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

Human microglia are central regulators and actors in brain infections and neuro-inflammatory pathologies. However,access to such cells is limited,and studies systematically mapping the spectrum of their inflammatory states are scarce. Here,we generated microglia-like cells (MGLCs) from human induced pluripotent stem cells and characterized them as a robust,accessible model system for studying inflammatory activation. We validated lineage identity through transcriptome profiling,revealing selective upregulation of microglial signature genes and enrichment of microglia/macrophage-related gene sets. MGLCs displayed distinct morphologies and produced stimulus- and time-dependent cytokine secretion profiles upon exposure to diverse inflammatory stimuli,including pro-inflammatory cytokines (TNFα,interferon-γ) and agonists of the Toll-like receptors TLR2 (FSL-1),TLR3 (Poly(I:C)),TLR4 (lipopolysaccharide,LPS),and TLR7 (imiquimod). Transcriptome profiling and bioinformatics analysis revealed distinct activation signatures. Functional assays demonstrated stimulus-specific engagement of NFκB and JAK-STAT signaling pathways. The shared NFκB nuclear translocation response of TLR ligands and TNFα was reflected in overlapping transcriptome profiles: they shared modules (e.g.,oxidative stress response and TNFα-related signaling) identified by weighted gene co-expression network analysis. Finally,the potential consequences of microglia activation for neighboring cells were studied on the example of microglia-astrocyte crosstalk. The capacity of MGLC supernatants to stimulate astrocytes was measured by quantifying astrocytic NFκB translocation. MGLCs stimulated with FSL-1,LPS,or Poly(I:C) indirectly activated astrocytes via a strictly TNFα-dependent mechanism,highlighting the role of soluble mediators in the signal propagation. Altogether,this platform enables a dissection of microglia activation states and multi-parametric characterization of subsequent neuroinflammation. View Publication

Radiotherapy represents the most effective nonsurgical treatments for gliomas. However,gliomas are highly radioresistant and recurrence is nearly universal. Results from our laboratory and other groups suggest that cancer stem cells contribute to radioresistance in gliomas and breast cancers. The Notch pathway is critically implicated in stem cell fate determination and cancer. In this study,we show that inhibition of Notch pathway with gamma-secretase inhibitors (GSIs) renders the glioma stem cells more sensitive to radiation at clinically relevant doses. GSIs enhance radiation-induced cell death and impair clonogenic survival of glioma stem cells but not non-stem glioma cells. Expression of the constitutively active intracellular domains of Notch1 or Notch2 protect glioma stem cells against radiation. Notch inhibition with GSIs does not alter the DNA damage response of glioma stem cells after radiation but rather reduces Akt activity and Mcl-1 levels. Finally,knockdown of Notch1 or Notch2 sensitizes glioma stem cells to radiation and impairs xenograft tumor formation. Taken together,our results suggest a critical role of Notch signaling to regulate radioresistance of glioma stem cells. Inhibition of Notch signaling holds promise to improve the efficiency of current radiotherapy in glioma treatment. View Publication

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state,and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics,these two reprogramming methods reset genomic methylation,an epigenetic modification of DNA that influences gene expression,leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin,which favours their differentiation along lineages related to the donor cell,while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming,or by treatment of iPSCs with chromatin-modifying drugs. In contrast,the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming,which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment. View Publication

CD4(+)Vβ5(+) peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process,cells lose surface Vβ5 expression and undergo RAG-dependent rearrangement of endogenous TCRβ genes,driving surface expression of novel TCRs. Although postrevision CD4(+)Vβ5(-)TCRβ(+) T cells accumulate with age in Vβ5 transgenic mice and bear a diverse TCR Vβ repertoire,it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ,and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly,postrevision cells do not proliferate in response to the tolerizing superantigen,implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal bacterial Ags and can generate I-A(b)-restricted responses to Ag by producing IFN-γ following Listeria monocytogenes challenge. These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host. View Publication

Differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hepatocyte-like cells provides a platform to study the molecular basis of human hepatocyte differentiation,to develop cell culture models of liver disease,and to potentially provide hepatocytes for treatment of end-stage liver disease. Additionally,hepatocyte-like cells generated from human pluripotent stem cells could serve as platforms for drug discovery,determination of pharmaceutical-induced hepatotoxicity,and evaluation of idiosyncratic drug-drug interactions. Here,we describe a step-wise protocol previously developed in our laboratory that facilitates the highly efficient and reproducible differentiation of human pluripotent stem cells into hepatocyte-like cells. Our protocol uses defined culture conditions and closely recapitulates key developmental events that are found to occur during hepatogenesis. View Publication

