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

Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells,whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3-/- pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues,whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability,DNA binding,and gene transcription. Moreover,NEUROG3 was highly unstable in the intestinal epithelium,explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships. View Publication

Naive human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation,but their mitochondrial regulators are poorly understood. Here we report that,proline dehydrogenase (PRODH),a mitochondria-localized proline metabolism enzyme,is dramatically upregulated in naive hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901,a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy,DNA damage,and apoptosis. Remarkably,MitoQ,a mitochondria-targeted antioxidant,effectively restored the pluripotency and proliferation of PRODH-knockdown naive hESCs,indicating that PRODH maintains naive pluripotency by preventing excessive ROS production. Concomitantly,PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus,we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production,and thereby safeguarding naive pluripotency of hESCs. Synopsis Downregulation of PRODH promotes oxidative phosphorylation and ROS production,which in turn impair pluripotency and proliferation of naive but not primed hESCs,revealing a crucial role of PRODH in safeguarding human naive pluripotency. PRODH is expressed in naive hESCs at a higher level compared to their primed counterparts.MEK inhibitor present in naive culture media upregulates PRODH by suppressing MYC.PRODH depletion boosts mtOXPHOS and ROS production in naive hESCs.PRODH promotes proteolytic degradation of the ETC complex components. Downregulation of PRODH promotes oxidative phosphorylation and ROS production,which in turn impair pluripotency and proliferation of naive but not primed hESCs,revealing a crucial role of PRODH in safeguarding human naive pluripotency. View Publication

Prion diseases,such as sporadic Creutzfeldt-Jakob Disease (sCJD),are neurodegenerative disorders caused by misfolding of the prion protein (PrP). The D178N mutation in the PrP gene causes Fatal Familial Insomnia (FFI). Here we show that both sCJD and FFI prions can infect human cerebral organoids with or without the D178N mutation,and that the resulting infection is dictated by the inoculating prion and not the host organoid genotype. View Publication

Nanoplastics (NPs) are emerging environmental pollutants that pose growing concerns due to their potential health risks. However,the effects of inhaled NP exposure during pregnancy on fetal brain development remain poorly understood. In this study,we investigated the impact of maternal exposure to polypropylene nanoplastics (PP-NPs) on fetal brain development and neurobehavioral outcomes in a mouse model and further explored its mechanism in human cerebral organoids. Maternal exposure to PP-NPs significantly impaired neuronal differentiation and proliferation in the fetal cortex. Neurobehavioral assessments revealed significant deficits in offspring following maternal exposure,including impaired spatial memory,reduced motor coordination,and heightened anxiety-like behavior. Furthermore,human brain organoids exposed to PP-NPs exhibited reduced growth and neuronal differentiation,with significant downregulation of key neuronal markers such as TUJ1,MAP2,and PAX6. Transcriptomic analysis identified alterations in gene expression,particularly in neuroactive ligand-receptor interaction pathway. Molecular docking and fluorescence co-localization analysis further suggested CYSLTR1 and PTH1R as key molecular targets of PP-NPs. These findings provide novel insights into the toxicological effects of NPs on the developing brain and emphasize the need for preventive measures to protect fetal neurodevelopment during pregnancy. The online version contains supplementary material available at 10.1186/s12951-025-03561-1. View Publication

This work focused on generating a three-dimensional (3D) in vitro dynamic model to study chronic lymphocytic leukemia (CLL) cell dissemination,homing,and mechanisms of therapy resistance. We used a gelatin-based,hard porous biomaterial as a support matrix to develop 3D tissue-like models of the human lymph node and bone marrow,which were matured inside bioreactors under dynamic perfusion of medium. Comparing static and dynamic cultures of these 3D constructs revealed that perfusion promoted a tissue-like internal organization of cells,characterized by the expression of specific functional markers and deposition of an intricate extracellular matrix protein network. Recirculation of CLL cells within the dynamic system led to changes in leukemic cell behavior and in the expression of key markers involved in tumor progression. These findings suggest that the model is well suited for investigating the pathophysiological mechanisms of CLL and potentially other hematological malignancies. View Publication

β-cell dysfunction and dedifferentiation towards an α-cell-like phenotype are hallmarks of type 2 diabetes. However,the cell subtypes involved in β-to-α-cell transition are unknown. Using single-cell and single-nucleus RNA-seq,RNA velocity,PAGA/cell trajectory inference,and gene commonality,we interrogated α-β-cell fate switching in human islets. We found five α-cell subclusters with distinct transcriptomes. PAGA analysis showed bifurcating cell trajectories in non-diabetic while unidirectional cell trajectories from β-to-α-cells in type 2 diabetes islets suggesting dedifferentiation towards α-cells. Ten genes comprised the common signature genes in trajectories towards α-cells. Among these,the α-cell gene SMOC1 was expressed in β-cells in type 2 diabetes. Enhanced SMOC1 expression in β-cells decreased insulin expression and secretion and increased β-cell dedifferentiation markers. Collectively,these studies reveal differences in α-β-cell trajectories in non-diabetes and type 2 diabetes human islets,identify signature genes for β-to-α-cell trajectories,and discover SMOC1 as an inducer of β-cell dysfunction and dedifferentiation. Subject terms: Cell signalling,Diabetes,Differentiation View Publication

