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

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts,perceptions,and emotions,usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however,most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells,derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient,presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS),when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia,contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening. View Publication

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

Peripheral blood was collected from a 39-year-old unaffected female carrier of an X-linked recessive mutation of Iduronate 2-sulfatase gene (NM000202.7(IDS):c.85CtextgreaterT) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC showed normal karyotype. The line offers a good platform to study MPS II pathophysiology,for drug testing,early biomarker discovery and gene therapy studies. View Publication

Genomic abnormalities may accumulate in human embryonic stem cells (hESCs) during in vitro maintenance. Characterization of the mechanisms enabling survival and expansion of abnormal hESCs is important due to consequences of genetic changes for the therapeutic utilization of stem cells. Furthermore,these cells provide an excellent model to study transformation in vitro. We report here that the histone deacetylase proteins,HDAC1 and HDAC2,are increased in karyotypically abnormal hESCs when compared to their normal counterparts. Importantly,similar to many cancer cell lines,we found that HDAC inhibitors repress proliferation of the karyotypically abnormal hESCs,whereas normal cells are more resistant to the treatment. The decreased proliferation correlates with downregulation of HDAC1 and HDAC2 proteins,induction of the proliferation inhibitor,cyclin-dependent kinase inhibitor 1A (CDKN1A),and altered regulation of tumor suppressor protein Retinoblastoma 1 (RB1). Through genome-wide transcriptome analysis we have identified genes with altered expression and responsiveness to HDAC inhibition in abnormal cells. Most of these genes are linked to severe developmental and neurological diseases and cancers. Our results highlight the importance of epigenetic mechanisms in the regulation of genomic stability of hESCs,and provide valuable candidates for targeted and selective growth inhibition of karyotypically abnormal cells. textcopyright 2013 Elsevier B.V. View Publication

In CD34(+) acute myeloid leukemia (AML),the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously,we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population,containing the CD34(+)CD38(-)CLL-1(+) cells,does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission,both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future. View Publication

It is known that umbilical cord blood (UCB) is a rich source of stem cells with practical and ethical advantages. Three important types of stem cells which can be harvested from umbilical cord blood and used in disease treatment are hematopoietic stem cells (HSCs),mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Since these stem cells have shown enormous potential in regenerative medicine,numerous umbilical cord blood banks have been established. In this study,we examined the ability of banked UCB collected to produce three types of stem cells from the same samples with characteristics of HSCs,MSCs and EPCs. We were able to obtain homogeneous plastic rapidly-adherent cells (with characteristics of MSCs),slowly-adherent (with characteristics of EPCs) and non-adherent cells (with characteristics of HSCs) from the mononuclear cell fractions of cryopreserved UCB. Using a protocol of 48 h supernatant transferring,we successfully isolated MSCs which expressed CD13,CD44 and CD90 while CD34,CD45 and CD133 negative,had typical fibroblast-like shape,and was able to differentiate into adipocytes; EPCs which were CD34,and CD90 positive,CD13,CD44,CD45 and CD133 negative,adherent with cobble-like shape; HSCs which formed colonies when cultured in MethoCult medium. View Publication

BACKGROUND Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However,a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to $\$-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in $\$-adrenergic signaling,we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. METHODS AND RESULTS First,we confirmed that PLB protein was differentially expressed in hESC (HES2,H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant,respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase,Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly,Ad-PLB-transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB-transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally,similar results were also seen with Ad-PLB-transduced and Ad-S16E-transduced hvCMTs. However,Ad-PLB altered neither the global transcriptome nor ICa,L,implicating a PLB-specific effect. CONCLUSIONS Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to $\$-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses. View Publication

There is an urgent need for new and advanced approaches to modeling the pathological mechanisms of complex human neurological disorders. This is underscored by the decline in pharmaceutical research and development efficiency resulting in a relative decrease in new drug launches in the last several decades. Induced pluripotent stem cells represent a new tool to overcome many of the shortcomings of conventional methods,enabling live human neural cell modeling of complex conditions relating to aberrant neurodevelopment,such as schizophrenia,epilepsy and autism as well as age-associated neurodegeneration. This review considers the current status of induced pluripotent stem cell-based modeling of neurological disorders,canvassing proven and putative advantages,current constraints,and future prospects of next-generation culture systems for biomedical research and translation. View Publication

Bone marrow-derived mesenchymal stromal cells (MSC) possess an immune plasticity manifested by either an immunosuppressive or,when activated with IFN-gamma,an APC phenotype. Herein,TLR expression by MSC and their immune regulatory role were investigated. We observed that human MSC and macrophages expressed TLR3 and TLR4 at comparable levels and TLR-mediated activation of MSC resulted in the production of inflammatory mediators such as IL-1beta,IL-6,IL-8/CXCL8,and CCL5. IFN-alpha or IFN-gamma priming up-regulated production of these inflammatory mediators and expression of IFNB,inducible NO synthase (iNOS),and TRAIL upon TLR activation in MSC and macrophages,but failed to induce IL-12 and TNF-alpha production in MSC. Nonetheless,TLR activation in MSC resulted in the formation of an inflammatory site attracting innate immune cells,as evaluated by human neutrophil chemotaxis assays and by the analysis of immune effectors retrieved from Matrigel-embedded MSC injected into mice after in vitro preactivation with cytokines and/or TLR ligands. Hence,TLR-activated MSC are capable of recruiting immune inflammatory cells. In addition,IFN priming combined with TLR activation may increase immune responses induced by Ag-presenting MSC through presentation of Ag in an inflammatory context,a mechanism that could be applied in a cell-based vaccine. View Publication

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope,since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but,in contrast to over-expression systems,cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8,in agreement with the observed reduction of VAPB in sporadic ALS. View Publication

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus,new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors,and Triple-Negative" Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor� View Publication