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

ABSTRACTThe gut microbiota and the immune system are closely connected,influencing early?life brain development. Brain?derived neurotrophic factor (BDNF),crucial for neuronal development,has been demonstrated to be produced by certain immune cells. However,the modulation of BDNF during bacterial antigen and metabolite challenge remains elusive. We investigate the effects of bacterial?derived antigens and metabolites on BDNF secretion in human PBMCs. Although BDNF levels were altered during stimulation,a specific cellular origin of BDNF within PBMCs was indeterminate. Positive magnetic separation of monocytes eliminated both the stimulant?induced BDNF secretion and reduced monocyte?platelet aggregates. Conversely,elevated platelet counts significantly increased BDNF levels,indicating that platelets,when interacting with monocytes and exposed to bacterial antigens,are likely the dominant source of BDNF in PBMC cultures. As previously described,platelets are a crucial source of circulating peripheral blood BDNF. Our findings emphasize the importance of the interplay between immune?blood cell complexes during microbial stimulation in regulating BDNF levels. This highlights the necessity of investigating such interactions to better understand the early?life gut?brain axis. Bacterial antigens primarily induce BDNF release from platelets interacting with monocytes in PBMCs. This interplay underscores how immune?blood cell complexes shape BDNF levels which may impact early human development. View Publication

Human induced pluripotent stem cells (hiPSC) provide an attractive tool to study disease mechanisms of neurodevelopmental disorders such as schizophrenia. A pertinent problem is the development of hiPSC-based assays to discriminate schizophrenia (SZ) from autism spectrum disorder (ASD) models. Healthy control individuals as well as patients with SZ and ASD were examined by a panel of diagnostic tests. Subsequently,skin biopsies were taken for the generation,differentiation,and testing of hiPSC-derived neurons from all individuals. SZ and ASD neurons share a reduced capacity for cortical differentiation as shown by quantitative analysis of the synaptic marker PSD95 and neurite outgrowth. By contrast,pattern analysis of calcium signals turned out to discriminate among healthy control,schizophrenia,and autism samples. Schizophrenia neurons displayed decreased peak frequency accompanied by increased peak areas,while autism neurons showed a slight decrease in peak amplitudes. For further analysis of the schizophrenia phenotype,transcriptome analyses revealed a clear discrimination among schizophrenia,autism,and healthy controls based on differentially expressed genes. However,considerable differences were still evident among schizophrenia patients under inspection. For one individual with schizophrenia,expression analysis revealed deregulation of genes associated with the major histocompatibility complex class II (MHC class II) presentation pathway. Interestingly,antipsychotic treatment of healthy control neurons also increased MHC class II expression. In conclusion,transcriptome analysis combined with pattern analysis of calcium signals appeared as a tool to discriminate between SZ and ASD phenotypes in vitro. View Publication

Overcoming the global health threat of HIV infection requires continuous pipelines of novel drug candidates. We identified the $\gamma$-pyrone polyketides Aureothin/Neoaureothin as potent hits by anti-HIV screening of an extensive natural compound collection. Total synthesis of a structurally diverse group of Aureothin-derivatives successfully identified a lead compound ({\#}7) superior to Aureothin that combines strong anti-HIV activity (IC90{\textless}45 nM),photostability and improved cell safety. Compound {\#}7 inhibited de novo virus production from integrated proviruses by blocking the accumulation of HIV RNAs that encode the structural components of virions and include viral genomic RNAs. Thus,the mode-of-action displayed by compound {\#}7 is different from those of all current clinical drugs. Proteomic analysis indicated that compound {\#}7 does not affect global protein expression in primary blood cells and may modulate cellular pathways linked to HIV infection. Compound {\#}7 inhibited multiple HIV genotypes,including HIV-type 1 and 2 and synergistically inhibited HIV in combination with clinical reverse transcriptase and integrase inhibitors. We conclude that compound {\#}7 represents a promising new class of HIV inhibitors that will facilitate the identification of new virus-host interactions exploitable for antiviral attack and holds promise for further drug development. View Publication

