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

BACKGROUND Oesophageal squamous cell carcinoma (ESCC) and colorectal cancer (CRC) are common types of human cancer. Spheroid colony formation is used to characterize cancer stem cell (CSCs). In the present study,we analyzed the significance of kinesin family 11 (KIF11 in human ESCC and CRC. MATERIALS AND METHODS Expression of KIF11 in 105 ESCC and 100 CRC cases was determined using immunohistochemistry. RNA interference was used to inhibit KIF11 expression in ESCC and CRC cell lines. RESULTS In total,61 out of 105 (58%) ESCC and 62 out of 100 (62%) CRC cases were positive for KIF11. Expression of KIF11 was not associated with any clinicopathological characteristics. Both the number and size of spheres produced by from TE-5 ESCC cells and DLD-1 CRC cells were significantly reduced upon KIF11 siRNA transfection compared to negative control siRNA transfection. CONCLUSION These results indicate that KIF11 plays an important role in CSCs of ESCC and CRC. View Publication

Mammalian synthetic biology may provide novel therapeutic strategies,help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet,our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design,construction and screening of synthetic gene networks. To address this problem,here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units,27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept,we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements,genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines. View Publication

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors,whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed,washed,immunomagnetically depleted of cells expressing glycophorin A and CD14,reacted for flow cytometric detection of ALDH,and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H,10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed,banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid,myeloid and megakaryocytic blood elements. 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

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

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

BACKGROUND: The radiation-induced bystander effect" (RIBE) was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR). RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC) are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However� 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

A method for the generation of human induced pluripotent stem (iPS) cells was established. This method employs adenovirus carrying the ecotropic retrovirus receptor mCAT1 and Moloney murine leukemia virus (MMLV)-based retroviral vectors carrying the four transcription factors POU5F1 (OCT3/4),KLF4,SOX2,and MYC (c-Myc) (Masaki H & Ishikawa T Stem Cell Res 1:105-15,2007). The differentiation of human iPS cells into hepatic cells was performed by a stepwise protocol (Song Z et al. Cell Res 19:1233-42,2009). These cells have potential as patient-specific in vitro models for studying disease etiology and could be used in drug discovery programs tailored to deal with genetic variations in drug efficacy and toxicity. View Publication