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

Increasing occurrence of moderate to severe intrauterine adhesion (IUA) is seriously affecting the quality of human life. The aim of the study was to establish IUA models in nonhuman primates and to explore the dual repair effects of human umbilical cord-derived mesenchymal stem cells (huMSCs) loaded on autocrosslinked hyaluronic acid gel (HA-GEL) on endometrial damage and adhesion. Here,we recorded the menstrual cycle data in detail with uterine cavities observed and endometrial tissues detected after intervention,and the thicker endometria,decreased amount of fibrotic formation,increased number of endometrium glands,etc.,suggested that both HA-GEL and huMSC/HA-GEL complexes could partially repair IUA caused by mechanical injury,but huMSC/HA-GEL complex transplantation had notable dual repair effects: a reliable antiadhesion property and the promotion of endometrial regeneration. View Publication

Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation,microcephaly,and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4),Artemis,and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders,we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity,but not Artemis deficiency,were associated with markedly reduced reprogramming efficiency,which could be partially rescued by genetic complementation. Moreover,we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest,particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-,but not Artemis- or DNA-PK-mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming,with a major role for LIG4 and DNA-PKcs and a minor,if any,for Artemis. View Publication

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture,the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study,we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells,including cardiomyocytes and fibroblasts,using a methionine-free culture condition. When cultured in the methionine-free condition,human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition,gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore,in fabricated cardiac cell sheets,spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination. View Publication

The recent development of induced pluripotent stem cells (iPSCs) by ectopic expression of defined reprogramming factors offers enormous therapeutic opportunity. To deliver these factors,murine leukemia virus (MLV)-based vectors have been broadly used in the setting of hematopoietic stem cell transplantation. However,MLV vectors have been implicated in malignancy induced by insertional mutagenesis,whereas lentiviral vectors have not. Furthermore,the infectivity of MLV vectors is limited to dividing cells,whereas lentiviral vectors can also transduce nondividing cells. One important characteristic of MLV vectors is a self-silencing property of the promoter element in pluripotent stem cells,allowing temporal transgene expression in a nonpluripotent state before iPSC derivation. Here we test iPSC generation using a novel chimeric vector carrying a mutant MLV promoter internal to a lentiviral vector backbone,thereby containing the useful properties of both types of vectors. Transgene expression of this chimeric vector was highly efficient compared with that of MLV vectors and was silenced specifically in human embryonic stem cells. Human fetal fibroblasts transduced with the vector encoding each factor were efficiently reprogrammed into a pluripotent state,and these iPSCs had potential to differentiate into a variety of cell types. To explore the possibility of iPSCs for gene therapy,we established iPSC clones expressing a short hairpin RNA (shRNA) targeting chemokine receptor 5 (CCR5),the main coreceptor for HIV-1. Using a reporter construct for CCR5 expression,we confirmed that CCR5 shRNA was expressed and specifically knocked down the reporter expression in iPSCs. These data indicate that our chimeric lentiviral vector is a valuable tool for generation of iPSCs and the combination with vectors encoding transgenes allows for rapid establishment of desired genetically engineered iPSC lines. View Publication

