搜索结果: 'NeuroCult Proliferation'

We used a panel of human and mouse fibroblasts with various abilities for supporting the prolonged growth of human embryonic stem cells (hESCs) to elucidate growth factors required for hESC survival,proliferation,and maintenance of the undifferentiated and pluripotent state (self-renewal). We found that supportive feeder cells secrete growth factors required for both hESC survival/proliferation and blocking hESC spontaneous differentiation to achieve self-renewal. The antidifferentiation soluble factor is neither leukemia inhibitory factor nor Wnt,based on blocking experiments using their antagonists. Because Wnt/beta-catenin signaling has been implicated in cell-fate determination and stem cell expansion,we further examined the effects of blocking or adding recombinant Wnt proteins on undifferentiated hESCs. In the absence of feeder cell-derived factors,hESCs cultured under a feeder-free condition survived/proliferated poorly and gradually differentiated. Adding recombinant Wnt3a stimulated hESC proliferation but also differentiation. After 4-5 days of Wnt3a treatment,hESCs that survived maintained the undifferentiated phenotype but few could form undifferentiated hESC colonies subsequently. Using a functional reporter assay,we found that the beta-catenin-mediated transcriptional activation in the canonical Wnt pathway was minimal in undifferentiated hESCs,but greatly upregulated during differentiation induced by the Wnt treatment and several other methods. Thus,Wnt/beta-catenin activation does not suffice to maintain the undifferentiated and pluripotent state of hESCs. We propose a new model for the role of Wnt/beta-catenin signaling in undifferentiated hESCs. View Publication

The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4,which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [(3)H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [(3)H]ERGO uptake. On the other hand,exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker βIII-tubulin,but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP),with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly,edaravone and ascorbic acid did not affect such differentiation of NPCs,in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP,but decreased the number immunoreactive for βIII-tubulin,with concomitant down-regulation of Math1 in P19-NPCs. Thus,OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress,and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action. View Publication

Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface,and their removal from CM impairs proliferation. Finally,we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography,immunoblotting,and mass spectrometry-based proteomic analysis,we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture. View Publication

BACKGROUND Valproic acid (VPA),a commonly used mood stabilizer that promotes neuronal differentiation,regulates multiple signaling pathways involving extracellular signal-regulated kinase (ERK) and glycogen synthase kinase3beta (GSK3beta). However,the mechanism by which VPA promotes differentiation is not understood. RESULTS We report here that 1 mM VPA simultaneously induces differentiation and reduces proliferation of basic fibroblast growth factor (bFGF)-treated embryonic day 14 (E14) rat cerebral cortex neural progenitor cells (NPCs). The effects of VPA on the regulation of differentiation and inhibition of proliferation occur via the ERK-p21Cip/WAF1 pathway. These effects,however,are not mediated by the pathway involving the epidermal growth factor receptor (EGFR) but via the pathway which stabilizes Ras through beta-catenin signaling. Stimulation of differentiation and inhibition of proliferation in NPCs by VPA occur independently and the beta-catenin-Ras-ERK-p21Cip/WAF1 pathway is involved in both processes. The independent regulation of differentiation and proliferation in NPCs by VPA was also demonstrated in vivo in the cerebral cortex of developing rat embryos. CONCLUSION We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects,as well as elucidate its role in the independent regulation of differentiation and inhibition of proliferation in the cerebral cortex of developing rat embryos. View Publication

Normal density granulocytes (NDGs) can suppress T-cell responses in a similar way as myeloid-derived suppressor cells (MDSCs). In this study,we tested the hypothesis that NDGs from healthy donors preferentially inhibit T helper 1 (Th1) cells and investigated the myeloid-derived suppressive effect in different T-cell populations. We found that NDG-induced suppression of T-cell proliferation was contact dependent,mediated by integrin CD11b,and dependent on NDG-production of reactive oxygen species (ROS). The suppression was rapid and occurred within the first few hours of coculture. The suppression did not influence the CD8+/CD4+ ratio indicating an equal sensitivity in these populations. We further analyzed the CD4+ T helper subsets and found that NDGs induced a loss of Th1 surface marker,CD183,that was unrelated to ligand-binding to CD183. In addition,we analyzed the Th1,Th2,and Th17 cytokine production and found that all cytokine groups were suppressed when T-cells were incubated with NDGs. We therefore concluded that NDGs do not preferentially suppress Th1-cells. Instead,NDGs generally suppress Th cells and cytotoxic T-cells but specifically downregulate the Th1 marker CD183. View Publication

Thiamine deficiency (TD) leads to Wernicke's encephalopathy (WE),in which focal histological lesions occur in periventricular areas of the brain. Recently,impaired neurogenesis has been reported in the hippocampus during the dietary form of TD,and in pyrithiamine-induced TD (PTD),a well-characterized model of WE. To further characterize the consequences of PTD on neural stem/progenitor cell (NSPC) activity,we have examined the effect of this treatment in the rat on both the subventricular zone (SVZ) of the rostral lateral ventricle and subgranular layer (SGL) of the hippocampus,and in the thalamus and inferior colliculus,two vulnerable brain regions in this disorder. In both the SVZ and SGL,PTD led to a decrease in the numbers of bromodeoxyuridine-stained cells,indicating that proliferation of NSPCs destined for neurogenesis in these areas was reduced. Doublecortin (DCX) immunostaining in the SGL was decreased,indicating a reduction in neuroblast formation,consistent with impaired NSPC activity. DCX labeling was not apparent in focal areas of vulnerability. In the thalamus,proliferation of cells was absent while in the inferior colliculus,numerous actively dividing cells were apparent,indicative of a differential response between these two brain regions. Exposure of cultured neurospheres to PTD resulted in decreased proliferation of NSPCs,consistent with our in vivo findings. Together,these results indicate that PTD considerably affects cell proliferation and neurogenesis activity in both neurogenic areas and parts of the brain known to display structural and functional vulnerability,confirming and extending recent findings on the effects of TD on neurogenesis. Future use of NSPCs in vitro may allow a closer and more detailed examination of the mechanism(s) underlying inhibition of these cells during TD. View Publication

