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

The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs,miR-375,was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally,miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly,motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly,SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons. View Publication

Human embryonic stem (hES) cells show an atypical cell-cycle regulation characterized by a high proliferation rate and a short G1 phase. In fact,a shortened G1 phase might protect ES cells from external signals inducing differentiation,as shown for certain stem cells. It has been suggested that self-renewal and pluripotency are intimately linked to cell-cycle regulation in ES cells,although little is known about the overall importance of the cell-cycle machinery in maintaining ES cell identity. An appealing model to address whether the acquisition of stem cell properties is linked to cell-cycle regulation emerged with the ability to generate induced pluripotent stem (iPS) cells by expression of defined transcription factors. Here,we show that the characteristic cell-cycle signature of hES cells is acquired as an early event in cell reprogramming. We demonstrate that induction of cell proliferation increases reprogramming efficiency,whereas cell-cycle arrest inhibits successful reprogramming. Furthermore,we show that cell-cycle arrest is sufficient to drive hES cells toward irreversible differentiation. Our results establish a link that intertwines the mechanisms of cell-cycle control with the mechanisms underlying the acquisition and maintenance of ES cell identity. View Publication

Appropriate culture methods for the interrogation of primary leukemic samples were hitherto lacking and current assays for compound screening are not adapted for large-scale investigation of synergistic combinations. In this study,we report a novel approach that efficiently distills synthetic lethal interactions between small molecules active on primary human acute myeloid leukemia (AML) specimens. In single-dose experiments and under culture conditions preserving leukemia stem cell activity,our strategy considerably reduces the number of tests needed for the identification of promising compound combinations. Initially conducted with a selected library of 5000 small molecules and 20 primary AML specimens,it reveals 5 broad classes of sensitized therapeutic target pathways along with their synergistic patient-specific fingerprints. This novel method opens new avenues for the development of AML personalized therapeutics and may be generalized to other tumor types,for which in vitro cancer stem cell cultures have been developed. View Publication

Although Th17 cells play critical roles in the pathogenesis of many inflammatory and autoimmune diseases,their prevalence among tumor-infiltrating lymphocytes (TILs) and function in human tumor immunity remains largely unknown. We have recently demonstrated high percentages of Th17 cells in TILs from ovarian cancer patients,but the mechanisms of accumulation of these Th17 cells in the tumor microenvironment are still unclear. In this study,we further showed elevated Th17 cell populations in the TILs obtained from melanoma and breast and colon cancers,suggesting that development of tumor-infiltrating CD4(+) Th17 cells may be a general feature in cancer patients. We then demonstrated that tumor microenvironmental RANTES and MCP-1 secreted by tumor cells and tumor-derived fibroblasts mediate the recruitment of Th17 cells. In addition to their recruitment,we found that tumor cells and tumor-derived fibroblasts produce a proinflammatory cytokine milieu as well as provide cell-cell contact engagement that facilitates the generation and expansion of Th17 cells. We also showed that inflammatory TLR and nucleotide oligomerization binding domain 2 signaling promote the attraction and generation of Th17 cells induced by tumor cells and tumor-derived fibroblasts. These results identify Th17 cells as an important component of human TILs,demonstrate mechanisms involved in the recruitment and regulation of Th17 cells in tumor microenvironments,and provide new insights relevant for the development of novel cancer immunotherapeutic approaches. View Publication

Dendritic cells (DCs) are potent mediators of the immune response,and can be activated by exogenous pathogen components. Galectin-1 is a member of the conserved beta-galactoside-binding lectin family that binds galactoside residues on cell surface glycoconjugates. Galectin-1 is known to play a role in immune regulation via action on multiple immune cells. However,its effects on human DCs are unknown. In this study,we show that galectin-1 induces a phenotypic and functional maturation in human monocyte-derived DCs (MDDCs) similar to but distinct from the activity of the exogenous pathogen stimuli,LPS. Immature human MDDCs exposed to galectin-1 up-regulated cell surface markers characteristic of DC maturation (CD40,CD83,CD86,and HLA-DR),secreted high levels of IL-6 and TNF-alpha,stimulated T cell proliferation,and showed reduced endocytic capacity,similar to LPS-matured MDDCs. However,unlike LPS-matured DCs,galectin-1-treated MDDCs did not produce the Th1-polarizing cytokine IL-12. Microarray analysis revealed that in addition to modulating many of the same DC maturation genes as LPS,galectin-1 also uniquely up-regulated a significant subset of genes related to cell migration through the extracellular matrix (ECM). Indeed,compared with LPS,galectin-1-treated human MDDCs exhibited significantly better chemotactic migration through Matrigel,an in vitro ECM model. Our findings show that galectin-1 is a novel endogenous activator of human MDDCs that up-regulates a significant subset of genes distinct from those regulated by a model exogenous stimulus (LPS). One unique effect of galectin-1 is to increase DC migration through the ECM,suggesting that galectin-1 may be an important component in initiating an immune response. View Publication

