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

BACKGROUND: Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds.backslashnbackslashnMETHODS AND RESULTS: Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome,familial hypertrophic cardiomyopathy,and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome,hypertrophic cardiomyopathy,and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs,but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations.backslashnbackslashnCONCLUSIONS: We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects,long QT syndrome,hypertrophic cardiomyopathy,and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays. View Publication

Cancer-targeting alkylphosphocholine (APC) analogs are being clinically developed for diagnostic imaging,intraoperative visualization,and therapeutic applications. These APC analogs derived from chemically-synthesized phospholipid ethers were identified and optimized for cancer-targeting specificity using extensive structure-activity studies. While they strongly label human brain cancers associated with disrupted blood-brain barriers (BBB),APC permeability across intact BBB remains unknown. Three of our APC analogs,CLR1404 (PET radiotracer),CLR1501 (green fluorescence),and CLR1502 (near infrared fluorescence),were tested for permeability across a BBB model composed of human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system has reproducibly consistent high barrier integrity marked by high transendothelial electrical resistance (TEERtextgreater1500 Ω-cm(2)) and functional expression of drug efflux transporters. Our radioiodinated and fluorescent APC analogs demonstrated fairly low permeability across the iPSC-BMEC (35±5.7 (CLR1404),54±3.2 (CLR1501),and 26±4.9 (CLR1502) x10(-5) cm/min) compared with BBB-impermeable sucrose (13±2.5) and BBB-permeable diazepam (170±29). Only our fluorescent APC analogs (CLR1501,CLR1502) underwent BCRP and MRP polarized drug efflux transport in the brain-to-blood direction of the BBB model and this efflux can be specifically blocked with pharmacological inhibition. None of our tested APC analogs appeared to undergo substantial P-gp transport. Limited permeability of our APC analogs across an intact BBB into normal brain likely contributes to the high tumor to background ratios observed in initial human trials. Moreover,addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP,and may affect fluorescence-guided applications. Overall,the characterization of APC analog permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents. View Publication

Spinocerebellar ataxia type3 (SCA3) is an autosomal dominant neurodegenerative disorder. Human embryonic stem cell line chHES-472 was derived from abnormal embryo donated by SCA3 patient after preimplantation genetic diagnosis (PGD) treatment. This cell line had a normal karyotype and retained the disease-causing mutant in ATXN3 gene. Characteristic tests proved that the embryonic stem cell line presented typical markers of pluripotency and had the capability to form the three germlayers in vivo. View Publication

Capsular polysaccharides are common virulence factors of extracellular,but not intracellular bacterial pathogens,due to the antiphagocytic properties of these surface structures. It is therefore paradoxical that Salmonella enterica subspecies enterica serovar Typhi,an intracellular pathogen,synthesizes a virulence-associated (Vi) capsule,which exhibits antiphagocytic properties. Here,we show that the Vi capsular polysaccharide has different functions when S. Typhi interacts with distinct subsets of host phagocytes. The Vi capsular polysaccharide allowed S. Typhi to selectively evade phagocytosis by human neutrophils while promoting human macrophage phagocytosis. A screen of C-type lectin receptors identified human DC-SIGN as the receptor involved in macrophage binding and phagocytosis of capsulated S. Typhi. Consistent with the anti-inflammatory activity of DC-SIGN,purified Vi capsular polysaccharide reduced inflammatory responses in macrophages. These data suggest that binding of the human C-type lectin receptor DC-SIGN by the Vi capsular polysaccharide contributes to the pathogenesis of typhoid fever. IMPORTANCE Salmonella enterica subspecies enterica serovar Typhi is the causative agent of typhoid fever. The recent emergence of S. Typhi strains which are resistant to antibiotic therapy highlights the importance of vaccination in managing typhoid fever. The virulence-associated (Vi) capsular polysaccharide is an effective vaccine against typhoid fever,but the role the capsule plays during pathogenesis remains incompletely understood. Here,we identify the human C-type lectin receptor DC-SIGN as the receptor for the Vi capsular polysaccharide. Binding of capsulated S. Typhi to DC-SIGN resulted in phagocytosis of the pathogen by macrophages and induction of an anti-inflammatory cytokine response. Thus,the interaction of the Vi capsular polysaccharide with human DC-SIGN contributes to the pathogenesis of typhoid fever and should be further investigated in the context of vaccine development. View Publication