Background: Phenotypically unstable Schwann cell-like cells (SCLCs),derived from mesenchymal stem cells (MSCs) require intercellular contact-mediated cues for Schwann cell (SCs)-fate commitment. Although rat dorsal root ganglion (DRG) neurons provide contact-mediated signals for the conversion of SCLCs into fate-committed SCs,the use of animal cells is clinically unacceptable. To overcome this problem,we previously acquired human induced pluripotent stem cell-derived sensory neurons (hiPSC-dSNs) as surrogates of rat DRG neurons that committed rat bone marrow SCLCs to the SC fate. In this study,we explored whether hiPSC-dSNs could mimic rat DRG neuron effects to obtain fate-committed SCs from hBMSC-derived SCLCs. Methods: hiPSCs were induced into hiPSC-dSNs using a specific chemical small molecule combination. hBMSCs were induced into hBMSC-derived SCLCs in a specific culture medium and then co-cultured with hiPSC-dSNs to generate SCs. The identity of hBMSC-derived SCs (hBMSC-dSCs) was examined by immunofluorescence,western bolt,electronic microscopy,and RNA-seq. Immunofluorescence was also used to detect the myelination capacity. Enzyme-linked immunosorbent assay and neurite outgrowth analysis were used to test the secretion of neurotrophic factors. Results: The hBMSC-dSCs exhibited bi-/tri-polar morphology of SCs and maintained the expression of the SC markers S100,p75NTR,p0,GFAP,and Sox10,even after withdrawing the glia-inducing factors or hiPSC-dSNs. Electronic microscopy and RNA-seq analysis provided evidence that hBMSC-dSCs were similar to the original human SCs in terms of their function and a variety of characteristics. Furthermore,these cells formed MBP-positive segments and secreted neurotrophic factors to facilitate the neurite outgrowth of Neuro2A. Conclusions: These results demonstrated that phenotypically stable and functionally mature hBMSC-dSCs were generated efficiently via the co-culture of hiPSC-dSNs and hBMSC-derived SCLCs. Our findings may provide a promising protocol through which stable and fully developed hBMSC-dSCs can be used for transplantation to regenerate myelin sheath. View Publication

BACKGROUND: Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However,relatively little is known about the mechanisms responsible for their immunomodulatory effects,which could be influenced by both the cell source and culture conditions. DESIGN AND METHODS: We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells,in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. RESULTS: The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology,immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency,umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition,these cells do not express hTERT and telomerase activity,do express p16(ink4a) protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses,umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation,(ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10,while decreasing interferon-gamma secretion,in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects,a prostaglandin E(2)-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. CONCLUSIONS: Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency,proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications. View Publication

The active form of vitamin D,1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)),has potent immunomodulatory properties that have promoted its potential use in the prevention and treatment of infectious disease and autoimmune conditions. A variety of immune cells,including macrophages,dendritic cells,and activated T cells express the intracellular vitamin D receptor and are responsive to 1,25(OH)(2)D(3.) Despite this,how 1,25(OH)(2)D(3) regulates adaptive immunity remains unclear and may involve both direct and indirect effects on the proliferation and function of T cells. To further clarify this issue,we have assessed the effects of 1,25(OH)(2)D(3) on human CD4(+)CD25(-) T cells. We observed that stimulation of CD4(+)CD25(-) T cells in the presence of 1,25(OH)(2)D(3) inhibited production of proinflammatory cytokines including IFN- gamma,IL-17,and IL-21 but did not substantially affect T cell division. In contrast to its inhibitory effects on inflammatory cytokines,1,25(OH)(2)D(3) stimulated expression of high levels of CTLA-4 as well as FoxP3,the latter requiring the presence of IL-2. T cells treated with 1,25(OH)(2)D(3) could suppress proliferation of normally responsive T cells,indicating that they possessed characteristics of adaptive regulatory T cells. Our results suggest that 1,25(OH)(2)D(3) and IL-2 have direct synergistic effects on activated T cells,acting as potent anti-inflammatory agents and physiologic inducers of adaptive regulatory T cells. View Publication

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential,but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic-scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34(-)CD19(+) (B-lymphoid) and CD34(+) (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains approximately 5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38(-) and CD38(+) subsets of CD34(+) CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34(+)CD38(-) human CB cells in serum-free medium containing flt-3 ligand,Steel factor,interleukin 3,interleukin 6,and granulocyte colony-stimulating factor for 5-8 days resulted in a 100-fold expansion of colony-forming cells,a 4-fold expansion of LTC-IC,and a 2-fold (but significant,P textless 0.02) increase in CRU. The culture-derived CRU,like the original CB CRU,generated pluripotent,erythroid,granulopoietic,megakaryopoietic,and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition,their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal. View Publication

Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a safe harbor" locus for inserting transgenes because of its open chromatin structure View Publication

