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

We mutated,by gene targeting,the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies,1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r,6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous,neor-containing,Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene. View Publication

Hematopoietic stem cells (HSC) are tightly regulated through,as yet,undefined mechanisms that balance self-renewal and differentiation. We have identified a role for the transcriptional coactivators CREB-binding protein (CBP) and p300 in such HSC fate decisions. A full dose of CBP,but not p300,is crucial for HSC self-renewal. Conversely,p300,but not CBP,is essential for proper hematopoietic differentiation. Furthermore,in chimeric mice,hematologic malignancies emerged from both CBP(-/-) and p300(-/-) cell populations. Thus,CBP and p300 play essential but distinct roles in maintaining normal hematopoiesis,and,in mice,both are required for preventing hematologic tumorigenesis. View Publication

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast,we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34,lineage markers,CD45 and CD133 (CD34⁻Lin⁻CD45⁻CD133⁻ cells),endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34⁻Lin⁻CD45⁻CD133⁻ cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo,CD34⁻Lin⁻CD45⁻CD133⁻ cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34⁻Lin⁻D45⁻CD133⁻ cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials,characterized by the expression of c-Kit and CXCR4 as well as EphB4,EphB2,and ephrinB2. Further molecular and functional characterization of CD34⁻Lin⁻CD45⁻CD133⁻ cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life. View Publication

Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here,we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition,SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore,through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether,our results indicate that even an extremely homogeneous HSC population,based on phenotype and dye efflux,cannot reconstitute mice at absolute efficiencies. View Publication

The regulatory pathways necessary for the maintenance of adult hematopoietic stem cells (HSCs) remain poorly defined. By using loss-of-function approaches,we report a selective and cell-autonomous requirement for the p300/CBP-binding transcriptional coactivator Cited2 in adult HSC maintenance. Conditional deletion of Cited2 in the adult mouse results in loss of HSCs causing multilineage bone marrow failure and increased lethality. In contrast,conditional ablation of Cited2 after lineage specification in lymphoid and myeloid lineages has no impact on the maintenance of these lineages. Additional deletion of Ink4a/Arf (encoding p16(Ink4a) and p19(Arf)) or Trp53 (encoding p53,a downstream target of p19(Arf)) in a Cited2-deficient background restores HSC functionality and rescues mice from bone marrow failure. Furthermore,we show that the critical role of Cited2 in primitive hematopoietic cells is conserved in humans. Taken together,our studies provide genetic evidence that Cited2 selectively maintains adult HSC functions,at least in part,via Ink4a/Arf and Trp53. View Publication

PTPN11 encodes the protein tyrosine phosphatase SHP-2,which relays signals from growth factor receptors to Ras and other effectors. Germline PTPN11 mutations underlie about 50% of Noonan syndrome (NS),a developmental disorder that is associated with an elevated risk of juvenile myelomonocytic leukemia (JMML). Somatic PTPN11 mutations were recently identified in about 35% of patients with JMML; these mutations introduce amino acid substitutions that are largely distinct from those found in NS. We assessed the functional consequences of leukemia-associated PTPN11 mutations in murine hematopoietic cells. Expressing an E76K SHP-2 protein induced a hypersensitive pattern of granulocyte-macrophage colony-forming unit (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells with high replating potential,perturbed erythroid growth,and impaired normal differentiation in liquid cultures. In addition,leukemia-associated SHP-2 mutations conferred a stronger phenotype than a germline mutation found in patients with NS. Mutant SHP-2 proteins induce aberrant growth in multiple hematopoietic compartments,which supports a primary role of hyperactive Ras in the pathogenesis of JMML. View Publication

Histone deacetylase inhibitors have attracted considerable attention because of their ability to overcome the differentiation block in leukemic blasts,an effect achieved either alone or in combination with differentiating agents,such as all-trans retinoic acid. We have previously reported favorable effects of the potent histone deacetylase inhibitor valproic acid in combination with all-trans retinoic acid in patients with advanced acute myeloid leukemia leading to blast cell reduction and improvement of hemoglobin. These effects were accompanied by hypergranulocytosis most likely due to an enhancement of nonleukemic myelopoiesis and the suppression of malignant hematopoiesis rather than enforced differentiation of the leukemic cells. These data prompted us to investigate the effect of valproic acid on normal hematopoietic stem cells (HSC). Here we show that valproic acid increases both proliferation and self-renewal of HSC. It accelerates cell cycle progression of HSC accompanied by a down-regulation of p21(cip-1/waf-1). Furthermore,valproic acid inhibits GSK3beta by phosphorylation on Ser9 accompanied by an activation of the Wnt signaling pathway as well as by an up-regulation of HoxB4,a target gene of Wnt signaling. Both are known to directly stimulate the proliferation of HSC and to expand the HSC pool. In summary,we here show that valproic acid,known to induce differentiation or apoptosis in leukemic blasts,stimulates the proliferation of normal HSC,an effect with a potential effect on its future role in the treatment of acute myeloid leukemia. View Publication

