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

Many respiratory viruses attack the airway epithelium and cause a wide spectrum of diseases for which we have limited therapies. To date,a few primary human stem cell-based models of the proximal airway have been reported for drug discovery but scaling them up to a higher throughput platform remains a significant challenge. As a result,most of the drug screening assays for respiratory viruses are performed on commercial cell line-based 2D cultures that provide limited translational ability. We optimized a primary human stem cell-based mucociliary airway epithelium model of SARS-CoV-2 infection,in 96-well air–liquid-interface (ALI) format,which is amenable to moderate throughput drug screening. We tested the model against SARS-CoV-2 parental strain (Wuhan) and variants Beta,Delta,and Omicron. We applied this model to screen 2100 compounds from targeted drug libraries using a high throughput-high content image-based quantification method. The model recapitulated the heterogeneity of infection among patients with SARS-CoV-2 parental strain and variants. While there were heterogeneous responses across variants for host factor targeting compounds,the two direct-acting antivirals we tested,Remdesivir and Paxlovid,showed consistent efficacy in reducing infection across all variants and donors. Using the model,we characterized a new antiviral drug effective against both the parental strain and the Omicron variant. This study demonstrates that the 96-well ALI model of primary human mucociliary epithelium can recapitulate the heterogeneity of infection among different donors and SARS-CoV-2 variants and can be used for moderate throughput screening. Compounds that target host factors showed variability among patients in response to SARS-CoV-2,while direct-acting antivirals were effective against SARS-CoV-2 despite the heterogeneity of patients tested. View Publication

The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However,investigators report studies of MSC using different methods of isolation and expansion,and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes,which hinders progress in the field. To begin to address this issue,the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First,MSC must be plastic-adherent when maintained in standard culture conditions. Second,MSC must express CD105,CD73 and CD90,and lack expression of CD45,CD34,CD14 or CD11b,CD79alpha or CD19 and HLA-DR surface molecules. Third,MSC must differentiate to osteoblasts,adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds,we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators. View Publication

Human sensory neurons are inaccessible for functional examination,and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell-derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro,we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor-specific genes and convert mechanical stimuli into electrical signals,their most salient characteristic in vivo. Strikingly,mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors,which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity. View Publication

Despite significant advances in commercially available media and kits and the differentiation approaches for human neural stem cell (NSC) generation,NSC production from the differentiation of human pluripotent stem cell (hPSC) is complicated by its time-consuming procedure,complex medium composition,and purification step. In this study,we developed a convenient and simplified NSC production protocol to meet the demand of NSC production. We demonstrated that NSCs can be generated efficiently without requirement of specific small molecules or embryoid body formation stage. Our experimental results suggest that a short suspension culture period may facilitate ectoderm lineage specification rather than endoderm or mesoderm lineage specification from hPSCs. The method developed in this study shortens the turnaround time of NSC production from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) differentiation. It provides a straightforward and useful strategy for generating NSCs that can benefit a wide range of research applications for human brain research. View Publication

There are several different technical approaches to the isolation of hematopoietic stem cells (HSCs) with long-term repopulating ability,but these have problems in terms of yield,complexity,or cell viability. Simpler strategies for HSC isolation are needed. We have enriched primitive hematopoietic progenitors from murine bone marrow of mice from different genetic backgrounds by lineage depletion followed by selection of cells with high aldehyde dehydrogenase activity using the Aldefluor reagent (BD Biosciences,Oxford,U.K.). Lin- ALDH(bright) cells comprised 26.8 +/- 1.0% of the total Lin- population of C57BL6 mice,and 23.5 +/- 1.0% of the Lin- population of BALB/c mice expressed certain cell-surface markers typical of primitive hematopoietic progenitors. In vitro hematopoietic progenitor function was substantially higher in the Lin- ALDH(bright) population compared with the Lin- ALDH(low) cells. These cells have higher telomerase activity and the lowest percentage of cells in S phase. These data strongly suggest that progenitor enrichment from Lin- cells on the basis of ALDH is a valid method whose simplicity of application makes it advantageous over conventional separations. View Publication

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here,we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro,both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing β-thalassemic phenotype. In an immunodeficient female mouse model,transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease. Subject terms: Targeted gene repair,Sickle cell disease View Publication

The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations,and their functional consequences. Here,single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated,including G-CSF. The G-CSF response was physiologically relevant-driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114(neg/lo) compartment,and almost completely absent in the CD114(pos) subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes,and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states. View Publication

