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

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However,HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C,in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29,CD73,CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2),OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also,the cells showed high expression of MSC markers CD29,CD73,CD44 and CD105,but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion,HUCB is a good source of MSC using this new technique. View Publication

The fabrication of high-density polymer microarray is described,allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance. View Publication

The angiotensin-converting enzyme 2 (ACE2) is a primary cell surface viral binding receptor for SARS-CoV-2,so finding new regulatory molecules to modulate ACE2 expression levels is a promising strategy against COVID-19. In the current study,we utilized islet organoids derived from human embryonic stem cells (hESCs),animal models and COVID-19 patients to discover that fibroblast growth factor 7 (FGF7) enhances ACE2 expression within the islets,facilitating SARS-CoV-2 infection and resulting in impaired insulin secretion. Using hESC-derived islet organoids,we demonstrated that FGF7 interacts with FGF receptor 2 (FGFR2) and FGFR1 to upregulate ACE2 expression predominantly in ? cells. This upregulation increases both insulin secretion and susceptibility of ? cells to SARS-CoV-2 infection. Inhibiting FGFR counteracts the FGF7-induced ACE2 upregulation,subsequently reducing viral infection and replication in the islets. Furthermore,retrospective clinical data revealed that diabetic patients with severe COVID-19 symptoms exhibited elevated serum FGF7 levels compared to those with mild symptoms. Finally,animal experiments indicated that SARS-CoV-2 infection increased pancreatic FGF7 levels,resulting in a reduction of insulin concentrations in situ. Taken together,our research offers a potential regulatory strategy for ACE2 by controlling FGF7,thereby protecting islets from SARS-CoV-2 infection and preventing the progression of diabetes in the context of COVID-19. View Publication

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by the selective and progressive death of motor neurons (MNs). Understanding the genetic and molecular factors influencing ALS survival is crucial for disease management and therapeutics. In this study,we introduce a deep learning-powered genetic analysis framework to link rare noncoding genetic variants to ALS survival. Using data from human induced pluripotent stem cell (iPSC)-derived MNs,this method prioritizes functional noncoding variants using deep learning,links cis-regulatory elements (CREs) to target genes using epigenomics data,and integrates these data through gene-level burden tests to identify survival-modifying variants,CREs,and genes. We apply this approach to analyze 6,715 ALS genomes,and pinpoint four novel rare noncoding variants associated with survival,including chr7:76,009,472:C>T linked to CCDC146. CRISPR-Cas9 editing of this variant increases CCDC146 expression in iPSC-derived MNs and exacerbates ALS-specific phenotypes,including TDP-43 mislocalization. Suppressing CCDC146 with an antisense oligonucleotide (ASO),showing no toxicity,completely rescues ALS-associated survival defects in neurons derived from sporadic ALS patients and from carriers of the ALS-associated G4C2-repeat expansion within C9ORF72. ASO targeting of CCDC146 may be a broadly effective therapeutic approach for ALS. Our framework provides a generic and powerful approach for studying noncoding genetics of complex human diseases. View Publication

Creation of disease models utilizing hiPSCs in combination with CRISPR/Cas9 gene editing enable mechanistic insights into differential pharmacological responses. This allows translation of efficacy and safety findings from a healthy to a diseased state and provides a means to predict clinical outcome sooner during drug discovery. Calcium handling disturbances including reduced expression levels of the type 2 ryanodine receptor (RYR2) are linked to cardiac dysfunction; here we have created a RYR2 deficient human cardiomyocyte model that mimics some aspects of heart failure. RYR2 deficient cardiomyocytes show differential pharmacological responses to L-type channel calcium inhibitors. Phenotypic and proteomic characterization reveal novel molecular insights with altered expression of structural proteins including CSRP3,SLMAP,and metabolic changes including upregulation of the pentose phosphate pathway and increased sensitivity to redox alterations. This genetically engineered in vitro cardiovascular model of RYR2 deficiency supports the study of pharmacological responses in the context of calcium handling and metabolic dysfunction enabling translation of drug responses from healthy to perturbed cellular states. 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

