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

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Stem cells derived from pre-implantation human embryos or from somatic cells by reprogramming are pluripotent and self-renew indefinitely in culture. Pluripotent stem cells are unique in being able to differentiate to any cell type of the human body. Differentiation towards the cardiac lineage has attracted significant attention,initially with a strong focus on regenerative medicine. Although an important research area,the heart has proven challenging to repair by cardiomyocyte replacement. However,the ability to reprogramme adult cells to pluripotent stem cells and genetically manipulate stem cells presented opportunities to develop models of human disease. The availability of human cardiomyocytes from stem cell sources is expected to accelerate the discovery of cardiac drugs and safety pharmacology by offering more clinically relevant human culture models than presently available. Here we review the state-of-the-art using stem cell-derived human cardiomyocytes in drug discovery,drug safety pharmacology,and regenerative medicine. View Publication

Although the importance of natural killer (NK) cells in innate immune responses against tumors or viral infections are well documented,their ability to directly recognize pathogens is less well defined. We have recently reported FimH,a bacterial fimbrial protein,as a novel Toll-like receptor (TLR)4 ligand that potently induces antiviral responses. Here,we investigated whether FimH either directly or indirectly can activate human and murine NK cells. We demonstrate that FimH potently activates both human and murine NK cells in vitro to induce cytokines [interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha] and cytotoxicity. Importantly,NK cells directly recognize FimH-expressing pathogens as FimH(+),but not FimH(-),bacteria were able to activate human NK cells. FimH activation of NK cells required TLR4 and MyD88 signaling,as NK cells from both TLR4(-/-) and MyD88(-/-) mice as well as human NK-92 cells,which lack TLR4,were all unresponsive to FimH. In addition,TLR4 neutralization significantly abrogated the response of human NK cells to FimH. Activation of purified NK cells by FimH was independent of lipopolysaccharide (LPS) or other bacterial contaminations. These data demonstrate for the first time that highly purified NK cells directly recognize and respond to FimH via TLR4-MyD88 pathways to aid innate protection against cancer or microbial infections. View Publication

BACKGROUND: Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects,including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments,with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However,current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover,FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific,whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations,we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs,and maintained the capacity to suppress conventional T cell responses directed against tyrosinase,as well as bystander T cell responses. Using this methodology in a model tumor system,murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging. CONCLUSIONS/SIGNIFICANCE: These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy. View Publication

Normal stem cells and cancer stem cells (CSCs) share similar properties,in that both have the capacity to self-renew and differentiate into multiple cell types. In both the normal stem cell and cancer stem cell fields,there has been a great need for a universal marker that can effectively identify and isolate these rare populations of cells in order to characterize them and use this information for research and therapeutic purposes. Currently,it would appear that certain isoenzymes of the aldehyde dehydrogenase (ALDH) superfamily may be able to fulfill this role as a marker for both normal and cancer stem cells. ALDH has been identified as an important enzyme in the protection of normal hematopoietic stem cells,and is now also widely used as a marker to identify and isolate various types of normal stem cells and CSCs. In addition,emerging evidence suggests that ALDH1 is not only a marker for stem cells,but may also play important functional roles related to self-protection,differentiation,and expansion. This comprehensive review discusses the role that ALDH plays in normal stem cells and CSCs,with focus on ALDH1 and ALDH3A1. Discrepancies in the functional themes between cell types and future perspectives for therapeutic applications will also be discussed. View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells and human-induced pluripotent stem cells,are a renewable cell source for a wide range of applications in regenerative medicine and useful tools for human disease modeling and drug discovery. For these purposes,large numbers of high-quality cells are essential. Recently,we showed that a biological substrate,recombinant E8 fragments of laminin isoforms,sustains long-term self-renewal of hPSCs in defined,xeno-free medium with dissociated single-cell passaging. Here,we describe a modified culture system with similar performance to efficiently expand hPSCs under defined,xeno-free conditions using a non-biological synthetic substrate. View Publication

Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood,aside from numerous congenital abnormalities. FA mouse models have been generated; however,they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero,a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology,we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential,disruption of the second allele causes a severe growth disadvantage. As a result,heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele,quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning,this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage. ?? 2014. View Publication

Nature Genetics 47,469 (2015). doi:10.1038/ng.3258 View Publication

Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs) to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5),hepatoblast (day 15) and hepatocyte-like cells (day 21) were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type,the hepatic cells (days 15 and 21) had significantly higher expression of acute phase protein genes including complement factors,coagulation factors,serum amyloid A and serpins. Furthermore,hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1,IL1R1,IL1RAP,IL2RG,IL6R,IL6ST and IL10RB. These cells also produced CCL14,CCL15,and CXCL- 1,2,3,16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors,CXCR4 and CXCR7,than that of hepatic cells. Sirtuin family of genes involved in aging,inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF) family as well as downstream signaling factors TRAF2,TRAF4,FADD,NFKB1 and NFKBIB were differentially expressed during hepatic differentiation. View Publication

Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here,we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7),a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2,a receptor that recognizes NAP-2,abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration. View Publication

Pluripotency of embryonic stem cells (ESCs) is defined by their ability to differentiate into three germ layers and derivative cell types and is established by an interactive network of proteins including OCT4 (also known as POU5F1; ref. ),NANOG (refs ,),SOX2 (ref. ) and their binding partners. The forkhead box O (FoxO) transcription factors are evolutionarily conserved regulators of longevity and stress response whose function is inhibited by AKT protein kinase. FoxO proteins are required for the maintenance of somatic and cancer stem cells; however,their function in ESCs is unknown. We show that FOXO1 is essential for the maintenance of human ESC pluripotency,and that an orthologue of FOXO1 (Foxo1) exerts a similar function in mouse ESCs. This function is probably mediated through direct control by FOXO1 of OCT4 and SOX2 gene expression through occupation and activation of their respective promoters. Finally,AKT is not the predominant regulator of FOXO1 in human ESCs. Together these results indicate that FOXO1 is a component of the circuitry of human ESC pluripotency. These findings have critical implications for stem cell biology,development,longevity and reprogramming,with potentially important ramifications for therapy. View Publication

The supply of human hepatic stem cells (hHpSCs) and other hepatic progenitors has been constrained by the limited availability of liver tissues from surgical resections,the rejected organs from organ donation programs,and the need to use cells immediately. To facilitate accessibility to these precious tissue resources,we have established an effective method for serum-free cryopreservation of the cells,allowing them to be stockpiled and stored for use as an off-the-shelf product for experimental or clinical programs. The method involves use of buffers,some serum-free,designed for cryopreservation and further supplemented with hyaluronans (HA) that preserve adhesion mechanisms facilitating postthaw culturing of the cells and preservation of functions. Multiple cryopreservation buffers were found to yield high viabilities (80-90%) of cells on thawing of the progenitor cells. Serum-free CS10 supplemented with 0.05% hyaluronan proved the most effective,both in terms of viabilities of cells on thawing and in yielding cell attachment and formation of expanding colonies of cells that stably maintain the stem/progenitor cell phenotype. Buffers to which 0.05 or 0.1% HAs were added showed cells postthaw to be phenotypically stable as stem/progenitors,as well as having a high efficiency of attachment and expansion in culture. Success correlated with improved expression of adhesion molecules,particularly CD44,the hyaluronan receptor,E-cadherin,β4 integrin in hHpSCs,and β1 integrins in hepatoblasts. The improved methods in cryopreservation offer more efficient strategies for stem cell banking in both research and potential therapy applications. View Publication