Cytotoxic chemotherapies could cause the dysregulation of hematopoiesis and even put patients at increased risk of hematopoietic malignancy. Therapy-related leukemia is mainly caused by cytotoxic chemotherapy-induced genetic mutations in hematopoietic stem/progenitor cells (HSPCs). In addition to the intrinsic mechanism,some extrinsic events occurring in the bone marrow (BM) microenvironment are also possible mechanisms involved in genetic alteration. In the present study,we investigated the damage to BM stromal cells induced by a chemotherapy drug,daunorubicin (DNR) and further identified the DNA damage in hematopoietic cells caused by drug-treated stromal cells. It was found that treatment with DNR in mice caused a temporary reduction in cell number in each BM stromal cell subpopulation and the impairment of clonal growth potential in BM stromal cells. DNR treatment led to a tendency of senescence,generation of intracellular reactive oxygen species,production of cytokines and chemokines,and dysfunction of mitochondrial in stromal cells. Transcriptome microarray data and gene ontology (GO) or gene set enrichment analysis (GSEA) showed that differentially expressed genes that were down-regulated in response to DNR treatment were significantly enriched in mitochondrion function,and negative regulators of reactive oxygen species. Surprisingly,it was found that DNR-treated stromal cells secreted high levels of H2O2 into the culture supernatant. Furthermore,coculture of hematopoietic cells with DNR-treated stromal cells led to the accumulation of DNA damage as determined by the levels of histone H2AX phosphorylation and 8-oxo-2'-deoxyguanosine in hematopoietic cells. Overall,our results suggest that DNR-induced BM stromal cell damage can lead to genomic instability in hematopoietic cells. View Publication

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases,both to study them and to explore therapies. However,a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study,we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV),adenoviruses and lentiviral vectors in hESC,hiPSC,and the derived cardiomyocytes. In undifferentiated cells,adenoviral and lentiviral vectors were superior,whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes,1,2,6,and 9,of which 2 and 6 were superior in their transduction efficiency. Interestingly,we observed that AAVs severely diminished the viability of undifferentiated cells,an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore,we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally,adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion,adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines,whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells. View Publication

SummaryInterleukin-33 (IL-33) is an immunoregulatory cytokine that moderately suppresses experimental autoimmune encephalomyelitis (EAE),a murine model of multiple sclerosis (MS). However,poor pharmacokinetics and toxicity hinder its clinical translation. To address these limitations,we develop an activity-attenuated IL-33 by recombinant fusion to serum albumin (SA). SA-IL-33 exhibits reduced toxicity and prolonged residence in the secondary lymphoid organs (SLOs),sites of T cell priming in autoimmunity,compared to wild-type (WT) IL-33. Prophylactic SA-IL-33 administration prevents EAE with superior efficacy to WT IL-33 and comparable efficacy to fingolimod (FTY720),a Food and Drug Administration (FDA)-approved MS drug. Therapeutic SA-IL-33 treatment also reduces disease severity in both chronic and relapsing-remitting EAE. SA-IL-33 modulates immunity in EAE by suppressing CD45+ cell infiltration (including myelin-reactive T helper 17 [TH17] cells) in the spinal cord,while expanding type 2 immune cells (including type 2 innate lymphoid cells [ILC2s],ST2+ regulatory T cells [Tregs],T helper 2 [TH2] cells,and M2-polarized macrophages) in the SLOs. These findings suggest that SA-IL-33 is a promising therapeutic for neuroinflammatory diseases. Graphical abstract Highlights•Fusion of serum albumin (SA) to interleukin-33 (IL-33) attenuates its activity and toxicity•Engineered SA-IL-33 exhibits prolonged residence in the secondary lymphoid organs (SLOs)•SA-IL-33 treatment both prevents the onset of and reduces established neuroinflammation in mice•Cytokine therapy suppresses TH17 cells in the CNS and promotes immunoregulation in the SLOs The clinical utility of interleukin-33 is hindered by poor pharmacokinetics and toxicity. Budina et al. develop a fusion of serum albumin and interleukin-33 (SA-IL-33) with reduced toxicity and prolonged lymph node residence. SA-IL-33 prevents the onset of and suppresses established inflammation-mediated paralysis in mice,demonstrating promise as a therapeutic for neuroinflammatory diseases. View Publication