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

BACKGROUND Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity,despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive,only cataloging heterogeneity at one point in time,and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity,and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS We introduce the programmable Polaris microfluidic lab-on-chip for single-cell sequencing,which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1,such as an altered oxidative stress response,have a large paracrine signaling component. Furthermore,we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation,both associated with the oxidative stress response and altered proteostasis. Interestingly,SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'),with no known co-regulation. CONCLUSION As single-cell methods continue to mature,so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture,live-cell imaging,and single-cell sequencing,we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages,for example,react to inflammation and form treatment resistant HIV reservoirs. View Publication

The use of an interleukin beta$ antibody is currently being investigated in the clinic for the treatment of acne,a dermatological disorder affecting 650M persons globally. Inhibiting the protease responsible for the cleavage of inactive pro-IL1beta$ into active IL-1beta$,caspase-1,could be an alternative small molecule approach. This report describes the discovery of uracil 20,a potent (38 nM in THP1 cells assay) caspase-1 inhibitor for the topical treatment of inflammatory acne. The uracil series was designed according to a published caspase-1 pharmacophore model involving a reactive warhead in P1 for covalent reversible inhibition and an aryl moiety in P4 for selectivity against the apoptotic caspases. Reversibility was assessed in an enzymatic dilution assay or by using different substrate concentrations. In addition to classical structure-activity-relationship exploration,topical administration challenges such as phototoxicity,organic and aqueous solubility,chemical stability in solution,and skin metabolic stability are discussed and successfully resolved. View Publication

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis,and gain-of-function mutations of the receptor are frequently seen in several malignancies,including acute myeloid leukemia,gastrointestinal stromal tumors,and testicular carcinoma. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V),leading to constitutive activation of the receptor. In this study,we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as of activation of downstream signaling pathways including receptor ubiquitination and the Ras/Mek/Erk pathway. Our data demonstrate that,unlike wild-type c-Kit,the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition,we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. We further present evidence that,in contrast to wild-type c-Kit,Src family kinases are dispensable for c-Kit/D816V cell survival,proliferation,and colony formation. Taken together,we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival,thereby contributing to the transforming potential of c-Kit/D816V. View Publication

Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson's disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1),a GPI-anchored cell surface molecule,specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a,a bona-fide mesDA lineage marker,during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development,as well as in ESC-derived mesDA lineage. FolR1(+) neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3,whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons. View Publication

Using a high-throughput chemical screen,we identified two small molecules that enhance the survival of human embryonic stem cells (hESCs). By characterizing their mechanisms of action,we discovered an essential role of E-cadherin signaling for ESC survival. Specifically,we showed that the primary cause of hESC death following enzymatic dissociation comes from an irreparable disruption of E-cadherin signaling,which then leads to a fatal perturbation of integrin signaling. Furthermore,we found that stability of E-cadherin and the resulting survival of ESCs were controlled by specific growth factor signaling. Finally,we generated mESC-like hESCs by culturing them in mESC conditions. And these converted hESCs rely more on E-cadherin signaling and significantly less on integrin signaling. Our data suggest that differential usage of cell adhesion systems by ESCs to maintain self-renewal may explain their profound differences in terms of morphology,growth factor requirement,and sensitivity to enzymatic cell dissociation. View Publication

Innate Defense Regulators (IDRs) are short synthetic peptides that target the host innate immune system via an intracellular adaptor protein which functions at key signaling nodes. In this work,further details of the mechanism of action of IDRs have been discovered. The studies reported here show that the lead clinical IDR,SGX94,has broad-spectrum activity against Gram-negative and Gram-positive bacterial infections caused by intracellular or extracellular bacteria and also complements the actions of standard of care antibiotics. Based on in vivo and primary cell culture studies,this activity is shown to result from the primary action of SGX94 on tissue-resident cells and subsequent secondary signaling to activate myeloid-derived cells,resulting in enhanced bacterial clearance and increased survival. Data from non-clinical and clinical studies also show that SGX94 treatment modulates pro-inflammatory and anti-inflammatory cytokine levels,thereby mitigating the deleterious inflammatory consequences of innate immune activation. Since they act through host pathways to provide both broad-spectrum anti-infective capability as well as control of inflammation,IDRs are unlikely to be impacted by resistance mechanisms and offer potential clinical advantages in the fight against emerging and antibiotic resistant bacterial infections. View Publication

5-Azacytidine was first synthesized almost 40 years ago. It was demonstrated to have a wide range of anti-metabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. However,because of 5-azacytidine's general toxicity,other nucleoside analogs were favored as therapeutics. The finding that 5-azacytidine was incorporated into DNA and that,when present in DNA,it inhibited DNA methylation,led to widespread use of 5-azacytidine and 5-aza-2'-deoxycytidine (Decitabine) to demonstrate the correlation between loss of methylation in specific gene regions and activation of the associated genes. There is now a revived interest in the use of Decitabine as a therapeutic agent for cancers in which epigenetic silencing of critical regulatory genes has occurred. Here,the current status of our understanding of the mechanism(s) by which 5-azacytosine residues in DNA inhibit DNA methylation is reviewed with an emphasis on the interactions of these residues with bacterial and mammalian DNA (cytosine-C5) methyltransferases. The implications of these mechanistic studies for development of less toxic inhibitors of DNA methylation are discussed. View Publication

There is evidence that neutrophil production is a balance between the proliferative action of granulocyte-colony-stimulating factor (G-CSF) and a negative feedback from mature neutrophils (the chalone). Two neutrophil serine proteases have been implicated in granulopoietic regulation: pro-proteinase 3 inhibits granulocyte macrophage-colony-forming unit (CFU-GM) growth,and elastase mutations cause cyclic and congenital neutropenia. We further studied the action of the neutrophil serine proteases (proteinase 3,elastase,azurocidin,and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM in methylcellulose culture. In serum-free suspension cultures of CD34+ cells,elastase completely abrogated the proliferation induced by G-CSF but not that of GM-CSF or stem cell factor (SCF). The blocking effect of elastase was prevented by inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride (PMSF) or heat treatment. When exposed to enzymatically active elastase,G-CSF,but not GM-CSF or SCF,was rapidly cleaved and rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of G-CSF. View Publication

Serotonin receptor affinity and photelectron spectral data were obtained on a number of substituted N,N-dimethyltryptamines. Evidence is presented that electron-donating substituents in the 5-position lead to enhanced behavioral disruption activity and serotonin receptor affinity as compared to unsubstituted N,N-dimethyltryptamine and analogues substituted in the 4- or 6-position. Some correlation was found between ionization potentials and behavioral activity,which may have implications concerning the mechanism of receptor binding. View Publication