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

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Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However,an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study,we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1,side population and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore,knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised non-obese diabetic/severe combined immunodeficiency mice,supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion,but not for CSC maintenance. Taken together,our study provides evidence that KLF4 has a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell-like features and by promoting cell migration and invasion. Thus,targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer. View Publication

Targeted nucleases,including zinc-finger nucleases (ZFNs),transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However,realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids,the direct delivery of nuclease proteins to cells provides rapid action and fast turnover,leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression,purification and delivery of ZFN proteins,which are intrinsically cell-permeable; TALEN proteins,which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein,whose nucleofection into cells facilitates rapid induction of multiplexed modifications,are described,along with procedures for evaluating nuclease protein activity. Once they are constructed,nuclease proteins can be expressed and purified within 6 d,and they can be used to induce genomic modifications in human cells within 2 d. View Publication

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach,often involving the use of viral vectors. Electroporation using square-wave generating devices,like Lonza's Nucleofector,is a widely used option,but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work,we show that our in-house developed buffers,termed Chicabuffers,can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device,we electroporated 14 different cell lines and also primary cells,like mesenchymal stem cells and cord blood CD34+,providing optimized protocols for each of them. Moreover,when combined with sleeping beauty-based transposon system,long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells,facilitating the widespread adoption of this technology. View Publication

Nanofibrous gelatin substrates are suited for long-term expansion of human pluripotent stem cells (hPSCs) under feeder- and serum-free culture conditions. A combinatorial library with different sets of processing parameters was established to assess the culture performance of hPSCs on nanofibrous substrates in terms of cell adhesion and growth rate,using Matrigel as control. Then,the optimal conditions were applied to long-term expansion of hPSCs with several cell lines,showing a maintained pluripotency over more than 20 passages without introducing any abnormal chromosome. In addition,this approach allowed us to avoid enzymatic disassociation and mechanic cutting during passages,thereby promoting a better hPSC culture and long-term expansion. ?? 2014 Elsevier Ltd. View Publication

Using endothelial cells for therapeutic angiogenesis/vasculogenesis of ischemia diseases has led to exploring human embryonic stem cells (hESCs) as a potentially unlimited source for endothelial progenitor cells. With their capacity for self-renewal and pluripotency,hESCs and their derived endothelial cells (hESC-ECs) may be more advantageous than other endothelial cells obtained from diseased populations. However,hESC-ECs' poor differentiation efficiency and poorly characterized in vivo function after transplantation present significant challenges for their future clinical application. This review will focus on the differentiation pathways of hESCs and their therapeutic potential for vascular diseases,as well as the monitoring of transplanted cells' fate via molecular imaging. Finally,cell enhancement strategies to improve the engraftment efficiency of hESC-ECs will be discussed. View Publication

Mesenchymal stem cells (MSCs) have the capability for renewal and differentiation into various lineages of mesenchymal tissues. These features of MSCs attract a lot of attention from investigators in the context of cell-based therapies of several human diseases. Despite the fact that bone marrow represents the main available source of MSCs,the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferative/differentiation capacity with age. Thus,the search for possible alternative MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells and does not contain mesenchymal progenitors. However,MSCs circulate in the blood of preterm fetuses and may be successfully isolated and expanded. Where these cells home at the end of gestation is not clear. In this investigation,we have made an attempt to isolate MSCs from the subendothelial layer of umbilical cord vein using two standard methodological approaches: the routine isolation of human umbilical vein endothelial cell protocol and culture of isolated cells under conditions appropriate for bone-marrow-derived MSCs. Our results suggest that cord vasculature contains a high number of MSC-like elements forming colonies of fibroblastoid cells that may be successfully expanded in culture. These MSC-like cells contain no endothelium- or leukocyte-specific antigens but express alpha-smooth muscle actin and several mesenchymal cell markers. Therefore,umbilical cord/placenta stroma could be regarded as an alternative source of MSCs for experimental and clinical needs. View Publication

