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Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. Maxadilan,a 61-amino acid vasodilatory peptide,specifically activates the PACAP type I receptor (PAC1). Although PAC1 has been identified in embryonic stem cells,little is known about its presence or effects in human induced pluripotent stem (iPS) cells. In the present study,we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. To study the physiological effects mediated by PAC1,we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC). After exposure to UVC,the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells,as demonstrated by WST-8 analysis,annexin V/propidium iodide (PI) analysis and the terminal transferase dUTP nick end labeling (TUNEL) assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly,immunofluorescence,western blot analysis,real-time quantitative polymerase chain reaction (RT-qPCR) analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover,karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary,these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype. View Publication

BACKGROUND: The histone variant H2A.Z has been implicated in nucleosome exchange,transcriptional activation and Polycomb repression. However,the relationships among these seemingly disparate functions remain obscure.backslashnbackslashnRESULTS: We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers,and correlates strongly with H3K4 methylation. Accordingly,H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation,but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z,including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin.backslashnbackslashnCONCLUSIONS: Our findings associate H2A.Z with functionally distinct genomic elements,and suggest that post-translational modifications may reconcile its contrasting locations and roles. View Publication

BACKGROUND/AIMS: Cell transplantation into the heart is a new therapy after myocardial infarction. Its success,however,is impeded by poor donor cell survival and by limited transdifferentiation of the transplanted cells into functional cardiomyocytes. A promising strategy to overcome these problems is the induction of cardiomyogenic properties in donor cells by small molecules. METHODS: Here we studied cardiomyogenic effects of the small molecule compound cardiogenol C (CgC),and structural derivatives thereof,on lineage-committed progenitor cells by various molecular biological,biochemical,and functional assays. RESULTS: Treatment with CgC up-regulated cardiac marker expression in skeletal myoblasts. Importantly,the compound also induced cardiac functional properties: first,cardiac-like sodium currents in skeletal myoblasts,and secondly,spontaneous contractions in cardiovascular progenitor cell-derived cardiac bodies. CONCLUSION: CgC induces cardiomyogenic function in lineage-committed progenitor cells,and can thus be considered a promising tool to improve cardiac repair by cell therapy. View Publication

Pluripotent stem cells,including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),have the potential to treat type 1 diabetes through cell replacement therapy. However,the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification,and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation,and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified,suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR,and the relationship between one of these miRNAs,miR-151a-5p,and its predicted target gene,SOX17,was investigated by luciferase assay,and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion,these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells. View Publication

Rapidly proliferating cells are highly sensitive to ionizing radiation and can undergo apoptosis if the oxidative and genotoxic injury exceed the defensive and regenerative capacity of the cell. Our earlier work has established the antiapoptotic action of the growth factor-like lipid mediator lysophosphatidic acid (LPA). Activation of the LPA2 GPCR has been hypothesized to elicit antiapoptotic and regenerative actions of LPA. Based on this hypothesis we developed a novel nonlipid agonist of LPA2,which we designated Radioprotectin-1 (RP-1). We tested RP-1 at the six murine LPA GPCR subtypes using the transforming growth factor alpha shedding assay and found that it had a 25 nM EC50 that is similar to that of LPA18:1 at 32 nM. RP-1 effectively reduced apoptosis induced by gamma-irradiation and the radiomimetic drug Adriamycin only in cells that expressed LPA2 either endogenously or after transfection. RP-1 reduced gamma-H2AX levels in irradiated mouse embryonic fibroblasts transduced with the human LPA2 GPCR but was ineffective in vector transduced MEF control cells and significantly increased clonogenic survival after gamma-irradiation. gamma-Irradiation induced the expression of lpar2 transcripts that was further enhanced by RP-1 exposure within 30 min after irradiation. RP-1 decreased the mortality of C57BL/6 mice in models of the hematopoietic and gastrointestinal acute radiation syndromes. Using Lgr5-EGFP-CreER;Tdtomatoflox transgenic mice,we found that RP-1 increased the survival and growth of intestinal enteroids via the enhanced survival of Lgr5+ intestinal stem cells. Taken together,our results suggest that the LPA2-specific agonist RP-1 exerts its radioprotective and radiomitigative action through specific activation of the upregulated LPA2 GPCR in Lgr5+ stem cells. View Publication

OBJECTIVE Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However,evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS Using complementary cell-based techniques,metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS Although TKI suppressed glycolysis,compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However,clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION Targeting glutamine metabolism with the FDA-approved drug,Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells. View Publication

