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

PURPOSE: Preclinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However,one of the challenges in screening anticancer therapies is the assessment of off-target human toxicity. There is a need for in vivo models that can simulate efficacy and toxicities of promising therapeutic regimens. For example,hematopoietic cells of human origin are particularly sensitive to a variety of chemotherapeutic regimens,but in vivo models to assess potential toxicities have not been developed. In this study,a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity. EXPERIMENTAL DESIGN: A proof-of-concept,temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing because it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD.Cg-Prkdc(scid)IL2rg(tm1Wjl)/Sz (NOD/SCID/γchain(null)),reconstituted with human hematopoietic cells,and the impact of treatment on human hematopoiesis was evaluated. RESULTS: The dose-intensive regimen resulted in significant decreases in growth of human glioblastoma xenografts. When this regimen was administered to mice containing humanized bone marrow,flow cytometric analyses indicated that the human bone marrow cells were significantly more sensitive to treatment than the murine bone marrow cells and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (progenitor,lymphoid,and myeloid). CONCLUSIONS: The humanized bone marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anticancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation. View Publication

BackgroundRecent studies highlight the critical role of microglia in neurodegenerative disorders,and emphasize the need for humanized models to accurately study microglial responses. Human-mouse microglia xenotransplantation models are a valuable platform for functional studies and for testing therapeutic approaches,yet currently those models are only available for academic research. This hampers their implementation for the development and testing of medication that targets human microglia.MethodsWe developed the hCSF1Bdes mouse line,which is suitable as a new transplantation model and available to be crossed to any disease model of interest. The hCSF1Bdes model created by CRISPR gene editing is RAG2 deficient and expresses human CSF1. Additionally,we crossed this model with two humanized App KI mice,the AppHu and the AppSAA. Flow cytometry,immunohistochemistry and bulk sequencing was used to study the response of microglia in the context of Alzheimer’s disease.ResultsOur results demonstrate the successful transplantation of iPSC-derived human microglia into the brains of hCSF1Bdes mice without triggering a NK-driven immune response. Furthermore,we confirmed the multipronged response of microglia in the context of Alzheimer’s disease. The hCSF1Bdes and the crosses with the Alzheimer’s disease knock-in model AppSAA and the humanized App knock-in control mice,AppHu are deposited with EMMA and fully accessible to the research community.ConclusionThe hCSF1Bdes mouse is available for both non-profit and for-profit organisations,facilitating the use of the xenotransplantation paradigm for human microglia to study complex human disease.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00823-2. View Publication

Adult hematopoietic stem cells (HSCs) are routinely used to reconstitute hematopoiesis after myeloablation; however,transplantation efficacy and multilineage reconstitution can be limited by inadequate HSC number,or poor homing,engraftment,or self-renewal. Here we report that mouse and human HSCs express prostaglandin E2 (PGE2) receptors,and that short-term ex vivo exposure of HSCs to PGE2 enhances their homing,survival,and proliferation,resulting in increased long-term repopulating cell (LTRC) and competitive repopulating unit (CRU) frequency. HSCs pulsed with PGE2 are more competitive,as determined by head-to-head comparison in a competitive transplantation model. Enhanced HSC frequency and competitive advantage is stable and maintained upon serial transplantation,with full multilineage reconstitution. PGE2 increases HSC CXCR4 mRNA and surface expression,enhances their migration to SDF-1 in vitro and homing to bone marrow in vivo,and stimulates HSC entry into and progression through cell cycle. In addition,PGE2 enhances HSC survival,associated with an increase in Survivin mRNA and protein expression and reduction in intracellular active caspase-3. Our results define novel mechanisms of action whereby PGE2 enhances HSC function and supports a strategy to use PGE2 to facilitate hematopoietic transplantation. View Publication

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy,but the overall metabolic properties of HSCs remain elusive. Using combined approaches,including single-cell RNA sequencing (RNA-seq),we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed,but not quiescent,HSCs relied readily on glycolysis. Notably,in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant. View Publication

Strategies to prevent organ transplant rejection whilst minimizing long-term immunosuppression are currently under intense investigation with regulatory T cells (Tregs) nearing clinical application. The clinical trial,ThRIL,recently commenced at King's College London,proposes to use Treg cell therapy to induce tolerance in liver transplant recipients,the success of which has the potential to revolutionize the management of these patients and enable a future of drug-free transplants. This is the first report of the manufacture of clinical grade Tregs from prospective liver transplant recipients via a CliniMACS-based GMP isolation technique and expanded using anti-CD3/CD28 beads,IL-2 and rapamycin. We report the enrichment of a pure,stable population of Tregs (textgreater95% CD4(+)CD25(+)FOXP3(+)),reaching adequate numbers for their clinical application. Our protocol proved successful in,influencing the expansion of superior functional Tregs,as compared to freshly isolated cells,whilst also preventing their conversion to Th17 cells under pro-inflammatory conditions. We conclude with the manufacture of the final Treg product in the clinical research facility (CRF),a prerequisite for the clinical application of these cells. The data presented in this manuscript together with the much-anticipated clinical results from ThRIL,will undoubtedly inform the improved management of the liver transplant recipient. View Publication

The establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism,genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs,the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells,which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation,metabolism,genetic network,and response to infection or other external stimuli. View Publication

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity,junctional complexes,integrin-dependent matrix adhesion,and E-cadherin-dependent cell-cell adhesion,all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures,programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies,their viability is significantly reduced. Here,we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase,downregulation of myosin heavy chain,and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain,suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. View Publication

Nano- and microscale topographical cues play critical roles in the induction and maintenance of various cellular functions,including morphology,adhesion,gene regulation,and communication. Recent studies indicate that structure and function at the heart tissue level is exquisitely sensitive to mechanical cues at the nano-scale as well as at the microscale level. Although fabrication methods exist for generating topographical features for cell culture,current techniques,especially those with nanoscale resolution,are typically complex,prohibitively expensive,and not accessible to most biology laboratories. Here,we present a tunable culture platform comprised of biomimetic wrinkles that simulate the heart's complex anisotropic and multiscale architecture for facile and robust cardiac cell alignment. We demonstrate the cellular and subcellular alignment of both neonatal mouse cardiomyocytes as well as those derived from human embryonic stem cells. By mimicking the fibrillar network of the extracellular matrix,this system enables monitoring of protein localization in real time and therefore the high-resolution study of phenotypic and physiologic responses to in-vivo like topographical cues. View Publication

Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth,while mechanical,enzymatic or chemical cell dissociation methods are used for cellular passaging. However,these methods are ill defined,thus introducing variability into the system,and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate,which support long-term human embryonic stem cell growth and pluripotency over a period of 2-6 months. The hydrogels permitted gentle,reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used,undefined biological substrates represent a flexible and scalable approach for improving the definition,efficacy and safety of human embryonic stem cell culture systems for research,industrial and clinical applications. View Publication

UNLABELLED: Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease-specific iPSCs have been generated,there has been limited progress in iPSC-based drug screening/discovery for liver diseases,and the low gene-targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency,for which there is currently no drug or gene therapy available,we established a platform to discover new drug candidates and correct disease-causing mutation with a high efficiency. A high-throughput format screening assay,based on our hepatic differentiation protocol,was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients,we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large-scale drug screening,five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition,using the recently developed transcription activator-like effector nuclease technology,we achieved high gene-targeting efficiency in AAT-deficiency patient iPSCs with 25%-33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications.backslashnbackslashnCONCLUSIONS: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs,both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases. View Publication

The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population,broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study,we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach,we identified two endoderm-specific antibodies,HDE1 and HDE2,which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential,whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination,the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line. View Publication