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

Recent methodological advances have improved the ease and efficiency of generating human induced pluripotent stem cells (hiPSCs),but this now typically results in a greater number of hiPSC clones being derived than can be wholly characterized. It is therefore imperative that methods are developed which facilitate rapid selection of hiPSC clones most suited for the downstream research aims. Here we describe a combination of procedures enabling the simultaneous screening of multiple clones to determine their genomic integrity as well as their cardiac differentiation potential within two weeks of the putative reprogrammed colonies initially appearing. By coupling splinkerette-PCR with Ion Torrent sequencing,we could ascertain the number and map the proviral integration sites in lentiviral-reprogrammed hiPSCs. In parallel,we developed an effective cardiac differentiation protocol that generated functional cardiomyocytes within 10 days without requiring line-specific optimization for any of the six independent human pluripotent stem cell lines tested. Finally,to demonstrate the scalable potential of these procedures,we picked 20 nascent iPSC clones and performed these independent assays concurrently. Before the clones required passaging,we were able to identify clones with a single integrated copy of the reprogramming vector and robust cardiac differentiation potential for further analysis. View Publication

Human induced pluripotent stem cell-derived sensory neuron (iPSC-dSN) models are a valuable resource for the study of neurotoxicity but are affected by poor replicability and reproducibility,often due to a lack of optimization. Here,we identify experimental factors related to culture conditions that substantially impact cellular drug response in vitro and determine optimal conditions for improved replicability and reproducibility. Treatment duration and cell seeding density were both found to be significant factors,while cell line differences also contributed to variation. A replicable dose–response in viability was demonstrated after 48-h exposure to docetaxel or paclitaxel. Additionally,a replicable dose-dependent reduction in neurite outgrowth was demonstrated,demonstrating the applicability of the model for the examination of additional phenotypes. Overall,we have established an optimized iPSC-dSN model for the study of taxane-induced neurotoxicity. View Publication

Monomeric HIV envelope vaccines fail to elicit broadly neutralizing antibodies or to protect against infection. Neutralizing antibodies against HIV bind to native functionally active Env trimers on the virion surface. Gag-Env pseudovirions recapitulate the native trimer and could serve as an effective epitope presentation platform for study of the neutralizing antibody response in HIV-infected individuals. To address if pseudovirions can recapitulate native HIV virion epitope structures,we carefully characterized these particles,concentrating on the antigenic structure of the coreceptor binding site. By blue native gel shift assays,Gag-Env pseudovirions were shown to contain native trimers that were competent for binding to neutralizing monoclonal antibodies. In enzyme-linked immunosorbent assay,pseudovirions exhibited increased binding of known CD4-induced antibodies after addition of CD4. Using flow cytometric analysis,fluorescently labeled pseudovirions specifically identified a subset of antigen-specific B cells in HIV-infected subjects. Interestingly,the sequence of one of these novel human antibodies,identified during cloning of single HIV-specific B cells and designated 2C6,exhibited homology to mAb 47e,a known anti-CD4-induced coreceptor binding site antibody. The secreted monoclonal antibody 2C6 did not bind monomeric gp120,but specifically bound envelope on pseudovirions. A recombinant form of the antibody 2C6 acted as a CD4-induced epitope-specific antibody in neutralization assays,yet did not bind monomeric gp120. These findings imply specificity against a quaternary epitope presented on the pseudovirion envelope spike. These data demonstrate that Gag-Env pseudovirions recapitulate CD4 and coreceptor binding pocket antigenic structures and can facilitate identification of B-cell clones that secrete neutralizing antibodies. View Publication

Importance Human motor neurons may be reliably derived from induced pluripotent stem cells (iPSCs). In vivo transplant studies of human iPSCs and their cellular derivatives are essential to gauging their clinical utility. Objective To determine whether human iPSC-derived motor neurons can engraft in an immunodeficient mouse model of sciatic nerve injury. Design,Setting,and Subjects This nonblinded interventional study with negative controls was performed at a biomedical research institute using an immunodeficient,transgenic mouse model. Induced pluripotent stem cell-derived motor neurons were cultured and differentiated. Cells were transplanted into 32 immunodeficient mice with sciatic nerve injury aged 6 to 15 weeks. Tissue analysis was performed at predetermined points after the mice were killed humanely. Animal experiments were performed from February 24,2015,to May 2,2016,and data were analyzed from April 7,2015,to May 27,2016. Interventions Human iPSCs were used to derive motor neurons in vitro before transplant. Main Outcomes and Measures Evidence of engraftment based on immunohistochemical analysis (primary outcome measure); evidence of neurite outgrowth and neuromuscular junction formation (secondary outcome measure); therapeutic effect based on wet muscle mass preservation and/or electrophysiological evidence of nerve and muscle function (exploratory end point). Results In 13 of the 32 mice undergoing the experiment,human iPSC-derived motor neurons successfully engrafted and extended neurites to target denervated muscle. Human iPSC-derived motor neurons reduced denervation-induced muscular atrophy (mean [SD] muscle mass preservation,54.2% [4.0%]) compared with negative controls (mean [SD] muscle mass preservation,33.4% [2.3%]) (P = .04). No electrophysiological evidence of muscle recovery was found. Conclusions and Relevance Human iPSC-derived motor neurons may have future use in the treatment of peripheral motor nerve injury,including facial paralysis. Level of Evidence NA. View Publication

