搜索结果: 'NeuroCult Proliferation'

Aberrant neural progenitor cell (NPC) proliferation and self-renewal have been linked to age-related neurodegeneration and neurodegenerative disorders including Alzheimer's disease (AD). Rhizoma Acori tatarinowii is a traditional Chinese herbal medicine against cognitive decline. In this study,we found that the extract of Rhizoma Acori tatarinowii (AT) and its active constituents,asarones,promote NPC proliferation. Oral administration of AT enhanced NPC proliferation and neurogenesis in the hippocampi of adult and aged mice as well as that of transgenic AD model mice. AT and its fractions also enhanced the proliferation of NPCs cultured in vitro. Further analysis identified α-asarone and β-asarone as the two active constituents of AT in promoting neurogenesis. Our mechanistic study revealed that AT and asarones activated extracellular signal-regulated kinase (ERK) but not Akt,two critical kinase cascades for neurogenesis. Consistently,the inhibition of ERK activities effectively blocked the enhancement of NPC proliferation by AT or asarones. Our findings suggest that AT and asarones,which can be orally administrated,could serve as preventive and regenerative therapeutic agents to promote neurogenesis against age-related neurodegeneration and neurodegenerative disorders. View Publication

Retrovirus-mediated transduction of Hoxb4 enhances hematopoietic stem cell (HSC) activity and enforced expression of Hoxb4 induces in vitro development of HSCs from differentiating mouse embryonic stem cells,but the underlying molecular mechanism remains unclear. We previously showed that the HSC activity was abrogated by accumulated Geminin,an inhibitor for the DNA replication licensing factor Cdt1 in mice deficient in Rae28 (also known as Phc1),which encodes a member of Polycomb-group complex 1. In this study we found that Hoxb4 transduction reduced accumulated Geminin in Rae28-deficient mice,despite increasing the mRNA,and restored the impaired HSC activity. Supertransduction of Geminin suppressed the HSC activity induced by Hoxb4 transduction,whereas knockdown of Geminin promoted the clonogenic and replating activities,indicating the importance of Geminin regulation in the molecular mechanism underlying Hoxb4 transduction-mediated enhancement of the HSC activity. This facilitated our investigation of how transduced Hoxb4 reduced Geminin. We showed in vitro and in vivo that Hoxb4 and the Roc1 (also known as Rbx1)-Ddb1-Cul4a ubiquitin ligase core component formed a complex designated as RDCOXB4,which acted as an E3 ubiquitin ligase for Geminin and down-regulated Geminin through the ubiquitin-proteasome system. Down-regulated Geminin and the resultant E2F activation may provide cells with proliferation potential by increasing a DNA prereplicative complex loaded onto chromatin. Here we suggest that transduced Hoxb4 down-regulates Geminin protein probably by constituting the E3 ubiquitin ligase for Geminin to provide hematopoietic stem and progenitor cells with proliferation potential. View Publication

We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27,a key regulator of cell cycle,in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly,expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly,when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients,increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition,when CLL cells were induced in vitro to enter cell cycle (eg,with cytosine phosphate guanine oligodeoxynucleotide),a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition. View Publication

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture,the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs,inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation,differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs,mainly as a consequence of increased replication. Furthermore,trisomy 12 increases the tumorigenicity of hPSCs in vivo,inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last,a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together,these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications. View Publication