Hematopoietic stem cells (HSC) can be harmed by disease,chemotherapy,radiation,and normal aging. We show in this study that damage also occurs in mice repeatedly treated with very low doses of LPS. Overall health of the animals was good,and there were relatively minor changes in marrow hematopoietic progenitors. However,HSC were unable to maintain quiescence,and transplantation revealed them to be myeloid skewed. Moreover,HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor. This phenomenon was previously seen in normal aging. Screening identified mAbs that resolve HSC subsets,and relative proportions of these HSC changed with age and/or chronic LPS treatment. For example,minor CD150(Hi)CD48(-) populations lacking CD86 or CD18 expanded. Simultaneous loss of CD150(Lo/-)CD48(-) HSC and gain of the normally rare subsets,in parallel with diminished transplantation potential,would be consistent with age- or TLR-related injury. In contrast,HSC in old mice differed from those in LPS-treated animals with respect to VCAM-1 or CD41 expression and lacked proliferation abnormalities. HSC can be exposed to endogenous and pathogen-derived TLR ligands during persistent low-grade infections. This stimulation might contribute in part to HSC senescence and ultimately compromise immunity. View Publication

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function,though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mphis) directs HSC platelet-bias. Mphis from the marrow of aged mice and humans exhibited an activated phenotype,with increased expression of inflammatory signals. Aged marrow Mphis also displayed decreased phagocytic function. Senescent neutrophils,typically cleared by marrow Mphis,were markedly increased in aged mice,consistent with functional defects in Mphi phagocytosis and efferocytosis. In aged mice,Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity,which can process pro-IL1B,was increased in marrow Mphis and neutrophils. Mechanistically,IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice,depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mphis induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mphis and IL1B in the age-associated lineage-skewing of HSCs,and reveals the therapeutic potential of their manipulation as antigeronic targets. View Publication

The corneal epithelium is maintained by stem cells located at the periphery of the cornea in a region known as the limbus. Depletion of limbal stem cells (LSCs) results in limbal stem cell deficiency. Treatments for this disease are based on limbal replacement or transplantation of ex vivo expanded LSCs. It is,therefore,crucial to identify cell surface markers for LSCs that can be used for their enrichment and characterization. Aldehyde dehydrogenases (ALDHs) are enzymes which protect cells from the toxic effects of peroxidic aldehydes. In this manuscript,we show for the first time that ALDH1 is absent from the basal cells of the limbal and corneal epithelium. We separated limbal epithelial cells on the basis of ALDH activity and showed that ALDH(dim) cells expressed significantly higher levels of DeltaNp63 and ABCG2 as well as having a greater colony forming efficiency (CFE) when compared to ALDH(bright) cells. Large scale transcriptional analysis of these two populations led to identification of a new cell surface marker,RHAMM/HMMR,which is located in all layers of corneal epithelium and in the suprabasal layers of the limbal epithelium but is completely absent from the basal layer of the limbus. Our studies indicate that absence of RHAMM/HMMR expression is correlated with properties associated with LSCs. RHAMM/HMMR- limbal epithelial cells are smaller in size,express negligible CK3,have higher levels of DeltaNp63 and have a higher CFE compared to RHAMM/HMMR+ cells. Taken together these results suggest a putative role for RHAMM/ HMMR as a negative marker of stem cell containing limbal epithelial cells. Cell selection based on Hoechst exclusion and lack of cell surface RHAMM/HMMR expression resulted in increased colony forming efficiency compared to negative selection using RHAMM/HMMR alone or positive selection using Hoechst on its own. Combination of these two cell selection methods presents a novel method for LSC enrichment and characterization. Disclosure of potential conflicts of interest is found at the end of this article. View Publication