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain,and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions,we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these,cysteine (at position 31) was highly (textgreater99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions,in contrast,the SC mutant was defective in both. Therefore,the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence,this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses. View Publication

Splitomicin is derived from beta-naphthol and is an inhibitor of Silent Information Regulator 2 (SIR2). Its naphthoic moiety might be responsible for its inhibitory effects on platelets. The major goal of our study was to examine possible mechanisms of action of splitomicin on platelet aggregation in order to promote development of a novel anti-platelet aggregation therapy for cardiovascular and cerebrovascular diseases. To study the inhibitory effects of splitomicin on platelet aggregation,we used washed human platelets,and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml),collagen (2 microg/ml),arachidonic acid (AA) (0.5 mM),U46619 (2 microM) or ADP (10 microM). Splitomicin inhibited platelet aggregation induced by thrombin,collagen,AA and U46619 with a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when splitomicin (150 microM) was combined with PGE1 (0.5 microM). It did not further increase cAMP when combined with IBMX. This data indicated that splitomicin increases cAMP by inhibiting activity of phosphodiestease. In addition,splitomicin (300 microM) attenuated intracellular Ca(++) mobilization,and production of thromboxane B2 (TXB2) in platelets that was induced by thrombin,collagen,AA or U46619. The inhibitory mechanism of splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca(++) mobilization,TXB2 formation and ATP release. View Publication

Induction of pluripotent stem cells from human fibroblasts has been achieved by the ectopic expression of two different sets of four genes. However,the mechanism of the pluripotent stem cell induction has not been elucidated. Here we identified a marked heterogeneity in colonies generated by the four-gene (Oct3/4,Sox2,c-Myc,and Klf4) transduction method in human neonatal skin-derived cells. The four-gene transduction gave a higher probability of induction for archetypal pluripotent stem cell marker genes (Nanog,TDGF,and Dnmt3b) than for marker genes that are less specific for pluripotent stem cells (CYP26A1 and TERT) in primary induction culture. This tendency may reflect the molecular mechanism underlying the induction of human skin-derived cells into pluripotent stem cells. Among the colonies induced by the four-gene transduction,small cells with a high nucleus-to-cytoplasm ratio could be established by repeated cloning. Subsequently established cell lines were similar to human embryonic stem cells as well as human induced pluripotent stem (iPS) cells derived from adult tissue in morphology,gene expression,long-term self-renewal ability,and teratoma formation. Genome-wide single-nucleotide polymorphism array analysis of the human iPS cell line indicates that the induction process did not induce DNA mutation. ?? 2008 Elsevier B.V. All rights reserved. View Publication

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) grown in engineered heart tissue (EHT) can be used for drug screening,disease modeling,and heart repair. However,the immaturity of hiPSC-CMs currently limits their use. Because mechanical loading increases during development and facilitates cardiac maturation,we hypothesized that afterload would promote maturation of EHTs. To test this we developed a system in which EHTs are suspended between a rigid post and a flexible one,whose resistance to contraction can be modulated by applying braces of varying length. These braces allow us to adjust afterload conditions over two orders of magnitude by increasing the flexible post resistance from 0.09 up to 9.2 mu$N/mu$m. After three weeks in culture,optical tracking of post deflections revealed that auxotonic twitch forces increased in correlation with the degree of afterload,whereas twitch velocities decreased with afterload. Consequently,the power and work of the EHTs were maximal under intermediate afterloads. When studied isometrically,the inotropy of EHTs increased with afterload up to an intermediate resistance (0.45 mu$N/mu$m) and then plateaued. Applied afterload increased sarcomere length,cardiomyocyte area and elongation,which are hallmarks of maturation. Furthermore,progressively increasing the level of afterload led to improved calcium handling,increased expression of several key markers of cardiac maturation,including a shift from fetal to adult ventricular myosin heavy chain isoforms. However,at the highest afterload condition,markers of pathological hypertrophy and fibrosis were also upregulated,although the bulk tissue stiffness remained the same for all levels of applied afterload tested. Together,our results indicate that application of moderate afterloads can substantially improve the maturation of hiPSC-CMs in EHTs,while high afterload conditions may mimic certain aspects of human cardiac pathology resulting from elevated mechanical overload. View Publication

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways,however,it remains unclear as to what extent these pathways can be exploited,either individually or in combination,in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro,but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways,we developed a high-density microbioreactor array (HDMA) - a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt,Hedgehog,IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation,inducing cell cycle activity marked by Ki67,and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration. View Publication

The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting,X-chromosome inactivation and genomic architecture. In this study,we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes,including NANOG,SOX2,cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation,but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs,CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors,such as Cohesin,differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly,the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology. View Publication