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

Zoledronic acid,a third-generation bisphosphonate,has been shown to reduce cell migration,invasion,and metastasis. However,the effects of zoledronic acid on the epithelial-mesenchymal transition (EMT),a cellular process essential to the metastatic cascade,remain unclear. Therefore,the effects of zoledronic acid on EMT,using triple-negative breast cancer (TNBC) cells as a model system,were examined in more detail. Zoledronic acid treatment decreased the expression of mesenchymal markers,N-cadherin,Twist,and Snail,and subsequently upregulated expression of E-cadherin. Zoledronic acid also inhibited cell viability,induced cell-cycle arrest,and decreased the proliferative capacity of TNBC,suggesting that zoledronic acid inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype,the effects of zoledronic acid on self-renewal in TNBC were also studied. Treatment with zoledronic acid decreased expression of self-renewal proteins,BMI-1 and Oct-4,and both prevented and eliminated mammosphere formation. To understand the mechanism of these results,the effect of zoledronic acid on established EMT regulator NF-$$B was investigated. Zoledronic acid inhibited phosphorylation of RelA,the active subunit of NF-$$B,at serine 536 and modulated RelA subcellular localization. Treatment with zoledronic acid reduced RelA binding to the Twist promoter,providing a direct link between inactivation of NF-$$B signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased,correlating modulation of EMT to decreased self-renewal. On the basis of these results,it is proposed that through inactivation of NF-$$B,zoledronic acid reverses EMT,which leads to a decrease in self-renewal. View Publication

The viral infection of higher vertebrates elicits potent innate and adaptive host immunity. However,an excessive or inappropriate immune response also may lead to host pathology that often is more severe than the direct effects of viral replication. Therefore,several mechanisms exist that regulate the magnitude and class of the immune response. Here,we have examined the potential involvement of regulatory T (Treg) cells in limiting pathology induced by influenza A virus (IAV) infection. Using lymphocyte-deficient mice as hosts,we showed that Treg cell reconstitution resulted in a significant delay in weight loss and prolonged survival following infection. The adoptively transferred Treg cells did not affect the high rate of IAV replication in the lungs of lymphocyte-deficient hosts,and therefore their disease-ameliorating effect was mediated through the suppression of innate immune pathology. Mechanistically,Treg cells reduced the accumulation and altered the distribution of monocytes/macrophages in the lungs of IAV-infected hosts. This reduction in lung monocytosis was associated with a specific delay in monocyte chemotactic protein-2 (MCP-2) induction in the infected lungs. Nevertheless,Treg cells failed to prevent the eventual development of severe disease in lymphocyte-deficient hosts,which likely was caused by the ongoing IAV replication. Indeed,using T-cell-deficient mice,which mounted a T-cell-independent B cell response to IAV,we further showed that the combination of virus-neutralizing antibodies and transferred Treg cells led to the complete prevention of clinical disease following IAV infection. Taken together,these results suggested that innate immune pathology and virus-induced pathology are the two main contributors to pathogenesis during IAV infection. View Publication

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs),mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine,numerous umbilical cord blood banks have been established. In this study,we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs,MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs),slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48 h supernatant transferring,we successfully isolated MSCs which expressed CD13,CD44 and CD90 while CD34,CD45 and CD133 negative,had typical fibroblast-like shape,and was able to differentiate into adipocytes; EPCs which were CD34,and CD90 positive,CD13,CD44,CD45 and CD133 negative,adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium. View Publication

