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

Hematopoiesis is dependent upon the bone marrow microenvironment,which is comprised of multiple mesenchymal cell types,including fibroblasts,endothelial cells,osteoblasts,and stroma progenitors. The canonical Wnt signaling pathway,which relies on the beta-catenin protein to mediate its signal,is necessary for the normal development of mesenchymal tissue. We hypothesized that canonical Wnt signaling regulates the cellular composition and function of the bone marrow microenvironment. We observed that a beta-catenin-deficient bone marrow microenvironment maintained hematopoietic stem cells but exhibited a decreased capacity to support primitive hematopoietic cells. These results correlated with decreased numbers of osteoblasts and with decreased production of basic fibroblast growth factor,stem cell factor,and vascular cell adhesion molecule-1. From these data,we propose a model in which beta-catenin in the microenvironment is required noncell autonomously for long-term maintenance of hematopoietic progenitors. View Publication

Maintaining a steady pool of self-renewing hematopoietic stem cells (HSCs) is critical for sustained production of multiple blood lineages. Many transcription factors and molecules involved in chromatin and epigenetic modifications have been found to be critical for HSC self-renewal and differentiation; however,their interplay is less understood. The transcription factor GA binding protein (GABP),consisting of DNA-binding subunit GABPα and transactivating subunit GABPβ,is essential for lymphopoiesis as shown in our previous studies. Here we demonstrate cell-intrinsic,absolute dependence on GABPα for maintenance and differentiation of hematopoietic stem/progenitor cells. Through genome-wide mapping of GABPα binding and transcriptomic analysis of GABPα-deficient HSCs,we identified Zfx and Etv6 transcription factors and prosurvival Bcl-2 family members including Bcl-2,Bcl-X(L),and Mcl-1 as direct GABP target genes,underlying its pivotal role in HSC survival. GABP also directly regulates Foxo3 and Pten and hence sustains HSC quiescence. Furthermore,GABP activates transcription of DNA methyltransferases and histone acetylases including p300,contributing to regulation of HSC self-renewal and differentiation. These systematic analyses revealed a GABP-controlled gene regulatory module that programs multiple aspects of HSC biology. Our studies thus constitute a critical first step in decoding how transcription factors are orchestrated to regulate maintenance and multipotency of HSCs. View Publication

Infection with a recombinant murine-feline gammaretrovirus,MoFe2,or with the parent virus,Moloney murine leukemia virus,caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective,in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state. View Publication

The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation,we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex,pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion,respectively,thereby demonstrating the potential of this forward screening approach in primary human stem cell populations. View Publication

Killer cell lectin-like receptor F1 (KLRF1) is an activating C-type lectin-like receptor expressed on human NK cells and subsets of T cells. In this study,we show that activation-induced C-type lectin (AICL) is a unique KLRF1 ligand expressed on tumor cell lines of hematopoietic and non-hematopoietic origins. We screened a panel of human tumor cell lines using the KLRF1 reporter cells and found that several tumor lines expressed KLRF1 ligands. We characterized a putative KLRF1 ligand expressed on the U937 cell line. The molecular mass for the deglycosylated ligand was 28 kDa under non-reducing condition and 17 kDa under reducing condition,suggesting that the KLRF1 ligand is a homodimer. By expression cloning from a U937 cDNA library,we identified AICL as a KLRF1 ligand. We generated mAbs against AICL to identify the KLRF1 ligands on non-hematopoietic tumor lines. The anti-AICL mAbs stained the tumor lines that express the KLRF1 ligands and importantly the interaction of KLRF1 with the KLRF1 ligand on non-hematopoietic tumors was completely blocked by the two anti-AICL mAbs. Moreover,NK cell degranulation triggered by AICL-expressing targets was partially inhibited by the anti-AICL mAb. Finally,we demonstrate that AICL is expressed in human primary liver cancers. These results suggest that AICL is expressed on tumor cells of non-hematopoietic origins and raise the possibility that AICL may contribute to NK cell surveillance of tumor cells. View Publication

Hematopoietic stem cells (HSCs) have the capacity to self-renew and the potential to differentiate into all of the mature blood cell types. The ability to prospectively identify and isolate HSCs has been the subject of extensive investigation since the first transplantation studies implying their existence almost 50 years ago. Despite significant advances in enrichment protocols,the continuous in vitro propagation of human HSCs has not yet been achieved. This chapter describes current procedures used to phenotypically and functionally characterize candidate human HSCs and initial efforts to derive permanent human HSC lines. View Publication

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However,the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study,we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow,in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells,NK progenitors,and NKG2A-negative NK cells),and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore,we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally,we show that acetylated histones are associated with the CpG-rich region in NKG2A positive,but not negative,cell lines,and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription,but cotreatment with both drugs resulted in a significantly greater induction,suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells. View Publication

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties,Sca-1 expression (Sca-1+),lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-),and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/- 0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in textgreater or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients,respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor,interleukin-6 (IL-6),and erythropoietin (with or without IL-3),a large increase in total cell number,including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly,this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo,as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum- and stroma cell-free culture,providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype. View Publication

