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

To better understand transcriptional regulation during human oogenesis and preimplantation development,we defined stage-specific transcription,which highlighted the cleavage stage as being highly distinctive. Here,we present multiple lines of evidence that a eutherian-specific multicopy retrogene,DUX4,encodes a transcription factor that activates hundreds of endogenous genes (for example,ZSCAN4,KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably,mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells,measured here by the reactivation of '2C' genes and repeat elements,the loss of POU5F1 (also known as OCT4) protein and chromocenters,and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus,we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state. View Publication

Alveolar macrophages (AM) perform a primary defense mechanism in the lung through phagocytosis of inhaled particles and microorganisms. AM are known to be relatively immunosuppressive consistent with the aim to limit alveolar inflammation and maintain effective gas exchange in the face of these constant challenges. How AM respond to T cell derived cytokine signals,which are critical to the defense against inhaled pathogens,is less well understood. For example,successful containment of Mycobacterium tuberculosis (Mtb) in lung macrophages is highly dependent on IFN-$\gamma$ secreted by Th-1 lymphocytes,however,the proteomic IFN-$\gamma$ response profile in AM remains mostly unknown. In this study,we measured IFN-$\gamma$ induced protein abundance changes in human AM and autologous blood monocytes (MN). AM cells were activated by IFN-$\gamma$ stimulation resulting in STAT1 phosphorylation and production of MIG/CXCL9 chemokine. However,the global proteomic response to IFN-$\gamma$ in AM was dramatically limited in comparison to that of MN (9 AM vs 89 MN differentially abundant proteins). AM hypo-responsiveness was not explained by reduced JAK-STAT1 signaling nor increased SOCS1 expression. These findings suggest that AM have a tightly regulated response to IFN-$\gamma$ which may prevent excessive pulmonary inflammation but may also provide a niche for the initial survival and growth of Mtb and other intracellular pathogens in the lung. View Publication

AbstractDespite the success of combination antiretroviral therapy (ART) for individuals living with HIV,mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation,neuronal dysfunction,and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia,we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1,ISG15,ISG20,IFI27,IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART,consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However,S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells,mature and immature ChP cells,neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons,accompanied by increased expression of genes promoting cellular senescence and cell death. Together,these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons. View Publication

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19,CD20,or CD22 can produce potent clinical responses for some B cell malignancies,but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller,simpler,and easier to recombine than single-chain variable fragments (scFvs) used in most CARs,but fewer sdAb-CARs have been reported. Thus,we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein,sdAb-cDNA library construction,and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro . CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro,although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies,a lead CD22sdCAR-T was selected,achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally,immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic. View Publication

During embryogenesis,human hematopoietic stem cells (HSCs) first emerge in the aorta-gonad-mesonephros (AGM) region via transformation of specialized hemogenic endothelial (HE) cells into premature HSC precursors. This process is termed endothelial-to-hematopoietic transition (EHT),in which the HE cells undergo drastic functional and morphological changes from flat,anchorage-dependent endothelial cells to free-floating round hematopoietic cells. Despite its essential role in human HSC development,molecular mechanisms underlying the EHT are largely unknown. This is due to lack of methods to visualize the emergence of human HSC precursors in real time in contrast to mouse and other model organisms. In this study,by inducing HE from human pluripotent stem cells in feeder-free monolayer cultures,we achieved real-time observation of the human EHT in vitro . By continuous observation and single-cell tracking in the culture,it was possible to visualize a process that a single endothelial cell gives rise to a hematopoietic cell and subsequently form a hematopoietic-cell cluster. The EHT was also confirmed by a drastic HE-to-HSC switching in molecular marker expressions. Notably,HSC precursor emergence was not linked to asymmetric cell division,whereas the hematopoietic cell cluster was formed through proliferation and assembling of the floating cells after the EHT. These results reveal unappreciated dynamics in the human EHT,and we anticipate that our human EHT model in vitro will provide an opportunity to improve our understanding of the human HSC development. View Publication

The upper airway epithelium,which is mainly composed of multiciliated,goblet,club and basal cells,ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases,defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference,we have unraveled trajectories from basal to luminal cells,providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells,which we have entitled deuterosomal cells,is defined by specific markers,such as DEUP1,FOXN4,YPEL1,HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells,thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process,such as keratins or components of the Notch,Wnt or BMP/TGF$\beta$ pathways,can be identified. Confirmation of our results on fresh human and pig airway samples,and on mouse tracheal cells,extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation. View Publication

Here a new,intrinsically pluripotent,CD45-negative population from human cord blood,termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts,chondroblasts,adipocytes,and hematopoietic and neural cells including astrocytes and neurons that express neurofilament,sodium channel protein,and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model,the preimmune fetal sheep,resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals. View Publication

Monitoring genome-wide,cell-specific responses to human disease,although challenging,holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements,including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations,combined with cell-specific pathway analyses,offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes),monocyte (2,801 genes),and total leukocyte (3,437 genes) transcriptomes,with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1,CD152,NRP-1,and Lag3) and concomitant decreases in stimulatory receptors (CD28,CD4,and IL-2Ralpha). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus,genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly,application of cell separation,genome-wide expression,and cell-specific pathway analyses can be used to discover pathway alterations in human disease. View Publication

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However,techniques to generate cell type-specific lineage reporters,as well as reliable tools to disrupt,repair or overexpress genes by gene targeting,are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First,using ZFNs specific for the OCT4 (POU5F1) locus,we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second,we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally,we targeted the PITX3 gene,demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs. View Publication

Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs),high interferon-gamma (IFN-gamma) production,but little cytotoxicity. CD56(dim) NK cells have high KIR expression,produce little IFN-gamma,yet display high cytotoxicity. We hypothesized that,if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype,an intermediary NK cell must exist,which demonstrates more functional overlap than these 2 subsets,and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L,CD2,and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively,our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that,in vivo,human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype. View Publication

Measuring spatial and temporal patterns of cytochemical variation in human embryonic stem cell (hESC) colonies is necessary for understanding the role of cellular communication in spontaneous differentiation,the mechanisms of biological niche creation,and structure-generating developmental processes. Such insights will ultimately facilitate directed differentiation and therewith promote advances in tissue engineering and regenerative medicine. However,the patterns of cytochemical inhomogeneities of hESC colonies are not well studied and their causes are not fully understood. We used Raman spectroscopic mapping to contrast supracellular variations in cytochemical composition across pluripotent and partly differentiated hESC colonies to gain a better understanding of the early-stage (i.e.,5 days) effects of the differentiation process on the nature and evolution of these patterns. Higher protein-to-nucleic acid ratios,a differentiation status indicator observed previously using Raman spectroscopy,confirmed reported results that spontaneous differentiation is more pronounced on the edges of a colony than elsewhere. In addition,pluripotent and partly differentiated colonies also showed higher lipid concentrations relative to nucleic acids at colony edges in contrast to relative glycogen concentrations,which were up to 400% more pronounced in the colony centers compared to their edges. Pluripotent and partly differentiated colonies differed,with the latter having higher average protein-to-nucleic acid and lipid-to-nucleic acid ratios but a lower glycogen-to-nucleic acid ratio. In both cases,cell density,pluripotency,and high glycogen appeared to vary in tandem. Spatial variations in glycogen- and protein-to-nucleic acid ratios have features on the order of 100 μm and larger. These dimensions are consistent with those reported for stem cell niches and suggest that cytochemical inhomogeneities may provide colony-level information about niches and niche formation. These results demonstrate Raman mapping to be a potentially useful technique for revealing the complexities in the spatial organization of hESC cultures and thus for monitoring the evolution of engineered hESC niches. View Publication