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

Teratogens,substances that may cause fetal abnormalities during development,are responsible for a significant number of birth defects. Animal models used to predict teratogenicity often do not faithfully correlate to human response. Here,we seek to develop a more predictive developmental toxicity model based on an in vitro method that utilizes both human embryonic stem (hES) cells and metabolomics to discover biomarkers of developmental toxicity. We developed a method where hES cells were dosed with several drugs of known teratogenicity then LC-MS analysis was performed to measure changes in abundance levels of small molecules in response to drug dosing. Statistical analysis was employed to select for specific mass features that can provide a prediction of the developmental toxicity of a substance. These molecules can serve as biomarkers of developmental toxicity,leading to better prediction of teratogenicity. In particular,our work shows a correlation between teratogenicity and changes of greater than 10% in the ratio of arginine to asymmetric dimethylarginine levels. In addition,this study resulted in the establishment of a predictive model based on the most informative mass features. This model was subsequently tested for its predictive accuracy in two blinded studies using eight drugs of known teratogenicity,where it correctly predicted the teratogenicity for seven of the eight drugs. Thus,our initial data shows that this platform is a robust alternative to animal and other in vitro models for the prediction of the developmental toxicity of chemicals that may also provide invaluable information about the underlying biochemical pathways. ?? 2010 Elsevier Inc. View Publication

Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study,we demonstrate that Y-27632,a specific inhibitor of Rho kinase (ROCK) activity,significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore,treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably,hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y-27632. Additionally,we determined that Y-27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus,Y-27632 provides a means to kick-start" slow-growing human pluripotent stem cells� View Publication

We studied leukemic stem cells (LSCs) in a Smad4(-/-) mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9. Although Hoxa9-Smad4 complexes accumulate in the cytoplasm of normal hematopoietic stem cells and progenitor cells (HSPCs) transduced with these oncogenes,there is no cytoplasmic stabilization of HOXA9 in Smad4(-/-) HSPCs,and as a consequence increased levels of Hoxa9 is observed in the nucleus leading to increased immortalization in vitro. Loss of Smad4 accelerates the development of leukemia in vivo because of an increase in transformation of HSPCs. Therefore,the cytoplasmic binding of Hoxa9 by Smad4 is a mechanism to protect Hoxa9-induced transformation of normal HSPCs. Because Smad4 is a potent tumor suppressor involved in growth control,we developed a strategy to modify the subcellular distribution of Smad4. We successfully disrupted the interaction between Hoxa9 and Smad4 to activate the TGF-β pathway and apoptosis,leading to a loss of LSCs. Together,these findings reveal a major role for Smad4 in the negative regulation of leukemia initiation and maintenance induced by HOXA9/NUP98-HOXA9 and provide strong evidence that antagonizing Smad4 stabilization by these oncoproteins might be a promising novel therapeutic approach in leukemia. View Publication

IntroductionImmune cells that contribute to the pathogenesis of systemic lupus erythematosus (SLE) derive from adult hematopoietic stem and progenitor cells (HSPCs) within the bone marrow (BM). For this reason,we reasoned that fundamental abnormalities in SLE can be traced to a BM-derived HSPC inflammatory signature.MethodsBM samples from four SLE patients,six healthy controls,and two umbilical cord blood (CB) samples were used. CD34+ cells were isolated from BM and CB samples,and single-cell RNA-sequencing was performed.ResultsA total of 426 cells and 24,473 genes were used in the analysis. Clustering analysis resulted in seven distinct clusters of cell types. Mutually exclusive markers,which were characteristic of each cell type,were identified. We identified three HSPC subpopulations,one of which consisted of proliferating cells (MKI67 expressing cells),one T-like,one B-like,and two myeloid-like progenitor subpopulations. Differential expression analysis revealed i) cell cycle-associated signatures,in healthy BM of HSPC clusters 3 and 4 when compared with CB,and ii) interferon (IFN) signatures in SLE BM of HSPC clusters 3 and 4 and myeloid-like progenitor cluster 5 when compared with healthy controls. The IFN signature in SLE appeared to be deregulated following TF regulatory network analysis and differential alternative splicing analysis between SLE and healthy controls in HSPC subpopulations.DiscussionThis study revealed both quantitative—as evidenced by decreased numbers of non-proliferating early progenitors—and qualitative differences—characterized by an IFN signature in SLE,which is known to drive loss of function and depletion of HSPCs. Chronic IFN exposure affects early hematopoietic progenitors in SLE,which may account for the immune aberrancies and the cytopenias in SLE. View Publication

Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging,however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS),DNA damage,senescence,and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC,as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically,CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress,as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC),improved their niche supporting activity,and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production,driving an HSC aging phenotype. Thus,modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function. View Publication

The human transferrin receptor (hTfR) is a target for cancer immunotherapy due to its overexpression on the surface of cancer cells. We previously developed an antibody-avidin fusion protein that targets hTfR (anti-hTfR IgG3-Av) and exhibits intrinsic cytotoxicity against certain malignant cells. Gambogic acid (GA),a drug that also binds hTfR,induces cytotoxicity in several malignant cell lines. We now report that anti-hTfR IgG3-Av and GA induce cytotoxicity in a new broader panel of hematopoietic malignant cell lines. Our results show that the effect of anti-hTfR IgG3-Av is iron-dependent whereas that of GA is iron-independent in all cells tested. In addition,we observed that GA exerts a TfR-independent cytotoxicity. We also found that GA increases the generation of reactive oxygen species that may play a role in the cytotoxicity induced by this drug. Additive cytotoxicity was observed by simultaneous combination treatment with these drugs and synergy by using anti-hTfR IgG3-Av as a chemosensitizing agent. In addition,we found a concentration of GA that is toxic to malignant hematopoietic cells but not to human hematopoietic progenitor cells. Our results suggest that these two compounds may be effective,alone or in combination,for the treatment of human hematopoietic malignancies. View Publication

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2),a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly,whereas normal HSCs were rapidly exhausted after serial transplantations,overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast,similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a genetic genomics" screen� View Publication

The transcription factor Runx1/AML1 is an important regulator of hematopoiesis and is critically required for the generation of the first definitive hematopoietic stem cells (HSCs) in the major vasculature of the mouse embryo. As a pivotal factor in HSC ontogeny,its transcriptional regulation is of high interest but is largely undefined. In this study,we used a combination of comparative genomics and chromatin analysis to identify a highly conserved 531-bp enhancer located at position + 23.5 in the first intron of the 224-kb mouse Runx1 gene. We show that this enhancer contributes to the early hematopoietic expression of Runx1. Transcription factor binding in vivo and analysis of the mutated enhancer in transient transgenic mouse embryos implicate Gata2 and Ets proteins as critical factors for its function. We also show that the SCL/Lmo2/Ldb-1 complex is recruited to the enhancer in vivo. Importantly,transplantation experiments demonstrate that the intronic Runx1 enhancer targets all definitive HSCs in the mouse embryo,suggesting that it functions as a crucial cis-regulatory element that integrates the Gata,Ets,and SCL transcriptional networks to initiate HSC generation. View Publication

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript,we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore,we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages,further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication

Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay,which can be explained,in part,by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes,display extensive hemorrhaging,and die by embryonic day 14.5. In addition,Meis1-deficient embryos lack well-formed capillaries,although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos,but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC. View Publication