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

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs),and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo,but is essential for normal HSC/HPC differentiation. In addition,Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased β1 integrin and Cxcr4 expression. Moreover,deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and,subsequently,improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult. View Publication

Stem cell-based tissue engineering is a promising technology in the effort to create functional tissues of choice. To establish an efficient approach for generating hematopoietic cell lineages directly from embryonic stem (ES) cells and to study the effects of three-dimensional (3D) biomaterials on ES cell differentiation,we cultured mouse ES cells on 3D,highly porous,biomimetic scaffolds. Cell differentiation was evaluated by microscopy and flow cytometry analysis with a variety of hematopoiesis- specific markers. Our data indicate that ES cells differentiated on porous 3D scaffold structures developed embryoid bodies (EBs) similar to those in traditional two-dimensional (2D) cultures; however,unlike 2D differentiation,these EBs integrated with the scaffold and appeared embedded in a network of extracellular matrix. Most significantly,the efficiency of hematopoietic precursor cell (HPC) generation on 3D,as indicated by the expression of various HPC-specific surface markers (CD34,Sca-1,Flk-1,and c-Kit) and colony-forming cell (CFC) assays,was reproducibly increased (about 2-fold) over their 2D counterparts. Comparison of static and dynamic 3D cultures demonstrated that spinner flask technology also contributed to the higher hematopoietic differentiation efficiency of ES cells seeded on scaffolds. Continued differentiation of 3D-derived HPCs into the myeloid lineage demonstrated increased efficiency (2-fold) of generating myeloid compared with differentiation from 2D-derived HPCs. View Publication

Hematopoiesis is tightly controlled by transcription regulatory networks,but how and when specific transcription factors control lineage commitment are still largely unknown. Within the hematopoietic stem cell (Lin(-)Sca-1(+)c-Kit(+)) compartment these lineage-specific transcription factors are expressed at low levels but are up-regulated with the process of lineage specification. CCAAT/enhancer binding protein α (C/EBPα) represents one of these factors and is involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor cells,which express C/EBPα,we developed a mouse model expressing Cre recombinase from the Cebpa promoter and a conditional EYFP allele. We show that Cebpa/EYFP(+) cells represent a significant subset of multipotent hematopoietic progenitors,which predominantly give rise to myeloid cells in steady-state hematopoiesis. C/EBPα induced a strong myeloid gene expression signature and down-regulated E2A-induced regulators of early lymphoid development. In addition,Cebpa/EYFP(+) cells compose a fraction of early thymic progenitors with robust myeloid potential. However,Cebpa/EYFP(+) multipotent hematopoietic progenitors and early thymic progenitors retained the ability to develop into erythroid and T-lymphoid lineages,respectively. These findings support an instructive but argue against a lineage-restrictive role of C/EBPα in multipotent hematopoietic and thymic progenitors. View Publication

PURPOSE: Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells,but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. METHODS: The seven osteosarcoma cell lines Cal72,SJSA-1,Saos-2,SK-ES-1,U2OS,143.98.2,and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). RESULTS: Although proliferation often was relatively low in serum-free media (X-vivo 10,X-vivo 15,X-vivo 20,Stem Span SFEM),some cell lines (Cal72,KHOS-32IH,Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However,all cell lines proliferated well in Stem Span with FCS,and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS),and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72,SJSA-1),and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1. CONCLUSIONS: Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines,but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation,cytokine release); and (iii) whether coculture experiments are included. View Publication

Vascular endothelial growth factor (VEGF) and its receptors play an essential role in the formation and maintenance of the hematopoietic and vascular compartments. The VEGF receptor-2 (VEGFR-2) is expressed on a population of hematopoietic cells,although its role in hematopoiesis is still unclear. In this report,we have utilized a strategy to selectively activate VEGFR-2 and study its effects in primary bone marrow cells. We found that VEGFR-2 can maintain the hematopoietic progenitor population in mouse bone marrow cultured in the absence of exogenous cytokines. Maintenance of the hematopoietic progenitor population is due to increased cell survival with minimal effect on proliferation. Progenitor survival is mainly mediated by activation of the phosphatidylinositol 3'-kinase/Akt pathway. Although VEGFR-2 also activated Erk1/2 mitogen-activated protein kinase,it did not induce cell proliferation,and blockade of this pathway only partially decreased VEGFR-2-mediated survival of hematopoietic progenitors. Thus,the role of VEGFR-2 in hematopoiesis is likely to maintain survival of hematopoietic progenitors through the activation of antiapoptotic pathways. View Publication

Efficient derivation and isolation of hematopoietic stem cells (HSCs) from human pluripotent stem cell (hPSC) populations remains a major goal in the field of developmental hematopoiesis. These enticing pluripotent stem cells (comprising both human embryonic stem cells and induced pluripotent stem cells) have been successfully used to generate a wide array of hematopoietic cells in vitro,from primitive hematoendothelial precursors to mature myeloid,erythroid,and lymphoid lineage cells. However,to date,PSC-derived cells have demonstrated only limited potential for long-term multilineage hematopoietic engraftment in vivo - the test by which putative HSCs are defined. Successful generation and characterization of HSCs from hPSCs not only requires an efficient in vitro differentiation system that provides insight into the developmental fate of hPSC-derived cells,but also necessitates an in vivo engraftment model that allows identification of specific mechanisms that hinder or promote hematopoietic engraftment. In this chapter,we will describe a method that utilizes firefly luciferase-expressing hPSCs and bioluminescent imaging to noninvasively track the survival,proliferation,and migration of transplanted hPSC-derived cells. Combined with lineage and functional analyses of engrafted cells,this system is a useful tool to gain insight into the in vivo potential of hematopoietic cells generated from hPSCs. View Publication

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However,the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34(-)c-kit(+)Sca-1(+)lineage(-) (34(-)KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34(-)KSL cells. Treatment of bone marrow (BM) 34(-)KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units,verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment,confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture,suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes. View Publication

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates,specifically,hematopoietic,mesodermal,and neurectodermal. In this study,we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore,we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells,expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo,and potentially for understanding and treating neurodegenerative and psychiatric diseases. View Publication

We have shown that a COOH-terminal peptide of p53 (amino acids 361-382,p53p),linked to the truncated homeobox domain of Antennapedia (Ant) as a carrier for transduction,induced rapid apoptosis in human premalignant and malignant cell lines. Here,we report that human and rat glioma lines containing endogenous mutant p53 or wild-type (WT) p53 were induced into apoptosis by exposure to this peptide called p53p-Ant. The peptide was comparatively nontoxic to proliferating nonmalignant human and rat glial cell lines containing WT p53 and proliferating normal human peripheral marrow blood stem cells. Degree of sensitivity to the peptide correlated directly with the level of endogenous p53 expression and mutant p53 conformation. Apoptosis induction by p53p-Ant was quantitated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and Annexin V staining in human glioma cells in vitro and in a syngeneic orthotopic 9L glioma rat model using convection-enhanced delivery in vivo. The mechanism of cell death by this peptide was solely through the Fas extrinsic apoptotic pathway. p53p-Ant induced a 3-fold increase in extracellular membrane Fas expression in glioma cells but no significant increase in nonmalignant glial cells. These data suggest that p53 function for inducing Fas-mediated apoptosis in gliomas,which express sufficient quantities of endogenous mutant or WT p53,may be restored or activated,respectively,by a cell-permeable peptide derived from the p53 COOH-terminal regulatory domain (p53p-Ant). p53p-Ant may serve as a prototypic model for the development of new anticancer agents with unique selectivity for glioma cancer cells and it can be successfully delivered in vivo into a brain tumor by a convection-enhanced delivery system,which circumvents the blood-brain barrier. View Publication

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway,which utilizes beta-catenin to relay the signal,have been demonstrated to regulate HSC function. In this study,we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation,such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G(0) state. From these data,we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway,resulting in a pool of quiescent HSCs. View Publication

Although practiced clinically for more than 40 years,the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative,StemRegenin 1 (SR1),that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy. View Publication