Innate lymphoid cells 2 (ILC2) play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). An important aspect of ILC2-mediated tumorigenesis is the expansion of the resident ILC2 and simultaneous recruitment of the peripheral ILC2. Here,we describe a protocol for isolation,enrichment,and DiD labeling of ILC2 for in vivo tracking of ILC2s in the mouse. Further,we describe steps for the adoptive transfer of ILC2 to a recipient mouse model of PDAC. For complete details on the use and execution of this protocol,please refer to Alam et al. (2022). View Publication

BACKGROUND Adoptive cell therapy (ACT) using genetically modified T cells has evolved into a promising treatment option for patients with cancer. However,even for the best-studied and clinically validated CD19-targeted chimeric antigen receptor (CAR) T-cell therapy,many patients face the challenge of lack of response or occurrence of relapse. There is increasing need to improve the efficacy of ACT so that durable,curative outcomes can be achieved in a broad patient population. METHODS Here,we investigated the impact of indomethacin (indo),a non-steroidal anti-inflammatory drug (NSAID),on the efficacy of ACT in multiple preclinical models. Mice with established B-cell lymphoma received various combinations of preconditioning chemotherapy,infusion of suboptimal dose of tumor-reactive T cells,and indo administration. Donor T cells used in the ACT models included CD4+ T cells expressing a tumor-specific T cell receptor (TCR) and T cells engineered to express CD19CAR. Mice were monitored for tumor growth and survival. The effects of indo on donor T cell phenotype and function were evaluated. The molecular mechanisms by which indo may influence the outcome of ACT were investigated. RESULTS ACT coupled with indo administration led to improved tumor growth control and prolonged mouse survival. Indo did not affect the activation status and tumor infiltration of the donor T cells. Moreover,the beneficial effect of indo in ACT did not rely on its inhibitory effect on the immunosuppressive cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis. Instead,indo-induced oxidative stress boosted the expression of death receptor 5 (DR5) in tumor cells,rendering them susceptible to donor T cells expressing TNF-related apoptosis-inducing ligand (TRAIL). Furthermore,the ACT-potentiating effect of indo was diminished against DR5-deficient tumors,but was amplified by donor T cells engineered to overexpress TRAIL. CONCLUSION Our results demonstrate that the pro-oxidative property of indo can be exploited to enhance death receptor signaling in cancer cells,providing rationale for combining indo with genetically modified T cells to intensify tumor cell killing through the TRAIL-DR5 axis. These findings implicate indo administration,and potentially similar use of other NSAIDs,as a readily applicable and cost-effective approach to augment the efficacy of ACT. View Publication

OBJECTIVES We sought to develop medium throughput standard operating procedures for screening cryopreserved human peripheral blood mononuclear cells (PBMCs) for CD4+ and CD8+ T cell responses to potential autoantigens. METHODS Dendritic cells were loaded with a peptide cocktail from ubiquitous viruses or full-length viral protein antigens and cocultured with autologous T cells. We measured expression of surface activation markers on T cells by flow cytometry and cytometry by time of flight 24-72 h later. We tested responses among T cells freshly isolated from healthy control PBMCs,cryopreserved T cells,and T cells derived from a variety of T cell expansion protocols. We also compared the transcriptional profile of CD8+ T cells rested with interleukin (IL)7 for 48 h after 1) initial thawing,2) expansion,and 3) secondary cryopreservation/thawing of expanded cells. To generate competent antigen presenting cells from PBMCs,we promoted differentiation of PBMCs into dendritic cells with granulocyte macrophage colony stimulating factor and IL-4. RESULTS We observed robust dendritic cell differentiation from human PBMCs treated with 50 ng/mL GM-CSF and 20 ng/mL IL-4 in as little as 3 days. Dendritic cell purity was substantially increased by magnetically enriching for CD14+ monocytes prior to differentiation. We also measured antigen-dependent T cell activation in DC-T cell cocultures. However,polyclonal expansion of T cells with anti-CD3/antiCD28 abolished antigen-dependent upregulation of CD69 in our assay despite minimal transcriptional differences between rested CD8+ T cells before and after expansion. Furthermore,resting these expanded T cells in IL-2,IL-7 or IL-15 did not restore the antigen dependent responses. In contrast,T cells that were initially expanded with IL-2 + IL-7 rather than plate bound anti-CD3 + anti-CD28 retained responsiveness to antigen stimulation and these responses strongly correlated with responses measured at initial thawing. SIGNIFICANCE While screening techniques for potential pathological autoantibodies have come a long way,comparable full-length protein target assays for screening patient T cells at medium throughput are noticeably lacking due to technical hurdles. Here we advance techniques that should have broad applicability to translational studies investigating cell mediated immunity in infectious or autoimmune diseases. Future studies are aimed at investigating possible CD8+ T cell autoantigens in MS and other CNS autoimmune diseases. View Publication

