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

Murine hematopoietic stem and progenitor cells (HSPCs) home to bone marrow in part by rolling on P-selectin and E-selectin expressed on endothelial cells. Human adult CD34(+) cells,which are enriched in HSPCs,roll on endothelial selectins in bone marrow vessels of nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Many human umbilical cord blood (CB) CD34(+) cells do not roll in these vessels,in part because of an uncharacterized defect in binding to P-selectin. Selectin ligands must be alpha1-3 fucosylated to form glycan determinants such as sialyl Lewis x (sLe(x)). We found that inadequate alpha1-3 fucosylation of CB CD34(+) cells,particularly CD34(+)CD38(-/low) cells that are highly enriched in HSPCs,caused them to bind poorly to E-selectin as well as to P-selectin. Treatment of CB CD34(+) cells with guanosine diphosphate (GDP) fucose and exogenous alpha1-3 fucosyltransferase VI increased cell-surface sLe(x) determinants,augmented binding to fluid-phase P- and E-selectin,and improved cell rolling on P- and E-selectin under flow. Similar treatment of CB mononuclear cells enhanced engraftment of human hematopoietic cells in bone marrows of irradiated NOD/SCID mice. These observations suggest that alpha1-3 fucosylation of CB cells might be a simple and effective method to improve hematopoietic cell homing to and engraftment in bone marrows of patients receiving CB transplants. View Publication

This study evaluated the hypothesis that smoke from harm reduction cigarettes impedes attachment and proliferation of H9 human embryonic stem cells (hESCs). Smoke from three harm reduction brands was compared with smoke from a conventional brand. Doses of smoke were measured in puff equivalents (PE) (1 PE = the amount of smoke in one puff that dissolves in 1 ml of medium). Cytotoxic doses were determined using morphological criteria and trypan blue staining,and apoptosis was confirmed using Magic Red staining. Attachment and proliferation of hESC were followed at a noncytotoxic dose in time-lapse videos collected using BioStation technology. Data were mined from videos either manually or using video bioinformatics subroutines developed with CL-Quant software. Mainstream (MS) and sidestream (SS) smoke from conventional and harm reduction cigarettes induced apoptosis in hESC colonies at 1 PE. At 0.1 PE (noncytotoxic),SS smoke from all brands inhibited attachment of hESC colonies to Matrigel with the strongest inhibition occurring in harm reduction brands. At 0.1 PE,SS smoke,but not MS smoke,from all brands inhibited hESC growth,and two harm reduction brands were more potent than the conventional brand. In general,hESC appeared more sensitive to smoke than their mouse ESC counterparts. Although harm reduction cigarettes are often marketed as safer than conventional brands,our assays show that SS smoke from harm reduction cigarettes was at least as potent or in some cases more potent than smoke from a conventional brand and that SS smoke was more inhibitory than MS smoke in all assays. View Publication

Retinoids are important molecules involved in the development and homeostasis of the nervous system. As such,various retinoid derivatives are often found in culture media and supplement formulations to support the growth and maintenance of neural cells. However,all-trans-retinoic acid (ATRA) and its associated derivatives are light sensitive and are highly susceptible to isomerisation. This can lead to variability in retinoid concentrations and the nature of the retinoid species present in culture solutions which in turn can influence biological activity and introduce inconsistency. We have previously described the development of the synthetic retinoid derivative,EC23,as a chemically and light stable alternative that does not degrade and has biological activity similar to ATRA. In this study we demonstrate that the addition of exogenous retinoid can significantly enhance neuronal differentiation of both human neuroprogenitor and human embryonic stem cells. In the former,both ATRA and EC23 induced increased maturation and stabilisation of the axonal cytoskeleton. However,EC23 was particularly potent at lower nanomolar concentrations resulting in significantly greater neurogenesis than ATRA. In ES cells enhanced motor neuron marker expression was also detected in response to both retinoids when incorporated into an established protocol for neuronal differentiation. We propose that synthetic retinoid EC23 represents a valuable addition to the formulation of new and existing culture supplements to enhance neuronal differentiation whilst enabling improved consistency. View Publication

