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

Gene therapy has proven its potential to cure diseases of the hematopoietic system. However,severe adverse events observed in clinical trials have demanded improved gene-transfer conditions. Whereas progress has been made to reduce the genotoxicity of integrating gene vectors,the role of pretransplantation cultivation is less well investigated. We observed that the STIF (stem cell factor [SCF],thrombopoietin [TPO],insulin-like growth factor-2 [IGF-2],and fibroblast growth factor-1 [FGF-1]) cytokine cocktail developed to effectively expand murine hematopoietic stem cells (HSCs) also supports the expansion of leukemia-initiating insertional mutants caused by gammaretroviral gene transfer. We compared 4 protocols to examine the impact of prestimulation and posttransduction culture in STIF in the context of lentiviral gene transfer. Observing 56 transplanted mice for up to 9.5 months,we found consistent engraftment and gene-marking rates after prolonged ex vivo expansion. Although a lentiviral vector with a validated insertional-mutagenic potential was used,longitudinal analysis identifying textgreater 7000 integration sites revealed polyclonal fluctuations,especially in expanded" groups� View Publication

A population of CD133(+)Lin(-)CD45(-) very small embryonic/epiblast-like stem cells (VSELs) has been purified by multiparameter sorting from umbilical cord blood (UCB). To speed up isolation of these cells,we employed anti-CD133-conjugated paramagnetic beads followed by staining with Aldefluor to detect aldehyde dehydrogenase (ALDH) activity; we subsequently sorted CD45(-)/GlyA(-)/CD133(+)/ALDH(high) and CD45(-)/GlyA(-)/CD133(+)/ALDH(low) cells,which are enriched for VSELs,and CD45(+)/GlyA /CD133(+)/ALDH(high) and CD45(+)/GlyA(-)/CD133(+)/ALDH(low) cells,which are enriched for hematopoietic stem/progenitor cells (HSPCs). Although freshly isolated CD45(-) VSELs did not grow hematopoietic colonies,the same cells,when activated/expanded over OP9 stromal support,acquired hematopoietic potential and grew colonies composed of CD45(+) hematopoietic cells in methylcellulose cultures. We also observed that CD45(-)/GlyA(-)/CD133(+)/ALDH(high) VSELs grew colonies earlier than CD45(-)/GlyA(-)/CD133(+)/ALDH(low) VSELs,which suggests that the latter cells need more time to acquire hematopoietic commitment. In support of this possibility,real-time polymerase chain reaction analysis confirmed that,whereas freshly isolated CD45(-)/GlyA(-)/CD133(+)/ALDH(high) VSELs express more hematopoietic transcripts (for example,c-myb),CD45(-)/GlyA(-)/CD133(+)/ALDH(low) VSELs exhibit higher levels of pluripotent stem cell markers (for example,Oct-4). More importantly,hematopoietic cells derived from VSELs that were co-cultured over OP9 support were able to establish human lympho-hematopoietic chimerism in lethally irradiated non-obese diabetic/severe combined immunodeficiency mice 4-6 weeks after transplantation. Overall,our data suggest that UCB-VSELs correspond to the most primitive population of HSPCs in UCB. View Publication

Realizing the potential that human embryonic stem cells (hESCs) hold,both for the advancement of biomedical science and the development of new treatments for many human disorders,will be greatly facilitated by the introduction of standardized methods for assessing and altering the biological properties of these cells. The 7-day in vitro alkaline phosphatase colony-forming cell (AP(+)-CFC) assay currently offers the most sensitive and specific method to quantify the frequency of undifferentiated cells present in a culture. In this regard,it is superior to any phenotypic assessment protocol. The AP(+)-CFC assay,thus,provides a valuable tool for monitoring the quality of hESC cultures,and also for evaluating quantitative changes in pluripotent cell numbers following manipulations that may affect the self-renewal and differentiation properties of the treated cells. Two other methods routinely used to evaluate hESC pluripotency involve either culturing the cells under conditions that promote the formation of nonadherent differentiating cell aggregates (termed embryoid bodies),or transplanting the cells into immunodeficient mice to obtain teratomas containing differentiated cells representative of endoderm,mesoderm,and ectoderm lineages. View Publication

Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate,proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs,there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis,graft vasculopathy and hyperlipidemia-induced atherosclerosis,bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably,purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs,and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization,homing,differentiation and proliferation of bone marrow-derived vascular progenitor cells. View Publication

The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC) must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however,the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely,the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that,once GADD45G is expressed,the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here,we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis. View Publication

C1q modulates the differentiation and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because the two C1q receptors found on the DC surface - gC1qR and cC1qR - lack a direct conduit into intracellular elements,we postulated that the receptors must form complexes with transmembrane partners. Here we show that DC-SIGN,a C-type lectin expressed on DCs,binds directly to C1q,as assessed by ELISA,flow cytometry and immuno-precipitation experiments. Surface plasmon resonance analysis revealed that the interaction was specific,and intact C1q,as well as the globular portion of C1q,bound to DC-SIGN. While IgG significantly reduced the binding; the Arg residues (162-163) of the C1q-A-chain,considered to contribute to C1q-IgG interaction,were not required for C1q binding to DC-SIGN. Binding was significantly reduced in the absence of Ca(2+) and by pre-incubation of DC-SIGN with mannan,suggesting that C1q binds to DC-SIGN at its principal Ca(2+)-binding pocket,which has increased affinity for mannose residues. Antigen-capture ELISA and immunofluorescence microscopy revealed that C1q and gC1qR associate with DC-SIGN on blood DC precursors and immature DCs. Thus the data suggest that C1q/gC1qR may regulate DC differentiation and function through DC-SIGN-mediated induction of cell signaling pathways. View Publication

BACKGROUND: Serum-free cultures supplemented with stem cell factor (SCF) and IL-6 is reported to support the extensive growth of less functional human cord blood-derived mast cells. OBJECTIVE: To obtain more functional mast cells from cord blood,we developed a culture system combining a serum-free condition for 0-8 weeks of culture,and followed by a serum-supplemented culture condition and examined the function of the cells compared to the cells cultured continuously in serum-free condition. METHODS: Human cord blood progenitors were purified with anti-CD133 antibody. They were cultured in a serum-free medium StemSpan supplemented with SCF at 100 ng/ml and IL-6 at 50 ng/ml for 8 weeks. Then,an aliquot of the cultured cells were cultured in the above condition but further supplemented with 10% fetal calf serum (FCS). RESULTS: The addition of FCS after 8 weeks of culture significantly increased the amount of histamine per mast cell (3.8 pg/cell) when compared to the serum-free condition (0.7 pg/cell). The cells cultured with FCS after 8 weeks expressed more FcvarepsilonRI alpha and released textgreater30% of the histamine content upon anti-IgE stimulation than those cultured without serum. CONCLUSION: It is uncertain why FCS enhanced the functional maturation of mast cells when added after week 8 of culture but suppressed mast cell development when added at day 0 of culture. Yet,the present method combining a serum-free culture system with a serum-supplemented culture system seems to be beneficial for most of the laboratories to obtain functional human mast cells. View Publication

Human pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization,but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line,where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter,we screened for textgreater 60 bioactive small molecules that would promote endothelial differentiation,and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated textgreater 50% conversion of hPSCs to endothelial cells (ECs),specifically VEC(+)CD31(+)CD34(+)CD14(-)KDR(high) endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling,in combination with VEGF-A treatment,resulted in efficient formation of EPs via KDR(+) mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods,which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs. View Publication

Improvement of methods to produce endoderm-derived cells from pluripotent stem cells is important to realize high-efficient induction of endodermal tissues such as pancreas and hepatocyte. Difficulties hampering such efforts include the low efficiency of definitive endoderm cell induction and establishing appropriate defined culture conditions to ensure a safe cell source for human transplantation. Based on previous studies,we revised the experimental condition of definitive endoderm induction in feeder- and serum-free culture. Our results suggested that CHIR99021 is more effective than Wnt3A ligand in feeder- and serum-free conditions. In addition,keeping cell density low during endoderm induction is important for the efficiency. On the other hand,we showed that overtreatment with CHIR99021 converted the cells into BRACHYURY-expressing posterior mesoderm cells rather than endoderm,indicating strict CHIR99021 treatment requirements for endoderm differentiation. Nevertheless,these results should enable better control in the production of definitive endoderm-derived cells. View Publication