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

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here,we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs.backslashnbackslashnEXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points,day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C.backslashnbackslashnKEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2,TUBB III,PAX6,TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2,PITX2,GATA5,MYL4,TNNT2,COL1A1 and COL1A2. In addition,no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated.backslashnbackslashnCONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments. View Publication

SummaryBackgroundMacrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However,the production and application of genetically edited,highly effective,and mass-produced CAR-modified macrophages (CAR-Ms) are challenging.MethodsHere,we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach,together with a simple differentiation protocol,produced stable and highly effective CAR-Ms without heterogeneity.FindingsThese engineered cells phagocytosed cancer cells,leading to significant inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore,the engineered CARs,which incorporated a combination of CD3? and Megf10 (referred to as FRP5M?),markedly enhanced the antitumour effect of CAR-Ms by promoting M1,but not M2,polarisation. FRP5M? promoted M1 polarisation via nuclear factor kappa B (NF-?B),ERK,and STAT1 signalling,and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory signalling,inducing M1 polarisation of bystander non-CAR macrophages,and enhancing the infiltration of T cells in cancer spheroids.InterpretationOur findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion,the guided insertion of CAR containing CD3? and Megf10 domains is an effective strategy for the immunotherapy of solid tumours.FundingThis work was supported by KRIBB Research Initiative Program Grant (KGM4562431,KGM5282423) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT,10.13039/501100003625Ministry of Health and Welfare) (22A0304L1-01). 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

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

Acute and chronic solid organ failures are costly disease processes with high mortality rates. Inflammation plays a central role in both acute and chronic organ failure,including heart,lung and kidney. In this regard,new therapies for these disorders have focused on inhibiting the mediators of inflammation,including cytokines and free radicals,with little or no success in clinical studies. Recent novel treatment strategies have been directed to cell-based rather than mediator-based approaches,designed to immunomodulate the deleterious effects of inflammation on organ function. One approach,cell therapy,replaces cells that were damaged in the acute or chronic disease process with stem/progenitor technology,to rebalance excessive inflammatory states. As an example of this approach,the use of an immunomodulatory role of renal epithelial progenitor cells to treat acute renal failure (ARF) and multiorgan failure arising from acute kidney injury is reviewed. A second therapeutic pathway,cell processing,does not incorporate stem/progenitor cells in the device,but rather biomimetic materials that remove and modulate the primary cellular components,which promote the worsening organ tissue injury associated with inflammation. The use of an immunomodulating leukocyte selective cytopheretic inhibitory device is also reviewed as an example of this cell processing approach. Both of these unconventional strategies have shown early clinical efficacy in pilot clinical trials and may transform the therapeutic approach to organ failure disorders. View Publication

A network of transcription factors (TFs) determines cell identity,but identity can be altered by overexpressing a combination of TFs. However,choosing and verifying combinations of TFs for specific cell differentiation have been daunting due to the large number of possible combinations of 2,000 TFs. Here,we report the identification of individual TFs for lineage-specific cell differentiation based on the correlation matrix of global gene expression profiles. The overexpression of identified TFs-Myod1,Mef2c,Esx1,Foxa1,Hnf4a,Gata2,Gata3,Myc,Elf5,Irf2,Elf1,Sfpi1,Ets1,Smad7,Nr2f1,Sox11,Dmrt1,Sox9,Foxg1,Sox2,or Ascl1-can direct efficient,specific,and rapid differentiation into myocytes,hepatocytes,blood cells,and neurons. Furthermore,transfection of synthetic mRNAs of TFs generates their appropriate target cells. These results demonstrate both the utility of this approach to identify potent TFs for cell differentiation,and the unanticipated capacity of single TFs directly guides differentiation to specific lineage fates. View Publication

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear,as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2,and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38,as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation,and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise,NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET,the positive regulator of IFN-gamma,and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC,and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively,our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC,and these effects are mediated via SMAD3. View Publication

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