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Pluripotent cells represent a powerful tool for tissue regeneration,but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study,we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated,bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting,we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate,thereby allowing for functional engraftment of pluripotent cells into regenerating tissue. View Publication

BACKGROUND Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits T-cell activation and is expressed on T-regulatory cells. Human CTLA-4 deficiency results in severe immune dysregulation. Abatacept (CTLA-4 Ig) is approved for the treatment of rheumatoid arthritis (RA) and its mechanism of action is attributed to effects on T-cells. It is known that CTLA-4 modulates the expression of its ligands CD80 and CD86 on antigen presenting cells (APC) by transendocytosis. As B-cells express CD80/CD86 and function as APC,we hypothesize that B-cells are a direct target of abatacept. OBJECTIVES To investigate direct effects of abatacept on human B-lymphocytes in vitro and in RA patients. METHODS The effect of abatacept on healthy donor B-cells' phenotype,activation and CD80/CD86 expression was studied in vitro. Nine abatacept-treated RA patients were studied. Seven of these were followed up to 24 months,and two up to 12 months only and treatment response,immunoglobulins,ACPA,RF concentrations,B-cell phenotype and ACPA-specific switched memory B-cell frequency were assessed. RESULTS B-cell development was unaffected by abatacept. Abatacept treatment resulted in a dose-dependent decrease of CD80/CD86 expression on B-cells in vitro,which was due to dynamin-dependent internalization. RA patients treated with abatacept showed a progressive decrease in plasmablasts and serum IgG. While ACPA-titers only moderately declined,the frequency of ACPA-specific switched memory B-cells significantly decreased. CONCLUSIONS Abatacept directly targets B-cells by reducing CD80/CD86 expression. Impairment of antigen presentation and T-cell activation may result in altered B-cell selection,providing a new therapeutic mechanism and a base for abatacept use in B-cell mediated autoimmunity. View Publication

A cDNA clone encoding a novel hematopoietic growth factor activity produced by a gibbon T cell line has been identified using a mammalian cell expression cloning system. The sequence of this cDNA proved to have significant homology to the sequence encoding murine interleukin 3 (IL-3). The human gene,which was readily identified because of its high degree of homology to the gibbon sequence,also displayed significant homology with the murine IL-3 sequence. The recombinant gibbon IL-3 protein proved to have multipotent colony stimulating activity when tested with normal human bone marrow cells,proving that this primate hematopoietin is not only structurally but also functionally related to murine IL-3. View Publication

Aldehyde dehydrogenases (ALDHs) belong to a superfamily of NAD(P)+-dependent enzymes,which catalyze the oxidation of endogenous and exogenous aldehydes to their corresponding acids. Increased expression and/or activity of ALDHs,particularly ALDH1A1,have been reported to occur in human cancers. It is proposed that the metabolic function of ALDH1A1 confers the stemness" properties to normal and cancer stem cells. Nevertheless� View Publication

The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs) have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here,iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD). Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs). USCs express the canonical reprogramming factors c-myc and klf4,and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry,RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery. ?? 2013. View Publication

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently,optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally,hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However,these methods have several major limitations,including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods,we have recently developed a new method,which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here,we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor),phenylbenzodioxane (ROCK II inhibitor),and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover,based on NCM,we have demonstrated efficient transfection or transduction of plasmid DNAs,lentiviral particles,and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture,and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies,stem cell research,and drug discovery. View Publication

DNA methylation regulation involves multi-layered chromatin interactions that require remodeling proteins like the helicase,lymphoid-specific (HELLS). Here,we generate HELLS and DNA methyltransferase 3A and B (DNMT3A/B) knockout human pluripotent stem cells and report telomere-to-telomere maps of whole genome bisulfite sequencing data combined with ATAC-sequencing. Disrupting HELLS induces a global loss of DNA methylation that is distinct from the DNMTs,in particular over peri/centromeric satellite repeats as defined in the telomere-to-telomere genome assembly. However,HELLS appears dispensable for local enhancer remodeling and the potential to differentiate into the three embryonic germ layers. Taken together,our results further clarify the genomic targets and role of HELLS in human cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03681-9. View Publication