Recent findings indicate that NK cells are involved in cardiac repair following myocardial infarction. The aim of this study is to investigate the role NK cells in infarct angiogenesis and cardiac remodeling. In normal C57BL/6 mice,myelomonocytic inflammatory cells invaded infarcted heart within 24 h followed by a lymphoid/NK cell infiltrate by day 6,accompanied by substantial expression of IL-2,TNF-α,and CCL2. In contrast,NOD SCID mice had virtually no lymphoid cells infiltrating the heart and did not upregulate IL-2 levels. In vitro and in vivo,IL-2-activated NK cells promoted TNF-α-stimulated endothelial cell proliferation,enhanced angiogenesis and reduced fibrosis within the infarcted myocardium. Adoptive transfer of IL-2-activated NK cells to NOD SCID mice improved post-myocardial infarction angiogenesis. RNA silencing technology and neutralizing Abs demonstrated that this process involved α4β7 integrin/VCAM-1 and killer cell lectin-like receptor 1/N-cadherin-specific binding. In this study,we show that IL-2-activated NK cells reduce myocardial collagen deposition along with an increase in neovascularization following acute cardiac ischemia through specific interaction with endothelial cells. These data define a potential role of activated NK cells in cardiac angiogenesis and open new perspectives for the treatment of ischemic diseases. View Publication

Cell enrichment methods that deal with larger volumes of peripheral blood and BM are needed for increased sensitivity of detection,characterization and quantification of isolated tumor cells (ITC). This study was designed to evaluate a new procedure,the RosetteSep-Applied Imaging Rare Event (RARE) detection method,which depletes the majority of the erythrocytes and leucocytes in a peripheral blood (PB) sample,thereby negatively enriching tumor cells if present. This enrichment procedure allows for increased sensitivity,by analyzing a 5-10 fold larger volume of blood,compared with a direct immunocytochemical (ICC) technique,with minimal impact on laboratory workload. Model experiments showed comparable tumor cell recoveries between the two tested methods,both in PB and BM. Clinical samples were evaluated using paired PB and BM samples from 95 carcinoma patients. Analysis of PB results showed that 25.3% had textgreater or = 1 tumor cell detected by the RARE procedure,compared with 5.2% after direct ICC analysis,analyzing a 10-fold larger volume by the RARE procedure. The direct ICC analysis of BM from the same patients revealed 16.8% positive. The ITC detection differed both quantitatively and qualitatively between BM and PB,as samples with high numbers of ITC in BM were still negative in PB. The clinical significance of ITC in blood still needs to be established. However,the easy access of peripheral blood,and the increased sensitivity obtained by increasing the sample volume with the RARE procedure,suggests that the value of peripheral blood analysis should be tested in parallel in studies where ITC detection in BM is performed. View Publication

Natural killer (NK) cells are thought to develop from common lymphoid progenitors in the bone marrow. However,immature thymocytes also retain NK potential. Currently,the contribution of the thymus-dependent pathway in normal steady-state NK-cell development is unknown. Here,we show that TCRgamma genes are rearranged in approximately 5% of neonatal and 1% of adult mouse splenic NK cells,and similar levels are detected in NK cells from TCRbeta,delta double-knockout mice,excluding the possibility of T-cell contamination. NK-cell TCRgamma gene rearrangement is thymus dependent because this rearrangement is undetectable in nude mouse NK cells. These results change the current view of NK-cell development and show that a subset of NK cells develops from immature thymocytes that have rearranged TCRgamma genes. View Publication

Interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are major factors for maintenance and expansion of hematopoietic stem cells (HSCs). Sensitivity of HSCs to IL-6 has been previously studied,in part by measuring the expression of IL-6R on the membrane (mIL-6R). Several studies have described the regulation of cell surface expression of IL-6R by several cytokines,but the role of glycoprotein 130 activation has not yet been investigated. In this study,CD133(+) cells were purified from adult peripheral blood and were precultured in the absence or presence of 5-fluorouracil (5-FU) for selection of quiescent HSCs. Cells were cultured with continuous or pulsed stimulations of an IL-6-sIL-6R fusion protein (hyperinterleukin-6 [HIL-6]) to 1) detect mIL-6R by flow cytometry,2) assess mIL-6R and sIL-6R RNAs by reverse transcription-polymerase chain reaction,3) measure sIL-6R in supernatants by enzyme-linked immunosorbent assay,4) analyze cell-cycle status,and 5) perform long-term culture-initiating cell assays. The level of mIL-6R(-) cells was preserved by 5-FU incubation. HIL-6 increased steady-state mIL-6R RNA and expression rate on HSCs,independently of treatment with 5-FU. Enhanced production of sIL-6R was observed with short pulses of HIL-6 on CD133(+) 5-FU-pretreated cells. This overproduction of sIL-6R was abrogated by tumor necrosis factor-alpha protease inhibitor-1,an inhibitor of a disintegrin and metalloprotease proteases,suggesting the shedding of mIL-6R. This phenomenon was mediated through the phosphatidylinositol-3'-kinase pathway and was involved in the maintenance of primitive HSCs. In conclusion,expression and production of IL-6R are tightly regulated and stage specific. We assume that sIL-6R produced by shedding should be involved in autocrine and paracrine loops in the HSC microenvironment. View Publication

