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

Primitive hematopoietic cells from several species are known to efflux both Hoechst 33342 and Rhodamine-123. We now show that murine hematopoietic stem cells (HSCs) defined by long-term multilineage repopulation assays efflux both dyes variably according to their developmental or activation status. In day 14.5 murine fetal liver,very few HSCs efflux Hoechst 33342 efficiently,and they are thus not detected as side population" (SP) cells. HSCs in mouse fetal liver also fail to efflux Rhodamine-123. Both of these features are retained by most of the HSCs present until 4 weeks after birth but are reversed by 8 weeks of age or after a new HSC population is regenerated in adult mice that receive transplants with murine fetal liver cells. Activation of adult HSCs in vivo following 5-fluorouracil treatment� View Publication

OBJECTIVE: Endothelial-like vascular progenitor cells (VPCs) can be collected in peripheral blood stem cell (PBSC) products that are used in hematopoietic stem cell transplantation (HSCT). The association between VPCs in PBSC products and transplant-related toxicity caused by high-dose chemo/radiotherapy was assessed to identify potential mediators of vascular repair. MATERIALS AND METHODS: PBSC grafts in 29 patients (mean age: 48 years; range,20-67 years) undergoing autologous HSCT were analyzed using a cell culture assay for VPC cluster formation in fibronectin-coated dishes in serum-rich angiogenic conditions. Transplant toxicity was estimated using total length of hospital stay (LOS) following HSCT and the Seattle criteria for transplant-related organ toxicity for 8 organ systems (grade 0-4). RESULTS: LOS following graft reinfusion was lower (14.7 vs 20.0 days,p = 0.002) and the mean number of organs with any toxicity (1.0 vs 2.4,p = 0.016) or with toxicity grade textgreater or = 2 was reduced (0.2 vs 1.6 organs,p = 0.007) in patients with high graft VPC content (n = 10,textgreater2.0 x 10(3) VPCs/kg) compared with reduced VPC content (n = 19,textless or = 2.0 x 10(3) VPCs/kg). An association between graft CD34(+) levels and LOS or organ toxicity was not observed. In addition,graft VPC levels were independent of graft CD34 counts,peripheral blood monocytes and hemoglobin levels,age,and disease (p = NS). CONCLUSION: PBSC products enriched for VPCs are associated with reduced toxicity following HSCT. Identifying specific factors that contribute to high graft VPC levels is needed. View Publication

Genome editing on human pluripotent stem cells (hPSCs) together with the development of protocols for organ decellularization opens the door to the generation of autologous bioartificial hearts. Here we sought to generate for the first time a fluorescent reporter human embryonic stem cell (hESC) line by means of Transcription activator-like effector nucleases (TALENs) to efficiently produce cardiomyocyte-like cells (CLCs) from hPSCs and repopulate decellularized human heart ventricles for heart engineering. In our hands,targeting myosin heavy chain locus (MYH6) with mCherry fluorescent reporter by TALEN technology in hESCs did not alter major pluripotent-related features,and allowed for the definition of a robust protocol for CLCs production also from human induced pluripotent stem cells (hiPSCs) in 14 days. hPSCs-derived CLCs (hPSCs-CLCs) were next used to recellularize acellular cardiac scaffolds. Electrophysiological responses encountered when hPSCs-CLCs were cultured on ventricular decellularized extracellular matrix (vdECM) correlated with significant increases in the levels of expression of different ion channels determinant for calcium homeostasis and heart contractile function. Overall,the approach described here allows for the rapid generation of human cardiac grafts from hPSCs,in a total of 24 days,providing a suitable platform for cardiac engineering and disease modeling in the human setting. View Publication

The number of colony forming unit-endothelial cells (CFU-EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study,the heterogeneous composition of immune cells in the CFU-ECs was investigated. We confirmed that monocytes are essential for the formation of CFU-ECs. Also,however,CD4(+) T cells were found to be indispensable for the induction of CFU-EC colonies,mainly through cell-cell contact. By blocking or activating CD3 receptors on CD4(+) T cells or blocking MHC class II molecules on monocytes,it was shown that TCR-MHCII interactions are required for induction of CFU-EC colonies. Because the supernatant from preactivated T cells could also induce colony formation from purified monocytes,the T cell support turned out to be cytokine mediated. Gene expression analysis of the endothelial-like colonies formed by CD14(+) cells showed that colony formation is a proangiogenic differentiation and might reflect the ability of monocytes to facilitate vascularization. This in vitro study is the first to reveal the role of TCR-MHC class II interactions between T cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization. View Publication

Poor recovery of cryopreserved human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells is a significant impediment to progress with pluripotent stem cells. In this study,we demonstrate that Y-27632,a specific inhibitor of Rho kinase (ROCK) activity,significantly enhances recovery of hES cells from cryopreserved stocks when cultured with or without a growth inactivated feeder layer. Furthermore,treatment with the ROCK inhibitor for several days increased the number of colonies and colony size of hES cells compared to shorter exposures. Remarkably,hES cells that had formed relatively few colonies 5 days after thawing exhibited rapid growth upon addition of Y-27632. Additionally,we determined that Y-27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture. Thus,Y-27632 provides a means to kick-start" slow-growing human pluripotent stem cells� View Publication