Overexpression of fibroblast growth factor receptor (FGFR) tyrosine kinases has been found in many human breast cancers and has been associated with poor patient prognosis. In order to understand the mechanism by which FGFR mediates breast cancer cell proliferation,we used a low molecular weight compound,PD173074,that selectively inhibits FGFR tyrosine kinase activity and autophosphorylation. This potential anticancer agent caused a G1 growth arrest of MDA-MB-415,MDA-MB-453 and SUM 52 breast cancer cells. Our analyses revealed that FGFR signaling links to the cell cycle machinery via D-type cyclins. PD173074-mediated inhibition of FGFR activity caused downregulation of cyclin D1 and cyclin D2 expression,inhibition of cyclin D/cdk4 activity and,as a consequence,reduction of pRB phosphorylation. Retroviral-mediated ectopic expression of cyclin D1 prevented pRB hypophosphorylation and the cell cycle G1 block in PD173074-treated cells,suggesting a central role for D cyclins in proliferation of FGFR-driven breast cancer cells. The repression of FGFR activity caused downregulation of MAPK in MDA-MB-415 and MDA-MB-453 cells. In SUM 52 cells,both MAPK and PI3K signaling pathways were suppressed. In conclusion,results shown here describe a mechanism by which FGFR promotes proliferation of breast cancer cells. View Publication

Sirt1,a NAD(+)-dependent histone deacetylase,may regulate senescence,metabolism,and apoptosis. In this study,primary pig preadipocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) with or without reagents affecting Sirt1 activity. The adipocyte differentiation process was visualized by light microscopy after Oil red O staining. Proliferation and differentiation of preadipocytes was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Oil red O extraction. Expression of Sirt1,FoxO1,and adipocyte specific genes was detected with semi-quantitive RT-PCR. The results showed that Sirt1 mRNA was widely expressed in various pig tissues from different developmental stages. Sirt1 mRNA was expressed throughout the entire differentiation process of pig preadipocytes. Resveratrol significantly increased Sirt1 mRNA expression,but decreased the expression of FoxO1 and adipocyte marker gene PPARgamma2. Resveratrol significantly inhibited pig preadipocyte proliferation and differentiation. Nicotinamide decreased the expression of Sirt1 mRNA,but increased the expression of FoxO1 and adipocyte specific genes. Nicotinamide greatly stimulated the proliferation and differentiation of pig preadipocytes. In conclusion,these results indicate that Sirt1 may modulate the proliferation and differentiation of pig preadipocytes. Sirt1 may down-regulate pig preadipocytes proliferation and differentiation through repression of adipocyte genes or FoxO1. View Publication

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages,adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine,but not those active on A1 (N6-(R)-phenylisopropyladenosine),A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine),or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors,induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact,both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover,the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1,a G1 cyclin-dependent kinase inhibitor,in a protein kinase A-dependent way. Moreover,the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor,the generation of cAMP,and the induction of p27kip-1 expression. View Publication

Neuronal precursor cells (NPCs) are temporally regulated and have the ability to proliferate and differentiate into mature neurons,oligodendrocytes,and astrocytes in the presence of growth factors (GFs). In the present study,the role of the Jak pathway in brain development was investigated in NPCs derived from neurosphere cultures using Jak2 and Jak3 small interfering RNAs and specific inhibitors. Jak2 inhibition profoundly decreased NPC proliferation,preventing further differentiation into neurons and glial cells. However,Jak3 inhibition induced neuronal differentiation accompanied by neurite growth. This phenomenon was due to the Jak3 inhibition-mediated induction of neurogenin (Ngn)2 and NeuroD in NPCs. Jak3 inhibition induced NPCs to differentiate into scattered neurons and increased the expression of Tuj1,microtubule associated protein 2 (MAP2),Olig2,and neuroglial protein (NG)2,but decreased glial fibrillary acidic protein (GFAP) expression,with predominant neurogenesis/polydendrogenesis compared with astrogliogenesis. Therefore,Jak2 may be important for NPC proliferation and maintenance,whereas knocking-down of Jak3 signaling is essential for NPC differentiation into neurons and oligodendrocytes but does not lead to astrocyte differentiation. These results suggest that NPC proliferation and differentiation are differentially regulated by the Jak pathway. View Publication

