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

The hematopoietic stem cell (HSC) gives rise to all mature,terminally differentiated cells of the blood. Here we show that calmodulin-dependent protein kinase IV (CaMKIV) is present in c-Kit+ ScaI+ Lin(-/low) hematopoietic progenitor cells (KLS cells) and that its absence results in hematopoietic failure,characterized by a diminished KLS cell population and by an inability of these cells to reconstitute blood cells upon serial transplantation. KLS cell failure in the absence of CaMKIV is correlated with increased apoptosis and proliferation of these cells in vivo and in vitro. In turn,these cell biological defects are correlated with decreases in CREB-serine 133 phosphorylation as well as in CREB-binding protein (CBP) and Bcl-2 levels. Re-expression of CaMKIV in Camk4-/- KLS cells results in the rescue of the proliferation defects in vitro as well as in the restoration of CBP and Bcl-2 to wild type levels. These studies show that CaMKIV is a regulator of HSC homeostasis and suggest that its effects may be in part mediated via regulation of CBP and Bcl-2. View Publication

In recent years,immunotherapy has shown considerable promise in the management of several malignancies. However,the majority of preclinical studies have been conducted in rodents,the results of which often translate poorly to patients given the substantial differences between murine and human immunology. As the porcine immune system is far more analogous to that of humans,pigs may serve as a supplementary preclinical model for future testing of such therapies. We have generated the genetically modified Oncopig with inducible tumor formation resulting from concomitant KRAS(G12D) and TP53(R167H) mutations under control of an adenoviral vector Cre-recombinase (AdCre). The objective of this study was to characterize the tumor microenvironment in this novel animal model with respect to T-cell responses in particular and to elucidate the potential use of Oncopigs for future preclinical testing of cancer immunotherapies. In this study,we observed pronounced intratumoral T-cell infiltration with a strong CD8$\beta$(+) predominance alongside a representation of highly differentiated $\gamma$$\delta$ T cells. The infiltrating CD8$\beta$(+) T cells displayed increased expression of the cytotoxic marker perforin when compared with the peripheral T-cell pool. Similarly,there was robust granzyme B staining localizing to the tumors; affirming the presence of cytotoxic immune cells within the tumor. In parallel with this antitumor immune response,the tumors displayed enrichment in FOXP3-expressing T cells and increased gene expression of indoleamine 2,3-dioxygenase 1 (IDO1),cytotoxic T-lymphocyte-associated protein 4 (CTLA4),and programmed death-ligand 1 (PDL1). Finally,we investigated the Oncopig immune system in mediating antitumor immunity. We observed pronounced killing of autologous tumor cells,which demonstrates the propensity of the Oncopig immune system to recognize and mount a cytotoxic response against tumor cells. Together,these findings suggest innate and adaptive recognition of the induced tumors with a concomitant in vivo suppression of T-cell effector functions. Combined,the data support that the Oncopig may serve as a valuable model for future preclinical testing of immunotherapies aimed at reactivating tumor-directed cytotoxicity in vivo. View Publication

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors,and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions,yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore,we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1,and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors,leading to target cell death. View Publication

AbstractObjectiveNatural killer (NK) cells are the largest innate lymphocyte subset with potent antitumour and antiviral functions. However,clinical utilisation of human NK cells is hampered due to a lack of reliable methods to augment their antitumour potential. We demonstrated technology in which human NK cells were cocultured with osteoclasts in the presence of probiotic bacteria. This approach significantly augmented the antitumour cytotoxicity and polyfunctionality of human NK cells,resulting in the generation of supercharged NK (sNK) cells.Methods and analysisWe explored the proteomic,transcriptomic and functional characterisation of sNK cells using cell imaging,flow cytometric analysis,51-chromium release cytotoxicity assay,ELISA,ELIspot,IsoPLexis single-cell secretome analysis,proteomic analysis,RNA analysis,western blot and enzyme kinetics.ResultsWe found that sNK cells were less susceptible to split anergy and tumour-induced exhaustion. Proteomic analyses revealed that sNK cells significantly increased their cell motility and proliferation. Single-cell transcriptomes uncovered sNK cells undertaking a unique differentiation trajectory and turning on STAT1,JUN,BHLHE40,ELF1,MAX and MYC regulons essential for augmenting antitumour effector functions and proliferation,respectively. Both proteomic and single-cell transcriptomes revealed that an increase in Cathepsin C helped to augment the quantity and function of Granzyme B.ConclusionsThese results support that this unique method produces potent NK cells for clinical utilisation and delineate the molecular mechanisms associated with this process. View Publication

Adult bone marrow-derived cells can participate in muscle regeneration after bone marrow transplantation. In recent studies a single hematopoietic stem cell (HSC) was shown to give rise to cells that not only reconstituted all of the lineages of the blood,but also contributed to mature muscle fibers. However,the relevant HSC derivative with this potential has not yet been definitively identified. Here we use fluorescence-activated cell sorter-based protocols to test distinct hematopoietic fractions and show that only fractions containing c-kit(+) immature myelomonocytic precursors are capable of contributing to muscle fibers after i.m. injection. Although these cells belong to the myeloid lineage,they do not include mature CD11b(+) myelomonocytic cells,such as macrophages. Of the four sources of mature macrophages tested that were derived either from monocytic culture,bone marrow,peripheral blood after granulocyte colony-stimulating factor mobilization,or injured muscle,none contributed to muscle. In addition,after transplantation of bone marrow isolated from CD11b-Cre-transgenic mice into the Cre-reporter strain (Z/EG),no GFP myofibers were detected,demonstrating that macrophages expressing CD11b do not fuse with myofibers. Irrespective of the underlying mechanisms,these data suggest that the HSC derivatives that integrate into regenerating muscle fibers exist in the pool of hematopoietic cells known as myelomonocytic progenitors. View Publication

