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

The histo-blood group i and I antigens have been characterized as straight and branched repeats of N-acetyllactosamine,respectively,and the conversion of the straight-chain i to the branched-chain I structure on red cells is regulated to occur after birth. It has been demonstrated that the human I locus expresses 3 IGnT transcripts,IGnTA,IGnTB,and IGnTC,and that the last of these is responsible for the I branching formation on red cells. In the present investigation,the K-562 cell line was used as a model to show that the i-to-I transition in erythroid differentiation is determined by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha),which enhances transcription of the IGnTC gene,consequently leading to formation of the I antigen. Further investigation suggested that C/EBPalpha IGnTC-activation activity is modulated at a posttranslational level,and that the phosphorylation status of C/EBPalpha may have a crucial effect. Results from studies using adult and cord erythropoietic cells agreed with those derived using the K-562 cell model,with lentiviral expression of C/EBPalpha in CD34(+) hemopoietic cells demonstrating the determining role of C/EBPalpha in the induction of the IGnTC gene as well as in I antigen expression. View Publication

Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23,MYB was a good candidate gene. Our molecular investigations,based on fluorescence in situ hybridization and rapid amplification of cDNA ends,revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together,these results establish,for the first time,a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia. View Publication

The future of safe cell-based therapy rests on overcoming teratoma/tumor formation,in particular when using human pluripotent stem cells (hPSCs),such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation,complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile,we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e.,survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors,leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular,a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g.,quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast,differentiated cell types (e.g.,dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together,these results provide the proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy." View Publication

Approximately 70% of KRAS-positive colorectal cancers (CRCs) have a CpG island methylator phenotype (CIMP) characterized by aberrant DNA hypermethylation and transcriptional silencing of many genes. The factors involved in,and the mechanistic basis of,CIMP is not understood. Among the CIMP genes are the tumor suppressors p14(ARF),p15(INK4B),and p16(INK4A),encoded by the INK4-ARF locus. In this study,we perform an RNA interference screen and identify ZNF304,a zinc-finger DNA-binding protein,as the pivotal factor required for INK4-ARF silencing and CIMP in CRCs containing activated KRAS. In KRAS-positive human CRC cell lines and tumors,ZNF304 is bound at the promoters of INK4-ARF and other CIMP genes. Promoter-bound ZNF304 recruits a corepressor complex that includes the DNA methyltransferase DNMT1,resulting in DNA hypermethylation and transcriptional silencing. KRAS promotes silencing through upregulation of ZNF304,which drives DNA binding. Finally,we show that ZNF304 also directs transcriptional silencing of INK4-ARF in human embryonic stem cells. DOI: http://dx.doi.org/10.7554/eLife.02313.001. View Publication

Understanding the mechanisms that sustain pluripotency in human embryonic stem cells (hESCs) is an active area of research that may prove useful in regenerative medicine and will provide fundamental information relevant to development and cancer. hESCs and cancer cells share the unique ability to proliferate indefinitely and rapidly. Because the protein survivin is uniquely overexpressed in virtually all human cancers and in hESCs,we sought to investigate its role in supporting the distinctive capabilities of these cell types. Results presented here suggest that survivin contributes to the maintenance of pluripotency and that post-transcriptional control of survivin isoform expression is selectively regulated by microRNAs. miR-203 has been extensively studied in human tumors,but has not been characterized in hESCs. We show that miR-203 expression and activity is consistent with the expression and subcellular localization of survivin isoforms that in turn modulate expression of the Oct4 and Nanog transcription factors to sustain pluripotency. This study contributes to understanding of the complex regulatory mechanisms that govern whether hESCs proliferate or commit to lineages. textcopyright 2014 Wiley Periodicals,Inc. View Publication

Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo,yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5,an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore,Ncx1 heartbeat mutants,as well as static cultures of AGM,exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures,transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. View Publication

Tools for rapid and efficient transgenesis in safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE� View Publication

Helper-dependent adenoviral vectors (HDAd) that express certain transgene products are impossible to produce because the transgene product is toxic to the producer cells,especially when made in large amounts during vector production. Downregulating transgene expression from the HDAd during vector production is a way to solve this problem. In this report,we show that this can be accomplished by inserting the target sequence for the adenoviral VA RNAI into the 3' untranslated region of the expression cassette in the HDAd. Thus during vector production,when the producer cells are coinfected with both the helper virus (HV) and the HDAd,the VA RNAI produced by the HV will target the transgene mRNA from the HDAd via the endogenous cellular RNAi pathway. Once the HDAd is produced and purified,transduction of the target cells results in unimpeded transgene expression because of the absence of HV. This simple and universal strategy permits for the robust production of otherwise recalcitrant HDAds. View Publication

Imatinib is currently the first line therapy for newly diagnosed patients with chronic myeloid leukemia. However,20-25% of patients do not achieve durable complete cytogenetic responses. The mechanism underlying this primary resistance is unknown,but variations in BCR-ABL1 kinase activity may play a role and can be investigated by measuring the autophosphorylation levels of BCR-ABL1 or of a surrogate target such as Crkl. In this study we used flow cytometry to investigate the in vitro inhibition of Crkl phosphorylation by imatinib in CD34(+) cells in diagnostic samples from two groups of patients distinguished by their cytogenetic response. No difference in inhibition of Crkl phosphorylation was observed in the two groups. The observation that increasing the dose of imatinib in vivo did not increase the level of cytogenetic response in some non-responders suggests that in at least a proportion of patients imatinib resistance may be due to activation of BCR-ABL1-independent pathway. View Publication

The immunodeficiency disorder,X-linked agammaglobulinemia (XLA),results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA,we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice,a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells,treated mice showed significant,albeit incomplete,rescue of mature B cells in the bone marrow,peripheral blood,spleen,and peritoneal cavity,and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression,viral integration,and partial functional responses,consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA. View Publication

Activating mutations in signaling molecules,such as JAK2-V617F,have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients,suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here,we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells,we demonstrated that Lnk expression is regulated by the TPO-signaling pathway,thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain,but also through an unrevealed JAK2-binding site within its N-terminal region. In addition,the presence of the V617F mutation causes a tighter association with Lnk. Finally,we found that the expression level of the Lnk protein can modulate JAK2-V617F-dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together,our results indicate that changes in Lnk expression and JAK2-V617F-binding regulate JAK2-mediated signals in MPNs. View Publication