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

BACKGROUND: Vascular trauma induced by surgical revascularization stimulates mobilization of hematopoietic and nonhematopoietic progenitor cells. However,it is not clear whether mobilized progenitors are functionally active and participate in peripheral homing. We have found no clinical studies available regarding the reaction of bone marrow to surgical revascularization. METHODS: This was an observational prospective study of 76 patients undergoing elective coronary artery bypass graft surgery. Bone marrow aspirates and blood samples were collected at baseline,at the end of surgery,and 24 hours postoperatively (blood samples only). The CD34+,CD34+CD133+,and CD34+CXCR4+ progenitor cell counts,CXCR4+ mononuclear cell counts,and CXCR4 expression on CD34+ cells were measured by flow cytometry. Progenitor cell functions were studied in vitro by clonogenic and migration assays. RESULTS: In response to coronary revascularization there was mobilization of CD34+ progenitors,having increased migratory and clonogenic function. The CD34+CXCR4+ subsets and CXCR4 expression on CD34+ cells in peripheral blood increased significantly 24 hours postoperatively. The CXCR4 expression on mobilized progenitors at the end of surgery was independently related to baseline CXCR4 expression on bone marrow resident CD34+ cells and duration of cardiopulmonary bypass in a multivariate model. At the end of surgery there was a significant fall in the expression of CXCR4 on CD34+ bone marrow cells,suggesting egress into peripheral circulation of the most active CXCR4-expressing progenitors. CONCLUSIONS: Coronary artery bypass graft surgery is associated with bone marrow release of functionally active progenitor cells. Further studies are needed to verify whether mobilized progenitors participate in regeneration of injured tissues. View Publication

In this study,we analyzed IL-2-activated polyclonal natural killer (NK) cells derived from 2 patients affected by leukocyte adhesion deficiency type I (LAD1),an immunodeficiency characterized by mutations of the gene coding for CD18,the beta subunit shared by major leukocyte integrins. We show that LAD1 NK cells express normal levels of various triggering NK receptors (and coreceptors) and that mAb-mediated engagement of these receptors results in the enhancement of both NK cytolytic activity and cytokine production. Moreover,these activating NK receptors were capable of recognizing their specific ligands on target cells. Thus,LAD1 NK cells,similarly to normal NK cells,were capable of killing most human tumor cells analyzed and produced high amounts of IFN-gamma when cocultured in presence of target cells. Murine target cells represented a common exception,as they were poorly susceptible to LAD1 NK cells. Finally,LAD1 NK cells could efficiently kill or induce maturation of monocyte-derived immature dendritic cells (DCs). Altogether our present study indicates that in LAD1 patients,3 important functions of NK cells (eg,cytotoxicity,IFN-gamma production,and DC editing) are only marginally affected and provides new insight on the cooperation between activating receptors and LFA-1 in the induction of NK cell activation and function. View Publication

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded new insight into brain cancer. However,the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs,which we refer to as the 'ground state',remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology,so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation,we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer. View Publication

The aldehyde dehydrogenase (ALDH) superfamily is composed of nicotinamide adenine dinucleotide (phosphate) (NAD(P)(+))-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. To date,24 ALDH gene families have been identified in the eukaryotic genome. In addition to aldehyde metabolizing capacity,ALDHs have additional catalytic (e.g. esterase and reductase) and non-catalytic activities. The latter include functioning as structural elements in the eye (crystallins) and as binding molecules to endobiotics and xenobiotics. Mutations in human ALDH genes and subsequent inborn errors in aldehyde metabolism are the molecular basis of several diseases. Most recently ALDH polymorphisms have been associated with gout and osteoporosis. Aldehyde dehydrogenase enzymes also play important roles in embryogenesis and development,neurotransmission,oxidative stress and cancer. This article serves as a comprehensive review of the current state of knowledge regarding the ALDH superfamily and the contribution of ALDHs to various physiological and pathophysiological processes. View Publication

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells,but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (textgreater60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. View Publication

Induced pluripotent stem cell (iPS cell) holds great potential for applications in regenerative medicine,drug discovery,and disease modeling. We describe here a practical method to generate human iPS cells from urine-derived cells (UCs) under feeder-free,virus-free,serum-free condition and without oncogene c-MYC. We showed that this approach could be applied in a large population with different genetic backgrounds. UCs are easily accessible and exhibit high reprogramming efficiency,offering advantages over other cell types used for the purpose of iPS generation. Using the approach described in this study,we have generated 93 iPS cell lines from 20 donors with diverse genetic backgrounds. The non-viral iPS cell bank with these cell lines provides a valuable resource for iPS cells research,facilitating future applications of human iPS cells. View Publication

