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

Canine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent,life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens--200 cGy total body irradiation (TBI) or 10 mg/kg busulfan--with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs,therapeutic levels of CD18(+) leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment,and immunosuppression was not required for efficacy. The percentage of CD18(+) leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification-mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific,large animal model using 2 clinically applicable conditioning regimens,and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD. View Publication

MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages,characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages,we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. MafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly,these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells,GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly,F4/80 expression in macrophages was suppressed in the homozygous mutant,although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells,MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages,whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation,renal tubule survival,and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages. View Publication

The in vitro 3D culture of intestinal epithelium is a valuable resource in the study of its function. Organoid culture exploits stem cells' ability to regenerate and produce differentiated epithelium. Intestinal organoid models from rodent or human tissue are widely available whereas large animal models are not. Livestock enteric and zoonotic diseases elicit significant morbidity and mortality in animal and human populations. Therefore,livestock species-specific models may offer novel insights into host-pathogen interactions and disease responses. Bovine and porcine jejunum were obtained from an abattoir and their intestinal crypts isolated,suspended in Matrigel,cultured,cryopreserved and resuscitated. 'Rounding' of crypts occurred followed by budding and then enlargement of the organoids. Epithelial cells were characterised using immunofluorescent staining and confocal microscopy. Organoids were successfully infected with Toxoplasma gondii or Salmonella typhimurium. This 3D organoid model offers a long-term,renewable resource for investigating species-specific intestinal infections with a variety of pathogens. View Publication

Inappropriate activation of the Hedgehog (Hh) signaling pathway has been implicated in a diverse spectrum of cancers,and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo),small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors. We report here four Hh pathway antagonists that are epistatic to the nucleocytoplasmic regulator Suppressor of Fused [Su(fu)],including two that can inhibit Hh target gene expression induced by overexpression of the Gli transcription factors. Each inhibitor has a unique mechanism of action,and their phenotypes reveal that Gli processing,Gli activation,and primary cilia formation are pharmacologically targetable. We further establish the ability of certain compounds to block the proliferation of cerebellar granule neuron precursors expressing an oncogenic form of Smo,and we demonstrate that Hh pathway inhibitors can have tissue-specific activities. These antagonists therefore constitute a valuable set of chemical tools for interrogating downstream Hh signaling mechanisms and for developing chemotherapies against Hh pathway-related cancers. View Publication

The ability to stimulate mammalian cells with light has significantly changed our understanding of electrically excitable tissues in health and disease,paving the way toward various novel therapeutic applications. Here,we demonstrate the potential of optogenetic control in cardiac cells using a hybrid experimental/computational technique. Experimentally,we introduced channelrhodopsin-2 into undifferentiated human embryonic stem cells via a lentiviral vector,and sorted and expanded the genetically engineered cells. Via directed differentiation,we created channelrhodopsin-expressing cardiomyocytes,which we subjected to optical stimulation. To quantify the impact of photostimulation,we assessed electrical,biochemical,and mechanical signals using patch-clamping,multielectrode array recordings,and video microscopy. Computationally,we introduced channelrhodopsin-2 into a classic autorhythmic cardiac cell model via an additional photocurrent governed by a light-sensitive gating variable. Upon optical stimulation,the channel opens and allows sodium ions to enter the cell,inducing a fast upstroke of the transmembrane potential. We calibrated the channelrhodopsin-expressing cell model using single action potential readings for different photostimulation amplitudes,pulse widths,and frequencies. To illustrate the potential of the proposed approach,we virtually injected channelrhodopsin-expressing cells into different locations of a human heart,and explored its activation sequences upon optical stimulation. Our experimentally calibrated computational toolbox allows us to virtually probe landscapes of process parameters,and identify optimal photostimulation sequences toward pacing hearts with light. ?? 2011 Biophysical Society. View Publication

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy,reliable,and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol,bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested,and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients,leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation,and the infiltration of AML cells was detected in the blood,bone marrow,spleen,and liver of recipients by flow cytometry,qPCR,and histological analyses. Thus,intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells. View Publication

Cerebral organoids offer significant potential for neuroscience research as complex in vitro models that mimic human brain development. However,challenges related to their quality and reproducibility hinder their reliability. Discrepancies in morphology,size,cellular composition,and cytoarchitectural organization limit their applications,particularly in disease modeling,drug screening,and neurotoxicity testing. Critically,current methods for organoid characterization often lack standardization,restricting their broader applicability. To address the need for standardized quality assessment of cerebral organoids,we developed a Quality Control (QC) methodology for 60-day cortical organoids,evaluating five key criteria using a scoring system: morphology,size and growth profile,cellular composition,cytoarchitectural organization,and cytotoxicity. We implemented a hierarchical approach,beginning with non-invasive assessments to exclude low-quality organoids,while reserving in-depth analyses for those that passed the initial evaluation. To validate this framework,we exposed 60-day cortical organoids to graded doses of hydrogen peroxide (H2O2),inducing a range of quality outcomes. The QC system demonstrated its robustness by accurately discriminating organoid qualities. Our proposed QC framework is designed to be user-friendly,flexible,and broadly applicable,making it suitable for routine assessment of cerebral organoid quality. Additionally,its scalability enables industrial applications,offering a valuable tool for advancing both fundamental and pre-clinical research.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-14425-x. View Publication