It has been proposed that bone marrow (BM) hematopoietic stem and progenitor cells are distributed along an oxygen (O2) gradient,where stem cells reside in the most hypoxic areas and proliferating progenitors are found in O2-rich areas. However,the effects of hypoxia on human hematopoietic stem cells (HSCs) have not been characterized. Our objective was to evaluate the functional and molecular responses of human BM progenitors and stem cells to hypoxic conditions. BM lineage-negative (Lin-) CD34+CD38- cells were cultured in serum-free medium under 1.5% O2 (hypoxia) or 20% O2 (normoxia) for 4 days. Using limiting dilution analysis,we demonstrate that the absolute number of SCID-repopulating cells (SRCs) increased by 5.8-fold in hypoxic cultures compared with normoxia,and by 4.2-fold compared with freshly isolated Lin-CD34+CD38- cells. The observed increase in BM-repopulating activity was associated with a preferential expansion of Lin-CD34+CD38- cells. We also demonstrate that,in response to hypoxia,hypoxia-inducible factor-1alpha protein was stabilized,surface expression of angiogenic receptors was upregulated,and VEGF secretion increased in BM Lin-CD34+ cultures. The use of low O2 levels to enhance the survival and/or self-renewal of human BM HSCs in vitro represents an important advance and could have valuable clinical implications. View Publication

Human embryonic stem cells (hESCs) provide great opportunities for regenerative medicine,pharmacological and toxicological investigation,and the study of human embryonic development. These applications require proper derivation,maintenance,and extensive characterization of undifferentiated cells before being used for differentiation into cells of interest. Undifferentiated hESCs possess several unique features,including their extensive proliferation capacity in the undifferentiated state,ability to maintain a normal karyotype after long-term culture,expression of markers characteristic of stem cells,high constitutive telomerase activity,and capacity to differentiate into essentially all somatic cell types. This chapter will summarize the current development in culture conditions and provide technical details for the evaluation and characterization of hESCs. View Publication

Recent observations indicate that,in several types of human cancer,only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the cancer stem cell" (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues� View Publication

Identification of new techniques to express proteins into mammal cells is of particular interest for both research and medical purposes. The present study describes the use of engineered vesicles to deliver exogenous proteins into human cells. We show that overexpression of the spike glycoprotein of the vesicular stomatitis virus (VSV-G) in human cells induces the release of fusogenic vesicles named gesicles. Biochemical and functional studies revealed that gesicles incorporated proteins from producer cells and could deliver them to recipient cells. This protein-transduction method allows the direct transport of cytoplasmic,nuclear or surface proteins in target cells. This was demonstrated by showing that the TetR transactivator and the receptor for the murine leukemia virus (MLV) envelope [murine cationic amino acid transporter-1 (mCAT-1)] were efficiently delivered by gesicles in various cell types. We further shows that gesicle-mediated transfer of mCAT-1 confers to human fibroblasts a robust permissiveness to ecotropic vectors,allowing the generation of human-induced pluripotent stem cells in level 2 biosafety facilities. This highlights the great potential of mCAT-1 gesicles to increase the safety of experiments using retro/lentivectors. Besides this,gesicles is a versatile tool highly valuable for the nongenetic delivery of functions such as transcription factors or genome engineering agents. View Publication

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells,identified by binding of the fluoresceinated peptide ligand,APELIN (APLN),or an anti-APLNR mAb,were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs),increased the expression of hematoendothelial genes in differentiating hESCs,and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore,APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells. View Publication

Magnetic resonance imaging (MRI) may emerge as an ideal non-invasive imaging modality to monitor stem cell therapy in the failing heart. This imaging modality generates any arbitrary tomographic view at high spatial and temporal resolution with exquisite intrinsic tissue contrast. This capability enables robust evaluation of both the cardiac anatomy and function. Traditionally,superparamagnetic iron oxide nanoparticle (SPIO) has been widely used for cellular MRI due to SPIO's ability to enhance sensitivity of MRI by inducing remarkable hypointense,negative signal,blooming effect" on T2*-weighted MRI acquisition. Recently� View Publication

Vogt-Koyanagi-Harada (VKH) disease (and sympathetic ophthalmia) is an ocular inflammatory disease that is considered to be a cell-mediated autoimmune disease against melanocytes. The purpose of this study was to determine the Ags specific to VKH disease and to develop an animal model of VKH disease. We found that exposure of lymphocytes from patients with VKH disease to peptides (30-mer) derived from the tyrosinase family proteins led to significant proliferation of the lymphocytes. Immunization of these peptides into pigmented rats induced ocular and extraocular changes that highly resembled human VKH disease,and we suggest that an experimental VKH disease was induced in these rats. We conclude that VKH disease is an autoimmune disease against the tyrosinase family proteins. View Publication