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition,recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL,classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore,the murine WEHI-3B(D+) cells and human leukemic cells classified as M2,M3,and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600. View Publication

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression,and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1,and mostly negative (textless3% positive) for CD14,31,49,144,c-kit,and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes,endothelial,and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting,CD34+/45- cells expressed only c-met mRNA,and did not express any other myogenic cell-related markers such as MyoD,myf-5,myf-6,myogenin,M-cadherin,Pax-3,and Pax-7. However,after 3 d of culture,these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells,as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al.,2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles,and that they can potentially contribute to postnatal skeletal muscle growth. View Publication

HIV-1 replication in simian cells is restricted at an early postentry step because of the presence of an inhibitory cellular factor. This block reduces the usefulness of HIV-1-based lentiviral vectors in primate animal models. Here,we demonstrate that substitution of the cyclophilin A (CyPA) binding region in the capsid of an HIV-1-based lentiviral vector (LV) with that of the macrophage tropic HIV-1 Ba-L resulted in a vector that was resistant to the inhibitory effect and efficiently transduced simian cells. Notably,the chimeric gag LV efficiently transduced primary simian hematopoietic progenitor cells,a critical cellular target in gene therapy. The alterations in the CyPA binding region did not affect CyPA incorporation; however,transduction by the gag chimeric LV seemed to be relatively insensitive to cyclosporin A,indicating that it does not require CyPA for early postentry steps. In dual infection experiments,the gag chimeric LV failed to remove the block to transduction of the WT LV,suggesting that the gag chimeric LV did not saturate the inhibitory simian cellular factor. These data suggest that the CyPA binding region of capsid contains a viral determinant involved in the postentry restriction of HIV-1-based lentiviral vectors. Overall,the findings demonstrate that the host range of HIV-1-based LV can be altered by modifications in the packaging construct. View Publication

A 4-kilobase and a 2-kilobase cDNA clone encoding a murine macrophage colony-stimulating factor have been isolated. Except for 2 amino acid residue differences,these two clones encode the same 520 amino acid residue protein,which is preceded by a 32-amino acid residue signal peptide. The two clones,whose molecular masses correspond to the two transcripts observed in murine L929 fibroblasts,contain 3' untranslated regions that are markedly different in sequence and length. Both clones can be expressed in COS cells and the recombinant protein is active in a mouse bone marrow colony assay. View Publication

BACKGROUND: Airway remodeling might explain lung function decline among asthmatic children. Extracellular matrix (ECM) deposition by human lung fibroblasts (HLFs) is implicated in airway remodeling. Airway epithelial cell (AEC) signaling might regulate HLF ECM expression. OBJECTIVES: We sought to determine whether AECs from asthmatic children differentially regulate HLF expression of ECM constituents. METHODS: Primary AECs were obtained from well-characterized atopic asthmatic (n = 10) and healthy (n = 10) children intubated during anesthesia for an elective surgical procedure. AECs were differentiated at an air-liquid interface for 3 weeks and then cocultured with HLFs from a healthy child for 96 hours. Collagen I (COL1A1),collagen III (COL3A1),hyaluronan synthase (HAS) 2,and fibronectin expression by HLFs and prostaglandin E2 synthase (PGE2S) expression by AECs were assessed by using RT-PCR. TGF-$$1 and TGF-$$2 concentrations in media were measured by using ELISA. RESULTS: COL1A1 and COL3A1 expression by HLFs cocultured with AECs from asthmatic patients was greater than that by HLFs cocultured with AECs from healthy subjects (2.2-fold,P textless .02; 10.8-fold,P textless .02). HAS2 expression by HLFs cocultured with AECs from asthmatic patients was 2.5-fold higher than that by HLFs cocultured with AECs from healthy subjects (P textless .002). Fibronectin expression by HLFs cocultured with AECs from asthmatic patients was significantly greater than that by HLFs alone. TGF-$$2 activity was increased in cocultures of HLFs with AECs from asthmatic patients (P textless .05),whereas PGES2 was downregulated in AEC-HLF cocultures (2.2-fold,P textless .006). CONCLUSIONS: HLFs cocultured with AECs from asthmatic patients showed differential expression of the ECM constituents COL1A1 and COL3A1 and HAS2 compared with HLFs cocultured with AECs from healthy subjects. These findings support a role for altered ECM production in asthmatic airway remodeling,possibly regulated by unbalanced AEC signaling. View Publication

It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study,we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors,by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner,which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors,in a 2-dimensional or 3-dimensional environment. However,Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices,we observed mature Purkinje-like cells with right morphology and marker expression patterns,which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. View Publication