In this study,we explore the therapeutic feasibility of globotriaosylceramide (Gb3) synthase (A4GALT)-specific siRNA-loaded polyhistidine (pHis)-incorporated lipid nanoparticles (HLNPs) for Fabry disease (FD). HLNPs were developed to deliver siRNAs targeting A4GALT using a microfluidic device,with pHis aiding in endosome escape. The therapy was tested on GLA-knockout human-induced pluripotent-stem-cell-derived endothelial cells (GLA-KO-hiPSC-ECs) and podocytes (GLA-KO-hiPSC-PCs). GLA-KO-hiPSCs-ECs or -PCs,upon differentiation,were treated with A4GALT-siRNA-HLNP. Successful intracellular uptake of A4GALT-siRNA-HLNP was confirmed through fluorescence and electron microscopy in both cell types. A4GALT-siRNA-HLNP treatment confirmed both cell types’ stability at 5 ?g/mL. Increased Gb3 deposition and zebra body formation were detected in both cell types,but A4GALT-siRNA-HLNP treatment attenuated these FD phenotypes,demonstrating reduced expression of A4GALT through western blot analysis. RNA sequencing analysis revealed that the expression of transcripts associated with FD was restored by A4GALT-siRNA-HLNP treatment in GLA-KO-hiPSCs-ECs,whereas in GLA-KO-hiPSCs-PCs,this effect was relatively less pronounced. Suppression of A4GALT via siRNA/HLNP treatment significantly rescued FD phenotypes especially in EC,presenting a novel therapeutic approach for FD. Graphical abstract This study highlights the therapeutic potential of A4GALT-siRNA delivered via HLNPs for Fabry disease (FD). In GLA-KO-hiPSC-derived endothelial cells and podocytes,treatment reduced Gb3 accumulation,restored transcriptomic changes,and mitigated FD phenotypes,with stronger effects in endothelial cells,supporting its promise as a novel FD therapy. View Publication

Heat shock protein 90 (Hsp90) is a molecular chaperone important for maintaining protein homeostasis (proteostasis) in the cell. Hsp90 inhibitors are being explored as cancer therapeutics because of their ability to disrupt proteostasis. Inhibiting Hsp90 increases surface density of the immunological receptor Major Histocompatibility Complex 1 (MHC1). Here we show that this increase occurs across multiple cancer cell lines and with both cytosol-specific and pan-Hsp90 inhibitors. We demonstrate that Hsp90 inhibition also alters surface expression of both IFNGR and PD-L1,two additional immunological receptors that play a significant role in anti-tumour or anti-immune activity in the tumour microenvironment. Hsp90 also negatively regulates IFN-γ activity in cancer cells,suggesting it has a unique role in mediating the immune system’s response to cancer. Our data suggests a strong link between Hsp90 activity and the pathways that govern anti-tumour immunity. This highlights the potential for the use of an Hsp90 inhibitor in combination with another currently available cancer treatment,immune checkpoint blockade therapy,which works to prevent immune evasion of cancer cells. Combination checkpoint inhibitor therapy and the use of an Hsp90 inhibitor may potentiate the therapeutic benefits of both treatments and improve prognosis for cancer patients. View Publication

Engineered T cells equipped with a chimeric antigen receptor (CAR) have shown tremendous clinical success,but tumor-mediated stimulation of T cell inhibitory receptors leads to exhaustion,hampering durable remission in patients. Mitigation of this effect via checkpoint inhibition or genome editing to knockout the genes encoding for these receptors has shown promise. Yet,the side effects of these procedures require better alternatives. Targeted epigenome editing offers a potent strategy to alter gene expression without DNA modifications. Its hit-and-run mechanism enables durable,multiplexed modulation of gene expression with greater safety. Here,we describe multiplexed epigenome editing inactivation of two critical-exhaustion-related genes,PDCD1 and LAG3,both in primary human T cells and in prostate-cancer-specific CAR T cells. Epigenetically modified CAR T cells are indistinguishable from parental cells across a range of functional assays. Although the model does not fully mimic T cell exhaustion,limiting functional assessment,gene silencing remains durable across multiple divisions and repeated CAR stimulations. Furthermore,transcriptomic analysis revealed minimal off-target effects not directly attributable to the effectors used. We demonstrate that targeted epigenome editing is effective and safe for multiplexed gene inhibition and holds potential in engineering CAR T cells with enhanced and customizable features. Graphical abstract Epigenome editing is used to engineer CAR T cells targeting prostate cancer by stably silencing the PDCD1 and LAG3 genes,which encode key inhibitory checkpoint receptors. This DNA break-free approach enhances safety by avoiding genomic damage and holds promise as a next-generation strategy for safer,more durable cancer immunotherapy. View Publication