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

The mechanisms that regulate the growth and survival of acute myeloid leukemia (AML) cells are largely unknown. We hypothesized that constitutive activation of phosphatidyl-inositide 3 kinase (PI3 kinase) could regulate survival in primary cells from patients with AML. Here we demonstrate that Akt,a critical substrate of PI3 kinase,is activated in AML blasts. In a short-term culture system,most AML patient samples showed a dose-dependent decrease in survival after incubation with the PI3 kinase inhibitor LY294002. This decrease in survival was partially due to the induction of apoptosis. Furthermore,we have shown that p70 S6 kinase and 4EBP-1,downstream mediators of Akt signaling,also are phosphorylated in AML blasts. Phosphorylation of these proteins is inhibited by the mTOR inhibitor RAD001. Incubation of AML blasts with RAD001 induces only a small decrease in survival of the cells; however,when combined with Ara-C,RAD001 enhances the toxicity of Ara-C. These results demonstrate that constitutive activation of the PI3 kinase pathway is necessary for the survival of AML blasts and that targeting of this pathway with pharmacologic inhibitors may be of clinical benefit in treatment of AML. View Publication

Recent technological advances in cell reprogramming by generation of induced pluripotent stem cells (iPSC) offer major perspectives in disease modelling and future hopes for providing novel stem cells sources in regenerative medicine. However,research on iPSC still requires refining the criteria of the pluripotency stage of these cells and exploration of their equivalent functionality to human embryonic stem cells (ESC). We report here on the use of infrared microspectroscopy to follow the spectral modification of somatic cells during the reprogramming process. We show that induced pluripotent stem cells (iPSC) adopt a chemical composition leading to a spectral signature indistinguishable from that of embryonic stem cells (ESC) and entirely different from that of the original somatic cells. Similarly,this technique allows a distinction to be made between partially and fully reprogrammed cells. We conclude that infrared microspectroscopy signature is a novel methodology to evaluate induced pluripotency and can be added to the tests currently used for this purpose. View Publication

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels,differentiation intermediates were investigated. ES cells generated progeny expressing CD34,which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7,the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells,and such lymphoid potential was highly enriched in the CD34+ population. Interestingly,the cytotoxic cells expressed the natural killer (NK) cell markers,such as NKR-P1,perforin,and granzymes,classified into two types,one of which showed target specificity of NK cells. Thus,ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells,and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate,on which VEGF may play an important role. View Publication

It is well accepted that umbilical cord blood has been a source for hematopoietic stem cells. However,controversy exists as to whether cord blood can serve as a source of mesenchymal stem cells,which can differentiate into cells of different connective tissue lineages such as bone,cartilage,and fat,and little success has been reported in the literature about the isolation of such cells from cord blood. Here we report a novel method to obtain single cell-derived,clonally expanded mesenchymal stem cells that are of multilineage differentiation potential by negative immunoselection and limiting dilution. The immunophenotype of these clonally expanded cells is consistent with that reported for bone marrow mesenchymal stem cells. Under appropriate induction conditions,these cells can differentiate into bone,cartilage,and fat. Surprisingly,these cells were also able to differentiate into neuroglial- and hepatocyte-like cells under appropriate induction conditions and,thus,these cells may be more than mesenchymal stem cells as evidenced by their ability to differentiate into cell types of all 3 germ layers. In conclusion,umbilical cord blood does contain mesenchymal stem cells and should not be regarded as medical waste. It can serve as an alternative source of mesenchymal stem cells to bone marrow. View Publication

Neural progenitor cells (NPCs) can be induced from somatic cells by defined factors. Here we report that NPCs can be generated from mouse embryonic fibroblasts by a chemical cocktail,namely VCR (V,VPA,an inhibitor of HDACs; C,CHIR99021,an inhibitor of GSK-3 kinases and R,Repsox,an inhibitor of TGF-β pathways),under a physiological hypoxic condition. These chemical-induced NPCs (ciNPCs) resemble mouse brain-derived NPCs re- garding their proliferative and self-renewing abilities,gene expression profiles,and multipotency for different neu- roectodermal lineages in vitro and in vivo. Further experiments reveal that alternative cocktails with inhibitors of histone deacetylation,glycogen synthase kinase,and TGF-β pathways show similar efficacies for ciNPC induction. Moreover,ciNPCs can also be induced from mouse tail-tip fibroblasts and human urinary cells with the same chemi- cal cocktail VCR. Thus our study demonstrates that lineage-specific conversion of somatic cells to NPCs could be achieved by chemical cocktails without introducing exogenous factors. View Publication