We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation,and by day 6,more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1,GATA-3,and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis,indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors,including interleukin-3 (IL-3),IL-1,IL-6,IL-11,erythropoietin,and Kit ligand. At days 10 and 14 of differentiation,EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first,approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next,and precursors of the mast cell lineage develop last. The kinetics of precursor development,as well as the growth factor responsiveness of these early cells,is similar to that found in the yolk sac and early fetal liver,indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo. View Publication

The mammalian embryonic zeta-globin genes,including that of humans,are expressed at the early embryonic stage and then switched off during erythroid development. This autonomous silencing of the zeta-globin gene transcription is probably regulated by the cooperative work of various protein-DNA and protein-protein complexes formed at the zeta-globin promoter and its upstream enhancer (HS-40). We present data here indicating that a protein-binding motif,ZF2,contributes to the repression of the HS-40-regulated human zeta-promoter activity in erythroid cell lines and in transgenic mice. Combined site-directed mutagenesis and EMSA suggest that repression of the human zeta-globin promoter is mediated through binding of the zinc finger factor RREB1 to ZF2. This model is further supported by the observation that human zeta-globin gene transcription is elevated in the human erythroid K562 cell line or the primary erythroid culture upon RNA interference (RNAi)(2) knockdown of RREB1 expression. These data together suggest that RREB1 is a putative repressor for the silencing of the mammalian zeta-globin genes during erythroid development. Because zeta-globin is a powerful inhibitor of HbS polymerization,our experiments have provided a foundation for therapeutic up-regulation of zeta-globin gene expression in patients with severe hemoglobinopathies. View Publication

The generation of liquefied poly-ɛ-caprolactone (PCL) droplets by means of a microfluidic device results in uniform-sized microspheres,which are validated as microcarriers for human embryonic stem cell culture. Formed droplet size and size distribution,as well as the resulting PCL microsphere size,are correlated with the viscosity and flow rate ratio of the dispersed (Q d) and continuous (Q c) phases. PCL in dichloromethane increases its viscosity with concentration and molecular weight. Higher viscosity and Q d/Q c lead to the formation of larger droplets,within two observed formation modes: dripping and jetting. At low viscosity of dispersed phase and Q d/Q c,the microfluidic device is operated in dripping mode,which generates droplets and microspheres with greater size uniformity. Solutions with lower molecular weight PCL have lower viscosity,resulting in a wider concentration range for the dripping mode. When coated with extracellular matrix (ECM) proteins,the fabricated PCL microspheres are demonstrated capable of supporting the expansion of human embryonic stem cells. View Publication

SummaryThe medial ganglionic eminence (MGE) gives rise to parvalbumin (PV)- and somatostatin (SST)-expressing cortical interneurons essential for regulating cortical excitability. Although PV interneurons are linked to various neurodevelopmental and neurodegenerative disorders,reliably generating them from human pluripotent stem cells (hPSCs) has been extremely challenging. We present a robust,reproducible protocol for generating single-rosette MGE organoids (MGEOs) from hPSCs. Transcriptomic analyses reveal that MGEOs exhibit MGE regional identity and faithfully model the developing human fetal MGE. As MGEOs mature,they generate abundant PV-expressing cortical interneurons,including putative basket and axoaxonic cells,at a scale not previously achieved in vitro. When fused with hPSC-derived cortical organoids,these interneurons rapidly migrate into cortical regions,integrate into excitatory networks,and contribute to complex electrophysiological patterns and the emergence of large numbers of fast-spiking neurons. MGEOs thus offer a powerful in vitro approach for probing human MGE-lineage cortical and subcortical GABAergic neuron development,modeling various neuropsychiatric disorders,and advancing cell-based therapies for neurodevelopmental and neurodegenerative disorders. Graphical abstract View Publication

Vascular abnormalities are a common component of eye diseases that often lead to vision loss. Vaso-obliteration is associated with inherited retinal degenerations,since photoreceptor atrophy lowers local metabolic demands and vascular support to those regions is no longer required. Given the degree of neurovascular crosstalk in the retina,it may be possible to use one cell type to rescue another cell type in the face of severe stress,such as hypoxia or genetically encoded cell-specific degenerations. Here,we show that intravitreally injected human endothelial colony-forming cells (ECFCs) that can be isolated and differentiated from cord blood in xeno-free media collect in the vitreous cavity and rescue vaso-obliteration and neurodegeneration in animal models of retinal disease. Furthermore,we determined that a subset of the ECFCs was more effective at anatomically and functionally preventing retinopathy; these cells expressed high levels of CD44,the hyaluronic acid receptor,and IGFBPs (insulin-like growth factor-binding proteins). Injection of cultured media from ECFCs or only recombinant human IGFBPs also rescued the ischemia phenotype. These results help us to understand the mechanism of ECFC-based therapies for ischemic insults and retinal neurodegenerative diseases. View Publication