搜索结果: 'NeuroCult Proliferation'

Insulin resistance is central to diabetes and metabolic syndrome. To define the consequences of genetic insulin resistance distinct from those secondary to cellular differentiation or in vivo regulation,we generated induced pluripotent stem cells (iPSCs) from individuals with insulin receptor mutations and age-appropriate control subjects and studied insulin signaling and gene expression compared with the fibroblasts from which they were derived. iPSCs from patients with genetic insulin resistance exhibited altered insulin signaling,paralleling that seen in the original fibroblasts. Insulin-stimulated expression of immediate early genes and proliferation were also potently reduced in insulin resistant iPSCs. Global gene expression analysis revealed marked differences in both insulin-resistant iPSCs and corresponding fibroblasts compared with control iPSCs and fibroblasts. Patterns of gene expression in patients with genetic insulin resistance were particularly distinct in the two cell types,indicating dependence on not only receptor activity but also the cellular context of the mutant insulin receptor. Thus,iPSCs provide a novel approach to define effects of genetically determined insulin resistance. This study demonstrates that effects of insulin resistance on gene expression are modified by cellular context and differentiation state. Moreover,altered insulin receptor signaling and insulin resistance can modify proliferation and function of pluripotent stem cell populations. View Publication

BACKGROUND Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors,our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin,chrysin,apigenin,emodin,aloe-emodin,rhein,cis-stilbene and trans-stilbene) were studied on cell proliferation,cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines,together with normal haematopoietic control cells. METHODS Cellular proliferation was measured by CellTiter-Glo(®) luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. RESULTS Emodin,quercetin,and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 μM,8-33 μM,and 25-85 μM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 μM,19-50 μM,and 8-50 μM respectively). Generally,lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines,however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. CONCLUSIONS These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly,the differential sensitivity of emodin,quercetin,and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia. View Publication

Mouse embryonic stem (mES) cells have short duration of their cell cycle and are capable of proliferating in the absence of growth factors. To find out which signaling pathways contribute to the regulation of the mES cell cycle,we used pharmacological inhibitors of MAP and PI3 kinase cascades. The MAP kinase inhibitors as well as serum withdrawal did not affect mES cell cycle distribution,whereas the inhibitor of PI3K activity,LY294002,induced accumulation of cells in G(1) phase followed by apoptotic cell death. Serum withdrawal also causes apoptosis,but it does not change the content and activity of cell cycle regulators. In contrast,in mES cells treated with LY294002,the activities of Cdk2 and E2F were significantly decreased. Interestingly,LY294002had a much stronger effect on cell cycle distribution in low serum conditions,implying that serum can promote G(1)--textgreaterS transition of mES cells by a LY294002-resistant mechanism. Thus,proliferation of mES cells is maintained by at least two separate mechanisms: a LY294002-sensitive pathway,which is active even in the absence of serum,and LY294002-resistant,but serum-dependent,pathway. View Publication

OBJECTIVES Histone deacetylase (HDAC) is an important therapeutic target in cancer. Two of the main anticancer mechanisms of HDAC inhibitors are induction of terminal differentiation and inhibition of cell proliferation. To investigate the role of HDAC in maintenance of self-renewal and cell proliferation,we treated mesenchymal stem cells (MSCs) that originated from adipose tissue or umbilical cord blood with valproic acid (VPA) and sodium butyrate (NaBu). MATERIALS AND METHODS Human MSCs were isolated from mammary fat tissue and cord blood. We performed MTT assay and flow cytometry-based cell cycle analysis to assess self-renewal of MSCs. In vitro differentiation assays into osteogenic,adipogenic,neurogenic and chondrogenic lineages were conducted to investigate MSC multipotency. Immunocytochemistry,Western blot and reverse transcription-polymerase chain reaction were used to interrogate molecular pathways. RESULTS VPA and NaBu flattened the morphology of MSCs and inhibited their growth. VPA and NaBu activated the transcription of p21(CIP1/WAF1) by increasing the acetylation of histone H3 and H4 and eventually blocked the cell cycle at G2/M phase. The expression level of p16(INK4A),a cdk inhibitor that is closely related to cellular senescence,was not changed by HDAC inhibitor treatment. We performed controlled differentiation into bone,fat,cartilage and nervous tissue to elucidate the role of HDAC in the pluripotency of MSC to differentiate into functional tissues. VPA and NaBu decreased the efficiency of adipogenic,chondrogenic,and neurogenic differentiation as visualized by specific staining and reverse transcription-polymerase chain reaction. In contrast,osteogenic differentiation was elevated by HDAC inhibitor treatment. CONCLUSION HDAC activity is essential for maintaining the self-renewal and pluripotency of MSCs. View Publication