PURPOSE:
Human embryonic stem cells (hESC) hold a great potential for regenerative medicine because,in principle,they can differentiate into any cell type found in the human body. In addition,studying the effect of ionizing radiation (IR) on hESC may provide valuable information about the response of human cells to IR exposure in their most naive state,as well as the consequences of IR exposure on the development of organisms. However,the effect of IR,in particular radionuclide uptake,on the pluripotency,proliferation and survival of hESC has not been extensively studied.
METHODS:
In this study we treated cultured hESC with 5-[(125)I]iodo-2'-deoxyuridine ((125)IdU),a precursor of DNA synthesis. Then we measured the expansion of colonies and expression of pluripotency markers in hESC.
RESULTS:
We found that uptake of (125)IdU was similar in both hESC and HT1080 human fibrosarcoma cells. However,treatment with 0.1 μCi/ml (125)IdU for 24 hours resulted in complete death of the hESC population; whereas HT1080 cancer cells continued to grow. Treatment with a 10-fold lower dose (125)IdU (0.01 μCi/ml) resulted in colonies of hESC becoming less defined with numerous cells growing in monolayer outside of the colonies showing signs of differentiation. Then we analyzed the expression of pluripotency markers (octamer-binding transcription factor 4 [Oct-4] and stage-specific embryonic antigen-4 [SSEA4]) in the surviving hESC. We found that hESC in the surviving colonies expressed pluripotency markers at levels comparable with those in the non-treated controls.
CONCLUSIONS:
Our results provide important initial insights into the sensitivity of hESC to IR,and especially that produced by the decay of an internalized radionuclide. View Publication

mRNA applications have undergone unprecedented applications—from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation,involving high costs,risks,and toxicity,emphasizing the need for alternative way as mRNA technology. We successfully developed,screened,and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100 nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells,KHYG-1 cells,and primary NK cells with high efficiency without cytotoxicity,while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover,the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably,the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether,our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies. Graphical abstract Pichon and colleagues developed imidazole lipids-based mRNA platforms very efficient to transfect both NK-92 cells,KHYG-1 cells and primary NK cells without cytotoxicity. They succeeded to replace IL-2 protein by IL-2 mRNA transfection and obtained NK cells with extended viability with preserved biomarkers and full functionalities to kill target cells. View Publication

Neurogenesis in the adult brain is important for memory and learning,and the alterations in neural stem cells (NSCs) may be an important part of Alzheimer's disease pathogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway has been suggested to play an important role in neuronal cell survival and is highly involved in adult neurogenesis. Recently,coenzyme Q10 (CoQ10) was found to affect the PI3K pathway. We investigated whether CoQ10 could restore amyloid β (Aβ)25-35 oligomer-inhibited proliferation of NSCs by focusing on the PI3K pathway. To evaluate the effects of CoQ10 on Aβ25-35 oligomer-inhibited proliferation of NSCs,NSCs were treated with several concentrations of CoQ10 and/or Aβ25-35 oligomers. BrdU labeling,Colony Formation Assays,and immunoreactivity of Ki-67,a marker of proliferative activity,showed that NSC proliferation decreased with Aβ25-35 oligomer treatment,but combined treatment with CoQ10 restored it. Western blotting showed that CoQ10 treatment increased the expression levels of p85α PI3K,phosphorylated Akt (Ser473),phosphorylated glycogen synthase kinase-3β (Ser9),and heat shock transcription factor,which are proteins related to the PI3K pathway in Aβ25-35 oligomers-treated NSCs. To confirm a direct role for the PI3K pathway in CoQ10-induced restoration of proliferation of NSCs inhibited by Aβ25-35 oligomers,NSCs were pretreated with a PI3K inhibitor,LY294002; the effects of CoQ10 on the proliferation of NSCs inhibited by Aβ25-35 oligomers were almost completely blocked. Together,these results suggest that CoQ10 restores Aβ25-35 oligomer-inhibited proliferation of NSCs by activating the PI3K pathway. View Publication

Insulin-like growth factor (IGF) is involved in regulating many processes during neural development,and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g.,via inhibiting Wnt/β-catenin signaling). In the present study,neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and β-catenin. Phosphorylation levels of Akt,Erk1,2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of β-catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1,2 and p38 were not significantly changed in IGFBP4-treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway. View Publication