Targeting of cancer stem cells (CSC) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here,we show for the first time that G2.2,a sulfated nonsaccharide GAG mimetic (NSGM) of heparin hexasaccharide,selectively inhibits colonic CSCs in vivo G2.2-reduced CSCs (CD133+/CXCR4+,Dual hi) induced HT-29 and HCT 116 colon xenografts' growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared with vehicle-treated xenograft controls. In fact,G2.2 robustly inhibited CSCs' abundance (measured by levels of CSC markers,e.g.,CD133,DCMLK1,LGR5,and LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly,G2.2 selectively induced apoptosis in the Dual hi CSCs in vivo eluding to its CSC targeting effects. More importantly,G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions,blood coagulation profile,and ex vivo analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive in vitro,in vivo,and ex vivo mechanistic studies,we showed that G2.2's inhibition of CSC self-renewal was mediated through activation of p38$\alpha$,uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence,G2.2 represents a first-in-class (NSGM) anticancer agent to reduce colorectal CSCs. View Publication

Osteoarthritis (OA) is characterized by cartilage degradation and chronic joint inflammation. Mesenchymal stem cells (MSCs) have shown promising results in OA,but their mechanism of action is not fully understood. We hypothesize that MSCs polarize macrophages,which are strongly associated with joint inflammation to more homeostatic sub-types. We tracked ferumoxytol (Feraheme™,iron oxide nanoparticle)-labeled murine MSCs (Fe-MSCs) in murine OA joints,and quantified changes to joint inflammation and fibrosis. 10-week-old C57BL/6 male mice (n = 5/group) were induced to undergo osteoarthritis by destabilization of medical meniscus (DMM) or sham surgery. 3 weeks post-surgery,mice were injected intra-articularly with either fluorescent dye-(DiR) labeled or DiR-Fe-MSC or saline to yield 4 groups (n = 5 per group for each timepoint [1,2 and 4weeks]). 4 weeks after injection,mice were imaged by MRI,and scored for i) OARSI (Osteoarthritis Research Society International) to determine cartilage damage; ii) immunohistochemical changes in iNOS,CD206,F4/80 and Prussian Blue/Sca-1 to detect pro-inflammatory,homeostatic and total macrophages and ferumoxytol -labeled MSCs respectively,and iii) Masson's Trichrome to detect changes in fibrosis. Ferumoxytol-labeled MSCs persisted at greater levels in DMM vs. SHAM-knee joints. We observed no difference in OARSI scores between MSC and vehicle groups. Sca-1 and Prussian Blue co-staining confirmed the ferumoxytol label resides in MSCs,although some ferumoxytol label was detected in proximity to MSCs in macrophages,likely due to phagocytosis of apoptotic MSCs,increasing functionality of these macrophages through MSC efferocytosis. MRI hypertintensity scores related to fluid edema decreased in MSC-treated vs. control animals. For the first time,we show that MSC-treated mice had increased ratios of {\%}CD206+: {\%}F4/80+ (homeostatic macrophages) (p{\textless}0.05),and decreased ratios of {\%}iNOS+: {\%}F4/80+ macrophages (p{\textless}0.01),supporting our hypothesis that MSCs may modulate synovial inflammation. View Publication

ABSTRACTRAD50?interacting protein1 (RINT1) deficiency has been implicated in recurrent acute liver failure (RALF) triggered by fever or infections. RINT1,together with neuroblastoma amplified sequence and Zeste White 10 (forming the NRZ complex),localizes at the interface between the endoplasmic reticulum and Golgi apparatus,where it plays a key role in vesicular trafficking. However,the mechanisms by which RINT1 deficiency leads to RALF remain unclear. This study aimed to describe a woman with RALF harboring a homozygous missense mutation in RINT1. Induced pluripotent stem cells (iPSCs) were generated from the patient's mononuclear cells and differentiated into hepatocyte?like cells (HLCs). Upon exposure to high temperature (40°C),RINT1?deficient HLCs exhibited cellular damage characteristic of RALF. Furthermore,these cells also demonstrated heightened sensitivity to cytokines and viral mimetics while showing comparatively lower responsiveness to bacterial infection?related stimuli. Transcriptome sequencing revealed dysregulated gene expression associated with the extracellular matrix (ECM). Additionally,glycosaminoglycan disaccharide analysis revealed abnormal levels of chondroitin sulfate,heparan sulfate,and hyaluronan in RINT1?deficient HLCs. In conclusion,HLCs derived from RINT1?deficient iPSCs serve as a valuable model for investigating RINT1?related liver pathogenesis. The results suggest that cytokine responses,particularly those triggered by viral infections,play a central role in the development of RALF. Furthermore,ECM alterations provided novel insights into the potential role of RINT1 defects in RALF. RAD50?interacting protein1 (RINT1) deficiency causes recurrent acute liver failure (RALF) during fever or infections. To investigate its underlying mechanism,induced pluripotent stem cells were generated from a patient with RINT1 deficiency and differentiated into hepatocyte?like cells (HLCs). RINT1?deficient HLCs exhibited damage resembling RALF when exposed to high temperatures and were more susceptible to cytokines and viral mimetics than to bacterial infection?related factors. Furthermore,RNA?seq and disaccharide analyses revealed dysregulation of extracellular matrix?related genes and abnormalities in extracellular matrix levels. View Publication