MYCN amplification without concurrent RB1 mutations characterizes a rare yet highly aggressive subtype of retinoblastoma; however,its precise developmental origins and therapeutic vulnerabilities remain incompletely understood. Here,we modeled this subtype by lentiviral-mediated MYCN overexpression in human pluripotent stem cell-derived retinal organoids,revealing a discrete developmental window (days 70–120) during which retinal progenitors showed heightened susceptibility to transformation. Tumors arising in this period exhibited robust proliferation,expressed SOX2,and lacked CRX,consistent with origin from primitive retinal progenitors. MYCN-overexpressing organoids generated stable cell lines that reproducibly gave rise to MYCN-driven tumors when xenografted into immunodeficient mice. Transcriptomic profiling demonstrated that MYCN-overexpressing organoids closely recapitulated molecular features of patient-derived MYCN-amplified retinoblastomas,particularly through activation of MYC/E2F and mTORC1 signaling pathways. Pharmacological screening further identified distinct therapeutic vulnerabilities,demonstrating distinct subtype-specific sensitivity of MYCN-driven cells to transcriptional inhibitors (THZ1,Flavopiridol) and the cell-cycle inhibitor Volasertib,indicative of a unique oncogene-addicted state compared to RB1-deficient retinoblastoma cells. Collectively,our study elucidates the developmental and molecular mechanisms underpinning MYCN-driven retinoblastoma,establishes a robust and clinically relevant human retinal organoid platform,and highlights targeted transcriptional inhibition as a promising therapeutic approach for this aggressive pediatric cancer subtype. View Publication

Solar ultraviolet (sUV) exposure is known to cause skin damage. However,the pathological mechanisms of sUV on hair follicles have not been extensively explored. Here,we established a model of sUV-exposed skin and its appendages using human induced pluripotent stem cell-derived skin organoids with planar morphology containing hair follicles. Our model closely recapitulated several symptoms of photodamage,including skin barrier disruption,extracellular matrix degradation,and inflammatory response. Specifically,sUV induced structural damage and catagenic transition in hair follicles. As a potential therapeutic agent for hair follicles,we applied exosomes isolated from human umbilical cord blood-derived mesenchymal stem cells to sUV-exposed organoids. As a result,exosomes effectively alleviated inflammatory responses by inhibiting NF-κB activation,thereby suppressing structural damage and promoting hair follicle regeneration. Ultimately,our model provided a valuable platform to mimic skin diseases,particularly those involving hair follicles,and to evaluate the efficacy and underlying mechanisms of potential therapeutics. View Publication

Drugs work by binding to a specific 3D structure on a protein. Drug discovery has historically been driven by prior knowledge of function,either of a protein or chemical. This knowledge of function then drives investigations to probe chemical/protein interactions. We undertook a different approach. We first identified unique 3D structures,agnostic of function,and investigated whether they could lead us to innovative therapeutics. Using a synchrotron-based X-ray source,we first determined high-resolution structures of hundreds of proteins. With a supercomputer running analytical programs created by us,we identified novel 3D structures and screened for chemicals binding them. We then tested their ability to inhibit cancer growth without damaging normal cells. We identified a potent inhibitor of a deadly cancer,melanoma. It was not toxic to normal cells even at 2100-fold higher doses. It worked by inducing anoikis,a fundamental process of known importance for cancer. Therapeutics that selectively induce anoikis are needed. In summary,we demonstrate the power of using a 3D protein structure as the starting point to discover new biology and drugs. Drug discovery historically starts with an established function,either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function,we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds,we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures,we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast,prostate,colon,or lung cancer,and for toxicity on normal human bone marrow stem cells,both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound,Dxr2-017,exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays,Dxr2-017 had an IC50 of 12 nM against melanoma cells,while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis,a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable. View Publication

