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

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure–function. This includes studies centered on disease etiology,drug development,and for potential clinical applications in heart regeneration/repair. Ultimately,for these applications to achieve success,a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state,and this represents a major hurdle for the field. To address this roadblock,we have developed a hiPSC-CMs (?-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere,the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile,the molecular motor of the heart,which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of ?-MyHC in ?-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis,we used gene editing with an inducible ?-MyHC expression cassette into isogeneic hiPSC-CMs,and separately by gene transfer,and then investigated the direct effects of increased ?-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with ?-MyHC. Positive inotropy and relaxation was evident in comparison to ?-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening,as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration,repair and re-muscularization applications. View Publication

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases,including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs,most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets,may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes,a human β cell line and human islet β cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet β cells. In exploring subsets and mechanisms that may explain this pattern,we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39− CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly,β cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased β cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet β cells in the setting of CAR immunotherapy. In summary,introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction. View Publication

CD8 + cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide–MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell–cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell–cell contact,promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux,providing the necessary energy through oxidative phosphorylation,and its own protein translation on 55S ribosomes. However,the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here,we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton,mitochondria,and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. View Publication

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS,but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore,we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2,KIF5A,NEK1,TBK1,and TUBA4A) which highlighted many novel binding partners,and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system,and of ALS-associated C21ORF2 variants is largely unknown. Therefore,we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First,our data show C21ORF2 expression in ALS-relevant mouse and human neurons,such as spinal and cortical motor neurons. Further,the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients,but not isogenic controls,show increased apoptosis,and changes in DNA damage response,mitochondria and neuronal excitability. In addition,C21ORF2-V58L induced post-transcriptional downregulation of NEK1,an ALS-associated protein implicated in apoptosis and DDR. In all,our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS. The online version contains supplementary material available at 10.1186/s40478-024-01852-6. View Publication

Extracellular vesicles (EVs) derived from human organoids are phospholipid bilayer-bound nanoparticles that carry therapeutic cargo. However,the low yield of EVs remains a critical bottleneck for clinical translation. Vertical-Wheel bioreactors (VWBRs),with unique design features,facilitate the scalable production of EVs secreted by human blood vessel organoids (BVOs) under controlled shear stress,using aggregate- and microcarrier-based culture systems. Human induced pluripotent stem cell-derived BVOs cultured as aggregates or on Synthemax II microcarriers within VWBRs (40 and 80 rpm) were compared to static controls. The organoids were characterized by metabolite profiling,flow cytometry,and gene expression of EV biogenesis markers. EVs were characterized by nanoparticle tracking analysis,electron microscopy,and Western blotting. Lipidomics provided insights into EV lipid composition,while functional assays assessed the impact of EVs in a D-galactose-induced senescence model. VWBR cultures showed more aerobic metabolism and higher expression of EV biogenesis genes compared to the static control. EVs from different conditions were comparable in size,but the yields were significantly higher for microcarrier and dynamic cultures than static aggregates. Lipidomic profiling revealed minimal variation (< 0.36%) in total lipid content; however,distinct differences were identified in lipid chain lengths and saturation levels,affecting key pathways such as sphingolipid and neurotrophin signaling. Human BVO EVs demonstrated the abilities of reducing oxidative stress and increasing cell proliferation in vitro. Human BVOs differentiated in VWBRs (in particular 40 rpm) produce 2–3 fold higher yield of EVs (per mL) than static control. The bio manufactured EVs from VWBRs have exosomal characteristics and therapeutic cargo,showing functional properties in in vitro assays. This innovative approach establishes VWBRs as a scalable platform for producing functional EVs with defined lipid profiles and therapeutic potential,paving the way for future in vivo studies. The online version contains supplementary material available at 10.1186/s13287-025-04317-2. View Publication

Chalcones are precursor compounds for flavonoid synthesis in plants,and they can also be synthesized in laboratory. Previous study has documented some of the pharmacological applications of these compounds. Estrogen has long been associated with the initiation and promotion of breast cancer. Inhibiting estrogen synthesis can be effective in the prevention and treatment of the disease. Since most breast cancers received estrogen supplied from local tissues,we employed a breast cancer cell line expressing aromatase to screen for the inhibitory potentials of five hydroxychalcones,i.e. 2-hydroxychalcone,2'-hydroxychalcone,4-hydroxychalcone,4,2',4'-trihydroxy-chalcone (isoquiritigenin),3,4,2',4'-tetrahydroxychalcone (butein). In the preliminary results,butein was found to be the strongest inhibitor among the tested compounds,and its IC(50) value was 3.75 microM. Subsequent enzyme kinetic study revealed that butein acted on aromatase with a mixed type of inhibition and the K(i) value was determined to be 0.32 microM. Cell proliferation assay indicated that the cell number increased by 10 nM-testosterone treatment was significantly reduced by 5 microM butein,and the administration of flutamide could not reverse the effect. The present study illustrated that butein was an aromatase inhibitor and a potential natural alternative for the chemoprevention or therapy of breast cancer. View Publication

