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

Mesenchymal stem cells (MSCs) have been isolated from various mesodermal and ectodermal tissues. While the phenotypic and functional heterogeneity of MSCs stemming from their developmental origins has been acknowledged,the genetic and environmental factors underpinning these differences are not well-understood. Here,we investigated whether substrate stiffness mediated mechanical cues can directly modulate the development of ectodermal MSCs (eMSCs) from a precursor human neural crest stem cell (NCSC) population. We showed that NCSC-derived eMSCs were transcriptionally and functionally distinct from mesodermal bone marrow MSCs. eMSCs derived on lower substrate stiffness specifically increased their expression of the MSC marker,CD44 in a Rho-ROCK signaling dependent manner,which resulted in a concomitant increase in the eMSCs' adipogenic and chondrogenic differentiation potential. This mechanically-induced effect can only be maintained for short-term upon switching back to a stiff substrate but can be sustained for longer-term when the eMSCs were exclusively maintained on soft substrates. We also discovered that CD44 expression modulated eMSC self-renewal and multipotency via the downregulation of downstream platelet-derived growth factor receptor beta (PDGFRbeta$) signaling. This is the first instance demonstrating that substrate stiffness not only influences the differentiation trajectories of MSCs but also their derivation from upstream progenitors,such as NCSCs. View Publication

Measles virus envelope pseudotyped LV (MV-LV) can achieve high B cell transduction rates (up to 50%),but suffers from low titers. To overcome current limitations,we developed an optimized MV-LV production protocol that achieved consistent B cell transduction efficiency up to 75%. We detail this protocol along with analytical assays to assess the results of MV-LV mediated B cell transduction,including flow cytometry for B cell phenotypic characterization and measurement of transduction efficiency,and ddPCR for VCN analysis. View Publication

During a heart attack,ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment,thereby shifting focus to cell-free therapies. Consequently,we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature,efficacy in previous studies,and easy obtainability from minimally invasive techniques. Nonetheless,using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome,regardless of donor health,would improve cardiovascular outcomes in the CM model of ischemia–reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors,passaged 6–10 times,underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content,independent of donor health status,had elevated expression of proteins involved in cell survival pathways. In IR conditions,iPSC-derived secretome increased cell survival as measured by metabolic activity (p < 0.05),cell viability (p < 0.001),and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently,only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p < 0.01). Heretofore,secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences. View Publication

Palmitic acid (PA) is the most common dietary saturated fatty acid,and is abundant in palm and cottonseed oil,butter,and cheese,whereas oleic acid (OA) is a monounsaturated omega-9 fatty acid found in olive oil. The differences in the cytotoxic and pro-inflammatory effects of PA and OA across endothelial cells (ECs) isolated from different vascular beds have not been investigated in detail. Here,we incubated primary human aortic valve (HAVEC),saphenous vein (HSaVEC),internal thoracic artery (HITAEC),and microvascular (HMVEC) ECs with albumin-bound PA or OA for 24 h and found that PA induced a considerable cytotoxic response,accompanied by an elevated expression of the genes encoding cell adhesion molecules (VCAM1,ICAM1,SELE,and SELP) and pro-inflammatory cytokines (MIF,PTX3,CSF2,CSF3,IL1A,IL6,CCL2,CCL5,CCL20,CSF2,CSF3,CXCL1,CXCL2,CXCL3,CXCL5,CXCL6,CXCL8,and CXCL10),followed by an increased release of interleukin-6 and interleukin-8. HAVEC and HSaVEC were more susceptible to PA,whereas OA had mild-to-moderate cytotoxic effects on HAVEC and HMVEC but did not induce generalized EC activation. Compared with other EC types,HITAEC was the most resistant to PA and OA treatment. Collectively,these results indicate considerable heterogeneity across the ECs of distinct origin in response to PA. View Publication

Histone lysine methylation is an important epigenetic mark that regulates gene expression and chromatin organization. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by methylating histone H3 Lys9. BIX-01294 was originally identified as a G9a inhibitor during a chemical library screen of small molecules and has previously been used in the generation of induced pluripotent stem cells. Here we present the crystal structure of the catalytic SET domain of GLP in complex with BIX-01294 and S-adenosyl-L-homocysteine. The inhibitor is bound in the substrate peptide groove at the location where the histone H3 residues N-terminal to the target lysine lie in the previously solved structure of the complex with histone peptide. The inhibitor resembles the bound conformation of histone H3 Lys4 to Arg8,and is positioned in place by residues specific for G9a and GLP through specific interactions. View Publication

The spatial organization of molecules in a cell is essential for their functions. While current methods focus on discerning tissue architecture,cell–cell interactions,and spatial expression patterns,they are limited to the multicellular scale. We present Bento,a Python toolkit that takes advantage of single-molecule information to enable spatial analysis at the subcellular scale. Bento ingests molecular coordinates and segmentation boundaries to perform three analyses: defining subcellular domains,annotating localization patterns,and quantifying gene–gene colocalization. We demonstrate MERFISH,seqFISH +,Molecular Cartography,and Xenium datasets. Bento is part of the open-source Scverse ecosystem,enabling integration with other single-cell analysis tools.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03217-7. View Publication

Baculovirus,an insect virus commonly used for recombinant protein expression in insect cells and gene delivery in mammalian systems,is often generated through bacmid-based engineering. To enable flexible and programmable bacmid engineering,we developed SHOT 2.0,an optimized CRISPR-associated transposon platform that mediates RNA-guided and customized bacmid editing in Escherichia coli. The edited bacmid can be transfected into insect cells to produce recombinant baculoviruses. SHOT 2.0 supported site-specific integration of large DNA cargos (at least 14 kb) into defined loci such as v-cath and ODVe56,with integration at ODVe56 markedly improving transgene stability during serial virus passaging. The system is fully compatible with the Bac-to-Bac® workflow,enabling dual-gene insertion into the bacmid and derived baculovirus. Leveraging this platform,we constructed an all-in-one baculovirus encoding the PE5max prime editor. This vector-mediated prime editing achieves efficiencies up to 85.6% in HEK293T cells and achieves robust prime editing in hard-to-transfect cell types,including iPSCs and liver cancer cells,with efficiencies up to 37.1%. These results demonstrate that SHOT 2.0 substantially expands the baculovirus engineering toolbox,providing a flexible platform for genome editing and future gene delivery. View Publication

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15,a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients,significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues,and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors,we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore,STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM,53BP1,and MDC1. Furthermore,protein levels of these DNA damage response molecules are reduced by IL-15,as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally,pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients. View Publication

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