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

The administration of antiretroviral therapy (ART) leads to a rapid reduction in plasma viral load in HIV-1 seropositive subjects. However,when ART is suspended,the virus rebounds due to the presence of a latent viral reservoir. Several techniques have been developed to characterize this latent viral reservoir. Of the various assay formats available presently,the Tat/Rev induced limiting dilution assay (TILDA) offers the most robust and technically simple assay strategy. The TILDA formats reported thus far are limited by being selective to one or a few HIV-1 genetic subtypes,thus,restricting them from a broader level application. The novel TILDA,labelled as U-TILDA ('U' for universal),can detect all the major genetic subtypes of HIV-1 unbiasedly,and with comparable sensitivity of detection. U-TILDA is well suited to characterize the latent reservoirs of HIV-1 and aid in the formulation of cure strategies. Graphical abstract. View Publication

Monoclonal antibodies are at the vanguard of the most promising cancer treatments. Whereas traditional therapeutic antibodies have been limited to extracellular antigens,T cell receptor mimic (TCRm) antibodies can target intracellular antigens presented by cell surface major histocompatibility complex (MHC) proteins. TCRm antibodies can therefore target a repertoire of otherwise undruggable cancer antigens. However,the consequences of off-target peptide/MHC recognition with engineered T cell therapies are severe,and thus there are significant safety concerns with TCRm antibodies. Here we explored the specificity and safety profile of a new TCRm-based T cell therapy for hepatocellular carcinoma (HCC),a solid tumor for which no effective treatment exists. We targeted an alpha-fetoprotein peptide presented by HLA-A*02 with a highly specific TCRm,which crystallographic structural analysis showed binds directly over the HLA protein and interfaces with the full length of the peptide. We fused the TCRm to the ? and ? subunits of a TCR,producing a signaling AbTCR construct. This was combined with an scFv/CD28 co-stimulatory molecule targeting glypican-3 for increased efficacy towards tumor cells. This AbTC + co-stimulatory T cell therapy showed potent activity against AFP-positive cancer cell lines in vitro and an in an in vivo model and undetectable activity against AFP-negative cells. In an in-human safety assessment,no significant adverse events or cytokine release syndrome were observed and evidence of efficacy was seen. Remarkably,one patient with metastatic HCC achieved a complete remission after nine months and ultimately qualified for a liver transplant. View Publication

Hydrogen Peroxide (H2O2) is a central oxidant in redox biology due to its pleiotropic role in physiology and pathology. However,real-time monitoring of H2O2 in living cells and tissues remains a challenge. We address this gap with the development of an optogenetic hydRogen perOxide Sensor (oROS),leveraging the bacterial peroxide binding domain OxyR. Previously engineered OxyR-based fluorescent peroxide sensors lack the necessary sensitivity and response speed for effective real-time monitoring. By structurally redesigning the fusion of Escherichia coli (E. coli) ecOxyR with a circularly permutated green fluorescent protein (cpGFP),we created a novel,green-fluorescent peroxide sensor oROS-G. oROS-G exhibits high sensitivity and fast on-and-off kinetics,ideal for monitoring intracellular H2O2 dynamics. We successfully tracked real-time transient and steady-state H2O2 levels in diverse biological systems,including human stem cell-derived neurons and cardiomyocytes,primary neurons and astrocytes,and mouse brain ex vivo and in vivo. These applications demonstrate oROS’s capabilities to monitor H2O2 as a secondary response to pharmacologically induced oxidative stress and when adapting to varying metabolic stress. We showcased the increased oxidative stress in astrocytes via A?-putriscine-MAOB axis,highlighting the sensor’s relevance in validating neurodegenerative disease models. Lastly,we demonstrated acute opioid-induced generation of H2O2 signal in vivo which highlights redox-based mechanisms of GPCR regulation. oROS is a versatile tool,offering a window into the dynamic landscape of H2O2 signaling. This advancement paves the way for a deeper understanding of redox physiology,with significant implications for understanding diseases associated with oxidative stress,such as cancer,neurodegenerative,and cardiovascular diseases. View Publication

Brain organoids offer unprecedented insights into brain development and disease modeling and hold promise for drug screening. Significant hindrances,however,are morphological and cellular heterogeneity,inter-organoid size differences,cellular stress,and poor reproducibility. Here,we describe a method that reproducibly generates thousands of organoids across multiple hiPSC lines. These High Quantity brain organoids (Hi-Q brain organoids) exhibit reproducible cytoarchitecture,cell diversity,and functionality,are free from ectopically active cellular stress pathways,and allow cryopreservation and re-culturing. Patient-derived Hi-Q brain organoids recapitulate distinct forms of developmental defects: primary microcephaly due to a mutation in CDK5RAP2 and progeria-associated defects of Cockayne syndrome. Hi-Q brain organoids displayed a reproducible invasion pattern for a given patient-derived glioma cell line. This enabled a medium-throughput drug screen to identify Selumetinib and Fulvestrant,as inhibitors of glioma invasion in vivo. Thus,the Hi-Q approach can easily be adapted to reliably harness brain organoids’ application for personalized neurogenetic disease modeling and drug discovery. Human brain organoids are plagued by heterogeneity and poor reproducibility,critical parameters for reliable disease modeling and drug testing. Here,the authors report on Hi-Q organoids which solve these limitations and can be cryopreserved in large quantities. View Publication

Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study,we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles,globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally,these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably,we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype,which reactivates fetal hemoglobin levels and rescues the disease phenotypes,thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether,we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling,drug screenings and cell and gene therapy-based applications. Sickle cell disease (SCD) and β-thalassemia (BT) are globally prevalent inherited blood disorders but,despite extensive research,no ex vivo system exists for SCD and BT. Here,the authors generate pathophysiologically relevant erythroid progenitor models of SCD and BT. View Publication

AbstractThere is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats),coupled with light’s unparalleled spatiotemporal resolution deliverable from a controllable source,makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here,we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs,BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR,including indels,CRISPRa,and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing,achieving optogenetic gene editing in T lymphocytes in vivo. View Publication

Enhanced glycolysis plays a pivotal role in fueling the aberrant proliferation,survival and therapy resistance of acute myeloid leukemia (AML) cells. Here,we aimed to elucidate the extent of glycolysis dependence in AML by focusing on the role of lactate dehydrogenase A (LDHA),a key glycolytic enzyme converting pyruvate to lactate coupled with the recycling of NAD + . We compared the glycolytic activity of primary AML patient samples to protein levels of metabolic enzymes involved in central carbon metabolism including glycolysis,glutaminolysis and the tricarboxylic acid cycle. To evaluate the therapeutic potential of targeting glycolysis in AML,we treated AML primary patient samples and cell lines with pharmacological inhibitors of LDHA and monitored cell viability. Glycolytic activity and mitochondrial oxygen consumption were analyzed in AML patient samples and cell lines post-LDHA inhibition. Perturbations in global metabolite levels and redox balance upon LDHA inhibition in AML cells were determined by mass spectrometry,and ROS levels were measured by flow cytometry. Among metabolic enzymes,we found that LDHA protein levels had the strongest positive correlation with glycolysis in AML patient cells. Blocking LDHA activity resulted in a strong growth inhibition and cell death induction in AML cell lines and primary patient samples,while healthy hematopoietic stem and progenitor cells remained unaffected. Investigation of the underlying mechanisms showed that LDHA inhibition reduces glycolytic activity,lowers levels of glycolytic intermediates,decreases the cellular NAD + pool,boosts OXPHOS activity and increases ROS levels. This increase in ROS levels was however not linked to the observed AML cell death. Instead,we found that LDHA is essential to maintain a correct NAD + /NADH ratio in AML cells. Continuous intracellular NAD + supplementation via overexpression of water-forming NADH oxidase from Lactobacillus brevis in AML cells effectively increased viable cell counts and prevented cell death upon LDHA inhibition. Collectively,our results demonstrate that AML cells critically depend on LDHA to maintain an adequate NAD + /NADH balance in support of their abnormal glycolytic activity and biosynthetic demands,which cannot be compensated for by other cellular NAD + recycling systems. These findings also highlight LDHA inhibition as a promising metabolic strategy to eradicate leukemic cells. The online version contains supplementary material available at 10.1186/s40170-025-00392-4. View Publication

Endogenous repair of osteochondral defect is usually limited by the insufficient number of cells in the early stage and incomplete cell differentiation in the later stage. The development of drug delivery systems for sequential release of pro-migratory and pro-chondrogenic molecules to induce endogenous bone marrow-derived mesenchymal stem cells (BMSCs) recruitment and chondrogenic differentiation is highly desirable for in situ osteochondral regeneration. In this study,a novel,all-silk-derived sequential delivery system was fabricated by incorporating the tunable drug-loaded silk fibroin (SF) nanospheres into a SF porous matrix. The loading efficiency and release kinetics of biomolecules depended on the initial SF/polyvinyl alcohol (PVA) concentrations (0.2{\%},1{\%} and 5{\%}) of the nanospheres,as well as the hydrophobicity of the loaded molecules,resulting in controllable and programmed delivery profiles. Our findings indicated that the 5{\%} nanosphere-incorporated matrix showed a rapid release of E7 peptide during the first 120 h,whereas the 0.2{\%} nanosphere-incorporated matrix provided a slow and sustained release of Kartogenin (KGN) longer than 30 days. During in vitro culture of BMSCs,this functional SF matrix incorporated with E7/KGN nanospheres showed good biocompatibility,as well as enhanced BMSCs migration and chondrogenic differentiation through the release of E7 and KGN. Furthermore,when implanted into rabbit osteochondral defect,the SF nanosphere matrix with sequential E7/KGN release promoted the regeneration of both cartilage and subchondral bone. This work not only provided a novel all-silk-derived drug delivery system for sequential release of molecules,but also a functional tissue-engineered scaffold for osteochondral regeneration. View Publication

Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response,as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells,we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast,rescue from AICD was not associated with a significant change of Bcl-2,an inhibitor of apoptosis induced by cell stress. Accordingly,MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis,a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall,MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state,providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

We have used in vitro and mouse xenograft models to examine the interaction between breast cancer stem cells (CSC) and bone marrow-derived mesenchymal stem cells (MSC). We show that both of these cell populations are organized in a cellular hierarchy in which primitive aldehyde dehydrogenase expressing mesenchymal cells regulate breast CSCs through cytokine loops involving IL6 and CXCL7. In NOD/SCID mice,labeled MSCs introduced into the tibia traffic to sites of growing breast tumor xenografts where they accelerated tumor growth by increasing the breast CSC population. With immunochemistry,we identified MSC-CSC niches in these tumor xenografts as well as in frozen sections from primary human breast cancers. Bone marrow-derived MSCs may accelerate human breast tumor growth by generating cytokine networks that regulate the CSC population. View Publication

C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However,Δ32 donors are scarce,heterologous bone marrow transplantation is not exempt of risks,and genetic engineering of autologous hHSCs is not trivial. Here,we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34(+) cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS. View Publication