α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet,taken up by cells and tissues,and subsequently reduced to dihydrolipoic acid (DHLA). Recently,DHLA has been used as the hydrophilic nanomaterial preparations,and therefore,determination of its bio-safety profile is essential. In this article,we show that DHLA (50-100 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5),but exerts no injury effects at treatment dosages below 50 μM. Higher concentrations of DHLA (50-100 μM) directly increased the reactive oxygen species (ROS) content in ESC-B5 cells,along with a significant increase in cytoplasmic free calcium and nitric oxide (NO) levels,loss of mitochondrial membrane potential (MMP),activation of caspases-9 and -3,and cell death. Pretreatment with NO scavengers suppressed the apoptotic biochemical changes induced by 100 μM DHLA and promoted the gene expression levels of p53 and p21 involved in apoptotic signaling. Our results collectively indicate that DHLA at concentrations of 50-100 μM triggers apoptosis of ESC-B5 cells,which involves both ROS and NO. Importantly,at doses of less than 50 μM (0-25 μM),DHLA does not exert hazardous effects on ESC-B5 cell properties,including viability,development and differentiation. These results provide important information in terms of dosage safety and biocompatibility of DHLA to facilitate its further use as a precursor for biomaterial preparation. View Publication

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation,a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation,as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data,possibly due to their crosstalk with Notch Signaling. Overall,this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs. View Publication

The sub-population of tumor cells termed 'cancer stem cells' (CSCs) possess the capability to generate tumors,undergo epithelial-mesenchymal transition (EMT) and are implicated in metastasis,making treatments to specifically target CSCs an attractive therapeutic strategy. Tumor hypoxia plays a key role in regulating EMT and cancer stem cell function. Carbonic anhydrase IX (CAIX) is a hypoxia-inducible protein that regulates cellular pH to promote cancer cell survival and invasion in hypoxic microenvironments and is a biomarker of poor prognosis for breast cancer metastasis and survival. Here,we demonstrate that inhibition of CAIX expression or activity with novel small-molecule inhibitors in breast cancer cell lines,or in primary metastatic breast cancer cells,results in the inhibition of breast CSC expansion in hypoxia. We identify the mTORC1 axis as a critical pathway downstream of CAIX in the regulation of cancer stem cell function. CAIX is also required for expression of EMT markers and regulators,as well as drivers of 'stemness',such as Notch1 and Jagged1 in isolated CSCs. In addition,treatment of mice bearing orthotopic breast tumors with CAIX-specific small-molecule inhibitors results in significant depletion of CSCs within these tumors. Furthermore,combination treatment with paclitaxel results in enhanced tumor growth delay and eradication of lung metastases. These data demonstrate that CAIX is a critical mediator of the expansion of breast CSCs in hypoxic niches by sustaining the mesenchymal and 'stemness' phenotypes of these cells,making CAIX an important therapeutic target for selectively depleting breast CSCs. View Publication

Prominin-1 (CD133) is commonly used to isolate stem and progenitor cells from the developing and adult nervous system and to identify cancer stem cells in brain tumors. However,despite extensive characterization of Prominin-1(+) precursor cells from the adult subventricular zone,no information about the expression of Prominin-1 by precursor cells in the subgranular zone (SGZ) of the adult hippocampus has been available. We show here that Prominin-1 is expressed by a significant number of cells in the SGZ of adult mice in vivo and ex vivo,including postmitotic astrocytes. A small subset of Prominin-1(+) cells coexpressed the nonspecific precursor cell marker Nestin as well as GFAP and Sox2. Upon fluorescence-activated cell sorting,only Prominin-1/Nestin double-positive cells fulfilled the defining stem cell criteria of proliferation,self-renewal,and multipotentiality as assessed by a neurosphere assay. In addition,isolated primary Prominin-1(+) cells preferentially migrated to the neurogenic niche in the SGZ upon transplantation in vivo. Finally,despite its expression by various stem and progenitor cells,Prominin-1 turned out to be dispensable for precursor cell proliferation in vitro and in vivo. Nevertheless,a net decrease in hippocampal neurogenesis,by ∼30% was found in Prominin-1 knock-out mice,suggesting other roles in controlling adult hippocampal neurogenesis. Remarkably,an upregulation of Prominin-2 was detected in Prominin-1-deficient mice highlighting a potential compensatory mechanism,which might explain the lack of severe symptoms in individuals carrying mutations in the Prom1 gene. View Publication

Aldehyde dehydrogenase (ALDH) has been identified in stem cells from both normal and cancerous tissues. This study aimed to evaluate the potential of ALDH as a universal brain tumour initiating cell (BTIC) marker applicable to primary brain tumours and their biological role in maintaining stem cell status. Cells from various primary brain tumours (24paediatric and 6 adult brain tumours) were stained with Aldefluor and sorted by flow cytometry. We investigated the impact of ALDH expression on BTIC characteristics in vitro and on tumourigenic potential in vivo. Primary brain tumours showed universal expression of ALDH,with 0.3-28.9% of the cells in various tumours identified as ALDH(+). The proportion of CD133(+) cells within ALDH(+) is higher than ALDH cells. ALDH(+) cells generate neurospheres with high proliferative potential,express neural stem cell markers and differentiate into multiple nervous system lineages. ALDH(+) cells tend to show high expression of induced pluripotent stem cell-related genes. Notably,targeted knockdown of ALDH1 by shRNA interference in BTICs potently disturbed their self-renewing ability. After 3months,ALDH(+) cells gave rise to tumours in 93% of mice whereas ALDH cells did not. The characteristic pathology of mice brain tumours from ALDH(+) cells was similar to that of human brain tumours,and these cells are highly proliferative in vivo. Our data suggest that primary brain tumours contain distinct subpopulations of cells that have high expression levels of ALDH and BTIC characteristics. ALDH might be a potential therapeutic target applicable to primary brain tumours. View Publication