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

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

Long-lived plasma cells are critical to humoral immunity as a lifelong source of protective antibodies. Antigen-activated B cells-with T-cell help-undergo affinity maturation within germinal centres and persist as long-lived IgG plasma cells in the bone marrow. Here we show that antigen-specific,induced IgM plasma cells also persist for a lifetime. Unlike long-lived IgG plasma cells,which develop in germinal centres and then home to the bone marrow,IgM plasma cells are primarily retained within the spleen and can develop even in the absence of germinal centres. Interestingly,their expressed IgV loci exhibit somatic mutations introduced by the activation-induced cytidine deaminase (AID). However,these IgM plasma cells are probably not antigen-selected,as replacement mutations are spread through the variable segment and not enriched within the CDRs. Finally,antibodies from long-lived IgM plasma cells provide protective host immunity against a lethal virus challenge. View Publication

Severe lupus often includes psychiatric and neurological sequelae,although the cellular contributors to CNS disease remain poorly defined. Using intravascular staining to discriminate tissue-localized from blood-borne cells,we find substantial accumulation of CD8+ T cells relative to other lymphocytes in brain tissue,which correlates with lupus disease and limited neuropathology. This is in contrast to all other affected organs,where infiltrating CD4+ cells are predominant. Brain-infiltrating CD8+ T cells represent an activated subset of those found in the periphery,having a resident-memory phenotype (CD69+CD122-PD1+CD44+CD62L-) and expressing adhesion molecules (VLA-4+LFA-1+) complementary to activated brain endothelium. Remarkably,infiltrating CD8+ T cells do not cause tissue damage in lupus-prone mice,as genetic ablation of these cells via $\beta$2 m deficiency does not reverse neuropathology,but exacerbates disease both in the brain and globally despite decreased serum IgG levels. Thus,lupus-associated inflammation disrupts the blood-brain barrier in a discriminating way biased in favor of non-pathogenic CD8+ T cells relative to other infiltrating leukocytes,perhaps preventing further tissue damage in such a sensitive organ. View Publication

The fate of pluripotent stem cells is tightly controlled during early embryonic development. Both the derivation and the maintenance of embryonic stem cells (ES cells) in vitro depend on feeder cell-derived growth factors that are largely unidentified. To dissect the mechanisms governing pluripotency,we conducted a screen to identify factors that are produced by mouse embryonic fibroblast STO cells and are required to maintain the pluripotency of ES cells. One of the factors is bone morphogenetic protein 4 (BMP4). Unexpectedly,the major effect of BMP4 on the self-renewal of ES cells is accomplished by means of the inhibition of both extracellular receptor kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways,and inhibitors of ERK and p38 MAPKs mimic the effect of BMP4 on ES cells. Importantly,inhibition of the p38 MAPK pathway by SB203580 overcomes the block in deriving ES cells from blastocysts lacking a functional Alk3,the BMP type IA receptor. These results uncover a paradigm for BMP signaling in the biology of pluripotent stem cells. View Publication

HIV-1 infected cells are eliminated in infected individuals by a variety of cellular mechanisms,the best characterized of which are cytotoxic T cell and NK cell-mediated killing. An additional antiviral mechanism is antibody-dependent cellular cytotoxicity. Here we use primary CD4(+) T cells infected with the BaL clone of HIV-1 as target cells and autologous NK cells,monocytes,and neutrophils as effector cells,to quantify the cytotoxicity mediated by the different effectors. This was carried out in the presence or absence of HIV-1-specific antiserum to assess antibody-dependent cellular cytotoxicity. We show that at the same effector to target ratio,NK cells and monocytes mediate similar levels of both antibody-dependent and antibody-independent killing of HIV-1-infected T cells. Neutrophils mediated significant antibody-dependent killing of targets,but were less effective than monocytes or NK cells. These data have implications for acquisition and control of HIV-1 in natural infection and in the context of vaccination. View Publication

Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately,with low efficiencies,from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model,elucidate,and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor,basic fibroblast growth factor,and bone morphogenetic protein 4 yields a higher percentage of CD31(+)CD34(+) cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media,these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells,respectively. Furthermore,we were able to expand the intermediate population more than a billion fold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations. View Publication

Self-renewing cell populations such as hematopoietic stem cells and memory B and T lymphocytes might be regulated by shared signaling pathways. The Wnt-beta-catenin pathway is an evolutionarily conserved pathway that promotes hematopoietic stem cell self-renewal and multipotency by limiting stem cell proliferation and differentiation,but its role in the generation and maintenance of memory T cells is unknown. We found that induction of Wnt-beta-catenin signaling by inhibitors of glycogen sythase kinase-3beta or the Wnt protein family member Wnt3a arrested CD8(+) T cell development into effector cells. By blocking T cell differentiation,Wnt signaling promoted the generation of CD44(low)CD62L(high)Sca-1(high)CD122(high)Bcl-2(high) self-renewing multipotent CD8(+) memory stem cells with proliferative and antitumor capacities exceeding those of central and effector memory T cell subsets. These findings reveal a key role for Wnt signaling in the maintenance of 'stemness' in mature memory CD8(+) T cells and have major implications for the design of new vaccination strategies and adoptive immunotherapies. View Publication

HIV causes a chronic infection characterized by depletion of CD4(+) T lymphocytes and the development of opportunistic infections. Despite drugs that inhibit viral spread,HIV infection has been difficult to cure because of uncharacterized reservoirs of infected cells that are resistant to highly active antiretroviral therapy (HAART) and the immune response. Here we used CD34(+) cells from infected people as well as in vitro studies of wild-type HIV to show infection and killing of CD34(+) multipotent hematopoietic progenitor cells (HPCs). In some HPCs,we detected latent infection that stably persisted in cell culture until viral gene expression was activated by differentiation factors. A unique reporter HIV that directly detects latently infected cells in vitro confirmed the presence of distinct populations of active and latently infected HPCs. These findings have major implications for understanding HIV bone marrow pathology and the mechanisms by which HIV causes persistent infection. View Publication

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs,but does not abrogate,V(D)J recombination activity. In spite of a severe block at the pro-B cell stage and profound B cell lymphopenia,significant serum levels of immunoglobulin (Ig) G,IgM,IgA,and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP-keyhole limpet hemocyanin were severely impaired,even after adoptive transfer of wild-type CD4(+) T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell-activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire,which is associated with defects in central and peripheral checkpoints of B cell tolerance,is an important,previously unrecognized,aspect of immunodeficiencies associated with hypomorphic RAG mutations. View Publication

More than 10 years after their first isolation,human embryonic stem cells are finally 'coming of age' in research and biotechnology applications as protocols for their differentiation and undifferentiated expansion in culture become robust and scalable,and validated commercial reagents become available. Production of human cardiomyocytes is now feasible on a daily basis for many laboratories with tissue culture expertise. An additional recent surge of interest resulting from the first production of human iPSCs (induced pluripotent stem cells) from somatic cells of patients now makes these technologies of even greater importance since it is likely that (genetic) cardiac disease phenotypes can be captured in the cardiac derivatives of these cells. Although cell therapy based on replacing cardiomyocytes lost or dysfunctional owing to cardiac disease are probably as far away as ever,biotechnology and pharmaceutical applications in safety pharmacology and drug discovery will probably impact this clinical area in the very near future. In the present paper,we review the cutting edge of this exciting area of translational research. View Publication

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