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

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene,predominantly expressed in the liver. Two compounds that knockdown TTR,comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx),are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background,this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown,siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (textgreater80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds. View Publication

Cancer stem cells (CSCs) play crucial roles in tumor metastasis,therapy resistance,and immune evasion. Identifying and understanding the factors that regulate the stemness of tumor cells presents promising opportunities for developing effective therapeutic strategies. In this study on oral squamous cell carcinoma (OSCC),we confirmed the key role of KLF7 in maintaining the stemness of OSCC. Using chromatin immunoprecipitation sequencing and dual-luciferase assays,we identified ITGA2,a membrane receptor,as a key downstream gene regulated by KLF7 in the maintenance of stemness. Tumor sphere formation assays,flow cytometry analyses,and in vivo limiting dilution tumorigenicity evaluations demonstrated that knocking down ITGA2 significantly impaired stemness. Upon binding to its extracellular matrix (ECM) ligand,type I collagen,ITGA2 activates stemness-associated signaling pathways,including PI3K-AKT,MAPK,and Hippo. TC-I 15,a small-molecule inhibitor of the ITGA2-collagen interaction,significantly sensitizes oral squamous cell carcinoma (OSCC) to cisplatin in xenograft models. In summary,we reveal that the KLF7/ITGA2 axis is a crucial modulator of stemness in OSCC. Our findings suggest that ITGA2 is a promising therapeutic target,offering a novel anti-CSC strategy. Subject terms: Cancer stem cells,Cancer therapeutic resistance View Publication

Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here,we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably,1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression,HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters,a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions. View Publication

Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) showed curative potential in patients affected by neurometabolic diseases treated in early stage. Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system (CNS) could allow enhancing further the therapeutic potential of this approach. Here we unveil that HSPCs haplo-insufficient at the Cx3cr1 (Cx3cr1 −/+ ) locus are favored in central nervous system (CNS) engraftment and generation of microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1 +/+ ) HSPCs upon transplantation in mice. Based on this evidence,we have developed a CRISPR-based targeted gene addition strategy at the human CX3CR1 locus resulting in an enhanced ability of the edited human HSPCs to generate mature MLCs upon transplantation in immunodeficient mice,and in lineage specific,regulated and robust transgene expression. This approach,which benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells,may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases. Subject terms: Targeted gene repair,Haematopoietic stem cells,Microglial cells View Publication

Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however,present methods to clonally grow them are inefficient and poorly defined for genetic manipulation and therapeutic purposes. Here we develop the first chemically defined,xeno-free,feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability,surface topography,surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure-function relationships between material properties and biological performance. These analyses show that optimal human embryonic stem cell substrates are generated from monomers with high acrylate content,have a moderate wettability and employ integrin alpha(v)beta(3) and alpha(v)beta(5) engagement with adsorbed vitronectin to promote colony formation. The structure-function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture. View Publication

Human pluripotent stem cells (PSCs),which include human embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs),represent an important source of cellular therapies in regenerative medicine and the study of early human development. As such,it is becoming increasingly important to develop methods for the large-scale banking of human PSC lines. There are several well-established methods for the propagation of human PSCs. The key to development of a good manufacturing practice (GMP) bank is to determine a manufacturing method that is amenable to large-scale production using materials that are fully documented. We have developed several banks of hESCs using animal feeder cells,animal-based matrices,or animal-free matrices. Protocols for growing hESCs on mouse embryonic fibroblasts (MEFs) are well established and are very helpful for producing research grade banks of cells. As most human ESCs cultured by research laboratories have been exposed to xenogeneic reagents,it is not imperative that all materials used in the production of a master cell bank be animal-free in origin. Nevertheless,as the field develops,it will no doubt become increasingly important to produce a bank of cells for clinical use without xenogeneic reagents,particularly nonhuman feeder cells which might harbor viruses with potential risk to human health or cell product integrity. Thus,even for cell lines previously exposed to xenogeneic reagents,it is important to minimize any subsequent exposure of the cell lines to additional adventitious agents. We have specifically described procedures for the growth of hESCs on Matrigel,an animal-matrix,and CELLstart,an animal-free matrix,and these can be used to produce hESCs as part of a clinical manufacturing process. View Publication

X-ALD is an inherited neurodegenerative disorder where mutations in the ABCD1 gene result in clinically diverse phenotypes: the fatal disorder of cerebral childhood ALD (cALD) or a milder disorder of adrenomyeloneuropathy (AMN). The various models used to study the pathobiology of X-ALD disease lack the appropriate presentation for different phenotypes of cALD vs AMN. This study demonstrates that induced pluripotent stem cells (IPSC) derived brain cells astrocytes (Ast),neurons and oligodendrocytes (OLs) express morphological and functional activities of the respective brain cell types. The excessive accumulation of saturated VLCFA,a hallmark" of X-ALD� View Publication

NK cells are key innate immune cells that play critical roles in host defense. Although NK cells have been shown to regulate immunity to some infectious diseases,their role in immunity to Trypanosoma congolense has not been investigated. NK cells are vital sources of IFN-gamma and TNF-alpha; two key cytokines that are known to play important roles in resistance to African trypanosomes. In this article,we show that infection with T. congolense leads to increased levels of activated and functional NK cells in multiple tissue compartments. Systemic depletion of NK cells with anti-NK1.1 mAb led to increased parasitemia,which was accompanied by significant reduction in IFN-gamma production by immune cells in the spleens and liver of infected mice. Strikingly,infected NFIL3-/- mice (which genetically lack NK cell development and function) on the normally resistant background were highly susceptible to T. congolense infection. These mice developed fulminating and uncontrolled parasitemia and died significantly earlier (13 ± 1 d) than their wild-type control mice (106 ± 26 d). The enhanced susceptibility of NFIL3-/- mice to infection was accompanied by significantly impaired cytokine (IFN-gamma and TNF-alpha) response by CD3+ T cells in the spleens and liver. Adoptive transfer of NK cells into NFIL3-/- mice before infection rescued them from acute death in a perforin-dependent manner. Collectively,these studies show that NK cells are critical for optimal resistance to T. congolense,and its deficiency leads to enhanced susceptibility in infected mice. View Publication

Epitope-specific CD4+ T cells can be labeled in complex cell mixtures from secondary lymphoid organs with fluorophore-labeled peptide:major histocompatibility complex class II (p:MHCII) tetramers and then detected by flow cytometry. Magnetic enrichment of tetramer-bound cells before flow cytometry increases the sensitivity of detection to the point where epitope-specific cells can be studied even when very rare at early and late times after the host has been exposed to the epitope. This method is very useful for studying polyclonal epitope-specific CD4+ T cells under physiological conditions. {\textcopyright} 2019 by John Wiley {\&} Sons,Inc. View Publication