ObjectivesMutations in Tissue Inhibitor of Metalloproteinases 3 (TIMP3) cause Sorsby's Fundus Dystrophy (SFD),a dominantly inherited,rare form of macular degeneration that results in vision loss. TIMP3 is synthesized primarily by retinal pigment epithelial (RPE) cells,which constitute the outer blood-retinal barrier. One major function of RPE is the synthesis and transport of vital nutrients,such as glucose,to the retina. Recently,metabolic dysfunction in RPE cells has emerged as an important contributing factor in retinal degenerations. We set out to determine if RPE metabolic dysfunction was contributing to SFD pathogenesis.MethodsQuantitative proteomics was conducted on RPE of mice expressing the S179C variant of TIMP3,known to be causative of SFD in humans. Proteins found to be differentially expressed (P < 0.05) were analyzed using statistical overrepresentation analysis to determine enriched pathways,processes,and protein classes using g:profiler and PANTHER Gene Ontology. We examined the effects of mutant TIMP3 on RPE metabolism using human ARPE-19 cells expressing mutant S179C TIMP3 and patient-derived induced pluripotent stem cell-derived RPE (iRPE) carrying the S204C TIMP3 mutation. RPE metabolism was directly probed using isotopic tracing coupled with GC/MS analysis. Steady state [U–13C6] glucose isotopic tracing was preliminarily conducted on S179C ARPE-19 followed by [U–13C6] glucose and [U–13C5] glutamine isotopic tracing in SFD iRPE cells.ResultsQuantitative proteomics and enrichment analysis conducted on RPE of mice expressing mutant S179C TIMP3 identified differentially expressed proteins that were enriched for metabolism-related pathways and processes. Notably these results highlighted dysregulated glycolysis and glucose metabolism. Stable isotope tracing experiments with [U–13C6] glucose demonstrated enhanced glucose utilization and glycolytic activity in S179C TIMP3 APRE-19 cells. Similarly,[U–13C6] glucose tracing in SFD iRPE revealed increased glucose contribution to glycolysis and the TCA cycle. Additionally,[U–13C5] glutamine tracing found evidence of altered malic enzyme activity.ConclusionsThis study provides important information on the dysregulation of RPE glucose metabolism in SFD and implicates a potential commonality with other retinal degenerative diseases,emphasizing RPE cellular metabolism as a therapeutic target. Highlights•SFD mice display alterations in proteins associated with metabolism.•SFD RPE cells have increased glycolytic activity and glucose contribution to the TCA cycle.•Glutamine contribution to energy metabolism is unaltered in SFD RPE cells however there is reduced malic enzyme activity.•SFD RPE cells display metabolic dysfunction potentially implicating metabolism as a viable therapeutic target. View Publication