Phosphoinositide 3-kinase (PI3K) p110$\delta$ signalling negatively regulates the production of mouse IgE. However,there are disparities between the mouse and human IgE biology,and the role of PI3K p110$\delta$ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110$\delta$ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114,a small molecule inhibitor of PI3K p110$\delta$. Using FACS,RT-PCR and ELISA we examined the effect of PI3K p110$\delta$ inhibition on IgE production and determined the mechanisms involved. Unlike in mice,we observed that PI3K p110$\delta$ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However,the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110$\delta$ inhibition. The expression levels of AID,$\epsilon$ and $\gamma$1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However,we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40,specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition,PI3K p110$\delta$ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion,PI3K p110$\delta$ signalling is required for the production of human IgE,which makes it a pharmacological target for the treatment of allergic disease. View Publication

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response,we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes on the basis of their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus,with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of advanced antibody discovery platforms. View Publication

Human induced pluripotent stem cells (hiPSCs),like embryonic stem cells,are under intense investigation for novel approaches to model disease and for regenerative therapies. Here,we describe the derivation and characterization of hiPSCs from a variety of sources and show that,irrespective of origin or method of reprogramming,hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium,CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage,we were able to demonstrate by single cell analysis (flow cytometry),that hiPSC-derived erythroblasts express alpha globin as previously described,and that a sub-population of these erythroblasts also express haemoglobin F (HbF),indicative of fetal definitive erythropoiesis. More notably however,we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner,but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover,the HbA expressing erythroblast population could be greatly enhanced (44textperiodcentered0 ± 6textperiodcentered04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine. View Publication

In pluripotent stem cell differentiation,embryoid bodies (EBs) provide a three-dimensional [3D] multicellular precursor in lineage specification. The internal structure of EBs is not well characterized yet is predicted to be an important parameter to differentiation. Here,we use custom SU-8 molds to generate transparent lithography-templated arrays of polydimethylsiloxane (LTA-PDMS) for high throughput analysis of human embryonic stem cell (hESC) EB formation and internal architecture. EBs formed in 200 and 500 $$m diameter microarray wells by use of single cells,2D clusters,or 3D early aggregates were compared. We observe that 200 $$m EBs are monocystic versus 500 $$m multicystic EBs that contain macro,meso and microsized cysts. In adherent differentiation of 500 $$m EBs,the multicystic character impairs the 3D to 2D transition creating non-uniform monolayers. Our findings reveal that EB core structure has a size-dependent character that influences its architecture and cell population uniformity during early differentiation. View Publication

OBJECTIVE The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors,such as 5-aza-2'-deoxycytidine,can reverse p15INK4B promoter methylation,but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies. METHODS We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples. RESULTS Zebularine treatment of AML193,which has a densely methylated p15INK4B promoter,results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation,arrests cells at G(2)/M,and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis. CONCLUSION Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies. View Publication

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel,a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here,we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin,fibronectin,and Matrigel but poorly to laminin + entactin and collagen IV. Integrin-blocking antibodies demonstrated that alphaVbeta5 integrins mediated adhesion to vitronectin,alpha5beta1 mediated adhesion to fibronectin,and alpha6beta1 mediated adhesion to laminin + entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices,but in defined,nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel,supporting sustained self-renewal and pluripotency in three independent hESC lines. View Publication

Growth factor independence-1B (Gfi-1B) is a transcriptional repressor essential for erythropoiesis and megakaryopoiesis. Targeted gene disruption of GFI1B in mice leads to embryonic lethality resulting from failure to produce definitive erythrocytes,hindering the study of Gfi-1B function in adult hematopoiesis. We here show that,in humans,Gfi-1B controls the development of erythrocytes and megakaryocytes by regulating the proliferation and differentiation of bipotent erythro-megakaryocytic progenitors. We further identify in this cell population the type III transforming growth factor-beta receptor gene,TGFBR3,as a direct target of Gfi-1B. Knockdown of Gfi-1B results in altered transforming growth factor-beta (TGF-beta) signaling as shown by the increase in Smad2 phosphorylation and its inability to associate to the transcription intermediary factor 1-gamma (TIF1-gamma). Because the Smad2/TIF1-gamma complex is known to specifically regulate erythroid differentiation,we propose that,by repressing TGF-beta type III receptor (TbetaRIotaII) expression,Gfi-1B favors the Smad2/TIF1-gamma interaction downstream of TGF-beta signaling,allowing immature progenitors to differentiate toward the erythroid lineage. View Publication

The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein,we report ARQ 197 as the first non-ATP-competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met-expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively,these data suggest that ARQ 197,currently in phase II clinical trials,is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation. View Publication