Disruption of pathways leading to programmed cell death plays a major role in most malignancies,including multiple myeloma (MM). ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-xL,preventing the sequestration of proapoptotic molecules and shifting the cell survival/apoptosis balance toward apoptosis induction. In this study,we show that ABT-737 is cytotoxic to MM cell lines,including those resistant to conventional therapies,and primary tumor cells. Flow cytometric analysis of intracellular levels of Bcl-2 family proteins demonstrates a clear inversion of the Bax/Bcl-2 ratio leading to induction of apoptosis. Activation of the mitochondrial apoptosis pathway was indicated by mitochondrial membrane depolarization and caspase cleavage. Additionally,several signaling pathways known to be important for MM cell survival are disrupted following treatment with ABT-737. The impact of ABT-737 on survival could not be overcome by the addition of interleukin-6,vascular endothelial growth factor or insulin-like growth factor,suggesting that ABT-737 may be effective in preventing the growth and survival signals provided by the microenvironment. These data indicate that therapies targeting apoptotic pathways may be effective in MM treatment and warrant clinical evaluation of ABT-737 and similar drugs alone or in combination with other agents in the setting of MM. View Publication

How tumor suppressor p53 selectively responds to specific signals,especially in normal cells,is poorly understood. We performed genome-wide profiling of p53 chromatin interactions and target gene expression in human embryonic stem cells (hESCs) in response to early differentiation,induced by retinoic acid,versus DNA damage,caused by adriamycin. Most p53-binding sites are unique to each state and define stimulus-specific p53 responses in hESCs. Differentiation-activated p53 targets include many developmental transcription factors and,in pluripotent hESCs,are bound by OCT4 and NANOG at chromatin enriched in both H3K27me3 and H3K4me3. Activation of these genes occurs with recruitment of p53 and H3K27me3-specific demethylases,UTX and JMJD3,to chromatin. In contrast,genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in cell death and cell cycle regulation are conserved in both DNA damage and differentiation. Comparative genomic analysis of p53-targets in mouse and human ESCs supports an inter-species divergence in p53 regulatory functions during evolution. Our findings expand the registry of p53-regulated genes to define p53-regulated opposition to pluripotency during early differentiation,a process highly distinct from stress-induced p53 response in hESCs. View Publication

An immunohistological study in the human breast and the rodent breast (from inbred Ludwig/Wistar/Olac rats) was conducted with the use of a murine monoclonal antibody,which reacts with the common acute lymphoblastic antigen,a glycosylated polypeptide of a molecular weight of 100,000. The epitope,as recognized by this antibody,is expressed on myoepithelial cells of the normal human and rat breasts and was studied in the developing rodent mammary gland. Ultrastructural studies in the normal human breast clearly demonstrated the presence of the antigen on the lateral membrane of the myoepithelial cells with no staining of luminal cells,blood vessels,or stromal elements. The antigen survived prolonged enzymatic digestion of human breast tissue and could be demonstrated on myoepithelial cells in single-cell suspensions of human breast where it stained approximately 3-14% of the total cell population. The presence of this antigen on myoepithelial cells is discussed in the context of myoepithelial differentiation in the breast and the potential utility of the antibodies for cell separation. View Publication

Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers,and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells,to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells,we examined the effect of varying initial cell seeding density from 1.3 x 104 cells/cm2 to 5.3 x 104 cells/cm2 followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1,PTF1a,NGN3,ARX,and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin,glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression,followed by pancreatic endocrine specification marker expression (BRN4,PAX4,ARX,NEUROD1,NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin,glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment,which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols. View Publication

There is a great deal of uncertainty on how low (≤ 0.1 Gy) doses of ionizing radiation (IR) affect human cells,partly due to a lack of suitable experimental model systems for such studies. The uncertainties arising from low-dose IR human data undermine practical societal needs to predict health risks emerging from diagnostic medical tests' radiation,natural background radiation,and environmental radiological accidents. To eliminate a variability associated with remarkable differences in radioresponses of hundreds of differentiated cell types,we established a novel,human embryonic stem cell (hESC)-based model to examine the radiobiological effects in human cells. Our aim is to comprehensively elucidate the gene expression changes in a panel of various hESC lines following low IR doses of 0.01; 0.05; 0.1 Gy; and,as a reference,relatively high dose of 1 Gy of IR. Here,we examined the dynamics of transcriptional changes of well-established IR-responsive set of genes,including CDKN1A,GADD45A,etc. at 2 and 16 h post-IR,representing early" and "late" radioresponses of hESCs. Our findings suggest the temporal- and hESC line-dependence of stress gene radioresponses with no statistically significant evidence for a linear dose-response relationship within the lowest doses of IR exposures." View Publication