Prime editing is a promising approach for correcting pathogenic variants,but its efficiency remains variable across genomic contexts. Here,we systematically evaluated 12 modifications of the PEmax system for correcting the CFTR F508del pathogenic variant that caused cystic fibrosis in patient-derived airway basal cells. We chose EXO1 and FEN1 nucleases to improve the original system. While all tested variants showed comparatively low efficiency in this AT-rich genomic region,4-FEN modification demonstrated significantly improved editing rates (up to 2.13 fold) compared to standard PEmax. Our results highlight two key findings: first,the persistent challenge of AT-rich target sequence correction even with optimized editors,and second,the performance of 4-FEN suggests its potential value for other genomic targets. View Publication

PURPOSE Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy,temozolomide. The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. EXPERIMENTAL DESIGN ZR2002 is a combi-molecule" designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs mesenchymal temozolomide-sensitive and resistant in vivo-derived GSC sublines and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. RESULTS ZR2002 induced submicromolar antiproliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR downstream Erk1/2 phosphorylation increased DNA strand breaks and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal temozolomide-resistant GSC line decreased EGFR Erk1/2 and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. CONCLUSIONS These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability blood-brain barrier permeability and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM." View Publication

Neural stem cell differentiation and the determination of lineage decision between neuronal and glial fates have important implications in the study of developmental,pathological,and regenerative processes. Although small molecule chemicals with the ability to control neural stem cell fate are considered extremely useful tools in this field,few were reported. AICAR is an adenosine analog and extensively used to activate AMP-activated protein kinase (AMPK),a metabolic fuel gauge" of the biological system. In the present study View Publication

MYH9 disorders such as May-Hegglin anomaly are characterized by macrothrombocytopenia and cytoplasmic granulocyte inclusion bodies that result from mutations in MYH9,the gene for nonmuscle myosin heavy chain-IIA (NMMHC-IIA). We examined the expression of mutant NMMHC-IIA polypeptide in peripheral blood cells from patients with MYH9 5770delG and 5818delG mutations. A specific antibody to mutant NMMHC-IIA (NT629) was raised against the abnormal carboxyl-terminal residues generated by 5818delG. NT629 reacted to recombinant 5818delG NMMHC-IIA but not to wild-type NMMHC-IIA,and did not recognize any cellular components of normal peripheral blood cells. Immunofluorescence and immunoblotting revealed that mutant NMMHC-IIA was present and sequestrated only in inclusion bodies within neutrophils,diffusely distributed throughout lymphocyte cytoplasm,sparsely localized on a diffuse cytoplasmic background in monocytes,and uniformly distributed at diminished levels only in large platelets. Mutant NMMHC-IIA did not translocate to lamellipodia in surface activated platelets. Wild-type NMMHC-IIA was homogeneously distributed among megakaryocytes derived from the peripheral blood CD34(+) cells of patients,but coarse mutant NMMHC-IIA was heterogeneously scattered without abnormal aggregates in the cytoplasm. We show the differential expression of mutant NMMHC-IIA and postulate that cell-specific regulation mechanisms function in MYH9 disorders. View Publication

Costimulatory signals are critical to T cell activation,but how their effects are mediated remains incompletely characterized. Here,we demonstrate that locally produced C5a and C3a anaphylatoxins interacting with their G protein-coupled receptors (GPCRs),C5aR and C3aR,on APCs and T cells both upstream and downstream of CD28 and CD40L signaling are integrally involved in T cell proliferation and differentiation. Disabling these interactions reduced MHC class II and costimulatory-molecule expression and dramatically diminished T cell responses. Importantly,impaired T cell activation by Cd80-/-Cd86-/- and Cd40-/- APCs was reconstituted by added C5a or C3a. C5aR and C3aR mediated their effects via PI-3 kinase-gamma-dependent AKT phosphorylation,providing a link between GPCR signaling,CD28 costimulation,and T cell survival. These local paracrine and autocrine interactions thus operate constitutively in naive T cells to maintain viability,and their amplification by cognate APC partners thus is critical to T cell costimulation. View Publication