Human adipose-derived stem cells (hASCs) become an appealing source for regenerative medicine. However,with the multi-passage or cryopreservation for large-scale growth procedures in terms of preclinical and clinical purposes,hASCs often reveal defective cell viability,which is a major obstacle for cell therapy. In our study,the effects of induced pluripotent stem cells-derived conditioned medium (iPS-CM) on the proliferation and anti-apoptosis in hASCs were investigated. hASCs at passage 1 were identified by the analysis of typical surface antigens with flow cytometry assay and adipogenic and osteogenic differentiation. The effect of iPS-CM on the proliferation in hASCs was analyzed by cell cycle assay and Ki67/P27 quantitative polymerase chain reaction analysis. The effect of iPS-CM on the anti-apoptosis of hASCs irradiated by 468 J/m(2) of ultraviolet C was investigated by annexin v/propidium iodide analysis,mitochondrial membrane potential assay,intracellular reactive oxygen species assay,Western blotting and caspase activity assays. The effect of iPS-CM on the surface antigen expressions of hASCs was analyzed using flow cytometry assay. The levels of Activin A and bFGF in culture supernatant of hASCs with different treatments were also detected by enzyme-linked immunosorbent assay. iPS-CM promoted proliferation and inhibited apoptosis of hASCs. This discovery demonstrates that iPS-CM might be used as one of the available means to overcome the propagation obstacle for hASCs and make for large-scale growth procedures in terms of preclinical and clinical purposes. View Publication

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed transmembrane protein whose cleavage product,the amyloid-beta (Abeta) protein,is deposited in amyloid plaques in neurodegenerative conditions such as Alzheimer disease,Down syndrome,and head injury. We recently reported that this protein,normally associated with neurodegenerative conditions,is expressed by human embryonic stem cells (hESCs). We now report that the differential processing of AbetaPP via secretase enzymes regulates the proliferation and differentiation of hESCs. hESCs endogenously produce amyloid-beta,which when added exogenously in soluble and fibrillar forms but not oligomeric forms markedly increased hESC proliferation. The inhibition of AbetaPP cleavage by beta-secretase inhibitors significantly suppressed hESC proliferation and promoted nestin expression,an early marker of neural precursor cell (NPC) formation. The induction of NPC differentiation via the non-amyloidogenic pathway was confirmed by the addition of secreted AbetaPPalpha,which suppressed hESC proliferation and promoted the formation of NPCs. Together these data suggest that differential processing of AbetaPP is normally required for embryonic neurogenesis. View Publication

BackgroundmicroRNAs (miRNAs) have been implicated in the control of many biological processes and their deregulation has been associated with many cancers. In recent years,the cancer stem cell (CSC) concept has been applied to many cancers including pediatric. We hypothesized that a common signature of deregulated miRNAs in the CSCs fraction may explain the disrupted signaling pathways in CSCs.Methodology/ResultsUsing a high throughput qPCR approach we identified 26 CSC associated differentially expressed miRNAs (DEmiRs). Using BCmicrO algorithm 865 potential CSC associated DEmiR targets were obtained. These potential targets were subjected to KEGG,Biocarta and Gene Ontology pathway and biological processes analysis. Four annotated pathways were enriched: cell cycle,cell proliferation,p53 and TGF-beta/BMP. Knocking down hsa-miR-21-5p,hsa-miR-181c-5p and hsa-miR-135b-5p using antisense oligonucleotides and small interfering RNA in cell lines led to the depletion of the CSC fraction and impairment of sphere formation (CSC surrogate assays).ConclusionOur findings indicated that CSC associated DEmiRs and the putative pathways they regulate may have potential therapeutic applications in pediatric cancers. View Publication

Retroviral overexpression of NF-Ya,the regulatory subunit of the transcription factor NF-Y,activates the transcription of multiple genes implicated in hematopoietic stem cell (HSC) self-renewal and differentiation and directs HSCs toward self-renewal. We asked whether TAT-NF-Ya fusion protein could be used to transduce human CD34(+) cells as a safer,more regulated alternative approach to gene therapy. Here we show that externally added recombinant protein was able to enter the cell nucleus and activate HOXB4,a target gene of NF-Ya,using real-time polymerase chain reaction RNA and luciferase-based protein assays. After TAT-NF-Ya transduction,the proliferation of human CD34(+) cells in the presence of myeloid cytokines was increased 4-fold. Moreover,TAT-NF-Ya-treated human primary bone marrow cells showed a 4-fold increase in the percentage of huCD45(+) cells recovered from the bone marrow of sublethally irradiated,transplanted NOD-Scid IL2Rγ(null) mice. These data demonstrate that TAT-peptide therapies are an alternative approach to retroviral stem cell therapies and suggest that NF-Ya peptide delivery should be further evaluated as a tool for HSC/progenitors ex vivo expansion and therapy. View Publication

On the basis of its inhibition by SB216763,we identified the multifunctional enzyme Glycogen Synthase Kinase 3beta (GSK3beta) as a central regulator for differentiation and cell survival of adult neural stem cells. Detected by proteomic approaches,members of the Wnt/beta-catenin signaling pathway appear to participate in enhanced neuronal differentiation and activated transcription of beta-catenin target genes during GSK3beta inhibition,associated with decreased apoptosis. View Publication