Thiazolidinediones (TZD) are widely prescribed for the treatment of Type 2 diabetes. Increased loss of bone mass and a higher incidence of fractures have been associated with the use of this class of drugs in post-menopausal women. In vitro studies performed in rodent cell models indicated that rosiglitazone (RGZ),one of the TZD,inhibited osteoblastogenesis and induced adipogenesis in bone marrow progenitor cells. The objective of the present study was to determine for the first time the RGZ-dependent shift from osteoblastogenesis toward adipogenesis using a human cell model. To this purpose,bone marrow-derived mesenchymal stem cells were characterized and induced to differentiate along osteogenic and adipogenic lineages. We found that the exposure to RGZ potentiated adipogenic differentiation and shifted the differentiation toward an osteogenic phenotype into an adipogenic phenotype,as assessed by the appearance of lipid droplets. Accordingly,RGZ markedly increased the expression of the typical marker of adipogenesis fatty-acid binding protein 4,whereas it reduced the expression of Runx2,a marker of osteoblastogenesis. This is the first demonstration that RGZ counteracts osteoblastogenesis and induces a preferential differentiation into adipocytes in human mesenchymal stem cells. View Publication

Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However,while classical,NAD(+)-independent HDACs are an established therapeutic target,the relevance of NAD(+)-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here,we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+)-lowering drug FK866,alone and in combination with traditional HDAC inhibitors. Primary leukemia cells,leukemia cell lines,healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol,cambinol,EX527) and with FK866,with or without addition of the HDAC inhibitors valproic acid,sodium butyrate,and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis,and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+) levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells,but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells,HDAC inhibitors were found to induce upregulation of Bax,a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result,leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion,NAD(+)-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited. View Publication

We developed a protocol of in vitro differentiation of human embryonic stem cells into three-dimensional structures histologically and molecularly similar to the developing retina. View Publication

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019),which is associated with familial and sporadic Parkinson's disease as well as impairment of adult neurogenesis in mice. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson's disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization,clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson's disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson's disease patients. Together,our results identify the nucleus as a previously unknown cellular organelle in Parkinson's disease pathology and may help to open new avenues for Parkinson's disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure. View Publication

The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find,unexpectedly,that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2,3,5,6,7 and 11),whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP,whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results,hESCs contain a previously undescribed complex comprising TAFs 2,6,7,11 and TBP. Altering the composition of hESC TAFs,either by depleting TAFs that are present or ectopically expressing TAFs that are absent,results in misregulated expression of pluripotency genes and induction of differentiation. Thus,the selective expression and use of TAFs underlies the ability of hESCs to self-renew.DOI:http://dx.doi.org/10.7554/eLife.00068.001. View Publication

Skin-derived precursor (SKP) cells are a valuable resource for tissue engineering and regenerative medicine,because they represent multipotent stem cells that differentiate into neural and mesodermal progenies. Previous studies suggest that the stem cell pool decreases with age. Here,we show that human multipotent SKP cells can be efficiently collected from adult cheek/chin skin,even in aged individuals of 70-78years. SKP cells were isolated from 38 skin samples by serum-free sphere culture and examined for the ability to differentiate into neural and mesodermal lineages. The number of spheres obtained from adult facial skin was significantly higher than that of trunk or extremity skin. SKP cells derived from cheek/chin skin exhibited a high ability to differentiate into neural and mesodermal cells relative to those derived from eyelid,trunk,or extremity skin. Furthermore,cheek/chin skin SKP cells were shown to express markers for undifferentiated stem cells,including a high expression level of the Sox9 gene. These results indicate that cheek/chin skin is useful for the recovery of multipotent stem cells for tissue engineering and regenerative therapy. View Publication