The ε4 isoform of apolipoprotein E (ApoE) is the most significant genetic risk factor for Alzheimer’s disease. Glial cells are the main source of ApoE in the brain,and in microglia,the ε4 isoform of ApoE has been shown to impair mitochondrial metabolism and the uptake of lipids and Aβ42. However,whether the ε4 isoform alters autophagy or lysosomal activity in microglia in basal and inflammatory conditions is unknown. Altogether,microglia-like cells (iMGs) from eight APOE 3/3 and six APOE 4/4 human induced pluripotent stem cell (iPSC) lines were used in this study. The responses of iMGs to Aβ42,LPS and IFNγ were studied by metabolomics,proteomics,and functional assays. Here,we demonstrate that iMGs with the APOE 4/4 genotype exhibit reduced basal pinocytosis levels compared to APOE 3/3 iMGs. Inflammatory stimulation with a combination of LPS and IFNγ or Aβ42 induced PI3K/AKT/mTORC signaling pathway,increased pinocytosis,and blocked autophagic flux,leading to the accumulation of sequestosome 1 (p62) in both APOE 4/4 and APOE 3/3 iMGs. Exposure to Aβ42 furthermore caused lysosomal membrane permeabilization,which was significantly stronger in APOE 4/4 iMGs and positively correlated with the secretion of the proinflammatory chemokine IL-8. Metabolomics analysis indicated a dysregulation in amino acid metabolism,primarily L-glutamine,in APOE 4/4 iMGs. Overall,our results suggest that inflammation-induced metabolic reprogramming places lysosomes under substantial stress. Lysosomal stress is more detrimental in APOE 4/4 microglia,which exhibit endo-lysosomal defects. The online version contains supplementary material available at 10.1186/s12974-025-03470-y. View Publication

Murine models of myeloid neoplasia show how leukemia infiltration alters the hematopoietic stem cell (HSC) niche to reinforce malignancy at the expense of healthy hematopoiesis. However,little is known about the bone marrow architecture in humans and its impact on clinical outcome. Here,we dissect the bone marrow niche in patients with acute myeloid leukemia (AML) at first diagnosis. We combined immunohistochemical stainings with global gene expression analyses from these AML patients and correlated them with clinical features. Mesenchymal stem and progenitor cells (MSPCs) lost quiescence and significantly expanded in the bone marrow of AML patients. Strikingly,their HSC- and niche-regulating capacities were impaired with significant inhibition of osteogenesis and bone formation in a cell contact-dependent manner through inhibition of cytoplasmic $\beta$-catenin. Assessment of bone metabolism by quantifying peripheral blood osteocalcin levels revealed 30{\%} lower expression in AML patients at first diagnosis than in non-leukemic donors. Furthermore,patients with osteocalcin levels ≤11 ng/mL showed inferior overall survival with a 1-year survival rate of 38.7{\%} whereas patients with higher osteocalcin levels reached a survival rate of 66.8{\%}. These novel insights into the human AML bone marrow microenvironment help translate findings from preclinical models and detect new targets which might pave the way for niche-targeted therapies in AML patients. View Publication

Neuronal differentiation requires extensive metabolic remodeling to support increased energetic and biosynthetic demands. Here,we present an integrated multi-omics and functional characterization of metabolic transitions during early differentiation of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons using doxycycline-inducible overexpression of neurogenin-2 (NGN2). We analyzed parental iPSCs and induced neurons (iNs) at days 7 and 14 of differentiation,integrating gene expression profiling,label-free quantitative proteomics,high-resolution respirometry,fluorescence lifetime imaging microscopy (FLIM),and 13C₆-glucose metabolic flux analysis. Our data reveal progressive metabolic remodeling associated with neuronal maturation,including enhanced oxidative phosphorylation,increased mitochondrial content,and respiratory capacity. Proteomic analyses showed upregulation of mitochondrial and antioxidant pathways,while FLIM indicated a progressive increase in enzyme-bound NAD(P)H,consistent with a shift toward oxidative metabolism. Notably,13C₆-glucose tracing revealed delayed labeling of the intracellular pool of fully labeled glucose and tricarboxylic acid cycle metabolites,together with enhanced labeling of pentose phosphate pathway intermediates and glutathione in iNs,indicating a shift toward biosynthetic and antioxidant glucose utilization during differentiation. Despite this enhancement in mitochondrial function,differentiated neurons maintained glycolytic activity,suggesting metabolic flexibility. Our results define the first week of differentiation as a critical window of metabolic specialization and establish NGN2-iPSC-derived cortical neurons as a versatile and well-characterized model system for investigating bioenergetic remodeling during early human neurodevelopment. It provides a robust foundation for mechanistic insights and high-throughput evaluation of metabolic pathways relevant to human disease. View Publication