Werner syndrome (WS) patients exhibit premature aging predominantly in mesenchyme-derived tissues,but not in neural lineages,a consequence of telomere dysfunction and accelerated senescence. The cause of this lineage-specific aging remains unknown. Here,we document that reprogramming of WS fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction. To obtain mechanistic insight into the origin of tissue-specific aging,we differentiated iPSCs to mesenchymal stem cells (MSCs) and neural stem/progenitor cells (NPCs). We observed recurrence of premature senescence associated with accelerated telomere attrition and defective synthesis of the lagging strand telomeres in MSCs,but not in NPCs. We postulate this aging" discrepancy is regulated by telomerase. Expression of hTERT or p53 knockdown ameliorated the accelerated aging phenotypein MSC� View Publication

X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known,in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containingTAF1,a large gene with at least 38 exons,and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 ofTAF1,as well as a neural-specific TAF1 isoform,N-TAF1,which showed decreased expression in post-mortem XDP brain compared with control tissue. Here,we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model,we compared expression ofTAF1and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells,XDP fibroblasts exhibited decreased expression ofTAF1transcript fragments derived from exons 32-36,a region spanning the SVA insertion site. N-TAF1,which incorporates an alternative exon (exon 34'),was not expressed in fibroblasts,but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP,but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration. View Publication

Asymmetry of cell fate is one fundamental property of stem cells,in which one daughter cell self-renews,whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms,such as plants,fungi,and mammals,has already been shown. However,before this current work,asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs),and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing,differentiating human and mouse ESCs. Moreover,we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3),indicating a molecular mechanism that regulates this phenomenon. Furthermore,our data support the hypothesis that retention of chromatids with the old" template DNA preserves the epigenetic memory of cell fate� View Publication

Individual variation in fetal hemoglobin (HbF,alpha(2)gamma(2)) response underlies the remarkable diversity in phenotypic severity of sickle cell disease and beta thalassemia. HbF levels and HbF-associated quantitative traits (e.g.,F cell levels) are highly heritable. We have previously mapped a major quantitative trait locus (QTL) controlling F cell levels in an extended Asian-Indian kindred with beta thalassemia to a 1.5-Mb interval on chromosome 6q23,but the causative gene(s) are not known. The QTL encompasses several genes including HBS1L,a member of the GTP-binding protein family that is expressed in erythroid progenitor cells. In this high-resolution association study,we have identified multiple genetic variants within and 5' to HBS1L at 6q23 that are strongly associated with F cell levels in families of Northern European ancestry (P = 10(-75)). The region accounts for 17.6% of the F cell variance in northern Europeans. Although mRNA levels of HBS1L and MYB in erythroid precursors grown in vitro are positively correlated,only HBS1L expression correlates with high F cell alleles. The results support a key role for the HBS1L-related genetic variants in HbF control and illustrate the biological complexity of the mechanism of 6q QTL as a modifier of fetal hemoglobin levels in the beta hemoglobinopathies. View Publication

Obesity has been suggested to be a low-grade systemic inflammatory state,therefore we studied the interaction between human adipocytes and monocytes via adipose tissue (AT)-derived capillary endothelium. Cells composing the stroma-vascular fraction (SVF) of human ATs were characterized by fluorescence-activated cell sorter (FACS) analysis and two cell subsets (resident macrophages and endothelial cells [ECs]) were isolated using antibody-coupled microbeads. Media conditioned by mature adipocytes maintained in fibrin gels were applied to AT-derived ECs. Thereafter,the expression of endothelial adhesion molecules was analyzed as well as the adhesion and transmigration of human monocytes. FACS analysis showed that 11% of the SVF is composed of CD14(+)/CD31(+) cells,characterized as resident macrophages. A positive correlation was found between the BMI and the percentage of resident macrophages,suggesting that fat tissue growth is associated with a recruitment of blood monocytes. Incubation of AT-derived ECs with adipocyte-conditioned medium resulted in the upregulation of EC adhesion molecules and the increased chemotaxis of blood monocytes,an effect mimicked by recombinant human leptin. These results indicate that adipokines,such as leptin,activate ECs,leading to an enhanced diapedesis of blood monocytes,and suggesting that fat mass growth might be linked to inflammatory processes. View Publication

Retinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach,we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor,retinoid-inducible nuclear factor (RINF),that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34(+) progenitors. Furthermore,short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly,RINF localizes to 5q31.3,a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene. View Publication

This work describes a genetic approach to isolate small,artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric,44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a scaffold to construct a retrovirus library expressing approximately 500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small,dimeric TM proteins that were structurally unrelated to erythropoietin (EPO),but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFbetaR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore,this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus,we have changed the specificity of a protein so that it no longer recognizes its natural target but,instead,modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word traptamer" for these transmembrane aptamers." View Publication