The retinoid X receptor (RXR) contributes to the regulation of diverse biological pathways via its role as a heterodimeric partner of several nuclear receptors. However,RXR has no established role in the regulation of hematopoietic stem cell (HSC) fate. In this study,we sought to determine whether direct modulation of RXR signaling could impact human HSC self-renewal or differentiation. Treatment of human CD34(+)CD38(-)lin(-) cells with LG1506,a selective RXR modulator,inhibited the differentiation of HSCs in culture and maintained long-term repopulating HSCs in culture that were otherwise lost in response to cytokine treatment. Further studies revealed that LG1506 had a distinct mechanism of action in that it facilitated the recruitment of corepressors to the retinoic acid receptor (RAR)/RXR complex at target gene promoters,suggesting that this molecule was functioning as an inverse agonist in the context of this heterodimer. Interestingly,using combinatorial peptide phage display,we identified unique surfaces presented on RXR when occupied by LG1506 and demonstrated that other modulators that exhibited these properties functioned similarly at both a mechanistic and biological level. These data indicate that the RAR/RXR heterodimer is a critical regulator of human HSC differentiation,and pharmacological modulation of RXR signaling prevents the loss of human HSCs that otherwise occurs in short-term culture. View Publication

The therapeutic potential of hematopoietic stem cell (HSC) gene therapy can be fully exploited only by reaching efficient gene transfer into HSCs without compromising their biologic properties. Although HSCs can be transduced by HIV-derived lentiviral vectors (LVs) in short ex vivo culture,they display low permissivity to the vector,requiring cytokine stimulation to reach high-frequency transduction. Using stringent assays of competitive xenograft repopulation,we show that early-acting cytokines synergistically enhanced human HSC gene transfer by LVs without impairing engraftment and repopulation capacity. Using S-phase suicide assays,we show that transduction enhancement by cytokines was not dependent on cell cycle progression and that LVs can transduce quiescent HSCs. Pharmacologic inhibition of the proteasome during transduction dramatically enhanced HSC gene transfer,allowing the reach of very high levels of vector integration in their progeny in vivo. Thus,LVs are effectively restricted at a postentry step by the activity of this proteolytic complex. Unexpectedly,cytokine stimulation rapidly and substantially down-regulated proteasome activity in hematopoietic progenitors,highlighting one mechanism by which cytokines may enhance permissiveness to LV gene transfer. These findings demonstrate that antiviral responses ultimately mediated by proteasomes strongly limit the efficiency of HSC transduction by LVs and establish improved conditions for HSC-based gene therapy. View Publication

Mesenchymal stromal cells (MSC) provide a supportive cellular microenvironment and are able to maintain the self-renewal capacity of hematopoietic progenitor cells (HPC). Isolation procedures for MSC vary extensively,and this may influence their biologic properties. In this study,we have compared human MSC isolated from bone marrow (BM) using two culture conditions,from cord blood (CB),and from adipose tissue (AT). The ability to maintain long-term culture-initiating cell frequency and a primitive CD34(+)CD38(-) immunophenotype was significantly higher for MSC derived from BM and CB compared with those from AT. These results were in line with a significantly higher adhesion of HPC to MSC from BM and CB versus MSC from AT. We have compared the cytokine production of MSC by cytokine antibody arrays,enzyme-linked immunosorbent assay,and a cytometric bead array. There were reproducible differences in the chemokine secretion profiles of various MSC preparations,but there was no clear concordance with differences in their potential to maintain primitive function of HPC. Global gene expression profiles of MSC preparations were analyzed and showed that adhesion proteins including cadherin-11,N-cadherin,vascular cell adhesion molecule 1,neural cell adhesion molecule 1,and integrins were highly expressed in MSC preparations derived from BM and CB. Thus,MSC from BM and CB are superior to MSC from AT for maintenance of primitive HPC. The latter property is associated with specific molecular profiles indicating the significance of cell-cell junctions but not with secretory profiles. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

A major goal of current hematopoiesis research is to develop in vitro methods suitable for the measurement and characterization of stem cells with long-term in vivo repopulating potential. Previous studies from several centers have suggested the presence in normal human or murine marrow of a population of very primitive cells that are biologically,physically,and pharmacologically different from cells detectable by short-term colony assays and that can give rise to the latter in long-term cultures (LTCs) containing a competent stromal cell layer. In this report,we show that such cultures can be used to provide a quantitative assay for human LTC-initiating cells" based on an assessment of the number of clonogenic cells present after 5-8 weeks. Production of derivative clonogenic cells is shown to be absolutely dependent on the presence of a stromal cell feeder. When this requirement is met� View Publication