Self-renewal and differentiation of hematopoietic stem cells (HSCs) are balanced by the concerted activities of the fibroblast growth factor (FGF),Wnt,and Notch pathways,which are tuned by enzyme-mediated remodeling of heparan sulfate proteoglycans (HSPGs). Sulfatase modifying factor 1 (SUMF1) activates the Sulf1 and Sulf2 sulfatases that remodel the HSPGs,and is mutated in patients with multiple sulfatase deficiency. Here,we show that the FGF signaling pathway is constitutively activated in Sumf1(-/-) HSCs and hematopoietic stem progenitor cells (HSPCs). These cells show increased p-extracellular signal-regulated kinase levels,which in turn promote beta-catenin accumulation. Constitutive activation of FGF signaling results in a block in erythroid differentiation at the chromatophilic erythroblast stage,and of B lymphocyte differentiation at the pro-B cell stage. A reduction in mature myeloid cells and an aberrant development of T lymphocytes are also seen. These defects are rescued in vivo by blocking the FGF pathway in Sumf1(-/-) mice. Transplantation of Sumf1(-/-) HSPCs into wild-type mice reconstituted the phenotype of the donors,suggesting a cell autonomous defect. These data indicate that Sumf1 controls HSPC differentiation and hematopoietic lineage development through FGF and Wnt signaling. View Publication

Prostate cancer (PCa) contains a small population of cancer stem cells (CSCs) that contribute to its initiation and progression. The development of specific markers for identification of the CSCs may lead to new diagnostic strategies of PCa. Increased aldehyde dehydrogenase 1A1 (ALDH1A1) activity has been found in the stem cell populations of leukemia and some solid tumors. The aim of the study was to investigate the stem-cell-related function and clinical significance of the ALDH1A1 in human PCa. ALDEFLUOR assay was used to isolate ALDH1A1(+) cells from PCa cell lines. Stem cell characteristics of the ALDH1A1(+) cells were then investigated by in vitro and in vivo approaches. The ALDH1A1 expression was also analyzed by immunohistochemistry in 18 normal prostate and 163 PCa tissues. The ALDH1A1(+) PCa cells showed high clonogenic and tumorigenic capacities,and serially reinitiated transplantable tumors that resembled histopathologic characteristics and heterogeneity of the parental PCa cells in mice. Immunohistochemical analysis of human prostate tissues showed that ALDH1A1(+) cells were sparse and limited to the basal component in normal prostates. However,in tumor specimens,increased ALDH1A1 immunopositivity was found not only in secretory type cancer epithelial cells but also in neuroendocrine tumor populations. Furthermore,the high ALDH1A1 expression in PCa was positively correlated with Gleason score (P=0.01) and pathologic stage (P=0.01),and inversely associated with overall survival and cancer-specific survival of the patients (P=0.00093 and 0.00017,respectively). ALDH1A1 could be a prostate CSC-related marker. Measuring its expression might provide a potential approach to study tumorigenesis of PCa and predict outcome of the disease. View Publication

The utility of measuring real-time cellular bioenergetics of peripheral blood mononuclear cells (PBMCs) as biomarkers in disease monitoring,such as the bioenergetic health index,is of emerging interest. However,various experimental factors can impact the accuracy and reproducibility of these measurements. Methods: : PBMC bioenergetics were probed in real-time using extracellular flux analysis to identify optimal seeding density and injection protocol. Using a modified protocol,we assessed the extent to which blood processing time and isolation method (SepMate™ vs. EasySep™ Direct) influence PBMC bioenergetics under basal and stimulated conditions. Advanced metabolic control analysis including mitochondrial and glycolytic ATP supply flux,respiratory control ratio,bioenergetic health index,and mitochondrial toxicity index were used to identify and quantify PBMC bioenergetics. Results: Measures of metabolic profiling such as mitochondrial respiration,glycolytic activity,ATP supply flux,and respiratory control ratio were significantly diminished in PBMCs due to blood processing delay (48–72 hours) and were influenced by isolation method. Extended blood processing time significantly lowered T cell activation capacity in PBMCs,evidenced by decreased responses of mitochondrial and glycolytic ATP supply to CD3/CD28 activation. Discussion/Conclusion: This study demonstrates that key experimental variables including blood processing time and isolation method critically affect the reliability and biological relevance of PBMC metabolic assessments,highlighting the importance of protocol standardisation for accurate bioenergetic biomarker measurements. View Publication

Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb,helper-dependent adenoviral vectors with long homology arms are used for gene editing. However,this makes vector construction and recombinant analysis difficult. Conversely,insufficient homology may compromise targeting efficiency. Thus,we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation. With 23.8 and 21.4 kb of homology,the frequencies of targeted recombinants were 50-64.6% after positive selection for vector integration,and 97.4-100% after negative selection against random integrations. With 14.8 kb,the frequencies were 26.9-57.1% after positive selection and 87.5-100% after negative selection. With 9.6 kb,the frequencies were 21.4 and 75% after positive and negative selection,respectively. With only 5.6 kb,the frequencies were 5.6-16.7% after positive selection and 50% after negative selection,but these were more than high enough for efficient identification and isolation of targeted clones. Furthermore,we demonstrate helper-dependent adenoviral vector-mediated footprintless correction of cystic fibrosis transmembrane conductance regulator mutations through piggyBac excision of the selectable marker. However,low frequencies (≤ 1 × 10(-3)) necessitated negative selection for piggyBac-excision product isolation. View Publication

Efficient tumor T-cell infiltration is crucial for the effectiveness of T-cell-based therapies against solid tumors. Eosinophils play crucial roles in recruiting T cells in solid tumors. Our group has previously generated induced eosinophils (iEOs) from human pluripotent stem cells and exhibited synergistic efficacy with CAR-T cells in solid tumor inhibition. However,administrated eosinophils might influx into inflammatory lungs,posing a potential safety risk. Mitigating the safety concern and enhancing efficacy is a promising development direction for further application of eosinophils.MethodsWe developed a new approach to generate eosinophils with enhanced potency from human chemically reprogrammed induced pluripotent stem cells (hCiPSCs) with the Toll-like receptor (TLR) 7/8 signaling agonist R848.ResultsR848-activated iEOs (R-iEOs) showed significantly decreased influx to the inflamed lungs,indicating a lower risk of causing airway disorders. Furthermore,these R-iEOs had enhanced anti-tumor functions,preferably accumulated at tumor sites,and further increased T-cell infiltration. The combination of R-iEOs and CAR-T cells suppressed tumor growth in mice. Moreover,the chemo-trafficking signaling increased in R-iEOs,which may contribute to the decreased lung influx of R-iEOs and the increased tumor recruitment of T cells.ConclusionOur study provides a novel approach to alleviate the potential safety concerns associated with eosinophils while increasing T-cell infiltration in solid tumors. This finding offers a prospective strategy for incorporating eosinophils to improve CAR-T-cell immunotherapy for solid tumors in the future. View Publication

Inhibition of cytosolic DNA sensing represents a strategy that tumor cells use for immune evasion,but the underlying mechanisms are unclear. Here we have shown that CD47-signal regulatory protein α (SIRPα) axis dictates the fate of ingested DNA in DCs for immune evasion. Although macrophages were more potent in uptaking tumor DNA,increase of DNA sensing by blocking the interaction of SIRPα with CD47 preferentially occurred in dendritic cells (DCs) but not in macrophages. Mechanistically,CD47 blockade enabled the activation of NADPH oxidase NOX2 in DCs,which in turn inhibited phagosomal acidification and reduced the degradation of tumor mitochondrial DNA (mtDNA) in DCs. mtDNA was recognized by cyclic-GMP-AMP synthase (cGAS) in the DC cytosol,contributing to type I interferon (IFN) production and antitumor adaptive immunity. Thus,our findings have demonstrated how tumor cells inhibit innate sensing in DCs and suggested that the CD47-SIRPα axis is critical for DC-driven antitumor immunity. View Publication

BackgroundSpinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study,we compared different isoforms of basic fibroblast growth factor 2 (FGF2),overexpressed in stably transfected Human embryonic stem cells (hESCs),following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB).MethodsIn the present work,hESCs bioengineered to overexpress 18,23,and 31 kD isoforms of FGF2,were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA,followed by Tukey’s or Dunnett’s multiple comparison tests. Significance was set at *p < 0.05,**p < 0.01,***p < 0.001,and ****p < 0.0001.ResultsFor the first set of experiments,rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival,glial reaction,and synaptic coverage,two weeks after the lesion,indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently,the second set of experiments was performed with that isoform,so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival,glial reaction,synaptic coverage,and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection,coupled with immunomodulation,attenuation of astrogliosis,and preservation of inputs to the rescued motoneurons. Behaviorally,the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery.ConclusionTransgenic hESCs were an effective delivery platform for neurotrophic factors,rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury,while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03676-6. View Publication