Heme oxygenase-1 (HO-1,HMOX1 ) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions,heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here,we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1 -deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1,the effect that was further enhanced in response to δ-aminolevulinic acid (ALA),a substrate in heme synthesis. This was associated with replication stress,as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1 -deficient patient. Interestingly,in the absence of HO-1,the speed of fork progression was higher,and the response to DNA conformational hindrance less stringent,indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead,we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53,an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin,which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1,presumably contributing to its widely recognized cytoprotective activity. View Publication

DNA methyltransferase inhibitors represent promising new drugs for cancer therapies. The first of these compounds (5-azacytidine,Vidaza) has recently been approved as an antitumor agent,and others are presently in various stages of their preclinical or clinical development. Most of the archetypal inhibitors have been established and characterized in different experimental systems,which has thus far precluded their direct comparison. We have now established defined experimental conditions that allowed a comparative analysis of the six most widely known DNA methyltransferase inhibitors: 5-azacytidine (5-aza-CR),5-aza-2'-deoxycytidine (5-aza-CdR),zebularine,procaine,(-)-epigallocatechin-3-gallate (EGCG),and RG108. Of these,5-aza-CR,5-aza-CdR,zebularine,and EGCG were found to exhibit significant cytotoxicity in human cancer cell lines. 5-aza-CdR and EGCG were also found to be genotoxic,as evidenced by the induction of micronuclei. In addition,5-aza-CR,5-aza-CdR,zebularine,and RG108 caused concentration-dependent demethylation of genomic DNA,whereas procaine and EGCG failed to induce significant effects. Finally,the experiments in cancer cell lines were complemented by a cell-free in vitro assay with purified recombinant DNA methyltransferase,which indicated that RG108 is the only drug capable of direct enzyme inhibition. These results show a substantial diversity in the molecular activities of DNA methyltransferase inhibitors and provide valuable insights into the developmental potential of individual drugs. View Publication

Given their self-renewing and pluripotent capabilities,human embryonic stem cells (hESCs) are well poised as a cellular source for tissue regeneration therapy. However,the host immune response against transplanted hESCs is not well characterized. In fact,controversy remains as to whether hESCs have immune-privileged properties. To address this issue,we used in vivo bioluminescent imaging to track the fate of transplanted hESCs stably transduced with a double-fusion reporter gene consisting of firefly luciferase and enhanced GFP. We show that survival after transplant is significantly limited in immunocompetent as opposed to immunodeficient mice. Repeated transplantation of hESCs into immunocompetent hosts results in accelerated hESC death,suggesting an adaptive donor-specific immune response. Our data demonstrate that transplanted hESCs trigger robust cellular and humoral immune responses,resulting in intragraft infiltration of inflammatory cells and subsequent hESC rejection. Moreover,we have found CD4(+) T cells to be an important modulator of hESC immune-mediated rejection. Finally,we show that immunosuppressive drug regimens can mitigate the anti-hESC immune response and that a regimen of combined tacrolimus and sirolimus therapies significantly prolongs survival of hESCs for up to 28 days. Taken together,these data suggest that hESCs are immunogenic,trigger both cellular and humoral-mediated pathways,and,as a result,are rapidly rejected in xenogeneic hosts. This process can be mitigated by a combined immunosuppressive regimen as assessed by molecular imaging approaches. View Publication

Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene account for approximately 95 % of all RTT cases. To model RTT in vitro,we generated induced pluripotent stem cells (iPSCs) from fibroblasts of two RTT patients with different mutations (MECP2 (R306C) and MECP2 (1155$$32)) in their MECP2 gene. We found that these iPSCs were capable of differentiating into functional neurons. Compared to control neurons,the RTT iPSC-derived cells had reduced soma size and a decreased amount of synaptic input,evident both as fewer Synapsin 1-positive puncta and a lower frequency of spontaneous excitatory postsynaptic currents. Supplementation of the culture media with choline rescued all of these defects. Choline supplementation may act through changes in the expression of choline acetyltransferase,an important enzyme in cholinergic signaling,and also through alterations in the lipid metabolite profiles of the RTT neurons. Our study elucidates the possible mechanistic pathways for the effect of choline on human RTT cell models,thereby illustrating the potential for using choline as a nutraceutical to treat RTT. View Publication

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions,resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration,these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals,as evidenced by two visual behavioral tests. Furthermore,the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease. View Publication

Human NK cell deficiencies are rare yet result in severe and often fatal disease,particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells,and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8,which encodes an interferon regulatory factor,as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells,and this impairment in terminal maturation was also observed in Irf8-/-,but not Irf8+/-,mice. We then determined that impaired maturation was NK cell intrinsic,and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together,these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease,thereby emphasizing a critical role for NK cells in human antiviral defense. View Publication