AIMS: Mesenchymal stem cells (MSCs) are widely used for cell therapy,particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity,critical elements for their survival and differentiation,have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and,more generally,to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. METHODS AND RESULTS: MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes,a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand,A-769662,a well-characterized AMPK activator,was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice,providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation,we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved,whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. CONCLUSION: MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation,p27 being involved in this regulation. View Publication

Ab-mediated lymphoablation is commonly used in solid organ and hematopoietic cell transplantation. However,these strategies fail to control pathogenic memory T cells efficiently and to improve long-term transplant outcomes significantly. Understanding the mechanisms of T cell reconstitution is critical for enhancing the efficacy of Ab-mediated depletion in sensitized recipients. Using a murine analog of anti-thymocyte globulin (mATG) in a mouse model of cardiac transplantation,we previously showed that peritransplant lymphocyte depletion induces rapid memory T cell proliferation and only modestly prolongs allograft survival. We now report that T cell repertoire following depletion is dominated by memory CD4 T cells. Additional depletion of these residual CD4 T cells severely impairs the recovery of memory CD8 T cells after mATG treatment. The CD4 T cell help during CD8 T cell recovery depends on the presence of B cells expressing CD40 and intact CD40/CD154 interactions. The requirement for CD4 T cell help is not limited to the use of mATG in heart allograft recipients,and it is observed in nontransplanted mice and after CD8 T cell depletion with mAb instead of mATG. Most importantly,limiting helper signals increases the efficacy of mATG in controlling memory T cell expansion and significantly extends heart allograft survival in sensitized recipients. Our findings uncover the novel role for helper memory CD4 T cells during homeostatic CD8 T cell proliferation and open new avenues for optimizing lymphoablative therapies in allosensitized patients. View Publication

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways,however,it remains unclear as to what extent these pathways can be exploited,either individually or in combination,in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro,but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways,we developed a high-density microbioreactor array (HDMA) - a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt,Hedgehog,IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation,inducing cell cycle activity marked by Ki67,and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration. View Publication

The placenta and umbilical cord are pre-eminent candidate sources of mesenchymal stem cells (MSCs). However,placenta-derived MSCs (P-MSCs) showed greater proliferation capacity than umbilical cord-derived MSCs (UC-MSCs) in our study. We investigated the drivers of this proliferation difference and elucidated the mechanisms of proliferation regulation. Proteomic profiling and Gene Ontology (GO) functional enrichment were conducted to identify candidate proteins that may influence proliferation. Using lentiviral or small interfering RNA infection,we established overexpression and knockdown models and observed changes in cell proliferation to examine whether a relationship exists between the candidate proteins and proliferation capacity. Real-time quantitative polymerase chain reaction,western blot analysis,and immunofluorescence assays were conducted to elucidate the mechanisms underlying proliferation. Six candidate proteins were selected based on the results of proteomic profiling and GO functional enrichment. Through further validation,yes-associated protein 1 (YAP1) and $\beta$-catenin were confirmed to affect MSCs proliferation rates. YAP1 and $\beta$-catenin showed increased nuclear colocalization during cell expansion. YAP1 overexpression significantly enhanced proliferation capacity and upregulated the expression of both $\beta$-catenin and the transcriptional targets of Wnt signaling,CCND1,and c-MYC,whereas silencing $\beta$-catenin attenuated this influence. We found that YAP1 directly interacts with $\beta$-catenin in the nucleus to form a transcriptional YAP/$\beta$-catenin/TCF4 complex. Our study revealed that YAP1 and $\beta$-catenin caused the different proliferation capacities of P-MSCs and UC-MSCs. Mechanism analysis showed that YAP1 stabilized the nuclear $\beta$-catenin protein,and also triggered the Wnt/$\beta$-catenin pathway,promoting proliferation. View Publication