The majority of BRCA1-associated breast cancers are basal cell-like,which is associated with a poor outcome. Using a spontaneous mouse mammary tumor model,we show that platinum compounds,which generate DNA breaks during the repair process,are more effective than doxorubicin in Brca1/p53-mutated tumors. At 0.5 mg/kg of daily cisplatin treatment,80% primary tumors (n = 8) show complete pathologic response. At greater dosages,100% show complete response (n = 19). However,after 2 to 3 months of complete remission following platinum treatment,tumors relapse and become refractory to successive rounds of treatment. Approximately 3.8% to 8.0% (mean,5.9%) of tumor cells express the normal mammary stem cell markers,CD29(hi)24(med),and these cells are tumorigenic,whereas CD29(med)24(-/lo) and CD29(med)24(hi) cells have diminished tumorigenicity or are nontumorigenic,respectively. In partially platinum-responsive primary transplants,6.6% to 11.0% (mean,8.8%) tumor cells are CD29(hi)24(med); these populations significantly increase to 16.5% to 29.2% (mean,22.8%; P textless 0.05) in platinum-refractory secondary tumor transplants. Further,refractory tumor cells have greater colony-forming ability than the primary transplant-derived cells in the presence of cisplatin. Expression of a normal stem cell marker,Nanog,is decreased in the CD29(hi)24(med) populations in the secondary transplants. Top2A expression is also down-regulated in secondary drug-resistant tumor populations and,in one case,was accompanied by genomic deletion of Top2A. These studies identify distinct cancer cell populations for therapeutic targeting in breast cancer and implicate clonal evolution and expansion of cancer stem-like cells as a potential cause of chemoresistance. View Publication

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However,scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard,suspension cultures are a viable alternative,because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However,the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here,we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production. ?? 2014 The Authors. View Publication

BACKGROUND: ALDH(br) cells express high aldehyde dehydrogenase (ALDH) activity and have progenitor cell activity in several contexts. We characterized human BM ALDH(br) cells to determine whether cell sorting based on ALDH activity isolates potentially useful populations for cell therapy. METHOD: We measured the expression of ALDH and cell-surface Ag by flow cytometry and compared the ability of sorted ALDH(br),and BM populations remaining after ALDH(br) cells were removed (ALDH(dim) populations),to develop into several cell lineages in culture. RESULTS: The ALDH(br) population comprised 1.2+/-0.8% (mean+/-SD,n=30) nucleated cells and was enriched in cells expressing CD34,CD117,CD105,CD127,CD133 and CD166,and in primitive CD34(+) CD38(-) and CD34(+) CD133(+) progenitors. Most of the CD34(+) and CD133(+) cells were ALDH(dim). ALDH(br) populations had 144-fold more hematopoietic colony-forming activity than ALDH(dim) cells and included all megakaryocyte progenitors. ALDH(br) populations readily established endothelial cell monolayers in cultures. Cells generating endothelial colonies in 7 days were 435-fold more frequent in ALDH(br) than ALDH(dim) populations. CFU-F were 9.5-fold more frequent in ALDH(br) than ALDH(dim) cells,and ALDH(br) cells gave rise to multipotential mesenchymal cell cultures that could be driven to develop into adipocytes,osteoblasts and chondrocytes. DISCUSSION: Hematopoietic,endothelial and mesenchymal progenitor cells can be isolated simultaneously from human BM by cell sorting based on ALDH activity. BM ALDH(br) populations may be useful in several cell therapy applications. View Publication

Heat-stable antigen (HSA)/CD24 is a cell surface molecule expressed by many cell types in the mouse. The molecule has an unusual structure because of its small protein core and extensive glycosylation. In order to study the functional role of the HSA-associated glycoconjugates we have isolated different forms of HSA. Using lectin analysis we provide evidence for extensive heterogeneity in carbohydrate composition and sialic acid linkage. Several HSA forms were recognized by mouse P-selectin-IgG but not E-selectin-IgG in ELISA. As expected,P-selectin-IgG also bound to L2/HNK-1-positive neural glycoproteins (L2-glycoproteins) and sulfatides but not to gangliosides and other control glycoproteins. The binding of P-selectin-IgG to L2-glycoproteins and HSA required bivalent cations. The reactivity to HSA was sensitive to sialidase treatment whereas the binding to L2-glycoproteins was not. Studies with alpha 2-6 sialytransferase indicated that alpha 2-6 linked sialic acid was not involved in the P-selectin binding to HSA. Surprisingly,an L2/HNK-1 specific antibody was found to cross-react with some HSA glycoforms and its binding correlated with P-selectin-IgG reactivity. L2/HNK-1-positive or L2/HNK-1-negative HSA glycoforms were also analyzed after coating to polystyrene beads. Only the L2/HNK-1-positive HSA coated beads were reactive with P-selectin-IgG and could bind to activated bend3 endothelioma cells expressing P-selectin whereas the L2/HNK-1-negative HSA beads did not. It is suggested that in its L2/HNK-1 modified form the HSA molecule on leukocytes could represent a ligand for P-selectin on endothelial cells or platelets. View Publication