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

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently,the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed,respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last,screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together,our results highlight the potential of hiPSCs for studying human tumourigenesis. View Publication

The aim of this study was to standardize in vitro culture conditions for human acute lymphoblastic leukemia (ALL) cells. The cells were cultured in medium containing 10% fetal calf serum (FCS) and in the four serum-free media X-vivo 10,X-vivo 15,X-vivo 20 and Stem Span. Native ALL blasts could proliferate in all four serum-free media,but the strongest responses were usually observed with Stem Span. Native leukemia blasts were also cultured in the presence of various single cytokines or cytokine combinations. The highest proliferation was usually observed in the presence of Flt3-Ligand (Flt3-L) when single cytokines were examined,and these responses could be further increased especially by combining Flt3-L with interleukin 3 (IL3),IL7 or stem cell factor (SCF). Proliferation could also be increased when ALL blasts were cultured in the presence of two commercially available fibroblast cell lines (Hs27 and HFL1). Based on these results we suggest that in vitro culture conditions for native human ALL blasts can be standardized by using serum-free culture media supplemented with exogenous Flt3-L+IL3+SCF,and the use of accessory cells can also be standardized by using well-characterized fibroblast cell lines. Detectable ALL blast proliferation can then be observed for most patients. Our experimental model can thereby be used for in vitro evaluation of possible antileukemic treatment strategies,and it will then allow comparison of experimental results between different studies. View Publication

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels,which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks,the gels contain bilayered mammary epithelial structures,including luminal and myoepithelial cells,their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations. View Publication

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Mesenchymal stem cells (MSCs) are highly useful in a variety of cell therapies owing to their multipotential differentiation capability. MSCs derived from umbilical cord blood are generally isolated by their plastic adherence without using specific cell surface markers and examined for their osteogenic,adipogenic,and chondrogenic differentiation properties retrospectively. Here,we report 2 subpopulations of MSCs,separated based on aldehyde dehydrogenase (ALDH) activity. MSCs with a high ALDH activity (Alde-High) proliferated more than those with a low ALDH activity (Alde-Low). Alde-High MSCs had a greater ability to differentiate than Alde-Low MSCs in in vitro culture. Transplantation of Alde-High MSCs into fractured mouse femurs enabled early repair of tissues and rapid bone substitution. Alde-High MSCs were also more responsive to hypoxia than Alde-Low MSCs,with the upregulation of Flt-1,CXCR4,and Angiopoietin-2. Thus,MSCs with a high ALDH activity might serve as an effective therapeutic tool for healing fractures within a short period of time. View Publication

Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers,including myeloid malignancies. We investigated the effects of an oncogenic Kras(G12D) allele on phosphorylated signaling molecules in primary c-kit(+) lin(-/low) hematopoietic stem/progenitor cells. Comparison of wild-type and Kras(G12D) c-kit(+) lin(-/low) cells shows that K-Ras(G12D) expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5,ERK,and S6 under basal and stimulated conditions. Whereas Kras(G12D) cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor,we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell-targeted interventions. View Publication

Acute myelogenous leukemia 1 (AML1; runt-related transcription factor 1 [Runx1]) is a member of Runx transcription factors and is essential for definitive hematopoiesis. Although AML1 possesses several subdomains of defined biochemical functions,the physiologic relevance of each subdomain to hematopoietic development has been poorly understood. Recently,the consequence of carboxy-terminal truncation in AML1 was analyzed by the hematopoietic rescue assay of AML1-deficient mouse embryonic stem cells using the gene knock-in approach. Nonetheless,a role for specific internal domains,as well as for mutations found in a human disease,of AML1 remains to be elucidated. In this study,we established an experimental system to efficiently evaluate the hematopoietic potential of AML1 using a coculture system of the murine embryonic para-aortic splanchnopleural (P-Sp) region with a stromal cell line,OP9. In this system,the hematopoietic defect of AML1-deficient P-Sp can be rescued by expressing AML1 with retroviral infection. By analysis of AML1 mutants,we demonstrated that the hematopoietic potential of AML1 was closely related to its transcriptional activity. Furthermore,we showed that other Runx transcription factors,Runx2/AML3 or Runx3/AML2,could rescue the hematopoietic defect of AML1-deficient P-Sp. Thus,this experimental system will become a valuable tool to analyze the physiologic function and domain contribution of Runx proteins in hematopoiesis. View Publication

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation,overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities,which indicates the presence of other mechanisms for Evi-1 activation. In this study,we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population,in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore,gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells. View Publication

The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein,which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis,we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells,as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients,even in the absence of t(8;21). On a functional level,knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly,self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies,serial replating capacity of primary cells,and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML. View Publication

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia,an aggressive CD4(+) malignancy. Although HTLV-2 is highly homologous to HTLV-1,infection with HTLV-2 has not been associated with lymphoproliferative disorders. Lentivirus-mediated transduction of CD34(+) cells with HTLV-1 Tax (Tax1) induced G(0)/G(1) cell cycle arrest and resulted in the concomitant suppression of multilineage hematopoiesis in vitro. Tax1 induced transcriptional upregulation of the cdk inhibitors p21(cip1/waf1) (p21) and p27(kip1) (p27),and marked suppression of hematopoiesis in immature (CD34(+)/CD38(-)) hematopoietic progenitor cells in comparison to CD34(+)/CD38(+) cells. HTLV-1 infection of CD34(+) cells also induced p21 and p27 expression. Tax1 also protected CD34(+) cells from serum withdrawal-mediated apoptosis. In contrast,HTLV-2 Tax (Tax2) did not detectably alter p21 or p27 gene expression,failed to induce cell cycle arrest,failed to suppress hematopoiesis in CD34(+) cells,and did not protect cells from programmed cell death. A Tax2/Tax1 chimera encoding the C-terminal 53 amino acids of Tax1 fused to Tax2 (Tax(221)) displayed a phenotype in CD34(+) cells similar to that of Tax1,suggesting that unique domains encoded within the C terminus of Tax1 may account for the phenotypes displayed in human hematopoietic progenitor cells. These remarkable differences in the activities of Tax1 and Tax2 in CD34(+) hematopoietic progenitor cells may underlie the sharp differences observed in the pathogenesis resulting from infection with HTLV-1 and HTLV-2. View Publication