Cystic fibrosis (CF) cells display a more cancer-like phenotype vs. non-CF cells. KLF4 overexpression has been described in CF and this transcriptional factor acts as a negative regulator of wt-CFTR. KLF4 is described as exerting its effects in a cell-context-dependent fashion,but it is generally considered a major regulator of proliferation,differentiation,and wound healing,all the processes that are also altered in CF. Therefore,it is relevant to characterize the differential role of KLF4 in these processes in CF vs. non-CF cells. To this end,we used wt- and F508del-CFTR CFBE cells and their respective KLF4 knockout (KO) counterparts to evaluate processes like cell proliferation,polarization,and wound healing,as well as to compare the expression of several epithelial differentiation markers. Our data indicate no major impact of KLF4 KO in proliferation and a differential impact of KLF4 KO in transepithelial electrical resistance (TEER) acquisition and wound healing in wt- vs. F508del-CFTR cells. In parallel,we also observed a differential impact on the levels of some differentiation markers and epithelial-mesencymal transition (EMT)-associated transcription factors. In conclusion,KLF4 impacts TEER acquisition,wound healing,and the expression of differentiation markers in a way that is partially dependent on the CFTR-status of the cell. 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

Granulocyte colony-stimulating factor (GCSF) administration has been linked to the development of monosomy 7 in severe congenital neutropenia and aplastic anemia. We assessed the effect of pharmacologic doses of GCSF on monosomy 7 cells to determine whether this chromosomal abnormality developed de novo or arose as a result of favored expansion of a preexisting clone. Fluorescence in situ hybridization (FISH) of chromosome 7 was used to identify small populations of aneuploid cells. When bone marrow mononuclear cells from patients with monosomy 7 were cultured with 400 ng/ml GCSF,all samples showed significant increases in the proportion of monosomy 7 cells. In contrast,bone marrow from karyotypically normal aplastic anemia,myelodysplastic syndrome,or healthy individuals did not show an increase in monosomy 7 cells in culture. In bone marrow CD34 cells of patients with myelodysplastic syndrome and monosomy 7,GCSF receptor (GCSFR) protein was increased. Although no mutation was found in genomic GCSFR DNA,CD34 cells showed increased expression of the GCSFR class IV mRNA isoform,which is defective in signaling cellular differentiation. GCSFR signal transduction via the Jak/Stat system was abnormal in monosomy 7 CD34 cells,with increased phosphorylated signal transducer and activation of transcription protein,STAT1-P,and increased STAT5-P relative to STAT3-P. Our results suggest that pharmacologic doses of GCSF increase the proportion of preexisting monosomy 7 cells. The abnormal response of monosomy 7 cells to GCSF would be explained by the expansion of undifferentiated monosomy 7 clones expressing the class IV GCSFR,which is defective in signaling cell maturation. View Publication

OBJECTIVE Pancreatic cancer continues to be an urgent clinical problem. We used the novel DNA methyltransferase inhibitor Zebularine and the histone deacetylase inhibitor SAHA to investigate the epigenetic influence on viability and differentiation of the pancreatic cancer cell lines YAP C,DAN G and Panc-89 in vitro and in vivo. MATERIAL AND METHODS Cell vitality,proliferation and expression of PDX-1,cytokeratin 7 and 20,chromogranin A,vimentin,bax and bcl-2 were determined on the protein and mRNA level in vitro and in a subcutaneous xenograft model. RESULTS A time- and dose-dependent increase of apoptosis,paralleled by decreased proliferation,was observed after incubation with single agents or a combination therapy with lower concentrations. This was associated with up-regulation of pro-apoptotic bax and a phenotypic stabilization by the enhanced expression of cytokeratin 7. In vivo,growth of xenografts was delayed with the most pronounced effect in Panc-89 after 1 week of daily intraperitoneal injections of Zebularine paralleled with CK7 up-regulation and down-regulation of dedifferentiation markers. CONCLUSIONS Epigenetic modulation via inhibition of DNA methyltransferase and histone deacetylase induces apoptosis in human pancreatic cancer cells in vitro and delays xenograft growth in vivo,which is associated with a morphological/molecular phenotypic stabilization. These compounds may therefore be suitable as adjunctive therapeutic agents in the treatment of pancreatic cancer. View Publication