In recent years,mesenchymal stem cells (MSCs) have been shown to inhibit T-lymphocyte proliferation induced by alloantigens or mitogens. However,no substantial information is available regarding their effect on natural killer (NK) cells. Here we show that MSCs sharply inhibit IL-2-induced proliferation of resting NK cells,whereas they only partially affect the proliferation of activated NK cells. In addition,we show that IL-2-activated NK cells (but not freshly isolated NK cells) efficiently lyse autologous and allogeneic MSCs. The activating NK receptors NKp30,NKG2D,and DNAM-1 represented the major receptors responsible for the induction of NK-mediated cytotoxicity against MSCs. Accordingly,MSCs expressed the known ligands for these activating NK receptors-ULBPs,PVR,and Nectin-2. Moreover,NK-mediated lysis was inhibited when IFN-gamma-exposed MSCs were used as target cells as a consequence of the up-regulation of HLA class I molecules at the MSC surface. The interaction between NK cells and MSCs resulted not only in the lysis of MSCs but also in cytokine production by NK cells. These results should be taken into account when evaluating the possible use of MSCs in novel therapeutic strategies designed to improve engraftment or to suppress graft-versus-host disease (GVHD) in bone marrow transplantation. View Publication

PURPOSE: The purpose of this study was to determine whether histone deacetylase (HDAC) inhibitors (HDACI) such as vorinostat or entinostat (SNDX-275) could increase the lethality of the dual Bcr/Abl-Aurora kinase inhibitor KW-2449 in various Bcr/Abl(+) human leukemia cells,including those resistant to imatinib mesylate (IM). EXPERIMENTAL DESIGN: Bcr/Abl(+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) cells,including those resistant to IM (T315I,E255K),were exposed to KW-2449 in the presence or absence of vorinostat or SNDX-275,after which apoptosis and effects on signaling pathways were examined. In vivo studies combining HDACIs and KW2449 were carried out by using a systemic IM-resistant ALL xenograft model. RESULTS: Coadministration of HDACIs synergistically increased KW-2449 lethality in vitro in multiple CML and Ph(+) ALL cell types including human IM resistant cells (e.g.,BV-173/E255K and Adult/T315I). Combined treatment resulted in inactivation of Bcr/Abl and downstream targets (e.g.,STAT5 and CRKL),as well as increased reactive oxygen species (ROS) generation and DNA damage (γH2A.X). The latter events and cell death were significantly attenuated by free radical scavengers (TBAP). Increased lethality was also observed in primary CD34(+) cells from patients with CML,but not in normal CD34(+) cells. Finally,minimally active vorinostat or SNDX275 doses markedly increased KW2449 antitumor effects and significantly prolonged the survival of murine xenografts bearing IM-resistant ALL cells (BV173/E255K). CONCLUSIONS: HDACIs increase KW-2449 lethality in Bcr/Abl(+) cells in association with inhibition of Bcr/Abl,generation of ROS,and induction of DNA damage. This strategy preferentially targets primary Bcr/Abl(+) hematopoietic cells and exhibits enhanced in vivo activity. Combining KW-2449 with HDACIs warrants attention in IM-resistant Bcr/Abl(+) leukemias. View Publication

As known from model organisms,such as frog,fish,mouse and chicken,the anterior-posterior patterning of the definitive endoderm (DE) into distinct domains is controlled by a variety of signaling interactions between the DE and its surrounding mesoderm. This includes Wnt/FGFs and BMPs in the posterior half and all-trans-retinoic acid,TGF-$$-ligands,Wnt- and BMP-inhibitors in the anterior half of the DE sheet. However,it is currently unclear how these embryonic tissue interactions can be translated into a defined differentiation protocol for human embryonic stem cells. Activin A has been proposed to direct DE into a SOX2-positive foregut-like cell type. Due to the pleiotropic nature of SOX2 in pluripotency and developing cells of the foregut we purified DE-cells by magnetic cell sorting and tested the effects of anteriorizing and posteriorizing factors on pure endoderm. We show in contrast to previous studies that the generation of the foregut marked by SOX2/FOXA2 double-positive cells does not depend on activin A/TGF-$$-signaling but is mediated by the inhibition of Wnt- and BMP-signaling. Retinoic acid can posteriorize and at the same time dorsalize the foregut towards a PDX1-positive pancreatic duodenal cell type whereas active Wnt/beta-catenin signaling synergistically with FGF-2,BMP-4 and RA induces the formation of CDX2-positive posterior endoderm. Thus,these results provide new insights into the mechanisms behind cell specification of human DE derived from pluripotent stem cells. This article is protected by copyright. All rights reserved. View Publication