搜索结果: 'NeuroCult Proliferation'

Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic,but it harbors cells that can behave as progenitor cells in vitro,including the ability to form new hair cells. Lgr5,a Wnt target gene,marks distinct supporting cell types in the neonatal cochlea. Here,we tested the hypothesis that Lgr5(+) cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior,Lgr5(+) cells isolated by flow cytometry from neonatal Lgr5(EGFP-CreERT2/+) mice proliferated and formed clonal colonies. After 10 d in culture,new sensory cells formed and displayed specific hair cell markers (myo7a,calretinin,parvalbumin,myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells,Lgr5(+) cells were enriched precursors to myo7a(+) cells,most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely,small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a(+) cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5(+) cells give rise to myo7a(+) hair cells in the neonatal Lgr5(EGFP-CreERT2/+) cochlea. In addition,overexpression of β-catenin initiated proliferation and led to transient expansion of Lgr5(+) cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development. View Publication

The aim of the present study was to determine the effect of AZD8055 on proliferation,apoptosis and glycolysis in the human cervical cancer cell line HeLa and to investigate the underlying mechanism(s) of action. HeLa human cervical cancer cells were treated with 10 nM AZD8055 for 24,48 or 72 h. MTT was used to determine cell proliferation. Annexin V/propidium iodide staining was used to determine cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. RNA and protein expression were examined by qRT-PCR and western blotting,respectively. Treatment with AZD8055 inhibited proliferation and glycolysis,and induced apoptosis in HeLa cells in a time-dependent manner. During the prolonged treatment with AZD8055,the phosphorylation of mammalian target of rapamycin (mTOR) C1 substrates p70S6K and phosphorylation of the mTORC2 substrate Akt were deregulated,suggesting that the activity of mTOR was downregulated. Furthermore,our study showed that the expression of miR-143 was upregulated in a time-dependent manner in HeLa cells treated with AZD8055. In summary,the present study reveals a novel antitumor mechanism of AZD8055 in HeLa human cervical cancer cells. View Publication

Neurogenesis persists in the adult brain subventricular zone where neural stem/progenitor cells (NSPCs) lie close to brain endothelial cells (BECs). We show in mouse that BECs produce bone morphogenetic proteins (BMPs). Coculture of embryonic and adult NSPCs with BECs activated the canonical BMP/Smad pathway and reduced their proliferation. We demonstrate that coculture with BECs in the presence of EGF and FGF2 induced a reversible cell cycle exit of NSPCs (LeX+) and an increase in the amount of GFAP/LeX-expressing progenitors thought to be stem cells. Levels of the phosphatidylinositol phosphatase PTEN were upregulated in NSPCs after coculture with BECs,or treatment with recombinant BMP4,with a concomitant reduction in Akt phosphorylation. Silencing Smad5 with siRNA or treatment with Noggin,a BMP antagonist,demonstrated that upregulation of PTEN in NSPCs required BMP/Smad signaling and that this pathway regulated cell cycle exit of NSPCs. Therefore,BECs may provide a feedback mechanism to control the proliferation of NSPCs. View Publication

Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors,the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors,however,may be needed to guide cell growth and differentiation. To provide access to such new materials,we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait,approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested,and seven cell-binding peptides were identified. Of these,six sequences possess EC cell-binding ability. Specifically,when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold,they mediate cell adhesion. The corresponding soluble peptides block this adhesion,indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR),they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells. View Publication

Sprouty (Spry) genes encode negative regulators of receptor tyrosine kinase (RTK) signaling,which plays important roles in human embryonic stem cells (hESCs). SPRY2 and SPRY4 are the two most highly expressed Sprouty family members in hESCs,suggesting that they may influence self-renewal. To test this hypothesis,we performed siRNA-mediated knock down (KD) studies. SPRY2 KD resulted in increased cell death and decreased proliferation,whereas SPRY4 KD enhanced survival. In both cases,after KD the cells were able to differentiate into cells of the three germ layers,although after SPRY2 KD there was a tendency toward increased ectodermal differentiation. SPRY2 KD cells displayed impaired mitochondrial fusion and cell membrane damage,explaining in part the increased cell death. These data indicate that Sprouty genes regulate pathways involved in proliferation and cell death in hESCs. View Publication

