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

Emerging evidence indicates that in myelodysplastic syndromes (MDS),the bone marrow (BM) microenvironment may also contribute to the ineffective,malignant haematopoiesis in addition to the intrinsic abnormalities of haematopoietic stem precursor cells (HSPCs). The BM microenvironment influences malignant haematopoiesis through indirect mechanisms,but the processes by which the BM microenvironment directly contributes to MDS initiation and progression have not yet been elucidated. Our previous data showed that BM-derived stromal cells (BMSCs) from MDS patients have an abnormal expression of focal adhesion kinase (FAK). In this study,we characterise the morpho-phenotypic features and the functional alterations of BMSCs from MDS patients and in FAK knock-downed HS-5 cells. The decreased expression of FAK or its phosphorylated form in BMSCs from low-risk (LR) MDS directly correlates with BMSCs' functional deficiency and is associated with a reduced level of haemoglobin. The downregulation of FAK in HS-5 cells alters their morphology,proliferation,and differentiation capabilities and impairs the expression of several adhesion molecules. In addition,we examine the CD34+ healthy donor (HD)-derived HSPCs' properties when co-cultured with FAK-deficient BMSCs. Both abnormal proliferation and the impaired erythroid differentiation capacity of HD-HSPCs were observed. Together,these results demonstrate that stromal adhesion mechanisms mediated by FAK are crucial for regulating HSPCs' homeostasis. View Publication

As a critical tumor suppressor,p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore,it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However,the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination,we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives,we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/386) in regulating human p53 responses to DNA damage. View Publication

UNLABELLED: Human bocavirus 1 (HBoV1) is a single-stranded DNA parvovirus that causes lower respiratory tract infections in young children worldwide. In this study,we identified novel splice acceptor and donor sites,namely,A1' and D1',in the large nonstructural protein (NS1)-encoding region of the HBoV1 precursor mRNA. The novel small NS proteins (NS2,NS3,and NS4) were confirmed to be expressed following transfection of an HBoV1 infectious proviral plasmid and viral infection of polarized human airway epithelium cultured at an air-liquid interface (HAE-ALI). We constructed mutant pIHBoV1 infectious plasmids which harbor silent mutations (sm) smA1' and smD1' at the A1' and D1' splice sites,respectively. The mutant infectious plasmids maintained production of HBoV1 progeny virions at levels less than five times lower than that of the wild-type plasmid. Importantly,the smA1' mutant virus that does not express NS3 and NS4 replicated in HAE-ALI as effectively as the wild-type virus; however,the smD1' mutant virus that does not express NS2 and NS4 underwent an abortive infection in HAE-ALI. Thus,our study identified three novel NS proteins,NS2,NS3,and NS4,and suggests an important function of the NS2 protein in HBoV1 replication in HAE-ALI. IMPORTANCE: Human bocavirus 1 infection causes respiratory diseases,including acute wheezing in infants,of which life-threatening cases have been reported. In vitro,human bocavirus 1 infects polarized human bronchial airway epithelium cultured at an air-liquid interface that mimics the environment of human lower respiratory airways. Viral nonstructural proteins are often important for virus replication and pathogenesis in infected tissues or cells. In this report,we identified three new nonstructural proteins of human bocavirus 1 that are expressed during infection of polarized human bronchial airway epithelium. Among them,we proved that one nonstructural protein is critical to the replication of the virus in polarized human bronchial airway epithelium. The creation of nonreplicating infectious HBoV1 mutants may have particular utility in vaccine development for this virus. View Publication

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function,developmental pathways,and disease mechanisms. Here,we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi,in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain,can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors,cardiomyocytes,and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn),CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types,dissect developmental pathways,and model disease. View Publication

Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling,we examined textgreater 680 colonies from 3 different preparations of cells over 5 days each,generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies,correlation of colony characteristics with Oct4 expression,and identification of rare events. View Publication

ABSTRACTProneural genes are conserved drivers of neurogenesis across the animal kingdom. How their functions have adapted to guide human-specific neurodevelopmental features is poorly understood. Here,we mined transcriptomic data from human fetal cortices and generated from human embryonic stem cell-derived cortical organoids (COs) to show that NEUROG1 and NEUROG2 are most highly expressed in basal neural progenitor cells,with pseudotime trajectory analyses indicating that NEUROG1-derived lineages predominate early and NEUROG2 lineages later. Using ChIP-qPCR,gene silencing and overexpression studies in COs,we show that NEUROG2 is necessary and sufficient to directly transactivate known target genes (NEUROD1,EOMES,RND2). To identify new targets,we engineered NEUROG2-mCherry knock-in human embryonic stem cells for CO generation. The mCherry-high CO cell transcriptome is enriched in extracellular matrix-associated genes,and two genes associated with human-accelerated regions: PPP1R17 and FZD8. We show that NEUROG2 binds COL1A1,COL3A1 and PPP1R17 regulatory elements,and induces their ectopic expression in COs,although NEUROG2 is not required for this expression. Neurog2 similarly induces Col3a1 and Ppp1r17 in murine P19 cells. These data are consistent with a conservation of NEUROG2 function across mammalian species. Summary: Analysis of human cortical organoids reveals that NEUROG1 lineages prevail early and NEUROG2 lineages later,and that NEUROG2 targets include COL genes and PPP1R17,a human-accelerated region-associated gene. View Publication

Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases,including inclusion body myositis (IBM),Sjögren’s syndrome,and systemic lupus erythematosus. While they have diagnostic relevance for IBM,little is known about the autoreactive B cells that produce these antibodies. To address this,we developed a robust protocol for the expression and site-specific biotinylation of recombinant human cN1A in Escherichia coli. The resulting antigen is suitable for generating double-labelled fluorescent baits for the isolation and characterisation of cN1A-specific B cells by flow cytometry. Site-specific biotinylation was achieved using the AviTag and BirA ligase,preserving the protein’s structure and immunoreactivity. Western blot analysis confirmed that the biotinylated cN1A was recognised by both human and rabbit anti-cN1A antibodies. Compared to conventional chemical biotinylation,this strategy minimises structural alterations that may affect antigen recognition. This approach provides a reliable method for producing biotinylated antigens for use in immunological assays. While demonstrated here for cN1A,the protocol can be adapted for other autoantigens to support studies of antigen-specific B cells in autoimmune diseases. View Publication

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-TCRα expression. Low circulating naïve αβ T cell counts at birth persisted over time,with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased,suggesting that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick,with infection,lymphoproliferation,and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naïve αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells,autoimmune conditions were more frequent in these patients than in the general population. View Publication

Astrocytes are essential for the formation and maintenance of neural networks. However,a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques,primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing,we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons,consistent with their further maturation. In coculture with human neurons,multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 ± 0.44 min −1 (hPSC-derived),2.86 ± 0.64 min −1 (rat); p = 0.19]. In comparison,we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 ± 8.56 Hz (hPSC-derived),24.77 ± 4.04 Hz (rat); p < 0.01],and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 ± 0.39 Hz (hPSC-derived),1.07 ± 0.14 Hz (rat); p < 0.001],consistent with a corresponding increase in synapse density [14.90 ± 1.27/100 μm 2 (hPSC-derived),8.39 ± 0.63/100 μm 2 (rat); p < 0.001]. Taken together,we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation,providing a fully human platform for investigating astrocyte function and neuronal-glial interactions. View Publication