The reconstruction of damaged neural circuits is critical for neurological repair after brain injury. Classical brain-computer interfaces (BCIs) allow direct communication between the brain and external controllers to compensate for lost functions. Importantly,there is increasing potential for generalized BCIs to input information into the brains to restore damage,but their effectiveness is limited when a large injured cavity is caused. Notably,it might be overcome by transplantation of brain organoids into the damaged region. Here,we construct innovative BCIs mediated by implantable organoids,coined as organoid-brain-computer interfaces (OBCIs). We assess the prolonged safety and feasibility of the OBCIs,and explore neuroregulatory strategies. OBCI stimulation promotes progressive differentiation of grafts and enhances structural-functional connections within organoids and the host brain,promising to repair the damaged brain via regenerating and regulating,potentially directing neurons to preselected targets and recovering functional neural networks in the future. Damaged neural circuits could be improved by generalized BCIs via inputting information into the brains,which is restricted when a large injured cavity caused. Here,the authors construct BCIs mediated by organoid grafts to repair the damaged brain View Publication

Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However,current QC methods,reliant on molecular staining,pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC),a machine learning-driven,multidimensional,high-content,and high-throughput flow cytometry approach,in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified cell count and viability of human peripheral blood mononuclear cells (PBMCs) and identified non-apoptotic live cells and early apoptotic/dead cells in PBMCs (ROC-AUC: area under receiver operating characteristic curve = 0.975),T cells and non-T cells in white blood cells (ROC-AUC = 0.969),activated T cells and quiescent T cells in PBMCs (ROC-AUC = 0.990),and particulate impurities in PBMCs (ROC-AUC ≧ 0.998). The results support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers,assess viability,identify specific cell subsets or phenotypic states,and remove impurities during cell therapy manufacturing. Thus,LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency. Subject terms: Biotechnology,Cell biology,Immunology,Biomedical engineering View Publication

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However,cellular and humoral immunogenicity of genome editing tools,which originate from bacteria,complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases,SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis,we identify putative immunogenic epitopes on each nuclease. Using computational modeling,we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components,including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses,yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9,but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection. Subject terms: Protein design,Immunogenetics,CRISPR-Cas9 genome editing View Publication

Respiratory organoids have emerged as a powerful in vitro model for studying respiratory diseases and drug discovery. However,the high-throughput analysis of organoid images remains a challenge due to the lack of automated and accurate segmentation tools. This study presents a semi-automatic algorithm for image analysis of respiratory organoids (nasal and lung organoids),employing the U-Net architecture and CellProfiler for organoids segmentation. The algorithm processes bright-field images acquired through z-stack fusion and stitching. The model demonstrated a high level of accuracy,as evidenced by an intersection-over-union metric (IoU) of 0.8856,F1-score = 0.937 and an accuracy of 0.9953. Applied to forskolin-induced swelling assays of lung organoids,the algorithm successfully quantified functional differences in Cystic Fibrosis Transmembrane conductance Regulator (CFTR)-channel activity between healthy donor and cystic fibrosis patient-derived organoids,without fluorescent dyes. Additionally,an open-source dataset of 827 annotated respiratory organoid images was provided to facilitate further research. Our results demonstrate the potential of deep learning to enhance the efficiency and accuracy of high-throughput respiratory organoid analysis for future therapeutic screening applications. Author summaryIn this study,we developed a semi-automated tool to analyze images of respiratory organoids—3D cell structures that mimic the human respiratory system. These organoids are vital for studying diseases like cystic fibrosis and testing potential drugs,but manually analyzing their images is time-consuming and prone to errors. Our tool uses artificial intelligence (AI) to quickly and accurately measure organoid size and shape from bright-field microscope images,eliminating the need for fluorescent dyes that can harm cells. We trained our AI model on a publicly shared dataset of 827 annotated organoid images,achieving high accuracy in detecting and quantifying organoids. When applied to cystic fibrosis research,the tool successfully measured differences in organoid swelling (forskolin-induced swelling - a key test for drug response) between healthy and patient-derived samples. By making our dataset and method openly available,we hope to support further research into respiratory diseases. Our work bridges the gap between complex lab techniques and practical applications,offering a faster,more reliable way to study human health and disease. View Publication

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways,including fatty acid oxidation,degradation of amino acids,and biosynthesis of ether lipids. Consequently,peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions,including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic,tissue-specific,and context-dependent nature of their biogenesis and function,live cell imaging of peroxisomes is essential for studying peroxisome regulation,as well as for the diagnosis of PBD-linked abnormalities. However,the peroxisomal imaging toolkit is lacking in many respects,with no reporters for substrate import,nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12,generating peroxisome-specific reagents,PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity,bright fluorescence in the red and far-red spectrum,and fast non-cytotoxic staining,making them ideal tools for live cell,whole organism,or tissue imaging of peroxisomes. Finally,we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines. The array of tools to image peroxisome regulation is still limited. Here,the authors develop improved fatty acid-based probes with high peroxisome specificity and bright fluorescence in the red/far-red spectrum,which makes them ideal to study peroxisomes in live cells and whole organisms. View Publication