AC133 antigen is a novel marker for human hematopoietic stem/progenitor cells. In this study,we examined the expression and proliferation potential of AC133(+) cells obtained from steady-state peripheral blood (PB). The proportion of AC133(+) cells in the CD34(+) subpopulation of steady-state PB was significantly lower than that of cord blood (CB),although that of cytokine-mobilized PB was higher than that of CB. The proliferation potential of AC133(+)CD34(+) and AC133(-)CD34(+) cells was examined by colony-forming analysis and analysis of long-term culture-initiating cells (LTC-IC). Although the total number of colony-forming cells was essentially the same in the AC133(+)CD34(+) fraction as in the AC133(-)CD34(+) fraction,the proportion of LTC-IC was much higher in the AC133(+)CD34(+) fraction. Virtually no LTC-IC were detected in the AC133(-)CD34(+) fraction. In addition,the features of the colonies grown from these two fractions were quite different. Approximately 70% of the colonies derived from the AC133(+)CD34(+) fraction were granulocyte-macrophage colonies,whereas more than 90% of the colonies derived from the AC133(-)CD34(+) fraction were erythroid colonies. Furthermore,an ex vivo expansion study observed expansion of colony-forming cells only in the AC133(+)CD34(+) population,and not in the AC133(-)CD34(+) population. These findings suggest that to isolate primitive hematopoietic cells from steady-state PB,selection by AC133 expression is better than selection by CD34 expression. View Publication

Glioblastoma multiforme (GBM) is a grade IV malignant brain tumor with high mortality and has been well known to involve many molecular pathways,including G-protein coupled receptor (GPCR)-mediated signaling (such as epithelial growth factor receptor [EGFR] and platelet derived growth factor receptor [PDGFR]). G protein-coupled receptor kinases (GRK) directly regulate GPCR activity by phosphorylating activated agonist-bound receptors to desensitize signaling and internalize receptors through beta-arrestins. Recent studies in various cancers,including prostate and breast cancer,have highlighted the role of change in GRK expression to oncogenesis and tumor proliferation. In this study,we evaluated the expression of GRK5 in grade II to grade IV glioma specimens using immunohistochemistry and found that GRK5 expression levels are highly correlated with aggressiveness of glioma. We used culture conditions to selectively promote the growth of either glioblastoma cells with stem cell markers (GSC) or differentiated glioblastoma cells (DGC) from fresh GBM specimens. GSC are known to be highly invasive and mobile,and have the capacity to self-renew and are more resistant to chemotherapy and radiation compared to differentiated populations of GBM. We examined the expression of GRK5 in these two sets of culturing conditions for GBM cells and found that GRK5 expression is upregulated in GSC compared to differentiated GBM cells. To better understand the role of GRK5 in GBM-derived stem cells,we created stable GRK5 knockdown and evaluated the proliferation rate. Using an ATP chemiluminescence assay,we show,for the first time,that knocking down the expression of GRK5 decreased the proliferation rate of GSC in contrast to control. View Publication

The intestinal epithelium plays an essential role in nutrient absorption,hormone release,and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by stem cells located in the intestinal crypts. The amount and type of diet influence this process and result in changes in the size and cellular make-up of the tissue. The mechanism underlying the nutrient-driven changes in proliferation is not known,but may involve a shift in intracellular metabolism that allows for more nutrients to be used to manufacture new cells. We hypothesized that nutrient availability drives changes in cellular energy metabolism of small intestinal epithelial crypts that could contribute to increases in crypt proliferation. We utilized primary small intestinal epithelial crypts from C57BL/6J mice to study (1) the effect of glucose on crypt proliferation and (2) the effect of glucose on crypt metabolism using an extracellular flux analyzer for real-time metabolic measurements. We found that glucose increased both crypt proliferation and glycolysis,and the glycolytic pathway inhibitor 2-deoxy-d-glucose (2-DG) attenuated glucose-induced crypt proliferation. Glucose did not enhance glucose oxidation,but did increase the maximum mitochondrial respiratory capacity,which may contribute to glucose-induced increases in proliferation. Glucose activated Akt/HIF-1alpha signaling pathway,which might be at least in part responsible for glucose-induced glycolysis and cell proliferation. These results suggest that high glucose availability induces an increase in crypt proliferation by inducing an increase in glycolysis with no change in glucose oxidation. View Publication

Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study,we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway,which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells,human CD34+,and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells,but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42),nuclear polyploidization levels,cell size,or cell survival. The downstream effectors of mTOR,p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1),are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion,these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors,without affecting differentiation or cell survival. View Publication

Human pluripotent stem cells (PSCs) are critical in vitro tools forbackslashnunderstanding mechanisms that regulate lineage differentiation inbackslashnthe human embryo as well as a potentially unlimited supply of stembackslashncells for regenerative medicine. Pluripotent human and mouse embryonicbackslashnstem cells (ESCs) derived from the inner cell mass of blastocystsbackslashnshare a similar transcription factor network to maintain pluripotencybackslashnand self-renewal,yet there are considerable molecular differencesbackslashnreflecting the diverse environments in which mouse and human ESCsbackslashnare derived. In the current study we evaluated the role of Proteinbackslashnarginine methyltransferase 5 (PRMT5) in human ESC (hESC) self-renewalbackslashnand pluripotency given its critical role in safeguarding mouse ESCbackslashnpluripotency. Unlike the mouse,we discovered that PRMT5 has no rolebackslashnin hESC pluripotency. Using microarray analysis we discovered thatbackslashna significant depletion in PRMT5 RNA and protein from hESCs changedbackslashnthe expression of only 78 genes,with the majority being repressed.backslashnFunctionally,we discovered that depletion of PRMT5 had no effectbackslashnon expression of OCT4,NANOG or SOX2,and did not prevent teratomabackslashnformation. Instead,we show that PRMT5 functions in hESCs to regulatebackslashnproliferation in the self-renewing state by regulating the fractionbackslashnof cells in Gap 1 (G1) of the cell cycle and increasing expressionbackslashnof the G1 cell cycle inhibitor P57. Taken together our data unveilsbackslashna distinct role for PRMT5 in hESCs and identifies P57 as new target. View Publication

Adipogenesis plays a key role in the pathogenesis of obesity. It begins with the commitment of mesenchymal stem cells (MSCs) to the adipocyte lineage,followed by terminal differentiation of preadipocytes to mature adipocytes. A critical,but poorly understood,component of adipogenesis involves proliferation of MSCs and preadipocytes. The present study was undertaken to examine the hypothesis that bone morphogenetic protein-3 (BMP-3) promotes adipogenesis using C3H10T1/2 MSCs and 3T3-L1 preadipocytes as in vitro model systems. We demonstrated that although it did not promote the commitment of MSCs to the adipocyte lineage or the differentiation of preadipocytes to adipocytes,BMP-3-stimulated proliferation by threefold in both cell types. Owing to a lack of information on MSC proliferation,we then delineated the molecular mechanisms underlying BMP-3-stimulated MSC proliferation. We showed that BMP-3 activated the transforming growth factor-beta (TGF-beta)/activin but not ERK1/2,p38 MAPK,or JNK signaling pathways in C3H10T1/2 cells. Furthermore,the TGF-beta/activin receptor kinase inhibitor SB-431542 blocked BMP-3-stimulated proliferation. Importantly,siRNA-mediated knockdown of the key TGF-beta/activin signaling pathway components,ActRIIB,ALK4,or Smad2,abrogated the mitogenic effects of BMP-3 on MSCs. Together,these results demonstrate that BMP-3 stimulates MSC proliferation via the TGF-beta/activin signaling pathway,thus revealing a novel role for this divergent and poorly understood member of the TGF-beta superfamily in regulating MSC proliferation. View Publication

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS,we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3),including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3 inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also,GSK3 phosphorylated PAX3-FKHR in vitro,suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies. View Publication