DNA repair mechanisms in human primary cells,including error-free repair,and,recurrent nuclease cleavage events,remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR),an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs,small indels,large deletions,and other aberrations in absolute terms in clinically relevant edited cells,including HSPCs,iPSCs,and T-cells. By quantifying up to 90% of loci with unresolved DSBs,CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore,we accurately quantify DNA repair precision,revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics,paving the way for mechanistic studies to advance gene therapy,designer editors,and small molecule discovery. Quantifying genomic aberrations resulting from designer nucleases activity is essential for gene therapy clinical translation. Here,the authors present a modular digital PCR technique that profiles DNA repair precision and cut-repair cycles at the edited loci,exposing current evaluation biases. View Publication

BACKGROUND: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury.backslashnbackslashnMETHODOLOGY AND PRINCIPAL FINDINGS: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both,rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis.backslashnbackslashnCONCLUSIONS/SIGNIFICANCE: These findings suggest that our differentiation protocol has the capacity to generate region-specific and electrophysiologically active neurons under in vitro conditions without embryoid body formation,co-culture with stromal cells and without presence of cells of mesodermal or endodermal lineages. View Publication

The NKG2D receptor activates natural killer (NK) cell cytotoxicity and cytokine production on recognition of self-molecules induced by cellular stress under different conditions such as viral infections. The importance of NKG2D in the immune response to human cytomegalovirus (HCMV) is supported by the identification of several viral molecules that prevent the expression of NKG2D ligands by infected cells. In this study we report that,paradoxically,a significant,selective,and transient reduction of NKG2D expression on NK cells is detected during HCMV infection of peripheral blood mononuclear cells if needed. Antagonizing type I interferon (IFN),interleukin-12 (IL-12),and IFNgamma prevented HCMV-induced down-regulation of surface NKG2D. Moreover,treatment of purified NK cells with recombinant IFNbeta1 and IL-12 mimicked the effect,supporting a direct role of these cytokines in regulating NKG2D surface expression in NK cells. The loss of NKG2D expression selectively impaired NK-cell cytotoxicity against cells expressing NKG2D ligands but preserved the response triggered through other activating receptors. These results support that down-regulation of NKG2D expression on NK cells by cytokines with a key role in antiviral immune response may constitute a physiologic mechanism to control NK-cell reactivity against normal cells expressing NKG2D ligands in the context of inflammatory responses to viral infections. View Publication

The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner,mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However,the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover,we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects,and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells,inducing a dose-dependent increase in SOX2,OCT4 and Nanog proteins,leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells. View Publication

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines,from 38 laboratories worldwide,for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal,but there was a progressive tendency to acquire changes on prolonged culture,commonly affecting chromosomes 1,12,17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants,determined from the SNP arrays,also appeared sporadically. No common variants related to culture were observed on chromosomes 1,12 and 17,but a minimal amplicon in chromosome 20q11.21,including three genes expressed in human ES cells,ID1,BCL2L1 and HM13,occurred in textgreater20% of the lines. Of these genes,BCL2L1 is a strong candidate for driving culture adaptation of ES cells. View Publication

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver,a preleukemic condition termed transient myeloproliferative disorder,and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins,human embryonic stem (hES),and induced pluripotent stem (iPS) cells,were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation,we isolated disomic and trisomic subclones from the same parental iPS line,thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like,γ-globin expressing,definitive hematopoiesis,we found that trisomic cells of hES,iPS,or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells,accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. View Publication

Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes,yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified,homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here,we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized,and defined growth factors were used to generate KDR+ EC progenitors. Magnetic purification of a KDR+ progenitor subpopulation resulted in an expanding,homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders. STEM Cells2013;31:92–103 View Publication

Many studies have compared the genetic and epigenetic profiles of human induced pluripotent stem cells (hiPSCs) to human embryonic stem cells (hESCs) and yet the picture remains unclear. To address this,we derived a population of neural precursor cells (NPCs) from the H1 (WA01) hESC line and generated isogenic iPSC lines by reprogramming. The gene expression and methylation profile of three lines were compared to the parental line and intermediate NPC population. We found no gene probe with expression that differed significantly between hESC and iPSC samples under undifferentiated or differentiated conditions. Analysis of the global methylation pattern also showed no significant difference between the two PSC populations. Both undifferentiated populations were distinctly different from the intermediate NPC population in both gene expression and methylation profiles. One point to note is that H1 is a male line and so extrapolation to female lines should be cautioned. However,these data confirm our previous findings that there are no significant differences between hESCs and hiPSCs at the gene expression or methylation level. View Publication