal stem cells (MSCs) and nerve tissue bridging. The MnO2 NPs alleviate the oxidative environment,thereby effectively improving the viability of MSCs. Transplantation of MSCs in the multifunctional gel generates a significant motor function restoration on a long-span rat spinal cord transection model and induces an in vivo integration as well as neural differentiation of the implanted MSCs,leading to a highly efficient regeneration of central nervous spinal cord tissue. Therefore,the MnO2 NP-dotted hydrogel represents a promising strategy for stem-cell-based therapies of central nervous system diseases through the comprehensive regulation of pathological microenvironment complications. View Publication

AbstractThe generation of chimeric antigen receptor?modified natural killer (CAR?NK) cells using induced pluripotent stem cells (iPSCs) has emerged as one of the paradigms for manufacturing off?the?shelf universal immunotherapy. However,there are still some challenges in enhancing the potency,safety,and multiple actions of CAR?NK cells. Here,iPSCs were site?specifically integrated at the ribosomal DNA (rDNA) locus with interleukin 24 (IL24) and CD19?specific chimeric antigen receptor (CAR19),and successfully differentiated into iPSC?derived NK (iNK) cells,followed by expansion using magnetic beads in vitro. Compared with the CAR19?iNK cells,IL24 armored CAR19?iNK (CAR19?IL24?iNK) cells showed higher cytotoxic capacity and amplification ability in vitro and inhibited tumor progression more effectively with better survival in a B?cell acute lymphoblastic leukaemia (B?ALL) (Nalm?6 (Luc1))?bearing mouse model. Interestingly,RNA?sequencing analysis showed that IL24 may enhance iNK cell function through nuclear factor kappa B (NF?B) pathway?related genes while exerting a direct effect on tumor cells. This study proved the feasibility and potential of combining IL24 with CAR?iNK cell therapy,suggesting a novel and promising off?the?shelf immunotherapy strategy. Zhang et al. successfully regenerated iNK cells from human iPSCs with rDNA locus gene editing. IL24 enhances the antitumor activity and proliferation of armored CAR?iNK cells,which may be involved in cellular?positive upregulation and adhesion pathways. View Publication

Autoantibodies against envelope (Env) protein encoded by human endogenous retrovirus group K (HERV-K) are prevalent in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE),but it remains unclear which proviruses are responsible for this autoantigen. It also remains poorly understood how the transcription of HERV-K loci is regulated in cells that can produce Env.ResultsWe aligned our neutrophil RNA sequencing data to the new telomere-to-telomere reference genome and found uniquely mapping transcripts from HERV-K101,K102,K104,K108,K109,K117 and ERVK5,of which only K102,K108,and K109 encode an intact Env. Expression of K102 and K108 were higher in SLE than in healthy donors or RA (padj < 0.05). Transcripts from these proviruses increased in response to interferon-α in monocytes and neutrophils from RA patients and healthy donors,but not in SLE,presumably because they have chronically elevated type I interferons in vivo. Indeed,HERV-K expression was significantly higher in SLE patients with high type I interferon gene signature. Tumor necrosis factor-α and other cytokines and TLR ligands also induced HERV-K102 and K108 transcripts. Interferon-α also increased detectable Env protein in monocytes,macrophages,and neutrophils from RA patients. Among the genes for epigenetic silencers of HERV-K,only TRIM28 was significantly decreased in SLE patients with high interferons (padj = 0.00024).ConclusionsOur data establish a role for interferons in maintaining increased HERV-K expression in SLE and suggest that interferons or other cytokines can upregulate HERV-K to similar levels in RA. A transient increase may also accompany normal immune responses,suggesting that endogenous retroviruses may have been co-opted for efficient immune responses.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00371-y. View Publication