The synthetic retinoid 6-[3-(adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437),which can bind to and activate the nuclear retinoic acid receptors beta and gamma (RARbeta/gamma),is a potent inducer of apoptosis in various cancer cell lines. However,this effect was reported to be independent of RARs. In this study,we compared and contrasted the potencies and mechanisms of action of CD437 and several other receptor-selective retinoids in induction of apoptosis and modulation of squamous differentiation in UMSCC22B human head and neck squamous cell carcinoma cell line. CD437 and the structurally related retinoid CD2325 exhibited almost equal potency in inducing apoptosis,whereas several other retinoids failed to induce apoptosis. The RAR-specific pan antagonist AGN193109 failed to suppress CD437-induced apoptosis,indicating that the induction of apoptosis by CD437 was RAR-independent. c-Fos expression was induced by CD437 and CD2325 that induced apoptosis in the cell line but not by other retinoids that failed to induce apoptosis,suggesting a role for c-Fos in CD437-induced apoptosis. At low concentration (0.01 microM),CD437 shared with several other receptor-selective retinoids the ability to suppress the mRNA levels of the squamous differentiation markers Spr1,involucrin,and cytokeratin 1. This effect of CD437 could be blocked by AGN193109. We conclude that CD437 can exert its effects in UMSCC22B human human head and neck squamous cell carcinoma cells by at least two mechanisms: RAR-mediated suppression of squamous differentiation and RAR-independent induction of apoptosis. View Publication

IL-12 is a potent proinflammatory cytokine. The effects of IL-12 are thought to be mediated by IFN-gamma production by NK,NKT,and T cells. In this study,we show that although IL-12 stimulates NK and NK1.1(+) T cells in bulk mouse splenocytes,it does not significantly stimulate purified NK cells,indicating that other cells are required. IL-12 stimulates T cell-deficient spleen cells and those depleted of macrophages. Unexpectedly,the depletion of dendritic cells also has little effect on the stimulation of spleen cells with IL-12. In contrast,B cell depletion almost completely inhibits IL-12-induced IFN-gamma production and B cell-deficient spleen cells are poorly stimulated with IL-12. Furthermore,purified NK cells are stimulated with IL-12 in the presence of purified B cells. Thus,B cells are necessary and also sufficient for the stimulation of purified NK cells with IL-12. Whereas spleen cells from IL-18-deficient mice are not stimulated with IL-12,NK cells purified from IL-18-deficient mice are stimulated with IL-12 in the presence of wild-type (WT) B cells,and WT NK cells are not stimulated with IL-12 in the presence of IL-18-deficient B cells. Cell contact between B and NK cells is also required for IL-12-induced IFN-gamma production. Finally,B cell-deficient mice injected with IL-12 produce significantly less IFN-gamma and IL-18 in the sera than WT mice do. Thus,stimulation of NK cells with IL-12 requires B cell cooperation in vitro as well as in vivo. View Publication

Pluripotent stem cells,both human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC),can give rise to multiple cell types and hence have tremendous potential for regenerative therapies. However,the tumorigenic potential of these cells remains a great concern,as reflected in the formation of teratomas by transplanted pluripotent cells. In clinical practice,most pluripotent cells will be differentiated into useful therapeutic cell types such as neuronal,cardiac,or endothelial cells prior to human transplantation,drastically reducing their tumorigenic potential. Our work investigated the extent to which these differentiated stem cell derivatives are truly devoid of oncogenic potential. In this study,we analyzed the gene expression patterns from three sets of hiPSC- and hESC-derivatives and the corresponding primary cells,and compared their transcriptomes with those of five different types of cancer. Our analysis revealed a significant gene expression overlap of the hiPSC- and hESC-derivatives with cancer,whereas the corresponding primary cells showed minimum overlap. Real-time quantitative PCR analysis of a set of cancer-related genes (selected on the basis of rigorous functional and pathway analyses) confirmed our results. Overall,our findings suggested that pluripotent stem cell derivatives may still bear oncogenic properties even after differentiation,and additional stringent functional assays to purify these cells should be done before they can be used for regenerative therapy. View Publication