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

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

West Nile virus (WNV),an arbovirus of emerging global interest,can cause neuroinvasive disease in humans. Currently,no protective vaccine or specific treatment is available for human WNV encephalitis. The virus induces neuronal cell death,while astrocytes and microglia cells are suspected to contribute to WNV pathology. Hence,understanding their role is crucial for future treatment approaches. In this study,we establish a WNV encephalitis model using human cerebral organoids,generated with male iPSCs. Infection results in heterogeneous kinetics with an early strong replication potentially leading to viral clearance,while a late peak was associated with more long-term infection. Viral foci are seen in cortical-like areas,rich in neurons and astrocytes,however void of microglia. Pro-inflammatory cytokines (IL-6,TNF-α,IL-18),chemokines (CXCL10,CCL17,CX3CL1,CCL2) and biomarkers (IL-1RA,sTREM-1,sRAGE,BDNF) are increasingly released. Conclusively,human cerebral organoids make suitable WNV encephalitis models with valuable properties to study acute and long-term infection. West Nile virus (WNV) can cause neuroinvasive disease. Here the authors develop a human cerebral organoid model for WNV infection and find heterogeneous viral kinetics with viral foci in neuron- and astrocyte-rich areas devoid of microglia,as well as increased release of cytokines and other biomarkers. 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

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

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis,biological activity,and further profiling of these compounds are described. This work resulted in the discovery of 17,GDC-0941,which is a potent,selective,orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer. View Publication

Imatinib mesylate has shown remarkable efficacy in the treatment of patients in the chronic phase of chronic myeloid leukemia. However,despite an overall significant hematological and cytogenetic response,imatinib therapy may favor the emergence of drug-resistant clones,ultimately leading to relapse. Some imatinib resistance mechanisms had not been fully elucidated yet. In this study we used sensitive and resistant sublines from a Bcr-Abl positive cell line to investigate the putative involvement of telomerase in the promotion of imatinib resistance. We showed that sensitivity to imatinib can be partly restored in imatinib-resistant cells by targeting telomerase expression,either by the introduction of a dominant-negative form of the catalytic protein subunit of the telomerase (hTERT) or by the treatment with all-trans-retinoic acid,a clinically used drug. Furthermore,we showed that hTERT overexpression favors the development of imatinib resistance through both its antiapoptotic and telomere maintenance functions. Therefore,combining antitelomerase strategies to imatinib treatment at the beginning of the treatment should be promoted to reduce the risk of imatinib resistance development and increase the probability of eradicating the disease. View Publication

Coinfection of HIV-1 patients with Plasmodium falciparum,the etiological agent of malaria,results in a raise of viral load and an acceleration of disease progression. The primary objective of this study was to investigate whether the malarial pigment hemozoin (HZ),a heme by-product of hemoglobin digestion by malaria parasites,can affect HIV-1 transmission by monocytes-derived dendritic cells (DCs) to CD4(+) T cells when HZ is initially internalized in monocytes before their differentiation in DCs. We demonstrate in this study that HZ treatment during the differentiation process induces an intermediate maturation phenotype when compared with immature and fully mature DCs. Furthermore,the DC-mediated transfer of HIV-1 is enhanced in presence of HZ,a phenomenon that may be linked with the capacity of HZ-loaded cells to interact and activate CD4(+) T cells. Altogether our findings suggest a new mechanism that could partially explain the increased HIV-1 virus production during a coinfection with P. falciparum. Understanding the multifaceted interactions between P. falciparum and HIV-1 is an important challenge that could lead to the development of new treatment strategies. View Publication

Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. Recently,a new pluripotent population of umbilical cord blood (UCB) cells,unrestricted somatic stem cells (USSCs),with intrinsic and directable potential to develop into mesodermal,endodermal,and ectodermal fates,has been identified. In this study,we evaluated the capacity of ex vivo expanded USSCs to influence the homing of UCB-derived CD34(+) cells into the marrow and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. USSCs induced a significant enhancement of CD34(+) cell homing to both bone marrow and spleen (2.2 +/- 0.3- and 2.4 +/- 0.6-fold,respectively; p textless .05),with a magnitude similar to that induced by USSCs that had been thawed prior to transplantation. The effect of USSCs was dose-dependent and detectable at USSC:CD34(+) ratios of 1:1 and above. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells,as similar enhancement was observed for both early-passage (passage 5 [p5]) and late-passage (p10) USSCs. The homing effect of USSCs was also reflected in an increased proportion of NOD/SCID mice exhibiting significant human cell engraftment 6 weeks after transplantation,with a similar distribution of myeloid and lymphoid components. USSCs enhanced the homing of cellular products of ex vivo expanded UCB lineage-negative (lin(-)) cells,generated in 14-day cultures by Selective Amplification. The relative proportion of homing CD34(+) cells within the culture-expanded cell population was unaltered by USSC cotransplantation. Production of stromal-derived factor-1 (SDF-1) by USSCs was detected by both gene expression and protein released into culture media of these cells. Knockdown of SDF-1 production by USSCs using lentiviral-SiRNA led to a significant (p textless .05) reduction in USSC-mediated enhancement of CD34(+) homing. Our findings thus suggest a clinical potential for using USSCs in facilitating homing and engraftment for cord blood transplant recipients. View Publication