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

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

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

This study reports the use of cell-type-specific in vivo bioluminescence to measure intraocular immune cell population dynamics during the course of inflammation in a mouse model of uveitis. Transgenic lines expressing luciferase in inflammatory cell subsets (myeloid cells,T cells,and B cells) were generated and ocular bioluminescence was measured serially for 35 days following uveitis induction. Ocular leukocyte populations were identified using flow cytometry and compared to the ocular bioluminescence profile. Acute inflammation is neutrophilic (75{\%} of ocular CD45 + cells) which is reflected by a significant increase in ocular bioluminescence in one myeloid reporter line on day 2. By day 7,the ocular T cell population increases to 50{\%} of CD45 + cells,leading to a significant increase in ocular bioluminescence in the T cell reporter line. While initially negligible ({\textless} 1{\%} of CD45 + cells),the ocular B cell population increases to {\textgreater} 4{\%} by day 35. This change is reflected by a significant increase in the ocular bioluminescence of the B cell reporter line starting on day 28. Our data demonstrates that cell-type-specific in vivo bioluminescence accurately detects changes in multiple intraocular immune cell populations over time in experimental uveitis. This assay could also be useful in other inflammatory disease models. 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

Huntington’s disease (HD) is the best-known example of a neurodegenerative disorder caused by the expansion of a glutamine-encoding CAG repeat in the causative gene. Growing evidence indicates that somatic CAG expansions play a key role in disease progression,providing a strong rationale for therapeutic strategies directly targeting the repeat tract. However,achieving sufficient efficacy while maintaining allele selectivity and minimizing off-target effects remains a major challenge. Here,we developed allele-selective,CAG-targeting artificial microRNA (amiRNA) molecules that exhibit significantly reduced off-target risk. This was achieved by introducing specific substitutions at selected positions within the guide strand. These molecules effectively downregulated polyglutamine (polyQ) proteins in cellular models of HD,spinocerebellar ataxias types 1 and 3,and dentatorubral pallidoluysian atrophy. The most promising candidate,amiR136-13A,reduced mutant huntingtin levels in different brain regions of the HD mouse model and did not induce toxicity up to 28 weeks following a single administration of an AAV5 vector. Transcriptomic profiling of human HD neural stem cells treated with amiR136-13A revealed minor changes in gene expression. Moreover,amiR136-13A reduced the level of HTT1a,a short pathogenic isoform of huntingtin. Collectively,these findings identify amiR136-13A as a potent,selective,and safe therapeutic candidate for HD and potentially other polyQ disorders. View Publication

INTRODUCTION: The physiological signals that direct the division and differentiation of the zygote to form a blastocyst,and subsequent embryonic stem cell division and differentiation during early embryogenesis,are unknown. Although a number of growth factors,including the pregnancy-associated hormone human chorionic gonadotropin (hCG) are secreted by trophoblasts that lie adjacent to the embryoblast in the blastocyst,it is not known whether these growth factors directly signal human embryonic stem cells (hESCs).backslashnbackslashnMETHODS: Here we used hESCs as a model of inner cell mass differentiation to examine the hormonal requirements for the formation of embryoid bodies (EB's; akin to blastulation) and neuroectodermal rosettes (akin to neurulation).backslashnbackslashnRESULTS: We found that hCG promotes the division of hESCs and their differentiation into EB's and neuroectodermal rosettes. Inhibition of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) signaling suppresses hESC proliferation,an effect that is reversed by treatment with hCG. hCG treatment rapidly upregulates steroidogenic acute regulatory protein (StAR)-mediated cholesterol transport and the synthesis of progesterone (P4). hESCs express P4 receptor A,and treatment of hESC colonies with P4 induces neurulation,as demonstrated by the expression of nestin and the formation of columnar neuroectodermal cells that organize into neural tubelike rosettes. Suppression of P4 signaling by withdrawing P4 or treating with the P4-receptor antagonist RU-486 inhibits the differentiation of hESC colonies into EB's and rosettes.backslashnbackslashnCONCLUSIONS: Our findings indicate that hCG signaling via LHCGR on hESC promotes proliferation and differentiation during blastulation and neurulation. These findings suggest that trophoblastic hCG secretion and signaling to the adjacent embryoblast could be the commencement of trophic support by placental tissues in the growth and development of the human embryo. View Publication

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study,we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM),Flk-1,tie-1,tie-2,vascular endothelial (VE) cadherin,MECA-32,and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4,whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1,PECAM,VE-cadherin,MECA-32,and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin,interleukin-6,fibroblast growth factor 2,and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis. View Publication

BACKGROUND Neuronal stem cells (NSCs) are promising for neurointestinal disease therapy. Although NSCs have been isolated from intestinal musclularis,their presence in mucosa has not been well described. Mucosa-derived NSCs are accessible endoscopically and could be used autologously. Brain-derived Nestin-positive NSCs are important in endogenous repair and plasticity. The aim was to isolate and characterize mucosa-derived NSCs,determine their relationship to Nestin-expressing cells and to demonstrate their capacity to produce neuroglial networks in vitro and in vivo. METHODS Neurospheres were generated from periventricular brain,colonic muscularis (Musc),and mucosa-submucosa (MSM) of mice expressing green fluorescent protein (GFP) controlled by the Nestin promoter (Nestin-GFP). Neuronal stem cells were also grown as adherent colonies from intestinal mucosal organoids. Their differentiation potential was assessed using immunohistochemistry using glial and neuronal markers. Brain and gut-derived neurospheres were transplanted into explants of chick embryonic aneural hindgut to determine their fate. KEY RESULTS Musc- and MSM-derived neurospheres expressed Nestin and gave rise to cells of neuronal,glial,and mesenchymal lineage. Although Nestin expression in tissue was mostly limited to glia co-labelled with glial fibrillary acid protein (GFAP),neurosphere-derived neurons and glia both expressed Nestin in vitro,suggesting that Nestin+/GFAP+ glial cells may give rise to new neurons. Moreover,following transplantation into aneural colon,brain- and gut-derived NSCs were able to differentiate into neurons. CONCLUSIONS & INFERENCES Nestin-expressing intestinal NSCs cells give rise to neurospheres,differentiate into neuronal,glial,and mesenchymal lineages in vitro,generate neurons in vivo and can be isolated from mucosa. Further studies are needed for exploring their potential for treating neuropathies. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication