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Sample records for survival proliferation differentiation

  1. Centriole Amplification in Zebrafish Affects Proliferation and Survival but Not Differentiation of Neural Progenitor Cells

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    Edo Dzafic

    2015-10-01

    Full Text Available In animal cells, supernumerary centrosomes, resulting from centriole amplification, cause mitotic aberrations and have been associated with diseases, including microcephaly and cancer. To evaluate how centriole amplification impacts organismal development at the cellular and tissue levels, we used the in vivo imaging potential of the zebrafish. We demonstrate that centriole amplification can induce multipolar anaphase, resulting in binucleated cells. Such binucleation causes substantial apoptosis in the neuroepithelium. Interestingly, not all epithelia are similarly sensitive to binucleation, as skin cells tolerate it without entering apoptosis. In the neuroepithelium, however, binucleation leads to tissue degeneration and subsequent organismal death. Notably, this tissue degeneration can be efficiently counterbalanced by compensatory proliferation of wild-type cells. Because the risk for generating a binucleated daughter recurs at every cell division, centriole amplification in the neuroepithelium is especially deleterious during progenitor proliferation. Once cells reach the differentiation phase, however, centriole amplification does not impair neuronal differentiation.

  2. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

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    Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

    2016-02-17

    Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an

  3. Plasma Rich in Growth Factors Induces Cell Proliferation, Migration, Differentiation, and Cell Survival of Adipose-Derived Stem Cells

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    Maravillas Mellado-López

    2017-01-01

    Full Text Available Adipose-derived stem cells (ASCs are a promising therapeutic alternative for tissue repair in various clinical applications. However, restrictive cell survival, differential tissue integration, and undirected cell differentiation after transplantation in a hostile microenvironment are complications that require refinement. Plasma rich in growth factors (PRGF from platelet-rich plasma favors human and canine ASC survival, proliferation, and delaying human ASC senescence and autophagocytosis in comparison with serum-containing cultures. In addition, canine and human-derived ASCs efficiently differentiate into osteocytes, adipocytes, or chondrocytes in the presence of PRGF. PRGF treatment induces phosphorylation of AKT preventing ASC death induced by lethal concentrations of hydrogen peroxide. Indeed, AKT inhibition abolished the PRGF apoptosis prevention in ASC exposed to 100 μM of hydrogen peroxide. Here, we show that canine ASCs respond to PRGF stimulus similarly to the human cells regarding cell survival and differentiation postulating the use of dogs as a suitable translational model. Overall, PRGF would be employed as a serum substitute for mesenchymal stem cell amplification to improve cell differentiation and as a preconditioning agent to prevent oxidative cell death.

  4. Long-term potentiation promotes proliferation/survival and neuronal differentiation of neural stem/progenitor cells.

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    Taesup Cho

    Full Text Available Neural stem cell (NSC replacement therapy is considered a promising cell replacement therapy for various neurodegenerative diseases. However, the low rate of NSC survival and neurogenesis currently limits its clinical potential. Here, we examined if hippocampal long-term potentiation (LTP, one of the most well characterized forms of synaptic plasticity, promotes neurogenesis by facilitating proliferation/survival and neuronal differentiation of NSCs. We found that the induction of hippocampal LTP significantly facilitates proliferation/survival and neuronal differentiation of both endogenous neural progenitor cells (NPCs and exogenously transplanted NSCs in the hippocampus in rats. These effects were eliminated by preventing LTP induction by pharmacological blockade of the N-methyl-D-aspartate glutamate receptor (NMDAR via systemic application of the receptor antagonist, 3-[(R-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP. Moreover, using a NPC-neuron co-culture system, we were able to demonstrate that the LTP-promoted NPC neurogenesis is at least in part mediated by a LTP-increased neuronal release of brain-derived neurotrophic factor (BDNF and its consequent activation of tropomysosin receptor kinase B (TrkB receptors on NSCs. Our results indicate that LTP promotes the neurogenesis of both endogenous and exogenously transplanted NSCs in the brain. The study suggests that pre-conditioning of the host brain receiving area with a LTP-inducing deep brain stimulation protocol prior to NSC transplantation may increase the likelihood of success of using NSC transplantation as an effective cell therapy for various neurodegenerative diseases.

  5. TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS.

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    Daniel Édgar Cortés

    2016-09-01

    Full Text Available Embryonic stem cells (ESC are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC that constitutively produce Glial cell-derived neurotrophic factor (GDNF and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic motor neurons. After lentiviral transduction, ESC lines integrated and expressed the human GDNF gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study motor neuron induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal motor neurons, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of motor neurons in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant human GDNF was added to control ESC, also resulting in enhanced motor neuron differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, motor neurons were selected for electrophysiological recordings. Motor neurons differentiated from GDNF-ESC, compared to control motor neurons, showed greater electrophysiological maturation, characterized by

  6. Survival, proliferation and differentiation enhancement of neural stem cells cultured in three-dimensional polyethylene glycol-RGD hydrogel with tenascin.

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    Naghdi, Pejman; Tiraihi, Taki; Ganji, Fariba; Darabi, Shehram; Taheri, Taher; Kazemi, Hadi

    2016-03-01

    Polyethylene glycol hydrogel (PEG) conjugated with arginyl glycyl aspartic acid (RGD) (PEG-RGD) has been considered to be a scaffold in three-dimensional (3D) culture that improves neurite outgrowth; on the other hand, tenascin C controls neural growth and differentiation. In this study, the effect of a combined RGD and tenascin C mixture in 3D culture (3D-PEG-RGD-TnC) on the survival, growth and differentiation of neural stem cells. The viability of the culture has been evaluated by live/dead assay and the results show that the viability of NSCs in 3D-PEG-RGD-TnC is significantly higher than its value in 3D-PEG-RGD. The proliferation was evaluated by MTS test and was found to be slightly improved but statistically not significant. Accordingly, the differentiation was evaluated by immunoreactivity to nestin, neurofilament 68, neurofilament 160, neurofilament 200 and GFAP; and the expression of nestin, neuro D, musashi1, β-tubulin III, GFAP, MBP and Oct4 was studied using RT-PCR. The results showed enhancement of the differentiation of NSCs into the neuronal phenotype in 3D-PEG-RGD-TnC. The morphology of NSCs cultured in 3D-PEG-RGD-TnC showed neurite outgrowths and increase in the contact between the differentiated cells' extensions. The conclusion of this study was that NSC survival, proliferation and differentiation are enhanced when the cells are cultured in 3D-PEG-RGD-TnC. Copyright © 2014 John Wiley & Sons, Ltd.

  7. Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases

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    Engstrom, Anna; Wang, Hao; Xia, Zhengui

    2015-01-01

    Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults. PMID:25967738

  8. Collagen Promotes Higher Adhesion, Survival and Proliferation of Mesenchymal Stem Cells.

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    Chinnapaka Somaiah

    Full Text Available Mesenchymal stem cells (MSC can differentiate into several cell types and are desirable candidates for cell therapy and tissue engineering. However, due to poor cell survival, proliferation and differentiation in the patient, the therapy outcomes have not been satisfactory. Although several studies have been done to understand the conditions that promote proliferation, differentiation and migration of MSC in vitro and in vivo, still there is no clear understanding on the effect of non-cellular bio molecules. Of the many factors that influence the cell behavior, the immediate cell microenvironment plays a major role. In this context, we studied the effect of extracellular matrix (ECM proteins in controlling cell survival, proliferation, migration and directed MSC differentiation. We found that collagen promoted cell proliferation, cell survival under stress and promoted high cell adhesion to the cell culture surface. Increased osteogenic differentiation accompanied by high active RHOA (Ras homology gene family member A levels was exhibited by MSC cultured on collagen. In conclusion, our study shows that collagen will be a suitable matrix for large scale production of MSC with high survival rate and to obtain high osteogenic differentiation for therapy.

  9. The research progress of MSCs proliferation and differentiation in ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-02-18

    MSCs) proliferation and differentiation in vitro and vivo. This review will introduce some newest articles mainly in China focusing on the growth and differentiation of MSCs, which were treated with a single herb or formulas.

  10. Learning and adult neurogenesis: survival with or without proliferation?

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    Prickaerts, Jos; Koopmans, Guido; Blokland, Arjan; Scheepens, Arjan

    2004-01-01

    Recent high quality papers have renewed interest in the phenomenon of neurogenesis within the adult mammalian brain. Many studies now show that neurogenesis can be modulated by environmental factors including physical activity, stress, and learning. These findings have considerable implications for neuroscience in general, including the study of learning and memory, neural network plasticity, aging, neurodegeneration, and the recovery from brain injury. Although new light has been shed on this field, many contradictory findings have been reported. Here we propose two principle issues which underlie these inconsistencies, with particular focus on the interaction between learning and neurogenesis. The first issue relates to the basic methodology of measuring the generation of new brain cells, i.e., proliferation, as compared to survival of the newly made cells. Mostly, measures of neurogenesis reported are a combination of proliferation and survival, making it impossible to distinguish between these separate processes. The second aspect is in regards to the role of environmental factors which can affect both proliferation and survival independently. Especially the interaction between stress and learning is of importance since these might counteract each other in some circumstances. Reviewing the literature while taking these issues into account indicates that, in contrast to some findings, cell proliferation in the dentate gyrus of the hippocampus as a result of learning cannot be ruled out yet. On the other hand, increased survival of granule cells in the dentate gyrus as a result of hippocampal-dependent learning has been clearly demonstrated. Moreover, this learning-induced survival of granule cells, which were born before the actual learning experience, might provide a molecular mechanism for the 'use it or lose it' principle.

  11. Proliferation, angiogenesis and differentiation related markers in ...

    African Journals Online (AJOL)

    The upregulation of these proliferation- and angiogenesis-related factors in endothelial cells and/or fibroblasts and not in follicular cells of compact carcinoma compared to healthy glands supports the relevance of stromal cells in cancer progression. Keywords: Canine, Histology, Immunohistochemistry, Thyroid carcinoma.

  12. Proliferation of differentiated glial cells in the brain stem

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    Barradas P.C.

    1998-01-01

    Full Text Available Classical studies of macroglial proliferation in muride rodents have provided conflicting evidence concerning the proliferating capabilities of oligodendrocytes and microglia. Furthermore, little information has been obtained in other mammalian orders and very little is known about glial cell proliferation and differentiation in the subclass Metatheria although valuable knowledge may be obtained from the protracted period of central nervous system maturation in these forms. Thus, we have studied the proliferative capacity of phenotypically identified brain stem oligodendrocytes by tritiated thymidine radioautography and have compared it with known features of oligodendroglial differentiation as well as with proliferation of microglia in the opossum Didelphis marsupialis. We have detected a previously undescribed ephemeral, regionally heterogeneous proliferation of oligodendrocytes expressing the actin-binding, ensheathment-related protein 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, that is not necessarily related to the known regional and temporal heterogeneity of expression of CNPase in cell bodies. On the other hand, proliferation of microglia tagged by the binding of Griffonia simplicifolia B4 isolectin, which recognizes an alpha-D-galactosyl-bearing glycoprotein of the plasma membrane of macrophages/microglia, is known to be long lasting, showing no regional heterogeneity and being found amongst both ameboid and differentiated ramified cells, although at different rates. The functional significance of the proliferative behavior of these differentiated cells is unknown but may provide a low-grade cell renewal in the normal brain and may be augmented under pathological conditions.

  13. Effect of excess dietary copper on proliferation and differentiation of ...

    African Journals Online (AJOL)

    It was found that the application of copper sulphate doses modulates the proliferation and differentiation of stem cell progenitors and erythrocytes. Several alterations were observed and these were time- and dosedependent. Of these alterations, the predominant existence of giant pro-erythroblasts and promyeloblasts ...

  14. Research progress on the proliferation and differentiation of

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    An, A.; Tan, B.

    Space environments such as microgravity magnetic field radiation and heavy metal ions affects the development and functions of human and mammalian cells To study these influences and the corresponding metabolisms is in favour of knowing about the development and differentiation process of organism cells In recent years researches on the differentiation of stem cells induced in vitro provide a new pathway for the repair of tissue lesion and therapy of human diseases Stem cells are potential in capable of differentiating into different functional cells But there has no reliable methods to induce the stem cells differentiating forward specific cells and to gain enough cells for transplantation which limited their application on clinical therapy It has been indicated that microgravity influenced embryonic development hematopoietic and mesenchymal stem cells and so on Hematopoietic stem cell migration and its differentiation were affected by microgravity The specific differentiation of hematopoietic stem cells was inhibited under microgravity The expression of proteins regulating cell cycle period also changed Mesenchymal stem cells provide a source of cells for the repair of musculoskeletal tissue in ground experiment While under microgravity the proliferation and differentiation of mesenchymal stem cells were influenced along with the differentiated cells function changed Furthermore in the differentiation process of stem cells under microgravity the mechanism of signal transport was also affected and the specific differentiation

  15. Micropatterning topology on soft substrates affects myoblast proliferation and differentiation.

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    Zatti, Susi; Zoso, Alice; Serena, Elena; Luni, Camilla; Cimetta, Elisa; Elvassore, Nicola

    2012-02-07

    Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell-cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 μm width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 μm wide lanes compared to (39 ± 7)% in 500 μm lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 μm, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models.

  16. Simulated Microgravity and 3D Culture Enhance Induction, Viability, Proliferation and Differentiation of Cardiac Progenitors from Human Pluripotent Stem Cells

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    Jha, Rajneesh; Wu, Qingling; Singh, Monalisa; Preininger, Marcela K.; Han, Pengcheng; Ding, Gouliang; Cho, Hee Cheol; Jo, Hanjoong; Maher, Kevin O.; Wagner, Mary B.; Xu, Chunhui

    2016-01-01

    Efficient generation of cardiomyocytes from human pluripotent stem cells is critical for their regenerative applications. Microgravity and 3D culture can profoundly modulate cell proliferation and survival. Here, we engineered microscale progenitor cardiac spheres from human pluripotent stem cells and exposed the spheres to simulated microgravity using a random positioning machine for 3 days during their differentiation to cardiomyocytes. This process resulted in the production of highly enriched cardiomyocytes (99% purity) with high viability (90%) and expected functional properties, with a 1.5 to 4-fold higher yield of cardiomyocytes from each undifferentiated stem cell as compared with 3D-standard gravity culture. Increased induction, proliferation and viability of cardiac progenitors as well as up-regulation of genes associated with proliferation and survival at the early stage of differentiation were observed in the 3D culture under simulated microgravity. Therefore, a combination of 3D culture and simulated microgravity can be used to efficiently generate highly enriched cardiomyocytes. PMID:27492371

  17. Effect of Ultrasonic Vibration on Proliferation and Differentiation of Cells

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    Haruka Hino

    2016-12-01

    Full Text Available The effect of mechanical stimulation of vibration on proliferation and differentiation of cells has been studied in vitro. To apply the vibration on the cells, a piezoelectric element was attached on the outside surface of the bottom of the culture plate of six wells. The piezoelectric element was vibrated by sinusoidally alternating voltage at 1.0 MHz generated by a function generator. Five kinds of cells were used in the experiment: C2C12 (mouse myoblast cell, L929 (fibroblast connective tissue of mouse, Hepa1-6 (mouse hepatoma cell, HUVEC (human umbilical vein endothelial cell, and Neuro-2a (mouse neural crest-derived cell line. After the incubation for 24 hours, cells were exposed to the ultrasonic vibration intermittently for three days: for thirty minutes per day. At the end of the experiment, the number of cells was counted by colorimetric method with a microplate photometer. In the case of Neuro-2a, the total length of the neurite was calculated at the microscopic image. The experimental study shows following results. Cells are exfoliated by the strong vibration. Proliferation and differentiation of cells are accelerated with mild vibration. The optimum intensity of vibration depends on the kind of cells.

  18. PDGFs regulate tooth germ proliferation and ameloblast differentiation.

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    Wu, Nan; Iwamoto, Tsutomu; Sugawara, Yu; Futaki, Masaharu; Yoshizaki, Keigo; Yamamoto, Shinya; Yamada, Aya; Nakamura, Takashi; Nonaka, Kazuaki; Fukumoto, Satoshi

    2010-06-01

    The purpose of this study was to elucidate the effects of platelet-derived growth factors (PDGFs) during tooth development, as well as the mechanisms underlying the interactions of growth factors with PDGF signalling during odontogenesis. We used an ex vivo tooth germ organ culture system and two dental cell lines, SF2 cells and mDP cells, as models of odontogenesis. AG17, a tyrosine kinase inhibitor, was utilised for blocking PDGF receptor signalling. To analyse the expressions of PDGFs, reverse transcriptase (RT)-PCR and immunohistochemistry were performed. Proliferation was examined using a BrdU incorporation assay for the organ cultures and a cell counting kit for the cell lines. The expressions of Fgf2 and ameloblastin were analysed by real-time RT-PCR. The PDGF ligands PDGF-A and PDGF-B, and their receptors, PDGFRalpha and PDGFRbeta, were expressed throughout the initial stages of tooth development. In the tooth germ organ cultures, PDGF-AA, but not PDGF-BB, accelerated cusp formation. Conversely, AG17 suppressed both growth and cusp formation of tooth germs. Exogenous PDGF-BB promoted mDP cell proliferation. Furthermore, PDGF-AA decreased Fgf2 expression and increased that of ameloblastin, a marker of differentiated ameloblasts. Our results indicate that PDGFs are involved in initial tooth development and regulate tooth size and shape, as well as ameloblast differentiation. Copyright 2010 Elsevier Ltd. All rights reserved.

  19. Hemopoietic stem cells: stochastic differentiation and humoral control of proliferation.

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    Ogawa, M

    1989-03-01

    The central feature of hemopoiesis is life-long, stable cell renewal. This process is supported by hemopoietic stem cells which, in the steady state, appear to be dormant in cell cycling. The entry into cell cycle of the dormant stem cells may be promoted by such factors as interleukin-1, interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). Once the stem cells leave G0 and begin proliferation, the subsequent process is characterized by continued proliferation and differentiation. While several models of stem cell differentiation have been proposed, micromanipulation studies of individual progenitors suggest that the commitment of multipotential progenitors to single lineages is a random (stochastic) process. The proliferation of early hemopoietic progenitors requires the presence of interleukin-3 (IL-3), and the intermediate process appears to be supported by granulocyte/macrophage colony-stimulating factor (GM-CSF). Once the progenitors are committed to individual lineages, the subsequent maturation process appears to be supported by late-acting, lineage-specific factors such as erythropoietin and G-CSF. Synthesis of a hemopoietic factor may take place in different cell types and is regulated by multiple factors. The physiological regulator of erythropoiesis is erythropoietin, which, by a feedback mechanism, provides fine control of erythrocyte production. Feedback mechanisms for leukocyte production have not been identified. It is possible that there is no feedback regulator of leukopoiesis. In this model, leukocyte production in the steady state is maintained at a genetically determined level. When an infection occurs, the bacterial lipopolysaccharides may augment the production of interleukin 1 alpha and beta, tumor necrosis factor, macrophage colony-stimulating factor, etc.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. B-cell lymphoma 6 promotes proliferation and survival of trophoblastic cells.

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    Muschol-Steinmetz, Cornelia; Jasmer, Britta; Kreis, Nina-Naomi; Steinhäuser, Kerstin; Ritter, Andreas; Rolle, Udo; Yuan, Juping; Louwen, Frank

    2016-01-01

    Preeclampsia is one of the leading causes of maternal and perinatal mortality and morbidity and its pathogenesis is not fully understood. B-cell lymphoma 6 (BCL6), a key regulator of B-lymphocyte development, is altered in preeclamptic placentas. We show here that BCL6 is present in all 3 studied trophoblast cell lines and it is predominantly expressed in trophoblastic HTR-8/SVneo cells derived from a 1(st) trimester placenta, suggestive of its involvement in trophoblast expansion in the early stage of placental development. BCL6 is strongly stabilized upon stress stimulation. Inhibition of BCL6, by administrating either small interfering RNA or a specific small molecule inhibitor 79-6, reduces proliferation and induces apoptosis in trophoblastic cells. Intriguingly, depletion of BCL6 in HTR-8/SVneo cells results in a mitotic arrest associated with mitotic defects in centrosome integrity, indicative of its involvement in mitotic progression. Thus, like in haematopoietic cells and breast cancer cells, BCL6 promotes proliferation and facilitates survival of trophoblasts under stress situation. Further studies are required to decipher its molecular roles in differentiation, migration and the fusion process of trophoblasts. Whether increased BCL6 observed in preeclamptic placentas is one of the causes or the consequences of preeclampsia warrants further investigations in vivo and in vitro.

  1. Growth, survival, proliferation and pathogenesis of Listeria monocytogenes under low oxygen or anaerobic conditions: a review.

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    Lungu, B; Ricke, S C; Johnson, M G

    2009-01-01

    Listeria monocytogenes is a Gram positive facultative anaerobe that causes listeriosis, a disease that mainly affects the immune-compromised, the elderly, infants and pregnant women. In the susceptible immune challenged population, listeriosis is very severe and has a fatality rate of up to 30%. Control of L. monocytogenes is difficult due to its: 1) widespread presence in the environment, 2) intrinsic physiological resistance, 3) ability to adapt to external stresses and 4) ability to grow at a wide range of temperatures. L. monocytogenes encounters anaerobic conditions in the external environment as well as during pathogenesis. Although L. monocytogenes is a facultative anaerobe, the differential effects of O(2) and oxidation-reduction potential on the multiplication of L. monocytogenes have not been established. In addition, most laboratory studies to determine the growth, survival and persistence of this pathogen in foods as well as in the environment have emphasized the response of this pathogen under aerobic conditions. Consequently, this has led to a limited understanding of the metabolic and physiological responses of L. monocytogenes in low oxygen environments. Therefore, the objective of our review was to highlight the progress that has been made in L. monocytogenes research with emphasis on the role of low oxygen and/or anaerobiosis in the growth, survival and proliferation of this pathogen in the environment as well as during pathogenesis.

  2. N-cadherin negatively regulates osteoblast proliferation and survival by antagonizing Wnt, ERK and PI3K/Akt signalling.

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    Eric Haÿ

    Full Text Available BACKGROUND: Osteoblasts are bone forming cells that play an essential role in osteogenesis. The elucidation of the mechanisms that control osteoblast number is of major interest for the treatment of skeletal disorders characterized by abnormal bone formation. Canonical Wnt signalling plays an important role in the control of osteoblast proliferation, differentiation and survival. Recent studies indicate that the cell-cell adhesion molecule N-cadherin interacts with the Wnt co-receptors LRP5/6 to regulate osteoblast differentiation and bone accrual. The role of N-cadherin in the control of osteoblast proliferation and survival remains unknown. METHODS AND PRINCIPAL FINDINGS: Using murine MC3T3-E1 osteoblastic cells and N-cadherin transgenic mice, we demonstrate that N-cadherin overexpression inhibits cell proliferation in vitro and in vivo. The negative effect of N-cadherin on cell proliferation results from decreased Wnt, ERK and PI3K/Akt signalling and is restored by N-cadherin neutralizing antibody that antagonizes N-cadherin-LRP5 interaction. Inhibition of Wnt signalling using DKK1 or Sfrp1 abolishes the ability of N-cadherin blockade to restore ERK and PI3K signalling and cell proliferation, indicating that the altered cell growth in N-cadherin overexpressing cells is in part secondary to alterations in Wnt signalling. Consistently, we found that N-cadherin overexpression inhibits the expression of Wnt3a ligand and its downstream targets c-myc and cyclin D1, an effect that is partially reversed by N-cadherin blockade. We also show that N-cadherin overexpression decreases osteoblast survival in vitro and in vivo. This negative effect on cell survival results from inhibition of PI3K/Akt signalling and increased Bax/Bcl-2, a mechanism that is rescued by Wnt3a. CONCLUSION: The data show that N-cadherin negatively controls osteoblast proliferation and survival via inhibition of autocrine/paracrine Wnt3a ligand expression and attenuation of Wnt

  3. EEN regulates the proliferation and survival of multiple myeloma cells by potentiating IGF-1 secretion

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    Huang, Er-Wen [Guangzhou Institute of Forensic Science, Guangzhou (China); Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou (China); Xue, Sheng-Jiang [Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou (China); Li, Xiao-Yan [Department of Pharmacy, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou (China); Xu, Suo-Wen [Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou (China); Cheng, Jian-Ding; Zheng, Jin-Xiang [Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou (China); Shi, He; Lv, Guo-Li; Li, Zhi-Gang; Li, Yue; Liu, Chang-Hui; Chen, Xiao-Hui; Liu, Hong [Guangzhou Institute of Forensic Science, Guangzhou (China); Li, Jie, E-mail: mdlijie@sina.com [Department of Anaesthesiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou (China); Liu, Chao, E-mail: liuchaogaj@21cn.com [Guangzhou Institute of Forensic Science, Guangzhou (China)

    2014-05-02

    Highlights: • Levels of EEN expression paralleled with the rate of cell proliferation. • EEN was involved in the proliferation and survival of multiple myeloma (MM) cells. • EEN regulated the activity of IGF-1-Akt/mTOR pathway. • EEN regulated proliferation and survival of MM cells by enhancing IGF-1 secretion. - Abstract: The molecular mechanisms of multiple myeloma are not well defined. EEN is an endocytosis-regulating molecule. Here we report that EEN regulates the proliferation and survival of multiple myeloma cells, by regulating IGF-1 secretion. In the present study, we observed that EEN expression paralleled with cell proliferation, EEN accelerated cell proliferation, facilitated cell cycle transition from G1 to S phase by regulating cyclin-dependent kinases (CDKs) pathway, and delayed cell apoptosis via Bcl2/Bax-mitochondrial pathway. Mechanistically, we found that EEN was indispensable for insulin-like growth factor-1 (IGF-1) secretion and the activation of protein kinase B-mammalian target of rapamycin (Akt-mTOR) pathway. Exogenous IGF-1 overcame the phenotype of EEN depletion, while IGF-1 neutralization overcame that of EEN over-expression. Collectively, these data suggest that EEN may play a pivotal role in excessive cell proliferation and insufficient cell apoptosis of bone marrow plasma cells in multiple myeloma. Therefore, EEN may represent a potential diagnostic marker or therapeutic target for multiple myeloma.

  4. Epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia modulates proliferation, cell survival and chemosensitivity

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    Thathia, Shabnam H.; Ferguson, Stuart; Gautrey, Hannah E.; van Otterdijk, Sanne D.; Hili, Michela; Rand, Vikki; Moorman, Anthony V.; Meyer, Stefan; Brown, Robert; Strathdee, Gordon

    2012-01-01

    Background Altered regulation of many transcription factors has been shown to be important in the development of leukemia. TWIST2 modulates the activity of a number of important transcription factors and is known to be a regulator of hematopoietic differentiation. Here, we investigated the significance of epigenetic regulation of TWIST2 in the control of cell growth and survival and in response to cytotoxic agents in acute lymphoblastic leukemia. Design and Methods TWIST2 promoter methylation status was assessed quantitatively, by combined bisulfite and restriction analysis (COBRA) and pyrosequencing assays, in multiple types of leukemia and TWIST2 expression was determined by quantitative reverse transcriptase polymerase chain reaction analysis. The functional role of TWIST2 in cell proliferation, survival and response to chemotherapy was assessed in transient and stable expression systems. Results We found that TWIST2 was inactivated in more than 50% of cases of childhood and adult acute lymphoblastic leukemia through promoter hypermethylation and that this epigenetic regulation was especially prevalent in RUNX1-ETV6-driven cases. Re-expression of TWIST2 in cell lines resulted in a dramatic reduction in cell growth and induction of apoptosis in the Reh cell line. Furthermore, re-expression of TWIST2 resulted in increased sensitivity to the chemotherapeutic agents etoposide, daunorubicin and dexamethasone and TWIST2 hypermethylation was almost invariably found in relapsed adult acute lymphoblastic leukemia (91% of samples hypermethylated). Conclusions This study suggests a dual role for epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia, initially through altering cell growth and survival properties and subsequently by increasing resistance to chemotherapy. PMID:22058208

  5. MiR-9, -31, and -182 deregulation promote proliferation and tumor cell survival in colon cancer.

    Science.gov (United States)

    Cekaite, Lina; Rantala, Juha K; Bruun, Jarle; Guriby, Marianne; Agesen, Trude H; Danielsen, Stine A; Lind, Guro E; Nesbakken, Arild; Kallioniemi, Olli; Lothe, Ragnhild A; Skotheim, Rolf I

    2012-09-01

    Several microRNAs (miRNAs) are known to be deregulated in colon cancer, but the mechanisms behind their potential involvement on proliferation and tumor cell survival are unclear. The present study aimed to identify miRNAs with functional implications for development of colon cancer. The cell proliferation and apoptosis were examined following perturbations of miRNA levels by employing a comprehensive miRNA library screen. miRNAs nominated for relevance to colon cancer were validated on expression and functional levels. By integrating the effect of miRNA up-regulation with the endogenous miRNA expression levels within the HT29, HCT116, and SW480 colon cancer cell lines, we identified miRNAs controlling cell proliferation (n = 53) and apoptosis (n = 93). From these functionally nominated miRNAs, we narrowed the list to 10 oncogene- and 20 tumor suppressor-like miRNAs that were also differentially expressed between colon cancer (n = 80) and normal colonic mucosa (n = 20). The differential expressions of miR-9, miR-31, and miR-182 were successfully validated in a series of colon carcinomas (n = 30) and polyps (n = 10) versus normal colonic mucosa (n = 10), whereas the functional effect was confirmed in an in-depth validation using different cell viability and apoptotic markers. Several transcription factors and genes regulating cell proliferation were identified as putative target genes by integrative miRNA/mRNA expression analysis obtained from the same colon cancer patient samples. This study suggests that deregulated expression of miR-9, miR-31, and miR-182 during carcinogenesis plays a significant role in the development of colon cancer by promoting proliferation and tumor cell survival.

  6. MiR-9, -31, and -182 Deregulation Promote Proliferation and Tumor Cell Survival in Colon Cancer12

    Science.gov (United States)

    Cekaite, Lina; Rantala, Juha K; Bruun, Jarle; Guriby, Marianne; Ågesen, Trude H; Danielsen, Stine A; Lind, Guro E; Nesbakken, Arild; Kallioniemi, Olli; Lothe, Ragnhild A; Skotheim, Rolf I

    2012-01-01

    Several microRNAs (miRNAs) are known to be deregulated in colon cancer, but the mechanisms behind their potential involvement on proliferation and tumor cell survival are unclear. The present study aimed to identify miRNAs with functional implications for development of colon cancer. The cell proliferation and apoptosis were examined following perturbations of miRNA levels by employing a comprehensive miRNA library screen. miRNAs nominated for relevance to colon cancer were validated on expression and functional levels. By integrating the effect of miRNA up-regulation with the endogenous miRNA expression levels within the HT29, HCT116, and SW480 colon cancer cell lines, we identified miRNAs controlling cell proliferation (n = 53) and apoptosis (n = 93). From these functionally nominated miRNAs, we narrowed the list to 10 oncogene- and 20 tumor suppressor-like miRNAs that were also differentially expressed between colon cancer (n = 80) and normal colonic mucosa (n = 20). The differential expressions of miR-9, miR-31, and miR-182 were successfully validated in a series of colon carcinomas (n = 30) and polyps (n = 10) versus normal colonic mucosa (n = 10), whereas the functional effect was confirmed in an in-depth validation using different cell viability and apoptotic markers. Several transcription factors and genes regulating cell proliferation were identified as putative target genes by integrative miRNA/mRNA expression analysis obtained from the same colon cancer patient samples. This study suggests that deregulated expression of miR-9, miR-31, and miR-182 during carcinogenesis plays a significant role in the development of colon cancer by promoting proliferation and tumor cell survival. PMID:23019418

  7. MiR-9, -31, and -182 Deregulation Promote Proliferation and Tumor Cell Survival in Colon Cancer

    Directory of Open Access Journals (Sweden)

    Lina Cekaite

    2012-09-01

    Full Text Available Several microRNAs (miRNAs are known to be deregulated in colon cancer, but the mechanisms behind their potential involvement on proliferation and tumor cell survival are unclear. The present study aimed to identify miRNAs with functional implications for development of colon cancer. The cell proliferation and apoptosis were examined following perturbations of miRNA levels by employing a comprehensive miRNA library screen. miRNAs nominated for relevance to colon cancer were validated on expression and functional levels. By integrating the effect of miRNA up-regulation with the endogenous miRNA expression levels within the HT29, HCT116, and SW480 colon cancer cell lines, we identified miRNAs controlling cell proliferation (n = 53 and apoptosis (n = 93. From these functionally nominated miRNAs, we narrowed the list to 10 oncogene- and 20 tumor suppressor-like miRNAs that were also differentially expressed between colon cancer (n = 80 and normal colonic mucosa (n = 20. The differential expressions of miR-9, miR-31, and miR-182 were successfully validated in a series of colon carcinomas (n = 30 and polyps (n = 10 versus normal colonic mucosa (n = 10, whereas the functional effect was confirmed in an in-depth validation using different cell viability and apoptotic markers. Several transcription factors and genes regulating cell proliferation were identified as putative target genes by integrative miRNA/mRNA expression analysis obtained from the same colon cancer patient samples. This study suggests that deregulated expression of miR-9, miR-31, and miR-182 during carcinogenesis plays a significant role in the development of colon cancer by promoting proliferation and tumor cell survival.

  8. Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells.

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    Opas Traitanon

    Full Text Available The direct effect of immunosuppressive drugs calcineurin inhibitor (Tacrolimus, TAC and mTOR inhibitor (Sirolimus, SRL on B cell activation, differentiation and proliferation is not well documented. Purified human B cells from healthy volunteers were stimulated through the B Cell Receptor with Anti-IgM + anti-CD40 + IL21 in the absence / presence of TAC or SRL. A variety of parameters of B cell activity including activation, differentiation, cytokine productions and proliferation were monitored by flow cytometry. SRL at clinically relevant concentrations (6 ng/ml profoundly inhibited CD19(+ B cell proliferation compared to controls whereas TAC at similar concentrations had a minimal effect. CD27(+ memory B cells were affected more by SRL than naïve CD27- B cells. SRL effectively blocked B cell differentiation into plasma cells (CD19(+CD138(+ and Blimp1(+/Pax5(low cells even at low dose (2 ng/ml, and totally eliminated them at 6 ng/ml. SRL decreased absolute B cell counts, but the residual responding cells acquired an activated phenotype (CD25(+/CD69(+ and increased the expression of HLA-DR. SRL-treated stimulated B cells on a per cell basis were able to enhance the proliferation of allogeneic CD4(+CD25(- T cells and induce a shift toward the Th1 phenotype. Thus, SRL and TAC have different effects on B lymphocytes. These data may provide insights into the clinical use of these two agents in recipients of solid organ transplants.

  9. Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2

    NARCIS (Netherlands)

    Dihal, A.A.; Woutersen, R.A.; Ommen, B.v.; Rietjens, I.M.C.M.; Stierum, R.H.

    2006-01-01

    The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001

  10. Distinct Effects of miR-210 Reduction on Neurogenesis: Increased Neuronal Survival of Inflammation But Reduced Proliferation Associated with Mitochondrial Enhancement.

    Science.gov (United States)

    Voloboueva, Ludmila A; Sun, Xiaoyun; Xu, Lijun; Ouyang, Yi-Bing; Giffard, Rona G

    2017-03-15

    Neurogenesis is essential to brain development and plays a central role in the response to brain injury. Stroke and head trauma stimulate proliferation of endogenous neural stem cells (NSCs); however, the survival of young neurons is sharply reduced by postinjury inflammation. Cellular mitochondria are critical to successful neurogenesis and are a major target of inflammatory injury. Mitochondrial protection was shown to improve survival of young neurons. This study tested whether reducing cellular microRNA-210 (miR-210) would enhance mitochondrial function and improve survival of young murine neurons under inflammatory conditions. Several studies have demonstrated the potential of miR-210 inhibition to enhance and protect mitochondrial function through upregulation of mitochondrial proteins. Here, miR-210 inhibition significantly increased neuronal survival and protected the activity of mitochondrial enzymes cytochrome c oxidase and aconitase in differentiating NSC cultures exposed to inflammatory mediators. Unexpectedly, we found that reducing miR-210 significantly attenuated NSC proliferation upon induction of differentiation. Further investigation revealed that increased mitochondrial function suppressed the shift to primarily glycolytic metabolism and reduced mitochondrial length characteristic of dividing cells. Activation of AMP-regulated protein kinase-retinoblastoma signaling is important in NSC proliferation and the reduction of this activation observed by miR-210 inhibition is one mechanism contributing to the reduced proliferation. Postinjury neurogenesis occurs as a burst of proliferation that peaks in days, followed by migration and differentiation over weeks. Our studies suggest that mitochondrial protective miR-210 inhibition should be delayed until after the initial burst of proliferation, but could be beneficial during the prolonged differentiation stage.SIGNIFICANCE STATEMENT Increasing the success of endogenous neurogenesis after brain injury

  11. Effect of dentin treatment on proliferation and differentiation of human dental pulp stem cells

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    Minjeong Park

    2015-11-01

    Full Text Available Objectives Sodium hypochlorite (NaOCl is an excellent bactericidal agent, but it is detrimental to stem cell survival, whereas intracanal medicaments such as calcium hydroxide (Ca[OH]2 promote the survival and proliferation of stem cells. This study evaluated the effect of sequential NaOCl and Ca[OH]2 application on the attachment and differentiation of dental pulp stem cells (DPSCs. Materials and Methods DPSCs were obtained from human third molars. All dentin specimens were treated with 5.25% NaOCl for 30 min. DPSCs were seeded on the dentin specimens and processed with additional 1 mg/mL Ca[OH]2, 17% ethylenediaminetetraacetic acid (EDTA treatment, file instrumentation, or a combination of these methods. After 7 day of culture, we examined DPSC morphology using scanning electron microscopy and determined the cell survival rate with 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay. We measured cell adhesion gene expression levels after 4 day of culture and odontogenic differentiation gene expression levels after 4 wk using quantitative real-time polymerase chain reaction. Results DPSCs did not attach to the dentin in the NaOCl-treated group. The gene expression levels of fibronectin-1 and secreted phosphoprotein-1 gene in both the Ca[OH]2- and the EDTA-treated groups were significantly higher than those in the other groups. All Ca[OH]2-treated groups showed higher expression levels of dentin matrix protein-1 than that of the control. The dentin sialophosphoprotein level was significantly higher in the groups treated with both Ca[OH]2 and EDTA. Conclusions The application of Ca[OH]2 and additional treatment such as EDTA or instrumentation promoted the attachment and differentiation of DPSCs after NaOCl treatment.

  12. MELK and EZH2 Cooperate to Regulate Medulloblastoma Cancer Stem-like Cell Proliferation and Differentiation.

    Science.gov (United States)

    Liu, Hailong; Sun, Qianwen; Sun, Youliang; Zhang, Junping; Yuan, Hongyu; Pang, Shuhuan; Qi, Xueling; Wang, Haoran; Zhang, Mingshan; Zhang, Hongwei; Yu, Chunjiang; Gu, Chunyu

    2017-09-01

    Medulloblastoma is the most common malignant brain tumor in children. Although accumulated research has suggested that cancer stem-like cells play a key role in medulloblastoma tumorigenesis, the specific molecular mechanism regarding proliferation remains elusive. Here, we reported more abundant expression of maternal embryonic leucine-zipper kinase (MELK) and enhancer of zeste homolog 2 (EZH2) in medulloblastoma stem-like cells than in neural stem cells and the interaction between the two proteins could mediate the self-renewal of sonic hedgehog subtype medulloblastoma. In human medulloblastoma, extensive nodularity and large-cell/anaplastic subgroups differed according to the staining levels of MELK and EZH2 from the other two subgroups. The proportion of MELK- or EZH2-positive staining status could be considered as a potential indicator for survival. Mechanistically, MELK bound to and phosphorylated EZH2, and its methylation was induced by EZH2 in medulloblastoma, which could regulate the proliferation of cancer stem-like cells. In xenografts, loss of MELK or EZH2 attenuated medulloblastoma stem-like cell-derived tumor growth and promoted differentiation. These findings indicate that MELK-induced phosphorylation and EZH2-mediated methylation in MELK/EZH2 pathway are essential for medulloblastoma stem-like cell-derived tumor proliferation, thereby identifying a potential therapeutic strategy for these patients.Implications: This study demonstrates that the interaction occurring between MELK and EZH2 promotes self-proliferation and stemness, thus representing an attractive therapeutic target and potential candidate for diagnosis of medulloblastoma. Mol Cancer Res; 15(9); 1275-86. ©2017 AACR. ©2017 American Association for Cancer Research.

  13. Differential ability of ectomycorrhizas to survive drying.

    Science.gov (United States)

    di Pietro, Magali; Churin, Jean-Louis; Garbaye, Jean

    2007-09-01

    To test the hypothesis that, depending on the fungal symbiont, ectomycorrhizas are differentially affected by severe drought stress, we developed a simple method to quantify the loss of vitality of excised ectomycorrhizal tips subjected to drying under controlled conditions. The method uses 96-well microtitration plates with one single ectomycorrhizal tip per well, and is based on measuring the loss of volume and the loss of electrolytes before and after the imposed stress. This approach very significantly discriminated the two ectomycorrhizal morphotypes formed with beech (Fagus silvatica) by Lactarius subdulcis and Cenococcum geophilum, which confirmed the ability of the latter fungal species to protect roots against desiccation already suggested by previous works. The new method should contribute to the present effort in deciphering the functional diversity of complex ectomycorrhizal communities.

  14. Integrin Signaling, Cell Survival, and Anoikis: Distinctions, Differences, and Differentiation

    Directory of Open Access Journals (Sweden)

    Pierre H. Vachon

    2011-01-01

    Full Text Available Cell survival and apoptosis implicate an increasing complexity of players and signaling pathways which regulate not only the decision-making process of surviving (or dying, but as well the execution of cell death proper. The same complex nature applies to anoikis, a form of caspase-dependent apoptosis that is largely regulated by integrin-mediated, cell-extracellular matrix interactions. Not surprisingly, the regulation of cell survival, apoptosis, and anoikis furthermore implicates additional mechanistic distinctions according to the specific tissue, cell type, and species. Incidentally, studies in recent years have unearthed yet another layer of complexity in the regulation of these cell processes, namely, the implication of cell differentiation state-specific mechanisms. Further analyses of such differentiation state-distinct mechanisms, either under normal or physiopathological contexts, should increase our understanding of diseases which implicate a deregulation of integrin function, cell survival, and anoikis.

  15. Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Suna, E-mail: wangs3@mail.nih.gov; Zhou, Yifu; Andreyev, Oleg; Hoyt, Robert F.; Singh, Avneesh; Hunt, Timothy; Horvath, Keith A.

    2014-04-15

    Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative {sup RT}PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions. - Highlights: • FABP3 expression pattern was studied in 12 human hypoxic-MSCs. • FABP3 mRNA and proteins are upregulated in the MSCs under hypoxic conditions.

  16. Enhancer of zeste homolog 2 regulates cell differentiation and proliferation in neuroblastoma

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    Amallia N. Setyawati

    2014-12-01

    Full Text Available Background Neuroblastoma (NB is one of the most common extracranial solid tumors occurring in infancy and childhood with highly variable outcomes. Polycomb group (PcG proteins are epigenetic gene silencers. Enhancer of zeste homolog 2 (EZH2 is a member of the polycomb repressor complex 2 (PRC2 group, with the main function to catalyze the polycomb repressor complex by methylating lysine 9 and 27 of histone H3. This study aimed to investigate the biological functionality of EZH2 in NB. Methods This was an experimental study with an analysis of correlation initially of the known prognostic factors of NB patients’ outcomes, by comparing the expression of v-myc avian myelocytomatosis viral oncogene neuroblastoma (MYCN with that of EZH2, on the basis of the patients’ overall and relapse free survival rates. This was followed with a biological functional study to assess the role of EZH2 expression in NB. Results EZH2 knockdown induces neurite extension and differentiation marker growth associated protein 43 (GAP43 in NB cells, although it does not affect cell cycle. By ectopic expression of EZH2, all-trans retinoic acid (ATRA induced neurite extension was suppressed and GAP43 was decreased. Overall, EZH2 seems to have an important role in NB cell differentiation. Although EZH2 did not alter cell proliferation, in the soft agar colony formation assay there was a significant increase in total colony number and number of large colonies. Conclusion Our result clarified the potential role of EZH2 in the regulation of cell differentiation and proliferation, which subsequently may play an important role in the poor prognosis of NB patients.

  17. Arctigenin protects against neuronal hearing loss by promoting neural stem cell survival and differentiation.

    Science.gov (United States)

    Huang, Xinghua; Chen, Mo; Ding, Yan; Wang, Qin

    2017-03-01

    Neuronal hearing loss has become a prevalent health problem. This study focused on the function of arctigenin (ARC) in promoting survival and neuronal differentiation of mouse cochlear neural stem cells (NSCs), and its protection against gentamicin (GMC) induced neuronal hearing loss. Mouse cochlea was used to isolate NSCs, which were subsequently cultured in vitro. The effects of ARC on NSC survival, neurosphere formation, differentiation of NSCs, neurite outgrowth, and neural excitability in neuronal network in vitro were examined. Mechanotransduction ability demonstrated by intact cochlea, auditory brainstem response (ABR), and distortion product optoacoustic emissions (DPOAE) amplitude in mice were measured to evaluate effects of ARC on GMC-induced neuronal hearing loss. ARC increased survival, neurosphere formation, neuron differentiation of NSCs in mouse cochlear in vitro. ARC also promoted the outgrowth of neurites, as well as neural excitability of the NSC-differentiated neuron culture. Additionally, ARC rescued mechanotransduction capacity, restored the threshold shifts of ABR and DPOAE in our GMC ototoxicity murine model. This study supports the potential therapeutic role of ARC in promoting both NSCs proliferation and differentiation in vitro to functional neurons, thus supporting its protective function in the therapeutic treatment of neuropathic hearing loss in vivo. © 2017 Wiley Periodicals, Inc.

  18. Asiatic Acid Prevents the Deleterious Effects of Valproic Acid on Cognition and Hippocampal Cell Proliferation and Survival

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    Jariya Umka Welbat

    2016-05-01

    Full Text Available Valproic acid (VPA is commonly prescribed as an anticonvulsant and mood stabilizer used in the treatment of epilepsy and bipolar disorder. A recent study has demonstrated that VPA reduces histone deacetylase (HDAC activity, an action which is believed to contribute to the effects of VPA on neural stem cell proliferation and differentiation which may explain the cognitive impairments produced in rodents and patients. Asiatic acid is a triterpenoid derived from the medicinal plant Centella asiatica. Our previous study has shown that Asiatic acid improves working spatial memory and increases cell proliferation in the sub granular zone of the hippocampal dentate gyrus. In the present study we investigate the effects of Asiatic acid in preventing the memory and cellular effects of VPA. Male Spraque-Dawley rats were orally administered Asiatic acid (30 mg/kg/day for 28 days, while VPA-treated animals received injections of VPA (300 mg/kg twice a day from Day 15 to Day 28 for 14 days. Spatial memory was determined using the novel object location (NOL test and hippocampal cell proliferation and survival was quantified by immuostaining for Ki-67 and Bromodeoxyuridine (BrdU, respectively. The results showed that VPA-treated animals were unable to discriminate between objects in familiar and novel locations. Moreover, VPA significantly reduced numbers of Ki-67 and BrdU positive cells. These results indicate that VPA treatment caused impairments of spatial working memory, cell proliferation and survival in the subgranular zone (SGZ of the hippocampal dentate gyrus (DG. However, these abnormalities were restored to control levels by co-treatment with Asiatic acid. These data demonstrate that Asiatic acid could prevent the spatial memory and neurogenesis impairments caused by VPA.

  19. Fibroblast growth factor receptor 2 (FGFR2) is required for corneal epithelial cell proliferation and differentiation during embryonic development.

    Science.gov (United States)

    Zhang, Jinglin; Upadhya, Dinesh; Lu, Lin; Reneker, Lixing W

    2015-01-01

    Fibroblast growth factors (FGFs) play important roles in many aspects of embryonic development. During eye development, the lens and corneal epithelium are derived from the same surface ectodermal tissue. FGF receptor (FGFR)-signaling is essential for lens cell differentiation and survival, but its role in corneal development has not been fully investigated. In this study, we examined the corneal defects in Fgfr2 conditional knockout mice in which Cre expression is activated at lens induction stage by Pax6 P0 promoter. The cornea in LeCre, Fgfr2(loxP/loxP) mice (referred as Fgfr2(CKO)) was analyzed to assess changes in cell proliferation, differentiation and survival. We found that Fgfr2(CKO) cornea was much thinner in epithelial and stromal layer when compared to WT cornea. At embryonic day 12.5-13.5 (E12.5-13.5) shortly after the lens vesicle detaches from the overlying surface ectoderm, cell proliferation (judged by labeling indices of Ki-67, BrdU and phospho-histone H3) was significantly reduced in corneal epithelium in Fgfr2(CKO) mice. At later stage, cell differentiation markers for corneal epithelium and underlying stromal mesenchyme, keratin-12 and keratocan respectively, were not expressed in Fgfr2(CKO) cornea. Furthermore, Pax6, a transcription factor essential for eye development, was not present in the Fgfr2(CKO) mutant corneal epithelial at E16.5 but was expressed normally at E12.5, suggesting that FGFR2-signaling is required for maintaining Pax6 expression in this tissue. Interestingly, the role of FGFR2 in corneal epithelial development is independent of ERK1/2-signaling. In contrast to the lens, FGFR2 is not required for cell survival in cornea. This study demonstrates for the first time that FGFR2 plays an essential role in controlling cell proliferation and differentiation, and maintaining Pax6 levels in corneal epithelium via ERK-independent pathways during embryonic development.

  20. ZONAB promotes proliferation and represses differentiation of proximal tubule epithelial cells.

    Science.gov (United States)

    Lima, Wânia R; Parreira, Kleber S; Devuyst, Olivier; Caplanusi, Adrian; N'kuli, Francisca; Marien, Benoit; Van Der Smissen, Patrick; Alves, Pedro M S; Verroust, Pierre; Christensen, Erik I; Terzi, Fabiola; Matter, Karl; Balda, Maria S; Pierreux, Christophe E; Courtoy, Pierre J

    2010-03-01

    Epithelial polarization modulates gene expression. The transcription factor zonula occludens 1 (ZO-1)-associated nucleic acid binding protein (ZONAB) can shuttle between tight junctions and nuclei, promoting cell proliferation and expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), but whether it also represses epithelial differentiation is unknown. Here, during mouse kidney ontogeny and polarization of proximal tubular cells (OK cells), ZONAB and PCNA levels decreased in parallel and inversely correlated with increasing apical differentiation, reflected by expression of megalin/cubilin, maturation of the brush border, and extension of the primary cilium. Conversely, ZONAB reexpression and loss of apical differentiation markers provided a signature for renal clear cell carcinoma. In confluent OK cells, ZONAB overexpression increased proliferation and PCNA while repressing megalin/cubilin expression and impairing differentiation of the brush border and primary cilium. Reporter and chromatin immunoprecipitation assays demonstrated that megalin and cubilin are ZONAB target genes. Sparsely plated OK cells formed small islands composed of distinct populations: Cells on the periphery, which lacked external tight junctions, strongly expressed nuclear ZONAB, proliferated, and failed to differentiate; central cells, surrounded by continuous junctions, lost nuclear ZONAB, stopped proliferating, and engaged in apical differentiation. Taken together, these data suggest that ZONAB is an important component of the mechanisms that sense epithelial density and participates in the complex transcriptional networks that regulate the switch between proliferation and differentiation.

  1. A Dual Program for Translation Regulation in Cellular Proliferation and Differentiation

    DEFF Research Database (Denmark)

    Gingold, Hila; Tehler, Disa; Christoffersen, Nanna R

    2014-01-01

    A dichotomous choice for metazoan cells is between proliferation and differentiation. Measuring tRNA pools in various cell types, we found two distinct subsets, one that is induced in proliferating cells, and repressed otherwise, and another with the opposite signature. Correspondingly, we found ...

  2. Inhibition of proliferation and induction of differentiation of glioma cells with Datura stramonium agglutinin.

    Science.gov (United States)

    Sasaki, T; Yamazaki, K; Yamori, T; Endo, T

    2002-10-07

    We found that a lectin, Datura stramonium agglutinin, induced irreversible differentiation in C6 glioma cells. The differentiated cells had long processes, a low rate of proliferation and a high content of glial fibrillary acidic protein. When the medium was replaced with Datura stramonium agglutinin-free medium after 1 h, cell proliferation continued to be inhibited. Experiments with several other lectins indicated that both recognition of linear N-acetyllactosamine repeats and recognition of multiantennary units of cell-surface glycans were required for the inhibition of C6 proliferation. Proliferation of four human glial tumour cells was also inhibited by Datura stramonium agglutinin. Further, these differentiated human glial tumour cells had long processes and a high content of glial fibrillary acidic protein similar to differentiated C6 glioma cells. Taken together, these observations suggest that Datura stramonium agglutinin may be useful as a new therapy for treating glioma without side effects. Copyright 2002 Cancer Research UK

  3. Effects of Thapsigargin on the Proliferation and Survival of Human Rheumatoid Arthritis Synovial Cells

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    Hui Wang

    2014-01-01

    Full Text Available A series of experiments have been carried out to investigate the effects of different concentrations of thapsigargin (0, 0.001, 0.1, and 1 μM on the proliferation and survival of human rheumatoid arthritis synovial cells (MH7A. The results showed that thapsigargin can block the cell proliferation in human rheumatoid arthritis synovial cells in a time- and dose-dependent manner. Results of Hoechst staining suggested that thapsigargin may induce cell apoptosis in MH7A cells in a time- and dose-dependent manner, and the percentages of cell death reached 44.6% at thapsigargin concentration of 1 μM treated for 4 days compared to the control. The protein and mRNA levels of cyclin D1 decreased gradually with the increasing of thapsigargin concentration and treatment times. Moreover, the protein levels of mTORC1 downstream indicators pS6K and p4EBP-1 were reduced by thapsigargin treatment at different concentrations and times, which should be responsible for the reduced cyclin D1 expressions. Our results revealed that thapsigargin may effectively impair the cell proliferation and survival of MH7A cells. The present findings will help to understand the molecular mechanism of fibroblast-like synoviocytes proliferations and suggest that thapsigargin is of potential for the clinical treatment of rheumatoid arthritis.

  4. microRNA-100 targets SMRT/NCOR2, reduces proliferation, and improves survival in glioblastoma animal models.

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    Bahauddeen M Alrfaei

    Full Text Available Glioblastoma (GBM is the most frequently diagnosed malignant human glioma, and current median patient survival is less than two years despite maximal surgery followed by temozolomide chemoradiation therapies. Novel microRNA-related therapies are now being developed for cancers such as GBM. Differential microRNA expression profiling revealed that miR-100 expression is down-regulated in GBM compared to normal controls. We report that miR-100 expression reduces GBM tumorigenicity. In vitro, four GBM lines (U87, U251, 22T, and 33T demonstrated reduced proliferation 24 hours after transient miR100 overexpression via transfection. miR-100 triggered cell death an average 70% more than scrambled miR controls 24 hours after transient transfection (p < 0.01. miR-100 targeted inhibition of the "silencing mediator of retinoid or thyroid hormone receptor-2" (SMRT/NCOR2 gene was confirmed via reporter assays. Ki67 proliferation index was decreased 40% in tumor xenografts generated from stable miR-100 transfected GBM lines versus controls (p < 0.01. Furthermore, treatment of tumor xenografts with a single pre-mir-100 injection (60 pmol significantly extended survival of mice bearing intracranial GBM xenografts 25% more than scrambled controls (p < 0.01; n=8. These studies establish miR-100's effect on tumor GBM growth, and suggest clinical potential for microRNA-related GBM therapy.

  5. microRNA-100 Targets SMRT/NCOR2, Reduces Proliferation, and Improves Survival in Glioblastoma Animal Models

    Science.gov (United States)

    Alrfaei, Bahauddeen M.; Vemuganti, Raghu; Kuo, John S.

    2013-01-01

    Glioblastoma (GBM) is the most frequently diagnosed malignant human glioma, and current median patient survival is less than two years despite maximal surgery followed by temozolomide chemoradiation therapies. Novel microRNA-related therapies are now being developed for cancers such as GBM. Differential microRNA expression profiling revealed that miR-100 expression is down-regulated in GBM compared to normal controls. We report that miR-100 expression reduces GBM tumorigenicity. In vitro, four GBM lines (U87, U251, 22T, and 33T) demonstrated reduced proliferation 24 hours after transient miR100 overexpression via transfection. miR-100 triggered cell death an average 70% more than scrambled miR controls 24 hours after transient transfection (p SMRT/NCOR2) gene was confirmed via reporter assays. Ki67 proliferation index was decreased 40% in tumor xenografts generated from stable miR-100 transfected GBM lines versus controls (p < 0.01). Furthermore, treatment of tumor xenografts with a single pre-mir-100 injection (60 pmol) significantly extended survival of mice bearing intracranial GBM xenografts 25% more than scrambled controls (p < 0.01; n=8). These studies establish miR-100’s effect on tumor GBM growth, and suggest clinical potential for microRNA-related GBM therapy. PMID:24244722

  6. on the proliferation, differentiation and tube formation of endothelial ...

    African Journals Online (AJOL)

    Yomi

    2011-12-21

    Dec 21, 2011 ... The proliferation, apoptosis, cell cycle, migration and tube formation of EPCs were detected after the cells expose to shear stress ... stem cells that can transfer between stem cell and endothelial cells, with the ..... hematopoietic stem cell antigen: isolation, characterization, and molecular cloning. Blood, 90: p ...

  7. Identification of a proliferation signature related to survival in nodal peripheral T-cell lymphomas.

    Science.gov (United States)

    Cuadros, Marta; Dave, Sandeep S; Jaffe, Elaine S; Honrado, Emiliano; Milne, Roger; Alves, Javier; Rodríguez, Jose; Zajac, Magdalena; Benitez, Javier; Staudt, Louis M; Martinez-Delgado, Beatriz

    2007-08-01

    Nodal peripheral T-cell lymphomas (PTCLs) constitute a heterogeneous group of neoplasms, suggesting the existence of molecular differences contributing to their histologic and clinical variability. Initial expression profiling studies of T-cell lymphomas have been inconclusive in yielding clinically relevant insights. We applied DNA microarrays to gain insight into the molecular signatures associated with prognosis. We analyzed the expression profiles of 35 nodal PTCLs (23 PTCLs unspecified and 12 angioimmunoblastic) using two different microarray platforms, the cDNA microarray developed at the Spanish National Cancer Centre and an oligonucleotide microarray. We identified five clusters of genes, the expression of which varied significantly among the samples. Genes in these clusters seemed to be functionally related to different cellular processes such as proliferation, inflammatory response, and T-cell or B-cell lineages. Regardless of the microarray platform used, overexpression of genes in the proliferation signature was associated significantly with shorter survival of patients. This proliferation signature included genes commonly associated with the cell cycle, such as CCNA, CCNB, TOP2A, and PCNA. Moreover the PTCL proliferation signature showed a statistically significant inverse correlation with clusters of the inflammatory response (P < .0001), as well as with the percentage of CD68(+) cells. Our findings indicate that proliferation could be an important factor in evaluating nodal PTCL outcome and may help to define a more aggressive phenotype.

  8. Mineralocorticoid receptor overexpression facilitates differentiation and promotes survival of embryonic stem cell-derived neurons.

    Science.gov (United States)

    Munier, Mathilde; Law, Frédéric; Meduri, Geri; Le Menuet, Damien; Lombès, Marc

    2012-03-01

    Mineralocorticoid receptor (MR), highly expressed in the hippocampus, binds corticosteroid hormones and coordinately participates, with the glucocorticoid receptor, to the control of stress responses, memorization, and behavior. To investigate the impact of MR in neuronal survival, we generated murine embryonic stem (ES) cells that overexpress human MR (hMR) (P1-hMR) and are induced to differentiate into mature neurons. We showed that recombinant MR expression increased throughout differentiation and is 2-fold higher in P1-hMR ES-derived neurons compared with wild-type controls, whereas glucocorticoid receptor expression was unaffected. Although proliferation and early neuronal differentiation were comparable in P1-hMR and wild-type ES cells, MR overexpression was associated with higher late neuronal marker expression (microtubule-associated protein 2 and β-tubulin III). This was accompanied by a shift towards neuron survival with an increased ratio of anti- vs. proapoptotic molecules and 50% decreased caspase 3 activity. Knocking down MR overexpression by small interfering RNA drastically reversed neuroprotective effects with reduced Bcl(2)/Bax ratio and decreased microtubule-associated protein 2 expression. P1-hMR neurons were protected against oxidative stress-induced apoptosis through reduced caspase 3 activation and drastically increased Bcl(2)/Bax ratio and β-tubulin III expression. We demonstrated the involvement of MR in neuronal differentiation and survival and identify MR as an important neuroprotective mediator opening potential pharmacological strategies.

  9. Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xin [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China); Dai, Hui [Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, Heilongjiang Province (China); Zhuang, Binyu [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China); Chai, Li; Xie, Yanguang [Institute of Dermatology of Heilongjiang Province, Harbin, 150001, Heilongjiang Province (China); Li, Yuzhen, E-mail: liyuzhen@medmail.com.cn [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China)

    2016-04-08

    The effects and the underlying mechanisms of hydrogen sulfide (H{sub 2}S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H{sub 2}S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H{sub 2}S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H{sub 2}S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. - Highlights: • Exogenous H{sub 2}S promotes keratinocyte proliferation and differentiation. • The effects of H{sub 2}S on proliferation and differentiation is modulated by autophagy. • Exogenous H{sub 2}S has no effect on keratinocyte apoptosis.

  10. In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Z.; Chen, J. [Liaocheng People' s Hospital, Department of Hepatobiliary Surgery, Liaocheng, Shandong, China, Department of Hepatobiliary Surgery, Liaocheng People’s Hospital, Liaocheng, Shandong (China); Li, L.; Ran, J.H.; Liu, J. [The First People' s Hospital of Kunming, Kunming, Yunnan, China, The First People’s Hospital of Kunming, Kunming, Yunnan (China); Gao, T.X.; Guo, B.Y. [Dongchangfu Hospital of Women and Child Health Care, Liaocheng, Shandong (China); Li, X.H.; Liu, Z.H.; Liu, G.J.; Gao, Y.C.; Zhang, X.L. [Liaocheng People' s Hospital, Department of Hepatobiliary Surgery, Liaocheng, Shandong, China, Department of Hepatobiliary Surgery, Liaocheng People’s Hospital, Liaocheng, Shandong (China)

    2013-07-30

    Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Student t-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

  11. The Fto Gene Regulates the Proliferation and Differentiation of Pre-Adipocytes in Vitro.

    Science.gov (United States)

    Jiao, Yang; Zhang, Jingying; Lu, Lunjie; Xu, Jiaying; Qin, Liqiang

    2016-02-19

    The highly regulated differentiation and proliferation of pre-adipocytes play a key role in the initiation of obesity. Fat mass and obesity associated (FTO) is a novel gene strongly associated with the risk of obesity. A deficiency of FTO may cause growth retardation in addition to fat mass and adipocyte size reduction in vivo. To investigate the potential role of Fto gene on the proliferation and differentiation of pre-adipocytes, we generated Fto-knockdown and overexpressed 3T3-L1 cells. Using numerous proliferation assays our results suggest that Fto knockdown leads to suppression of proliferation, lower mitochondrial membrane potential, less cellular ATP, and decreased and smaller intracellular lipid droplets compared with controls (p < 0.05). Western blot analysis demonstrated that Fto knockdown can significantly suppress peroxisome proliferator-activated receptor gamma (PPARγ) and glucose transporter type 4 (GLUT4) expression and inhibit Akt phosphorylation. By contrast, overexpression of Fto had the opposing effect on proliferation, mitochondrial membrane potential, ATP generation, in vitro differentiation, Akt phosphorylation, and PPARγ and GLUT4 expression. Moreover, we demonstrated that Wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, could inhibit phospho-Akt in Fto overexpressed 3T3-L1 cells. Taken together, the results suggest that Fto regulates the proliferation and differentiation of 3T3-L1 cells via multiple mechanisms, including PPARγ and PI3K/Akt signaling.

  12. The Fto Gene Regulates the Proliferation and Differentiation of Pre-Adipocytes in Vitro

    Directory of Open Access Journals (Sweden)

    Yang Jiao

    2016-02-01

    Full Text Available The highly regulated differentiation and proliferation of pre-adipocytes play a key role in the initiation of obesity. Fat mass and obesity associated (FTO is a novel gene strongly associated with the risk of obesity. A deficiency of FTO may cause growth retardation in addition to fat mass and adipocyte size reduction in vivo. To investigate the potential role of Fto gene on the proliferation and differentiation of pre-adipocytes, we generated Fto-knockdown and overexpressed 3T3-L1 cells. Using numerous proliferation assays our results suggest that Fto knockdown leads to suppression of proliferation, lower mitochondrial membrane potential, less cellular ATP, and decreased and smaller intracellular lipid droplets compared with controls (p < 0.05. Western blot analysis demonstrated that Fto knockdown can significantly suppress peroxisome proliferator-activated receptor gamma (PPARγ and glucose transporter type 4 (GLUT4 expression and inhibit Akt phosphorylation. By contrast, overexpression of Fto had the opposing effect on proliferation, mitochondrial membrane potential, ATP generation, in vitro differentiation, Akt phosphorylation, and PPARγ and GLUT4 expression. Moreover, we demonstrated that Wortmannin, a phosphoinositide 3-kinase (PI3K inhibitor, could inhibit phospho-Akt in Fto overexpressed 3T3-L1 cells. Taken together, the results suggest that Fto regulates the proliferation and differentiation of 3T3-L1 cells via multiple mechanisms, including PPARγ and PI3K/Akt signaling.

  13. The Effect of Valproic Acid on Mesenchymal Pluripotent Cell Proliferation and Differentiation in Extracellular Matrices

    Directory of Open Access Journals (Sweden)

    Yuji Hatakeyama

    2011-01-01

    Full Text Available Valproic acid (2- n -propylpentanoic acid, VPA is a widely used antiepileptic and anticonvulsant drug. Previous studies have reported that VPA effects osteogenesis in vivo and in vitro, yet it remains unclear whether VPA promotes cell differentiation of osteoblasts derived from mesenchymal cells. The purpose of this study was to clarify the effect of VPA on undifferentiated pluripotent mesenchymal cell proliferation and differentiation into osteoblasts while analyzing the impact of the absence or presence of extracellular matrices (ECMs. Mouse mesenchymal cells were cultured on non-coated plastic, type I collagen-coated, and fibronectin-coated plates in the absence or presence of VPA. A cell proliferation assay was performed in which modified formazan dye content was analyzed and proliferation nuclear antigen (PCNA-positive cells were counted at various concentrations of VPA. A high concentration of VPA did not clearly alter cell morphology, but large numbers of stress fibers were observed in these cells and the cell proliferation ratio was decreased with positive PCNA counts. In the presence of matrices, the cell proliferation ratio decreased at low VPA concentrations compared with the ratio obtained in the absence of these ECMs. On the other hand, VPA promoted osteoblastic differentiation in the presence of type I collagen. These findings indicate that for undifferentiated mesenchymal cells, VPA promotes a decrease in the cell proliferation rate in the presence of ECMs and promotes osteoblastic differentiation, both of which could provide insight into additional mechanisms of osteoblastic cell differentiation caused by VPA.

  14. Cell proliferating and differentiating role of H2O2 in Sclerotium rolfsii and Sclerotinia sclerotiorum.

    Science.gov (United States)

    Papapostolou, Ioannis; Sideri, Marina; Georgiou, Christos D

    2014-01-01

    This study shows that the oxidant and also signal transducing H2O2 exerts a cell proliferating action at certain intracellular concentrations (around 80 nM), by inhibiting the lateral-chained and terminal sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii and S. sclerotiorum, respectively. H2O2 also promotes sclerotial differentiation in these fungi at higher intracellular concentrations (approx. 130 nM). A cell proliferating and differentiation inhibiting effect was exerted also by the inhibitor of catalase activity aminotriazole via increase of intracellular H2O2. Copyright © 2013 Elsevier GmbH. All rights reserved.

  15. Arabidopsis and Tobacco superman regulate hormone signalling and mediate cell proliferation and differentiation.

    Science.gov (United States)

    Nibau, Candida; Di Stilio, Verónica S; Wu, Hen-Ming; Cheung, Alice Y

    2011-01-01

    Arabidopsis thaliana superman (SUP) plays an important role during flower development by maintaining the boundary between stamens and carpels in the inner two whorls. It was proposed that SUP maintains this boundary by regulating cell proliferation in both whorls, as loss-of-function superman mutants produce more stamens at the expense of carpels. However, the cellular mechanism that underlies SUP function remains unknown. Here Arabidopsis or tobacco (Nicotiana tabacum) SUP was overexpressed in tobacco plants to substantiate SUP's role as a regulator of cell proliferation and boundary definition and provide evidence that its biological role may be mediated via hormonal changes. It was found that moderate levels of SUP stimulated cell growth and proliferation, whereas high levels were inhibitory. SUP stimulated auxin- and cytokinin-regulated processes, and cells overexpressing SUP displayed reduced hormone dependency for proliferation and regeneration into plants. SUP also induced proliferation of female traits in the second and third flower whorls and promoted differentiation of petaloid properties in sepals, further supporting a role for SUP as a boundary regulator. Moreover, cytokinin suppressed stamen development and promoted differentiation of carpeloid tissues, suggesting that SUP may regulate male and female development via its effect on cytokinin signalling. Taken together, these observations suggest a model whereby the effect of SUP on cell growth and proliferation involves the modulation of auxin- and cytokinin-regulated processes. Furthermore, differential SUP expression or different sensitivities of different cell types to SUP may determine whether SUP stimulates or suppresses their proliferation.

  16. Modelling T cell proliferation: Dynamics heterogeneity depending on cell differentiation, age, and genetic background.

    Directory of Open Access Journals (Sweden)

    Julien Vibert

    2017-03-01

    Full Text Available Cell proliferation is the common characteristic of all biological systems. The immune system insures the maintenance of body integrity on the basis of a continuous production of diversified T lymphocytes in the thymus. This involves processes of proliferation, differentiation, selection, death and migration of lymphocytes to peripheral tissues, where proliferation also occurs upon antigen recognition. Quantification of cell proliferation dynamics requires specific experimental methods and mathematical modelling. Here, we assess the impact of genetics and aging on the immune system by investigating the dynamics of proliferation of T lymphocytes across their differentiation through thymus and spleen in mice. Our investigation is based on single-cell multicolour flow cytometry analysis revealing the active incorporation of a thymidine analogue during S phase after pulse-chase-pulse experiments in vivo, versus cell DNA content. A generic mathematical model of state transition simulates through Ordinary Differential Equations (ODEs the evolution of single cell behaviour during various durations of labelling. It allows us to fit our data, to deduce proliferation rates and estimate cell cycle durations in sub-populations. Our model is simple and flexible and is validated with other durations of pulse/chase experiments. Our results reveal that T cell proliferation is highly heterogeneous but with a specific "signature" that depends upon genetic origins, is specific to cell differentiation stages in thymus and spleen and is altered with age. In conclusion, our model allows us to infer proliferation rates and cell cycle phase durations from complex experimental 5-ethynyl-2'-deoxyuridine (EdU data, revealing T cell proliferation heterogeneity and specific signatures.

  17. Modelling T cell proliferation: Dynamics heterogeneity depending on cell differentiation, age, and genetic background.

    Science.gov (United States)

    Vibert, Julien; Thomas-Vaslin, Véronique

    2017-03-01

    Cell proliferation is the common characteristic of all biological systems. The immune system insures the maintenance of body integrity on the basis of a continuous production of diversified T lymphocytes in the thymus. This involves processes of proliferation, differentiation, selection, death and migration of lymphocytes to peripheral tissues, where proliferation also occurs upon antigen recognition. Quantification of cell proliferation dynamics requires specific experimental methods and mathematical modelling. Here, we assess the impact of genetics and aging on the immune system by investigating the dynamics of proliferation of T lymphocytes across their differentiation through thymus and spleen in mice. Our investigation is based on single-cell multicolour flow cytometry analysis revealing the active incorporation of a thymidine analogue during S phase after pulse-chase-pulse experiments in vivo, versus cell DNA content. A generic mathematical model of state transition simulates through Ordinary Differential Equations (ODEs) the evolution of single cell behaviour during various durations of labelling. It allows us to fit our data, to deduce proliferation rates and estimate cell cycle durations in sub-populations. Our model is simple and flexible and is validated with other durations of pulse/chase experiments. Our results reveal that T cell proliferation is highly heterogeneous but with a specific "signature" that depends upon genetic origins, is specific to cell differentiation stages in thymus and spleen and is altered with age. In conclusion, our model allows us to infer proliferation rates and cell cycle phase durations from complex experimental 5-ethynyl-2'-deoxyuridine (EdU) data, revealing T cell proliferation heterogeneity and specific signatures.

  18. Effects of IGFBP-2 on proliferation and differentiation in neural stem cell line C17.2

    Directory of Open Access Journals (Sweden)

    Deng Y

    2017-07-01

    Full Text Available Yujia Deng,1 Lei Wang,1,2 Lite Ge,1,3 Da Duan,1 Yi Zhuo,1 Ting Yuan,1 Weiping Yan,1 Peiqi Huang,1 Xiaohua Teng,1 Ming Lu1,3 1Department of Neurosurgery, The Second Affiliated Hospital of Hunan Normal University (163 Hospital of the People’s Liberation Army, Changsha, 2Department of Neurosurgery, Affiliated Haikou Hospital, Xiangya School of Central South University, Haikou, 3Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, People’s Republic of China Objective: Insulin-like growth factor binding protein-2 (IGFBP-2, a member of a highly conserved family of six insulin-like growth factor binding proteins (IGFBPs, can regulate several cellular processes through IGF-dependent or IGF-independent pathway. Recent studies have provided solid evidence for the importance to delineate that olfactory ensheathing cells (OEC-conditioned medium (OCM can not only facilitate the differentiation of neural stem cell line (C17.2 into neurons, but also promote the survival and proliferation. We have previously reported that IGFBP-2 was detected in OCM. This study is designed to investigate the roles of IGFBP-2 for the regulation of C17.2 differentiation and proliferation.Methods and results: IGFBP-2 was identified and upregulated in OCM to compare with astrocytes-conditioned medium by shotgun proteomics and semiquantitative proteomic analysis. In order to investigate whether exogenous IGFBP-2 could stimulate proliferation in C17.2 cells and differentiate it into glia or neuron, we used various concentrations of IGFBP-2 to induce C17.2 cells which were cultured in DMEM/F12. The results showed that exogenous IGFBP-2 can promote proliferation in C17.2 cells, but had little effect on differentiation. Interestingly, we also found that IGFBP-2 could induce C17.2 cells to differentiate into astrocytes, while inhibiting their differentiation into neurons in a dose

  19. [Effects of basic orange II on proliferation and differentiation of limb bud cells in rat embryos].

    Science.gov (United States)

    Zheng, Lixin; Feng, Jiawang; Tian, Shimin

    2015-01-01

    To explore the effects of basic orange II on proliferation and differentiation of limb bud cells. Limb bud cell were separated from SD rat embryo at 13-day gestational age, limb bud cell were exposed to basic orange II at concentrations of 0.0, 12.5, 25.0, 50.0, 100.0, 200, 0 and 400.0 mg/L in the culture medium. The effect of basic orange II on limb bud cell proliferation was detected by Cell Counting Kit-8, the effect of basic orange II on limb bud cell differentiation was assessed by Alcian Blue 8GX. With the increasing of basic orange II concentration, the proliferation and differentiation of embryo limb bud cells were poorer and poorer in vitro, and there was the dose-effect relationship. The pID50 and dLD50 of basic orange II on limb bud cells were 240.6 mg/L and 69.3 mg/L respectively. The inhibition of basic orange II on cell differentiation might exceed that on cell proliferation. Basic orange II could inhibit proliferation and differentiation of embryo limb bud cells. It might be a potential developmental toxic substance in rat embryo.

  20. [Comprehensive regulation effect of traditional Chinese medicine on proliferation and differentiation of neural stem cells].

    Science.gov (United States)

    Wang, Hong-Jin; Li, Jing-Jing; Ke, Hui; Xu, Xiao-Yu

    2017-11-01

    Since the discovery of neural stem cells(NSCs) in embryonic and adult mammalian central nervous systems, new approaches for proliferation and differentiation of NSCs have been put forward. One of the approaches to promote the clinical application of NSCs is to search effective methods to regulate the proliferation and differentiation. This problem is urgently to be solved in the medical field. Previous studies have shown that traditional Chinese medicine could promote the proliferation and differentiation of NSCs by regulating the relevant signaling pathway in vivo and in vitro. Domestic and foreign literatures for regulating the proliferation and differentiation of neural stem cells in recent 10 years and the reports for their target and signaling pathways were analyzed in this paper. Traditional Chinese medicine could regulate the proliferation and differentiation of NSCs through signaling pathways of Notch, PI3K/Akt, Wnt/β-catenin and GFs. However, studies about NSCs and traditional Chinese medicine should be further deepened; the mechanism of multiple targets and the comprehensive regulation function of traditional Chinese medicine should be clarified. Copyright© by the Chinese Pharmaceutical Association.

  1. The effects and mechanisms of clinorotation on proliferation and differentiation in bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Ming [Department of Orthopaedic Surgery, XiJing Hospital, The Fourth Military Medical University, Xi' an 710032 (China); Wang, Yongchun [Department of Aerospace Biodynamics, School of Aerospace Medicine, Fourth Military Medical University, Xi' an 710032 (China); Yang, Min; Liu, Yanwu [Department of Orthopaedic Surgery, XiJing Hospital, The Fourth Military Medical University, Xi' an 710032 (China); Qu, Bo [Chengdu Military General Hospital, Chengdu, 610083 (China); Ye, Zhengxu; Liang, Wei [Department of Orthopaedic Surgery, XiJing Hospital, The Fourth Military Medical University, Xi' an 710032 (China); Sun, Xiqing, E-mail: sunxiqing@fmmu.edu.cn [Department of Aerospace Biodynamics, School of Aerospace Medicine, Fourth Military Medical University, Xi' an 710032 (China); Luo, Zhuojing, E-mail: zjluo@fmmu.edu.cn [Department of Orthopaedic Surgery, XiJing Hospital, The Fourth Military Medical University, Xi' an 710032 (China)

    2015-05-01

    Data from human and rodent studies have demonstrated that microgravity induces observed bone loss in real spaceflight or simulated experiments. The decrease of bone formation and block of maturation may play important roles in bone loss induced by microgravity. The aim of this study was to investigate the changes of proliferation and differentiation in bone marrow mesenchymal stem cells (BMSCs) induced by simulated microgravity and the mechanisms underlying it. We report here that clinorotation, a simulated model of microgravity, decreased proliferation and differentiation in BMSCs after exposure to 48 h simulated microgravity. The inhibited proliferation are related with blocking the cell cycle in G2/M and enhancing the apoptosis. While alterations of the osteoblast differentiation due to the decreased SATB2 expression induced by simulated microgravity in BMSCs. - Highlights: • Simulated microgravity inhibited proliferation and differentiation in BMSCs. • The decreased proliferation due to blocked cell cycle and enhanced the apoptosis. • The inhibited differentiation accounts for alteration of SATB2, Hoxa2 and Cbfa1.

  2. Filamin B regulates chondrocyte proliferation and differentiation through Cdk1 signaling.

    Directory of Open Access Journals (Sweden)

    Jianjun Hu

    Full Text Available Humans who harbor loss of function mutations in the actin-associated filamin B (FLNB gene develop spondylocarpotarsal syndrome (SCT, a disorder characterized by dwarfism (delayed bone formation and premature fusion of the vertebral, carpal and tarsal bones (premature differentiation. To better understand the cellular and molecular mechanisms governing these seemingly divergent processes, we generated and characterized FlnB knockdown ATDC5 cell lines. We found that FlnB knockdown led to reduced proliferation and enhanced differentiation in chondrocytes. Within the shortened growth plate of postnatal FlnB(-/- mice long bone, we observed a similarly progressive decline in the number of rapidly proliferating chondrocytes and premature differentiation characterized by an enlarged prehypertrophic zone, a widened Col2a1(+/Col10a1(+ overlapping region, but relatively reduced hypertrophic zone length. The reduced chondrocyte proliferation and premature differentiation were, in part, attributable to enhanced G2/M phase progression, where fewer FlnB deficient ATDC5 chondrocytes resided in the G2/M phase of the cell cycle. FlnB loss reduced Cdk1 phosphorylation (an inhibitor of G2/M phase progression and Cdk1 inhibition in chondrocytes mimicked the null FlnB, premature differentiation phenotype, through a β1-integrin receptor- Pi3k/Akt (a key regulator of chondrocyte differentiation mediated pathway. In this context, the early prehypertrophic differentiation provides an explanation for the premature differentiation seen in this disorder, whereas the progressive decline in proliferating chondrocytes would ultimately lead to reduced chondrocyte production and shortened bone length. These findings begin to define a role for filamin proteins in directing both cell proliferation and differentiation through indirect regulation of cell cycle associated proteins.

  3. A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Hatkevich, Talia; Ramos, Joseph; Santos-Sanchez, Idalys; Patel, Yashomati M., E-mail: ympatel@uncg.edu

    2014-10-01

    Since over 60% of breast cancers are estrogen receptor positive (ER+), many therapies have targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth factor promoting phosphorylation of the ER. Other proliferation pathways such as the phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting multiple cellular proliferation and survival pathways at the onset of treatment is critical for the development of more effective therapies. The grapefruit flavanone naringenin (Nar) is an inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use serum containing medium in order to retain all the potential inducers of cell proliferation so as not to exclude any targets of Nar. Here we show that a Nar–Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar–Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar–Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar–Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells. - Highlights: • Nar–Tam impairs cell viability more effectively than

  4. miR-381 Regulates Neural Stem Cell Proliferation and Differentiation via Regulating Hes1 Expression.

    Directory of Open Access Journals (Sweden)

    Xiaodong Shi

    Full Text Available Neural stem cells are self-renewing, multipotent and undifferentiated precursors that retain the capacity for differentiation into both glial (astrocytes and oligodendrocytes and neuronal lineages. Neural stem cells offer cell-based therapies for neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and spinal cord injuries. However, their cellular behavior is poorly understood. MicroRNAs (miRNAs are a class of small noncoding RNAs involved in cell development, proliferation and differentiation through regulating gene expression at post-transcriptional level. The role of miR-381 in the development of neural stem cells remains unknown. In this study, we showed that overexpression of miR-381 promoted neural stem cells proliferation. It induced the neural stem cells differentiation to neurons and inhibited their differentiation to astrocytes. Furthermore, we identified HES1 as a direct target of miR-381 in neural stem cells. Moreover, re-expression of HES1 impaired miR-381-induced promotion of neural stem cells proliferation and induce neural stem cells differentiation to neurons. In conclusion, miR-381 played important role in neural stem cells proliferation and differentiation.

  5. Knockdown of tissue nonspecific alkaline phosphatase impairs neural stem cell proliferation and differentiation.

    Science.gov (United States)

    Kermer, Vanessa; Ritter, Mathias; Albuquerque, Boris; Leib, Christoph; Stanke, Matthias; Zimmermann, Herbert

    2010-11-26

    In the adult mammalian brain the subependymal layer of the lateral ventricles houses neural stem cells giving rise to young neurons migrating towards the olfactory bulb. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, differentiation and functional integration are poorly understood. Neural progenitors in situ express the tissue nonspecific form of alkaline phosphatase (TNAP), a cell surface-located nonspecific phosphomonoesterase capable of hydrolyzing extracellular nucleotides. To gain insight into the functional role of TNAP in cultured multipotent neural stem cells we applied a knockdown protocol using RNA interference with shRNA and retroviral infection. We show that TNAP knockdown reduces cell proliferation and differentiation into neurons or oligodendrocytes. This effect is abrogated by addition of alkaline phosphatase to the culture medium. Our results suggest that TNAP is essential for NSC proliferation and differentiation in vitro and possibly also in vivo. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  6. Proliferation and osteoblastic differentiation of hMSCs on cellulose-based hydrogels.

    Science.gov (United States)

    Raucci, Maria Grazia; Alvarez-Perez, Marco Antonio; Demitri, Christian; Sannino, Alessandro; Ambrosio, Luigi

    2012-01-01

    The aim of this project was to study the proliferation and differentiation of human Mesenchymal Stem Cells (hMSCs) onto a cellulose-based hydrogel for bone tissue engineering. Modified-cellulose hydrogel was prepared via double esterification crosslinking using citric acid. The response of human Mesenchymal Stem Cells (hMSCs) in terms of cell proliferation and differentiation into osteoblastic phenotype was evaluated by using Alamar blue assay and Alkaline phosphatase activity. The results showed that CMCNa and CMCNa_CA have no negative effect on hMSC, adhesion and proliferation. Moreover, the increase of the ALP expression for CMCNa_CA confirms the ability of the hydrogels to support the osteoblastic differentiation. The cellulose-based hydrogels have a potential application as filler in bone tissue regeneration.

  7. Proliferation and osteogenic differentiation of mesenchymal stromal cells in a novel porous hydroxyapatite scaffold.

    Science.gov (United States)

    Krishnamurithy, Genasan; Murali, Malliga Raman; Hamdi, Mohd; Abbas, Azlina Amir; Raghavendran, Hanumantharao Balaji; Kamarul, Tunku

    2015-01-01

    To compare the effect of bovine bone derived porous hydroxyapatite (BDHA) scaffold on proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hMSCs) compared with commercial hydroxyapatite (CHA) scaffold. The porosity and pore size were analyzed using micro-CT. The biocompatibility was demonstrated by alamar blue assay, and cell attachment through SEM and Hoechst staining. The osteogenic differentiation was demonstrated using biochemical assay and osteogenic gene expression. BDHA and CHA scaffolds showed porosity of 76.6 ± 0.6 and 64.3 ± 0.3% and pore size diameter of 0.04-0.25 and 0.1-2.6 mm, respectively. hMSCs proliferation, ALP activity, osteocalcin secretion and osteogenic gene expression are comparable in both the scaffolds. These results demonstrated that BDHA is biocompatible, supports cell adhesion and promotes proliferation and osteogenic differentiation.

  8. MANF Is Indispensable for the Proliferation and Survival of Pancreatic β Cells

    Directory of Open Access Journals (Sweden)

    Maria Lindahl

    2014-04-01

    Full Text Available All forms of diabetes mellitus (DM are characterized by the loss of functional pancreatic β cell mass, leading to insufficient insulin secretion. Thus, identification of novel approaches to protect and restore β cells is essential for the development of DM therapies. Mesencephalic astrocyte-derived neurotrophic factor (MANF is an endoplasmic reticulum (ER-stress-inducible protein, but its physiological role in mammals has remained obscure. We generated MANF-deficient mice that strikingly develop severe diabetes due to progressive postnatal reduction of β cell mass, caused by decreased proliferation and increased apoptosis. Additionally, we show that lack of MANF in vivo in mouse leads to chronic unfolded protein response (UPR activation in pancreatic islets. Importantly, MANF protein enhanced β cell proliferation in vitro and overexpression of MANF in the pancreas of diabetic mice enhanced β cell regeneration. We demonstrate that MANF specifically promotes β cell proliferation and survival, thereby constituting a therapeutic candidate for β cell protection and regeneration.

  9. Activation of proliferation and differentiation of dental follicle stem cells (DFSCs) by heat stress.

    Science.gov (United States)

    Rezai Rad, M; Wise, G E; Brooks, H; Flanagan, M B; Yao, S

    2013-02-01

    Adult stem cells (ASCs) remain in a slowly cycling/quiescent state under normal physiological conditions, but they can be awakened from this by certain factors, such as injury signals. Previously, our group has shown that dental follicle stem cells (DFSCs) appear to proliferate more rapidly than their non-stem cell counterparts at elevated temperatures. The study described here has aimed to (i) elucidate optimal temperature in which to culture DFSCs, (ii) determine whether elevated temperatures could enhance differentiation capability of DFSCs and (iii) characterize stem cell and osteogenic marker expression of DFSCs at elevated temperatures. DFSCs obtained from rat first molars were cultured at 37 (control), 38, 39, 40 and 41 ºC. Cell proliferation was evaluated by Alamar blue reduction assay and mean numbers of viable dissociated cells. Osteogenic differentiation was evaluated after 7 or 14 days osteogenic induction. Expression of selected marker genes was also assessed during proliferation and differentiation of the cells. Increased cell proliferation was seen at heat-stress temperatures of 38º, 39º and 40 ºC. DFSCs revealed maximal osteogenesis when cultured at 39 and 40 ºC. Moreover, some stem cell and osteogensis-associated markers had elevated expression in heat-stress conditions. Under determined heat-stress conditions, DFSCs increased their proliferation, osteogenic differentiation and expression of some marker genes. Thus, it is likely that elevated temperature could serve as a factor to activate adult stem cells. © 2013 Blackwell Publishing Ltd.

  10. Roles of TRPM8 Ion Channels in Cancer: Proliferation, Survival, and Invasion

    Directory of Open Access Journals (Sweden)

    Nelson S. Yee

    2015-10-01

    Full Text Available The goal of this article is to provide a critical review of the transient receptor potential melastatin-subfamily member 8 (TRPM8 in cancers, with an emphasis on its roles in cellular proliferation, survival, and invasion. The TRPM8 ion channels regulate Ca²⁺ homeostasis and function as a cellular sensor and transducer of cold temperature. Accumulating evidence has demonstrated that TRPM8 is aberrantly expressed in a variety of malignant solid tumors. Clinicopathological analysis has shown that over-expression of TRPM8 correlates with tumor progression. Experimental data have revealed important roles of TRPM8 channels in cancer cells proliferation, survival, and invasion, which appear to be dependent on the cancer type. Recent reports have begun to reveal the signaling mechanisms that mediate the biological roles of TRPM8 in tumor growth and metastasis. Determining the mechanistic roles of TRPM8 in cancer is expected to elucidate the impact of thermal and chemical stimuli on the formation and progression of neoplasms. Translational research and clinical investigation of TRPM8 in malignant diseases will help exploit these ion channels as molecular biomarkers and therapeutic targets for developing precision cancer medicine.

  11. Collagen-lactoferrin fibrillar coatings enhance osteoblast proliferation and differentiation.

    Science.gov (United States)

    Vandrovcova, Marta; Douglas, Timothy E L; Heinemann, Sascha; Scharnweber, Dieter; Dubruel, Peter; Bacakova, Lucie

    2015-02-01

    Lactoferrin is a milk-derived glycoprotein with anabolic effects on the bone tissue. In this study, artificial extracellular matrices (aECM) consisting of collagen type I fibrils formed in the presence of lactoferrin at two different concentrations (0.5 and 1 mg mL(-1) ) were prepared on the surface of poly(lactic-co-glycolic acid) (PLGA) foils. The aim of the study was to investigate the effects of aECM on the adhesion, growth and osteogenic differentiation of human osteoblast-like Saos-2 cells. On days 1 and 3 after seeding, higher numbers of cells were found on samples with collagen and collagen-lactoferrin coatings (particularly on those formed at the higher concentration of lacroferrin) than on control microscopic glass coverslips. Cells on coatings formed in the presence of lactoferrin had more numerous and better developed vinculin-containing focal adhesion plaques. On day 7, cells on coatings with and without lactoferrin produced significantly higher levels of osteocalcin than cells on control polystyrene cell culture dishes, the highest average values being found on samples with the lower concentration of lactoferrin. Expression of collagen I and alkaline phosphatase was on a similar level in cells on all tested samples and control polystyrene. Thus, lactoferrin promotes adhesion, growth and osteogenic differentiation of Saos-2 cells and is promising as a bone implant coating component. © 2014 Wiley Periodicals, Inc.

  12. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available During retina development, retinal progenitor cell (RPC proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC self-renewal, as well as betacellulin (BTC, an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

  13. FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

    Energy Technology Data Exchange (ETDEWEB)

    Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki, E-mail: yasukiishizaki@gunma-u.ac.jp

    2015-08-07

    The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytes and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.

  14. Regulation of the Balance Between Proliferation and Differentiation in Germ Line Stem Cells.

    Science.gov (United States)

    Singh, Ramya; Hansen, Dave

    In many animals, reproductive fitness is dependent upon the production of large numbers of gametes over an extended period of time. This level of gamete production is possible due to the continued presence of germ line stem cells. These cells can produce two types of daughter cells, self-renewing daughter cells that will maintain the stem cell population and differentiating daughter cells that will become gametes. A balance must be maintained between the proliferating self-renewing cells and those that differentiate for long-term gamete production to be maintained. Too little proliferation can result in depletion of the stem cell population, while too little differentiation can lead to a lack of gamete formation and possible tumor formation. In this chapter, we discuss our current understanding of how the balance between proliferation and differentiation is achieved in three well-studied germ line model systems: the Drosophila female, the mouse male, and the C. elegans hermaphrodite. While these three systems have significant differences in how this balance is regulated, including differences in stem cell population size, signaling pathways utilized, and the use of symmetric and/or asymmetric cell divisions, there are also similarities found between them. These similarities include the reliance on a predominant signaling pathway to promote proliferation, negative feedback loops to rapidly shutoff proliferation-promoting cues, close association of the germ line stem cells with a somatic niche, cytoplasmic connections between cells, projections emanating from the niche cell, and multiple mechanisms to limit the spatial influence of the niche. A comparison between different systems may help to identify elements that are essential for a proper balance between proliferation and differentiation to be achieved and elements that may be achieved through various mechanisms.

  15. Controlling Redox Status for Stem Cell Survival, Expansion, and Differentiation

    Directory of Open Access Journals (Sweden)

    Sébastien Sart

    2015-01-01

    Full Text Available Reactive oxygen species (ROS have long been considered as pathological agents inducing apoptosis under adverse culture conditions. However, recent findings have challenged this dogma and physiological levels of ROS are now considered as secondary messengers, mediating numerous cellular functions in stem cells. Stem cells represent important tools for tissue engineering, drug screening, and disease modeling. However, the safe use of stem cells for clinical applications still requires culture improvements to obtain functional cells. With the examples of mesenchymal stem cells (MSCs and pluripotent stem cells (PSCs, this review investigates the roles of ROS in the maintenance of self-renewal, proliferation, and differentiation of stem cells. In addition, this work highlights that the tight control of stem cell microenvironment, including cell organization, and metabolic and mechanical environments, may be an effective approach to regulate endogenous ROS generation. Taken together, this paper indicates the need for better quantification of ROS towards the accurate control of stem cell fate.

  16. Stem cell proliferation and differentiation a multitype branching process model

    CERN Document Server

    Macken, Catherine A

    1988-01-01

    The body contains many cellular systems that require the continuous production of new, fully functional, differentiated cells to replace cells lacking or having limited self-renewal capabilities that die or are damaged during the lifetime of an individual. Such systems include the epidermis, the epithelial lining of the gut, and the blood. For example, erythrocytes (red blood cells) lack nuclei and thus are incapable of self-replication. They have a life span in the circulation of about 120 days. Mature granulocytes, which also lack proliferative capacity, have a much shorter life span - typically 12 hours, though this may be reduced to only two or three hours in times of serious tissue infection. Perhaps a more familiar example is the outermost layer of the skin. This layer is composed of fully mature, dead epidermal cells that must be replaced by the descendants of stem cells lodged in lower layers of the epidermis (cf. Alberts et al. , 1983). In total, to supply the normal steady-state demands of cells, an...

  17. Hepatocyte growth factor modulates in vitro survival and proliferation of germ cells during postnatal testis development.

    Science.gov (United States)

    Catizone, A; Ricci, G; Del Bravo, J; Galdieri, M

    2006-04-01

    The hepatocyte growth factor (HGF) is a pleiotropic cytokine that influences mitogenesis, motility and differentiation of many different cell types by its tyrosine kinase receptor c-Met. We previously demonstrated that the c-Met/HGF system is present and functionally active during postnatal testis development. We found also that spermatozoa express c-Met and that HGF has a positive effect on the maintenance of sperm motility. In the present paper, we extend our study on the germ cells at different stages of differentiation during the postnatal development of the testis. We demonstrate that c-met is present in rat spermatogonia, pachytene spermatocytes and round spermatids and that HGF significantly increases spermatogonial proliferation in 8- to 10-day-old pre-pubertal rats. At this age HGF does not affect Sertoli cells and peritubular myoid cells proliferation. In addition, we studied the effect of the factor on germ cell apoptosis and we show that HGF prevents the germ cell apoptotic process. We also studied the effect of HGF on 18- to 20-day-old and 28- to 30-day-old rat testes. At these ages also the factor significantly increases germ cell duplication and decreases the number of apoptotic cells. However, the effect on programmed cell death is higher in the 8- to 10-day-old rats and declines in the older animals. In conclusion, we report that rat germ cells (spermatogonia, pachytene spermatocytes and round spermatids) express c-met and that HGF modulates germ cell proliferating activity and apoptosis in vitro. These data indicate that the c-Met/HGF system is involved in male germ cell homeostasis and, consequently, has a role in male fertility.

  18. Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells.

    Science.gov (United States)

    Sun, Bo Kyung; Kim, Ji Hye; Choi, Joon-Seok; Hwang, Sung-Joo; Sung, Jong-Hyuk

    2015-07-22

    Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs) or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA), significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation.

  19. Fluoxetine Decreases the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Bo Kyung Sun

    2015-07-01

    Full Text Available Fluoxetine was originally developed as an antidepressant, but it has also been used to treat obesity. Although the anti-appetite effect of fluoxetine is well-documented, its potential effects on human adipose-derived stem cells (ASCs or mature adipocytes have not been investigated. Therefore, we investigated the mechanisms underlying the inhibitory effects of fluoxetine on the proliferation of ASCs. We also investigated its inhibitory effect on adipogenic differentiation. Fluoxetine significantly decreased ASC proliferation, and signal transduction PCR array analysis showed that it increased expression of autophagy-related genes. In addition, fluoxetine up-regulated SQSTM1 and LC3B protein expression as detected by western blotting and immunofluorescence. The autophagy inhibitor, 3-methyladenine (3-MA, significantly attenuated fluoxetine-mediated effects on ASC proliferation and SQSTM1/LC3B expression. In addition, 3-MA decreased the mRNA expression of two autophagy-related genes, beclin-1 and Atg7, in ASCs. Fluoxetine also significantly inhibited lipid accumulation and down-regulated the levels of PPAR-γ and C/EBP-α in ASCs. Collectively, these results indicate that fluoxetine decreases ASC proliferation and adipogenic differentiation. This is the first in vitro evidence that fluoxetine can reduce fat accumulation by inhibiting ASC proliferation and differentiation.

  20. Morphology, proliferation, and osteogenic differentiation of mesenchymal stem cells cultured on titanium, tantalum, and chromium surfaces.

    Science.gov (United States)

    Stiehler, Maik; Lind, Martin; Mygind, Tina; Baatrup, Anette; Dolatshahi-Pirouz, Alireza; Li, Haisheng; Foss, Morten; Besenbacher, Flemming; Kassem, Moustapha; Bünger, Cody

    2008-08-01

    Metallic implants are widely used in orthopedic surgery and dentistry. Durable osseous fixation of an implant requires that osteoprogenitor cells attach and adhere to the implant, proliferate, differentiate into osteoblasts, and produce mineralized matrix. In the present study, we investigated the interactions between human mesenchymal stem cells (MSCs) and smooth surfaces of titanium (Ti), tantalum (Ta), and chromium (Cr). Mean cellular area was quantified using fluorescence microscopy (4 h). Cellular proliferation was assessed by (3)H-thymidine incorporation and methylene blue cell counting assays (4 days). Osteogenic differentiation response was quantified by cell-specific alkaline phosphatase activity (ALP) assay (4 days), expression analysis of bone-related genes (4 days), and mineralization assay (21 days). Undifferentiated and osteogenically stimulated MSCs cultured on the different surfaces showed the same tendencies for proliferation and differentiation. MSCs exposed to Ti surfaces demonstrated enhanced proliferation compared with Ta and Cr surfaces. Cultivation of MSCs on Ta surfaces resulted in significantly increased mean cellular area and cell-specific ALP activity compared with the other surfaces tested. Cells cultured on Cr demonstrated reduced spreading and proliferation. In conclusion, Ta metal, as an alternative for Ti, can be considered as a promising biocompatible material, whereas further studies are needed to fully understand the role of Cr and its alloys in bone implants.

  1. Retinoic acid-loaded polymeric nanoparticles enhance vascular regulation of neural stem cell survival and differentiation after ischaemia

    Science.gov (United States)

    Ferreira, R.; Fonseca, M. C.; Santos, T.; Sargento-Freitas, J.; Tjeng, R.; Paiva, F.; Castelo-Branco, M.; Ferreira, L. S.; Bernardino, L.

    2016-04-01

    Stroke is one of the leading causes of death and disability worldwide. However, current therapies only reach a small percentage of patients and may cause serious side effects. We propose the therapeutic use of retinoic acid-loaded nanoparticles (RA-NP) to safely and efficiently repair the ischaemic brain by creating a favourable pro-angiogenic environment that enhances neurogenesis and neuronal restitution. Our data showed that RA-NP enhanced endothelial cell proliferation and tubule network formation and protected against ischaemia-induced death. To evaluate the effect of RA-NP on vascular regulation of neural stem cell (NSC) survival and differentiation, endothelial cell-conditioned media (EC-CM) were collected. EC-CM from healthy RA-NP-treated cells reduced NSC death and promoted proliferation while EC-CM from ischaemic RA-NP-treated cells decreased cell death, increased proliferation and neuronal differentiation. In parallel, human endothelial progenitor cells (hEPC), which are part of the endogenous repair response to vascular injury, were collected from ischaemic stroke patients. hEPC treated with RA-NP had significantly higher proliferation, which further highlights the therapeutic potential of this formulation. To conclude, RA-NP protected endothelial cells from ischaemic death and stimulated the release of pro-survival, proliferation-stimulating factors and differentiation cues for NSC. RA-NP were shown to be up to 83-fold more efficient than free RA and to enhance hEPC proliferation. These data serve as a stepping stone to use RA-NP as vasculotrophic and neurogenic agents for vascular disorders and neurodegenerative diseases with compromised vasculature.

  2. Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Li, Zubing, E-mail: lizubing0827@163.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China)

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Different PTH administration exerts different effects on condylar chondrocyte. Black-Right-Pointing-Pointer Intermittent PTH administration suppresses condylar chondrocyte proliferation. Black-Right-Pointing-Pointer Continuous PTH administration maintains condylar chondrocyte proliferating. Black-Right-Pointing-Pointer Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular

  3. Lipocalin-2 inhibits osteoclast formation by suppressing the proliferation and differentiation of osteoclast lineage cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun-Ju, E-mail: biohjk@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Hye-Jin [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Kyung-Ae [Department of Orthopedic Surgery, Skeletal Diseases Genome Research Center, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Gwon, Mi-Ri; Jin Seong, Sook [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Suk, Kyoungho [Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, Shin-Yoon [Department of Orthopedic Surgery, Skeletal Diseases Genome Research Center, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Yoon, Young-Ran, E-mail: yry@knu.ac.kr [Department of Molecular Medicine, Cell and Matrix Research Institute, Clinical Trial Center, BK21 Plus KNU Biomedical Convergence Program, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of)

    2015-06-10

    Lipocalin-2 (LCN2) is a member of the lipocalin superfamily and plays a critical role in the regulation of various physiological processes, such as inflammation and obesity. In this study, we report that LCN2 negatively modulates the proliferation and differentiation of osteoclast precursors, resulting in impaired osteoclast formation. The overexpression of LCN2 in bone marrow-derived macrophages or the addition of recombinant LCN2 protein inhibits the formation of multinuclear osteoclasts. LCN2 suppresses macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursor cells without affecting their apoptotic cell death. Interestingly, LCN2 decreases the expression of the M-CSF receptor, c-Fms, and subsequently blocks its downstream signaling cascades. In addition, LCN2 inhibits RANKL-induced osteoclast differentiation and attenuates the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important modulators in osteoclastogenesis. Mechanistically, LCN2 inhibits NF-κB signaling pathways, as demonstrated by the suppression of IκBα phosphorylation, nuclear translocation of p65, and NF-κB transcriptional activity. Thus, LCN2 is an anti-osteoclastogenic molecule that exerts its effects by retarding the proliferation and differentiation of osteoclast lineage cells. - Highlights: • LCN2 expression is regulated during osteoclast development. • LCN2 suppresses M-CSF-mediated osteoclast precursor proliferation. • LCN2 inhibits RANKL-induced osteoclast differentiation.

  4. Inhibition of TRPM7 channels prevents proliferation and differentiation of human lung fibroblasts.

    Science.gov (United States)

    Yu, Mingzhe; Huang, Cheng; Huang, Yan; Wu, Xiaoqin; Li, Xiaohui; Li, Jun

    2013-11-01

    Transient receptor potential melastatin 7 (TRPM7) is involved in both normal physiological processes and pathology of various diseases. The purpose of this study was to explore the function and underlying mechanisms of TRPM7 channels in human lung fibroblast (MRC5) proliferation and differentiation induced by transforming growth factor β1 (TGF-β1) in vitro. We determined the expression of TRPM7 in MRC5 cells in response to TGF-β1 treatment in vitro. Chemical inhibitors (Gd(3+) and 2-APB) and specific siRNA for TRPM7 were used to study the role of TRPM7 in MRC5 cell proliferation and differentiation. The phosphorylation of Akt was determined by Western blotting. The expression of TRPM7 was significantly potentiated in response to TGF-β1. Co-incubation of MRC5 cells with Gd(3+), 2-APB or TRPM7-siRNA decreased cell proliferation and differentiation. Furthermore, we found that suppression of TRPM7 channels also reduced the p-Akt in MRC5 cells induced by TGF-β1. We conclude that suppression of TRPM7 channels may decrease fibroblast proliferation and differentiation stimulated by TGF-β1 in vitro and this is associated with Akt phosphorylation.

  5. Epidermis-type lipoxygenase 3 regulates adipocyte differentiation and peroxisome proliferator-activated receptor gamma activity

    DEFF Research Database (Denmark)

    Hallenborg, Philip; Jørgensen, Claus; Petersen, Rasmus K

    2010-01-01

    The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR gamma, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differenti...

  6. Activation of the proliferation and differentiation of dental follicle stem cells (DFSCs) by heat-stress

    Science.gov (United States)

    Rad, Maryam Rezai; Wise, Gary E.; Brooks, Hunter; Flanagan, Michael B.; Yao, Shaomian

    2012-01-01

    Objectives Adult stem cells (ASCs) are maintained in a slow cycling and quiescent state under normal physiological conditions. This state could be awakened by certain factors, such as injury signals. Previously, we have shown that dental follicle stem cells (DFSCs) appear to grow more rapidly than their non-stem cell counterparts at elevated temperatures. This study aimed to (a) elucidate the optimal temperature to grow DFSCs, (b) determine if the elevated temperatures could enhance the differentiation capability of DFSCs, and (c) characterize the stem cell and osteogenic markers expression in DFSCs under elevated temperatures. Materials and methods DFSCs obtained from rat first molar were cultured at 37 (control), 38, 39, 40, and 41°C. Cell proliferation was evaluated by Alamar blue reduction assay and mean number of viable dissociated cells. Osteogenic differentiation was evaluated after 7 or 14 days of osteogenic induction. Expression of selected marker genes was also assessed during proliferation and differentiation of the DFSCs. Results Increased cell proliferation was seen at heat-stress temperatures of 38, 39 and 40 °C, DFSCs showed maximal osteogenesis when cultured at 39 and 40°C. Moreover, some stem cell and osteogenic associated markers increased their expression under heat-stress conditions. Conclusions Under proper heat-stress conditions, DFSCs increased proliferation, osteogenic differentiation, and expression of some marker genes. Thus, it is likely that elevated temperature could serve as a factor to activate ASCs. PMID:23278983

  7. Proliferation and differentiation of Trypanosoma cruzi inside its vector have a new trigger: redox status.

    Directory of Open Access Journals (Sweden)

    Natália P Nogueira

    Full Text Available Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS. We tested the influence of the pro-oxidant molecules H2O2 and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. β-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle.

  8. The Clinicopathologic Heterogeneity of Grade 3 Gastroenteropancreatic Neuroendocrine Neoplasms: Morphological Differentiation and Proliferation Identify Different Prognostic Categories.

    Science.gov (United States)

    Milione, Massimo; Maisonneuve, Patrick; Spada, Francesca; Pellegrinelli, Alessio; Spaggiari, Paola; Albarello, Luca; Pisa, Eleonora; Barberis, Massimo; Vanoli, Alessandro; Buzzoni, Roberto; Pusceddu, Sara; Concas, Laura; Sessa, Fausto; Solcia, Enrico; Capella, Carlo; Fazio, Nicola; La Rosa, Stefano

    2017-01-01

    Gastroenteropancreatic (GEP) neuroendocrine carcinomas (NECs) are defined as neuroendocrine neoplasms (NENs) with a Ki-67 index >20% according to the 2010 WHO classification. Some reports suggest that this category is heterogeneous. We retrospectively studied a series of 136 patients affected by grade 3 GEP-NECs with the aim to clarify the prognostic role of tumor morphological differentiation, proliferation, defect in mismatch repair proteins (MMRd), CD117 expression, and site of origin. The primary endpoint was the correlation between these parameters and the overall survival (OS). Univariate and multivariable Cox proportional hazards regression analyses were used to assess the prognostic significance of various clinical and histopathologic features. With a median follow-up of 81 months, the median OS was 12.9 months. At multivariate analysis, morphological differentiation, Ki-67 index, MMRd, stage, and CD117 expression were independent prognostic markers in NECs. Three different prognostic categories of NECs were identified according to the degree of morphologic differentiation (well vs. poorly differentiated) and Ki-67 index (neoplasms with a Ki-67 index 20-55% (named type A), 24.5 months in poorly differentiated neoplasms with a Ki-67 index 20-55% (type B), and 5.3 months (p neoplasms with a Ki-67 index ≥55% (type C). The present study suggests that GEP-NECs represent a heterogeneous group of neoplasms which can be better classified in different prognostic categories using both tumor morphology and Ki-67 index. © 2016 S. Karger AG, Basel.

  9. Dehydroepiandrosterone exerts antiglucocorticoid action on human preadipocyte proliferation, differentiation, and glucose uptake

    Science.gov (United States)

    McNelis, Joanne C.; Manolopoulos, Konstantinos N.; Gathercole, Laura L.; Bujalska, Iwona J.; Stewart, Paul M.; Tomlinson, Jeremy W.

    2013-01-01

    Glucocorticoids increase adipocyte proliferation and differentiation, a process underpinned by the local reactivation of inactive cortisone to active cortisol within adipocytes catalyzed by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). The adrenal sex steroid precursor dehydroepiandrosterone (DHEA) has been shown to inhibit 11β-HSD1 in murine adipocytes; however, rodent adrenals do not produce DHEA physiologically. Here, we aimed to determine the effects and underlying mechanisms of the potential antiglucocorticoid action of DHEA and its sulfate ester DHEAS in human preadipocytes. Utilizing a human subcutaneous preadipocyte cell line, Chub-S7, we examined the metabolism and effects of DHEA in human adipocytes, including adipocyte proliferation, differentiation, 11β-HSD1 expression, and activity and glucose uptake. DHEA, but not DHEAS, significantly inhibited preadipocyte proliferation via cell cycle arrest in the G1 phase independent of sex steroid and glucocorticoid receptor activation. 11β-HSD1 oxoreductase activity in differentiated adipocytes was inhibited by DHEA. DHEA coincubated with cortisone significantly inhibited preadipocyte differentiation, which was assessed by the expression of markers of early (LPL) and terminal (G3PDH) adipocyte differentiation. Coincubation with cortisol, negating the requirement for 11β-HSD1 oxoreductase activity, diminished the inhibitory effect of DHEA. Further consistent with glucocorticoid-opposing effects of DHEA, insulin-independent glucose uptake was significantly enhanced by DHEA treatment. DHEA increases basal glucose uptake and inhibits human preadipocyte proliferation and differentiation, thereby exerting an antiglucocorticoid action. DHEA inhibition of the amplification of glucocorticoid action mediated by 11β-HSD1 contributes to the inhibitory effect of DHEA on human preadipocyte differentiation. PMID:24022868

  10. The effect of interferon-{beta} on mouse neural progenitor cell survival and differentiation

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    Hirsch, Marek [Neurology Department, University of Vermont College of Medicine, Burlington, VT (United States); Knight, Julia [Neuroscience Department, University of Vermont College of Medicine, Burlington, VT (United States); Tobita, Mari; Soltys, John; Panitch, Hillel [Neurology Department, University of Vermont College of Medicine, Burlington, VT (United States); Mao-Draayer, Yang, E-mail: yang.mao-draayer@vtmednet.org [Neurology Department, University of Vermont College of Medicine, Burlington, VT (United States)

    2009-10-16

    Interferon-{beta} (IFN-{beta}) is a mainstay therapy for relapse-remitting multiple sclerosis (MS). However, the direct effects of IFN-{beta} on the central nervous system (CNS) are not well understood. To determine whether IFN-{beta} has direct neuroprotective effects on CNS cells, we treated adult mouse neural progenitor cells (NPCs) in vitro with IFN-{beta} and examined the effects on proliferation, apoptosis, and differentiation. We found that mouse NPCs express high levels of IFN{alpha}/{beta} receptor (IFNAR). In response to IFN-{beta} treatment, no effect was observed on differentiation or proliferation. However, IFN-{beta} treated mouse NPCs demonstrated decreased apoptosis upon growth factor withdrawal. Pathway-specific polymerase chain reaction (PCR) arrays demonstrated that IFN-{beta} treatment upregulated the STAT 1 and 2 signaling pathway, as well as GFRA2, NOD1, Caspases 1 and 12, and TNFSF10. These results suggest that IFN-{beta} can directly affect NPC survival, possibly playing a neuroprotective role in the CNS by modulating neurotrophic factors.

  11. Melanoma-initiating cells exploit M2 macrophage TGFβ and arginase pathway for survival and proliferation

    Science.gov (United States)

    Tham, Muly; Tan, Kar Wai; Keeble, Jo; Wang, Xiaojie; Hubert, Sandra; Barron, Luke; Tan, Nguan Soon; Kato, Masashi; Prevost-Blondel, Armelle; Angeli, Veronique; Abastado, Jean-Pierre

    2014-01-01

    M2 macrophages promote tumor growth and metastasis, but their interactions with specific tumor cell populations are poorly characterized. Using a mouse model of spontaneous melanoma, we showed that CD34− but not CD34+ tumor-initiating cells (TICs) depend on M2 macrophages for survival and proliferation. Tumor-associated macrophages (TAMs) and macrophage-conditioned media protected CD34− TICs from chemotherapy in vitro. In vivo, while inhibition of CD115 suppressed the macrophage-dependent CD34− TIC population, chemotherapy accelerated its development. The ability of TICs to respond to TAMs was acquired during melanoma progression and immediately preceded a surge in metastatic outgrowth. TAM-derived transforming growth factor-β (TGFβ) and polyamines produced via the Arginase pathway were critical for stimulation of TICs and synergized to promote their growth. PMID:25294815

  12. Lipotoxicity in the Pancreatic Beta Cell: Not Just Survival and Function, but Proliferation as Well?

    Science.gov (United States)

    Sharma, Rohit B.; Alonso, Laura C.

    2014-01-01

    Free fatty acids (FFAs) exert both positive and negative effects on beta cell survival and insulin secretory function, depending on concentration, duration, and glucose abundance. Lipid signals are mediated not only through metabolic pathways, but also through cell surface and nuclear receptors. Toxicity is modulated by positive signals arising from circulating factors such as hormones, growth factors and incretins, as well as negative signals such as inflammatory mediators and cytokines. Intracellular mechanisms of lipotoxicity include metabolic interference and cellular stress responses such as oxidative stress, endoplasmic reticulum (ER) stress, and possibly autophagy. New findings strengthen an old hypothesis that lipids may also impair compensatory beta cell proliferation. Clinical observations continue to support a role for lipid biology in the risk and progression of both type 1 (T1D) and type 2 diabetes (T2D). This review summarizes recent work in this important, rapidly evolving field. PMID:24740729

  13. G-CSFR ubiquitination critically regulates myeloid cell survival and proliferation.

    Directory of Open Access Journals (Sweden)

    Jing Ai

    Full Text Available The granulocyte colony-stimulating factor receptor (G-CSFR is a critical regulator of granulopoiesis. Mutations in the G-CSFR in patients with severe congenital neutropenia (SCN transforming to acute myelogenous leukemia (AML have been shown to induce hypersensitivity and enhanced growth responses to G-CSF. Recent studies have demonstrated the importance of the ubiquitin/proteasome system in the initiation of negative signaling by the G-CSFR. To further investigate the role of ubiquitination in regulating G-CSFR signaling, we generated a mutant form of the G-CSFR (K762R/G-CSFR which abrogates the attachment of ubiquitin to the lysine residue at position 762 of the G-CSFR that is deleted in the Delta716 G-CSFR form isolated from patients with SCN/AML. In response to G-CSF, mono-/polyubiquitination of the G-CSFR was impaired in cells expressing the mutant K762R/G-CSFR compared to cells transfected with the WT G-CSFR. Cells stably transfected with the K762R/G-CSFR displayed a higher proliferation rate, increased sensitivity to G-CSF, and enhanced survival following cytokine depletion, similar to previously published data with the Delta716 G-CSFR mutant. Activation of the signaling molecules Stat5 and Akt were also increased in K762R/G-CSFR transfected cells in response to G-CSF, and their activation remained prolonged after G-CSF withdrawal. These results indicate that ubiquitination is required for regulation of G-CSFR-mediated proliferation and cell survival. Mutations that disrupt G-CSFR ubiquitination at lysine 762 induce aberrant receptor signaling and hyperproliferative responses to G-CSF, which may contribute to leukemic transformation.

  14. Hedgehog pathway regulators influence cervical cancer cell proliferation, survival and migration

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    Samarzija, Ivana [Ecole Polytechnique Federale Lausanne (EPFL), Department of Life Sciences, Swiss Institute for Experimental Cancer Research (ISREC), 1015 Lausanne (Switzerland); Beard, Peter, E-mail: peter.beard@epfl.ch [Ecole Polytechnique Federale Lausanne (EPFL), Department of Life Sciences, Swiss Institute for Experimental Cancer Research (ISREC), 1015 Lausanne (Switzerland)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Unknown cellular mutations complement papillomavirus-induced carcinogenesis. Black-Right-Pointing-Pointer Hedgehog pathway components are expressed by cervical cancer cells. Black-Right-Pointing-Pointer Hedgehog pathway activators and inhibitors regulate cervical cancer cell biology. Black-Right-Pointing-Pointer Cell immortalization by papillomavirus and activation of Hedgehog are independent. -- Abstract: Human papillomavirus (HPV) infection is considered to be a primary hit that causes cervical cancer. However, infection with this agent, although needed, is not sufficient for a cancer to develop. Additional cellular changes are required to complement the action of HPV, but the precise nature of these changes is not clear. Here, we studied the function of the Hedgehog (Hh) signaling pathway in cervical cancer. The Hh pathway can have a role in a number of cancers, including those of liver, lung and digestive tract. We found that components of the Hh pathway are expressed in several cervical cancer cell lines, indicating that there could exists an autocrine Hh signaling loop in these cells. Inhibition of Hh signaling reduces proliferation and survival of the cervical cancer cells and induces their apoptosis as seen by the up-regulation of the pro-apoptotic protein cleaved caspase 3. Our results indicate that Hh signaling is not induced directly by HPV-encoded proteins but rather that Hh-activating mutations are selected in cells initially immortalized by HPV. Sonic Hedgehog (Shh) ligand induces proliferation and promotes migration of the cervical cancer cells studied. Together, these results indicate pro-survival and protective roles of an activated Hh signaling pathway in cervical cancer-derived cells, and suggest that inhibition of this pathway may be a therapeutic option in fighting cervical cancer.

  15. High MRPS23 expression contributes to hepatocellular carcinoma proliferation and indicates poor survival outcomes.

    Science.gov (United States)

    Pu, Meng; Wang, Jianlin; Huang, Qike; Zhao, Ge; Xia, Congcong; Shang, Runze; Zhang, Zhuochao; Bian, Zhenyuan; Yang, Xishegn; Tao, Kaishan

    2017-07-01

    Hepatocellular carcinoma is one of the most prevalent neoplasms and the leading cause of cancer-related mortality worldwide. Mitochondrial ribosomal protein S23 is encoded by a nuclear gene and participates in mitochondrial protein translation. Mitochondrial ribosomal protein S23 overexpression has been found in many types of cancer. In this study, we explored mitochondrial ribosomal protein S23 expression in primary hepatocellular carcinoma tissues compared with matched adjacent non-tumoral liver tissues using mitochondrial ribosomal protein S23 messenger RNA and protein levels collected from public databases and clinical samples. Immunohistochemistry was performed to analyze the relationship between mitochondrial ribosomal protein S23 and various clinicopathological features. The results indicated that mitochondrial ribosomal protein S23 was significantly overexpressed in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 expression was correlated with the tumor size and tumor-metastasis-node stage. Moreover, patients with high mitochondrial ribosomal protein S23 expression levels presented poorer survival rates. Mitochondrial ribosomal protein S23 was an independent prognostic factor for survival, especially at the early stage of hepatocellular carcinoma. In addition, the downregulation of mitochondrial ribosomal protein S23 decreased the proliferation of hepatocellular carcinoma in vitro and in vivo. In conclusion, we verified for the first time that mitochondrial ribosomal protein S23 expression was upregulated in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 levels can predict poor clinical outcomes in hepatocellular carcinoma, and this protein plays a key role in tumor proliferation. Therefore, mitochondrial ribosomal protein S23 may be a potential therapeutic target for hepatocellular carcinoma.

  16. Domain of dentine sialoprotein mediates proliferation and differentiation of human periodontal ligament stem cells.

    Directory of Open Access Journals (Sweden)

    Alkan Ozer

    Full Text Available Classic embryological studies have documented the inductive role of root dentin on adjacent periodontal ligament differentiation.  The biochemical composition of root dentin includes collagens and cleavage products of dentin sialophosphoprotein (DSPP, such as dentin sialoprotein (DSP.  The high abundance of DSP in root dentin prompted us to ask the question whether DSP or peptides derived thereof would serve as potent biological matrix components to induce periodontal progenitors to further differentiate into periodontal ligament cells. Here, we test the hypothesis that domain of DSP influences cell fate. In situ hybridization and immunohistochemical analyses showed that the COOH-terminal DSP domain is expressed in mouse periodontium at various stages of root development. The recombinant COOH-terminal DSP fragment (rC-DSP enhanced attachment and migration of human periodontal ligament stem cells (PDLSC, human primary PDL cells without cell toxicity. rC-DSP induced PDLSC cell proliferation as well as differentiation and mineralization of PDLSC and PDL cells by formation of mineralized tissue and ALPase activity. Effect of rC-DSP on cell proliferation and differentiation was to promote gene expression of tooth/bone-relate markers, transcription factors and growth factors. The results for the first time showed that rC-DSP may be one of the components of cell niche for stimulating stem/progenitor cell proliferation and differentiation and a natural scaffold for periodontal regeneration application.

  17. Catechins Variously Affect Activities of Conjugation Enzymes in Proliferating and Differentiated Caco-2 Cells

    Directory of Open Access Journals (Sweden)

    Kateřina Lněničková

    2016-09-01

    Full Text Available The knowledge of processes in intestinal cells is essential, as most xenobiotics come into contact with the small intestine first. Caco-2 cells are human colorectal adenocarcinoma that once differentiated, exhibit enterocyte-like characteristics. Our study compares activities and expressions of important conjugation enzymes and their modulation by green tea extract (GTE and epigallocatechin gallate (EGCG using both proliferating (P and differentiated (D caco-2 cells. The mRNA levels of the main conjugation enzymes were significantly elevated after the differentiation of Caco-2 cells. However, no increase in conjugation enzymes’ activities in differentiated cells was detected in comparison to proliferating ones. GTE/EGCG treatment did not affect the mRNA levels of any of the conjugation enzymes tested in either type of cells. Concerning conjugation enzymes activities, GTE/EGCG treatment elevated glutathione S-transferase (GST activity by approx. 30% and inhibited catechol-O-methyltransferase (COMT activity by approx. 20% in differentiated cells. On the other hand, GTE as well as EGCG treatment did not significantly affect the activities of conjugation enzymes in proliferating cells. Administration of GTE/EGCG mediated only mild changes of GST and COMT activities in enterocyte-like cells, indicating a low risk of GTE/EGCG interactions with concomitantly administered drugs. However, a considerable chemo-protective effect of GTE via the pronounced induction of detoxifying enzymes cannot be expected as well.

  18. In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish.

    Directory of Open Access Journals (Sweden)

    Sergey V Prykhozhij

    Full Text Available BACKGROUND: Sonic hedgehog (Shh signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Analysis of the zebrafish shh(-/- mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh(-/- mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh(-/- mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh(-/- mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53(-/-shh(-/- mutant retina suggesting the effect of p53 on retinal differentiation. CONCLUSIONS: Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.

  19. Sox2 activates cell proliferation and differentiation in the respiratory epithelium.

    Science.gov (United States)

    Tompkins, David H; Besnard, Valérie; Lange, Alexander W; Keiser, Angela R; Wert, Susan E; Bruno, Michael D; Whitsett, Jeffrey A

    2011-07-01

    Sox2, a transcription factor critical for the maintenance of embryonic stem cells and induction of pluripotent stem cells, is expressed exclusively in the conducting airway epithelium of the lung, where it is required for differentiation of nonciliated, goblet, and ciliated cells. To determine the role of Sox2 in respiratory epithelial cells, Sox2 was selectively and conditionally expressed in nonciliated airway epithelial cells and in alveolar type II cells in the adult mouse. Sox2 induced epithelial cell proliferation within 3 days of expression. Epithelial cell proliferation was associated with increased Ki-67 and cyclin D1 staining. Expression of cell cycle genes, including FoxM1, Ccna2 (Cyclin A2), Ccnb2 (Cyclin B2), and Ccnd1 (Cyclin D1), was increased. Consistent with a role in cell proliferation, Sox2 activated the transcription of FoxM1 in vitro. In alveoli, Sox2 caused hyperplasia and ectopic differentiation of epithelial cells to those with morphologic and molecular characteristics of conducting airway epithelium. Sox2 induced the expression of conducting airway epithelial specific genes, including Scgb1a1, Foxj1, Tubb3, and Cyp2f2. Although prolonged expression of Sox2 caused cell proliferation and epithelial hyperplasia, Sox2 did not induce pulmonary tumors. Sox2 induces proliferation of respiratory epithelial cells and, subsequently, partially reprograms alveolar epithelial cells into cells with characteristics of the conducting airways.

  20. Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Peng-Yeh [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China); Tsai, Chong-Bin [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China); Department of Ophthalmology, Chiayi Christian Hospital, Chiayi 600, Taiwan, ROC (China); Tseng, Min-Jen, E-mail: biomjt@ccu.edu.tw [Institute of Molecular Biology and Department of Life Science, National Chung Cheng University, Chiayi 621, Taiwan, ROC (China)

    2013-01-18

    Highlights: ► Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ► Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ► Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ► Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBPδ and PPARγ was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

  1. Pannexin 3 regulates proliferation and differentiation of odontoblasts via its hemichannel activities.

    Science.gov (United States)

    Iwamoto, Tsutomu; Nakamura, Takashi; Ishikawa, Masaki; Yoshizaki, Keigo; Sugimoto, Asuna; Ida-Yonemochi, Hiroko; Ohshima, Hayato; Saito, Masahiro; Yamada, Yoshihiko; Fukumoto, Satoshi

    2017-01-01

    Highly coordinated regulation of cell proliferation and differentiation contributes to the formation of functionally shaped and sized teeth; however, the mechanism underlying the switch from cell cycle exit to cell differentiation during odontogenesis is poorly understood. Recently, we identified pannexin 3 (Panx3) as a member of the pannexin gap junction protein family from tooth germs. The expression of Panx3 was predominately localized in preodontoblasts that arise from dental papilla cells and can differentiate into dentin-secreting odontoblasts. Panx3 also co-localized with p21, a cyclin-dependent kinase inhibitor protein, in preodontoblasts. Panx3 was expressed in primary dental mesenchymal cells and in the mDP dental mesenchymal cell line. Both Panx3 and p21 were induced during the differentiation of mDP cells. Overexpression of Panx3 in mDP cells reduced cell proliferation via up-regulation of p21, but not of p27, and promoted the Bone morphogenetic protein 2 (BMP2)-induced phosphorylation of Smad1/5/8 and the expression of dentin sialophosphoprotein (Dspp), a marker of differentiated odontoblasts. Furthermore, Panx3 released intracellular ATP into the extracellular space through its hemichannel and induced the phosphorylation of AMP-activated protein kinase (AMPK). 5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR), an activator of AMPK, reduced mDP cell proliferation and induced p21 expression. Conversely, knockdown of endogenous Panx3 by siRNA inhibited AMPK phosphorylation, p21 expression, and the phosphorylation of Smad1/5/8 even in the presence of BMP2. Taken together, our results suggest that Panx3 modulates intracellular ATP levels, resulting in the inhibition of odontoblast proliferation through the AMPK/p21 signaling pathway and promotion of cell differentiation by the BMP/Smad signaling pathway.

  2. miR-429 Inhibits Differentiation and Promotes Proliferation in Porcine Preadipocytes

    Directory of Open Access Journals (Sweden)

    Ying Peng

    2016-12-01

    Full Text Available MicroRNAs (miRNAs are crucial regulatory molecules for adipogenesis. They contribute to the controlling of proliferation and differentiation of preadipocytes. Previous studies revealed an important role of miR-429 in cell invasion, migration, and apoptosis. Our previous work has shown that the expression of miR-429 in subcutaneous fat can be observed in newly born (3-day-old Rongchang piglets rather than their adult counterparts (180-day-old. This expression pattern suggests that miR-429 might be functionally related to postnatal adipogenesis. However, we currently lack a mechanistic understanding of miR-429 within the context of preadipocyte differentiation. In this study, we investigated the function of miR-429 in porcine subcutaneous and intramuscular preadipocyte proliferation and differentiation. In our porcine preadipocyte differentiation model, miR-429 expression decreased remarkably upon adipogenic induction. Overexpression of miR-429 notably down-regulated the expression of adipogenic marker genes: PPARγ, aP2, FAS and impaired the triglyceride accumulation, while the expression of lipolytic gene ATGL was not affected. In addition, we observed that miR-429 significantly promoted the proliferation of porcine preadipocytes. We also found that miR-429 could directly bind to the 3′-UTRs of KLF9 and p27, which have been well documented to promote preadipocyte differentiation and repress cell cycle progression. Taken together, our data support a novel role of miR-429 in regulating porcine preadipocyte differentiation and proliferation, and KLF9 and p27 are potent targets of miR-429 during these processes.

  3. Numerical modeling of cell differentiation and proliferation in force-induced substrates via encapsulated magnetic nanoparticles.

    Science.gov (United States)

    Mousavi, Seyed Jamaleddin; Doweidar, Mohamed Hamdy

    2016-07-01

    Cell migration, differentiation, proliferation and apoptosis are the main processes in tissue regeneration. Mesenchymal Stem Cells have the potential to differentiate into many cell phenotypes such as tissue- or organ-specific cells to perform special functions. Experimental observations illustrate that differentiation and proliferation of these cells can be regulated according to internal forces induced within their Extracellular Matrix. The process of how exactly they interpret and transduce these signals is not well understood. A previously developed three-dimensional (3D) computational model is here extended and employed to study how force-free substrates and force-induced substrate control cell differentiation and/or proliferation during the mechanosensing process. Consistent with experimental observations, it is assumed that cell internal deformation (a mechanical signal) in correlation with the cell maturation state directly triggers cell differentiation and/or proliferation. The Extracellular Matrix is modeled as Neo-Hookean hyperelastic material assuming that cells are cultured within 3D nonlinear hydrogels. In agreement with well-known experimental observations, the findings here indicate that within neurogenic (0.1-1kPa), chondrogenic (20-25kPa) and osteogenic (30-45kPa) substrates, Mesenchymal Stem Cells differentiation and proliferation can be precipitated by inducing the substrate with an internal force. Therefore, cells require a longer time to grow and maturate within force-free substrates than within force-induced substrates. In the instance of Mesenchymal Stem Cells differentiation into a compatible phenotype, the magnitude of the net traction force increases within chondrogenic and osteogenic substrates while it reduces within neurogenic substrates. This is consistent with experimental studies and numerical works recently published by the same authors. However, in all cases the magnitude of the net traction force considerably increases at the

  4. Prokineticin receptor 1 as a novel suppressor of preadipocyte proliferation and differentiation to control obesity.

    Directory of Open Access Journals (Sweden)

    Cécilia Szatkowski

    Full Text Available BACKGROUND: Adipocyte renewal from preadipocytes occurs throughout the lifetime and contributes to obesity. To date, little is known about the mechanisms that control preadipocyte proliferation and differentiation. Prokineticin-2 is an angiogenic and anorexigenic hormone that activate two G protein-coupled receptors (GPCRs: PKR1 and PKR2. Prokineticin-2 regulates food intake and energy metabolism via central mechanisms (PKR2. The peripheral effect of prokineticin-2 on adipocytes/preadipocytes has not been studied yet. METHODOLOGY/PRINCIPAL FINDINGS: Since adipocytes and preadipocytes express mainly prokineticin receptor-1 (PKR1, here, we explored the role of PKR1 in adipose tissue expansion, generating PKR1-null (PKR1(-/- and adipocyte-specific (PKR1(ad-/- mutant mice, and using murine and human preadipocyte cell lines. Both PKR1(-/- and PKR1(ad-/- had excessive abdominal adipose tissue, but only PKR1(-/- mice showed severe obesity and diabetes-like syndrome. PKR1(ad-/- mice had increased proliferating preadipocytes and newly formed adipocyte levels, leading to expansion of adipose tissue. Using PKR1-knockdown in 3T3-L1 preadipocytes, we show that PKR1 directly inhibits preadipocyte proliferation and differentiation. These PKR1 cell autonomous actions appear targeted at preadipocyte cell cycle regulatory pathways, through reducing cyclin D, E, cdk2, c-Myc levels. CONCLUSIONS/SIGNIFICANCE: These results suggest PKR1 to be a crucial player in the preadipocyte proliferation and differentiation. Our data should facilitate studies of both the pathogenesis and therapy of obesity in humans.

  5. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Brady, Robert T. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); O' Brien, Fergal J. [Tissue Engineering Research Group, Dept. of Anatomy, Royal College of Surgeons in Ireland (Ireland); Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Advanced Materials and BioEngineering Research Centre (AMBER), Trinity College Dublin & Royal College of Surgeons in Ireland (Ireland); Hoey, David A., E-mail: david.hoey@ul.ie [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin (Ireland); Dept. of Mechanical, Aeronautical and Biomedical Engineering, University of Limerick (Ireland); The Centre for Applied Biomedical Engineering Research, University of Limerick (Ireland); Materials & Surface Science Institute, University of Limerick (Ireland)

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  6. Gel Microstructure Regulates Proliferation and Differentiation of MC3T3-E1 Cells Encapsulated in Alginate Beads

    Science.gov (United States)

    Lee, Baek-Hee; Li, Bing; Guelcher, Scott A.

    2012-01-01

    For cell transplantation into damaged tissues, viable cells must be delivered to the defect site in a suitable carrier. However, the hypoxic and nutrient-limited environment in the carrier can induce massive cell death. The aims of this study were to increase the viability and regulate the behavior of osteoprogenitor cells encapsulated in alginate hydrogels through control of the gel microstructure. Cell survivability in alginate beads was improved through the use of α-MEM as the solvent for alginic acid sodium salt and CaCl2 solutions, which supplied additional nutrients for the cells compared to water or buffer. The mesh size and shear modulus of the hydrogel were hypothesized to regulate proliferation and differentiation of osteoprogenitor cells. MC3T3-E1 cells demonstrated enhanced osteoblast differentiation when encapsulated in high-density alginate with smaller mesh size and more rigid mechanical properties, as confirmed by increased alkaline phosphatase activity and osteocalcin secretion. However, MC3T3-E1 cells encapsulated in low-density alginate beads with a larger mesh size and more compliant mechanical properties exhibited increased proliferation. These results demonstrate that the microstructure of alginate hydrogels can regulate the behavior of osteoprogenitor cells, thus suggesting that the tuning the properties of the gel may be a useful approach for enhancing new bone formation. PMID:22306825

  7. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    Energy Technology Data Exchange (ETDEWEB)

    Manceur, Aziza P. [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Tseng, Michael [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Holowacz, Tamara [Donnelly Centre, University of Toronto, Toronto, Ontario (Canada); Witterick, Ian [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Department of Otolaryngology, Head and Neck Surgery, University of Toronto, ON (Canada); Weksberg, Rosanna [Institute of Medical Science, University of Toronto, Toronto, ON (Canada); The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); McCurdy, Richard D. [The Hospital for Sick Children, Research Institute, Program in Genetics and Genomic Biology, Toronto, Ontario Canada (Canada); Warsh, Jerry J. [Laboratory of Cellular and Molecular Pathophysiology, Centre for Addiction and Mental Health (CAMH), University of Toronto, Toronto, Ontario (Canada); Department of Psychiatry, University of Toronto, Toronto, ON (Canada); Institute of Medical Science, University of Toronto, Toronto, ON (Canada); Audet, Julie, E-mail: julie.audet@utoronto.ca [Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, Ontario (Canada); Donnelly Centre, University of Toronto, Toronto, Ontario (Canada)

    2011-09-10

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  8. The ROCK/GGTase Pathway Are Essential to the Proliferation and Differentiation of Neural Stem Cells Mediated by Simvastatin.

    Science.gov (United States)

    Zhang, Chan; Wu, Jian-Min; Liao, Min; Wang, Jun-Ling; Xu, Chao-Jin

    2016-12-01

    Simvastatin, a lipophilic and fermentation-derived natural statin, is reported to treat neurological disorders, such as traumatic brain injury, Parkinson's disease (PD), Alzheimer disease (AD), etc. Recently, research also indicated that simvastatin could promote regeneration in the dentate gyrus of adult mice by Wnt/β-catenin signaling (Robin et al. in Stem Cell Reports 2:9-17, 2014). However, the effect and mechanisms by which simvastatin may affect the neural stem cells (NSCs; from the embryonic day 14.5 (E14.5) SD rat brain) are not fully understood. Here, we investigated the effects of different doses of simvastatin on the survival, proliferation, differentiation, migration, and cell cycle of NSCs as well as underlying intracellular signaling pathways. The results showed that simvastatin not only inhibits the proliferation of NSCs but also enhances the βIII-tubulin(+) neuron differentiation rate. Additionally, we find that simvastatin could also promote NSC migration and induce cell cycle arrest at M2 phrase. All these effects of simvastatin on NSCs were mimicked with an inhibitor of Rho kinase (ROCK) and a specific inhibitor of geranylgeranyl transferase (GGTase). In conclusion, these data indicate that simvastatin could promote neurogenesis of neural stem cells, and these effects were mediated through the ROCK/GGTase pathway.

  9. Differential survival of leukocyte subsets mediated by synovial, bone marrow, and skin fibroblasts: site-specific versus activation-dependent survival of T cells and neutrophils.

    Science.gov (United States)

    Filer, Andrew; Parsonage, Greg; Smith, Emily; Osborne, Chloe; Thomas, Andrew M C; Curnow, S John; Rainger, G Ed; Raza, Karim; Nash, Gerard B; Lord, Janet; Salmon, Mike; Buckley, Christopher D

    2006-07-01

    Synovial fibroblasts share a number of phenotype markers with fibroblasts derived from bone marrow. In this study we investigated the role of matched fibroblasts obtained from 3 different sources (bone marrow, synovium, and skin) to test the hypothesis that synovial fibroblasts share similarities with bone marrow-derived fibroblasts in terms of their ability to support survival of T cells and neutrophils. Matched synovial, bone marrow, and skin fibroblasts were established from 8 different patients with rheumatoid arthritis who were undergoing knee or hip surgery. Resting or activated fibroblasts were cocultured with either CD4 T cells or neutrophils, and the degree of leukocyte survival, apoptosis, and proliferation were measured. Fibroblasts derived from all 3 sites supported increased survival of CD4 T cells, mediated principally by interferon-beta. However, synovial and bone marrow fibroblasts shared an enhanced site-specific ability to maintain CD4 T cell survival in the absence of proliferation, an effect that was independent of fibroblast activation or proliferation but required direct T cell-fibroblast cell contact. In contrast, fibroblast-mediated neutrophil survival was less efficient, being independent of the site of origin of the fibroblast but dependent on prior fibroblast activation, and mediated solely by soluble factors, principally granulocyte-macrophage colony-stimulating factor. These results suggest an important functional role for fibroblasts in the differential accumulation of leukocyte subsets in a variety of tissue microenvironments. The findings also provide a potential explanation for site-specific differences in the pattern of T cell and neutrophil accumulation observed in chronic inflammatory diseases.

  10. MiR-371-5p facilitates pancreatic cancer cell proliferation and decreases patient survival.

    Directory of Open Access Journals (Sweden)

    De He

    Full Text Available microRNAs (miRNAs play a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. To date, scientists have obtained a substantial amount of knowledge with regard to miRNAs in pancreatic cancer. However, the expression and function of miR-371-5p in pancreatic cancer has not been clearly elucidated. The aim of this study was to investigate the roles of miR-371-5p in pancreatic cancer and its association with the survival of patients with pancreatic cancer.The expression of miR-371-5p was examined in pancreatic duct adenocarcinoma (PDAC and their adjacent normal pancreatic tissues (ANPT or in pancreatic cancer cell lines by qRT-PCR. The association of miR-371-5p expression with overall survival was determined. The proliferation and apoptosis of SW-1990 and Panc-1 cells, transfected with miR-371-5p mimics or inhibitor, were assessed using MTT assay and flow cytometry, respectively. The tumorigenicity was evaluated via mice xenograft experiments. miR-371-5p promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP. Protein expression was analyzed by Western blot.The expression level of miR-371-5p was dramatically upregulated in clinical PDAC tissues compared with ANPT. Patients with high miR-371-5p expression had a significantly shorter survival than those with low miR-371-5p expression. The in vitro and in vivo assays showed that overexpression of miR-371-5p resulted in cell proliferation and increased tumor growth, which was associated with inhibitor of growth 1 (ING1 downregulation. Interestingly, we also found that ING1, in turn, inhibited expression of miR-371-5p in the promoter region.our study demonstrates a novel ING1-miR-371-5p regulatory feedback loop, which may have a critical role in PDAC. Thus miR-371-5p can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.

  11. Low-dose X-ray irradiation promotes osteoblast proliferation, differentiation and fracture healing.

    Directory of Open Access Journals (Sweden)

    Ming Chen

    Full Text Available Great controversy exists regarding the biologic responses of osteoblasts to X-ray irradiation, and the mechanisms are poorly understood. In this study, the biological effects of low-dose radiation on stimulating osteoblast proliferation, differentiation and fracture healing were identified using in vitro cell culture and in vivo animal studies. First, low-dose (0.5 Gy X-ray irradiation induced the cell viability and proliferation of MC3T3-E1 cells. However, high-dose (5 Gy X-ray irradiation inhibited the viability and proliferation of osteoblasts. In addition, dynamic variations in osteoblast differentiation markers, including type I collagen, alkaline phosphatase, Runx2, Osterix and osteocalcin, were observed after both low-dose and high-dose irradiation by Western blot analysis. Second, fracture healing was evaluated via histology and gene expression after single-dose X-ray irradiation, and low-dose X-ray irradiation accelerates fracture healing of closed femoral fractures in rats. In low-dose X-ray irradiated fractures, an increase in proliferating cell nuclear antigen (PCNA-positive cells, cartilage formation and fracture calluses was observed. In addition, we observed more rapid completion of endochondral and intramembranous ossification, which was accompanied by altered expression of genes involved in bone remodeling and fracture callus mineralization. Although the expression level of several osteoblast differentiation genes was increased in the fracture calluses of high-dose irradiated rats, the callus formation and fracture union were delayed compared with the control and low-dose irradiated fractures. These results reveal beneficial effects of low-dose irradiation, including the stimulation of osteoblast proliferation, differentiation and fracture healing, and highlight its potential translational application in novel therapies against bone-related diseases.

  12. Estrogen Stimulates Proliferation and Differentiation of Neural Stem/Progenitor Cells through Different Signal Transduction Pathways

    Directory of Open Access Journals (Sweden)

    Makiko Okada

    2010-10-01

    Full Text Available Our previous study indicated that both 17β-estradiol (E2, known to be an endogenous estrogen, and bisphenol A (BPA, known to be a xenoestrogen, could positively influence the proliferation or differentiation of neural stem/progenitor cells (NS/PCs. The aim of the present study was to identify the signal transduction pathways for estrogenic activities promoting proliferation and differentiation of NS/PCs via well known nuclear estrogen receptors (ERs or putative membrane-associated ERs. NS/PCs were cultured from the telencephalon of 15-day-old rat embryos. In order to confirm the involvement of nuclear ERs for estrogenic activities, their specific antagonist, ICI-182,780, was used. The presence of putative membrane-associated ER was functionally examined as to whether E2 can activate rapid intracellular signaling mechanism. In order to confirm the involvement of membrane-associated ERs for estrogenic activities, a cell-impermeable E2, bovine serum albumin-conjugated E2 (E2-BSA was used. We showed that E2 could rapidly activate extracellular signal-regulated kinases 1/2 (ERK 1/2, which was not inhibited by ICI-182,780. ICI-182,780 abrogated the stimulatory effect of these estrogens (E2 and BPA on the proliferation of NS/PCs, but not their effect on the differentiation of the NS/PCs into oligodendroglia. Furthermore, E2-BSA mimicked the activity of differentiation from NS/PCs into oligodendroglia, but not the activity of proliferation. Our study suggests that (1 the estrogen induced proliferation of NS/PCs is mediated via nuclear ERs; (2 the oligodendroglial generation from NS/PCs is likely to be stimulated via putative membrane‑associated ERs.

  13. Enhancer of zeste homolog 2 regulates cell differentiation and proliferation in neuroblastoma

    Directory of Open Access Journals (Sweden)

    Amallia N. Setyawati

    2015-12-01

    Full Text Available BACKGROUND Neuroblastoma  (NB  is  one  of  the  most  common  extracranial  solid  tumors occurring in infancy and childhood with highly variable outcomes. Polycomb group (PcG proteins are epigenetic gene silencers. Enhancer of zeste homolog 2 (EZH2 is a member of the polycomb repressor complex 2 (PRC2 group, with  the  main  function  to  catalyze  the  polycomb  repressor  complex  by methylating lysine 9 and 27 of histone H3. This study aimed to investigate the biological functionality of EZH2 in NB.   METHODS This was an experimental study with an analysis of correlation initially of the known prognostic factors of NB patients’ outcomes, by comparing the expression of v-myc avian myelocytomatosis viral oncogene neuroblastoma (MYCN with that of EZH2, on the basis of the patients’ overall and relapse free survival rates. This was followed with a biological functional study to assess the role of EZH2 expression in NB.   RESULTS EZH2 knockdown induces neurite extension and differentiation marker growth associated  protein  43  (GAP43  in  NB  cells,  although  it  does  not  affect  cell cycle. By ectopic expression of EZH2, all-trans retinoic acid (ATRA inducedneurite extension was suppressed and GAP43 was decreased. Overall, EZH2 seems to have an important role in NB cell differentiation. Although EZH2 did not alter cell proliferation, in the soft agar colony formation assay there was a significant increase in total colony number and number of large colonies.   CONCLUSION Our  result  clarified  the  potential  role  of  EZH2  in  the  regulation  of  cell differentiation and proliferation, which subsequently may play an important role in the poor prognosis of NB patients.

  14. ErbB3 drives mammary epithelial survival and differentiation during pregnancy and lactation.

    Science.gov (United States)

    Williams, Michelle M; Vaught, David B; Joly, Meghan Morrison; Hicks, Donna J; Sanchez, Violeta; Owens, Philip; Rahman, Bushra; Elion, David L; Balko, Justin M; Cook, Rebecca S

    2017-09-08

    During pregnancy, as the mammary gland prepares for synthesis and delivery of milk to newborns, a luminal mammary epithelial cell (MEC) subpopulation proliferates rapidly in response to systemic hormonal cues that activate STAT5A. While the receptor tyrosine kinase ErbB4 is required for STAT5A activation in MECs during pregnancy, it is unclear how ErbB3, a heterodimeric partner of ErbB4 and activator of phosphatidyl inositol-3 kinase (PI3K) signaling, contributes to lactogenic expansion of the mammary gland. We assessed mRNA expression levels by expression microarray of mouse mammary glands harvested throughout pregnancy and lactation. To study the role of ErbB3 in mammary gland lactogenesis, we used transgenic mice expressing WAP-driven Cre recombinase to generate a mouse model in which conditional ErbB3 ablation occurred specifically in alveolar mammary epithelial cells (aMECs). Profiling of RNA from mouse MECs isolated throughout pregnancy revealed robust Erbb3 induction during mid-to-late pregnancy, a time point when aMECs proliferate rapidly and undergo differentiation to support milk production. Litters nursed by ErbB3 KO dams weighed significantly less when compared to litters nursed by ErbB3 WT dams. Further analysis revealed substantially reduced epithelial content, decreased aMEC proliferation, and increased aMEC cell death during late pregnancy. Consistent with the potent ability of ErbB3 to activate cell survival through the PI3K/Akt pathway, we found impaired Akt phosphorylation in ErbB3 KO samples, as well as impaired expression of STAT5A, a master regulator of lactogenesis. Constitutively active Akt rescued cell survival in ErbB3-depleted aMECs, but failed to restore STAT5A expression or activity. Interestingly, defects in growth and survival of ErbB3 KO aMECs as well as Akt phosphorylation, STAT5A activity, and expression of milk-encoding genes observed in ErbB3 KO MECs progressively improved between late pregnancy and lactation day 5. We found a

  15. Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane

    Energy Technology Data Exchange (ETDEWEB)

    Rodríguez-Fuentes, Nayeli; Rodríguez-Hernández, Ana G. [Depto. Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), Mexico City 04510 (Mexico); Enríquez-Jiménez, Juana [Depto. Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), México City 14000 (Mexico); Alcántara-Quintana, Luz E. [Subd. de Investigación, Centro Nacional de la Transfusión Sanguínea, Secretaria de Salud, Mexico City 07370 (Mexico); Fuentes-Mera, Lizeth [Depto. Biología Molecular e Histocompatibilidad, Hospital General “Dr. Manuel Gea González”, México City 4800 (Mexico); Piña-Barba, María C. [Depto. Materiales Metálicos y Cerámicos, Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); Zepeda-Rodríguez, Armando [Depto. Biología Celular y Tisular, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), México City 04510 (Mexico); and others

    2013-05-10

    Highlights: •Nukbone showed to be a good scaffold for adhesion, proliferation and differentiation of stem cells. •Nukbone induced osteoblastic differentiation of human mesenchymal stem cells. •Results showed that Nukbone offer an excellent option for bone tissue regeneration due to properties. -- Abstract: Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.

  16. Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation

    Directory of Open Access Journals (Sweden)

    Resende Rodrigo R

    2009-08-01

    Full Text Available Abstract Acetylcholine (ACh has been shown to modulate neuronal differentiation during early development. Both muscarinic and nicotinic acetylcholine receptors (AChRs regulate a wide variety of physiological responses, including apoptosis, cellular proliferation and neuronal differentiation. However, the intracellular mechanisms underlying these effects of AChR signaling are not fully understood. It is known that activation of AChRs increase cellular proliferation and neurogenesis and that regulation of intracellular calcium through AChRs may underlie the many functions of ACh. Intriguingly, activation of diverse signaling molecules such as Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, protein kinase C and c-Src is modulated by AChRs. Here we discuss the roles of ACh in neuronal differentiation, cell proliferation and apoptosis. We also discuss the pathways involved in these processes, as well as the effects of novel endogenous AChRs agonists and strategies to enhance neuronal-differentiation of stem and neural progenitor cells. Further understanding of the intracellular mechanisms underlying AChR signaling may provide insights for novel therapeutic strategies, as abnormal AChR activity is present in many diseases.

  17. Novel function of keratins 5 and 14 in proliferation and differentiation of stratified epithelial cells.

    Science.gov (United States)

    Alam, Hunain; Sehgal, Lalit; Kundu, Samrat T; Dalal, Sorab N; Vaidya, Milind M

    2011-11-01

    Keratins are cytoplasmic intermediate filament proteins preferentially expressed by epithelial tissues in a site-specific and differentiation-dependent manner. The complex network of keratin filaments in stratified epithelia is tightly regulated during squamous cell differentiation. Keratin 14 (K14) is expressed in mitotically active basal layer cells, along with its partner keratin 5 (K5), and their expression is down-regulated as cells differentiate. Apart from the cytoprotective functions of K14, very little is known about K14 regulatory functions, since the K14 knockout mice show postnatal lethality. In this study, K14 expression was inhibited using RNA interference in cell lines derived from stratified epithelia to study the K14 functions in epithelial homeostasis. The K14 knockdown clones demonstrated substantial decreases in the levels of the K14 partner K5. These cells showed reduction in cell proliferation and delay in cell cycle progression, along with decreased phosphorylated Akt levels. K14 knockdown cells also exhibited enhanced levels of activated Notch1, involucrin, and K1. In addition, K14 knockdown AW13516 cells showed significant reduction in tumorigenicity. Our results suggest that K5 and K14 may have a role in maintenance of cell proliferation potential in the basal layer of stratified epithelia, modulating phosphatidylinositol 3-kinase/Akt-mediated cell proliferation and/or Notch1-dependent cell differentiation.

  18. Slow and sustained nitric oxide releasing compounds inhibit multipotent vascular stem cell proliferation and differentiation without causing cell death

    Energy Technology Data Exchange (ETDEWEB)

    Curtis, Brandon M.; Leix, Kyle Alexander [Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48859 (United States); Ji, Yajing [Department of Biomedical Science and Medicine, Michigan State University, East Lansing, MI 48824 (United States); Glaves, Richard Samuel Elliot [Department of Biology, Central Michigan University, Mount Pleasant, MI 48859 (United States); Ash, David E. [Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48859 (United States); Mohanty, Dillip K., E-mail: Mohan1dk@cmich.edu [Department of Chemistry, Central Michigan University, Mount Pleasant, MI 48859 (United States)

    2014-07-18

    Highlights: • Multipotent vascular stem cells (MVSCs) proliferate and differentiate. • Nitric oxide inhibits proliferation of MVSCs. • Nitric oxide inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs). • Smooth muscle cells (SMCs) neither de-differentiate nor proliferate. - Abstract: Atherosclerosis is the leading cause of cerebral and myocardial infarction. It is believed that neointimal growth common in the later stages of atherosclerosis is a result of vascular smooth muscle cell (SMC) de-differentiation in response to endothelial injury. However, the claims of the SMC de-differentiation theory have not been substantiated by monitoring the fate of mature SMCs in response to such injuries. A recent study suggests that atherosclerosis is a consequence of multipotent vascular stem cell (MVSC) differentiation. Nitric oxide (NO) is a well-known mediator against atherosclerosis, in part because of its inhibitory effect on SMC proliferation. Using three different NO-donors, we have investigated the effects of NO on MVSC proliferation. Results indicate that NO inhibits MVSC proliferation in a concentration dependent manner. A slow and sustained delivery of NO proved to inhibit proliferation without causing cell death. On the other hand, larger, single-burst NO concentrations, inhibits proliferation, with concurrent significant cell death. Furthermore, our results indicate that endogenously produced NO inhibits MVSC differentiation to mesenchymal-like stem cells (MSCs) and subsequently to SMC as well.

  19. In vitro proliferation and osteogenic differentiation of endometrial stem cells and dental pulp stem cells.

    Science.gov (United States)

    Tabatabaei, Fahimeh Sadat; Torshabi, Maryam

    2017-06-01

    Stem-cell-based therapies were introduced aiming to overcome the limitations of the existing procedures for regeneration of mineralized tissues. Stem cells isolated from the endometrial tissue and dental pulp have the capacity to differentiate into various functional cells including osteoblasts. However, studies comparing their ability to regenerate mineralized tissue are lacking. The purpose of this study was to compare the proliferation and osteogenic differentiation potential of endometrial stem cells (EnSCs) and dental pulp stem cells (DPSCs) using in vitro cell culture technique. The DPSCs and EnSCs were isolated from human dental pulp and endometrium, respectively. Their proliferation and osteogenic potential were compared in the same osteogenic medium (OM) after 3, 5, 7 and 10 days using the methyl thiazol tetrazolium assay, alizarin red staining, and real-time quantitative reverse transcription polymerase chain reaction (Real-Time qRT-PCR). The EnSCs showed higher proliferation rate compared to DPSCs. Regarding osteogenesis, alizarin red-positive colonies appeared earlier and in greater amounts in DPSCs group. The real-time qRT-PCR demonstrated significantly greater osteogenic potential of DPSCs compared to EnSCs. Our findings revealed significant differences in stem cell properties based on the tissue source. The EnSCs had greater proliferation rate than DPSCs, while DPSCs showed greater osteogenic potential compared to EnSCs in the same OM.

  20. Equol promotes rat osteoblast proliferation and differentiation through activating estrogen receptor.

    Science.gov (United States)

    Wang, J; Xu, J; Wang, B; Shu, F R; Chen, K; Mi, M T

    2014-07-04

    Phytoestrogens have been suggested as alternative treatment for postmenopausal osteoporosis. Equol, a metabolite of daidzein, has been shown to inhibit bone loss in ovariectomized mice and rats. However, whether or not equol influences the formation of bone has not yet been investigated. Therefore, we investigated the effect of equol on the proliferation and differentiation of rat primary osteoblasts and explored the involved mechanisms. Different equol concentrations significantly promoted the proliferation of osteoblasts after 48- and 72-h incubations. The alkaline phosphatase (ALP) activity also increased significantly in all of the equol and 17β-estradiol (E2) groups, except for the lowest (0.01 μM) equol group. Equol also significantly elevated the osteocalcin levels. The effects of equol on osteoblast proliferation, ALP activity, and osteocalcin levels were blocked by the estrogen receptor (ER) antagonist ICI182780. After a 24-h incubation, the expression of protein kinase C alpha (PKCα) in osteoblasts was significantly increased by equol. In conclusion, our study demonstrated that equol could promote the proliferation and differentiation of rat osteoblasts through activating the ER-PKCα-related signaling pathway, suggesting that equol could promote bone formation. These results suggest that equol could be a potential alternative agent for the management of postmenopausal osteoporosis.

  1. Cell density-dependent differential proliferation of neural stem cells on omnidirectional nanopore-arrayed surface.

    Science.gov (United States)

    Cha, Kyoung Je; Kong, Sun-Young; Lee, Ji Soo; Kim, Hyung Woo; Shin, Jae-Yeon; La, Moonwoo; Han, Byung Woo; Kim, Dong Sung; Kim, Hyun-Jung

    2017-10-12

    Recently, the importance of surface nanotopography in the determination of stem cell fate and behavior has been revealed. In the current study, we generated polystyrene cell-culture dishes with an omnidirectional nanopore arrayed surface (ONAS) (diameter: 200 nm, depth: 500 nm, center-to-center distance: 500 nm) and investigated the effects of nanotopography on rat neural stem cells (NSCs). NSCs cultured on ONAS proliferated better than those on the flat surface when cell density was low and showed less spontaneous differentiation during proliferation in the presence of mitogens. Interestingly, NSCs cultured on ONAS at clonal density demonstrated a propensity to generate neurospheres, whereas those on the flat surface migrated out, proliferated as individuals, and spread out to attach to the surface. However, the differential patterns of proliferation were cell density-dependent since the distinct phenomena were lost when cell density was increased. ONAS modulated cytoskeletal reorganization and inhibited formation of focal adhesion, which is generally observed in NSCs grown on flat surfaces. ONAS appeared to reinforce NSC-NSC interaction, restricted individual cell migration and prohibited NSC attachment to the nanopore surface. These data demonstrate that ONAS maintains NSCs as undifferentiated while retaining multipotency and is a better topography for culturing low density NSCs.

  2. microRNA-10b Is Overexpressed and Critical for Cell Survival and Proliferation in Medulloblastoma

    Science.gov (United States)

    Pal, Rekha; Greene, Stephanie

    2015-01-01

    This study demonstrates the effects of miRNA-10b on medulloblastoma proliferation through transcriptional induction of the anti-apoptotic protein BCL2. Using a cancer specific miRNA-array, high expression of miRNA-10b in medulloblastoma cell lines compared to a normal cerebellar control was shown, and this was confirmed with real time PCR (RT-PCR). Two medulloblastoma cell lines (DAOY and UW228) were transiently transfected with control miRNA, miRNA-10b inhibitor or miRNA-10b mimic and subjected to RT-PCR, MTT, apoptosis, clonogenic assay and western blot analysis. Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression, inhibited proliferation, and induced apoptosis, while miRNA-10b mimic exerted an opposite effect. Inhibition of miRNA-10b abrogated the colony-forming capability of medulloblastoma cells, and markedly down-regulated the expression of BCL2. Down-regulation of BCL2 by antisense oligonucleotides or siRNA also significantly down-regulated miRNA-10b, suggesting that BCL2 is a major mediator of the effects of miRNA-10b. ABT-737 and ABT-199, potent inhibitors of BCL2, downregulated the expression of miRNA-10b and increased apoptosis. Analysis of miRNA-10b levels in 13 primary medulloblastoma samples revealed that the 2 patients with the highest levels of miRNA-10b had multiple recurrences (4.5) and died within 8 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and nearly 40% long-term survival. The data presented here indicate that miRNA-10b may act as an oncomir in medulloblastoma tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as a mediator of the effects of miRNA-10b upon medulloblastoma cell survival. PMID:26394044

  3. microRNA-10b Is Overexpressed and Critical for Cell Survival and Proliferation in Medulloblastoma.

    Directory of Open Access Journals (Sweden)

    Rekha Pal

    Full Text Available This study demonstrates the effects of miRNA-10b on medulloblastoma proliferation through transcriptional induction of the anti-apoptotic protein BCL2. Using a cancer specific miRNA-array, high expression of miRNA-10b in medulloblastoma cell lines compared to a normal cerebellar control was shown, and this was confirmed with real time PCR (RT-PCR. Two medulloblastoma cell lines (DAOY and UW228 were transiently transfected with control miRNA, miRNA-10b inhibitor or miRNA-10b mimic and subjected to RT-PCR, MTT, apoptosis, clonogenic assay and western blot analysis. Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression, inhibited proliferation, and induced apoptosis, while miRNA-10b mimic exerted an opposite effect. Inhibition of miRNA-10b abrogated the colony-forming capability of medulloblastoma cells, and markedly down-regulated the expression of BCL2. Down-regulation of BCL2 by antisense oligonucleotides or siRNA also significantly down-regulated miRNA-10b, suggesting that BCL2 is a major mediator of the effects of miRNA-10b. ABT-737 and ABT-199, potent inhibitors of BCL2, downregulated the expression of miRNA-10b and increased apoptosis. Analysis of miRNA-10b levels in 13 primary medulloblastoma samples revealed that the 2 patients with the highest levels of miRNA-10b had multiple recurrences (4.5 and died within 8 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and nearly 40% long-term survival. The data presented here indicate that miRNA-10b may act as an oncomir in medulloblastoma tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as a mediator of the effects of miRNA-10b upon medulloblastoma cell survival.

  4. Influence of abiotic factors on bacterial proliferation and anoxic survival of the sea mussel Mytilus edulis L.

    NARCIS (Netherlands)

    Babarro, J.M.F.; De Zwaan, A.

    2002-01-01

    The effect of several abiotic factors (salinity, temperature and pH) on bacterial proliferation and survival time of the sea mussel Mytilus edulis L. were studied under anoxic incubations. In addition, the presence in the incubation media of ammonium and the volatile fatty acids propionate and

  5. Effects of protein kinase C activators and staurosporine on protein kinase activity, cell survival, and proliferation in Tetrahymena thermophila

    DEFF Research Database (Denmark)

    Straarup, EM; Schousboe, P; Hansen, HQ

    1997-01-01

    Autocrine factors prevent cell death in the ciliate Tetrahymena thermophila, a unicellular eukaryote, in a chemically defined medium. At certain growth conditions these factors are released at a sufficient concentration by > 500 cells ml-1 to support cell survival and proliferation. The protein k...

  6. [Effects of MAPK antagonist on TPO stimulated UT2 cells proliferation and differentiation].

    Science.gov (United States)

    Li, Wen-lin; Shi, Xiao-yu; Li, Rong; Tang, Hong-lin

    2005-05-01

    To explore the effects of MAPK antagonist on TPO stimulated UT7 cell proliferation and differentiation, and to elucidate the mechanism of TPO functioning on UT7 cells. EGFP pMSCV and MEK 1 pMSCV MEK 1 plasmids were transferred into UT7 cells. Phosphorylated MEK1 of UT7 cells was examined by Western blot. The proliferation and CD41 expression of UT7 cells transfected with mutant (ser222A) MEK1 or exposed to PD98059 were examined. (1) 60.73% EGFP positive cells were obtained in retroviral vector MEK1 pMSCV transfected UT7cells. (2) In different time of TPO stimulating UT7 cells, the level of phosphorylated MEK1 was lower in experiment group than in control group. In experiment group, the level of phosphorylated MEK1 was decreased after stimulated by TPO for 1 hour, and almost disappeared after stimulated for 3 hours. (3) The effect of TPO on UT7 cell proliferation was inhibited by PD98059 and the transfected mutation MEK1 gene. The proliferation rate was 98.58% in DMSO control group, 39.00% in PD98059 group (P TPO. There was a relationship between the TPO stimulating time and phosphorylation of MEK1. The effects of TPO on UT7 cell proliferation and CD41 expression is mediated by MAPK signal transduction pathway.

  7. Effects of high glucose on mesenchymal stem cell proliferation and differentiation

    DEFF Research Database (Denmark)

    Li, Yu-Ming; Schilling, Tatjana; Benisch, Peggy

    2007-01-01

    High glucose (HG) concentrations impair cellular functions and induce apoptosis. Exposition of mesenchymal stem cells (MSC) to HG was reported to reduce colony forming activity and induce premature senescence. We characterized the effects of HG on human MSC in vitro using telomerase...... was not influenced by HG in both cell types. MSC treatment with HG favored osteogenic differentiation. MSC are resistant to HG toxicity, depending on the stemness of MSC. Proliferation and osteogenic differentiation are stimulated by HG. Effects of HG on the transient amplifying compartment of MSC may differ from...... those in mature cells. Further research is needed to unravel the molecular mechanisms of HG resistance of MSC...

  8. Review of Differentiation and Proliferation of Primordial Germ Cells in Culture

    Directory of Open Access Journals (Sweden)

    Zohreh Makoolati

    2011-12-01

    Full Text Available Primordial germ cells (PGCs are highly specialized cell population that arises from the epiblast in vivo. There are three critical steps in the life cycle of these cells: 1-Specification 2-migration and proliferation 3-prenatal and postnatal sex specific development. Specification of germ cells in epiblast occurs due to signals secreted from extraembryonic tissues. Primordial germ cells are required for continuation and development of the species. Thus, differentiation and purification of these cells from different cell sources is valuable for research, genetical analysis of germ cell development, epigenetic eveluation and infertility treatment. The most important part in the germ cell differentiation includes; optimum media selection, distinguishing and purification of differentiated cell. Several studies about in vitro PGC differentiation have been reported. In order to distinguish PGCs in vitro, specific markers which are expressed in these cells are used. Furthermore, functional ability of these cells for production of offspring can be employed for this purpose.

  9. ADP-Ribosylation Factor 1 Regulates Proliferation, Migration, and Fusion in Early Stage of Osteoclast Differentiation

    Directory of Open Access Journals (Sweden)

    Min Jae Kim

    2015-12-01

    Full Text Available Small G-protein adenosine diphosphate (ADP-ribosylation factors (ARFs regulate a variety of cellular functions, including actin cytoskeleton remodeling, plasma membrane reorganization, and vesicular transport. Here, we propose the functional roles of ARF1 in multiple stages of osteoclast differentiation. ARF1 was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL-induced osteoclast differentiation and transiently activated in an initial stage of their differentiation. Differentiation of ARF1-deficient osteoclast precursors into mature osteoclasts temporarily increased in pre-maturation stage of osteoclasts followed by reduced formation of mature osteoclasts, indicating that ARF1 regulates the osteoclastogenic process. ARF1 deficiency resulted in reduced osteoclast precursor proliferation and migration as well as increasing cell-cell fusion. In addition, ARF1 silencing downregulated c-Jun N-terminal kinase (JNK, Akt, osteopontin, and macrophage colony-stimulating factor (M-CSF-receptor c-Fms as well as upregulating several fusion-related genes including CD44, CD47, E-cadherin, and meltrin-α. Collectively, we showed that ARF1 stimulated proliferation and migration of osteoclast precursors while suppressing their fusion, suggesting that ARF1 may be a plausible inter-player that mediates the transition to osteoclast fusion at multiple steps during osteoclast differentiation

  10. Polyesteramide-derived nonwovens as innovative degradable matrices support preadipocyte adhesion, proliferation, and differentiation.

    Science.gov (United States)

    Hemmrich, K; Meersch, M; Wiesemann, U; Salber, J; Klee, D; Gries, Th; Pallua, N

    2006-12-01

    Extended soft tissue defects resulting from injuries or tumor resections are still an unresolved problem in plastic and reconstructive surgery because adequate reconstruction is difficult. Immature adipogenic precursor cells, called preadipocytes, which are located between mature adipocytes in adipose tissue, represent a powerful tool for soft tissue engineering because of their ability to proliferate and differentiate into adipose tissue after transplantation. In previous studies, we compared preadipocyte-loaded hyaluronan or collagen biomaterials and their applicability for adipose tissue engineering. Our findings demonstrated successful de novo formation of adipose tissue in vivo but pore size and stiffness were limiting factors not allowing for sufficient cell distribution in the construct. This study presents a nonwoven made of novel bioabsorbable co-poly(ester amide) based on e-caprolactam, adipic acid, and 1,4-butanediol in an innovative 3-dimensional architecture. The material was formed into nonwovens by textile manufacturing using an aerodynamic web formation process and a needle felting technique. Carriers were seeded with human preadipocytes and examined for cellular proliferation and differentiation. In addition, methods of preparing scaffolds for optimal cell interaction were evaluated. Our findings show that polyesteramide-derived nonwovens allow good adherence, proliferation, and differentiation of preadipocytes. These results are promising guidance toward an optimally designed scaffold for in vivo use.

  11. VEGF-mediated angiogenesis stimulates neural stem cell proliferation and differentiation in the premature brain

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jinqiao, E-mail: jinqiao1977@163.com [Institute of Pediatrics, Children' s Hospital of Fudan University (China); Sha, Bin [Department of Neonatology, Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China); Zhou, Wenhao, E-mail: zhou_wenhao@yahoo.com.cn [Department of Neonatology, Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China); Yang, Yi [Institute of Pediatrics, Children' s Hospital of Fudan University (China)

    2010-03-26

    This study investigated the effects of angiogenesis on the proliferation and differentiation of neural stem cells in the premature brain. We observed the changes in neurogenesis that followed the stimulation and inhibition of angiogenesis by altering vascular endothelial growth factor (VEGF) expression in a 3-day-old rat model. VEGF expression was overexpressed by adenovirus transfection and down-regulated by siRNA interference. Using immunofluorescence assays, Western blot analysis, and real-time PCR methods, we observed angiogenesis and the proliferation and differentiation of neural stem cells. Immunofluorescence assays showed that the number of vWF-positive areas peaked at day 7, and they were highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at every time point. The number of neural stem cells, neurons, astrocytes, and oligodendrocytes in the subventricular zone gradually increased over time in the VEGF up-regulation group. Among the three groups, the number of these cells was highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at the same time point. Western blot analysis and real-time PCR confirmed these results. These data suggest that angiogenesis may stimulate the proliferation of neural stem cells and differentiation into neurons, astrocytes, and oligodendrocytes in the premature brain.

  12. The hippo pathway promotes Notch signaling in regulation of cell differentiation, proliferation, and oocyte polarity.

    Directory of Open Access Journals (Sweden)

    Jianzhong Yu

    2008-03-01

    Full Text Available Specification of the anterior-posterior axis in Drosophila oocytes requires proper communication between the germ-line cells and the somatically derived follicular epithelial cells. Multiple signaling pathways, including Notch, contribute to oocyte polarity formation by controlling the temporal and spatial pattern of follicle cell differentiation and proliferation. Here we show that the newly identified Hippo tumor-suppressor pathway plays a crucial role in the posterior follicle cells in the regulation of oocyte polarity. Disruption of the Hippo pathway, including major components Hippo, Salvador, and Warts, results in aberrant follicle-cell differentiation and proliferation and dramatic disruption of the oocyte anterior-posterior axis. These phenotypes are related to defective Notch signaling in follicle cells, because misexpression of a constitutively active form of Notch alleviates the oocyte polarity defects. We also find that follicle cells defective in Hippo signaling accumulate the Notch receptor and display defects in endocytosis markers. Our findings suggest that the interaction between Hippo and classic developmental pathways such as Notch is critical to spatial and temporal regulation of differentiation and proliferation and is essential for development of the body axes in Drosophila.

  13. JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation.

    Science.gov (United States)

    Besancenot, Rodolphe; Roos-Weil, Damien; Tonetti, Carole; Abdelouahab, Hadjer; Lacout, Catherine; Pasquier, Florence; Willekens, Christophe; Rameau, Philippe; Lecluse, Yann; Micol, Jean-Baptiste; Constantinescu, Stefan N; Vainchenker, William; Solary, Eric; Giraudier, Stéphane

    2014-09-25

    Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses. © 2014 by The American Society of Hematology.

  14. Epithelial Xbp1 Is Required for Cellular Proliferation and Differentiation during Mammary Gland Development

    Science.gov (United States)

    Hasegawa, Daisuke; Calvo, Veronica; Avivar-Valderas, Alvaro; Lade, Abigale; Chou, Hsin-I; Lee, Youngmin A.; Farias, Eduardo F.; Aguirre-Ghiso, Julio A.

    2015-01-01

    Xbp1, a key mediator of the unfolded protein response (UPR), is activated by IRE1α-mediated splicing, which results in a frameshift to encode a protein with transcriptional activity. However, the direct function of Xbp1 in epithelial cells during mammary gland development is unknown. Here we report that the loss of Xbp1 in the mammary epithelium through targeted deletion leads to poor branching morphogenesis, impaired terminal end bud formation, and spontaneous stromal fibrosis during the adult virgin period. Additionally, epithelial Xbp1 deletion induces endoplasmic reticulum (ER) stress in the epithelium and dramatically inhibits epithelial proliferation and differentiation during lactation. The synthesis of milk and its major components, α/β-casein and whey acidic protein (WAP), is significantly reduced due to decreased prolactin receptor (Prlr) and ErbB4 expression in Xbp1-deficient mammary epithelium. Reduction of Prlr and ErbB4 expression and their diminished availability at the cell surface lead to reduced phosphorylated Stat5, an essential regulator of cell proliferation and differentiation during lactation. As a result, lactating mammary glands in these mice produce less milk protein, leading to poor pup growth and postnatal death. These findings suggest that the loss of Xbp1 induces a terminal UPR which blocks proliferation and differentiation during mammary gland development. PMID:25713103

  15. Further studies on the survival of non-proliferating human diploid fibroblasts irradiated with ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Chan, G.L.; Little, J.B. (Harvard Univ., Boston, MA (USA). School of Public Health)

    1982-04-01

    Labelling index data showed that in AG1518 cells, a diploid human fibroblast strain, there was a lag period of at least 14 hours between subculture from the density-inhibited plateau phase of growth and entry into DNA synthesis. Cells irradiated with 254nm wavelength U.V. light 8 hours after subculture did not exhibit the same degree of resistance as cells irradiated in plateau phase and subcultured immediately. When cells were arrested from proliferation by maintenance in an arginine and glutamine deficient medium for 72 hours, they were nearly as resistant to U.V. light as plateau phase cells although maintenance in this medium for 24 hours after irradiation supported further recovery only after low U.V. doses. One strain of Cockayne syndrome fibroblasts was found to be resistant to U.V. light in plateau phase while another strain was found to have the same survival response whether it was irradiated in the plateau or log phase of growth.

  16. Melanoma tumors frequently acquire LRP2/megalin expression, which modulates melanoma cell proliferation and survival rates.

    Science.gov (United States)

    Andersen, Rikke K; Hammer, Katrine; Hager, Henrik; Christensen, Julie N; Ludvigsen, Maja; Honoré, Bent; Thomsen, Mai-Britt H; Madsen, Mette

    2015-05-01

    We show that the multiligand receptor megalin, known to mediate uptake and trafficking of nutrients and signaling molecules, is frequently expressed in malignant melanoma samples. Expression of megalin-encoding mRNA was investigated in 65 samples of nevi, melanomas, and melanoma metastases and was observed in more than 60% of the malignant samples, while only in 20% of the benign counterparts. Megalin expression in nevus and melanoma samples was additionally investigated by immunohistochemistry, which confirmed our mRNA-based observations. We furthermore show that a panel of tumor-derived melanoma cell lines express LRP2/megalin endogenously. In these cells, megalin is internalized from the cell surface and localizes extensively to intracellular vesicles, confirming receptor activity and pointing toward association with the endocytic apparatus. Groundbreaking, our results indicate that sustained megalin expression in melanoma cells is crucial for cell maintenance, as siRNA-mediated reduction in melanoma cell expression of LRP2/megalin significantly decreases melanoma cell proliferation and survival rates. © 2015 The Authors. Pigment Cell & Melanoma Research Published by John Wiley & Sons Ltd.

  17. Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation.

    Directory of Open Access Journals (Sweden)

    Fen Li

    Full Text Available Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs.The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10(-8 to 10(-5 M and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin and smooth muscle protein 22α (SM22α protein expressions and inhibits CASMC migration induced by growth medium.GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.

  18. Effects of celecoxib on proliferation and tenocytic differentiation of tendon-derived stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Kairui; Zhang, Sheng [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Li, Qianqian [Cancer Research Institute, Southern Medical University, Guangzhou 510515 (China); Yang, Jun [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Department of Orthopaedics, 421 Hospital of PLA, Guangzhou 510318 (China); Dong, Weiqiang [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Department of Orthopaedics, The First Affiliated Hospital to Guangzhou Medical University, Guangzhou 510120 (China); Wang, Shengnan; Cheng, Yirong; Al-Qwbani, Mohammed [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China); Wang, Qiang, E-mail: 1780468505@qq.com [Department of Orthopaedics, Subei People’s Hospital of Jiangsu Province (Clinical Medical College of Yangzhou University), Yangzhou, Jiangsu Province 225001 (China); Yu, Bin, E-mail: carryzhang1985@live.com [Department of Orthopaedics and Traumatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515 (China)

    2014-07-18

    Highlights: • Celecoxib has no effects on TDSCs cell proliferation in various concentrations. • Celecoxib reduced mRNAs levels of tendon associated transcription factor. • Celecoxib reduced mRNAs levels of main tendon associated collagen. • Celecoxib reduced mRNAs levels of tendon associated molecules. - Abstract: NSAIDs are often ingested to reduce the pain and improve regeneration of tendon after tendon injury. Although the effects of NSAIDs in tendon healing have been reported, the data and conclusions are not consistent. Recently, tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and has been suggested involved in tendon repair. Our study aims to determine the effects of COX-2 inhibitor (celecoxib) on the proliferation and tenocytic differentiation of TDSCs. TDSCs were isolated from mice Achilles tendon and exposed to celecoxib. Cell proliferation rate was investigated at various concentrations (0.1, 1, 10 and 100 μg/ml) of celecoxib by using hemocytometer. The mRNA expression of tendon associated transcription factors, tendon associated collagens and tendon associated molecules were determined by reverse transcription-polymerase chain reaction. The protein expression of Collagen I, Collagen III, Scleraxis and Tenomodulin were determined by Western blotting. The results showed that celecoxib has no effects on TDSCs cell proliferation in various concentrations (p > 0.05). The levels of most tendon associated transcription factors, tendon associated collagens and tendon associated molecules genes expression were significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). Collagen I, Collagen III, Scleraxis and Tenomodulin protein expression were also significantly decreased in celecoxib (10 μg/ml) treated group (p < 0.05). In conclusion, celecoxib inhibits tenocytic differentiation of tendon-derived stem cells but has no effects on cell proliferation.

  19. Inhibition of glutamate regulated calcium entry into leukemic megakaryoblasts reduces cell proliferation and supports differentiation.

    Science.gov (United States)

    Kamal, Tania; Green, Taryn N; Morel-Kopp, Marie-Christine; Ward, Christopher M; McGregor, Ailsa L; McGlashan, Susan R; Bohlander, Stefan K; Browett, Peter J; Teague, Lochie; During, Matthew J; Skerry, Timothy M; Josefsson, Emma C; Kalev-Zylinska, Maggie L

    2015-09-01

    Human megakaryocytes release glutamate and express glutamate-gated Ca(2+)-permeable N-methyl-D-aspartate receptors (NMDARs) that support megakaryocytic maturation. While deregulated glutamate pathways impact oncogenicity in some cancers, the role of glutamate and NMDARs in megakaryocytic malignancies remains unknown. The aim of this study was to determine if NMDARs participate in Ca(2+) responses in leukemic megakaryoblasts and if so, whether modulating NMDAR activity could influence cell growth. Three human cell lines, Meg-01, Set-2 and K-562 were used as models of leukemic megakaryoblasts. NMDAR components were examined in leukemic cells and human bone marrow, including in megakaryocytic disease. Well-established NMDAR modulators (agonists and antagonists) were employed to determine NMDAR effects on Ca(2+) flux, cell viability, proliferation and differentiation. Leukemic megakaryoblasts contained combinations of NMDAR subunits that differed from normal bone marrow and the brain. NMDAR agonists facilitated Ca(2+) entry into Meg-01 cells, amplified Ca(2+) responses to adenosine diphosphate (ADP) and promoted growth of Meg-01, Set-2 and K-562 cells. Low concentrations of NMDAR inhibitors (riluzole, memantine, MK-801 and AP5; 5-100μM) were weakly cytotoxic but mainly reduced cell numbers by suppressing proliferation. The use-dependent NMDAR inhibitor, memantine (100μM), reduced numbers and proliferation of Meg-01 cells to less than 20% of controls (IC50 20μM and 36μM, respectively). In the presence of NMDAR inhibitors cells acquired morphologic and immunophenotypic features of megakaryocytic differentiation. In conclusion, NMDARs provide a novel pathway for Ca(2+) entry into leukemic megakaryoblasts that supports cell proliferation but not differentiation. NMDAR inhibitors counteract these effects, suggesting a novel opportunity to modulate growth of leukemic megakaryoblasts. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. The Effect of Laser Irradiation on Adipose Derived Stem Cell Proliferation and Differentiation

    Science.gov (United States)

    Abrahamse, H.; de Villiers, J.; Mvula, B.

    2009-06-01

    There are two fundamental types of stem cells: Embryonic Stem cells and Adult Stem cells. Adult Stem cells have a more restricted potential and can usually differentiate into a few different cell types. In the body these cells facilitate the replacement or repair of damaged or diseased cells in organs. Low intensity laser irradiation was shown to increase stem cell migration and stimulate proliferation and it is thought that treatment of these cells with laser irradiation may increase the stem cell harvest and have a positive effect on the viability and proliferation. Our research is aimed at determining the effect of laser irradiation on differentiation of Adipose Derived Stem Cells (ADSCs) into different cell types using a diode laser with a wavelength of 636 nm and at 5 J/cm2. Confirmation of stem cell characteristics and well as subsequent differentiation were assessed using Western blot analysis and cellular morphology supported by fluorescent live cell imaging. Functionality of subsequent differentiated cells was confirmed by measuring adenosine triphosphate (ATP) production and cell viability.

  1. Blue-light irradiation regulates proliferation and differentiation in human skin cells.

    Science.gov (United States)

    Liebmann, Joerg; Born, Matthias; Kolb-Bachofen, Victoria

    2010-01-01

    Sunlight influences the physiology of the human skin in beneficial as well as harmful ways, as has been shown for UV light. However, little is known about the effects of other wavelengths of solar irradiation. In this study we irradiated human keratinocytes and skin-derived endothelial cells with light-emitting-diode devices of distinct wavelengths to study the effects on cell physiology. We found that light at wavelengths of 632-940 nm has no effect, but irradiation with blue light at 412-426 nm exerts toxic effects at high fluences. Light at 453 nm is nontoxic up to a fluence of 500 J/cm(2). At nontoxic fluences, blue light reduces proliferation dose dependently by up to 50%, which is attributable to differentiation induction as shown by an increase of differentiation markers. Experiments with BSA demonstrate that blue-light irradiation up to 453 nm photolytically generates nitric oxide (NO) from nitrosated proteins, which is known to initiate differentiation in skin cells. Our data provide evidence for a molecular mechanism by which blue light may be effective in treating hyperproliferative skin conditions by reducing proliferation due to the induction of differentiation. We observed a photolytic release of NO from nitrosated proteins, indicating that they are light acceptors and signal transducers up to a wavelength of 453 nm.

  2. Neurogenesis and Increase in Differentiated Neural Cell Survival via Phosphorylation of Akt1 after Fluoxetine Treatment of Stem Cells

    Directory of Open Access Journals (Sweden)

    Anahita Rahmani

    2013-01-01

    Full Text Available Fluoxetine (FLX is a selective serotonin reuptake inhibitor (SSRI. Its action is possibly through an increase in neural cell survival. The mechanism of improved survival rate of neurons by FLX may relate to the overexpression of some kinases such as Akt protein. Akt1 (a serine/threonine kinase plays a key role in the modulation of cell proliferation and survival. Our study evaluated the effects of FLX on mesenchymal stem cell (MSC fate and Akt1 phosphorylation levels in MSCs. Evaluation tests included reverse transcriptase polymerase chain reaction, western blot, and immunocytochemistry assays. Nestin, MAP-2, and β-tubulin were detected after neurogenesis as neural markers. Ten μM of FLX upregulated phosphorylation of Akt1 protein in induced hEnSC significantly. Also FLX did increase viability of these MSCs. Continuous FLX treatment after neurogenesis elevated the survival rate of differentiated neural cells probably by enhanced induction of Akt1 phosphorylation. This study addresses a novel role of FLX in neurogenesis and differentiated neural cell survival that may contribute to explaining the therapeutic action of fluoxetine in regenerative pharmacology.

  3. Proliferation and differentiation potential of mouse adult hepatic progenitor cells cultured in vitro.

    Science.gov (United States)

    Song, Lujun; Wang, Hongshan; Gao, Xiaodong; Shen, Kuntang; Niu, Weixin; Qin, Xinyu

    2010-02-01

    This study aimed to isolate the stem cells or progenitors, if exist, from normal adult mouse liver and investigate their potential of proliferation and differentiation. Hepatocytes were isolated by modified two-step liver perfusion method and centrifugation, and then cultured in modified serumcontaining DMEM for observation more than 60 days. Immunofluorescence technique was applied to check the hepatocytes and to examine the formation of colonies with albumin, alpha-fetoprotein (AFP) and cytokeratin 19 (CK19). Results showed that some hepatocytes that were strongly positive for hepatocyte specific markers albumin on Day 1 in culture, could be activated at Days 2-3, followed by rapid proliferation and formation of colonies. The colonies could expand continually for more than 60 days. On Day 5, all the cells in the colony expressed hepatic stem cell (HSC) markers AFP. With the time of culture, some cells in colonies lost ability to divide at Days 13-15, and differentiated into cells which had a large cytoplasm and some two nuclei, similar to the appearance of mature hepatocytes morphologically. These differentiated cells demonstrated strong expression of albumin. Around Day 30, some big cells appeared in colonies and expressed bile duct cell marker CK19. Therefore, this subpopulation of mouse hepatocytes could acquire some characteristics of immature hepatocytes and showed the profile of hepatic progenitor cells with a high proliferating ability and bi-potential of differentiation. They were isolated from normal adult mouse, hence, named adult hepatic progenitor cells (AHPCs). Mouse AHPCs may be used as an HSC model for hepatocytes transplantation and hepatopathy study.

  4. Effects of Wnt-10b on proliferation and differentiation of murine melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Misu, Masayasu [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ouji, Yukiteru, E-mail: oujix@naramed-u.ac.jp [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Kawai, Norikazu [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Nishimura, Fumihiko [Department of Neurosurgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Nakamura-Uchiyama, Fukumi [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Yoshikawa, Masahide, E-mail: myoshika@naramed-u.ac.jp [Department of Pathogen, Infection and Immunity, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan)

    2015-08-07

    In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cells in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. - Highlights: • Wnt-10b inhibited proliferation and migration of melanoma cells. • Wnt-10b induced tyrosinase activity and senescence of melanoma cells. • Wnt-10b suppressed growth of cell aggregates in collagen 3D-gel cultures. • Wnt-10b represses tumor cell properties, driving them toward a more differentiated state along a melanocyte lineage.

  5. Differential gene expression in apoptosis: identification of ribosomal protein 23K, a cell proliferation inhibitor.

    Science.gov (United States)

    Chen, F W; Davies, J P; Ioannou, Y A

    1998-08-01

    Gene expression during the camptothecin-induced apoptotic death of human leukemic U937 cells and mouse T-cell hybridoma QW4.1 cells was studied by the mRNA differential display technique. Ten clones were confirmed to be differentially expressed, nine of which encoded novel sequences. One clone, U3.2, was induced approximately 10-fold in camptothecin-treated cells and was found to be identical to a highly basic 23-kDa human protein which contains basic leucine zipper-like motifs and has recently been identified as the human homologue of the rat ribosomal protein L13a. Northern blot analysis revealed a major mRNA of approximately 0.9 kb and a minor mRNA of approximately 1.3 kb. Overexpression of a full-length 23K cDNA, tagged with a FLAG sequence, in COS-7 cells revealed a predominantly nucleolar localization and the absence of any 23K protein from the cytoplasm. Subsequent transfection studies, using antisense phosphorothioate-modified oligonucleotides, revealed that inhibition of 23K expression results in an increased cell proliferation and greater sensitivity of U937 cells to the effects of camptothecin-induced cell death. Upregulation of 23K expression using a cDNA construct resulted in a decrease in cell proliferation and growth arrest, suggesting a role for 23K protein as a proliferation checkpoint following a cellular insult. Copyright 1998 Academic Press.

  6. Selective estrogen receptor down-regulator and selective estrogen receptor modulators differentially regulate lactotroph proliferation.

    Directory of Open Access Journals (Sweden)

    Sanjay Kansra

    2010-04-01

    Full Text Available We recently reported that estrogen receptor alpha (ERalpha, even in absence of estrogen (E2, plays a critical role in lactotroph homeostasis. The anti-estrogen ICI 182780 (ICI, but not tamoxifen or raloxifene, rapidly promoted the degradation of ERalpha, and inhibited cell proliferation. However, all three ER antagonists suppressed PRL release, suggesting that receptor occupation is sufficient to inhibit prl gene expression whereas receptor degradation is required to suppress lactotroph proliferation. In this study our objective was to determine whether ERalpha degradation versus occupation, differentially modulates the biological outcome of anti-estrogens.Using the rat lactotroph cell line, GH3 cells, we report that ICI induced proteosome mediated degradation of ERalpha. In contrast, an ERalpha specific antagonist, MPP, that does not promote degradation of ERalpha, did not inhibit cell proliferation. Further, ICI, but not MPP, abolished anchorage independent growth of GH3 cells. Yet, both ICI and MPP were equally effective in suppressing prl expression and release, as well as ERE-mediated transcriptional activity.Taken together, our results demonstrate that in lactotrophs, ERalpha degradation results in decreased cell proliferation, whereas ERalpha occupation by an antagonist that does not promote degradation of ERalpha is sufficient to inhibit prl expression.

  7. Human fibroblasts treated with hydrogen peroxide stimulate human melanoblast proliferation and melanocyte differentiation, but inhibit melanocyte proliferation in serum-free co-culture system.

    Science.gov (United States)

    Hirobe, Tomohisa; Shibata, Tatako; Sato, Kiyoshi

    2016-12-01

    Oxidative stress caused by hydrogen peroxide (H2O2) elicits harmful effects on human melanocytes such as DNA damage and cell death. On the contrary, H2O2 is known to possess beneficial effects on melanocytes. However, mechanisms of the beneficial effects of H2O2 on melanocytes have not been fully understood, especially the indirect effects on melanocyte proliferation and differentiation from cells constituting surrounding tissue environment such as fibroblasts. The aim of this study was to clarify whether H2O2-treated human fibroblasts affect the proliferation and differentiation of human melanocytes using serum-free co-culture system. Epidermal melanoblasts and melanocytes were co-cultured with H2O2-treated or control fibroblasts in serum-free culture media. The effects of H2O2-treated fibroblasts were detected by changes in the proliferation and differentiation of melanoblasts/melanocytes. H2O2-treated fibroblasts stimulated the proliferation of melanoblasts and the differentiation, melanogenesis, and dendritogenesis of melanocytes, but inhibited the proliferation of melanocytes. In the melanocytes co-cultured with H2O2-treated fibroblasts, the expression of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and KIT was increased, whereas TYRP2 and microphthalmia-associated transcription factor showed no change. These results suggest that H2O2-treated fibroblasts can produce and release some mitogenic and melanogenic factors toward melanoblasts in addition to some proliferation-inhibiting factors toward melanocytes. The stimulation of melanocyte differentiation seems to be performed through the upregulation of TYR, TYRP1, and KIT. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  8. Differential expression of store-operated calcium- and proliferation-related genes in hepatocellular carcinoma cells following TRPC1 ion channel silencing.

    Science.gov (United States)

    Selli, Cigdem; Pearce, Dominic A; Sims, Andrew H; Tosun, Metiner

    2016-09-01

    TRPC1 and store-operated Ca(2+) (SOC) entry have previously been associated with hepatocellular carcinoma cell proliferation. The aim of the study was to determine genes and processes associated with TRPC1 down-regulation and the resulting increase of SOC entry and decrease in hepatocellular carcinoma cell proliferation. For this purpose, transcriptome analysis was performed to determine differentially expressed genes in TRPC1-silenced Huh7 cells. SOC entry- and proliferation-related genes correlated with TRPC1 down-regulation were also examined. Changes in SOC entry and cell proliferation were monitored in the TRPC1-silenced and parental cells and found to be significantly increased and decreased, respectively, in TRPC1-silenced cells. A total of 71 genes were significantly differentially expressed (40 up- and 31 down-regulated), including four mitogen-activated protein kinase (MAPK) signalling-associated genes. STIM1 levels were significantly up-regulated and negatively correlated with TRPC1 levels. In addition, expression of two cell cycle regulation genes, CDK11A/11B and URGCP, was observed to decrease, whereas ERBB3 and FGFR4, pro-survival genes, increased significantly in TRPC1-silenced cells. In conclusion, these results suggest reciprocal alterations in TRPC1 and STIM1 levels and a role for STIM1 in the regulation of SOC entry in TRPC1-silenced Huh7 cells. In addition to TRPC1, STIM1 may participate in Huh7 cell proliferation by regulating SOC entry. Alterations in MAPK signalling genes may be involved in diminished cell proliferation in TRPC1-silenced Huh7 cells. Similarly, changes in cell cycle regulating genes in TRPC1-silenced cells indicate possible cell cycle arrest along with compensatory up-regulation of ERBB3 growth factor receptor-amongst others-to maintain hepatocellular carcinoma cell proliferation.

  9. Epidermal cell proliferation and terminal differentiation in skin organ culture after topical exposure to sodium dodecyl sulphate

    NARCIS (Netherlands)

    Sandt, J.J.M. van de; Bos, T.A.; Rutten, A.A.J.J.L.

    1995-01-01

    Epidermal cell proliferation and differentiation were investigated in vitro after exposure to the anionic surfactant sodium dodecyl sulfate (SDS). Human skin organ cultures were exposed topically to various concentrations of SDS for 22 h, after which the irritant was removed. Cell proliferation was

  10. Virgin olive oil phenols inhibit proliferation of human promyelocytic leukemia cells (HL60) by inducing apoptosis and differentiation

    National Research Council Canada - National Science Library

    Fabiani, Roberto; De Bartolomeo, Angelo; Rosignoli, Patrizia; Servili, Maurizio; Selvaggini, Roberto; Montedoro, Gian Francesco; Di Saverio, Cristina; Morozzi, Guido

    2006-01-01

    .... In the present study, we investigated the effect of a virgin olive oil phenol extract (PE) on proliferation, the cell cycle distribution profile, apoptosis, and differentiation of the human promyelocytic cell line HL60...

  11. Survival and proliferation characteristics of the microalga Chlamydomonas sp. ICE-L after hypergravitational stress pretreatment

    Science.gov (United States)

    Gao, Zhengquan; Li, Demao; Meng, Chunxiao; Xu, Dong; Zhang, Xiaowen; Ye, Naihao

    2013-09-01

    Seeking extraterrestrial life, transferring between planets, even migrating to other planets attracts more and more attention of public and scientists. However, to make it clear for the ability to survive the forces studies is prerequisite to enable the speculations by natural means. Gravity is a critical force involved in all the life on Earth and, possibly, others planets. Organisms have been grown in microgravity habitats and in centrifuges to characterize the biological response to a range of gravitational forces and radiation levels in space and on Earth. However, little is known about the profiles of eukaryotic life under conditions of hyperacceleration attributable to extreme gravities. In this study, a eukaryotic extremophile, the Antarctic green microalga Chlamydomonas sp. ICE-L, showed amazing proliferation capacity during and after hypergravitational stress for 30 min to 48 h at 110,200, 423,400, and 670,800g. These extreme gravities also had profound effects on viability, reproduction rate, photosynthesis efficiency, and gene transcriptional expression of this microalga. Most notably, all three supergravities efficiently stimulated algal cell division, but the greater the centrifugal force and the longer the duration of treatment, the lower the viable rate and breeding potential of samples in the following incubation. These results illustrated Chlamydomonas sp. ICE-L is a useful eukaryotic model system candidate for space research. Further studies could provide new insight into the physical limits of life and its evolution and enhance the possibility for interstellar space travel and the quest for extraterrestrial life according to panspermia theory. Also, it indicated that life come from the outer space is not always prokaryotes but may be eukaryotes.

  12. Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production

    Directory of Open Access Journals (Sweden)

    Benson Heather L

    2012-03-01

    Full Text Available Abstract Background Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs and plasmacytoid (pDCs are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown. Methods Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2d prior to transplanting into C57BL/6 mice (H-2b, followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+. Results Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production. Conclusion Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.

  13. The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway

    Directory of Open Access Journals (Sweden)

    María F. Organista

    2012-10-01

    The expression of the spalt genes is regulated by the Decapentaplegic signalling pathway in the Drosophila wing. These genes participate in the patterning of the longitudinal wing veins by regulating the expression of vein-specific genes, and in the establishment of cellular affinities in the central region of the wing blade epithelium. The Spalt proteins act as transcription factors, most likely regulating gene expression by repression, but the identity of their target genes in the wing is still unknown. As a preliminary step to unravel the genetic hierarchy controlled by the Spalt proteins, we have analysed their requirements during wing development, and addressed to what extent they mediate all the functions of the Decapentaplegic pathway in this developmental system. We identify additional functions for Spalt in cell division, survival, and maintenance of epithelial integrity. Thus, Spalt activity is required to promote cell proliferation, acting in the G2/M transition of the cell cycle. The contribution of Spalt to cell division is limited to the central region of the wing blade, as they do not mediate the extra growth triggered by Decapentaplegic signalling in the peripheral regions of the wing disc. In addition, Spalt function is required to maintain cell viability in cells exposed to high levels of Decapentaplegic signalling. This aspect of Spalt function is related to the repression of JNK signalling in the spalt domain of expression. Finally, we further characterise the requirements of Spalt to maintain epithelial integrity by regulating cellular affinities between cells located in the central wing region. Our results indicate that Spalt function mediates most of the requirements identified for Decapentaplegic signalling, contributing to establish the cellular qualities that differentiate central versus peripheral territories in the wing blade.

  14. Critical roles of hMAGEA2 in induced pluripotent stem cell pluripotency, proliferation, and differentiation.

    Science.gov (United States)

    Park, Song; Sung, Yonghun; Jeong, Jain; Choi, Minjee; Lee, Jinhee; Kwon, Wookbong; Jang, Soyoung; Park, Si Jun; Kim, Jae Young; Kim, Sung Hyun; Yoon, Duhak; Ryoo, Zae Young; Kim, Myoung Ok

    2017-09-11

    Induced pluripotent stem (iPS) cells are important for clinical application and stem cell research. Although human melanoma-associated antigen A2 (hMAGEA2) expression is known to affect differentiation in embryonic stem cells, its specific role in iPS cells remains unclear. To evaluate the function of hMAGEA2 and its characteristics in iPS cells, we produced hMAGEA2-overexpressing iPS cells from hMAGEA2-overexpressing transgenic mice. Although the iPS cells with overexpressed hMAGEA2 did not differ in morphology, their pluripotency, and self-renewal related genes (Nanog, Oct3/4, Sox2, and Stat3), expression level was significantly upregulated. Moreover, hMAGEA2 contributed to the promotion of cell cycle progression, thereby accelerating cell proliferation. Through embryoid body formation in vitro and teratoma formation in vivo, we demonstrated that hMAGEA2 critically decreases the differentiation ability of iPS cells. These data indicate that hMAGEA2 intensifies the self-renewal, pluripotency, and degree of proliferation of iPS cells, while significantly repressing their differentiation efficiency. Therefore, our findings prove that hMAGEA2 plays key roles in iPS cells. Copyright © 2017 John Wiley & Sons, Ltd.

  15. Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Handgretinger Rupert

    2010-01-01

    Full Text Available Abstract Background Human multipotent mesenchymal stromal cells (MSC can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity. Results After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation. Conclusion Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

  16. Suppressing Pitx2 inhibits proliferation and promotes differentiation of iHepSCs.

    Science.gov (United States)

    Chen, Fei; Yao, Hao; Wang, Minjun; Yu, Bing; Liu, Qinggui; Li, Jianxiu; He, Zhiying; Hu, Yi-Ping

    2016-11-01

    Induced hepatic stem cells (iHepSCs) have great potential as donors for liver cell therapy due to their abilities for self-renewal and bi-potential differentiation. However, the molecular mechanism regulating proliferation and differentiation of iHepSCs is poorly understood. In this study, we provide evidence that the homeodomain transcription factor, Pitx2, is essential to maintain iHepSCs stem cell characteristics. Suppressing Pitx2 expression in iHepSCs by lentivirus mediated specific shRNA markedly reduced the expression of the hepatic stem cell-associated genes (Lgr5, EpCAM, and Sox9) with concomitant inhibition of proliferation by blocking the G1/S phase transition, and these phenotypic changes were reversed upon re-expression of Pitx2. Pitx2 knockdown also resulted in up-regulation of the p53-induced Cdk inhibitor p21, and down-regulation of its downstream effector CDK2-Cyclin E kinase complex. Furthermore, we observed that iHepSCs were more efficiently induced to differentiate into both hepatocytes and cholangiocytes when Pitx2 expression was suppressed, as compared to unmanipulated iHepSCs. These findings reveal that Pitx2 expression may be leveraged to control the status of iHepSCs during expansion in vitro to provide a strategy for further application of iHepSCs in liver cell therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Neuron-NG2 Cell Synapses: Novel Functions for Regulating NG2 Cell Proliferation and Differentiation

    Directory of Open Access Journals (Sweden)

    Qian-Kun Yang

    2013-01-01

    Full Text Available NG2 cells are a population of CNS cells that are distinct from neurons, mature oligodendrocytes, astrocytes, and microglia. These cells can be identified by their NG2 proteoglycan expression. NG2 cells have a highly branched morphology, with abundant processes radiating from the cell body, and express a complex set of voltage-gated channels, AMPA/kainate, and GABA receptors. Neurons notably form classical and nonclassical synapses with NG2 cells, which have varied characteristics and functions. Neuron-NG2 cell synapses could fine-tune NG2 cell activities, including the NG2 cell cycle, differentiation, migration, and myelination, and may be a novel potential therapeutic target for NG2 cell-related diseases, such as hypoxia-ischemia injury and periventricular leukomalacia. Furthermore, neuron-NG2 cell synapses may be correlated with the plasticity of CNS in adulthood with the synaptic contacts passing onto their progenies during proliferation, and synaptic contacts decrease rapidly upon NG2 cell differentiation. In this review, we highlight the characteristics of classical and nonclassical neuron-NG2 cell synapses, the potential functions, and the fate of synaptic contacts during proliferation and differentiation, with the emphasis on the regulation of the NG2 cell cycle by neuron-NG2 cell synapses and their potential underlying mechanisms.

  18. Slit/Robo1 signaling regulates neural tube development by balancing neuroepithelial cell proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Guang; Li, Yan; Wang, Xiao-yu [Key Laboratory for Regenerative Medicine of The Ministry of Education, Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou 510632 (China); Han, Zhe [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Chuai, Manli [College of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH (United Kingdom); Wang, Li-jing [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Ho Lee, Kenneth Ka [Stem Cell and Regeneration Thematic Research Programme, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin (Hong Kong); Geng, Jian-guo, E-mail: jgeng@umich.edu [Institute of Vascular Biological Sciences, Guangdong Pharmaceutical University, Guangzhou 510224 (China); Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109 (United States); Yang, Xuesong, E-mail: yang_xuesong@126.com [Key Laboratory for Regenerative Medicine of The Ministry of Education, Department of Histology and Embryology, School of Medicine, Jinan University, Guangzhou 510632 (China)

    2013-05-01

    development by tightly coordinating cell proliferation and differentiation during neurulation. - Highlights: ► The role of Slit/Robo1 signaling was investigated with chick and mouse models. ► Disturbance of Slit/Robo1 signaling resulted in neural tube defects. ► Slit/Robo1 signaling regulated the proliferation of neural tube cells. ► Slit/Robo1 signaling modulated the differentiation of neural tube cells. ► Slit/Robo1 signaling balanced the proliferation and differentiation of neural tube.

  19. Lack of potassium channel induces proliferation and survival causing increased neurogenesis and two-fold hippocampus enlargement

    DEFF Research Database (Denmark)

    Almgren, Malin; Persson, Ann-Sophie; Fenghua, Chen

    2007-01-01

    and the second week of life. To investigate the hyperplasia, cell proliferation was studied within the subgranular zone of the DG using BrdU and Ki67. There was a three-fold increase in proliferation in mceph/mceph mice compared to wild type mice at an age before onset of epileptic symptoms (3 weeks...... was lower in mceph/mceph supporting additional overgrowth mechanism than induced by seizures. In conclusion, lack of a functional Kv1.1 ion channel subunit in the mceph/mceph mice causes a unique neuronal hyperplasia in distinct hippocampal regions and consequently hippocampal enlargement from 2 to 3 weeks...... of age. This phenotype is a result, at least in DG, from increased proliferation, neurogenesis, and enhanced general hippocampal cell survival. Udgivelsesdato: 2007-null...

  20. Effects of strontium on proliferation and differentiation of rat bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yunfeng; Li, Jihua; Zhu, Songsong; Luo, En; Feng, Ge; Chen, Qianming [State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, No. 14, Section 3, Southern Renmin Road, Chengdu 610041 (China); Hu, Jing, E-mail: drhu@vip.sohu.com [State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, No. 14, Section 3, Southern Renmin Road, Chengdu 610041 (China)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer Strontium ranelate (SrR) inhibits proliferation of BMMSCs. Black-Right-Pointing-Pointer SrR increases osteoblastic but decreases adipocytic differentiation of BMMSCs. Black-Right-Pointing-Pointer SrR increases expression of Runx2, BSP and OCN by BMMSCs in osteogenic medium. Black-Right-Pointing-Pointer SrR decreases expression of PPAR{gamma}, aP2/ALBP and LPL by BMMSCs in adipogenic medium. -- Abstract: Strontium ranelate (SrR) was an effective anti-osteoporotic drug to increase bone formation and decrease bone resorption. However, reports about the effect of SR on osteoblastic and adipocytic differentiation from bone marrow mesenchymal stem cells (BMMSCs) are limited. The purpose of this study is to evaluate whether SrR affects the ability of BMMSCs to differentiate into osteoblasts or adipocytes. Rat BMMSCs were identified by flow cytometry and exposed to SR (0.1 and 1.0 mM Sr{sup 2+}) under osteogenic or adipogenic medium for 1 and 2 weeks. The proliferation and differentiation of BMMSCs were analyzed by MTT, alkaline phosphatase (ALP), Oil red O staining, quantitative real-time RT-PCR and Western blot assays. SrR significantly inhibited the proliferation, increased osteoblastic but decreased adipocytic differentiation of rat BMMSCs dose-dependently. In osteogenic medium, SrR increased the expression of ALP, the mRNA levels of Cbfa1/Runx2, bone sialoprotein, and osteocalcin by RT-PCR, and the protein levels of Cbfa1/Runx2 by Western blot. In adipogenic medium, SrR decreased the mRNA levels of PPAR{gamma}2, adipocyte lipid-binding protein 2 (aP2/ALBP), and lipoprotein lipase (LPL) by RT-PCR, and the protein expression of PPAR{gamma} in Western blot analysis. These results indicated that the effects of SrR to promote osteoblastic but inhibit adipocytic differentiation of BMMSCs might contribute to its effect on osteoporosis treatment.

  1. MicroRNA-378 regulates neural stem cell proliferation and differentiation in vitro by modulating Tailless expression

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yanxia [Department of Psychology and Psychiatry, The Second Affiliated Hospital of Xi' an Jiaotong University, Xi' an 710004 (China); Department of Rehabilitation, Xi' an Children' s Hospital, Xi' an 710003 (China); Liu, Xiaoguai [The 3rd Department of Infectious Diseases, Xi' an Children' s Hospital, Xi' an 710003 (China); Wang, Yaping, E-mail: yapwangyy@163.com [Department of Psychology and Psychiatry, The Second Affiliated Hospital of Xi' an Jiaotong University, Xi' an 710004 (China)

    2015-10-16

    Previous studies have suggested that microRNAs (miRNAs) play an important role in regulating neural stem cell (NSC) proliferation and differentiation. However, the precise role of miRNAs in NSC remains largely unexplored. In this study, we showed that miR-378 can target Tailless (TLX), a critical regulator of NSC, to regulate NSC proliferation and differentiation. By bioinformatic algorithms, miR-378 was found to have a predicted target site in the 3′-untranslated region of TLX, which was verified by a dual-luciferase reporter assay. The expression of miR-378 was increased during NSC differentiation and inversely correlated with TLX expression. qPCR and Western blot analysis also showed that miR-378 negatively regulated TLX mRNA and protein expression in neural stem cells (NSCs). Intriguingly, overexpression of miR-378 increased NSC differentiation and reduced NSC proliferation, whereas suppression of miR-378 led to decreased NSC differentiation and increased NSC proliferation. Moreover, the downstream targets of TLX, including p21, PTEN and Wnt/β-catenin were also found to be regulated by miR-378. Additionally, overexpression of TLX rescued the NSC proliferation deficiency induced by miR-378 overexpression and abolished miR-378-promoted NSC differentiation. Taken together, our data suggest that miR-378 is a novel miRNA that regulates NSC proliferation and differentiation via targeting TLX. Therefore, manipulating miR-378 in NSCs could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders. - Highlights: • miR-378 targeted and regulated TLX. • miR-378 was increased during NSC differentiation. • miR-378 regulated NSC proliferation and differentiation. • miR-378 regulated NSC self-renew through TLX.

  2. Neuronal adhesion, proliferation and differentiation of embryonic stem cells on hybrid scaffolds made of xanthan and magnetite nanoparticles.

    Science.gov (United States)

    Glaser, Talita; Bueno, Vânia B; Cornejo, Daniel R; Petri, Denise F S; Ulrich, Henning

    2015-07-08

    Hybrid scaffolds made of xanthan and magnetite nanoparticles (XCA/mag) were prepared by dipping xanthan membranes (XCA) into dispersions of magnetic nanoparticles for different periods of time. The resulting hybrid scaffolds presented magnetization values ranging from 0.25 emu g(-1) to 1.80 emu g(-1) at 70 kOe and corresponding iron contents ranging from 0.25% to 2.3%, respectively. They were applied as matrices for in vitro embryoid body adhesion and neuronal differentiation of embryonic stem cells; for comparison, neat XCA and commercial plastic plates were also used. Adhesion rates were more pronounced when cells were seeded on XCA/mag than on neat XCA or plastic dishes; however, proliferation levels were independent from those of the scaffold type. Embryonic stem cells showed similar differentiation rates on XCA/mag scaffolds with magnetization of 0.25 and 0.60 emu g(-1), but did not survive on scaffolds with 1.80 emu g(-1). Differentiation rates, expressed as the number of neurons obtained on the chosen scaffolds, were the largest on neat XCA, which has a high density of negative charge, and were smallest on the commercial plastic dishes. The local magnetic field inherent of magnetite particles present on the surface of XCA/mag facilitates synapse formation, because synaptophysin expression and electrical transmission were increased when compared to the other scaffolds used. We conclude that XCA/mag and XCA hydrogels are scaffolds with distinguishable performance for adhesion and differentiation of ESCs into neurons.

  3. The transition from proliferation to differentiation in colorectal cancer is regulated by the calcium activated chloride channel A1.

    Directory of Open Access Journals (Sweden)

    Bo Yang

    Full Text Available Breaking the balance between proliferation and differentiation in animal cells can lead to cancer, but the mechanisms maintaining this balance remain largely undefined. The calcium activated chloride channel A1 (CLCA1 is a member of the calcium sensitive chloride conductance family of proteins and is expressed mainly in the colon, small intestine and appendix. We show that CLCA1 plays a functional role in differentiation and proliferation of Caco-2 cells and of intestinal tissue. Caco-2 cells spontaneously differentiate either in confluent culture or when treated with butyrate, a molecule present naturally in the diet. Here, we compared CLCA1 expressional levels between patients with and without colorectal cancer (CRC and determined the functional role of CLCA1 in differentiation and proliferation of Caco-2 cells. We showed that: 1 CLCA1 and CLCA4 expression were down-regulated significantly in CRC patients; 2 CLCA1 expression was up-regulated in Caco-2 cells induced to differentiate by confluent culture or by treatment with sodium butyrate (NaBT; 3 Knockdown of CLCA1 with siRNA significantly inhibited cell differentiation and promoted cell proliferation in Caco-2 confluent cultures, and 4 In Caco-2 3D culture, suppression of CLCA1 significantly increased cell proliferation and compromised NaBT-induced inhibition of proliferation. In conclusion, CLCA1 may contribute to promoting spontaneous differentiation and reducing proliferation of Caco-2 cells and may be a target of NaBT-induced inhibition of proliferation and therefore a potential diagnostic marker for CRC prognosis.

  4. Blockage of Wnt/β-Catenin Signaling by Nanoparticles Reduces Survival and Proliferation of CLL Cells In Vitro-Preliminary Study.

    Science.gov (United States)

    Franiak-Pietryga, Ida; Maciejewski, Henryk; Ziemba, Barbara; Appelhans, Dietmar; Voit, Brigitte; Robak, Tadeusz; Jander, Magdalena; Treliński, Jacek; Bryszewska, Maria; Borowiec, Maciej

    2017-11-01

    The Wnt/β-catenin signaling pathway is shown to play a significant role in the control of the survival, proliferation, and differentiation of hematopoietic cells. Studies have confirmed that aberrant activation of canonical Wnt signaling occurs in various forms of leukemia, and is crucial for chronic lymphocytic leukemia (CLL) pathogenesis. The aim of the study is to evaluate the influence of maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) on Wnt/β-catenin pathway gene expression in CLL (MEC-1) cells and to compare these findings with those obtained with fludarabine (FA). Microarray data analysis reveals seven of 19 Wnt/β-catenin pathway genes whose expression changes significantly during dendrimer and FA treatment: WNT10A, WNT6, and CDH1 among others. PPI-G4-M3 is already known to influence MEC-1 cell apoptosis and proliferation. The obtained results suggest that the reduction in cell survival under the influence of glycodendrimers and FA may be due to loss of Wnt signaling. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. In situ normoxia enhances survival and proliferation rate of human adipose tissue-derived stromal cells without increasing the risk of tumourigenesis.

    Directory of Open Access Journals (Sweden)

    Jane Ru Choi

    Full Text Available Adipose tissue-derived stromal cells (ASCs natively reside in a relatively low-oxygen tension (i.e., hypoxic microenvironment in human body. Low oxygen tension (i.e., in situ normoxia, has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O2 concentration (21% O2 or in situ normoxia (2% O2. We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O2 concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O2 as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.

  6. Effect of matrix stiffness on the proliferation and differentiation of umbilical cord mesenchymal stem cells.

    Science.gov (United States)

    Xu, Juanjuan; Sun, Meiyu; Tan, Ye; Wang, Haowei; Wang, Heping; Li, Pengdong; Xu, Ziran; Xia, Yuhan; Li, Lisha; Li, Yulin

    Mesenchymal stem cells (MSCs) are a compatible cellular alternative for regenerative medicine and tissue engineering because of their powerful multipotency. Matrix stiffness plays a profound role on stem cell behavior. Nevertheless, the effect of matrix stiffness on umbilical cordmesenchymal stem cells (UC-MSCs) remains unexplored. To conduct an in-depth exploration, we cultured UC-MSCs on different stiffness (Young's modulus: 13-16, 35-38, 48-53, and 62-68 kPa) polyacrylamide gels coated with fibronectin. We found that the proliferation and adhesion of UC-MSCs varied when cultured on the different matrices, and the spreading capacity was stronger as the stiffness increased (*Pmatrix promoted adipogenic differentiation, with higher expression levels of adipocytic markers like PPARγ and C/EBPα (*Pmatrix, which was validated by increased expression of myogenic makers like desminand MOYG (*Pmatrix. Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  7. Sox2 Is Essential for Oligodendroglial Proliferation and Differentiation during Postnatal Brain Myelination and CNS Remyelination.

    Science.gov (United States)

    Zhang, Sheng; Zhu, Xiaoqing; Gui, Xuehong; Croteau, Christopher; Song, Lanying; Xu, Jie; Wang, Aijun; Bannerman, Peter; Guo, Fuzheng

    2018-02-14

    In the CNS, myelination and remyelination depend on the successful progression and maturation of oligodendroglial lineage cells, including proliferation and differentiation of oligodendroglial progenitor cells (OPCs). Previous studies have reported that Sox2 transiently regulates oligodendrocyte (OL) differentiation in the embryonic and perinatal spinal cord and appears dispensable for myelination in the postnatal spinal cord. However, the role of Sox2 in OL development in the brain has yet to be defined. We now report that Sox2 is an essential positive regulator of developmental myelination in the postnatal murine brain of both sexes. Stage-specific paradigms of genetic disruption demonstrated that Sox2 regulated brain myelination by coordinating upstream OPC population supply and downstream OL differentiation. Transcriptomic analyses further supported a crucial role of Sox2 in brain developmental myelination. Consistently, oligodendroglial Sox2-deficient mice developed severe tremors and ataxia, typical phenotypes indicative of hypomyelination, and displayed severe impairment of motor function and prominent deficits of brain OL differentiation and myelination persisting into the later CNS developmental stages. We also found that Sox2 was required for efficient OPC proliferation and expansion and OL regeneration during remyelination in the adult brain and spinal cord. Together, our genetic evidence reveals an essential role of Sox2 in brain myelination and CNS remyelination, and suggests that manipulation of Sox2 and/or Sox2-mediated downstream pathways may be therapeutic in promoting CNS myelin repair. SIGNIFICANCE STATEMENT Promoting myelin formation and repair has translational significance in treating myelin-related neurological disorders, such as periventricular leukomalacia and multiple sclerosis in which brain developmental myelin formation and myelin repair are severely affected, respectively. In this report, analyses of a series of genetic conditional

  8. Proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells.

    Science.gov (United States)

    Vogetseder, Alexander; Picard, Nicolas; Gaspert, Ariana; Walch, Michael; Kaissling, Brigitte; Le Hir, Michel

    2008-01-01

    We investigated the proliferative capacity of renal proximal tubular cells in healthy rats. Previously, we observed that tubular cells originate from differentiated cells. We now found 1) by application of bromo-deoxyuridine (BrdU) for 14 days and costaining for BrdU, and the G(1)-phase marker cyclin D1 that the bulk of cells in the S3 segment of juvenile rats were involved in proliferation; 2) that although the proliferation rate was about 10-fold higher in juvenile rats compared with adult rats, roughly 40% of S3 cells were in G(1) in both groups; 3) that after a strong mitotic stimulus (lead acetate), proliferation was similar in juveniles and adults; 4) that there was a high incidence of cyclin D1-positive cells also in the healthy human kidney; and 5) by labeling dividing cells with BrdU for 2 days before the application of lead acetate and subsequent costaining for BrdU and cell cycle markers, that, although a strong mitotic stimulus does not abolish the period of quiescence following division, it shortens it markedly. Thus the capacity of the proximal tubule to rapidly recruit cells into division relies on a large reserve pool of cells in G(1) and on the shortening of the obligatory period of quiescence that follows division.

  9. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Stefania Bruno

    2016-01-01

    Full Text Available Human liver stem cells (HLSCs are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs, and dendritic cells (DCs in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2 and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs, HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  10. miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells.

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    Emilio Satoshi Hara

    Full Text Available Dental pulp cells (DPCs are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP cells from human DPCs and periodontal ligament cells (PDLCs, and performed a locked nucleic acid (LNA-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs, which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.

  11. Retinoic acid and cAMP inhibit rat hepatocellular carcinoma cell proliferation and enhance cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ionta, M. [Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Alfenas MG (Brazil); Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo SP (Brazil); Rosa, M.C.; Almeida, R.B.; Freitas, V.M.; Rezende-Teixeira, P.; Machado-Santelli, G.M. [Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo SP (Brazil)

    2012-05-25

    Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation [E-cadherin, connexin 26 (Cx26), and connexin 32 (Cx32)]. RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.

  12. The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation.

    Directory of Open Access Journals (Sweden)

    Marie Smith

    Full Text Available The endocannabinoid system is expressed in bone, although its role in the regulation of bone growth is controversial. Many studies have examined the effect of endocannabinoids directly on osteoclast function, but few have examined their role in human osteoblast function, which was the aim of the present study. Human osteoblasts were treated from seeding with increasing concentrations of anandamide or 2-arachidonoylglycerol for between 1 and 21 days. Cell proliferation (DNA content and differentiation (alkaline phosphatase (ALP, collagen and osteocalcin secretion and calcium deposition were measured. Anandamide and 2-arachidonoylglycerol significantly decreased osteoblast proliferation after 4 days, associated with a concentration-dependent increase in ALP. Inhibition of endocannabinoid degradation enzymes to increase endocannabinoid tone resulted in similar increases in ALP production. 2-arachidonoylglycerol also decreased osteocalcin secretion. After prolonged (21 day treatment with 2-arachidonoylglycerol, there was a decrease in collagen content, but no change in calcium deposition. Anandamide did not affect collagen or osteocalcin, but reduced calcium deposition. Anandamide increased levels of phosphorylated CREB, ERK 1/2 and JNK, while 2-arachidonoylglycerol increased phosphorylated CREB and Akt. RT-PCR demonstrated the expression of CB2 and TRPV1, but not CB1 in HOBs. Anandamide-induced changes in HOB differentiation were CB1 and CB2-independent and partially reduced by TRPV1 antagonism, and reduced by inhibition of ERK 1/2 and JNK. Our results have demonstrated a clear involvement of anandamide and 2-arachidonoylglycerol in modulating the activity of human osteoblasts, with anandamide increasing early cell differentiation and 2-AG increasing early, but decreasing late osteoblast-specific markers of differentiation.

  13. Retinoic acid and cAMP inhibit rat hepatocellular carcinoma cell proliferation and enhance cell differentiation

    Directory of Open Access Journals (Sweden)

    M. Ionta

    2012-08-01

    Full Text Available Hepatocellular carcinoma (HCC is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM and cAMP (1 mM, an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β and liver differentiation (E-cadherin, connexin 26 (Cx26, and Cx32. RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form expression while the expression of Cx43, Tyr216-GSK-3β (active form and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.

  14. Effects of glutamine on proliferation, migration, and differentiation of human dental pulp cells.

    Science.gov (United States)

    Kim, Duck-Su; Jue, Seong-Suk; Lee, So-Youn; Kim, Young-Suk; Shin, Seung-Yun; Kim, Eun-Cheol

    2014-08-01

    Although glutamine (Gln) is mitogenic in various cell types, little is known about its role in human dental pulp cells (HDPCs). This study investigated the effects of Gln on proliferation, migration, and odontoblastic differentiation of HDPCs and the underlying signal pathway mechanisms. Growth and migration were assessed by cell counting and colorimetric cell migration kits. Differentiation was measured as alkaline phosphatase activity, calcified nodule formation by alizarin red staining, and marker mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Chemokine expression was also evaluated by RT-PCR. Signal transduction pathways were examined by RT-PCR and Western blot analysis. Gln dose-dependently increased proliferation, migration, alkaline phosphatase activity, mineralized nodule formation, and odontoblast-marker mRNA of HDPCs. Gln also up-regulated expression of interleukin-6, interleukin-8, MCP-1, MIP-3α, CCL2, CCL20, and CXCL1. Gln increased BMP-2 and BMP-4 mRNA, phosphorylation of Smad 1/5/8, β-catenin, and key proteins of the Wnt signaling pathway. Furthermore, Gln resulted in up-regulation of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. In addition, noggin, DKK1, inhibitors of p38, ERK, and JNK significantly attenuatted Gln-induced growth, migration, and odontoblastic differentiation. Collectively, this study demonstrated that Gln promoted growth, migration, and differentiation in HDPCs through the BMP-2, Wnt, and MAPK pathways, leading to improved pulp repair and regeneration. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  15. Individual and Complementary Effects of Human Papillomavirus Oncogenes on Epithelial Cell Proliferation and Differentiation.

    Science.gov (United States)

    Bergner, Sven; Halec, Gordana; Schmitt, Markus; Aubin, François; Alonso, Angel; Auvinen, Eeva

    2016-01-01

    Previous studies on human papillomavirus (HPV) type 16 protein functions have established the oncogenic nature of three viral proteins: E5, E6 and E7. Here we have studied the functions of these proteins by functional deletion of the individual E5, E6 or E7, or both E6 and E7 oncogenes in the context of the whole viral genome. These mutants, or the intact wild-type genome, were expressed from the natural viral promoters along with differentiation of epithelial HaCaT cells in three-dimensional collagen raft cultures. High episomal viral copy numbers were obtained using a transfection-based loxp-HPV16-eGFP-N1 vector system. All epithelial equivalents carrying the different HPV type 16 genomes showed pronounced hyperplastic and dysplastic morphology. Particularly the E7 oncogene, with contribution of E6, was shown to enhance cell proliferation. Specifically, the crucial role of E7 in HPV-associated hyperproliferation was clearly manifested. Based on morphological characteristics, immunohistochemical staining for differentiation and proliferation markers, and low expression of E1^E4, we propose that our raft culture models produce cervical intraepithelial neoplasia (CIN)1 and CIN2-like tissue. Our experimental setting provides an alternative tool to study concerted functions of HPV proteins in the development of epithelial dysplasia. © 2015 S. Karger AG, Basel.

  16. miR-141-3p inhibits human stromal (mesenchymal) stem cell proliferation and differentiation

    DEFF Research Database (Denmark)

    Qiu, Weimin; Kassem, Moustapha

    2014-01-01

    Wnt signaling determines human stromal (mesenchymal) stem cell (hMSC) differentiation fate into the osteoblast or adipocyte lineage. microRNAs (miRNAs) are small RNA molecules of 21-25 nucleotides that regulate many aspects of osteoblast biology. Thus, we examined miRNAs regulated by Wnt signaling...... activity, gene expression and in vitro mineralized matrix formation. Bioinformatic studies, Western blot analysis and 3'UTR reporter assay demonstrated that cell division cycle 25A (CDC25A) is a direct target of miR-141-3p. siRNA-mediated knock-down of CDC25A inhibited hMSC proliferation and osteoblast...... in hMSC. We identified miRNA (miR)-141-3p as a Wnt target which in turn inhibited Wnt signaling. Moreover, miR-141-3p inhibited hMSC proliferation by arresting cells at the G1 phase of the cell cycle. miR-141-3p inhibited osteoblast differentiation of hMSC as evidenced by reduced alkaline phosphatase...

  17. Microglia and microglia-like cell differentiated from DC inhibit CD4 T cell proliferation.

    Directory of Open Access Journals (Sweden)

    Bo Bai

    Full Text Available The central nervous system (CNS is generally regarded as a site of immune privilege, whether the antigen presenting cells (APCs are involved in the immune homeostasis of the CNS is largely unknown. Microglia and DCs are major APCs in physiological and pathological conditions, respectively. In this work, primary microglia and microglia-like cells obtained by co-culturing mature dendritic cells with CNS endothelial cells in vitro were functional evaluated. We found that microglia not only cannot prime CD4 T cells but also inhibit mature DCs (maDCs initiated CD4 T cells proliferation. More importantly, endothelia from the CNS can differentiate maDCs into microglia-like cells (MLCs, which possess similar phenotype and immune inhibitory function as microglia. Soluble factors including NO lie behind the suppression of CD4 T cell proliferation induced by both microglia and MLCs. All the data indicate that under physiological conditions, microglia play important roles in maintaining immune homeostasis of the CNS, whereas in a pathological situation, the infiltrated DCs can be educated by the local microenvironment and differentiate into MLCs with inhibitory function.

  18. Astilbin decreases proliferation and improves differentiation in HaCaT keratinocytes.

    Science.gov (United States)

    Zhang, Chunhong; Xu, Qingqing; Tan, Xi; Meng, Liya; Wei, Guo; Liu, Ying; Zhang, Chunmin

    2017-09-01

    Psoriasis is a common chronic dermatosis characterized by keratinocyte hyperproliferation accompanied by inflammatory reactions. Pathological changes upset the balance between keratinocyte proliferation, differentiation, and death in psoriatic lesions, suggesting that molecules with topical anti-inflammatory, anti-proliferation and anti-angiogenesis abilities may be useful for its treatment. The flavonoid astilbin is the major active component extracted from the rhizome of Smilax glabra, which has been widely used in China to treat inflammatory and autoimmune diseases. Here, we investigate the potential of astilbin as a treatment for psoriasis. We reveal that astilbin inhibits the growth of HaCaT keratinocytes. Detailed study shows that astilbin leads to S phase arrest of the cell cycle by induction of p53 and p21 and activated-AMPK. Additionally, astilbin induced keratinocyte differentiation correlated with suppression of keratin 5 (KRT5) and KRT14 proteins (the markers of epidermal basal layer) and induction KRT1 and KRT10 proteins (occurring in the upper layers). Moreover, astilbin regulates the expression of VEGF in human HaCaT keratinocytes. These results suggest that astilbin may be a promising agent for psoriasis treatment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  19. Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

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    Zhao, Ningbo, E-mail: curl-zhao@163.com; Wang, Xin, E-mail: 394041230@qq.com; Qin, Lei, E-mail: qinlei30@126.com; Guo, Zhengze, E-mail: zhzeguo@163.com; Li, Dehua, E-mail: lidehuafmmu@163.com

    2015-09-25

    Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased by increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.

  20. Hugl1 and Hugl2 in mammary epithelial cells: polarity, proliferation, and differentiation.

    Directory of Open Access Journals (Sweden)

    Atlantis Russ

    Full Text Available Loss of epithelial polarity is described as a hallmark of epithelial cancer. To determine the role of Hugl1 and Hugl2 expression in the breast, we investigated their localization in human mammary duct tissue and the effects of expression modulation in normal and cancer cell lines on polarity, proliferation and differentiation. Expression of Hugl1 and Hugl2 was silenced in both MCF10A cells and Human Mammary Epithelial Cells and cell lines were grown in 2-D on plastic and in 3-D in Matrigel to form acini. Cells in monolayer were compared for proliferative and phenotypic changes while acini were examined for differences in size, ability to form a hollow lumen, nuclear size and shape, and localization of key domain-specific proteins as a measure of polarity. We detected overlapping but distinct localization of Hugl1 and Hugl2 in the human mammary gland, with Hugl1 expressed in both luminal and myoepithelium and Hugl2 largely restricted to myoepithelium. On a plastic surface, loss of Hugl1 or Hugl2 in normal epithelium induced a mesenchymal phenotype, and these cells formed large cellular masses when grown in Matrigel. In addition, loss of Hugl1 or Hugl2 expression in MCF10A cells resulted in increased proliferation on Matrigel, while gain of Hugl1 expression in tumor cells suppressed proliferation. Loss of polarity was also observed with knockdown of either Hugl1 or Hugl2, with cells growing in Matrigel appearing as a multilayered epithelium, with randomly oriented Golgi and multiple enlarged nuclei. Furthermore, Hugl1 knock down resulted in a loss of membrane identity and the development of cellular asymmetries in Human Mammary Epithelial Cells. Overall, these data demonstrate an essential role for both Hugl1 and Hugl2 in the maintenance of breast epithelial polarity and differentiated cell morphology, as well as growth control.

  1. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration.

    Science.gov (United States)

    Zhang, Lei; Wang, Huaxi; Yang, Yan; Liu, Hui; Zhang, Qihao; Xiang, Qi; Ge, Renshan; Su, Zhijian; Huang, Yadong

    2013-06-28

    Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful information for developing new potential therapies for PADAM (Partial Androgen Deficiency in the Aging Male). Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Elongation, proliferation & migration differentiate endothelial cell phenotypes and determine capillary sprouting

    Directory of Open Access Journals (Sweden)

    Popel Aleksander S

    2009-01-01

    Full Text Available Abstract Background Angiogenesis, the growth of capillaries from preexisting blood vessels, has been extensively studied experimentally over the past thirty years. Molecular insights from these studies have lead to therapies for cancer, macular degeneration and ischemia. In parallel, mathematical models of angiogenesis have helped characterize a broader view of capillary network formation and have suggested new directions for experimental pursuit. We developed a computational model that bridges the gap between these two perspectives, and addresses a remaining question in angiogenic sprouting: how do the processes of endothelial cell elongation, migration and proliferation contribute to vessel formation? Results We present a multiscale systems model that closely simulates the mechanisms underlying sprouting at the onset of angiogenesis. Designed by agent-based programming, the model uses logical rules to guide the behavior of individual endothelial cells and segments of cells. The activation, proliferation, and movement of these cells lead to capillary growth in three dimensions. By this means, a novel capillary network emerges out of combinatorially complex interactions of single cells. Rules and parameter ranges are based on literature data on endothelial cell behavior in vitro. The model is designed generally, and will subsequently be applied to represent species-specific, tissue-specific in vitro and in vivo conditions. Initial results predict tip cell activation, stalk cell development and sprout formation as a function of local vascular endothelial growth factor concentrations and the Delta-like 4 Notch ligand, as it might occur in a three-dimensional in vitro setting. Results demonstrate the differential effects of ligand concentrations, cell movement and proliferation on sprouting and directional persistence. Conclusion This systems biology model offers a paradigm closely related to biological phenomena and highlights previously

  3. Comparison of calcium supplementation or low-fat dairy foods on epithelial cell proliferation and differentiation.

    Science.gov (United States)

    Holt, P R; Wolper, C; Moss, S F; Yang, K; Lipkin, M

    2001-01-01

    Epidemiological evidence suggests that dietary calcium and vitamin D intake are inversely related to incidence of colon cancer. Previous studies have demonstrated that supplementation of the diet with calcium in the form of calcium tablets or low-fat dairy foods alters colonic epithelial cell proliferation from a higher- to a lower-risk pattern. The present study compared relative effects of administration of calcium carbonate at approximately 900 mg/day (calcium) with those of a low-fat dairy food diet providing about the same amount of calcium (dairy) in a cross-over "head-to-head" study of 40 subjects at risk for colonic neoplasia. Dietary intake of macronutrients was similar in the two study periods, except for a slight increase in protein intake during dairy calcium supplementation. Rectal epithelial cell proliferation was studied in flat endoscopically normal-appearing mucosa at baseline and at the end of each of the two study periods and showed a significant reduction in epithelial crypt cell labeling index from 12.5% to 9.1% (calcium) or 9.3% (dairy) as well as in proliferating cells in the upper 40% of the crypt from 0.09 to 0.03 in the calcium- and low-fat dairy-supplemented intervention groups. No significant changes in two epithelial cell differentiation markers, cytokeratin AE1 and acidic mucins, were found. Furthermore, there were no differences in epithelial cell apoptosis or expression of the proapoptotic gene product BAK. These data indicate that increased dietary calcium given as supplements or in the diet in low-fat dairy foods lowers epithelial cell proliferation indexes from a higher- to a lower-risk pattern. Because supplemental calcium has been shown to reduce the recurrence of colonic adenomatous polyps in patients at increased risk for colonic neoplasia, our data suggest that supplemental low-fat dairy foods may also be effective.

  4. Survival and proliferation of alginate encapsulated Trichoderma spp. in Egyptian soil in comparison with allyl alcohol soil fumigation.

    Science.gov (United States)

    Shaban, G M; El-Komy, H M

    2001-01-01

    Conidia of Trichoderma harzianum and T. pseudokoningii (Rifai) were formulated to make alginate pellets with or without 10% cellulose as a food-base material. The formulations were compared for their ability for survival and proliferation of Trichoderma spp. in clay-loamy soil (50% moisture content) with allyl alcohol fumigation (0.05, 0.1 and 0.2 ml/1,000 ml space). Trichoderma medium E (TME) containing 100 microg/ml pentachloronitrobenzene (PCNB) was valuable for isolation and counting of Trichoderma spp. from the tested soil than the Glucose-Czapek's agar medium containing 1:15,000 Rose-bengal. The promotive effect of Trichoderma by different doses of allyl alcohol fumigation still enhanced after two-month incubation period. Conidia entrapped in alginate with or without cellulose and introduced into the soil survived better than conidia added directly to the same soil after three months incubation period. Sterile soil provided a more favorable environment for the proliferation and survival of immobilized conidia than the non-sterile soil, and the addition of 10% cellulose increased the survival of the entrapped conidia more than those prepared without cellulose. Soil fumigation inhibited the occurrence of other fungal species; however, inoculation of the soil with alginate immobilized conidia or conidial suspension had such inhibitory effect but in a less extent.

  5. Functional dissection of HOXD cluster genes in regulation of neuroblastoma cell proliferation and differentiation.

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    Yunhong Zha

    Full Text Available Retinoic acid (RA can induce growth arrest and neuronal differentiation of neuroblastoma cells and has been used in clinic for treatment of neuroblastoma. It has been reported that RA induces the expression of several HOXD genes in human neuroblastoma cell lines, but their roles in RA action are largely unknown. The HOXD cluster contains nine genes (HOXD1, HOXD3, HOXD4, and HOXD8-13 that are positioned sequentially from 3' to 5', with HOXD1 at the 3' end and HOXD13 the 5' end. Here we show that all HOXD genes are induced by RA in the human neuroblastoma BE(2-C cells, with the genes located at the 3' end being activated generally earlier than those positioned more 5' within the cluster. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 is sufficient to induce both growth arrest and neuronal differentiation, which is associated with downregulation of cell cycle-promoting genes and upregulation of neuronal differentiation genes. However, induction of other HOXD genes either has no effect (HOXD1 or has partial effects (HOXD3, HOXD4, HOXD11 and HOXD13 on BE(2-C cell proliferation or differentiation. We further show that knockdown of HOXD8 expression, but not that of HOXD9 expression, significantly inhibits the differentiation-inducing activity of RA. HOXD8 directly activates the transcription of HOXC9, a key effector of RA action in neuroblastoma cells. These findings highlight the distinct functions of HOXD genes in RA induction of neuroblastoma cell differentiation.

  6. 3-hydroxykynurenine suppresses CD4+ T-cell proliferation, induces T-regulatory-cell development, and prolongs corneal allograft survival.

    Science.gov (United States)

    Zaher, Sarah S; Germain, Conrad; Fu, Hongmei; Larkin, Daniel F P; George, Andrew J T

    2011-04-01

    IDO (indoleamine 2,3-dioxygenase) modulates the immune response by depletion of the essential amino acid tryptophan, and IDO overexpression has been shown to prolong corneal allograft survival. This study was conducted to examine the effect of kynurenines, the products of tryptophan breakdown and known to act directly on T lymphocytes, on corneal graft survival. The effects of kynurenines on T-cell proliferation and death, T-regulatory-cell development, and dendritic cell function, phenotype, and viability were analyzed in vitro. The effect of topical and systemic administration of 3-hydroxykynurenine (3HK) on orthotopic murine corneal allograft survival was examined. T-lymphocyte proliferation was inhibited by two of the four different kynurenines: 3HK and 3-hydroxyanthranilic acid (3HAA). This effect was accompanied by significant T-cell death. Neither 3HK nor 3HAA altered dendritic cell function, nor did they induce apoptosis or pathogenicity to corneal endothelial cells. Administration of systemic and topical 3HK to mice receiving a fully mismatched corneal graft resulted in significant prolongation of graft survival (median survival of control grafts, 12 days; of treated, 19 and 15 days, respectively; P < 0.0003). While systemic administration of 3HK was associated with a significant depletion of CD4(+) T, CD8(+) T, and B lymphocytes in peripheral blood, no depletion was found after topical administration. The production of kynurenines, in particular 3HK and 3HAA, may be one mechanism (in addition to tryptophan depletion) by which IDO prolongs graft survival. These molecules have potential as specific agents for preventing allograft rejection in patients at high rejection risk.

  7. Nanomaterial scaffolds for stem cell proliferation and differentiation in tissue engineering.

    Science.gov (United States)

    Zhao, Chunyan; Tan, Aaron; Pastorin, Giorgia; Ho, Han Kiat

    2013-01-01

    Tissue engineering is a clinically driven field and has emerged as a potential alternative to organ transplantation. The cornerstone of successful tissue engineering rests upon two essential elements: cells and scaffolds. Recently, it was found that stem cells have unique capabilities of self-renewal and multilineage differentiation to serve as a versatile cell source, while nanomaterials have lately emerged as promising candidates in producing scaffolds able to better mimic the nanostructure in natural extracellular matrix and to efficiently replace defective tissues. This article, therefore, reviews the key developments in tissue engineering, where the combination of stem cells and nanomaterial scaffolds has been utilized over the past several years. We consider the high potential, as well as the main issues related to the application of stem cells and nanomaterial scaffolds for a range of tissues including bone, cartilage, nerve, liver, eye etc. Promising in vitro results such as efficient attachment, proliferation and differentiation of stem cells have been compiled in a series of examples involving different nanomaterials. Furthermore, the merits of the marriage of stem cells and nanomaterial scaffolds are also demonstrated in vivo, providing early successes to support subsequent clinical investigations. This progress simultaneously drives mechanistic research into the mechanotransduction process responsible for the observations in order to optimize the process further. Current understanding is chiefly reported to involve the interaction of stem cells and the anchoring nanomaterial scaffolds by activating various signaling pathways. Substrate surface characteristics and scaffold bulk properties are also reported to influence not only short term stem cell adhesion, spreading and proliferation, but also longer term lineage differentiation, functionalization and viability. It is expected that the combination of stem cells and nanomaterials will develop into an

  8. Enterococcus faecalis affects the proliferation and differentiation of ovine osteoblast-like cells.

    Science.gov (United States)

    Karygianni, Lamprini; Wiedmann-Al-Ahmad, Margit; Finkenzeller, Günter; Sauerbier, Sebastian; Wolkewitz, Martin; Hellwig, Elmar; Al-Ahmad, Ali

    2012-06-01

    Enterococcus faecalis (E. faecalis) is a Gram-positive bacterium, mostly recovered from root-filled teeth with persistent periapical lesions. Bacterial contamination of root canals inevitably results in interaction between E. faecalis and periapical tissues during the dynamic process of periapical inflammation. This study investigated the impact of heat-inactivated endodontic E. faecalis on the proliferation and the differentiation of ovine osteoblast-like cells, in an attempt to elucidate its putative enhanced pathogenicity mechanisms. Therefore, two different concentrations of a heat-inactivated endodontic E. faecalis isolate (2 × 10(6) or 2 × 10(8) CFU/ml) were incubated with ovine osteoblast-like cells for 7 and 14 days, respectively. Cells without antigen served as control. The effects of antigen on cell growth were evaluated by a proliferation assay (EZ4U). Furthermore, the assessment of alkaline phosphatase (ALP) activity, calcium deposition, and osteocalcin (OCN) gene expression through quantitative real-time PCR determined the degree of osteogenic cell differentiation. Scanning electron microscopy (SEM) was also performed to detect alterations in cell morphology. Interestingly, although highly concentrated E. faecalis increased cellular reproduction after 14 days, ALP activity and OCN gene expression decreased in an antigen concentration-dependent and incubation time-independent way. SEM images revealed E. faecalis adhesion on cells, a fact that might contribute to its virulence. These results suggest that E. faecalis stimulated cell multiplication, whereas it likely restrained cell differentiation of ovine osteoblast-like cells. In conclusion, the presence of E. faecalis in root canals may negatively affect periapical new bone formation, and thus, the healing of periapical lesions.

  9. Effects of Aging on the Proliferation and Differentiation Capacity of Human Periodontal Ligament Stem Cells.

    Science.gov (United States)

    Du, Tingting; Liu, Na; Gu, Bin; Li, Ying; Yuan, Yifang; Zhang, Wei; Zhang, Tong

    2017-06-10

    Objective The aim of this study is to investigate the proliferation, differentiation and apoptosis of periodontal ligament stem cells (PDLSC) derived from different aged donors, and to evaluate the effects of aging on the biological characteristics of PDLSC.Methods Periodontal ligament tissues were obtained from 24 surgically extracted human premolars during orthodontics therapy. The specimens were divided into three groups according to the donor's age. Group A: 18-20 years, group B: 30-35 years, group C: 45-50 years. PDLSC were isolated and cultured using a tissue-block-based enzymolytic method by limiting dilution assay. The colony forming efficiency of PDLSC for three experimental groups was determined. Senescence-Associated β-Galactosidase (SA-β-G) expression in the three groups was examined using β-galactosidase staining working solution. Cell cycle and apoptosis of the PDLSC were examined by the flow cytometry. Alkaline phosphatase (ALP) activity was evaluated by ALP staining. The expression of osteoplastic differentiation related genes Runt-related transcription factor-2 (Runx-2), Collagen Type 1 (col-1), and ALP of PDLSC were examined by quantitative real-time RT-PCR.Results The colony forming efficiency of PDLSC in Group A, B and C was 36.67%, 22.67% and 9.33%, respectively, which decreased with donors' age (Pages, and the expression of Runx-2, col-1 and ALP decreased gradually from group A to group C (all Paging.Conclusion Human PDLSC could be successfully isolated from periodontal ligament tissues of different aged donors. However, the proliferation and osteogenic differentiation capacity of PDLSC decreased while donor aging.

  10. RELM-β promotes human pulmonary artery smooth muscle cell proliferation via FAK-stimulated surviving

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Chunlong, E-mail: lclmd@sina.com; Li, Xiaohui; Luo, Qiong; Yang, Hui; Li, Lun; Zhou, Qiong; Li, Yue; Tang, Hao; Wu, Lifu

    2017-02-01

    Resistin-like molecule-β (RELM-β), focal adhesion kinase (FAK), and survivin may be involved in the proliferation of cultured human pulmonary artery smooth muscle cells (HPAMSCs), which is involved in pulmonary hypertension. HPAMSCs were treated with human recombinant RELM-β (rhRELM-β). siRNAs against FAK and survivin were transfected into cultured HPASMCs. Expression of FAK and survivin were examined by RT-PCR and western blot. Immunofluorescence was used to localize FAK. Flow cytometry was used to examine cell cycle distribution and cell death. Compared to the control group, all rhRELM-β-treated groups demonstrated significant increases in the expression of FAK and survivin (P<0.05). rhRELM-β significantly increased the proportion of HPASMCs in the S phase and decreased the proportion in G0/G1. FAK siRNA down-regulated survivin expression while survivin siRNA did not affect FAK expression. FAK siRNA effectively inhibited FAK and survivin expression in RELM-β-treated HPASMCs and partially suppressed cell proliferation. RELM-β promoted HPASMC proliferation and upregulated FAK and survivin expression. In conclusion, results suggested that FAK is upstream of survivin in the signaling pathway mediating cell proliferation. FAK seems to be important in RELM-β-induced HPASMC proliferation, partially by upregulating survivin expression. - Highlights: • rhRELM-β increased the expression of FAK and survivin. • rhRELM-β increased the proportion of HPASMCs in the S phase. • FAK is upstream of survivin in the signaling pathway mediating cell proliferation. • FAK is important in RELM-β-induced HPASMC proliferation, partly via survivin.

  11. Diurnal variations in proliferation and crypt survival suggest a small target cell population in mouse colon

    Energy Technology Data Exchange (ETDEWEB)

    Dobbin, J.; Hamilton, E.

    1986-01-01

    Male C57BLasup(t) mice of two ages, 3-5 months (young) and 14-15 months (old) were given 11 or 15Gy whole body irradiation at different times through the day. The mice were killed after 4.5 days and the number of surviving crypts per circumference of jejunum, ileum, transverse colon and descending colon were scored. These results show crypt survival in the small and large intestine of 15-month-old mice. In the ileum the maximum crypt survival was found at 04.00 h and the minimum at 08.00 h. In the jejunum and both regions of the colon the maximum crypt survival occurred at 16.00 h. The nadir of crypt survival after 15 Gy was at 04.00 h in the jejunum and at 20.00 and 24.00 h in the transverse and descending colon, respectively. In young mice, crypt survival levels were similar to those found in old animals except at 04.00 h. when survival in the jejunum and ileum fell to 0.0004+-0.0002 and 0.0007+-0.0004, respectively. The lowest crypt survival in the colon of young mice also occurred at 04.00 h and in all four tissues the greatest number of crypts survived irradiation at 24.00 h.

  12. StearoylCoA desaturase-5: a novel regulator of neuronal cell proliferation and differentiation.

    Directory of Open Access Journals (Sweden)

    Debora I Sinner

    Full Text Available Recent studies have demonstrated that human stearoylCoA desaturase-1 (SCD1, a Δ9-desaturase that converts saturated fatty acids (SFA into monounsaturated fatty acids, controls the rate of lipogenesis, cell proliferation and tumorigenic capacity in cancer cells. However, the biological function of stearoylCoA desaturase-5 (SCD5, a second isoform of human SCD that is highly expressed in brain, as well as its potential role in human disease, remains unknown. In this study we report that the constitutive overexpression of human SCD5 in mouse Neuro2a cells, a widely used cell model of neuronal growth and differentiation, displayed a greater n-7 MUFA-to-SFA ratio in cell lipids compared to empty-vector transfected cells (controls. De novo synthesis of phosphatidylcholine and cholesterolesters was increased whereas phosphatidylethanolamine and triacylglycerol formation was reduced in SCD5-expressing cells with respect to their controls, suggesting a differential use of SCD5 products for lipogenic reactions. We also observed that SCD5 expression markedly accelerated the rate of cell proliferation and suppressed the induction of neurite outgrowth, a typical marker of neuronal differentiation, by retinoic acid indicating that the desaturase plays a key role in the mechanisms of cell division and differentiation. Critical signal transduction pathways that are known to modulate these processes, such epidermal growth factor receptor (EGFRAkt/ERK and Wnt, were affected by SCD5 expression. Epidermal growth factor-induced phosphorylation of EGFR, Akt and ERK was markedly blunted in SCD5-expressing cells. Furthermore, the activity of canonical Wnt was reduced whereas the non-canonical Wnt was increased by the presence of SCD5 activity. Finally, SCD5 expression increased the secretion of recombinant Wnt5a, a non-canonical Wnt, whereas it reduced the cellular and secreted levels of canonical Wnt7b. Our data suggest that, by a coordinated modulation of key

  13. Survival at the Center - The Stability of Minimum Differentiation

    OpenAIRE

    Hehenkamp, Burkhard; Wambach, Achim

    2010-01-01

    International audience; We model a Hotelling market with multidimensional product differentiation in an evolutionary framework. Both evolutionary stability (in the sense of Schaffer, 1989) and stochastic stability (following Kandori et al., 1993, and Young, 1993) are analysed. It is shown that firms move towards the center in product space, i.e. a "principle of minimum differentiation" on all dimensions of the product space applies.

  14. Int6/eIF3e Is Essential for Proliferation and Survival of Human Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Julie Sesen

    2014-01-01

    Full Text Available Glioblastomas (GBM are very aggressive and malignant brain tumors, with frequent relapses despite an appropriate treatment combining surgery, chemotherapy and radiotherapy. In GBM, hypoxia is a characteristic feature and activation of Hypoxia Inducible Factors (HIF-1α and HIF-2α has been associated with resistance to anti-cancer therapeutics. Int6, also named eIF3e, is the “e” subunit of the translation initiation factor eIF3, and was identified as novel regulator of HIF-2α. Eukaryotic initiation factors (eIFs are key factors regulating total protein synthesis, which controls cell growth, size and proliferation. The functional significance of Int6 and the effect of Int6/EIF3E gene silencing on human brain GBM has not yet been described and its role on the HIFs is unknown in glioma cells. In the present study, we show that Int6/eIF3e suppression affects cell proliferation, cell cycle and apoptosis of various GBM cells. We highlight that Int6 inhibition induces a diminution of proliferation through cell cycle arrest and increased apoptosis. Surprisingly, these phenotypes are independent of global cell translation inhibition and are accompanied by decreased HIF expression when Int6 is silenced. In conclusion, we demonstrate here that Int6/eIF3e is essential for proliferation and survival of GBM cells, presumably through modulation of the HIFs.

  15. Insulin-like growth factor 1 regulation of proliferation and differentiation of Xenopus laevis myogenic cells in vitro.

    Science.gov (United States)

    Miyata, Sairi; Yada, Tomotaka; Ishikawa, Natsuko; Taheruzzaman, Kazi; Hara, Ryohei; Matsuzaki, Takashi; Nishikawa, Akio

    2017-03-01

    To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1's cell differentiation promotion, suggesting the possibility that IGF-1's differentiation-promotion effect is an indirect effect via IGF-1's cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100-500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-L-thyronine (T3) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T3. In larval tail cells, cell count was 76% lower in the presence of T3, and IGF-1 did not promote proliferation and differentiation in T3-containing medium. In larval dorsal cells, cell count was also lower in the presence of T3, but IGF-1 enhanced proliferation and differentiation in T3-containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T3 and helps accelerate dorsal muscle remodeling during metamorphosis.

  16. Heterogeneous activation of a slow myosin gene in proliferating myoblasts and differentiated single myofibers.

    Science.gov (United States)

    Wang, Jing-Hua; Wang, Qiao-Jing; Wang, Chao; Reinholt, Brad; Grant, Alan L; Gerrard, David E; Kuang, Shihuan

    2015-06-01

    Each skeletal muscle contains a fixed ratio of fast and slow myofibers that are distributed in a stereotyped pattern to achieve a specific motor function. How myofibers are specified during development and regeneration is poorly understood. Here we address this question using transgenic reporter mice that indelibly mark the myofiber lineages based on activation of fast or slow myosin. Lineage tracing indicates that during development all muscles have activated the fast myosin gene Myl1, but not the slow myosin gene Myh7, which is activated in all slow but a subset of fast myofibers. Similarly, most nascent myofibers do not activate Myh7 during fast muscle regeneration, but the ratio and pattern of fast and slow myofibers are restored at the completion of regeneration. At the single myofiber level, most mature fast myofibers are heterogeneous in nuclear composition, manifested by mosaic activation of Myh7. Strikingly, Myh7 is activated in a subpopulation of proliferating myoblasts that co-express the myogenic progenitor marker Pax7. When induced to differentiate, the Myh7-activated myoblasts differentiate more readily than the non-activated myoblasts, and have a higher tendency, but not restricted, to become slow myotubes. Together, our data reveal significant nuclear heterogeneity within a single myofiber, and challenge the conventional view that myosin genes are only expressed after myogenic differentiation. These results provide novel insights into the regulation of muscle fiber type specification. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Immunohistochemical study of cell proliferation and differentiation in epidermis of mice after administration of cholera toxin.

    Science.gov (United States)

    Rahman, S A; Tsuyama, S

    1993-01-01

    Cholera toxin causes reversible epidermal hyperplasia. We observed maximal thickness of the epidermis on the fourth day after treatment and a return to pretreatment values by day 7. The increase in thickness occurred in the basal and intermediate layers, with these layers becoming two to three times thicker than those of normal epidermis. The time sequence of epidermal proliferation was studied using bromodeoxyuridine (BrdU) labelling. We observed a maximum number of labelled basal cells within the first 24 h. Only a few cells were labelled 7 days after toxin injection. Griffonia simplicifolia-IB4 (GSA-IB4), Ulex europaeus-I (UEA-I) and Griffonia simplicifolia-II (GSA-II) lectins were used for the analysis of epidermal cell differentiation in the tissue sections. To study keratinocyte differentiation, further immunological staining was performed using two anticytokeratin antibodies, PKK2 and PKK3 mouse monoclonal antibodies. From the immunocytochemical results, we conclude that synchronous differentiation of the epidermis occurs after cholera toxin administration.

  18. MicroRNA-378 regulates neural stem cell proliferation and differentiation in vitro by modulating Tailless expression.

    Science.gov (United States)

    Huang, Yanxia; Liu, Xiaoguai; Wang, Yaping

    2015-10-16

    Previous studies have suggested that microRNAs (miRNAs) play an important role in regulating neural stem cell (NSC) proliferation and differentiation. However, the precise role of miRNAs in NSC remains largely unexplored. In this study, we showed that miR-378 can target Tailless (TLX), a critical regulator of NSC, to regulate NSC proliferation and differentiation. By bioinformatic algorithms, miR-378 was found to have a predicted target site in the 3'-untranslated region of TLX, which was verified by a dual-luciferase reporter assay. The expression of miR-378 was increased during NSC differentiation and inversely correlated with TLX expression. qPCR and Western blot analysis also showed that miR-378 negatively regulated TLX mRNA and protein expression in neural stem cells (NSCs). Intriguingly, overexpression of miR-378 increased NSC differentiation and reduced NSC proliferation, whereas suppression of miR-378 led to decreased NSC differentiation and increased NSC proliferation. Moreover, the downstream targets of TLX, including p21, PTEN and Wnt/β-catenin were also found to be regulated by miR-378. Additionally, overexpression of TLX rescued the NSC proliferation deficiency induced by miR-378 overexpression and abolished miR-378-promoted NSC differentiation. Taken together, our data suggest that miR-378 is a novel miRNA that regulates NSC proliferation and differentiation via targeting TLX. Therefore, manipulating miR-378 in NSCs could be a novel strategy to develop novel interventions for the treatment of relevant neurological disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Metabolic and protein interaction sub-networks controlling the proliferation rate of cancer cells and their impact on patient survival.

    Science.gov (United States)

    Feizi, Amir; Bordel, Sergio

    2013-10-24

    Cancer cells can have a broad scope of proliferation rates. Here we aim to identify the molecular mechanisms that allow some cancer cell lines to grow up to 4 times faster than other cell lines. The correlation of gene expression profiles with the growth rate in 60 different cell lines has been analyzed using several genome-scale biological networks and new algorithms. New possible regulatory feedback loops have been suggested and the known roles of several cell cycle related transcription factors have been confirmed. Over 100 growth-correlated metabolic sub-networks have been identified, suggesting a key role of simultaneous lipid synthesis and degradation in the energy supply of the cancer cells growth. Many metabolic sub-networks involved in cell line proliferation appeared also to correlate negatively with the survival expectancy of colon cancer patients.

  20. Glucagon-like peptide 2 dose-dependently activates intestinal cell survival and proliferation in neonatal piglets

    DEFF Research Database (Denmark)

    Burrin, Douglas G; Stoll, Barbara; Guan, Xinfu

    2005-01-01

    saline or GLP-2 at three rates (2.5, 5.0, and 10.0 nmol.kg(-1).d(-1)) for 7 d. Plasma GLP-2 concentrations ranged from 177 +/- 27 to 692 +/- 85 pM in the low- and high-infusion groups, respectively. GLP-2 infusion dose-dependently increased small intestinal weight, DNA and protein content, and villus...... of caspase-3 and -6 and active caspase-3 abundance decreased, yet procaspase-3 abundance increased markedly with increasing infusion rate and plasma concentration of GLP-2. The GLP-2-dose-dependent suppression of intestinal apoptosis and caspase-3 activity was associated with increased protein kinase B...... is concentration dependent at physiological GLP-2 concentrations; however, induction of cell proliferation and protein synthesis is a pharmacological response. Moreover, we show that GLP-2 stimulates intestinal cell survival and proliferation in association with induction of protein kinase B and glycogen...

  1. Meclozine facilitates proliferation and differentiation of chondrocytes by attenuating abnormally activated FGFR3 signaling in achondroplasia.

    Directory of Open Access Journals (Sweden)

    Masaki Matsushita

    Full Text Available Achondroplasia (ACH is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8 cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.

  2. Polyunsaturated Fatty Acids Differentially Modulate Cell Proliferation and Endocannabinoid System in Two Human Cancer Lines.

    Science.gov (United States)

    Gastón, Repossi; María Eugenia, Pasqualini; Das, Undurti N; Eynard, Aldo R

    2017-01-01

    Evidence suggests that quantity and quality of dietary polyunsaturated fatty acids (PUFAs) play a role in the development of cancer. However, the mechanisms involved in this interaction(s) are not clear. Endocannabinoids are lipid metabolites known to have growth modulatory actions. We studied the effect of supplementation with PUFAs ω-6 and ω-3 (essential fatty acids, EFAs), saturated and monounsaturated fatty acids (non-EFAs) on the growth of tumor cells and modifications in their endocannabinoid content. Cell cultures of human glioblastoma (T98G) and breast cancer (MCF7) were supplemented with 50 or 100 mmol EFAs and non-EFAs for 72 h. Cell proliferation was then determined by MTT, anandamide (AEA) levels by HPLC, total fatty acids profiles by GLC, CB1 receptor expression by WB and FAAH activity by spectrophotometric method. Fatty acids profile reflected the incorporation of the lipids supplemented in each assay. Arachidonic acid (EFA ω-6) supplementation increased AEA levels and inhibited the growth of T98G, whereas palmitic acid (non-EFA) enhanced their proliferation. In breast cancer (MCF7) cells, eicosapentaenoic acid (EFA ω-3) reduced and oleic acid (non-EFA) enhanced their proliferation. CB1 expression was higher in T98G and no differences were observed in FAAH activity. The growth of tumor cells can be differentially modulated by fatty acids and, at least in part, can be attributed to their ability to act on the components of the endocannabinoid system. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

  3. Meclozine Facilitates Proliferation and Differentiation of Chondrocytes by Attenuating Abnormally Activated FGFR3 Signaling in Achondroplasia

    Science.gov (United States)

    Matsushita, Masaki; Kitoh, Hiroshi; Ohkawara, Bisei; Mishima, Kenichi; Kaneko, Hiroshi; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

    2013-01-01

    Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias. PMID:24324705

  4. Effects of radiofrequency exposure emitted from a GSM mobile phone on proliferation, differentiation, and apoptosis of neural stem cells

    OpenAIRE

    Eghlidospour, Mahsa; Ghanbari, Amir; Mortazavi, Seyyed Mohammad Javad; Azari, Hassan

    2017-01-01

    Due to the importance of neural stem cells (NSCs) in plasticity of the nervous system and treating neurodegenerative diseases, the main goal of this study was to evaluate the effects of radiofrequency radiation emitted from a GSM 900-MHz mobile phone with different exposure duration on proliferation, differentiation and apoptosis of adult murine NSCs in vitro. We used neurosphere assay to evaluate NSCs proliferation, and immunofluorescence assay of neural cell markers to examine NSCs differen...

  5. Histone demethylase JMJD2B is required for tumor cell proliferation and survival and is overexpressed in gastric cancer

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wenjuan; Zhao, Li; Zang, Wen; Liu, Zhifang; Chen, Long; Liu, Tiantian [Department of Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education, School of Medicine, Shandong University, 44 Wenhua Xi Road, Jinan 250012 (China); Xu, Dawei, E-mail: Dawei.Xu@ki.se [Department of Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education, School of Medicine, Shandong University, 44 Wenhua Xi Road, Jinan 250012 (China); Department of Medicine, Division of Hematology, Karolinska University Hospital, Solna and Karolinska Institutet, Stockholm (Sweden); Jia, Jihui, E-mail: jiajihui@sdu.edu.cn [Department of Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education, School of Medicine, Shandong University, 44 Wenhua Xi Road, Jinan 250012 (China)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer JMJD2B is required for cell proliferation and in vivo tumorigenesis. Black-Right-Pointing-Pointer JMJD2B depletion induces apoptosis and/or cell cycle arrest. Black-Right-Pointing-Pointer JMJD2B depletion activates DNA damage response and enhances p53 stabilization. Black-Right-Pointing-Pointer JMJD2B is overexpressed in human primary gastric cancer. -- Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Jumonji domain containing 2B (JMJD2B) is a newly identified histone demethylase that regulates chromatin structure or gene expression by removing methyl residues from trimethylated lysine 9 on histone H3. Recent observations have shown oncogenic activity of JMJD2B. We explored the functional role of JMJD2B in cancer cell proliferation, survival and tumorigenesis, and determined its expression profile in gastric cancer. Knocking down JMJD2B expression by small interfering RNA (siRNA) in gastric and other cancer cells inhibited cell proliferation and/or induced apoptosis and elevated the expression of p53 and p21{sup CIP1} proteins. The enhanced p53 expression resulted from activation of the DNA damage response pathway. JMJD2B knockdown markedly suppressed xenograft tumor growth in vivo in mice. Moreover, JMJD2B expression was increased in primary gastric-cancer tissues of humans. Thus, JMJD2B is required for sustained proliferation and survival of tumor cells in vitro and in vivo, and its aberrant expression may contribute to the pathogenesis of gastric cancer.

  6. Normal proliferation and differentiation of Hoxc-8 transgenic chondrocytes in vitro

    Directory of Open Access Journals (Sweden)

    Mello Maria

    2003-04-01

    Full Text Available Abstract Background Hox genes encode transcription factors that are involved in pattern formation in the skeleton, and recent evidence suggests that they also play a role in the regulation of endochondral ossification. To analyze the role of Hoxc-8 in this process in more detail, we applied in vitro culture systems, using high density cultures of primary chondrocytes from neonatal mouse ribs. Results Cultured cells were characterized on the basis of morphology (light microscopy and production of cartilage-specific extracellular matrix (sulfated proteoglycans and type II Collagen. Hypertrophy was demonstrated by increase in cell size, alkaline phosphatase activity and type X Collagen immunohistochemistry. Proliferation was assessed by BrdU uptake and flow cytometry. Unexpectedly, chondrocytes from Hoxc-8 transgenic mice, which exhibit delayed cartilage maturation in vivo 1, were able to proliferate and differentiate normally in our culture systems. This was the case even though freshly isolated Hoxc-8 transgenic chondrocytes exhibited significant molecular differences as measured by real-time quantitative PCR. Conclusions The results demonstrate that primary rib chondrocytes behave similar to published reports for chondrocytes from other sources, validating in vitro approaches for studies of Hox genes in the regulation of endochondral ossification. Our analysis of cartilage-producing cells from Hoxc-8 transgenic mice provides evidence that the cellular phenotype induced by Hoxc-8 overexpression in vivo is reversible in vitro.

  7. Fluoxetine Induces Proliferation and Inhibits Differentiation of Hypothalamic Neuroprogenitor Cells In Vitro

    Science.gov (United States)

    Sousa-Ferreira, Lígia; Aveleira, Célia; Botelho, Mariana; Álvaro, Ana Rita; Pereira de Almeida, Luís; Cavadas, Cláudia

    2014-01-01

    A significant number of children undergo maternal exposure to antidepressants and they often present low birth weight. Therefore, it is important to understand how selective serotonin reuptake inhibitors (SSRIs) affect the development of the hypothalamus, the key center for metabolism regulation. In this study we investigated the proliferative actions of fluoxetine in fetal hypothalamic neuroprogenitor cells and demonstrate that fluoxetine induces the proliferation of these cells, as shown by increased neurospheres size and number of proliferative cells (Ki-67+ cells). Moreover, fluoxetine inhibits the differentiation of hypothalamic neuroprogenitor cells, as demonstrated by decreased number of mature neurons (Neu-N+ cells) and increased number of undifferentiated cells (SOX-2+ cells). Additionally, fluoxetine-induced proliferation and maintenance of hypothalamic neuroprogenitor cells leads to changes in the mRNA levels of appetite regulator neuropeptides, including Neuropeptide Y (NPY) and Cocaine-and-Amphetamine-Regulated-Transcript (CART). This study provides the first evidence that SSRIs affect the development of hypothalamic neuroprogenitor cells in vitro with consequent alterations on appetite neuropeptides. PMID:24598761

  8. In vitro effects of pirfenidone on cardiac fibroblasts: proliferation, myofibroblast differentiation, migration and cytokine secretion.

    Directory of Open Access Journals (Sweden)

    Qiang Shi

    Full Text Available Cardiac fibroblasts (CFs are the primary cell type responsible for cardiac fibrosis during pathological myocardial remodeling. Several studies have illustrated that pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone attenuates cardiac fibrosis in different animal models. However, the effects of pirfenidone on cardiac fibroblast behavior have not been examined. In this study, we investigated whether pirfenidone directly modulates cardiac fibroblast behavior that is important in myocardial remodeling such as proliferation, myofibroblast differentiation, migration and cytokine secretion. Fibroblasts were isolated from neonatal rat hearts and bioassays were performed to determine the effects of pirfenidone on fibroblast function. We demonstrated that treatment of CFs with pirfenidone resulted in decreased proliferation, and attenuated fibroblast α-smooth muscle actin expression and collagen contractility. Boyden chamber assay illustrated that pirfenidone inhibited fibroblast migration ability, probably by decreasing the ratio of matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1. Furthermore, pirfenidone attenuated the synthesis and secretion of transforming growth factor-β1 but elevated that of interleukin-10. These direct and pleiotropic effects of pirfenidone on cardiac fibroblasts point to its potential use in the treatment of adverse myocardial remodeling.

  9. Novel Polypyrrole-Coated Polylactide Scaffolds Enhance Adipose Stem Cell Proliferation and Early Osteogenic Differentiation

    Science.gov (United States)

    Pelto, Jani; Björninen, Miina; Pälli, Aliisa; Talvitie, Elina; Hyttinen, Jari; Mannerström, Bettina; Suuronen Seppanen, Riitta; Kellomäki, Minna; Miettinen, Susanna; Haimi, Suvi

    2013-01-01

    An electrically conductive polypyrrole (PPy) doped with a bioactive agent is an emerging functional biomaterial for tissue engineering. We therefore used chondroitin sulfate (CS)-doped PPy coating to modify initially electrically insulating polylactide resulting in novel osteogenic scaffolds. In situ chemical oxidative polymerization was used to obtain electrically conductive PPy coating on poly-96L/4D-lactide (PLA) nonwoven scaffolds. The coated scaffolds were characterized and their electrical conductivity was evaluated in hydrolysis. The ability of the coated and conductive scaffolds to enhance proliferation and osteogenic differentiation of human adipose stem cells (hASCs) under electrical stimulation (ES) in three-dimensional (3D) geometry was compared to the noncoated PLA scaffolds. Electrical conductivity of PPy-coated PLA scaffolds (PLA-PPy) was evident at the beginning of hydrolysis, but decreased during the first week of incubation due to de-doping. PLA-PPy scaffolds enhanced hASC proliferation significantly compared to the plain PLA scaffolds at 7 and 14 days. Furthermore, the alkaline phosphatase (ALP) activity of the hASCs was generally higher in PLA-PPy seeded scaffolds, but due to patient variation, no statistical significance could be determined. ES did not have a significant effect on hASCs. This study highlights the potential of novel PPy-coated PLA scaffolds in bone tissue engineering. PMID:23126228

  10. Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

    Directory of Open Access Journals (Sweden)

    Shaohui Yuan

    2013-01-01

    Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

  11. Improved Isolation, Proliferation, and Differentiation Capacity of Mouse Ovarian Putative Stem Cells.

    Science.gov (United States)

    Yazdekhasti, Hossein; Hosseini, Marzieh Agha; Rajabi, Zahra; Parvari, Soraya; Salehnia, Mojdeh; Koruji, Morteza; Izadyar, Fariborz; Aliakbari, Fereshte; Abbasi, Mehdi

    2017-04-01

    The recent discovery of ovarian stem cells in postnatal mammalian ovaries, also referred to as putative stem cells (PSCs), and their roles in mammalian fertility has challenged the long-existing theory that women are endowed with a certain number of germ cells. The rare amount of PSCs is the major limitation for utilizing them through different applications. Therefore, this study was conducted in six phases to find a way to increase the number of Fragilis- and mouse vasa homolog (MVH)-positive sorted cells from 14-day-old NMRI strain mice. Results showed that there is a population of Fragilis- and MVH-positive cells with pluripotent stem cell characteristics, which can be isolated and expanded for months in vitro. PSCs increase their proliferation capacity under the influence of some mitogenic agents, and our results showed that different doses of stem cell factor (SCF) induce PSC proliferation with the maximum increase observed at 50 ng/mL. SCF was also able to increase the number of Fragilis- and MVH-positive cells after sorting by magnetic-activated cell sorting and enhance colony formation efficiency in sorted cells. Differentiation capacity assay indicated that there is a basic level of spontaneous differentiation toward oocyte-like cells during 3 days of culture. However, relative gene expression was significantly higher in the follicle-stimulating hormone-treated groups, especially in the Fragilis- sorted PSCs. We suggest that higher number of PSCs provides us either a greater source of energy that can be injected into energy-impaired oocytes in women with a history of repeat IVF failure or a good source for research.

  12. Effects of BIO on proliferation and chondrogenic differentiation of mouse marrow derived mesenchymal stem cells

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    Nasrin Fallah

    2013-06-01

    Full Text Available In vitroexpansion of mesenchymal stem cell (MSCs into large number is necessary fortheir application in cell-based treatment of articular cartilage defects. On the other hand,some studies have indicated that BIO (6-Bromoindirubin-3-Oxime possesses mitogeniceffects on cell culture. The objective of the present study was to examine the effect of BIO onin vitro expansion and chondrogenic differentiation of mouse marrow-derived MSCs. Theculture was established using bone marrow tissue obtained from 10 NMRI mice. MSC natureof the isolated cells was verified according to the minimal criteria proposed for MSC.Passaged-3 cells were seeded in 24-well culture plates and treated by 0.05, 0.01, 0.1, 1.0 and1.5 μM BIO forsevendays. The culture without BIO was taken as the control. At the end ofcultivation period, the cultures were examinedfor viable cell number which was then used tocalculate population doubling time (PDT. The BIO with higher proliferation-promoting effectwas investigated for its chondrogenic effect on MSC culture. There was significantly moreviable cells at the cultures treated by 0.1 μM BIO. At this culture the cells tended to doubletheir population in rapid rate (each 43.07 hr than the cells treated with the other BIOconcentrations (p< 0.05. Interestingly treatment of MSC chondrogenic culture with 0.1 μMBIO ledto the up-regulation of cartilage specific genes including aggrecan, collagen II andSox9. In conclusion BIO at 0.1 μM could enhance mouse MSC in vitro proliferation as well astheir chondrogenic differentiation. These findings would be of great importance for the fieldof regenerative medicine.

  13. Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells

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    Zaminy Arash

    2008-01-01

    Full Text Available Background: Osteogenesis driven by adipose-derived stem cells (ADSCs is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs. Materials and Methods: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM. After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium (MTT assay and flow cytometry, respectively. Results: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased. Conclusion: These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.

  14. Preparation and Properties of Soluble Pig Skin Fractions Influencing on Human Keratinocyte Proliferation and Differentiation.

    Science.gov (United States)

    Belova, Olga V.; Arion, Vitaly A.; Lopukhin, Yuri M.; Orlova, Valeria F.; Kapitanov, Alexandr B.; Korotkova, Marina N.; Dvorczova, Valentina V.; Rabovskij, Alexandr B.; Beckman, Edit M.; Baranova, Olga A.; Zimina, Irina V.

    1999-07-01

    Three fractions with different molecular weights were isolated from pig skin. Fraction 1 (F1) and fraction 3 (F3) stimulated human keratinocyte proliferation in primary and regenerating cultures, but didn't affect their differentiation. Fraction 2 (F2) inhibited keratinocyte proliferation and stimulated their differentiation. The content of the proteins, carbohydrates, ribonucleic acids (RNA) and total lipids were determined in the fractions. The content of proteins and total lipids decreased from F1 to F3, but the content of carbohydrates and RNA increased. Physico-chemical properties of the fractions were studied by the methods of isoelectric focusing and vertical electrophoresis in polyacrilamide gel with sodium dodecyl sulphate and 7 M urea. The authors found that F1 contained 2-3 main proteins with molecular weight of 58 kDa and pI from 4.9 to 5.1 and RNA with molecular weight of 44 kDa. F2 was more heterogeneous. It contained a few main proteins with molecular weights from 6.9 to 61 kDa and pI from 4.4 to 8.3 and RNA with approximate molecular weights from 5.0 to 7.5 kDa. F3 contained a few main proteins with molecular weights from 3.5 to 59 kDa and pI from 4.8 to 5.1 and RNA with approximate molecular weights from 4.8 to 7.0 kDa.

  15. Activation of the Aryl Hydrocarbon Receptor during Different Critical Windows in Pregnancy Alters Mammary Epithelial Cell Proliferation and Differentiation

    Science.gov (United States)

    Lew, Betina J.; Collins, Loretta L.; O'Reilly, Michael A.; Lawrence, B. Paige

    2009-01-01

    Exposure to the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy causes severe defects in mammary gland development and function; however, the underlying mechanism remains unclear. Alterations in epithelial cell proliferation, differentiation, and apoptosis during pregnancy-related mammary development can lead to failed lactogenesis. To determine which of these processes are affected and at what time periods, we examined proliferation, differentiation and apoptosis in mammary glands following exposure to TCDD during early, mid or throughout pregnancy. Although AhR activation throughout pregnancy did not cause early involution, there was a 50% decrease in cell proliferation, which was observed as early as the sixth day of pregnancy (DP). TCDD treatment on the day of impregnation only reduced development and proliferation in early and mid-pregnancy, followed by partial recovery by DP17. However, when AhR activation was delayed to DP7, developmental impairment was not observed in mid-pregnancy, but became evident by DP17, whereas proliferation was reduced at all times. Thus, early exposure to TCDD was neither necessary nor sufficient to cause persistent defects in lactogenesis. These varying outcomes in mammary development due to exposure at different times in pregnancy suggest there are critical windows during which AhR activation impairs mammary epithelial cell proliferation and differentiation. PMID:19502548

  16. Effect of melatonin on the proliferation and differentiation of chondrocytes from rat vertebral body growth plate in vitro.

    Science.gov (United States)

    Zhong, Zhao-Ming; Li, Tao; Xu, Zi-Xing; Meng, Ting-Ting; Zeng, Ji-Huan; Zheng, Shuai; Ye, Wen-Bin; Wu, Qian; Chen, Jian-Ting

    2013-01-01

    Abnormal growth of vertebral body growth plate (VBGP) is considered as one of the etiologic factors in the adolescent idiopathic scoliosis (AIS). It was well-known that melatonin was correlated with the emergence and development of AIS. This study aimed to investigate the effect of melatonin on rat VBGP chondrocytes in vitro. Chondrocytes were isolated from rat VBGP, and treated with or without melatonin. Cell proliferation was measured by the Alamar Blue assay. Gene expression of collagen type II and aggrecan were evaluated by real-time PCR. Expression of the melatonin receptors (MT1, MT2), proliferating cell nuclear antigen (PCNA, a cell proliferation marker), Sox9 (a chondrocytic differentiation marker) and Smad4 (a common mediator in regulating the proliferation and differentiation of chondrocytes) were detected by Western blotting. Expression of melatonin receptors (MT1, MT2) were detected in the rat VBGP chondrocytes. Melatonin, at 10 and 100 µg/mL concentration, significantly inhibited the proliferation of VBGP-chondrocytes and the gene expression of collagen type II and aggrecan, and down-regulated the protein expression of PCNA, Sox9 and Smad4. In addition, the inhibitory effect of melatonin was reversed by luzindole, a melatonin receptor antagonist. These results suggest that melatonin at high concentrations can inhibit the proliferation and differentiation of VBGP chondrocytes, which might give some new insight into the pathogenic mechanism of AIS.

  17. Clonal characterization of rat muscle satellite cells: proliferation, metabolism and differentiation define an intrinsic heterogeneity.

    Directory of Open Access Journals (Sweden)

    Carlo A Rossi

    2010-01-01

    Full Text Available Satellite cells (SCs represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair and maintenance of skeletal muscle. Distinguished on the basis of their unique position in mature skeletal muscle, SCs were considered unipotent stem cells with the ability of generating a unique specialized phenotype. Subsequently, it was demonstrated in mice that opposite differentiation towards osteogenic and adipogenic pathways was also possible. Even though the pool of SCs is accepted as the major, and possibly the only, source of myonuclei in postnatal muscle, it is likely that SCs are not all multipotent stem cells and evidences for diversities within the myogenic compartment have been described both in vitro and in vivo. Here, by isolating single fibers from rat flexor digitorum brevis (FDB muscle we were able to identify and clonally characterize two main subpopulations of SCs: the low proliferative clones (LPC present in major proportion (approximately 75% and the high proliferative clones (HPC, present instead in minor amount (approximately 25%. LPC spontaneously generate myotubes whilst HPC differentiate into adipocytes even though they may skip the adipogenic program if co-cultured with LPC. LPC and HPC differ also for mitochondrial membrane potential (DeltaPsi(m, ATP balance and Reactive Oxygen Species (ROS generation underlying diversities in metabolism that precede differentiation. Notably, SCs heterogeneity is retained in vivo. SCs may therefore be comprised of two distinct, though not irreversibly committed, populations of cells distinguishable for prominent differences in basal biological features such as proliferation, metabolism and differentiation. By these means, novel insights on SCs heterogeneity are provided and evidences for biological readouts potentially relevant for diagnostic purposes described.

  18. The potential functional interaction of biglycan and WISP-1 in controlling differentiation and proliferation of osteogenic cells.

    Science.gov (United States)

    Inkson, Colette A; Ono, Mitsuaki; Bi, Yanming; Kuznetsov, Sergei A; Fisher, Larry W; Young, Marian F

    2009-01-01

    Biglycan (BGN) and WISP-1 are 2 extracellular matrix proteins that bind to each other and colocalize in mineralizing tissue. Here we show that WISP-1 abrogates the repression of proliferation in bone marrow stromal cells induced by BGN. We also demonstrate that WISP-1 and its variant WISP-1va can alleviate the repressed osteogenic differentiation caused by the absence of BGN. These preliminary data suggest that WISP-1 and BGN may functionally interact and control each other's activity, thus regulating the differentiation and proliferation of osteogenic cells. Copyright 2008 S. Karger AG, Basel.

  19. Functionalized carbon nanotubes as suitable scaffold materials for proliferation and differentiation of canine mesenchymal stem cells.

    Science.gov (United States)

    Das, Kinsuk; Madhusoodan, A P; Mili, Bhabesh; Kumar, Ajay; Saxena, A C; Kumar, Kuldeep; Sarkar, Mihir; Singh, Praveen; Srivastava, Sameer; Bag, Sadhan

    2017-01-01

    In the field of regenerative medicine, numerous potential applications of mesenchymal stem cells (MSCs) can be envisaged, due to their ability to differentiate into a range of tissues on the basis of the substrate on which they grow. With the advances in nanotechnology, carbon nanotubes (CNTs) have been widely explored for use as cell culture substrate in tissue engineering applications. In this study, canine bone marrow-derived MSCs were considered as the cellular model for an in vitro study to elucidate the collective cellular processes, using three different varieties of thin films of functionalized carbon nanotubes (COOH-single-walled CNTs [SWCNTs], COOH-multiwalled CNTs [MWCNTs] and polyethylene glycol [PEG]-SWCNTs), which were spray dried onto preheated cover slips. Cells spread out better on the CNT films, resulting in higher cell surface area and occurrence of filopodia, with parallel orientation of stress fiber bundles. Canine MSCs proliferated at a slower rate on all types of CNT substrates compared to the control, but no decline in cell number was noticed during the study period. Expression of apoptosis-associated genes decreased on the CNT substrates as time progressed. On flow cytometry after AnnexinV-fluorescein isothiocyanate/propidium iodide (PI) staining, total number of apoptotic and necrotic cells remained lower in COOH-functionalized films compared to PEG-functionalized ones. Collectively, these results indicate that COOH-MWCNT substrate provided an environment of low cytotoxicity. Canine MSCs were further induced to differentiate along osteogenic, chondrogenic, and neuronal lineages by culturing under specific differentiation conditions. The cytochemical and immunocytochemical staining results, as well as the expression of the bone marker genes, led us to hypothesize that the COOH-MWCNT substrate acted as a better cue, accelerating the osteogenic differentiation process. However, while chondrogenesis was promoted by COOH-SWCNT, neuronal

  20. MicroRNA-194 regulates keratinocyte proliferation and differentiation by targeting Grainyhead-like 2 in psoriasis.

    Science.gov (United States)

    Yu, Xiaoyun; An, Jingang; Hua, Yunhui; Li, Zihai; Yan, Ning; Fan, Weixin; Su, Chuan

    2017-02-01

    MicroRNAs (miRNAs) are currently emerged as important regulators in psoriasis. Psoriasis is characterized by hyperproliferation and impaired differentiation of keratinocytes in skin lesions. miR-194 is a well-known regulator of cell proliferation and differentiation. However, the role of miR-194 in psoriasis pathogenesis remains unclear. In this study we aimed to investigate the role of miR-194 in keratinocyte hyperproliferation and differentiation. We found that miR-194 was significantly downregulated in psoriasis lesional skin. Overexpression of miR-194 inhibited the proliferation and promoted the differentiation of primary human keratinocytes, whereas miR-194 suppression promoted the proliferation and inhibited their differentiation. Bioinformatic analysis predicted that the Grainyhead-like 2 (GRHL2) was a target gene of miR-194, which we further validated with a dual-luciferase reporter assay, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot analysis. The effect of miR-194 on cell proliferation and differentiation was significantly reversed by overexpression of GRHL2. Moreover, the expression of miR-194 and GRHL2 was inversely correlated in psoriasis lesional skin. Taken together, our results suggest that miR-194 inhibits the proliferation and promotes the differentiation of keratinocytes through targeting GRHL2. The downregulation of miR-194 expression may contribute to the pathogenesis of psoriasis and targeting miR-194 may represent a novel and potential therapeutic strategy for psoriasis. Copyright © 2016 Elsevier GmbH. All rights reserved.

  1. Peroxisome Proliferator-Activated Receptor (PPAR): Balance for Survival in Parasitic Infections

    OpenAIRE

    Chan, Marion M.; Evans, Kyle W.; Moore, Andrea R.; Dunne Fong

    2010-01-01

    Parasitic infections induce a magnitude of host responses. At the opposite ends of the spectrum are those that ensure the host's needs to eliminate the invaders and to minimize damage to its own tissues. This review analyzes how parasites would manipulate immunity by activating the immunosuppressive nuclear factor, peroxisome proliferator-activated receptors (PPARs) with type 2 cytokines and free fatty acids from arachidonic acid metabolism. PPARs limit the action of type 1 immunity, in w...

  2. Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation

    Energy Technology Data Exchange (ETDEWEB)

    Herzmann, Nicole; Salamon, Achim [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany); Fiedler, Tomas [Institute for Medical Microbiology, Virology and Hygiene, University Medicine Rostock, Schillingallee 70, D-18057 Rostock (Germany); Peters, Kirsten, E-mail: kirsten.peters@med.uni-rostock.de [Department of Cell Biology, University Medicine Rostock, Schillingallee 69, D-18057 Rostock (Germany)

    2017-01-01

    Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.

  3. Enhancement of Dopaminergic Differentiation in Proliferating Midbrain Neuroblasts by Sonic Hedgehog and Ascorbic Acid

    Science.gov (United States)

    Volpicelli, Floriana; Consales, Claudia; Caiazzo, Massimiliano; Colucci-D'Amato, Luca; Perrone-Capano, Carla; di Porzio, Umberto

    2004-01-01

    We analyzed the molecular mechanisms involved in the acquisition and maturation of dopaminergic (DA) neurons generated in vitro from rat ventral mesencephalon (MES) cells in the presence of mitogens or specific signaling molecules. The addition of basic fibroblast growth factor (bFGF) to MES cells in serum-free medium stimulates the proliferation of neuroblasts but delays DA differentiation. Recombinant Sonic hedgehog (SHH) protein increases up to three fold the number of tyrosine hydroxylase (TH)-positive cells and their differentiation, an effect abolished by anti-SHH antibodies. The expanded cultures are rich in nestin-positive neurons, glial cells are rare, all TH+ neurons are DA, and all DA and GABAergic markers analyzed are expressed. Adding ascorbic acid to bFGF/SHH-treated cultures resulted in a further five- to seven-fold enhancement of viable DA neurons. This experimental system also provides a powerful tool to generate DA neurons from single embryos. Our strategy provides an enriched source of MES DA neurons that are useful for analyzing molecular mechanisms controlling their function and for experimental regenerative approaches in DA dysfunction. PMID:15303305

  4. [Impact of different degree pulpitis on cell proliferation and osteoblastic differentiation of dental pulp stem cell in Beagle immature premolars].

    Science.gov (United States)

    Ling, L; Zhao, Y M; Ge, L H

    2016-10-18

    To compare the proliferation and osteoblastic differentiation of dental pulp stem cell (DPSC) isolated from normal and inflamed pulps of different degrees in Beagle immature premolars, and provide evidence for the use of inflammatory DPSC (IDPSC). This study evaluated 14 Beagle's young premolars (21 roots). In the experiment group, irreversible pulpitis was induced by pulp exposure and the inflamed pulps were extracted 2 weeks and 6 weeks after the pulp chamber opening.For the control group, normal pulps were extracted immediately after the exposure. HE staining and real-time PCR were performed to confirm the inflammation. The cells were isolated from the inflamed and normal pulps (IDPSC and DPSC). Cell proliferation and osteoblastic differentiation potentials of the two cells were compared. Inflammation cells infiltration was observed in the inflamed pulps by HE staining. The expression of inflammatory factor was much higher in the 6 week inflamed pulp. IDPSC had higher potential of cell proliferation and osteoblastic differentiation potentials. Furthermore, the osteoblastic differentiation potentials of IDPSC from 2 week inflamed pulp were higher than those from 6 week inflamed pulp. The potential of cell proliferation and osteoblastic differentiation of DPSC was enhanced at early stage of irreversible pulpitis, and reduced at late stage in Beagle immature premolars.

  5. Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression

    Science.gov (United States)

    Ruijtenberg, Suzan; van den Heuvel, Sander

    2016-01-01

    ABSTRACT Cell proliferation and differentiation show a remarkable inverse relationship. Precursor cells continue division before acquiring a fully differentiated state, while terminal differentiation usually coincides with proliferation arrest and permanent exit from the division cycle. Mechanistic insight in the temporal coordination between cell cycle exit and differentiation has come from studies of cells in culture and genetic animal models. As initially described for skeletal muscle differentiation, temporal coordination involves mutual antagonism between cyclin-dependent kinases that promote cell cycle entry and transcription factors that induce tissue-specific gene expression. Recent insights highlight the contribution of chromatin-regulating complexes that act in conjunction with the transcription factors and determine their activity. In particular SWI/SNF chromatin remodelers contribute to dual regulation of cell cycle and tissue-specific gene expression during terminal differentiation. We review the concerted regulation of the cell cycle and cell type-specific transcription, and discuss common mutations in human cancer that emphasize the clinical importance of proliferation versus differentiation control. PMID:26825227

  6. Chronic treatment with the GLP1 analogue liraglutide increases cell proliferation and differentiation into neurons in an AD mouse model.

    Directory of Open Access Journals (Sweden)

    Vadivel Parthsarathy

    Full Text Available Neurogenesis is a life long process, but the rate of cell proliferation and differentiation decreases with age. In Alzheimer's patients, along with age, the presence of Aβ in the brain inhibits this process by reducing stem cell proliferation and cell differentiation. GLP-1 is a growth factor that has neuroprotective properties. GLP1 receptors are present on neuronal progenitor cells, and the GLP-1 analogue liraglutide has been shown to increase cell proliferation in an Alzheimer's disease (AD mouse model. Here we investigated acute and chronic effects of liraglutide on progenitor cell proliferation, neuroblast differentiation and their subsequent differentiation into neurons in wild type and APP/PS-1 mice at different ages. APP/PS1 and their littermate controls, aged 3, 6, 12, 15 months were injected acutely or chronically with 25 nmol/kg liraglutide. Acute treatment with liraglutide showed an increase in cell proliferation in APP/PS1 mice, but not in controls whereas chronic treatment increased cell proliferation at all ages (BrdU and Ki67 markers. Moreover, numbers of immature neurons (DCX were increased in both acute and chronic treated animals at all ages. Most newly generated cells differentiated into mature neurons (NeuN marker. A significant increase was observed with chronically treated 6, 12, 15 month APP/PS1 and WT groups. These results demonstrate that liraglutide, which is currently on the market as a treatment for type 2 diabetes (Victoza(TM, increases neurogenesis, which may have beneficial effects in neurodegenerative disorders like AD.

  7. Fullerene mediates proliferation and cardiomyogenic differentiation of adipose-derived stem cells via modulation of MAPK pathway and cardiac protein expression.

    Science.gov (United States)

    Hao, Tong; Zhou, Jin; Lü, Shuanghong; Yang, Boguang; Wang, Yan; Fang, Wancai; Jiang, Xiaoxia; Lin, Qiuxia; Li, Junjie; Wang, Changyong

    2016-01-01

    Zero-dimensional fullerenes can modulate the biological behavior of a variety of cell lines. However, the effects and molecular mechanisms of proliferation and cardiomyogenic differentiation in brown adipose-derived stem cells (BADSCs) are still unclear. In this study, we report the initial biological effects of fullerene-C60 on BADSCs at different concentrations. Results suggest that fullerene-C60 has no cytotoxic effects on BADSCs even at a concentration of 100 μg/mL. Fullerene-C60 improves the MAPK expression level and stem cell survival, proliferation, and cardiomyogenesis. Further, we found that the fullerene-C60 modulates cardiomyogenic differentiation. Fullerene-C60 improves the expression of cardiomyocyte-specific proteins (cTnT and α-sarcomeric actinin). At elevated concentration, fullerene-C60 reduces the incidence of diminished spontaneous cardiac differentiation of BADSCs with time. At the genetic level, fullerene-C60 (5 μg/mL) also improves the expression of cTnT. In addition, fullerene-C60 promotes the formation of gap junction among cells. These findings have important implications for clinical application of fullerenes in the treatment of myocardial infarction.

  8. Tenovin-6 inhibits proliferation and survival of diffuse large B-cell lymphoma cells by blocking autophagy.

    Science.gov (United States)

    Yuan, Hongfeng; He, Meilan; Cheng, Fan; Bai, Rosemary; da Silva, Suzane Ramos; Aguiar, Ricardo C T; Gao, Shou-Jiang

    2017-02-28

    Diffuse large B-cell lymphoma (DLBCL) is one of the most aggressive non-Hodgkin lymphomas. It is curable but one-third of cases are refractory to therapy or relapse after initial response highlighting the urgent need for developing novel therapeutic approaches. Targeting sirtuins, particularly SIRT1 by genetic approaches or using pharmaceutical inhibitor tenovin-6, has shown promising therapeutic potential in various hematopoietic malignancies. However, it remains unknown whether these approaches are effective for DLBCL. In this study, we have found that tenovin-6 potently inhibits the proliferation and survival of DLBCL cells. Surprisingly, specific knockdown of SIRT1/2/3 has no effect on DLBCL. Mechanistically, tenovin-6 increases the level of microtubule-associated protein 1 light chain 3B (LC3B)-II in a SIRT1/2/3- and p53-independent manner in DLBCL cell lines. Tenovin-6-mediated increase of LC3B-II is through inhibition of classical autophagy pathway. Furthermore, inhibition of the autophagy pathway by using other inhibitors or by knocking down key genes in the pathway impairs cell proliferation and survival of DLBCL cells. These results indicate that targeting the autophagic pathway could be a novel therapeutic strategy for DLBCL and that precaution should be taken to interpret data where tenovin-6 was used as an inhibitor of sirtuins.

  9. Bmi1 overexpression in the cerebellar granule cell lineage of mice affects cell proliferation and survival without initiating medulloblastoma formation

    Directory of Open Access Journals (Sweden)

    Hourinaz Behesti

    2013-01-01

    BMI1 is a potent inducer of neural stem cell self-renewal and neural progenitor cell proliferation during development and in adult tissue homeostasis. It is overexpressed in numerous human cancers – including medulloblastomas, in which its functional role is unclear. We generated transgenic mouse lines with targeted overexpression of Bmi1 in the cerebellar granule cell lineage, a cell type that has been shown to act as a cell of origin for medulloblastomas. Overexpression of Bmi1 in granule cell progenitors (GCPs led to a decrease in cerebellar size due to decreased GCP proliferation and repression of the expression of cyclin genes, whereas Bmi1 overexpression in postmitotic granule cells improved cell survival in response to stress by altering the expression of genes in the mitochondrial cell death pathway and of Myc and Lef-1. Although no medulloblastomas developed in ageing cohorts of transgenic mice, crosses with Trp53−/− mice resulted in a low incidence of medulloblastoma formation. Furthermore, analysis of a large collection of primary human medulloblastomas revealed that tumours with a BMI1high TP53low molecular profile are significantly enriched in Group 4 human medulloblastomas. Our data suggest that different levels and timing of Bmi1 overexpression yield distinct cellular outcomes within the same cellular lineage. Importantly, Bmi1 overexpression at the GCP stage does not induce tumour formation, suggesting that BMI1 overexpression in GCP-derived human medulloblastomas probably occurs during later stages of oncogenesis and might serve to enhance tumour cell survival.

  10. Ex Vivo Expanded Human NK Cells Survive and Proliferate in Humanized Mice with Autologous Human Immune Cells.

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    Vahedi, Fatemeh; Nham, Tina; Poznanski, Sophie M; Chew, Marianne V; Shenouda, Mira M; Lee, Dean; Ashkar, Ali A

    2017-09-21

    Adoptive immune cell therapy is emerging as a promising immunotherapy for cancer. Particularly, the adoptive transfer of NK cells has garnered attention due to their natural cytotoxicity against tumor cells and safety upon adoptive transfer to patients. Although strategies exist to efficiently generate large quantities of expanded NK cells ex vivo, it remains unknown whether these expanded NK cells can persist and/or proliferate in vivo in the absence of exogenous human cytokines. Here, we have examined the adoptive transfer of ex vivo expanded human cord blood-derived NK cells into humanized mice reconstituted with autologous human cord blood immune cells. We report that ex vivo expanded NK cells are able to survive and possibly proliferate in vivo in humanized mice without exogenous cytokine administration, but not in control mice that lack human immune cells. These findings demonstrate that the presence of autologous human immune cells supports the in vivo survival of ex vivo expanded human NK cells. These results support the application of ex vivo expanded NK cells in cancer immunotherapy and provide a translational humanized mouse model to test the lifespan, safety, and functionality of adoptively transferred cells in the presence of autologous human immune cells prior to clinical use.

  11. Induction of cell proliferation and survival genes by estradiol-repressed microRNAs in breast cancer cells

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    Yu Xinfeng

    2012-01-01

    Full Text Available Abstract Background In estrogen responsive MCF-7 cells, estradiol (E2 binding to ERα leads to transcriptional regulation of genes involved in the control of cell proliferation and survival. MicroRNAs (miRNAs have emerged as key post-transcriptional regulators of gene expression. The aim of this study was to explore whether miRNAs were involved in hormonally regulated expression of estrogen responsive genes. Methods Western blot and QPCR were used to determine the expression of estrogen responsive genes and miRNAs respectively. Target gene expression regulated by miRNAs was validated by luciferase reporter assays and transfection of miRNA mimics or inhibitors. Cell proliferation was evaluated by MTS assay. Results E2 significantly induced bcl-2, cyclin D1 and survivin expression by suppressing the levels of a panel of miRNAs (miR-16, miR-143, miR-203 in MCF-7 cells. MiRNA transfection and luciferase assay confirmed that bcl-2 was regulated by miR-16 and miR-143, cyclinD1 was modulated by miR-16. Importantly, survivin was found to be targeted by miR-16, miR-143, miR-203. The regulatory effect of E2 can be either abrogated by anti-estrogen ICI 182, 780 and raloxifene pretreatment, or impaired by ERα siRNA, indicating the regulation is dependent on ERα. In order to investigate the functional significance of these miRNAs in estrogen responsive cells, miRNAs mimics were transfected into MCF-7 cells. It revealed that overexpression of these miRNAs significantly inhibited E2-induced cell proliferation. Further study of the expression of the miRNAs indicated that miR-16, miR-143 and miR-203 were highly expressed in triple positive breast cancer tissues, suggesting a potential tumor suppressing effect of these miRNAs in ER positive breast cancer. Conclusions These results demonstrate that E2 induces bcl-2, cyclin D1 and survivin by orchestrating the coordinate downregulation of a panel of miRNAs. In turn, the miRNAs manifest growth suppressive effects

  12. Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Michael Lohmann

    Full Text Available The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS or other serum supplements such as human platelet lysate (HPL. In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1, but it was significantly higher with HPLs from younger donors (45 years. Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal. HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1 or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3 were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

  13. Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Lohmann, Michael; Walenda, Gudrun; Hemeda, Hatim; Joussen, Sylvia; Drescher, Wolf; Jockenhoevel, Stefan; Hutschenreuter, Gabriele; Zenke, Martin; Wagner, Wolfgang

    2012-01-01

    The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

  14. The alpha subunit of Go modulates cell proliferation and differentiation through interactions with Necdin.

    Science.gov (United States)

    Ju, Hyunhee; Lee, Sujin; Kang, Sunghak; Kim, Sung-Soo; Ghil, Sungho

    2014-07-10

    Heterotrimeric GTP-binding proteins (G-proteins) play an important role in mediating signal transduction generated by neurotransmitters or hormones. Go, a member of the Gi/Go subfamily, is the most abundant G-protein found in the brain. Recently, the alpha subunit of Go (Gαo) was characterized as an inducer of neuronal differentiation. However, its underlying molecular mechanisms have remained unclear to date, since the downstream effectors of Gαo are ambiguous. A neurally differentiated embryonal carcinoma-derived protein (Necdin) was isolated as an interacting partner for Gαo from a mouse brain cDNA library using yeast two-hybrid screening. Interactions between the proteins were confirmed with several affinity binding assays, both in vitro and in vivo. Necdin interacted directly and preferentially with activated Gαo, compared to wild-type protein. Interestingly, Gαo did not interact with Gαi, despite high sequence homology between the two proteins. We subsequently analyzed whether Gαo modulates the cellular activities of Necdin. Notably, expression of Gαo significantly augmented Necdin-mediated cellular responses, such as proliferation and differentiation. Moreover, activation of type 1 cannabinoid receptor (CB1R), a Gi/oα-coupled receptor, augmented cell growth suppression, which was mediated by Gαo and Necdin in U87MG cells containing CB1R, Gαo, and Necdin as normal components. These results collectively suggest that Necdin is a candidate downstream effector for Gαo. Our findings provide novel insights into the cellular roles of Gαo and its coupled receptor.

  15. Folic acid is necessary for proliferation and differentiation of C2C12 myoblasts.

    Science.gov (United States)

    Hwang, Seong Y; Kang, Yong J; Sung, Bokyung; Jang, Jung Y; Hwang, Na L; Oh, Hye J; Ahn, Yu R; Kim, Hong J; Shin, Jin H; Yoo, Mi-Ae; Kim, Cheol M; Chung, Hae Y; Kim, Nam D

    2017-05-04

    Folic acid, a water soluble B vitamin, plays an important role in cellular metabolic activities, such as functioning as a cofactor in one-carbon metabolism for DNA and RNA synthesis as well as nucleotide and amino acid biosynthesis in the body. A lack of dietary folic acid can lead to folic acid deficiency and result in several health problems, including macrocytic anemia, elevated plasma homocysteine, cardiovascular disease, birth defects, carcinogenesis, muscle weakness, and walking difficulty. However, the effect of folic acid deficiency on skeletal muscle development and its molecular mechanisms are unknown. We, therefore, investigated the effect of folic acid deficiency on myogenesis in skeletal muscle cells and found that folic acid deficiency induced proliferation inhibition and cell cycle breaking as well as cellular senescence in C2C12 myoblasts, implying that folic acid deficiency influences skeletal muscle development. Folic acid deficiency also inhibited differentiation of C2C12 myoblasts and induced deregulation of the cell cycle exit and many cell cycle regulatory genes. It inhibited expression of muscle-specific marker MyHC as well as myogenic regulatory factor (myogenin). Moreover, immunocytochemistry and Western blot analyses revealed that DNA damage was more increased in folic acid-deficient medium-treated differentiating C2C12 cells. Furthermore, we found that folic acid resupplementation reverses the effect on the cell cycle and senescence in folic acid-deficient C2C12 myoblasts but does not reverse the differentiation of C2C12 cells. Altogether, the study results suggest that folic acid is necessary for normal development of skeletal muscle cells. © 2017 Wiley Periodicals, Inc.

  16. RANK Overexpression in Transgenic Mice with Mouse Mammary Tumor Virus Promoter-Controlled RANK Increases Proliferation and Impairs Alveolar Differentiation in the Mammary Epithelia and Disrupts Lumen Formation in Cultured Epithelial Acini▿ †

    Science.gov (United States)

    Gonzalez-Suarez, Eva; Branstetter, Daniel; Armstrong, Allison; Dinh, Huyen; Blumberg, Hal; Dougall, William C.

    2007-01-01

    RANK and RANKL, the key regulators of osteoclast differentiation and activation, also play an important role in the control of proliferation and differentiation of mammary epithelial cells during pregnancy. Here, we show that RANK protein expression is strictly regulated in a spatial and temporal manner during mammary gland development. RANK overexpression under the control of the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model results in increased mammary epithelial cell proliferation during pregnancy, impaired differentiation of lobulo-alveolar structures, decreased expression of the milk proteins β-casein and whey acidic protein, and deficient lactation. We also show that treatment of three-dimensional in vitro cultures of primary mammary cells from MMTV-RANK mice with RANKL results in increased proliferation and decreased apoptosis in the luminal area, resulting in bigger acini with filled lumens. Taken together, these results suggest that signaling through RANK not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells. Moreover, the increased proliferation and survival observed in a three-dimensional culture system suggests a role for aberrant RANK signaling during breast tumorigenesis. PMID:17145767

  17. RANK overexpression in transgenic mice with mouse mammary tumor virus promoter-controlled RANK increases proliferation and impairs alveolar differentiation in the mammary epithelia and disrupts lumen formation in cultured epithelial acini.

    Science.gov (United States)

    Gonzalez-Suarez, Eva; Branstetter, Daniel; Armstrong, Allison; Dinh, Huyen; Blumberg, Hal; Dougall, William C

    2007-02-01

    RANK and RANKL, the key regulators of osteoclast differentiation and activation, also play an important role in the control of proliferation and differentiation of mammary epithelial cells during pregnancy. Here, we show that RANK protein expression is strictly regulated in a spatial and temporal manner during mammary gland development. RANK overexpression under the control of the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model results in increased mammary epithelial cell proliferation during pregnancy, impaired differentiation of lobulo-alveolar structures, decreased expression of the milk proteins beta-casein and whey acidic protein, and deficient lactation. We also show that treatment of three-dimensional in vitro cultures of primary mammary cells from MMTV-RANK mice with RANKL results in increased proliferation and decreased apoptosis in the luminal area, resulting in bigger acini with filled lumens. Taken together, these results suggest that signaling through RANK not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells. Moreover, the increased proliferation and survival observed in a three-dimensional culture system suggests a role for aberrant RANK signaling during breast tumorigenesis.

  18. TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling.

    Science.gov (United States)

    He, Xiaoqing; Wang, Hai; Jin, Tao; Xu, Yongqing; Mei, Liangbin; Yang, Jun

    2016-01-01

    Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.

  19. Akt- and Erk-mediated regulation of proliferation and differentiation during PDGFRβ-induced MSC self-renewal.

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    Gharibi, Borzo; Ghuman, Mandeep S; Hughes, Francis J

    2012-11-01

    Understanding the mechanisms that direct mesenchymal stem cell (MSC) self-renewal fate decisions is a key to most tissue regenerative approaches. The aim of this study here was to investigate the mechanisms of action of platelet-derived growth factor receptor β (PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF-BB together with inhibitors of second messenger pathways. Cell proliferation was assessed using ethynyl-2'-deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT-PCR, and by long-term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K/Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K/Akt and PDGFR-β expression. In addition, PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E-BP1 were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ-induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self-renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self-renewal strategies. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  20. c-Myb Regulates Proliferation and Differentiation of Adventitial Sca1+ Vascular Smooth Muscle Cell Progenitors by Transactivation of Myocardin.

    Science.gov (United States)

    Shikatani, Eric A; Chandy, Mark; Besla, Rickvinder; Li, Cedric C; Momen, Abdul; El-Mounayri, Omar; Robbins, Clinton S; Husain, Mansoor

    2016-07-01

    Vascular smooth muscle cells (VSMCs) are believed to dedifferentiate and proliferate in response to vessel injury. Recently, adventitial progenitor cells were implicated as a source of VSMCs involved in vessel remodeling. c-Myb is a transcription factor known to regulate VSMC proliferation in vivo and differentiation of VSMCs from mouse embryonic stem cell-derived progenitors in vitro. However, the role of c-Myb in regulating specific adult vascular progenitor cell populations was not known. Our objective was to examine the role of c-Myb in the proliferation and differentiation of Sca1(+) adventitial VSMC progenitor cells. Using mice with wild-type or hypomorphic c-myb (c-myb(h/h)), BrdU (bromodeoxyuridine) uptake and flow cytometry revealed defective proliferation of Sca1(+) adventitial VSMC progenitor cells at 8, 14, and 28 days post carotid artery denudation injury in c-myb(h/h) arteries. c-myb(h/h) cKit(+)CD34(-)Flk1(-)Sca1(+)CD45(-)Lin(-) cells failed to proliferate, suggesting that c-myb regulates the activation of specific Sca1(+) progenitor cells in vivo and in vitro. Although expression levels of transforming growth factor-β1 did not vary between wild-type and c-myb(h/h) carotid arteries, in vitro differentiation of c-myb(h/h) Sca1(+) cells manifested defective transforming growth factor-β1-induced VSMC differentiation. This is mediated by reduced transcriptional activation of myocardin because chromatin immunoprecipitation revealed c-Myb binding to the myocardin promoter only during differentiation of Sca1(+) cells, myocardin promoter mutagenesis identified 2 specific c-Myb-responsive binding sites, and adenovirus-mediated expression of myocardin rescued the phenotype of c-myb(h/h) progenitors. These data support a role for c-Myb in the regulation of VSMC progenitor cells and provide novel insight into how c-myb regulates VSMC differentiation through myocardin. © 2016 American Heart Association, Inc.

  1. Serum-free spheroid suspension culture maintains mesenchymal stem cell proliferation and differentiation potential.

    Science.gov (United States)

    Alimperti, Stella; Lei, Pedro; Wen, Yuan; Tian, Jun; Campbell, Andrew M; Andreadis, Stelios T

    2014-01-01

    There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several disease states such as graft-versus-host disease, acute myocardial infarction, Crohn's disease, and multiple sclerosis. The use of MSCs for therapy is expected to become more prevalent as clinical progress is demonstrated. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the MSC-based indications further exacerbates this manufacturing challenge. In contrast, expanding MSCs as spheroids does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In the present study, we developed and optimized serum-free media for culturing MSC spheroids. We used Design of Experiment (DoE)-based strategies to systematically evaluate media mixtures and a panel of different components for effects on cell proliferation. The optimization yielded two prototype serum-free media that enabled MSCs to form aggregates and proliferate in both static and dynamic cultures. MSCs from spheroid cultures exhibited the expected immunophenotype (CD73, CD90, and CD105) and demonstrated similar or enhanced differentiation potential toward all three lineages (osteogenic, chondrogenic, adipogenic) as compared with serum-containing adherent MSC cultures. Our results suggest that serum-free media for MSC spheroids may pave the way for scale-up production of MSCs in clinically relevant manufacturing platforms such as stirred tank bioreactors. © 2014 American Institute of Chemical Engineers.

  2. Polycomb group protein Ezh2 regulates hepatic progenitor cell proliferation and differentiation in murine embryonic liver.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Koike

    Full Text Available In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. Enhancer of zeste homolog 2 (Ezh2, a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3, which trigger the gene suppression. In the present study, we investigated a role of Ezh2 in the regulation of the expanding hepatic progenitor population in vivo. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of Ezh2, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from in vivo fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion in vivo, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3.

  3. Improved osteoblast proliferation, differentiation and mineralization on nanophase Ti6Al4V.

    Science.gov (United States)

    Han, Pei; Ji, Wei-Ping; Zhao, Chang-Li; Zhang, Xiao-Nong; Jiang, Yao

    2011-01-01

    Previous studies have demonstrated increased functions of osteoblasts on nanophase materials compared to conventional ceramics or composites. Nanophase materials are unique materials that simulate dimensions of constituent components of bone as they possess particle or grain sizes less than 100 nm. However, to date, interactions of osteoblasts on nanophase materials compared to conventional metals remain to be elucidated. The objective of the present in vitro study was to synthesize nanophase metals (Ti6Al4V), characterize, and evaluate osteoblast functions on Ti6Al4V. Such metals in conventional form are widely used in orthopedic applications. In this work, nanophase Ti6Al4V surfaces were processed by the severe plastic deformation (SPD) principle and used to investigate osteoblast long-term functions. Primary cultured osteoblasts from neonatal rat calvaria were cultured on both nanophase and conventional Ti6Al4V substrates. Cell proliferation, total protein content, and alkaline phosphatase (ALP) activity were evaluated after 1, 3, 7, 10 and 14 days. Calcium deposition, gene expression of type I collagen (Col-I), osteocalcin (OC), osteopontin (OP) and the production of insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-β1) were also investigated after 14 days of culture. Functions of osteoblasts including proliferation, synthesis of protein, and ALP activity were improved on the nanophase compared to the conventional Ti6Al4V. The expression of Col-I, OC and OP mRNA was also increased on nanophase Ti6Al4V after 14 days of culture. Calcium deposition was the same; the average number of the calcified nodules on the two Ti6Al4V surfaces was similar after 14 days of culture; however, highly significant size calcified nodules on the nanophase Ti6Al4V was observed. Of the growth factors examined, only TGF-β1 showed a difference in production on the nanophase surface. Nanophase Ti6Al4V surfaces improve proliferation, differentiation and

  4. The effect of human platelet-rich plasma on adipose-derived stem cell proliferation and osteogenic differentiation.

    Science.gov (United States)

    Tavakolinejad, Sima; Khosravi, Mohsen; Mashkani, Baratali; Ebrahimzadeh Bideskan, Alireza; Sanjar Mossavi, Nasser; Parizadeh, Mohammad Reza Seyyed; Hamidi Alamdari, Daryoush

    2014-07-01

    The cultured mesenchymal stem cells (MSC) have been used in many clinical trials; however, there are still some concerns about the cultural conditions. One concern is related to the use of FBS as a widely used xenogeneic supplement in the culture system. Human platelet-rich plasma (hPRP) is a candidate replacement for FBS. In this study, the effect of hPRP on MSC proliferation and osteogenic differentiation has been evaluated. Human adipose-derived stem cells (hADSC) were expanded. Cells from the third passage were characterized by flow cytometric analysis and used for in vitro experiments. Resazurin and alizarin red stains were used for cell proliferation and osteogenic differentiation assays, respectively. Treatment with hPRP resulted in a statistically significant increase in cell proliferation compare to the negative control group (Pinvestigation.

  5. Prostaglandin receptor EP3 regulates cell proliferation and migration with impact on survival of endometrial cancer patients.

    Science.gov (United States)

    Zhu, Junyan; Trillsch, Fabian; Mayr, Doris; Kuhn, Christina; Rahmeh, Martina; Hofmann, Simone; Vogel, Marianne; Mahner, Sven; Jeschke, Udo; von Schönfeldt, Viktoria

    2018-01-02

    Prostaglandin E2 (PGE2) receptor 3 (EP3) regulates tumor cell proliferation, migration, and invasion in numerous cancers. The role of EP3 as a prognostic biomarker in endometrial cancer remains unclear. The primary aim of this study was to analyze the prognostic significance of EP3 expression in endometrial cancer. We analyzed the EP3 expression of 140 endometrial carcinoma patients by immunohistochemistry. RL95-2 endometrial cancer cell line was chosen from four endometrial cancer cell lines (RL95-2, Ishikawa, HEC-1-A, and HEC-1-B) according to EP3 expression level. Treated with PGE2 and EP3 antagonist, RL95-2 cells were investigated by MTT, BrdU, and wound healing assay for functional assessment of EP3. EP3 staining differed significantly according to WHO tumor grading in both whole cohort (p = 0.01) and the subgroup of endometrioid carcinoma (p = 0.01). Patients with high EP3 expression in their respective tumors had impaired progression-free survival as well as overall survival in both cohorts above. EP3 expression in the overall cohort was identified as an independent prognostic marker for progression-free survival (HR 1.014, 95%CI 1.003-1.024, p = 0.01) when adjusted for age, stage, grading, and recurrence. Treatment with EP3 antagonists induced upregulation of estrogen receptor β and decreased activity of Ras and led to attenuated proliferation and migration of RL95-2 cells. EP3 seems to play a crucial role in endometrial cancer progression. In the context of limited systemic treatment options for endometrial cancer, this explorative analysis identifies EP3 as a potential target for diagnostic workup and therapy.

  6. Correct timing of proliferation and differentiation is necessary for normal inner ear development and auditory hair cell viability.

    Science.gov (United States)

    Kopecky, Benjamin J; Jahan, Israt; Fritzsch, Bernd

    2013-02-01

    Hearing restoration through hair cell regeneration will require revealing the dynamic interactions between proliferation and differentiation during development to avoid the limited viability of regenerated hair cells. Pax2-Cre N-Myc conditional knockout (CKO) mice highlighted the need of N-Myc for proper neurosensory development and possible redundancy with L-Myc. The late-onset hair cell death in the absence of early N-Myc expression could be due to mis-regulation of genes necessary for neurosensory formation and maintenance, such as Neurod1, Atoh1, Pou4f3, and Barhl1. Pax2-Cre N-Myc L-Myc double CKO mice show that proliferation and differentiation are linked together through Myc and in the absence of both Mycs, altered proliferation and differentiation result in morphologically abnormal ears. In particular, the organ of Corti apex is re-patterned into a vestibular-like organization and the base is truncated and fused with the saccule. These data indicate that therapeutic approaches to restore hair cells must take into account a dynamic interaction of proliferation and differentiation regulation of basic Helix-Loop-Helix transcription factors in attempts to stably replace lost cochlear hair cells. In addition, our data indicate that Myc is an integral component of the evolutionary transformation process that resulted in the organ of Corti development. Copyright © 2012 Wiley Periodicals, Inc.

  7. Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stiehler, Maik; Bünger, Cody; Baatrup, Anette

    2009-01-01

    Ex vivo engineering of autologous bone tissue as an alternative to bone grafting is a major clinical need. In the present study, we evaluated the effect of 3-D dynamic spinner flask culture on the proliferation, distribution, and differentiation of human mesenchymal stem cells (MSCs). Immortalize...

  8. Comparative Analysis of Spontaneous and Stimulus-Evoked Calcium Transients in Proliferating and Differentiating Human Midbrain-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Torben Johansen

    2017-01-01

    Full Text Available Spontaneous cytosolic calcium transients and oscillations have been reported in various tissues of nonhuman and human origin but not in human midbrain-derived stem cells. Using confocal microfluorimetry, we studied spontaneous calcium transients and calcium-regulating mechanisms in a human ventral mesencephalic stem cell line undergoing proliferation and neuronal differentiation. Spontaneous calcium transients were detected in a large fraction of both proliferating (>50% and differentiating (>55% cells. We provide evidence for the existence of intracellular calcium stores that respond to muscarinic activation of the cells, having sensitivity for ryanodine and thapsigargin possibly reflecting IP3 receptor activity and the presence of ryanodine receptors and calcium ATPase pumps. The observed calcium transient activity potentially supports the existence of a sodium-calcium antiporter and the existence of calcium influx induced by depletion of calcium stores. We conclude that the cells have developed the most important mechanisms governing cytosolic calcium homeostasis. This is the first comparative report of spontaneous calcium transients in proliferating and differentiating human midbrain-derived stem cells that provides evidence for the mechanisms that are likely to be involved. We propose that the observed spontaneous calcium transients may contribute to mechanisms involved in cell proliferation, phenotypic differentiation, and general cell maturation.

  9. The effect of hypothyroidism on Sertoli cell proliferation and differentiation and hormone levels during testicular development in the rat

    NARCIS (Netherlands)

    van Haaster, L. H.; de Jong, F. H.; Docter, R.; de rooij, D. G.

    1992-01-01

    In this study we show that 6-propyl-2-thiouracil (PTU) treatment of Wistar rats from birth up to day 26 p.p. retards the morphological differentiation of Sertoli cells, and prolongs the proliferation of these cells up to day 30. Sertoli cell numbers per testis, determined at day 36, were increased

  10. The regulation of the proliferation and differentiation of rat Leydig cell precursor cells after EDS administration or daily HCG treatment

    NARCIS (Netherlands)

    Teerds, K. J.; de rooij, D. G.; Rommerts, F. F.; Wensing, C. J.

    1988-01-01

    The proliferation and differentiation of possible Leydig cell precursors in adult rats were studied after destruction of the existing Leydig cells with EDS or after daily treatment with hCG. After 2 days with either treatment, a 12- to 16-fold increase in the number of [3H]thymidine-incorporating

  11. Haemodynamic unloading increases the survival and affects the differentiation of cardiac stem cells after implantation into an infarcted heart.

    Science.gov (United States)

    Kurazumi, Hiroshi; Li, Tao-Sheng; Takemoto, Yoshihiro; Suzuki, Ryo; Mikamo, Akihito; Guo, Chang-Ying; Murata, Tomoaki; Hamano, Kimikazu

    2014-06-01

    It has been anticipated that stem cell therapy is capable of repairing an injured heart but is currently limited by its marginal efficacy. We believe that mechanical stress due to haemodynamic loading may negate the therapeutic potency of stem cells and therefore investigated how haemodynamic unloading affects the survival and differentiation of stem cells after implantation into an infarcted heart. A left ventricular (LV) haemodynamic unloading model was implemented by heterotopic transplantation of an infarcted donor heart into another healthy mouse. An in situ infarcted heart with general haemodynamic loading was used as control. A total of 5 million cardiac stem cells expanded from green fluorescence protein (GFP)-transgenic mouse were intramyocardially implanted into the infarcted LVs of haemodynamically unloaded donor heart or general haemodynamic loaded heart. The survival and differentiation of the implanted cardiac stem cells were evaluated by histological analyses at 3 and 21 days after cell implantation (n = 5-6 in each time points per group). Compared with the general haemodynamic loading condition, haemodynamic unloading of the infarcted hearts significantly improved the survival, increased the proliferation and inhibited the apoptosis of cardiac stem cells at 21 days after cell implantation (P cells was much higher in the unloaded hearts than in the loaded hearts at 21 days after cell implantation, although the difference was not statistically significant (5.67 ± 5.10 vs 0.75 ± 0.50, P = 0.051). Among the surviving GFP(+) donor cells 21 days after implantation, the expressions of platelet endothelial cell adhesion molecule-1, smooth muscle actin and sarcomeric alpha actin were ~7, 38 and 27% in the loaded heart and ~19, 14 and 55% in the unloaded heart, respectively. Haemodynamic unloading favours the survival/engraftment of donor stem cells and affects their differentiation after implantation into an infarcted heart. Although further studies in a

  12. Effect of all-trans retinoic acid on the proliferation and differentiation of brain tumor stem cells

    Directory of Open Access Journals (Sweden)

    Niu Chao

    2010-08-01

    Full Text Available Abstract Objective To investigate the effect of all-trans retinoic acid(ATRA on the proliferation and differentiation of brain tumor stem cells(BTSCs in vitro. Methods Limiting dilution and clonogenic assay were used to isolate and screen BTSCs from the fresh specimen of human brain glioblastoma. The obtained BTSCs, which were cultured in serum-free medium, were classified into four groups in accordance with the composition of the different treatments. The proliferation of the BTSCs was evaluated by MTT assay. The BTSCs were induced to differentiate in serum-containing medium, and classified into the ATRA group and control group. On the 10th day of induction, the expressions of CD133 and glial fibrillary acidic protein (GFAP in the differentiated BTSCs were detected by immunofluorescence. The differentiated BTSCs were cultured in serum-free medium, the percentage and the time required for formation of brain tumor spheres (BTS were observed. Results BTSCs obtained by limiting dilution were all identified as CD133-positive by immunofluorescence. In serum-free medium, the proliferation of BTSCs in the ATRA group was observed significantly faster than that in the control group, but slower than that in the growth factor group and ATRA/growth factor group, and the size of the BTS in the ATRA group was smaller than that in the latter two groups(P P P P Conclusion ATRA can promote the proliferation and induce the differentiation of BTSCs, but the differentiation is incomplete, terminal differentiation cannot be achieved and BTSs can be formed again.

  13. Macrophages improve survival, proliferation and migration of engrafted myogenic precursor cells into MDX skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Pierre-François Lesault

    Full Text Available Transplantation of muscle precursor cells is of therapeutic interest for focal skeletal muscular diseases. However, major limitations of cell transplantation are the poor survival, expansion and migration of the injected cells. The massive and early death of transplanted myoblasts is not fully understood although several mechanisms have been suggested. Various attempts have been made to improve their survival or migration. Taking into account that muscle regeneration is associated with the presence of macrophages, which are helpful in repairing the muscle by both cleansing the debris and deliver trophic cues to myoblasts in a sequential way, we attempted in the present work to improve myoblast transplantation by coinjecting macrophages. The present data showed that in the 5 days following the transplantation, macrophages efficiently improved: i myoblast survival by limiting their massive death, ii myoblast expansion within the tissue and iii myoblast migration in the dystrophic muscle. This was confirmed by in vitro analyses showing that macrophages stimulated myoblast adhesion and migration. As a result, myoblast contribution to regenerating host myofibres was increased by macrophages one month after transplantation. Altogether, these data demonstrate that macrophages are beneficial during the early steps of myoblast transplantation into skeletal muscle, showing that coinjecting these stromal cells may be used as a helper to improve the efficiency of parenchymal cell engraftment.

  14. Regulation of neural progenitor proliferation and survival by beta1 integrins

    DEFF Research Database (Denmark)

    Leone, Dino P; Relvas, João B; Campos, Lia S

    2005-01-01

    Neural stem cells give rise to undifferentiated nestin-positive progenitors that undergo extensive cell division before differentiating into neuronal and glial cells. The precise control of this process is likely to be, at least in part, controlled by instructive cues originating from...

  15. Effect of cryopreservation on proliferation and differentiation of periodontal ligament stem cell sheets.

    Science.gov (United States)

    Li, Mengying; Feng, Cheng; Gu, Xiuge; He, Qin; Wei, Fulan

    2017-04-17

    Cryopreservation has been extensively applied to the long-term storage of a diverse range of biological materials. However, no comprehensive study is currently available on the cryopreservation of periodontal ligament stem cell (PDLSC) sheets which have been suggested as excellent transplant materials for periodontal tissue regeneration. The aim of this study is to investigate the effect of cryopreservation on the structural integrity and functional viability of PDLSC sheets. PDLSC sheets prepared from extracted human molars were divided into two groups: the cryopreservation group (cPDLSC sheets) and the freshly prepared control group (fPDLSC sheets). The cPDLSC sheets were cryopreserved in a solution consisting of 90% fetal bovine serum and 10% dimethyl sulfoxide for 3 months. Cell viability and cell proliferation rates of PDLSCs in both groups were evaluated by cell viability assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. The multilineage differentiation potentials of the cells were assessed by von Kossa staining and Oil Red O staining. The chromosomal stability was examined by karyotype analysis. Moreover, the cell sheets in each group were transplanted subcutaneously into the dorsal site of nude mice, after which Sirius Red staining was performed to analyze the efficiency of tissue regeneration. The PDLSCs derived from both groups of cell sheets showed no significant difference in their viability, proliferative capacities, and multilineage differentiation potentials, as well as chromosomal stability. Furthermore, transplantation experiments based on a mouse model demonstrated that the cPDLSC sheets were equally effective in generating viable osteoid tissues in vivo as their freshly prepared counterparts. In both cases, the regenerated tissues showed similar network patterns of bone-like matrix. Our results offer convincing evidence that cryopreservation does not alter the biological properties of PDLSC sheets

  16. Peroxisome Proliferator-Activated Receptor (PPAR: Balance for Survival in Parasitic Infections

    Directory of Open Access Journals (Sweden)

    Marion M. Chan

    2010-01-01

    Full Text Available Parasitic infections induce a magnitude of host responses. At the opposite ends of the spectrum are those that ensure the host's needs to eliminate the invaders and to minimize damage to its own tissues. This review analyzes how parasites would manipulate immunity by activating the immunosuppressive nuclear factor, peroxisome proliferator-activated receptors (PPARs with type 2 cytokines and free fatty acids from arachidonic acid metabolism. PPARs limit the action of type 1 immunity, in which classically activated macrophages act through the production of proinflammatory signals, to spare the parasites. They also favor the development of alternately activated macrophages which control inflammation so the host would not be destroyed. Possibly, the nuclear factors hold a pivotal role in the establishment of chronic infection by delicately balancing the pro- and anti-inflammatory signaling mechanisms and their ligands may be used as combination therapeutics to limit host pathology.

  17. Differential sexual survival of Drosophila melanogaster on copper sulfate.

    Science.gov (United States)

    Balinski, Michael A; Woodruff, Ronny C

    2017-04-01

    Based on studies of the influence of X-chromosomes on the viability of Drosophila melanogaster exposed to cadmium, and on the role of X-linked genes on copper homeostasis, we examined the effect of copper sulfate (CuSO4) on offspring viability using three independent, inbred D. melanogaster crosses (ensuring identical autosomes for males and females within each cross). Each cross was performed with attached X-chromosome females and males with a single X-chromosome. As female D. melanogaster have less metallothionein RNA expression than males, we predicted fewer female offspring than male offspring in crosses exposed to CuSO4, even though females have two copies of X-chromosome genes, possibly resulting in overdominant heterozygosity. In two of three crosses, CuSO4 caused significantly higher numbers of male offspring compared to female offspring. We hypothesized that these gender-based viability differences to copper exposure are caused by X-chromosome ploidy and X-linked genetic variation affecting metallothionein expression. Observed differential offspring viability responses among crosses to copper exposure also showed that different genetic backgrounds (autosomal and/or X-chromosome) can result in significant differences in heavy metal and metallothionein regulation. These results suggest that the effect of copper on offspring viability depends on both genetic background and gender, as both factors can affect the regulation of metallothionein proteins as well as homeostasis of biologically necessary heavy metals.

  18. Activation of peroxisome proliferator-activated receptor gamma bypasses the function of the retinoblastoma protein in adipocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Jacob B.; Petersen, R K; Larsen, B M

    1999-01-01

    The retinoblastoma protein (pRB) is an important regulator of development, proliferation, and cellular differentiation. pRB was recently shown to play a pivotal role in adipocyte differentiation, to interact physically with adipogenic CCAAT/enhancer-binding proteins (C/EBPs), and to positively......RB. The differentiated RB-/- cells accumulate lipid and express adipocyte markers, including C/EBPalpha and PPARgamma2. Interestingly, adipose conversion of pRB-deficient cells occurs in the absence of compensatory up-regulations of the other pRB family members p107 and p130. RB+/+ as well as RB-/- cells efficiently...

  19. Progesterone modulates the proliferation and differentiation of human periodontal ligament cells.

    Science.gov (United States)

    Yuan, Gongjie; Cai, Chuan; Dai, Juan; Liu, Yali; Zhang, Rui; Dai, Yuanyuan; Wen, Li; Ding, Yin

    2010-08-01

    Hormone deficiency has been recognized as a risk factor for periodontal disease in postmenopausal women. However, the anabolic effects of progesterone on human periodontal ligament cells (hPDLCs) are still unclear. Therefore, the objective of this study was to detect the expression of progesterone receptor (PgR) in hPDLCs and investigate the bone-sparing effects of progesterone. We detected PgR expression in hPDLCs by reverse transcriptase-polymerase chain reaction and immunocytochemistry. After progesterone stimulation, the percentage of hPDLCs entering the S + G2M phase of the cell cycle increased significantly, accompanied by an increased cell growth curve. In both basic culture medium and osteogenic medium, progesterone activated alkaline phosphatase-positive cells and alizarin red-positive nodules. Moreover, mineralization-related markers were up-regulated by progesterone in both time-dependent and dose-dependent manners. In contrast, these effects of progesterone were blocked by the PgR antagonist (RU486). Our results demonstrated that the PgR is expressed in hPDLCs at the gene and protein level, and that progesterone can stimulate the proliferation and differentiation of the hPDLCs. These findings suggest that progesterone may play a significant role in osteoblastic function of hPDLCs and may influence the maintenance of alveolar bone mass.

  20. Melatonin decreases cell proliferation, impairs myogenic differentiation and triggers apoptotic cell death in rhabdomyosarcoma cell lines.

    Science.gov (United States)

    Codenotti, Silvia; Battistelli, Michela; Burattini, Sabrina; Salucci, Sara; Falcieri, Elisabetta; Rezzani, Rita; Faggi, Fiorella; Colombi, Marina; Monti, Eugenio; Fanzani, Alessandro

    2015-07-01

    Melatonin is a small indole produced by the pineal gland and other tissues, and has numerous functions that aid in the maintenance of the whole body homeostasis, ranging from the regulation of circadian rhythms and sleep to protection from oxidative stress. Melatonin has also been reported to counteract cell growth and chemoresistance in different types of cancer. In the present study, we investigated the effects of exogenous melatonin administration on different human cell lines and primary mouse tumor cultures of rhabdomyosarcoma (RMS), the most frequent soft tissue sarcoma affecting childhood. The results showed that melatonin significantly affected the behavior of RMS cells, leading to inhibition of cell proliferation and impairment of myogenic differentiation followed by increased apoptotic cell death, as observed by immunoblotting analysis of apoptosis-related markers including Bax, Bcl-2 and caspase-3. Similar findings were observed using a combination of microscopy techniques, including scanning/transmission electron and confocal microscopy. Furthermore, melatonin in combination with doxorubicin or cisplatin, two compounds commonly used for the treatment of solid tumors, increased the sensitivity of RMS cells to apoptosis. These data indicated that melatonin may be effective in counteracting RMS tumor growth and chemoresistance.

  1. Wnt-11 promotes neuroendocrine-like differentiation, survival and migration of prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Diez Soraya

    2010-03-01

    Full Text Available Abstract Background Wnt-11 is a secreted protein that modulates cell growth, differentiation and morphogenesis during development. We previously reported that Wnt-11 expression is elevated in hormone-independent prostate cancer and that the progression of prostate cancer from androgen-dependent to androgen-independent proliferation correlates with a loss of mutual inhibition between Wnt-11- and androgen receptor-dependent signals. However, the prevalence of increased expression of Wnt-11 in patient tumours and the functions of Wnt-11 in prostate cancer cells were not known. Results Wnt-11 protein levels in prostate tumours were determined by immunohistochemical analysis of prostate tumour tissue arrays. Wnt-11 protein was elevated in 77/117 of tumours when compared with 27 benign prostatic hypertrophy specimens and was present in 4/4 bone metastases. In addition, there was a positive correlation between Wnt-11 expression and PSA levels above 10 ng/ml. Androgen-depleted LNCaP prostate cancer cells form neurites and express genes associated with neuroendocrine-like differentiation (NED, a feature of prostate tumours that have a poor prognosis. Since androgen-depletion increases expression of Wnt-11, we examined the role of Wnt-11 in NED. Ectopic expression of Wnt-11 induced expression of NSE and ASCL1, which are markers of NED, and this was prevented by inhibitors of cyclic AMP-dependent protein kinase, consistent with the known role of this kinase in NED. In contrast, Wnt-11 did not induce NSE expression in RWPE-1 cells, which are derived from benign prostate, suggesting that the role of Wnt-11 in NED is specific to prostate cancer. In addition, silencing of Wnt-11 expression in androgen-depleted LNCaP cells prevented NED and resulted in apoptosis. Silencing of Wnt-11 gene expression in androgen-independent PC3 cells also reduced expression of NSE and increased apoptosis. Finally, silencing of Wnt-11 reduced PC3 cell migration and ectopic

  2. The effect of TRPM7 suppression on the proliferation, migration and osteogenic differentiation of human dental pulp stem cells.

    Science.gov (United States)

    Cui, L; Xu, S M; Ma, D D; Wu, B L

    2014-06-01

    To investigate the role of the Ca(2+) -Mg(2+) ion channel TRPM7 in the proliferation, migration and osteogenic differentiation of human dental pulp stem cells (hDPSCs). Immunohistochemistry was used to localize expression of TRPM7 in human dental pulp tissues and in cultured hDPSCs. Isolated hDPSCs were infected with recombinant lentiviruses expressing short hairpin RNA (shRNA) specific for TRPM7, or control shRNA, in order to suppress TRPM7 mRNA expression and investigate its functional role. The proliferation of the shRNA-infected hDPSCs was evaluated using both an MTT assay to measure viable cell numbers and cell cycle analysis. Cell migration was assessed using a transwell assay. The dynamic mRNA expression of TRPM7 during osteogenic differentiation of hDPSCs and the effect of shRNA specific for TRPM7 on hDPSC osteogenic differentiation were evaluated by real-time PCR. TRPM7 expression was widespread in human dental pulp tissue and was detected mainly in the cytomembrane and cytoplasm of hDPSCs. Suppression of TRPM7 inhibited both the proliferation and the migratory capacity of hDPSCs. TRPM7 mRNA expression was elevated during osteogenic differentiation of hDPSCs. TRPM7-specific shRNA inhibited osteogenic differentiation of hDPSCs, with downregulated mRNA expression of the osteogenic markers alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP), bone sialoprotein (BSP), runt-related transcription factor (RUNX2) and osterix (OSX). TRPM7 was involved in the regulation of hDPSC proliferation, migration and osteogenic differentiation and may play a role in the dental pulp repair process. © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  3. The role of integrin-α5 in the proliferation and odontogenic differentiation of human dental pulp stem cells.

    Science.gov (United States)

    Cui, Li; Xu, Shuaimei; Ma, Dandan; Gao, Jie; Liu, Ying; Yue, Jing; Wu, Buling

    2014-02-01

    It has been reported that integrin-α5 (ITGA5) activity is related to cell proliferation, differentiation, migration, and organ development. However, the involvement of ITGA5 in the biological functions of human dental pulp stem cells (hDPSCs) has not been explored. The aim of this study was to investigate the role of ITGA5 in the proliferation and odontogenic differentiation of hDPSCs. We knocked down ITGA5 in hDPSCs using lentivirus-mediated ITGA5 short hairpin RNA (shRNA). Changes in the proliferation in hDPSCs infected with lentiviruses expressing ITGA5-specific shRNA or negative control shRNA were examined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine labeling. Both ITGA5 knockdown cells and shMock cells were cultured in mineralization medium for 3 weeks, and the differentiation of cells was detected with alizarin red S staining. The expression of odontogenic differentiation-related molecular markers was assessed using real-time polymerase chain reaction and Western blot assays. The knockdown of ITGA5 decreased the proliferation capacity of hDPSCs. ITGA5 shRNA promoted odontogenic differentiation of hDPSCs with the enhanced formation of mineralized nodules. It also up-regulated the messenger RNA expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. These findings suggest that ITGA5 plays an important role in maintaining hDPSCs in a proliferative state. The inhibition of ITGA5 signaling promotes the odontogenic differentiation of hDPSCs. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  4. 2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM).

    Science.gov (United States)

    Hoffrogge, Raimund; Mikkat, Stefan; Scharf, Christian; Beyer, Susanne; Christoph, Hilmar; Pahnke, Jens; Mix, Eilhard; Berth, Matthias; Uhrmacher, Adelinde; Zubrzycki, Igor Z; Miljan, Erik; Völker, Uwe; Rolfs, Arndt

    2006-03-01

    The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.

  5. Inhibition of Delta1 promotes differentiation of odontoblasts and inhibits proliferation of human dental pulp stem cell in vitro.

    Science.gov (United States)

    Wang, Xuefei; He, Fei; Tan, Yinghui; Tian, Weidong; Qiu, Songbo

    2011-09-01

    Dental pulp stem cells (DPSCs) have been receiving more attentions recently as an important biomaterial for tissue engineering. Notch signalling plays a key role in regulating self-renewal and differentiation of a variety of cells. The objective of this study is to investigate the effects of Notch-Delta1 RNA interference (RNAi) on the proliferation and differentiation of human dental pulp stem cells in vitro. In the present study, we performed gene knockdown of Notch ligand Delta1 in DPSCs using lentivirus-mediated Delta1-RNAi. Changes of proliferation in DPSCs/Delta1-RNAi were examined by cell cycle analysis, Cell viability assay (CCK-8) and Western blot analysis of proliferating cell nuclear antigen (PCNA). Cells were cultured in odontoblast differentiation-inducing medium, and the differentiation of cells was detected with Alkaline phosphatase ALP activity assay, Alizarin red S staining, calcium concentration measurement, and Western blot analysis of Dentine sialophosphoprotein (DSPP). Lentivirus-mediated Delta1-RNAi stably knocked-down the expression of Delta1 and Notch signalling, and some of DPSCs/Delta1-RNAi displayed changes in morphology or DSPP expression. The growth rate of Delta1-deficient DPSCs was significantly suppressed as compared with wild type DPSCs and control lentivirus vector transfected DPSCs. Furthermore, the differentiating capability of DPSCs/Delta1-RNAi into odontoblasts is much higher than the two control groups. Notch signalling plays a crucial role in regulating self-renewal and differentiation in DPSCs. The deficient Notch signalling inhibits the self-renewal capacity of DPSCs and tends to induce DPSCs differentiation under odontoblast differentiation-inducing conditions. These findings suggested that DPSCs/Delta1-RNAi might be applicable to stem cell therapies and tooth tissue engineering. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. The DNA glycosylases OGG1 and NEIL3 influence differentiation potential, proliferation, and senescence-associated signs in neural stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Reis, Amilcar [Linnaeus Center in Developmental Biology for Regenerative Medicine (DBRM), Department of Neuroscience, Karolinska Institutet, SE 17177 Stockholm (Sweden); Hermanson, Ola, E-mail: ola.hermanson@ki.se [Linnaeus Center in Developmental Biology for Regenerative Medicine (DBRM), Department of Neuroscience, Karolinska Institutet, SE 17177 Stockholm (Sweden)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer DNA glycosylases OGG1 and NEIL3 are required for neural stem cell state. Black-Right-Pointing-Pointer No effect on cell viability by OGG1 or NEIL3 knockdown in neural stem cells. Black-Right-Pointing-Pointer OGG1 or NEIL3 RNA knockdown result in decreased proliferation and differentiation. Black-Right-Pointing-Pointer Increased HP1{gamma} immunoreactivity after NEIL3 knockdown suggests premature senescence. -- Abstract: Embryonic neural stem cells (NSCs) exhibit self-renewal and multipotency as intrinsic characteristics that are key parameters for proper brain development. When cells are challenged by oxidative stress agents the resulting DNA lesions are repaired by DNA glycosylases through the base excision repair (BER) pathway as a means to maintain the fidelity of the genome, and thus, proper cellular characteristics. The functional roles for DNA glycosylases in NSCs have however remained largely unexplored. Here we demonstrate that RNA knockdown of the DNA glycosylases OGG1 and NEIL3 decreased NSC differentiation ability and resulted in decreased expression of both neuronal and astrocytic genes after mitogen withdrawal, as well as the stem cell marker Musashi-1. Furthermore, while cell survival remained unaffected, NEIL3 deficient cells displayed decreased cell proliferation rates along with an increase in HP1{gamma} immunoreactivity, a sign of premature senescence. Our results suggest that DNA glycosylases play multiple roles in governing essential neural stem cell characteristics.

  7. Nuclear trafficking of secreted factors and cell-surface receptors: new pathways to regulate cell proliferation and differentiation, and involvement in cancers

    Directory of Open Access Journals (Sweden)

    Planque Nathalie

    2006-10-01

    Full Text Available Abstract Secreted factors and cell surface receptors can be internalized by endocytosis and translocated to the cytoplasm. Instead of being recycled or proteolysed, they sometimes translocate to the nucleus. Nuclear import generally involves a nuclear localization signal contained either in the secreted factor or its transmembrane receptor, that is recognized by the importins machinery. In the nucleus, these molecules regulate transcription of specific target genes by direct binding to transcription factors or general coregulators. In addition to the transcription regulation, nuclear secreted proteins and receptors seem to be involved in other important processes for cell life and cellular integrity such as DNA replication, DNA repair and RNA metabolism. Nuclear secreted proteins and transmembrane receptors now appear to induce new signaling pathways to regulate cell proliferation and differentiation. Their nuclear localization is often transient, appearing only during certain phases of the cell cycle. Nuclear secreted and transmembrane molecules regulate the proliferation and differentiation of a large panel of cell types during embryogenesis and adulthood and are also potentially involved in wound healing. Secreted factors such as CCN proteins, EGF, FGFs and their receptors are often detected in the nucleus of cancer cells. Nuclear localization of these molecules has been correlated with tumor progression and poor prognosis for patient survival. Nuclear growth factors and receptors may be responsible for resistance to radiotherapy.

  8. Krüppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells

    Directory of Open Access Journals (Sweden)

    Mandayam O. Nandan

    2015-01-01

    Full Text Available Krüppel-like factor 5 (KLF5 is a pro-proliferative transcription factor that is expressed in dividing epithelial cells of the intestinal crypt. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5 has been identified as a stem cell marker in both small intestinal and colonic epithelial cells. To determine whether KLF5 regulates proliferation of intestinal stem cells, we investigated the effects of Klf5 deletion specifically from the intestinal stem cells in adult mice. Mice with inducible intestinal stem cell-specific deletion of Klf5 (Lgr5-Klf5fl/fl were injected with tamoxifen for 5 consecutive days to induce Lgr5-driven Cre expression. Intestinal and colonic tissues were examined by immunohistochemistry at various time points up to 112 days following start of tamoxifen treatment. Klf5 is co-localized in the crypt-based columnar (CBC cells that express Lgr5. By 11 days following the start of tamoxifen treatment, Lgr5-positive crypts from which Klf5 was deleted exhibited a loss of proliferation that was accompanied by an increase in apoptosis. Beginning at 14 days following the start of tamoxifen treatment, both Klf5 expression and proliferation were re-established in the transit-amplifying epithelial cells but not in the Lgr5-positive CBC cells. By 112 days post-treatment, up to 90% of the Lgr5-positive cells from which Klf5 was deleted were lost from the intestinal crypts. These results indicate a critical role for KLF5 in the survival and maintenance of intestinal stem cells.

  9. Anti-MHC Class I Antibody Activation of Proliferation and Survival Signaling in Murine Cardiac Allografts1

    Science.gov (United States)

    Jindra, Peter T.; Hsueh, Aileen; Hong, Longshen; Gjertson, David; Shen, Xiu-Da; Gao, Feng; Dang, Julie; Mischel, Paul S.; Baldwin, William M.; Fishbein, Michael C.; Kupiec-Weglinski, Jerzy W.; Reed, Elaine F.

    2013-01-01

    Anti-MHC class I alloantibodies have been implicated in the process of acute and chronic rejection because these Abs can bind to endothelial cells and transduce signals leading to the activation of cell survival and proliferation pathways. To characterize the role of the MHC class I-signaling pathway in the pathogenesis of Ab-mediated rejection, we developed a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2Kb/Db) received a fully MHC-incompatible BALB/c (H-2Kd/Dd) heart transplant and were passively transfused with anti-donor MHC class I Ab. We demonstrate that cardiac allografts of mice treated with anti-MHC class I Abs show characteristic features of Ab-mediated rejection including microvascular changes accompanied by C4d deposition. Phosphoproteomic analysis of signaling molecules involved in the MHC class I cell proliferation and survival pathways were elevated in anti-class I-treated mice compared with the isotype control-treated group. Pairwise correlations, hierarchical clustering, and multidimensional scaling algorithms were used to dissect the class I-signaling pathway in vivo. Treatment with anti-H-2Kd Ab was highly correlated with the activation of Akt and p70S6Kinase (S6K). When measuring distance as a marker of interrelatedness, multidimensional scaling analysis revealed a close association between members of the mammalian target of rapamycin pathway including mammalian target of rapamycin, S6K, and S6 ribosomal protein. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experiments. PMID:18250428

  10. ICT1 predicts a poor survival and correlated with cell proliferation in diffuse large B-cell lymphoma.

    Science.gov (United States)

    Xie, Wenjun; Wu, Meijuan; Fu, Tianhong; Li, Xiaohong; Wang, Zhaoming; Hu, Yongxian; Zhu, Liyan; Zhang, Gu

    2017-09-05

    Immature colon carcinoma transcript-1 (ICT1) is a crucial member of the large mitoribosomal subunit in mitochondrial ribosome, which has been shown to be closely related to tumorigenesis. Its expression and function in human diffuse large B-cell lymphoma (DLBCL), however, remained elusive. In this study, analysis of public available Oncomine database suggested that the expression levels of ICT1 mRNA was significantly upregulated in DLBCL tissues. Consistently, we described ICT1 was remarkably upregulated in fresh DLBCL samples compared with the corresponding normal tissues using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting. Moreover, ICT1 overexpression was associated with the poor overall survival (OS) of DLBCL patients. Finally, we used DLBCL cell lines to further probe the potential mechanisms, and found shRNA-mediated knockdown of ICT1 significantly suppressed DLBCL cell proliferation, induced cell cycle arrest at G0/G1 phase and apoptosis in vitro. Further verification showed that inhibition of ICT1 gene expression caused the upregulation of the p21, Bad and caspase-3, and downregulation of PCNA, Survivin, CDK4, CDK6 and Cyclin D1. Taken together, this study suggested that ICT1 may play an oncogenic role in human DLBCL by promoting cell proliferation and it might be a biomarker of unfavorable prognosis in DLBCL patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Serratia marcescens Is Able to Survive and Proliferate in Autophagic-Like Vacuoles inside Non-Phagocytic Cells

    Science.gov (United States)

    Colombo, María Isabel; García Véscovi, Eleonora

    2011-01-01

    Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell. PMID:21901159

  12. Selection, proliferation and differentiation of bone marrow-derived liver stem cells with a culture system containing cholestatic serum in vitro.

    Science.gov (United States)

    Cai, Yun-Feng; Zhen, Zuo-Jun; Min, Jun; Fang, Tian-Ling; Chu, Zhong-Hua; Chen, Ji-Sheng

    2004-11-15

    To explore the feasibility of direct separation, selective proliferation and differentiation of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. Whole bone marrow cells of rats cultured in routine medium were replaced with conditioning selection media containing 20 mL/L, 50 mL/L, 70 mL/L, and 100 mL/L cholestatic sera, respectively, after they attached to the plates. The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures. Then the selected BDLSC were induced to proliferate and differentiate with the addition of hepatocyte growth factor (HGF). The morphology and phenotypic markers of BDLSC were characterized using immunohistochemistry, RT-PCR and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. Bone marrow cells formed fibroblast-like but not hepatocyte-like colonies in the presence of 20 mL/L cholestatic serum. In 70 mL/L cholestatic serum, BDLSC colonies could be selected but could not maintain good growth status. In 100 mL/L cholestatic serum, all of the bone marrow cells were unable to survive. A 50 mL/L cholestatic serum was the optimal concentration for the selection of BDLSC at which BDLSC could survive while the other populations of the bone marrow cells could not. The selected BDLSC proliferated and differentiated after HGF was added. Hepatocyte-like colony-forming units (H-CFU) then were formed. H-CFU expressed markers of embryonic hepatocytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors (HNF-1alpha and HNF-3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. The selected medium containing cholestatic serum can select BDLSC from whole bone marrow cells. It

  13. [Effects of alginate/collagen scaffold on cell proliferation and differentiation of human adipose-derived mesenchymal stem cells].

    Science.gov (United States)

    Cheng, W; Han, X P; Mou, S L; Yang, F; Liu, L P

    2017-04-09

    Objective: To build scaffold materials with different concentrations of alginate and collagen, and to observe the effects of alginate/collagen ratio on the proliferation of human adipose-derived mesenchymal stem cells (hAMSC) and osteogenic differentiation. The optimal concentration of alginate/collagen will be chosen for constructing hydrogel that will be used for bone tissue engineering. Methods: Soluble hydrogel scaffold materials containing alginate/collagen were prepared, and the following groups were established based on different alginate/collagen ratio: 4∶1 (group A), 2∶1 (group B), and 1∶1 (group C). Cell proliferation on the material surface was observed using the cell counting kit-8 (CCK-8) assay, while cell viability in each material group were observed using live/dead staining. Quantitative real-time PCR(qPCR) was used to measure the differential expression of osteogenesis-related genes on and in the materials. Immunofluorescence staining was used to measure the differential gene expression of osteogenesis-related proteins in each group. Results: The results from the CCK-8 assay showed increasing cell proliferation rate on the lyophilized hydrogel material surface as the collagen concentration increased, and the highest cell proliferation was observed in group C. Live/dead staining assay indicated that cells were able to proliferate in all three types of hydrogel materials, and the highest cell viability was found in material from group B ([87.50±2.65]%). qPCR showed that the expression of osteogenesis-related genes in group C was the highest, among the three groups, while the expression of osteocalcin in group B was significantly higher than those in the other two groups (Palginate/collagen scaffold materials did not show adverse effects on the cell proliferation of hAMSC and osteogenenic differentiation. Bone tissue engineering can use 10% hydrogel material, and when the sodium alginate and collagen have a ratio of 2∶1, the hydrogel can be

  14. Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function.

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    Kwok Yeung Tsang

    2007-03-01

    Full Text Available In protein folding and secretion disorders, activation of endoplasmic reticulum (ER stress signaling (ERSS protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del, misfolded alpha1(X chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

  15. The Role of Heme and Reactive Oxygen Species in Proliferation and Survival of Trypanosoma cruzi

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    Marcia Cristina Paes

    2011-01-01

    Full Text Available Trypanosoma cruzi, the protozoan responsible for Chagas disease, has a complex life cycle comprehending two distinct hosts and a series of morphological and functional transformations. Hemoglobin degradation inside the insect vector releases high amounts of heme, and this molecule is known to exert a number of physiological functions. Moreover, the absence of its complete biosynthetic pathway in T. cruzi indicates heme as an essential molecule for this trypanosomatid survival. Within the hosts, T. cruzi has to cope with sudden environmental changes especially in the redox status and heme is able to increase the basal production of reactive oxygen species (ROS which can be also produced as byproducts of the parasite aerobic metabolism. In this regard, ROS sensing is likely to be an important mechanism for the adaptation and interaction of these organisms with their hosts. In this paper we discuss the main features of heme and ROS susceptibility in T. cruzi biology.

  16. Metformin Acts on Two Different Molecular Pathways to Enhance Adult Neural Precursor Proliferation/Self-Renewal and Differentiation

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    Michael Fatt

    2015-12-01

    Full Text Available The recruitment of endogenous adult neural stem cells for brain repair is a promising regenerative therapeutic strategy. This strategy involves stimulation of multiple stages of adult neural stem cell development, including proliferation, self-renewal, and differentiation. Currently, there is a lack of a single therapeutic approach that can act on these multiple stages of adult neural stem cell development to enhance neural regeneration. Here we show that metformin, an FDA-approved diabetes drug, promotes proliferation, self-renewal, and differentiation of adult neural precursors (NPCs. Specifically, we show that metformin enhances adult NPC proliferation and self-renewal dependent upon the p53 family member and transcription factor TAp73, while it promotes neuronal differentiation of these cells by activating the AMPK-aPKC-CBP pathway. Thus, metformin represents an optimal candidate neuro-regenerative agent that is capable of not only expanding the adult NPC population but also subsequently driving them toward neuronal differentiation by activating two distinct molecular pathways.

  17. Effects of radiofrequency exposure emitted from a GSM mobile phone on proliferation, differentiation, and apoptosis of neural stem cells.

    Science.gov (United States)

    Eghlidospour, Mahsa; Ghanbari, Amir; Mortazavi, Seyyed Mohammad Javad; Azari, Hassan

    2017-06-01

    Due to the importance of neural stem cells (NSCs) in plasticity of the nervous system and treating neurodegenerative diseases, the main goal of this study was to evaluate the effects of radiofrequency radiation emitted from a GSM 900-MHz mobile phone with different exposure duration on proliferation, differentiation and apoptosis of adult murine NSCs in vitro. We used neurosphere assay to evaluate NSCs proliferation, and immunofluorescence assay of neural cell markers to examine NSCs differentiation. We also employed alamarBlue and caspase 3 apoptosis assays to assess harmful effects of mobile phone on NSCs. Our results showed that the number and size of resulting neurospheres and also the percentage of cells differentiated into neurons decreased significantly with increasing exposure duration to GSM 900-MHz radiofrequency (RF)-electromagnetic field (EMF). In contrast, exposure to GSM 900-MHz RF-EMF at different durations did not influence cell viability and apoptosis of NSCs and also their astrocytic differentiation. It is concluded that accumulating dose of GSM 900-MHz RF-EMF might have devastating effects on NSCs proliferation and neurogenesis requiring more causations in terms of using mobile devices.

  18. Nuclear Factor I-C promotes proliferation and differentiation of apical papilla-derived human stem cells in vitro

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    Zhang, Jing [State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, Xi' an (China); Stomatologic Hospital & College, Anhui Medical University, Key Lab of Oral Diseases Research of Anhui Province, Hefei (China); Wang, Zhihua; Jiang, Yong [State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, Xi' an (China); Niu, Zhongying [Treatment center of oral diseases, The 306th Hospital of People' s Liberation Army, Beijing (China); Fu, Lei; Luo, Zhirong [State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, Xi' an (China); Cooper, Paul R.; Smith, Anthony J. [Oral Biology, School of Dentistry, University of Birmingham, B4 6NN (United Kingdom); He, Wenxi, E-mail: hewenxi@fmmu.edu.cn [State Key Laboratory of Military Stomatology, Department of Operative Dentistry & Endodontics, School of Stomatology, The Fourth Military Medical University, Xi' an (China)

    2015-03-15

    The transcription factor Nuclear Factor I-C (NFIC) has been implicated in the regulation of tooth root development, where it may be anticipated to impact on the behavior of stem cells from the apical papilla (SCAPs) and root odontoblast activity. We hypothesized that NFIC may provide an important target for promoting dentin/root regeneration. In the present study, the effects of NFIC on the proliferation and differentiation of SCAPs were investigated. Over-expression of NFIC increased cell proliferation, mineralization nodule formation and alkaline phosphatase (ALP) activity in SCAPs. Furthermore, NFIC up-regulated the mRNA levels of odontogenic-related markers, ALP, osteocalcin and collagen type I as well as dentin sialoprotein protein levels. In contrast, knockdown of NFIC by si-RNA inhibited the mineralization capacity of SCAPs and down-regulated the expression of odontogenic-related markers. In conclusion, the results indicated that upregulation of NFIC activity in SCAPs may promote osteo/odontoblastic differentiation of SCAPs. - Highlights: • NFIC promotes the proliferation of SCAPs in vitro. • NFIC promotes osteo/odontogenic differentiation of SCAPs in vitro. • Knockdown of NFIC inhibits odontogenic differentiation in SCAPs.

  19. The Characteristics and Function of S100A7 Induction in Squamous Cell Carcinoma: Heterogeneity, Promotion of Cell Proliferation and Suppression of Differentiation.

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    Zhi Qi

    Full Text Available S100A7 is highly expressed in squamous cell carcinomas (SCC and is related to the terminal differentiation of keratinocytes. However, its characteristic and function in SCC is not very known. In this present study, we used immunohistochemistry to examine the expression of S100A7 in 452 SCC specimens, including the lung, esophagus, oral cavity, skin, cervix, bladder, and three SCC cell lines. We found that S100A7-positive staining showed significant heterogeneity in six types of SCC specimen and three SCC cell lines. Further examination found that S100A7-positive cells and its expression at mRNA and protein levels could be induced in HCC94, FaDu, and A-431 cells both in vitro and in vivo using immunohistochemistry, real-time PCR, and Western blotting. Notably, the upregulation of squamous differentiation markers, including keratin-4, keratin-13, TG-1, and involucrin, also accompanied S100A7 induction, and a similar staining pattern of S100A7 and keratin-13 was found in HCC94 cells both in vitro and in vivo. Further study revealed that the overexpression of S100A7 significantly increased proliferation and inhibited squamous differentiation in A-431 cells both in vitro and in vivo. Conversely, silencing S100A7 inhibited cell growth and survival and increased the expression of keratin-4, keratin-13, TG-1, and involucrin in HCC94 cells. Therefore, these results demonstrate that S100A7 displays heterogeneous and inducible characteristic in SCC and also provide novel evidence that S100A7 acts as a dual regulator in promoting proliferation and suppressing squamous differentiation of SCC.

  20. Essential roles of G9a in cell proliferation and differentiation during tooth development.

    Science.gov (United States)

    Kamiunten, Taichi; Ideno, Hisashi; Shimada, Akemi; Arai, Yoshinori; Terashima, Tatsuo; Tomooka, Yasuhiro; Nakamura, Yoshiki; Nakashima, Kazuhisa; Kimura, Hiroshi; Shinkai, Yoichi; Tachibana, Makoto; Nifuji, Akira

    2017-08-15

    control mice at E15.5. Localization of Shh and related signaling components, including Gli1, Ptch1, and Ptch2, in the tooth mesenchyme of cKO mice was generally similar to that at earlier stages in control mice. In addition, expression of Fgf3 and Fgf10 in the mesenchyme was decreased in G9a cKO mice at P0. Expression levels of Fgf9 and p21, both of which were expressed in the secondary enamel-knot, were also decreased. Thus, the expression of genes associated with tooth development was delayed in cKO mice. Our results suggest that H3K9MTase G9a regulates cell proliferation and timing of differentiation and that G9a expression in the tooth mesenchyme is required for proper tooth development. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Conditioned media from differentiating craniofacial bone marrow stromal cells influence mineralization and proliferation in periodontal ligament stem cells.

    Science.gov (United States)

    Jin, Zhenyu; Feng, Yuan; Liu, Hongwei

    2016-10-01

    Previous reports have mainly focused on the behavioral responses of human periodontal ligament stem cells (hPDLSCs) in interaction with tibia bone marrow stromal cells (BMSCs). However, there is little study on the biologic features of hPDLSCs under the induction of maxilla BMSCs (M-BMSCs) at different phases of osteogenic differentiation. We hypothesized that M-BMSCs undergoing osteogenic differentiation acted on the proliferation, differentiation, and bone-forming capacity of hPDLSCs. In this paper, primary hPDLSCs and human M-BMSCs (hM-BMSCs) were expanded in vitro. After screening of surface markers for characterization, hPDLSCs were cocultured with different phases of differentiating hM-BMSCs. Cell proliferation and alkaline phosphatase activity were examined, and mineralization-associated markers such as osteocalcin and runt-related transcription factor 2 of hPDLSCs in coculture with uninduced/osteoinduced hM-BMSCs were evaluated. hPDLSCs in hM-BMSCs-conditioned medium (hM-BMSCs-CM) group showed a reduction in proliferation compared with untreated hPDLSCs, while osteoinduced hM-BMSCs for 10 day-conditioned medium (hM-BMSCs-CM-10ds) and osteoinduced hM-BMSCs for 15 day-conditioned medium (hM-BMSCs-CM-15ds) enhance the proliferation of hPDLSCs. hM-BMSCs of separate differentiation stages temporarily inhibited osteogenesis of hPDLSCs in the early days. Upon extending time periods, uninduced/osteoinduced hM-BMSCs markedly enhanced osteogenesis of hPDLSCs to different degrees. The transplantation results showed hM-BMSCs-CM-15ds treatment promoted tissue regeneration to generate cementum/periodontal ligament-like structure characterized by hard-tissue formation. This research supported the notion that hM-BMSCs triggered osteogenesis of hPDLSCs suggesting important implications for periodontal engineering.

  2. Adult sertoli cells are not terminally differentiated in the Djungarian hamster: effect of FSH on proliferation and junction protein organization.

    Science.gov (United States)

    Tarulli, Gerard A; Stanton, Peter G; Lerchl, Alexander; Meachem, Sarah J

    2006-05-01

    Sertoli cell number is considered to be stable and unmodifiable by hormones after puberty in mammals, although recent data using the seasonal breeding adult Djungarian hamster (Phodopus sungorus) model challenged this assertion by demonstrating a decrease in Sertoli cell number after gonadotropin depletion and a return to control levels following 7 days of FSH replacement. The present study aimed to determine whether adult Sertoli cells are terminally differentiated using known characteristics of cellular differentiation, including proliferation, junction protein localization, and expression of particular maturational markers, in the Djungarian hamster model. Adult long-day (LD) photoperiod (16L:8D) hamsters were exposed to short-day (SD) photoperiod (8L:16D) for 11 wk to suppress gonadotropins and then received exogenous FSH for up to 10 days. Sertoli cell proliferation was assessed by immunofluorescence by the colocalization of GATA4 and proliferating cell nuclear antigen and quantified by stereology. Markers of Sertoli cell maturation (immature, cytokeratin 18 [KRT18]; mature, GATA1) and junction proteins (actin, espin, claudin 11 [CLDN11], and tight junction protein 1 [TJP1, also known as ZO-1]) also were localized using confocal immunofluorescence. In response to FSH treatment, proliferation was upregulated within 2 days compared with SD controls (90% vs. 0.2%, P < 0.001) and declined gradually thereafter. In LD hamsters, junction proteins colocalized at the basal aspect of Sertoli cells, consistent with inter-Sertoli cell junctions, and were disordered within the Sertoli cell cytoplasm in SD animals. Exogenous FSH treatment promptly restored localization of these junction markers to the LD phenotype. Protein markers of maturity remain consistent with those of adult Sertoli cells. It is concluded that adult Sertoli cells are not terminally differentiated in the Djungarian hamster and that FSH plays an important role in governing the differentiation process. It

  3. Gold Nanoparticles Promote Proliferation of Human Periodontal Ligament Stem Cells and Have Limited Effects on Cells Differentiation

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    Chen Li

    2016-01-01

    Full Text Available Gold nanoparticles (AuNPs had been widely applied in the practice and advancement of chemistry, biology, and medicine due to facility of synthesis and versatility in surface functionalization. Recent studies had shown that AuNPs can be applied to cells, affecting cellular physiological processes such as proliferation and differentiation. In this study, four diameters of AuNPs (20, 40, 60, and 80 nm were cocultured with human periodontal ligament cells (hPDLCs at six different concentrations. The optimal size and concentration of AuNPs were selected to treat human periodontal ligament stem cells (hPDLSCs to evaluate proliferation. Moreover, the influence of AuNPs on multiple differentiation capacity of hPDLSCs was clarified. The results revealed that AuNPs (60 nm, 56 μM can effectively promote the proliferation of hPDLCs/hPDLSCs in vitro, slightly enhance osteoblastic differentiation, and have no effect on adipogenic differentiation. In addition, the expression of COL-1, Runx2, BSP, and OCN was upregulated in the presence of AuNPs (60 nm, 56 μM. These results indicated that AuNPs (60 nm, 56 μM can effectively promote the proliferation of hPDLCs/hPDLSCs and have no significant effect on the differentiation of hPDLSCs. These results provide an insight on the advantage of implementing of AuNPs on hPDLSCs culture and expose the influence of these materials on periodontal tissue engineering.

  4. In vivo knockdown of TAK1 accelerates bone marrow proliferation/differentiation and induces systemic inflammation.

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    Paul M Vink

    Full Text Available TAK1 (TGF-β Activated Kinase 1 is a MAPK kinase kinase, which activates the p38- and JNK-MAPK and NF-κB pathways downstream of receptors such as Toll-Like-, cytokine- and T-cell and B-cell receptors. Representing such an important node in the pro-inflammatory signal-transduction network, the function of TAK1 has been studied extensively. TAK1 knock-out mice are embryonic lethal, while conditional knock-out mice demonstrated either a pro- or anti-inflammatory function. To study the function of TAK1 protein in the adult immune system, we generated and characterized a transgenic mouse expressing TAK1 shRNA under the control of a doxycycline-inducible promoter. Following treatment of TAK-1 shRNA transgenic mice with doxycycline an effective knockdown of TAK1 protein levels was observed in lymphoid organs and cells in the peritoneal cavity (>50% down regulation. TAK1 knockdown resulted in significant changes in leukocyte populations in blood, bone marrow, spleen and peritoneal cavity. Upon TAK1 knockdown mice demonstrated splenomegaly, signs of systemic inflammation (increased levels of circulating cytokines and increase in cellularity of the B-cell areas and in germinal center development in the follicles and degenerative changes in heart, kidneys and liver. Not surprisingly, TAK1-Tg mice treated with LPS or anti-CD3 antibodies showed enhanced cytokine/chemokine secretion. Finally, analysis of progenitor cells in the bone marrow upon doxycycline treatment showed increased proliferation and differentiation of myeloid progenitor cells. Given the similarity of the phenotype with TGF-β genetic models, our data suggest that in our model the function of TAK1 in TGF-β signal-transduction is overruling its function in pro-inflammatory signaling.

  5. Cholinesterases in development: AChE as a firewall to inhibit cell proliferation and support differentiation.

    Science.gov (United States)

    Layer, Paul G; Klaczinski, Janine; Salfelder, Anika; Sperling, Laura E; Thangaraj, Gopenath; Tuschl, Corina; Vogel-Höpker, Astrid

    2013-03-25

    Acetylcholinesterase (AChE) is a most remarkable protein, not only because it is one of the fastest enzymes in nature, but also since it appears in many molecular forms and is regulated by elaborate genetic networks. AChE is expressed in many tissues during development and in mature organisms, as well as in healthy and diseased states. In search for alternative, "non-classical" functions of cholinesterases (ChEs), AChE could either work within the frame of classic cholinergic systems, but in non-neural tissues ("non-synaptic function"), or act non-enzymatically. Here, we review briefly some of the major ideas and advances of this field, and report on some recent progress from our own experimental work, e.g. that (i) non-neural ChEs have pronounced, predominantly enzymatic effects on early embryonic (limb) development in chick and mouse, that (ii) retinal R28 cells of the rat overexpressing synaptic AChE present a significantly decreased cell proliferation, and that (iii) in developing chick retina ACh-synthesizing and ACh-degrading cells originate from the same postmitotic precursor cells, which later form two locally opposing cell populations. We suggest that such distinct distributions of ChAT(+) vs. AChE(+) cells in the inner half retina provide graded distributions of ACh, which can direct cell differentiation and network formation. Thus, as corroborated by works from many labs, AChE can be considered a highly co-opting protein, which can combine enzymatic and non-enzymatic functions within one molecule. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  6. Electrospun tilapia collagen nanofibers accelerating wound healing via inducing keratinocytes proliferation and differentiation.

    Science.gov (United States)

    Zhou, Tian; Wang, Nanping; Xue, Yang; Ding, Tingting; Liu, Xin; Mo, Xiumei; Sun, Jiao

    2016-07-01

    The development of biomaterials with the ability to induce skin wound healing is a great challenge in biomedicine. In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4(+)/CD8(+) lymphocytes, and the level of IgG or IgM in Sprague-Dawley rats. The tensile strength and contact angle of collagen nanofibers were 6.72±0.44MPa and 26.71±4.88°, respectively. They also had good thermal stability and swelling property. Furthermore, the nanofibers could significantly promote the proliferation of human keratinocytes (HaCaTs) and stimulate epidermal differentiation through the up-regulated gene expression of involucrin, filaggrin, and type I transglutaminase in HaCaTs. The collagen nanofibers could also facilitate rat skin regeneration. In the present study, electrospun biomimetic tilapia skin collagen nanofibers were succesfully prepared, were proved to have good bioactivity and could accelerate rat wound healing rapidly and effectively. These biological effects might be attributed to the biomimic extracellular matrix structure and the multiple amino acids of the collagen nanofibers. Therefore, the cost-efficient tilapia collagen nanofibers could be used as novel wound dressing, meanwhile effectively avoiding the risk of transmitting animal disease in the future clinical apllication. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activates promyogenic signaling pathways, thereby promoting myoblast differentiation

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    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Seo, Dong-Wan [College of Pharmacy, Dankook University, Cheonan 330-714 (Korea, Republic of); Kang, Jong-Sun [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 440-746 (Korea, Republic of); Bae, Gyu-Un, E-mail: gbae@sookmyung.ac.kr [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2016-01-29

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) regulates postnatal myogenesis by alleviating myostatin activity, but the molecular mechanisms by which it regulates myogenesis are not fully understood. In this study, we investigate molecular mechanisms of PPARβ/δ in myoblast differentiation. C2C12 myoblasts treated with a PPARβ/δ agonist, GW0742 exhibit enhanced myotube formation and muscle-specific gene expression. GW0742 treatment dramatically activates promyogenic kinases, p38MAPK and Akt, in a dose-dependent manner. GW0742-stimulated myoblast differentiation is mediated by p38MAPK and Akt, since it failed to restore myoblast differentiation repressed by inhibition of p38MAPK and Akt. In addition, GW0742 treatment enhances MyoD-reporter activities. Consistently, overexpression of PPARβ/δ enhances myoblast differentiation accompanied by elevated activation of p38MAPK and Akt. Collectively, these results suggest that PPARβ/δ enhances myoblast differentiation through activation of promyogenic signaling pathways. - Highlights: • A PPARβ/δ agonist, GW0742 promotes myoblast differentiation. • GW0742 activates both p38MAPK and Akt activation in myogenic differentiation. • GW0742 enhances MyoD activity for myogenic differentiation. • Overexpression of PPARβ/δ enhances myoblast differentiation via activating promyogenic signaling pathways. • This is the first finding for agonistic mechanism of PPARβ/δ in myogenesis.

  8. Effect of ternary phosphate-based glass compositions on osteoblast and osteoblast-like proliferation, differentiation and death in vitro.

    Science.gov (United States)

    Skelton, K L; Glenn, J V; Clarke, S A; Georgiou, G; Valappil, S P; Knowles, J C; Nazhat, S N; Jordan, G R

    2007-07-01

    There is currently a need to expand the range of graft materials available to orthopaedic surgeons. This study investigated the effect of ternary phosphate-based glass (PBG) compositions on the behaviour of osteoblast and osteoblast-like cells. PBGs of the formula (in mol.%) P(2)O(5)(50)-CaO(50-X)-Na(2)O(X), where X is either 2, 4, 6, 8 or 10, were produced and their influence on the proliferation, differentiation and death in vitro of adult human bone marrow stromal cells (hBMSCs) and human fetal osteoblast 1.19 (HFOB 1.19) cells were assessed. Tissue culture plastic (TCP) and hydroxyapatite (HA) were used as controls. Exposure to PBGs in culture inhibited cell adhesion and proliferation and increased cell death in both cell types studied. There was no significant difference in percentage cell death between the PBGs, which was significantly greater than the controls. However, compared with other PBGs, a greater number of cells were found on the 48mol.% CaO which may have been due to either increased adherence or proliferation, or both. This composition was capable of supporting osteogenic proliferation and early differentiation, and supports the notion that chemical modification of the glass could lead to a more biologically compatible substrate with the potential to support osteogenic grafting. Realisation of this potential should lead to the development of novel grafting strategies for the treatment of problematic bone defects.

  9. High glucose improves healing of periodontal wound by inhibiting proliferation and osteogenetic differentiation of human PDL cells.

    Science.gov (United States)

    Li, Min; Li, Cheng-Zhang

    2016-02-01

    Periodontal ligament (PDL) cells play an important role in wound healing of periodontal tissues. Response of PDL cells' cellular activity to high-glucose concentration levels may be the key in understanding the relationship between periodontal disease and diabetes mellitus. We studied the effect of high-glucose medium on proliferation of PDL cells in vitro. PDL cells were cultured for 1, 4, 7, 10, 14 and 17 days in normal (1100 mg/l) glucose or in high (4500 mg/l) glucose medium. The 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for proliferation was performed. In order to evaluate the osteogenetic differentiation of human PDL cells, the cells were induced with normal- or high-glucose medium for 1, 7, 14, 21 and 28 days. The results indicated that high glucose significantly inhibited proliferation of PDL cells. Concerning the mineralised nodule formation, the percentage of calcified area to total culture dish of PDL cells in high glucose level was lower than that in normal glucose medium. The increase in alkaline phosphatase activity and collagen expression could be observed in high-glucose-containing osteogenetic factor. In conclusion, high glucose improves healing of periodontal wound by inhibiting proliferation and differentiation of PDL cells, which could explain for delayed periodontal regeneration and healing in diabetic patients. © 2014 The Authors. International Wound Journal © 2014 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  10. Stochastic cellular automata model of cell migration, proliferation and differentiation: validation with in vitro cultures of muscle satellite cells.

    Science.gov (United States)

    Garijo, N; Manzano, R; Osta, R; Perez, M A

    2012-12-07

    Cell migration and proliferation has been modelled in the literature as a process similar to diffusion. However, using diffusion models to simulate the proliferation and migration of cells tends to create a homogeneous distribution in the cell density that does not correlate to empirical observations. In fact, the mechanism of cell dispersal is not diffusion. Cells disperse by crawling or proliferation, or are transported in a moving fluid. The use of cellular automata, particle models or cell-based models can overcome this limitation. This paper presents a stochastic cellular automata model to simulate the proliferation, migration and differentiation of cells. These processes are considered as completely stochastic as well as discrete. The model developed was applied to predict the behaviour of in vitro cell cultures performed with adult muscle satellite cells. Moreover, non homogeneous distribution of cells has been observed inside the culture well and, using the above mentioned stochastic cellular automata model, we have been able to predict this heterogeneous cell distribution and compute accurate quantitative results. Differentiation was also incorporated into the computational simulation. The results predicted the myotube formation that typically occurs with adult muscle satellite cells. In conclusion, we have shown how a stochastic cellular automata model can be implemented and is capable of reproducing the in vitro behaviour of adult muscle satellite cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Effects of the enamel matrix derivative on the proliferation and odontogenic differentiation of human dental pulp cells.

    Science.gov (United States)

    Wang, Yuanyuan; Zhao, Yuming; Ge, Lihong

    2014-01-01

    The enamel matrix derivative (EMD) has a positive effect on the proliferation of human periodontal ligament cells and the healing of periodontal tissues. The aim of this study was to evaluate the effects of EMD on the proliferation and differentiation of human dental pulp cells (hDPCs) in vitro. hDPCs were isolated from human impacted third molars and cultured in vitro. After treatment with100μg/mL EMD, the proliferation of hDPCs was determined by a cell counting kit 8 (CCK8) assay. After incubation in EMD osteogenic induction medium for 14 days, the osteogenic differentiation of hDPCs was evaluated by alkaline phosphatase (ALP) activity, alizarin staining and the expression of osteogenesis-related genes. The EMD osteogenic induction medium enhanced the proliferation of hDPCs. After osteogenic induction, EMD increased the osteogenic potential of hDPCs, as measured by alkaline phosphatase activity and calcium accumulation; the expression levels of osteogenesis-related genes, such as ALP, DSPP, BMP, and OPN were also upregulated. In addition, the expression levels of odontogenesis-related transcription factors Osterix and Runx2 were upregulated. EMD could enhance the mineralization of hDPSCs upregulated the expression of markers for odontoblast/osteoblast-like cells. Further studies are required to determine if EMD can improve pulp tissue repair and regeneration. Copyright © 2013. Published by Elsevier Ltd.

  12. Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.

    Science.gov (United States)

    Chen, Wan-Chun; Lin, Hsi-Hui; Tang, Ming-Jer

    2014-09-15

    To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis. Copyright © 2014 the American Physiological Society.

  13. ErbB3 binding protein-1 (Ebp1) controls proliferation and myogenic differentiation of muscle stem cells.

    Science.gov (United States)

    Figeac, Nicolas; Serralbo, Olivier; Marcelle, Christophe; Zammit, Peter S

    2014-02-01

    Satellite cells are resident stem cells of skeletal muscle, supplying myoblasts for post-natal muscle growth, hypertrophy and repair. Many regulatory networks control satellite cell function, which includes EGF signalling via the ErbB family of receptors. Here we investigated the role of ErbB3 binding protein-1 (Ebp1) in regulation of myogenic stem cell proliferation and differentiation. Ebp1 is a well-conserved DNA/RNA binding protein that is implicated in cell growth, apoptosis and differentiation in many cell types. Of the two main Ebp1 isoforms, only p48 was expressed in satellite cells and C2C12 myoblasts. Although not present in quiescent satellite cells, p48 was strongly induced during activation, remaining at high levels during proliferation and differentiation. While retroviral-mediated over-expression of Ebp1 had only minor effects, siRNA-mediated Ebp1 knockdown inhibited both proliferation and differentiation of satellite cells and C2C12 myoblasts, with a clear failure of myotube formation. Ebp1-knockdown significantly reduced ErbB3 receptor levels, yet over-expression of ErbB3 in Ebp1 knockdown cells did not rescue differentiation. Ebp1 was also expressed by muscle cells during developmental myogenesis in mouse. Since Ebp1 is well-conserved between mouse and chick, we switched to chick to examine its role in muscle formation. In chick embryo, Ebp1 was expressed in the dermomyotome, and myogenic differentiation of muscle progenitors was inhibited by specific Ebp1 down-regulation using shRNA electroporation. These observations demonstrate a conserved function of Ebp1 in the regulation of embryonic muscle progenitors and adult muscle stem cells, which likely operates independently of ErbB3 signaling. © 2013 Published by Elsevier Inc.

  14. Effect of estradiol on proliferation and differentiation of side population stem/progenitor cells from murine endometrium

    Directory of Open Access Journals (Sweden)

    Xu Jing

    2011-07-01

    Full Text Available Abstract Background In our previous study, endometrium side population cells (SP cells were isolated from postpartum murine uterus, and characterized by a heterogeneous population of stem/progenitor cells. In this study, we investigated the effect of estrogen on the proliferation and differentiation of SP cells. Methods SP and non-SP cells of postpartum murine endometrium were isolated by DNA dye Hoechst 33342. The expression of estrogen receptor 1 (ESR1 was measured by reverse transcription polymerase chain reaction (RT-PCR, Real-time PCR, Western blot, immunofluorescence and immunohistochemistry. The proliferation and differentiation of SP cells treated with different concentrations [10(-8 M-10(-6 M] of estradiol (E2 and E2+ ICI182780 (Faslodex, inhibitor of ESR1 were measured by 3-(4, 5-dimethylthiazoly1-2-2,5-diphenyltetrazolium bromide(MTT and clonogenic assays. Results (1 SP cells expressed ESR1 at a higher level than non-SP cells. (2 The level of E2 in the serum and the expression of ESR1 in the uterus of postpartum murine changed in the same manner with the ratio of SP cells to total uterus cells at a different postpartum time point. ESR1, as ABCG2 is also predominantly located in the stroma and the glandular epithelium of the uterus. (3 10(-6 M E2 notably promoted the proliferation of SP cells after treatment for 24 h. This effect could be inhibited by ICI182780. E2 at the concentration of 10(-7 M or 10(-8 M was sent to impair the large cloning efficiency (CE of SP cells. Conclusions The effect of estrogen on the proliferation and differentiation of endometrium SP cells via ESR1 was observed and it was in a concentration dependent fashion. Clearly, more work is needed to understand the in vivo effect of E2 at the physiological concentration on the differentiation of SP cells.

  15. Effects of high-mobility group box 1 on the proliferation and odontoblastic differentiation of human dental pulp cells.

    Science.gov (United States)

    Qi, S C; Cui, C; Yan, Y H; Sun, G H; Zhu, S R

    2013-12-01

    To investigate the expression of high-mobility group box 1 (HMGB1) in human dental pulp tissues and the effects of HMGB1 on proliferation and odontoblastic differentiation of human dental pulp cells (hDPCs). Immunohistochemical assay, immunofluorescence staining and flow cytometric analysis were used to detect the expression of HMGB1 in the human dental pulp and hDPCs, respectively. The proliferation of hDPCs was examined by CCK-8 after culturing human primary hDPCs in the presence of HMGB1 with different doses. Odontoblastic differentiation of hDPCs was determined using alkaline phosphatase (ALPase) activity assay and mineralized nodule formation. Important mineralization-related genes such as ALP, dental sialophosphoprotein (DSPP) and dental matrix protein-1 (DMP-1) were determined by real-time polymerase chain reaction. Western blot analysis was performed to determine the difference in expressions of DMP-1 and DSP with or without the presence of exogenous HMGB1. Simultaneously, messenger RNA and protein levels of HMGB1 and RAGE were also detected. The protein level of HMGB1 in the supernatants was quantified using ELISA analysis. HMGB1 was found in human dental pulp tissue and in the nuclei of hDPCs. During hDPC odontoblastic differentiation, HMGB1 translocated from the nuclei to the cytoplasm and then secreted out from hDPCs. Exogenous HMGB1 promoted hDPC proliferation and mineralized nodule formation. It up-regulated the activity of ALPase and the mRNA and protein levels of dentine matrix protein-1 (DMP-1), alkaline phosphatase (ALP), dentine sialophosphoprotein (DSPP) and receptor for advance glycation end (RAGE) of hDPCs. HMGB1 promoted the proliferation and odontoblastic differentiation of hDPCs. © 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  16. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xin, Ying [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Lu, Qingxian [Tumor Immunobiology Group, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Li, Qiutang, E-mail: q.li@louisville.edu [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States)

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  17. Cytoprotection, proliferation and epidermal differentiation of adipose tissue-derived stem cells on emu oil based electrospun nanofibrous mat.

    Science.gov (United States)

    Pilehvar-Soltanahmadi, Younes; Nouri, Mohammad; Martino, Mikaël M; Fattahi, Amir; Alizadeh, Effat; Darabi, Masoud; Rahmati-Yamchi, Mohammad; Zarghami, Nosratollah

    2017-08-15

    Electrospun nanofibrous scaffolds containing natural substances with wound healing properties such as Emu oil (EO) may have a great potential for increasing the efficiency of stem cell-based skin bioengineering. For this purpose, EO blended PCL/PEG electrospun nanofibrous mats were successfully fabricated and characterized using FE-SEM, FTIR and Universal Testing Machine. The efficiency of the scaffolds in supporting the adherence, cytoprotection, proliferation and differentiation of adipose tissue-derived stem cells (ADSCs) to keratinocyte was evaluated. GC/MS and HPLC were used to determine the composition of pure EO, which revealed to be mainly fatty acids and carotenoids. FE-SEM and cell proliferation assays showed that adhesion and proliferation of ADSCs on EO-PCL/PEG nanofibers was significantly higher than on PCL/PEG nanofibers. Additionally, EO-PCL/PEG nanofibers with free radical scavenging properties conferred a cytoprotective effect against cell-damaging free radicals, while the ability to support cell adhesion and growth was maintained or even improved. Immunostaining of ADSCs on EO-PCL/PEG nanofibers confirmed the change in morphology of ADSCs from spindle to polygonal shape suggesting their differentiation toward an epidermal linage. Moreover, the expression levels of the keratin 10, filaggrin, and involucrin that are involved in epidermal differentiation were upregulated in a stage-specific manner. This preliminary study shows that EO-PCL/PEG nanofibers could be a good candidate for the fabrication of wound dressings and skin bioengineered substitutes with ADSCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Zirconium ions up-regulate the BMP/SMAD signaling pathway and promote the proliferation and differentiation of human osteoblasts.

    Directory of Open Access Journals (Sweden)

    Yongjuan Chen

    Full Text Available Zirconium (Zr is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2 or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV oxynitrate (ZrO(NO32 at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.

  19. The high dosage of earthworm (Eisenia andrei) extract decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus

    Science.gov (United States)

    Yan, Bing Chun; Yoo, Ki-Yeon; Park, Joon Ha; Lee, Choong Hyun; Choi, Jung Hoon

    2011-01-01

    Earthworm extract has shown anticancer characteristics. In the present study, we examined the effect of chronic treatment with a high dose of earthworm (Eisenia andrei) extract (EE) on cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus (DG) of 3-week-old mice using 5-bromo-2'-deoxyuridine (BrdU) and Ki-67 immunohistochemistry for cell proliferation and doublecortin (DCX) immunohistochemistry for neuroblast differentiation, respectively. BrdU-, Ki-67-, and DCX-immunoreactive cells were easily detected in the subgranular zone of the DG in vehicle (saline)-treated mice. However, BrdU-, Ki-67-, and DCX-immunoreactive cells in the 500 mg/kg EE-treated mice decreased distinctively compared to those in the vehicle-treated mice. In addition, brain-derived neurotrophic factor (BDNF) immunoreactivity and its protein level decreased markedly in the DG of the EE-treated group compared to those in the vehicle-treated group. These results indicate that chronic treatment with high dose EE decreased cell proliferation and neuroblast differentiation, and that BDNF immunoreactivity decreased in the DG of EE-treated mice. PMID:22025974

  20. Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics.

    Science.gov (United States)

    Sun, Hongli; Wu, Chengtie; Dai, Kerong; Chang, Jiang; Tang, Tingting

    2006-11-01

    In the present study, the effects of a calcium magnesium silicate bioactive ceramic (akermanite) on proliferation and osteoblastic differentiation of human bone marrow stromal cells (hBMSC) have been investigated and compared with the classical ceramic (beta-tricalcium phosphate, beta-TCP). Akermanite and beta-TCP disks were seeded with hBMSC and kept in growth medium or osteogenic medium for 10 days. Proliferation and osteoblastic differentiation were evaluated on day 1, 4, 7 and 10. The data from the Alamar Blue assay and lactic acid production assay showed that hBMSC proliferated more significantly on akermanite than on beta-TCP. The analysis of osteoblast-related genes, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), indicated that akermanite ceramics enhanced the expression of osteoblast-related genes, but type I collagen (COL I) showed no noticeable difference among akermanite and beta-TCP ceramics. Furthermore, this stimulatory effect was observed not only in osteogenic medium, but also in normal growth medium without osteogenic reagents such as l-ascorbic acid, glycerophosphate and dexamethasone. This result suggests that akermanite can promote osteoblastic differentiation of hBMSC in vitro even without osteogenic reagents, and may be used as a bioactive material for bone regeneration and tissue engineering applications.

  1. Merlin, a "magic" linker between extracellular cues and intracellular signaling pathways that regulate cell motility, proliferation, and survival.

    Science.gov (United States)

    Stamenkovic, Ivan; Yu, Qin

    2010-09-01

    Genetic alterations of neurofibromatosis type 2 (NF2) gene lead to the development of schwannomas, meningiomas, and ependymomas. Mutations of NF2 gene were also found in thyroid cancer, mesothelioma, and melanoma, suggesting that it functions as a tumor suppressor in a wide spectrum of cells. The product of NF2 gene is merlin (moesin-ezrin-radixin-like protein), a member of the Band 4.1 superfamily proteins. Merlin shares significant sequence homology with the ERM (Ezrin-Radixin-Moesin) family proteins and serves as a linker between transmembrane proteins and the actin-cytoskeleton. Merlin is a multifunctional protein and involved in integrating and regulating the extracellular cues and intracellular signaling pathways that control cell fate, shape, proliferation, survival, and motility. Recent studies showed that merlin regulates the cell-cell and cell-matrix adhesions and functions of the cell surface adhesion/extracellular matrix receptors including CD44 and that merlin and CD44 antagonize each other's function and work upstream of the mammalian Hippo signaling pathway. Furthermore, merlin plays important roles in stabilizing the contact inhibition of proliferation and in regulating activities of several receptor tyrosine kinases. Accumulating data also suggested an emerging role of merlin as a negative regulator of growth and progression of several non-NF2 associated cancer types. Together, these recent advances have improved our basic understanding about merlin function, its regulation, and the major signaling pathways regulated by merlin and provided the foundation for future translation of these findings into the clinic for patients bearing the cancers in which merlin function and/or its downstream signaling pathways are impaired or altered.

  2. Physiological and hypoxic oxygen concentration differentially regulates human c-Kit+ cardiac stem cell proliferation and migration.

    Science.gov (United States)

    Bellio, Michael A; Rodrigues, Claudia O; Landin, Ana Marie; Hatzistergos, Konstantinos E; Kuznetsov, Jeffim; Florea, Victoria; Valasaki, Krystalenia; Khan, Aisha; Hare, Joshua M; Schulman, Ivonne Hernandez

    2016-12-01

    Cardiac stem cells (CSCs) are being evaluated for their efficacy in the treatment of heart failure. However, numerous factors impair the exogenously delivered cells' regenerative capabilities. Hypoxia is one stress that contributes to inadequate tissue repair. Here, we tested the hypothesis that hypoxia impairs cell proliferation, survival, and migration of human CSCs relative to physiological and room air oxygen concentrations. Human endomyocardial biopsy-derived CSCs were isolated, selected for c-Kit expression, and expanded in vitro at room air (21% O2). To assess the effect on proliferation, survival, and migration, CSCs were transferred to physiological (5%) or hypoxic (0.5%) O2 concentrations. Physiological O2 levels increased proliferation (P air and hypoxia, a significant reduction of β-galactosidase activity (-4,203 fluorescent units, P air and hypoxia, and treatment with mesenchymal stem cell-conditioned media rescued CSC migration under hypoxia to levels comparable to physiological O2 migration (2-fold, P air may diminish cell regenerative potential. This study provides novel insights into the modulatory effects of O2 concentration on CSC biology and has important implications for refining stem cell therapies. Copyright © 2016 the American Physiological Society.

  3. Piperlongumine inhibits the proliferation and survival of B-cell acute lymphoblastic leukemia cell lines irrespective of glucocorticoid resistance

    Energy Technology Data Exchange (ETDEWEB)

    Han, Seong-Su, E-mail: seong-su-han@uiowa.edu [Division of Pediatric Hematology-Oncology, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Han, Sangwoo [Health and Human Physiology, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Kamberos, Natalie L. [Division of Pediatric Hematology-Oncology, University of Iowa Carver College of Medicine, Iowa City, IA (United States)

    2014-09-26

    Highlights: • PL inhibits the proliferation of B-ALL cell lines irrespective of GC-resistance. • PL selectively kills B-ALL cells by increasing ROS, but not normal counterpart. • PL does not sensitize majority of B-ALL cells to DEX. • PL represses the network of constitutively activated TFs and modulates their target genes. • PL may serve as a new therapeutic molecule for GC-resistant B-ALL. - Abstract: Piperlongumine (PL), a pepper plant alkaloid from Piper longum, has anti-inflammatory and anti-cancer properties. PL selectively kills both solid and hematologic cancer cells, but not normal counterparts. Here we evaluated the effect of PL on the proliferation and survival of B-cell acute lymphoblastic leukemia (B-ALL), including glucocorticoid (GC)-resistant B-ALL. Regardless of GC-resistance, PL inhibited the proliferation of all B-ALL cell lines, but not normal B cells, in a dose- and time-dependent manner and induced apoptosis via elevation of ROS. Interestingly, PL did not sensitize most of B-ALL cell lines to dexamethasone (DEX). Only UoC-B1 exhibited a weak synergistic effect between PL and DEX. All B-ALL cell lines tested exhibited constitutive activation of multiple transcription factors (TFs), including AP-1, MYC, NF-κB, SP1, STAT1, STAT3, STAT6 and YY1. Treatment of the B-ALL cells with PL significantly downregulated these TFs and modulated their target genes. While activation of AURKB, BIRC5, E2F1, and MYB mRNA levels were significantly downregulated by PL, but SOX4 and XBP levels were increased by PL. Intriguingly, PL also increased the expression of p21 in B-ALL cells through a p53-independent mechanism. Given that these TFs and their target genes play critical roles in a variety of hematological malignancies, our findings provide a strong preclinical rationale for considering PL as a new therapeutic agent for the treatment of B-cell malignancies, including B-ALL and GC-resistant B-ALL.

  4. The Role of Lipid Metabolism in T Lymphocyte Differentiation and Survival

    Directory of Open Access Journals (Sweden)

    Duncan Howie

    2018-01-01

    Full Text Available The differentiation and effector functions of both the innate and adaptive immune system are inextricably linked to cellular metabolism. The features of metabolism which affect both arms of the immune system include metabolic substrate availability, expression of enzymes, transport proteins, and transcription factors which control catabolism of these substrates, and the ability to perform anabolic metabolism. The control of lipid metabolism is central to the appropriate differentiation and functions of T lymphocytes, and ultimately to the maintenance of immune tolerance. This review will focus on the role of fatty acid (FA metabolism in T cell differentiation, effector function, and survival. FAs are important sources of cellular energy, stored as triglycerides. They are also used as precursors to produce complex lipids such as cholesterol and membrane phospholipids. FA residues also become incorporated into hormones and signaling moieties. FAs signal via nuclear receptors and their channeling, between storage as triacyl glycerides or oxidation as fuel, may play a role in survival or death of the cell. In recent years, progress in the field of immunometabolism has highlighted diverse roles for FA metabolism in CD4 and CD8 T cell differentiation and function. This review will firstly describe the sensing and modulation of the environmental FAs and lipid intracellular signaling and will then explore the key role of lipid metabolism in regulating the balance between potentially damaging pro-inflammatory and anti-inflammatory regulatory responses. Finally the complex role of extracellular FAs in determining cell survival will be discussed.

  5. Mucinous and Signet Ring Cell Differentiation Affect Patterns of Metastasis in Colorectal Carcinoma and Influence Survival.

    Science.gov (United States)

    Kermanshahi, Taher Reza; Magge, Deepa; Choudry, Haroon; Ramalingam, Leksmi; Zhu, Benjamin; Pingpank, James; Ahrendt, Steven; Holtzman, Matthew; Zeh, Herbert; Bartlett, David; Zureikat, Amer; Pai, Reetesh K

    2017-04-01

    Peritoneal metastasis in colorectal carcinoma is associated with a dismal prognosis; however, features that correlate with patterns of metastatic spread are not well characterized. We analyzed the clinicopathologic and molecular features of 166 patients with colorectal carcinomas stratified by metastases to the peritoneum or liver. Mucinous and signet ring cell differentiation were more frequently observed in colorectal carcinoma with peritoneal dissemination compared to colorectal carcinoma with liver metastasis (mucinous differentiation: 62% vs 23%, P metastasis was identified in patients with both synchronous and metachronous development of metastasis ( P metastasis were more frequently low-grade (90% vs 72%, P = .005) and associated with dirty necrosis (81% vs 56%, P = .001) compared with colorectal carcinomas with peritoneal dissemination. No significant differences were identified between colorectal carcinoma with peritoneal metastasis versus liver metastasis with respect to KRAS mutations, BRAF mutation, or high levels of microsatellite instability. Patients with tumors involving the peritoneum had a significantly worse overall survival in comparison to patients with liver metastasis lacking peritoneal involvement ( P = .02). When including only those patients with peritoneal metastasis, the presence of any mucinous or signet ring cell differentiation was associated with a significantly worse overall survival ( P = .006). Our findings indicate that mucinous and signet ring cell differentiation may be histologic features that are associated with an increased risk of peritoneal dissemination and poor overall survival in patients with peritoneal metastasis.

  6. The role of Zic family zinc finger transcription factors in the proliferation and differentiation of retinal progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Watabe, Yui [Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo (Japan); Department of Ophthalmology, Teikyo University School of Medicine, Tokyo (Japan); Division of Orthoptics, Teikyo University School of Medical Care and Technology, Tokyo (Japan); Baba, Yukihiro [Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo (Japan); Nakauchi, Hiromitsu [Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo (Japan); Mizota, Atsushi [Department of Ophthalmology, Teikyo University School of Medicine, Tokyo (Japan); Watanabe, Sumiko, E-mail: sumiko@ims.u-tokyo.ac.jp [Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo (Japan)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Zic transcription factors expressed early retinal progenitor cells. Black-Right-Pointing-Pointer Zics sustain proliferation activity of retinal progenitor cells. Black-Right-Pointing-Pointer Overexpression of Zic in retinal progenitors perturbed rod differentiation. Black-Right-Pointing-Pointer Fate determination to rod photoreceptor was not affected. -- Abstract: Members of the Zic family of zinc finger transcription factors play critical roles in a variety of developmental processes. Using DNA microarray analysis, we found that Zics are strongly expressed in SSEA-1-positive early retinal progenitors in the peripheral region of the mouse retina. Reverse-transcription polymerase chain reaction using mRNA from the retina at various developmental stages showed that Zic1 and Zic2 are expressed in the embryonic retina and then gradually disappear during retinal development. Zic3 is also expressed in the embryonic retina; its expression level slightly decreases but it is expressed until adulthood. We overexpressed Zic1, Zic2, or Zic3 in retinal progenitors at embryonic day 17.5 and cultured the retina as explants for 2 weeks. The number of rod photoreceptors was fewer than in the control, but no other cell types showed significant differences between control and Zic overexpressing cells. The proliferation activity of normal retinal progenitors decreased after 5 days in culture, as observed in normal in vivo developmental processes. However, Zic expressing retinal cells continued to proliferate at days 5 and 7, suggesting that Zics sustain the proliferation activities of retinal progenitor cells. Since the effects of Zic1, 2, and 3 are indistinguishable in terms of differentiation and proliferation of retinal progenitors, the redundant function of Zics in retinal development is suggested.

  7. Lck is involved in interleukin-2 induced proliferation but not cell survival in human T cells through a MAP kinase-independent pathway

    DEFF Research Database (Denmark)

    Brockdorff, J; Nielsen, M; Kaltoft, K

    2000-01-01

    The role of Lck in IL-2-induced proliferation and cell survival is still controversial. Here, we show that the Src family kinase inhibitor, PP1, reduced the IL-2-induced proliferation of human T cells significantly without inhibiting the anti-apoptotic effect of IL-2. As Lck is the only Src family...... kinase activated upon IL-2 stimulation in T cells, this indicates that Lck is involved in IL-2-induced proliferation but not survival. IL-2-induced MAP kinase activation was only slightly inhibited by PP1, suggesting that Lck is not essential for IL-2-induced MAP kinase activation in human T cells. We...... found that an IL-2-sensitive, human mycosis fungoides-derived tumor T cell line is Lck negative, and that the IL-2-induced MAP kinase activation is comparable to non-cancerous T cells, although a little delayed in kinetics. An Lck expressing clone was established by transfecting Lck into mycosis...

  8. The aryl hydrocarbon receptor suppresses osteoblast proliferation and differentiation through the activation of the ERK signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Haitao; Du, Yuxuan; Zhang, Xulong; Sun, Ying; Li, Shentao; Dou, Yunpeng [Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069 (China); Li, Zhanguo [Department of Rheumatology and Immunology, Clinical Immunology Center, Peking University People' s Hospital, No. 11 Xizhimen South Street, Beijing 100044 (China); Yuan, Huihui, E-mail: huihui_yuan@163.com [Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069 (China); Zhao, Wenming, E-mail: zhao-wenming@163.com [Department of Immunology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069 (China)

    2014-11-01

    Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity of Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5 days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice. - Highlights: • The upregulation of Ahr was localized in osteoblasts of CIA mice. • The overexpression of Ahr suppressed osteoblast development. • The Ahr activated ERK signaling pathway to exacerbate bone erosion.

  9. Prox1 Inhibits Proliferation and Is Required for Differentiation of the Oligodendrocyte Cell Lineage in the Mouse.

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    Kentaro Kato

    Full Text Available Central nervous system injury induces a regenerative response in ensheathing glial cells comprising cell proliferation, spontaneous axonal remyelination, and limited functional recovery, but the molecular mechanisms are not fully understood. In Drosophila, this involves the genes prospero and Notch controlling the balance between glial proliferation and differentiation, and manipulating their levels in glia can switch the response to injury from prevention to promotion of repair. In the mouse, Notch1 maintains NG2 oligodendrocyte progenitor cells (OPCs in a progenitor state, but what factor may enable oligodendrocyte (OL differentiation and functional remyelination is not understood. Here, we asked whether the mammalian homologue of prospero, Prox1, is involved. Our data show that Prox1 is distributed in NG2+ OPCs and in OLs in primary cultured cells, and in the mouse spinal cord in vivo. siRNA prox1 knockdown in primary OPCs increased cell proliferation, increased NG2+ OPC cell number and decreased CC1+ OL number. Prox1 conditional knockout in the OL cell lineage in mice increased NG2+ OPC cell number, and decreased CC1+ OL number. Lysolecithin-induced demyelination injury caused a reduction in CC1+ OLs in homozygous Prox1-/- conditional knockout mice compared to controls. Remarkably, Prox1-/- conditional knockout mice had smaller lesions than controls. Altogether, these data show that Prox1 is required to inhibit OPC proliferation and for OL differentiation, and could be a relevant component of the regenerative glial response. Therapeutic uses of glia and stem cells to promote regeneration and repair after central nervous system injury would benefit from manipulating Prox1.

  10. Bovine collagen peptides compounds promote the proliferation and differentiation of MC3T3-E1 pre-osteoblasts.

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    JunLi Liu

    Full Text Available OBJECTIVE: Collagen peptides (CP compounds, as bone health supplements, are known to play a role in the treatment of osteoporosis. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of bovine CP compounds on the proliferation and differentiation of MC3T3-E1 cells. METHODS: Mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells were treated with bovine CP compounds. Cell proliferation was analyzed by MTT assays and the cell cycle was evaluated by flow cytometry scanning. Furthermore, MC3T3-E1 cell differentiation was analyzed at the RNA level by real-time PCR and at the protein level by western blot analysis for runt-related transcription factor 2 (Runx2, a colorimetric p-nitrophenyl phosphate assay for alkaline phosphatase (ALP, and ELISA for osteocalcin (OC. Finally, alizarin red staining for mineralization was measured using Image Software Pro Plus 6.0. RESULTS: Cell proliferation was very efficient after treatment with different concentrations of bovine CP compounds, and the best concentration was 3 mg/mL. Bovine CP compounds significantly increased the percentage of MC3T3-E1 cells in G2/S phase. Runx2 expression, ALP activity, and OC production were significantly increased after treatment with bovine CP compounds for 7 or 14 days. Quantitative analyses with alizarin red staining showed significantly increased mineralization of MC3T3-E1 cells after treatment with bovine CP compounds for 14 or 21 days. CONCLUSIONS: Bovine CP compounds increased osteoblast proliferation, and played positive roles in osteoblast differentiation and mineralized bone matrix formation. Taking all the experiments together, our study indicates a molecular mechanism for the potential treatment of osteoarthritis and osteoporosis.

  11. A Flap Endonuclease (TcFEN1) Is Involved in Trypanosoma cruzi Cell Proliferation, DNA Repair, and Parasite Survival.

    Science.gov (United States)

    Ponce, Ivan; Aldunate, Carmen; Valenzuela, Lucia; Sepúlveda, Sofia; Garrido, Gilda; Kemmerling, Ulrike; Cabrera, Gonzalo; Galanti, Norbel

    2017-07-01

    FLAP endonucleases (FEN) are involved both in DNA replication and repair by processing DNA intermediaries presenting a nucleotide flap using its phosphodiesterase activity. In spite of these important functions in DNA metabolism, this enzyme was not yet studied in Trypanosomatids. Trypanosoma cruzi, the ethiological agent of Chagas disease, presents two dividing cellular forms (epimastigote and amastigote) and one non-proliferative, infective form (trypomastigote). The parasite survives DNA damage produced by reactive species generated in its hosts. The activity of a T. cruzi FLAP endonuclease (TcFEN1) was determined in the three cellular forms of the parasite using a DNA substrate generated by annealing three different oligonucleotides to form a double-stranded DNA with a 5' flap in the middle. This activity showed optimal pH and temperature similar to other known FENs. The substrate cut by the flap endonuclease activity could be ligated by the parasite generating a repaired DNA product. A DNA flap endonuclease coding sequence found in the T. cruzi genome (TcFEN1) was cloned, inserted in parasite expression vectors and transfected to epimastigotes. The purified native recombinant protein showed DNA flap endonuclease activity. This endonuclease was found located in the parasite nucleus of transfected epimastigotes and its over-expression increased both parasite proliferation and survival to H 2 O 2 . The presence of a flap endonuclease activity in T. cruzi and its nuclear location are indicative of the participation of this enzyme in DNA processing of flap fragments during DNA replication and repair in this parasite of ancient evolutive origin. J. Cell. Biochem. 118: 1722-1732, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Differential effects of a complex organochlorine mixture on the proliferation of breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Aube, Michel, E-mail: 4aubem@videotron.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Larochelle, Christian, E-mail: christian.larochelle@inspq.qc.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Ayotte, Pierre, E-mail: pierre.ayotte@inspq.qc.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Laboratoire de Toxicologie, Institut national de sante publique du Quebec, 945 avenue Wolfe, Quebec, QC, Canada G1V 5B3 (Canada)

    2011-04-15

    Organochlorine compounds (OCs) are a group of persistent chemicals that accumulate in fatty tissues with age. Although OCs has been tested individually for their capacity to induce breast cancer cell proliferation, few studies examined the effect of complex mixtures that comprise compounds frequently detected in the serum of women. We constituted such an OC mixture containing 15 different components in environmentally relevant proportions and assessed its proliferative effects in four breast cancer cell lines (MCF-7, T47D, CAMA-1, MDAMB231) and in non-cancerous CV-1 cells. We also determined the capacity of the mixture to modulate cell cycle stage of breast cancer cells and to induce estrogenic and antiandrogenic effects using gene reporter assays. We observed that low concentrations of the mixture (100x10{sup 3} and 50x10{sup 3} dilutions) stimulated the proliferation of MCF-7 cells while higher concentrations (10x10{sup 3} and 5x10{sup 3} dilutions) had the opposite effect. In contrast, the mixture inhibited the proliferation of non-hormone-dependent cell lines. The mixture significantly increased the number of MCF-7 cells entering the S phase, an effect that was blocked by the antiestrogen ICI 182,780. Low concentrations of the mixture also caused an increase in CAMA-1 cell proliferation but only in the presence estradiol and dihydrotestosterone (p<0.05 at the 50x10{sup 3} dilution). DDT analogs and polychlorinated biphenyls all had the capacity to stimulate the proliferation of CAMA-1 cells in the presence of sex steroids. Reporter gene assays further revealed that the mixture and several of its constituents (DDT analogs, aldrin, dieldrin, {beta}-hexachlorocyclohexane, toxaphene) induced estrogenic effects, whereas the mixture and several components (DDT analogs, aldrin, dieldrin and PCBs) inhibited the androgen signaling pathway. Our results indicate that the complex OC mixture increases the proliferation of MCF-7 cells due to its estrogenic potential. The

  13. The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells

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    J.Z. Fan

    2015-07-01

    Full Text Available We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α. Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

  14. The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells.

    Science.gov (United States)

    Fan, J Z; Yang, X; Bi, Z G

    2015-07-01

    We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

  15. 3D Bioprinting Human Induced Pluripotent Stem Cell Constructs for In Situ Cell Proliferation and Successive Multilineage Differentiation.

    Science.gov (United States)

    Gu, Qi; Tomaskovic-Crook, Eva; Wallace, Gordon G; Crook, Jeremy M

    2017-09-01

    The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine, including individualized, patient-specific stem cell-based treatments. There are, however, few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues, ideally comprising direct-write printing of cells for encapsulation, proliferation, and differentiation. Here, such a method, employing a clinically amenable polysaccharide-based bioink, is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically, we have extrusion printed the bioink including iPSCs, alginate (Al; 5% weight/volume [w/v]), carboxymethyl-chitosan (5% w/v), and agarose (Ag; 1.5% w/v), crosslinked the bioink in calcium chloride for a stable and porous construct, proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm, ectoderm, and mesoderm, or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined, scalable, and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Three-step method for proliferation and differentiation of human embryonic stem cell (hESC-derived male germ cells.

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    Jung Jin Lim

    Full Text Available The low efficiency of differentiation into male germ cell (GC-like cells and haploid germ cells from human embryonic stem cells (hESCs reflects the culture method employed in the two-dimensional (2D-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC-like cells and haploid germ cells. In the first step, embryoid bodies (EBs were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.

  17. The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells

    Energy Technology Data Exchange (ETDEWEB)

    Fan, J.Z.; Yang, X.; Bi, Z.G. [Department of Orthopedic Surgery, First Affiliated Hospital, Harbin Medicine University, Harbin (China)

    2015-04-28

    We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

  18. Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

    Science.gov (United States)

    Lim, Jung Jin; Shim, Myung Sun; Lee, Jeoung Eun; Lee, Dong Ryul

    2014-01-01

    The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.

  19. Cardiomyocyte Differentiation Promotes Cell Survival During Nicotinamide Phosphoribosyltransferase Inhibition Through Increased Maintenance of Cellular Energy Stores.

    Science.gov (United States)

    Kropp, Erin M; Broniowska, Katarzyna A; Waas, Matthew; Nycz, Alyssa; Corbett, John A; Gundry, Rebekah L

    2017-04-01

    To address concerns regarding the tumorigenic potential of undifferentiated human pluripotent stem cells (hPSC) that may remain after in vitro differentiation and ultimately limit the broad use of hPSC-derivatives for therapeutics, we recently described a method to selectively eliminate tumorigenic hPSC from their progeny by inhibiting nicotinamide phosphoribosyltransferase (NAMPT). Limited exposure to NAMPT inhibitors selectively removes hPSC from hPSC-derived cardiomyocytes (hPSC-CM) and spares a wide range of differentiated cell types; yet, it remains unclear when and how cells acquire resistance to NAMPT inhibition during differentiation. In this study, we examined the effects of NAMPT inhibition among multiple time points of cardiomyocyte differentiation. Overall, these studies show that in vitro cardiomyogenic commitment and continued culturing provides resistance to NAMPT inhibition and cell survival is associated with the ability to maintain cellular ATP pools despite depletion of NAD levels. Unlike cells at earlier stages of differentiation, day 28 hPSC-CM can survive longer periods of NAMPT inhibition and maintain ATP generation by glycolysis and/or mitochondrial respiration. This is distinct from terminally differentiated fibroblasts, which maintain mitochondrial respiration during NAMPT inhibition. Overall, these results provide new mechanistic insight into how regulation of cellular NAD and energy pools change with hPSC-CM differentiation and further inform how NAMPT inhibition strategies could be implemented within the context of cardiomyocyte differentiation. Stem Cells Translational Medicine 2017;6:1191-1201. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  20. Asiatic acid protects against cognitive deficits and reductions in cell proliferation and survival in the rat hippocampus caused by 5-fluorouracil chemotherapy.

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    Pornthip Chaisawang

    Full Text Available The chemotherapy drug, 5-fluorouracil (5-FU, has been reported to cause cognitive impairments in cancer patients. The drug also reduces cell proliferation and survival in the brain. Asiatic acid (AA is a triterpene compound found in Centella asiatica that can protect against reduction of neurogenesis in the hippocampus and memory deficits induced by valproic acid (VPA. In the present study, we investigated the preventive effects of AA on the deficits in spatial working memory and cell proliferation and survival caused by 5-FU chemotherapy in a rat model. Male Sprague Dawley rats received 5-FU (5 i.v. injections, 25 mg/kg on day 8, 11, 14, 17 and 20 of the study. This was co-administered with AA (30 mg/kg, oral gavage tube either 20 days before receiving 5-FU (preventive, after receiving 5-FU (recovery, or for the entire period of the experiment (throughout. Spatial working memory was determined using the novel object location (NOL test and hippocampal cell proliferation and survival of dividing cells were quantified using immunohistochemistry. Rats in the 5-FU alone and recovery groups showed memory deficits in the NOL test and reductions in cell proliferation and cell survival in the subgranular zone (SGZ of the hippocampal dentate gyrus. Rats in the control, AA alone, and both preventive and throughout co-administration groups, however, did not exhibit these characteristics. The results showed that 5-FU chemotherapy impaired memory and reduced cell proliferation and cell survival in the SGZ of the hippocampal dentate gyrus. However, these impairments in the animals receiving 5-FU chemotherapy were restored to control levels when AA was co-administered before and during 5-FU treatment. These data demonstrate that AA can prevent the spatial working memory and hippocampal neurogenesis impairments caused by 5-FU chemotherapy.

  1. Glycolysis and the pentose phosphate pathway are differentially associated with the dichotomous regulation of glioblastoma cell migration versus proliferation.

    Science.gov (United States)

    Kathagen-Buhmann, Annegret; Schulte, Alexander; Weller, Jonathan; Holz, Mareike; Herold-Mende, Christel; Glass, Rainer; Lamszus, Katrin

    2016-09-01

    The dichotomy between glioblastoma cell migration and proliferation is regulated by various parameters including oxygen tension. In glioblastoma stem-like cells, hypoxia induces downregulation of pentose phosphate pathway (PPP) enzymes and a flux shift towards glycolysis. We investigated whether the 2 parallel glucose metabolic pathways are intrinsically linked with cell function and whether these pathways are mechanistically involved in regulating functional programs. Enzyme expression, migration, and proliferation under hypoxia were studied in multiple cell types. Rapidly and slowly dividing or migrating glioblastoma cells were separated, and enzyme profiles were compared. Glucose-6-phosphate dehydrogenase (G6PD) and Aldolase C (ALDOC), the most strongly inversely regulated PPP and glycolysis enzymes, were knocked down by short hairpin RNA. Hypoxia caused downregulation of PPP enzymes and upregulation of glycolysis enzymes in a broad spectrum of cancer and nonneoplastic cells and consistently stimulated migration while reducing proliferation. PPP enzyme expression was increased in rapidly dividing glioblastoma cells, whereas glycolysis enzymes were decreased. Conversely, glycolysis enzymes were elevated in migrating cells, whereas PPP enzymes were diminished. Knockdown of G6PD reduced glioblastoma cell proliferation, whereas ALDOC knockdown decreased migration. Enzyme inhibitors had similar effects. G6PD knockdown in a highly proliferative but noninvasive glioblastoma cell line resulted in prolonged survival of mice with intracerebral xenografts, whereas ALDOC knockdown shortened survival. In a highly invasive glioblastoma xenograft model, tumor burden was unchanged by either knockdown. Cell function and metabolic state are coupled independently of hypoxia, and glucose metabolic pathways are causatively involved in regulating "go or grow" cellular programs. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro

  2. Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Jeffrey eKim

    2014-03-01

    Full Text Available Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25µM of arachidonate (AA or docosahexaenoate (DHA, 25µM of EC [anandamide (AEA, 2-arachidonoylglycerol (2-AG, docosahexaenoylethanolamide (DHEA], 1µM of CB1 antagonist NESS0327, and CB2 antagonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts.Key Words: endocannabinoid system •C2C12 myoblasts cannabinoid receptors glucose uptake gene expression DHEA • polyunsaturated fatty acids

  3. Typha latifolia L. fruit polysaccharides induce the differentiation and stimulate the proliferation of human keratinocytes in vitro.

    Science.gov (United States)

    Gescher, Kirsten; Deters, Alexandra M

    2011-09-01

    In Northern America Typha latifolia L. (Typhaceae) fruits are used for more than 4000 years for treatment of skin disorders, burns and as wound dressing to absorb the ichors. The following studies attempted to characterize water-soluble polysaccharides from aqueous Typha latifolia extracts and to investigate the influence of the polymers on cell physiology of human dermal fibroblasts (NHDF) and epidermal keratinocytes (NHEK). Water-soluble raw polysaccharides (RPS) were isolated from Typha latifolia fruits and fractionated by anion exchange chromatography (AEC) and size exclusion chromatography (GPC). Fractions obtained were characterized concerning monosaccharide composition by HPAEC-PAD. The bioactivity of the polysaccharides was investigated on cell viability, proliferation, differentiation and gene expression NHDF of NHEK. RPS was fractionated into 5 heterodisperse fractions (TL1-TL5). The polysaccharides were composed mainly of glucose (more than 50% in RPS and TL4), galactose, xylose, mannose, glucuronic acid, galacturonic acid, arabinose, ribose, fucose, rhamnose, and fructose with differing amounts concerning to RPS and AEC-fractions. Proteins were detected in the RPS (10%) and to a less extend in TL1-TL3 (1-3%). TL1-TL3 significantly increased the proliferation of keratinocytes, whereas TL4 was shown to be a potent inductor of the early differentiation process of keratinocytes. Gene expression analysis supported these results since Smad3 and PKC-α, known to be part of signal pathways leading to cell differentiation, were significantly up regulated. Effects on fibroblasts were not observed, indicating cell specific activity of the polysaccharides. The results clearly indicate a rationale for the traditional use of Typha latifolia fruits extracts for wound healing to the strong stimulatory activity of the polysaccharides on keratinocytes proliferation and early differentiation, major activities necessary for potent wound-healing agents. Copyright © 2011

  4. Spreading, proliferation and differentiation of human dental pulp stem cells on chitosan scaffolds immobilized with RGD or fibronectin.

    Science.gov (United States)

    Asghari Sana, Farzin; Çapkın Yurtsever, Merve; Kaynak Bayrak, Gökçe; Tunçay, Ekin Özge; Kiremitçi, Arlin S; Gümüşderelioğlu, Menemşe

    2017-08-01

    Nowadays, human dental pulp stem cells (hDPSCs) became more attractive for therapeutic purposes because of their high proliferation and differentiation potential. Thus, coupling the desired cellular characteristics of hDPSCs with good biomaterial properties of the chitosan scaffolds provide an interesting approach for tissue engineering applications. On the other hand, scaffold surface modification is also needed to promote stem cell adhesion since chitosan lacks adhesion motifs to support direct cell anchorage. In this study, hDPSCs were isolated from third molars of healthy female individuals (aged 16-25) with enzymatic digestion. For cell culture studies, the chitosan scaffolds which have approximately 9 mm diameter and 2 mm thickness with interconnected structure were prepared by freeze-drying. To support cellular attachment the scaffolds were covalently immobilized with either RGD (arginine-glycine-aspartic acid) or fibronectin (Fn) molecules. Cells were seeded on chitosan scaffolds with or without immobilized RGD and fibronectin. Cell attachment, spreading, adhesion behaviors and proliferation capacity were examined by scanning electron microscopy, immunofluorescence staining and PrestoBlue ® assays, respectively. In addition, differentiation potential of hDPSCs on Fn immobilized chitosan scaffolds was determined with real time reverse transcriptase polymerase chain reaction analysis. The results showed that chitosan scaffolds were not able to support stem cell attachment. hDPSCs on chitosan scaffolds formed spheroids more quickly and the size of spheroids were smaller than on chitosan-RGD while Fn-immobilized chitosan scaffolds strongly supported cellular attachment but not odontogenic differentiation. The results suggest that the Fn-immobilized chitosan scaffolds may serve as good three-dimensional substrates for dental pulp stem cell attachment and proliferation. In the case of dental regeneration, they must be supported by appropriate biosignals to

  5. mTORC1 promotes proliferation of immature Schwann cells and myelin growth of differentiated Schwann cells

    Science.gov (United States)

    Milbrandt, Jeffrey

    2017-01-01

    The myelination of axons in peripheral nerves requires precisely coordinated proliferation and differentiation of Schwann cells (SCs). We found that the activity of the mechanistic target of rapamycin complex 1 (mTORC1), a key signaling hub for the regulation of cellular growth and proliferation, is progressively extinguished as SCs differentiate during nerve development. To study the effects of different levels of sustained mTORC1 hyperactivity in the SC lineage, we disrupted negative regulators of mTORC1, including TSC2 or TSC1, in developing SCs of mutant mice. Surprisingly, the phenotypes ranged from arrested myelination in nerve development to focal hypermyelination in adulthood, depending on the level and timing of mTORC1 hyperactivity. For example, mice lacking TSC2 in developing SCs displayed hyperproliferation of undifferentiated SCs incompatible with normal myelination. However, these defects and myelination could be rescued by pharmacological mTORC1 inhibition. The subsequent reconstitution of SC mTORC1 hyperactivity in adult animals resulted in focal hypermyelination. Together our data suggest a model in which high mTORC1 activity promotes proliferation of immature SCs and antagonizes SC differentiation during nerve development. Down-regulation of mTORC1 activity is required for terminal SC differentiation and subsequent initiation of myelination. In distinction to this developmental role, excessive SC mTORC1 activity stimulates myelin growth, even overgrowth, in adulthood. Thus, our work delineates two distinct functions of mTORC1 in the SC lineage essential for proper nerve development and myelination. Moreover, our studies show that SCs retain their plasticity to myelinate and remodel myelin via mTORC1 throughout life. PMID:28484008

  6. Differential diagnosis of benign epithelial proliferations and carcinomas of the breast using antibodies to cytokeratins.

    Science.gov (United States)

    Jarasch, E D; Nagle, R B; Kaufmann, M; Maurer, C; Böcker, W J

    1988-03-01

    The immunohistochemical reactivity on frozen sections of diverse benign and malignant epithelial proliferations of human breast tissue from 156 patients was examined using antibodies to different cytokeratins. Antibodies recognizing cytokeratins 18 and 19 reacted with luminal epithelial cells but not with myoepithelial cells of normal mammary gland, cystic disease, adenosis, papilloma, and fibroadenoma or with a subpopulation of proliferating cells in sclerosing adenosis and epitheliosis. These antibodies reacted with the tumor cells of all in situ and invasive carcinomas. KA1 antibody, which by one- and two-dimensional gel electrophoresis and immunoblotting was shown to bind preferentially to cytokeratin 14 in a complex with cytokeratin 5, reacted with the nonproliferating myoepithelium of normal gland, cystic disease, adenosis, papilloma, fibroadenoma, and in situ carcinoma; it also reacted with a subpopulation of proliferating cells in sclerosing adenosis and epitheliosis (papillomatosis) but was negative with the tumor cells of all preinvasive and most invasive carcinomas. In adenotic and epitheliotic proliferations, groups of cells were identified that reacted strongly with KA1 antibody in addition to antibodies to cytokeratins 18 and 19. The data are discussed with respect to epithelial cell heterogeneity in the breast. We show that by using such antibodies, benign epithelial proliferations are clearly distinguished from carcinomas.

  7. Role of PDGF-BB in proliferation, differentiation and maintaining stem cell properties of PDL cells in vitro.

    Science.gov (United States)

    Mihaylova, Zornitsa; Tsikandelova, Rozaliya; Sanimirov, Pavel; Gateva, Natalia; Mitev, Vanyo; Ishkitiev, Nikolay

    2018-01-01

    Platelet-derived growth factor-BB (PDGF-BB) is one of the most abundant growth factors in platelet derived products and has been shown to stimulate regeneration after tissue injury. There is a population of mesenchymal stem cells (MSC) in human periodontal ligament (PDL) which can contribute to tissue regeneration under appropriate conditions. PDL cells were isolated and characterized using stem cell and differentiation markers via immunofluorescence and flow cytometry and then cultured in vitro and treated with different concentrations of PDGF-BB. The effect of PDGF-BB on cell proliferation, stem cell and differentiation markers expression, soluble collagen production, lysyl oxidase (LOX) activity, alkaline phosphatase (ALP) activity and calcium nodules formation was assessed. PDGF-BB stimulated the proliferation of cells with the maximum effect at 50ng/mL. The growth factor increased the expression of stem cell markers and SPARC; Col1a2 expression was decreased, whereas the expression of Col3a1 remain unchanged. Soluble collagen production, ALP activity and calcium nodules formation were also significantly decreased by PDGF-BB; LOX activity was significantly increased. PDGF-BB is a powerful promoter of cell proliferation and increases the expression of stem cell markers; inhibites collagen production and mineraliration but accelerates the maturation of collagen chains through increased LOX activity and SPARC expression. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. The effect of perfusion culture on proliferation and differentiation of human mesenchymal stem cells on biocorrodible bone replacement material

    Energy Technology Data Exchange (ETDEWEB)

    Farack, J., E-mail: jana.farack@tu-dresden.de [Technische Universitaet Dresden, Max Bergmann Center of Biomaterials, Budapester Str. 27, D-01069 Dresden (Germany); Wolf-Brandstetter, C. [Technische Universitaet Dresden, Max Bergmann Center of Biomaterials, Budapester Str. 27, D-01069 Dresden (Germany); Glorius, S.; Nies, B. [InnoTERE GmbH, Tatzberg 47-49, D-01307 Dresden (Germany); Standke, G. [Fraunhofer Institute for Ceramic Technologies and Systems (IKTS), Winterbergstr. 28, D-01277 Dresden (Germany); Quadbeck, P. [Fraunhofer Institute for Manufacturing Technology and Advanced Materials (IFAM), Winterbergstr. 28, D-01277 Dresden (Germany); Worch, H.; Scharnweber, D. [Technische Universitaet Dresden, Max Bergmann Center of Biomaterials, Budapester Str. 27, D-01069 Dresden (Germany)

    2011-12-15

    Biocorrodible iron foams were coated with different calcium phosphate phases (CPP) to obtain a bioactive surface and controlled degradation. Further adhesion, proliferation and differentiation of SaOs-2 and human mesenchymal stem cells were investigated under both static and dynamic culture conditions. Hydroxyapatite (HA; [Ca{sub 10}(PO{sub 4}){sub 6}OH{sub 2}]) coated foams released 500 {mu}g/g iron per day for Dulbecco's modified eagle medium (DMEM) and 250 {mu}g/g iron per day for McCoys, the unmodified reference 1000 {mu}g/g iron per day for DMEM and 500 {mu}g/g iron per day for McCoys, while no corrosion could be detected on brushite (CaHPO{sub 4}) coated foams. Using a perfusion culture system with conditions closer to the in vivo situation, cells proliferated and differentiated on iron foams coated with either brushite or HA while in static cell culture cells could proliferate only on Fe-brushite. We conclude that the degradation behaviour of biocorrodible iron foams can be varied by different calcium phosphate coatings, offering opportunities for design of novel bone implants. Further studies will focus on the influence of different modifications of iron foams on the expression of oxidative stress enzymes. Additional information about in vivo reactions and remodelling behaviour are expected from testing in implantation studies.

  9. Interleukin 22 early affects keratinocyte differentiation, but not proliferation, in a three-dimensional model of normal human skin

    Energy Technology Data Exchange (ETDEWEB)

    Donetti, Elena, E-mail: elena.donetti@unimi.it [Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan (Italy); Cornaghi, Laura; Arnaboldi, Francesca; Landoni, Federica [Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan (Italy); Romagnoli, Paolo [Department of Experimental and Clinical Medicine, Università degli Studi di Firenze, 50125 Florence (Italy); Mastroianni, Nicolino [Department of Biomedical Sciences for Health, Università degli Studi di Milano, 20133 Milan (Italy); Pescitelli, Leonardo [Department of Surgery and Translational Medicine, Università degli Studi di Firenze, 50125 Florence (Italy); Baruffaldi Preis, Franz W. [I.R.C.C.S. Istituto Ortopedico Galeazzi, 20161 Milan (Italy); Prignano, Francesca [Department of Surgery and Translational Medicine, Università degli Studi di Firenze, 50125 Florence (Italy)

    2016-07-15

    Interleukin (IL)-22 is a pro-inflammatory cytokine driving the progression of the psoriatic lesion with other cytokines, as Tumor Necrosis Factor (TNF)-alpha and IL-17. Our study was aimed at evaluating the early effect of IL-22 alone or in combination with TNF-alpha and IL-17 by immunofluorescence on i) keratinocyte (KC) proliferation, ii) terminal differentiation biomarkers as keratin (K) 10 and 17 expression, iii) intercellular junctions. Transmission electron microscopy (TEM) analysis was performed. A model of human skin culture reproducing a psoriatic microenvironment was used. Plastic surgery explants were obtained from healthy young women (n=7) after informed consent. Fragments were divided before adding IL-22 or a combination of the three cytokines, and harvested 24 (T24), 48 (T48), and 72 (T72) h later. From T24, in IL-22 samples we detected a progressive decrease in K10 immunostaining in the spinous layer paralleled by K17 induction. By TEM, after IL-22 incubation, keratin aggregates were evident in the perinuclear area. Occludin immunostaining was not homogeneously distributed. Conversely, KC proliferation was not inhibited by IL-22 alone, but only by the combination of cytokines. Our results suggest that IL-22 affects keratinocyte terminal differentiation, whereas, in order to induce a proliferation impairment, a more complex psoriatic-like microenvironment is needed.

  10. Ethanol extract of Oenanthe javanica increases cell proliferation and neuroblast differentiation in the adolescent rat dentate gyrus

    Directory of Open Access Journals (Sweden)

    Bai Hui Chen

    2015-01-01

    Full Text Available Oenanthe javanica is an aquatic perennial herb that belongs to the Oenanthe genus in Apiaceae family, and it displays well-known medicinal properties such as protective effects against glutamate-induced neurotoxicity. However, few studies regarding effects of Oenanthe javanica on neurogenesis in the brain have been reported. In this study, we examined the effects of a normal diet and a diet containing ethanol extract of Oenanthe javanica on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus of adolescent rats using Ki-67 (an endogenous marker for cell proliferation and doublecortin (a marker for neuroblast. Our results showed that Oenanthe javanica extract significantly increased the number of Ki-67-immunoreactive cells and doublecortin-immunoreactive neuroblasts in the subgranular zone of the dentate gyrus in the adolescent rats. In addition, the immunoreactivity of brain-derived neurotrophic factor was significantly increased in the dentate gyrus of the Oenanthe javanica extract-treated group compared with the control group. However, we did not find that vascular endothelial growth factor expression was increased in the Oenanthe javanica extract-treated group compared with the control group. These results indicate that Oenanthe javanica extract improves cell proliferation and neuroblast differentiation by increasing brain-derived neurotrophic factor immunoreactivity in the rat dentate gyrus.

  11. The effect of enamel matrix proteins on the spreading, proliferation and differentiation of osteoblasts cultured on titanium surfaces.

    Science.gov (United States)

    Miron, Richard J; Oates, Christine J; Molenberg, Aart; Dard, Michel; Hamilton, Douglas W

    2010-01-01

    Modifications of implant surface topography and chemistry have proven a means to enhance osseointegration, a process that ensures the stability of bone-contacting devices, including titanium dental implants. The commercial product Emdogain is an enamel matrix derivative (EMD) extracted from porcine teeth commonly used in periodontal surgery, where it has been shown to potentiate regeneration of bone. The aim of the present study was to evaluate the effect of EMD on the attachment, proliferation and differentiation of osteoblasts on titanium surfaces in vitro. Pickled (smooth) and SLA (roughened) titanium discs were coated with EMD or left uncoated. Primary rat calvarial osteoblasts were cultured on each surface from 1h to 4 weeks. EMD significantly increased cell spreading and proliferation at time points ranging from 3 to 7 days on both topographies. Alkaline phosphatase activity was significantly increased on EMD-coated titanium compared with titanium alone. Moreover, there was a 6 fold increase in levels of mRNA encoding bone sialoprotein and osteocalcin in osteoblasts cultured on EMD-coated titanium surfaces compared with uncoated surfaces. We conclude that coating of titanium with EMD enhances the proliferation and differentiation of osteoblasts irrespective of the titanium substratum topography.

  12. 17β-Estradiol modulates cell proliferation of medullary cords during ovarian differentiation of the Lepidochelys olivacea sea turtle.

    Science.gov (United States)

    Díaz-Hernández, Verónica; Vázquez-Gómez, Alma; Marmolejo-Valencia, Alejandro; Montaño, Luis Manuel; Merchant-Larios, Horacio

    2017-11-15

    In turtles undergoing temperature sex determination (TSD), bipotential gonads express Sox9 in medullary cords at both female- (FPT) and male-producing temperatures (MPT). Subsequently, when the sex fate of medullary cords becomes dimorphic, at FPT, Sox9 is downregulated, whereas at MPT, its expression is maintained. Medullary cords in the ovary turn into ovarian lacuna, whereas in the testis they differentiate as seminiferous cords. When embryos of Lepidochelys olivacea sea turtle are incubated at MPT and treated with estradiol, Sox9 expression persists in the medullary cords in the form of tiny ovotestis-like formations. The perturbed development of the treated gonads is due to a significant decrease in the number of proliferating cells. This suggests that the disturbed effect caused by exogenous estradiol may be due to a conflict between the gene networks regulated by temperature and the increased level of endogenous estrogens, induced by the treatment. Here, we decided to use fadrozole and fulvestrant, an aromatase inhibitor and an estrogen-receptor antagonist, respectively, to provide insights into the role played by endogenous estrogens in regulating the cell proliferation of the two main gonadal compartments: the medullary cords and the cortex. Comparing cell proliferation patterns, our current results suggest that the endogenous estrogens are involved in determining the sex fate of medullary cords, by repressing proliferation. Interestingly, our results showed that endogenous estradiol levels are unnecessary for the thickening of the ovarian cortex. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. 3'UTR Shortening Potentiates MicroRNA-Based Repression of Pro-differentiation Genes in Proliferating Human Cells.

    Directory of Open Access Journals (Sweden)

    Yonit Hoffman

    2016-02-01

    Full Text Available Most mammalian genes often feature alternative polyadenylation (APA sites and hence diverse 3'UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3'UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3'UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3'UTR might become more active upon 3'UTR shortening. To address this conjecture we performed 3' sequencing to determine the 3' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3'UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes.

  14. Stem cell survival is severely compromised by the thymidineanalog EdU (5-ethynyl-2'-deoxyuridine), an alternative to BrdU for proliferation assays and stem cell tracing

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Skovrind, Ida; Christensen, Marlene Louise

    2013-01-01

    Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment...... and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2'-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent...... tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation...

  15. Morphology, proliferation, and osteogenic differentiation of mesenchymal stem cells cultured on titanium, tantalum, and chromium surfaces

    DEFF Research Database (Denmark)

    Stiehler, Maik; Lind, M.; Mygind, Tina

    2007-01-01

    the interactions between human mesenchymal stem cells (MSCs) and smooth surfaces of titanium (Ti), tantalum (Ta), and chromium (Cr). Mean cellular area was quantified using fluorescence microscopy (4 h). Cellular proliferation was assessed by (3)H-thymidine incorporation and methylene blue cell counting assays (4...... with the other surfaces tested. Cells cultured on Cr demonstrated reduced spreading and proliferation. In conclusion, Ta metal, as an alternative for Ti, can be considered as a promising biocompatible material, whereas further studies are needed to fully understand the role of Cr and its alloys in bone implants...

  16. APP regulates NGF receptor trafficking and NGF-mediated neuronal differentiation and survival.

    Directory of Open Access Journals (Sweden)

    Yun-wu Zhang

    Full Text Available β-Amyloid precursor protein (APP is a key factor in Alzheimer's disease (AD but its physiological function is largely undetermined. APP has been found to regulate retrograde transport of nerve growth factor (NGF, which plays a crucial role in mediating neuronal survival and differentiation. Herein, we reveal the mechanism underlying APP-mediated NGF trafficking, by demonstrating a direct interaction between APP and the two NGF receptors, TrkA and p75NTR. Downregulation of APP leads to reduced cell surface levels of TrkA/p75NTR and increased endocytosis of TrkA/p75NTR and NGF. In addition, APP-deficient cells manifest defects in neurite outgrowth and are more susceptible to Aβ-induced neuronal death at physiological levels of NGF. However, APP-deficient cells show better responses to NGF-stimulated differentiation and survival than control cells. This may be attributed to increased receptor endocytosis and enhanced activation of Akt and MAPK upon NGF stimulation in APP-deficient cells. Together, our results suggest that APP mediates endocytosis of NGF receptors through direct interaction, thereby regulating endocytosis of NGF and NGF-induced downstream signaling pathways for neuronal survival and differentiation.

  17. Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP.

    Science.gov (United States)

    Zhang, Dawei; Yang, Shanzheng; Toledo, Enrique M; Gyllborg, Daniel; Saltó, Carmen; Carlos Villaescusa, J; Arenas, Ernest

    2017-08-22

    Parkinson's disease (PD) is a neurodegenerative disorder in which the loss of dopaminergic neurons in the midbrain (mDA neurons) causes progressive loss of motor control and function. Using embryonic and mDA neurons, midbrain tissue from mice, and differentiated human neural stem cells, we investigated the mechanisms controlling the survival of mDA neurons. We found that the extracellular matrix protein laminin-511 (LM511) promoted the survival and differentiation of mDA neurons. LM511 bound to integrin α3β1 and activated the transcriptional cofactor YAP. LM511-YAP signaling enhanced cell survival by inducing the expression of the microRNA miR-130a, which suppressed the synthesis of the cell death-associated protein PTEN. In addition, LM511-YAP signaling increased the expression of transcription factors critical for mDA identity, such as LMX1A and PITX3, and prevented the loss of mDA neurons in response to oxidative stress, a finding that warrants further investigation to assess therapeutic potential for PD patients. We propose that by enhancing LM511-YAP signaling, it may be possible to prevent mDA neuron degeneration in PD or enhance the survival of mDA neurons in cell replacement therapies. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  18. Classic Ras Proteins Promote Proliferation and Survival Via Distinct Phosphoproteome Alterations in Neurofibromin-Null Malignant Peripheral Nerve Sheath Tumor Cells

    Science.gov (United States)

    Brossier, Nicole M.; Prechtl, Amanda M.; Longo, Jody Fromm; Barnes, Stephen; Wilson, Landon S.; Byer, Stephanie J.; Brosius, Stephanie N.; Carroll, Steven L.

    2015-01-01

    Neurofibromin, the tumor suppressor encoded by the neurofibromatosis type 1 (NF1) gene, potentially suppresses the activation of H-Ras, N-Ras and K-Ras. However, it is not known whether these classic Ras proteins are hyperactivated in NF1-null nerve sheath tumors, how they contribute to tumorigenesis and what signaling pathways mediate their effects. Here we show that H-Ras, N-Ras and K-Ras are coexpressed with their activators, (guanine nucleotide exchange factors), in neurofibromin-null malignant peripheral nerve sheath tumor (MPNST) cells and that all 3 Ras proteins are activated. Dominant negative (DN) H-Ras, a pan-inhibitor of the classic Ras family, inhibited MPNST proliferation and survival, but not migration. However, NF1-null MPNST cells were variably dependent on individual Ras proteins. In some lines, ablation of H-Ras, N-Ras and/or K-Ras inhibited mitogenesis. In others, ablation of a single Ras protein had no effect on proliferation; in these lines, ablation of a single Ras protein resulted in compensatory increases in the activation and/or expression of other Ras proteins. Using mass spectrometry-based phosphoproteomics, we identified 7 signaling networks affecting morphology, proliferation and survival that are regulated by DN H-Ras. Thus, neurofibromin loss activates multiple classic Ras proteins that promote proliferation and survival by regulating several distinct signaling cascades. PMID:25946318

  19. The effect of electrospun poly(lactic acid and nanohydroxyapatite nanofibers’ diameter on proliferation and differentiation of mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Amir Doustgani

    2016-10-01

    Full Text Available Objective(s: Electrospun nanofibrous mats of poly(lactic acid (PLA and nanohydroxyapatite (nano-HA were prepared and proliferation and differentiation of mesenchymal stem cells on the prepared nanofibers were investigated in this study. Materials and Methods: PLA/nano-HA nanofibers were prepared by electrospinning. The effects of process parameters, such as nano-HA concentration, distance, applied voltage, and flow rate on the mean diameter of electrospun nanofibers were investigated. Scanning electron microscopy (SEM was used to determine the mean fiber diameter of produced nanofibers. Mechanical propertes of nanofibrous mats were evaluated using a universal testing machine. Response surface methodology was used to model the fiber diameter of electrospun PLA/nano-HA nanofibers. Results: The average fiber diameter for optimized nanofibers was 125 ± 11 nm. MTT and ALP results showed that optimization of fiber diameter increased the osteogenic differentiation of stem cells. Conclusion: It could be concluded that optimization of fiber diameter has beneficial effect on cell proliferation and differentiation. Optimized nanofibers of PLA/nano-HA could be good candidates for bone tissue engineering.

  20. Epigenetic regulation of miR-184 by MBD1 governs neural stem cell proliferation and differentiation.

    Science.gov (United States)

    Liu, Changmei; Teng, Zhao-Qian; Santistevan, Nicholas J; Szulwach, Keith E; Guo, Weixiang; Jin, Peng; Zhao, Xinyu

    2010-05-07

    Methyl-CpG binding protein 1 (MBD1) regulates gene expression via a DNA methylation-mediated epigenetic mechanism. We have previously demonstrated that MBD1 deficiency impairs adult neural stem/progenitor cell (aNSC) differentiation and neurogenesis, but the underlying mechanism was unclear. Here, we show that MBD1 regulates the expression of several microRNAs in aNSCs and, specifically, that miR-184 is directly repressed by MBD1. High levels of miR-184 promoted proliferation but inhibited differentiation of aNSCs, whereas inhibition of miR-184 rescued the phenotypes associated with MBD1 deficiency. We further found that miR-184 regulates the expression of Numblike (Numbl), a known regulator of brain development, by binding to the 3'-UTR of Numbl mRNA and affecting its translation. Expression of exogenous Numbl could rescue the aNSC defects that result from either miR-184 overexpression or MBD1 deficiency. Therefore, MBD1, miR-184, and Numbl form a regulatory network that helps control the balance between proliferation and differentiation of aNSCs.

  1. Involvement of the D-type cyclins in germ cell proliferation and differentiation in the mouse

    NARCIS (Netherlands)

    Beumer, T. L.; Roepers-Gajadien, H. L.; Gademan, I. S.; Kal, H. B.; de rooij, D. G.

    2000-01-01

    Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D(1) is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D(1) in

  2. Cooperative effects in differentiation and proliferation between PDGF-BB and matrix derived synthetic peptides in human osteoblasts

    Directory of Open Access Journals (Sweden)

    Vordemvenne Thomas

    2011-11-01

    Full Text Available Abstract Background Enhancing osteogenic capabilities of bone matrix for the treatment of fractures and segmental defects using growth factors is an active area of research. Recently, synthetic peptides like AC- 100, TP508 or p-15 corresponding to biologically active sequences of matrix proteins have been proven to stimulate bone formation. The platelet-derived growth factor (PDGF BB has been identified as an important paracrine factor in early bone healing. We hypothesized that the combined use of PDGF-BB with synthetic peptides could result in an increase in proliferation and calcification of osteoblast-like cells. Methods Osteoblast-like cell cultures were treated with PDGF and synthetic peptides, singly and as combinations, and compared to non-treated control cell cultures. The cultures were evaluated at days 2, 5, and 10 in terms of cell proliferation, calcification and gene expression of alkaline phosphate, collagen I and osteocalcin. Results Experimental findings revealed that the addition of PDGF, p-15 and TP508 and combinations of PDGF/AC-100, PDGF/p-15 and PDGF/TP508 resulted in an increase in proliferating osteoblasts, especially in the first 5 days of cultivation. Proliferation did not significantly differ between single factors and factor combinations (p > 0.05. The onset of calcification in osteoblasts occurred earlier and was more distinct compared to the corresponding control or PDGF stimulation alone. Significant difference was found for the combined use of PDGF/p-15 and PDGF/AC-100 (p Conclusions Our findings indicate that PDGF exhibits cooperative effects with synthetic peptides in differentiation and proliferation. These cooperative effects cause a significant early calcification of osteoblast-like cells (p

  3. Effects of TGFβ1, PDGF-BB, and bFGF, on human corneal fibroblasts proliferation and differentiation during stromal repair.

    Science.gov (United States)

    Gallego-Muñoz, Patricia; Ibares-Frías, Lucía; Valsero-Blanco, María Cruz; Cantalapiedra-Rodriguez, Roberto; Merayo-Lloves, Jesús; Martínez-García, M Carmen

    2017-08-01

    In an effort to improve the regenerative nature of corneal repair, this study reports the use of an in vitro human corneal fibroblasts (HCFs) wound model after treatment with three of the main growth factors (GFs) involved in corneal healing: transforming growth factor beta 1 (TGFβ1), platelet-derived growth factor BB-isoform (PDGF-BB), and basic fibroblast growth factor (bFGF) in order to delve in cell proliferation and differentiation processes. HCFs were mechanically wounded. The individual effect of TGFβ1, PDGF-BB, and bFGF on cell proliferation and differentiation during the repair process was studied at different time points until wound closure. Wound dimensions and morphological changes were evaluated by microscopy. Cell proliferation and myofibroblast differentiation were analyzed by immunofluorescence cytochemistry. Changes in cell morphology were apparent at Day 4. PDGF-BB- and bFGF-treated cells had fibroblast-like morphology. TGFβ1 stimulated proliferation in the wound edge and surrounding area, induced myofibroblast differentiation and inhibited cellular migration. PDGF-BB induced rapid wound closure due to proliferation, high motility, and late myofibroblast differentiation. The time course of closure induced by bFGF was similar to that for PDGF-BB, but was mostly due to proliferation in the wound area, and inhibited myofibroblast differentiation. Each of the GFs induced increases in responses promoting stromal repair differently. This study provides insight regarding how to optimize the outcome of stromal repair following corneal injury. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. [PDGFRα Participates in Basic Fibroblast Growth Factor-mediated Recovery of Human Bone Marrow Mesenchymal Stem Cell Proliferation and Osteogenic Differentiation after Irradiation].

    Science.gov (United States)

    Dai, Kai; Yang, Zhi; Xu, Shuang-Nian; Zhang, Jian-Min; Chen, Jie-Ping

    2015-12-01

    To explore the effects of basic fibroblast growth factor (bFGF) on human bone marrow mesenchymal stem cell (hBMMSC) damaged by irradiation and its underlying mechanisms. hBMMSC was irradiated with 0, 6, 12 Gy X ray, then flow cytometry, cell counting kit-8 (CCK-8), Western blot and alizarin red staining were used to detect the effects of X ray on apoptosis, proliferation and osteogenic differentiation of hBMMSC; 0, 1, 5, 10, 20 ng/ml bFGF was added to hBMMSC irradiated with X ray for selecting the suitable bFGF reaction concentration; then the Western blot was used to detect the expression of PDGFRα so as to evaluate whether the expression of PDGFRα participated in bFGF-mediated recovery of hBMMSC proliferation and osteogenic differentiation after irradiation. The proliferation and osteogenic differentiation of hBMMSC decreased remarkably after irradiation. bFGF promoted the recovery of proliferation and osteogenic differentiation of irradiated hBMMSC compared with untreated irradiated hBMMSC (P recovery of hBMMSC proliferation and osteogenic differentiation. The damage of hBMMSC proliferation and osteogenic differentiation associates with downregulation of PDGFRα expression induced by irrediation. PDGFRα involves in repairing effect of bFGF on irradiation damage of hBMMSC.

  5. Pathological and immunological responses associated with differential survival of Chinook salmon following Renibacterium salmoninarum challenge

    Science.gov (United States)

    Metzger, David C.; Elliott, Diane G.; Wargo, Andrew; Park, Linda K.; Purcell, Maureen K.

    2010-01-01

    Chinook salmon Oncorhynchus tshawytscha are highly susceptible to Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). Previously we demonstrated that introduced Chinook salmon from Lake Michigan, Wisconsin (WI), USA, have higher survival following R. salmoninarum challenge relative to the progenitor stock from Green River, Washington, USA. In the present study, we investigated the pathological and immunological responses that are associated with differential survival in the 2 Chinook salmon stocks following intra-peritoneal R. salmoninarum challenge of 2 different cohort years (2003 and 2005). Histological evaluation revealed delayed appearance of severe granulomatous lesions in the kidney and lower overall prevalence of membranous glomerulopathy in the higher surviving WI stock. The higher survival WI stock had a lower bacterial load at 28 d post-infection, as measured by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). However, at all other time points, bacterial load levels were similar despite higher mortality in the more susceptible Green River stock, suggesting the possibility that the stocks may differ in their tolerance to infection by the bacterium. Interferon-γ, inducible nitric oxide synthase (iNOS), Mx-1, and transferrin gene expression were up-regulated in both stocks following challenge. A trend of higher iNOS gene expression at later time points (≥28 d post-infection) was observed in the lower surviving Green River stock, suggesting the possibility that higher iNOS expression may contribute to greater pathology in that stock.

  6. Inhibition of miR-29c promotes proliferation, and inhibits apoptosis and differentiation in P19 embryonic carcinoma cells

    OpenAIRE

    CHEN, BIN; SONG, GUIXIAN; LIU, MING; QIAN, LINGMEI; WANG, LIHUA; GU, HAITAO; SHEN, YAHUI

    2016-01-01

    In our previous study, the upregulation of microRNA (miR)-29c was identified in the mother of a fetus with a congenital heart defect. However, the functional and regulatory mechanisms of miR-29c in the development of the heart remain to be elucidated. In the present study, the role and mechanism of miR-29c inhibition in heart development were investigated in an embryonic carcinoma cell model. Inhibition of miR-29c promoted proliferation, and suppressed the apoptosis and differentiation of P19...

  7. Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy.

    Science.gov (United States)

    Amini, Afshin; Masoumi-Moghaddam, Samar; Ehteda, Anahid; Morris, David Lawson

    2014-11-12

    Bromelain and N-acetylcysteine are two natural, sulfhydryl-containing compounds with good safety profiles which have been investigated for their benefits and application in health and disease for more than fifty years. As such, the potential values of these agents in cancer therapy have been variably reported in the literature. In the present study, the efficacy of bromelain and N-acetylcysteine in single agent and combination treatment of human gastrointestinal carcinoma cells was evaluated in vitro and the underlying mechanisms of effect were explored. The growth-inhibitory effects of bromelain and N-acetylcysteine, on their own and in combination, on a panel of human gastrointestinal carcinoma cell lines, including MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T, were assessed by sulforhodamine B assay. Moreover, the influence of the treatment on the expression of a range of proteins involved in the regulation of cell cycle and survival was investigated by Western blot. The presence of apoptosis was also examined by TUNEL assay. Bromelain and N-acetylcysteine significantly inhibited cell proliferation, more potently in combination therapy. Drug-drug interaction in combination therapy was found to be predominantly synergistic or additive. Mechanistically, apoptotic bodies were detected in treated cells by TUNEL assay. Furthermore, Western blot analysis revealed diminution of cyclins A, B and D, the emergence of immunoreactive subunits of caspase-3, caspase-7, caspase-8 and cleaved PARP, withering or cleavage of procaspase-9, overexpression of cytochrome c, reduced expression of anti-apoptotic Bcl-2 and pro-survival phospho-Akt, the emergence of the autophagosomal marker LC3-II and deregulation of other autophagy-related proteins, including Atg3, Atg5, Atg7, Atg12 and Beclin 1. These results were more prominent in combination therapy. We report for the first time to our knowledge the growth-inhibitory and cytotoxic effects of bromelain and N-acetylcysteine, in

  8. Ciliary neurotrophic factor: a survival and differentiation inducer in human retinal progenitors.

    Science.gov (United States)

    Dutt, Kamla; Cao, Yang; Ezeonu, Ifeoma

    2010-07-01

    Retinitis pigmentosa, age-related macular degeneration, and Parkinson's disease remain major problems in the field of medicine. Some of the strategies being explored for treatment include replacement of damaged tissue by transplantation of healthy tissues or progenitor cells and delivery of neurotrophins to rescue degenerating tissue. One of the neurotrophins with promise is the ciliary neurotrophic factor (CNTF). In this study, we report the role played by CNTF in retinal cell differentiation and survival in retinal progenitors. We found that CNTF is a survival factor for multipotential human retinal cells and increased cell survival by 50%, over a 7-d period, under serum-free conditions, as determined by apoptotic assays (immunohistochemistry and flow cytometry). This effect is dose dependent with a maximum survival at a CNTF concentration of 20 ng/ml. We also report that CNTF might be a cell commitment factor, directing the differentiation mainly toward large multipolar cells with ganglionic and amacrine phenotype. These cells express tyrosine hydroxylase (amacrine cells) as well as, thy 1.1 and neuron-specific enolase (ganglionic cells). Additionally, there was also an increase in protein kinase C alpha, a protein expressed in rod and cone bipolars as well as cone photoreceptors and calbindin, a protein expressed in cone photoreceptors and horizontal cells. In our studies, CNTF doubled the number of cells with ganglionic phenotypes, and basic fibroblast growth factor doubled the number of cells with photoreceptor phenotype. Additionally, CNTF induced a subset of progenitors to undergo multiple rounds of cell division before acquiring the large multipolar ganglionic phenotype. Our conclusion is that CNTF could be an agent that has therapeutic potential and possibly induces differentiation of large multipolar ganglionic phenotype in a subset of progenitors.

  9. Effects of novel hydroxyapatite-based 3D biomaterials on proliferation and osteoblastic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Karadzic, Ivana; Vucic, Vesna; Jokanovic, Vukoman; Debeljak-Martacic, Jasmina; Markovic, Dejan; Petrovic, Snjezana; Glibetic, Marija

    2015-01-01

    The aim of this study was to examine the differential capacity of isolated dental pulp stem cells (SHED) cultured onto four different scaffold materials. The differential potential of isolated SHED was examined on the following scaffolds: porous hydroxyapatite (pHAP) alone or combined with three polymers [polylactic-co-glycolic acid (PLGA), alginate, and ethylene vinylacetate / ethylene vinylversatate (EVA/EVV)]. SHED were isolated by "outgrowth" method and characterized by the flow cytometry. Viability of cells grown with scaffolds was assessed by MTT and LDH assays. No significant cytotoxic effect of any of the tested materials was shown. Staining with alizarin red and estimated alkaline phosphatase activity to identify differentiation, demonstrated osteoblastic phenotype of SHED and newly deposited and mineralized extra cellular matrix (ECM) in presence of all tested scaffolds. The developed ECM seen at scanning electronic micrographs additionally confirmed the osteogenic differentiation and biocompatibility between cells and materials. In summary, all studied biomaterials are suitable carriers for proliferation and osteoblastic differentiation of dental pulp mesenchymal stem cells in vitro. © 2014 Wiley Periodicals, Inc.

  10. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis

    DEFF Research Database (Denmark)

    Müller, H; Bracken, A P; Vernell, R

    2001-01-01

    The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. We have used high-density oligonucleotide arrays to identify genes in which expression changed in response to activation...... to the variety of phenotypes observed as a consequence of a deregulated pRB/E2F pathway....

  11. The Use Of Laser Irradiation To Stimulate Adipose Derived Stem Cell Proliferation And Differentiation For Use In Autologous Grafts

    Science.gov (United States)

    Abrahamse, Heidi

    2009-09-01

    Stem cells are characterized by the qualities of self-renewal, long term viability, and the ability to differentiate into various cell types. Historically, stem cells have been isolated from the inner cell mass of blastocysts and harvesting these cells resulted in the death of the embryo leading to religious, political and ethical issues. The identification and subsequent isolation of adult stem cells from bone marrow stroma have been welcomed as an alternate source for stem cells. The clinical use of Mesenchymal Stem Cells (MSCs) presented problems such as limited cell number, pain and morbidity upon isolation. Adipose tissue is derived from the mesenchyme, is easily isolated, a reliable source of stem cells and able to differentiate into different cell types including smooth muscle. Over the past few years, the identification and characterization of stem cells has led the potential use of these cells as a promising alternative to cell replacement therapy. Smooth muscle is a major component of human tissues and is essential for the normal functioning of many different organs. Low intensity laser irradiation has been shown to increase viability, protein expression and migration of stem cells in vitro, and to stimulate proliferation of various types of stem cells. In addition, the use of laser irradiation to stimulate differentiation in the absence of growth factors has also been demonstrated in normal human neural progenitor cells (NHNPCs) in vitro where NHNPCs are not only capable of being sustained by light in the absence of growth factors, but that they are also able to differentiate normally as assessed by neurite formation. Our work has focused on the ability of laser irradiation to proliferate adipose derived stem cells (ADSCs), maintain ADSC character and increase the rate and maintenance of differentiation of ADSCs into smooth muscle and skin fibroblast cells. Current studies are also investigating the effect of different irradiation wavelengths and

  12. Gender differentials and old age survival in the Nairobi slums, Kenya.

    Science.gov (United States)

    Bennett, Rachel; Chepngeno-Langat, Gloria; Evandrou, Maria; Falkingham, Jane

    2016-08-01

    This paper examines gender differentials in survival amongst older people (50+ years) in the Nairobi slums and to the best of our knowledge is the first study of its kind in an urban African setting. The results provide evidence contrary to the expected paradox of poorer self-rated health yet better survival amongst older women. Older women in the Nairobi slums have poorer self-rated health and poorer circumstances across other factors, including disability and socio-economic status. Further, older women in the slums do not have better survival. The conventional female advantage in mortality only becomes apparent after accounting for the cumulative influence of individual characteristics, social networks, health and socio-economic status, suggesting the female advantage in unadjusted old-age mortality does not apply to contexts where women experience significant disadvantage across multiple life domains. This highlights the urgent need to redress the support, status and opportunities available for women across the life course in contexts such as the Nairobi slums. In addition, a greater number of factors differentiate mortality risk amongst men than amongst women, suggesting inequality amongst slum dwelling older men and highlighting the need for gender sensitive interventions which account for the particular needs of both genders in old age. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Xi [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Zhang, Kunshan [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Wang, Yanlu; Wang, Junbang; Cui, Yaru [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China); Li, Siguang, E-mail: siguangli@163.com [Department of Regenerative Medicine, Stem Cell Center, Tongji University School of Medicine, Shanghai 200092 (China); Luo, Yuping, E-mail: luoyuping@163.com [State Key Laboratory of Food Science and Technology, College of Life Sciences and Food Engineering, Nanchang University, Nanchang 330047 (China)

    2013-10-04

    Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fate specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome.

  14. Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures.

    Science.gov (United States)

    Kolind, K; Kraft, D; Bøggild, T; Duch, M; Lovmand, J; Pedersen, F S; Bindslev, D A; Bünger, C E; Foss, M; Besenbacher, F

    2014-02-01

    The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell's capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 μm for surfaces with small pillar sizes of 1 and 2 μm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 μm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  15. Rapamycin attenuates BAFF-extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B-lymphoid cells.

    Science.gov (United States)

    Zeng, Qingyu; Qin, Shanshan; Zhang, Hai; Liu, Beibei; Qin, Jiamin; Wang, Xiaoxue; Zhang, Ruijie; Liu, Chunxiao; Dong, Xiaoqing; Zhang, Shuangquan; Huang, Shile; Chen, Long

    2018-01-01

    B cell activating factor from the TNF family (BAFF) stimulates B-cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)-stimulated B-cell proliferation/survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF-promoted B cell proliferation/survival is also related to blocking hsBAFF-stimulated phosphorylation of Akt, S6K1, and 4E-BP1, as well as expression of survivin in normal and B-lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF-induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr-Akt) or constitutively active S6K1 (S6K1-ca), or downregulation of 4E-BP1 conferred resistance to rapamycin's attenuation of hsBAFF-induced survivin expression and B-cell proliferation/viability, whereas overexpression of dominant negative Akt (dn-Akt) or constitutively hypophosphorylated 4E-BP1 (4EBP1-5A), or downregulation of S6K1, or co-treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF-induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B-lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF-evoked aggressive B-cell malignancies and autoimmune diseases. © 2017 Wiley Periodicals, Inc.

  16. Neuroprotective Effect of Uncaria rhynchophylla in Kainic Acid-Induced Epileptic Seizures by Modulating Hippocampal Mossy Fiber Sprouting, Neuron Survival, Astrocyte Proliferation, and S100B Expression

    Directory of Open Access Journals (Sweden)

    Chung-Hsiang Liu

    2012-01-01

    Full Text Available Uncaria rhynchophylla (UR, which is a traditional Chinese medicine, has anticonvulsive effect in our previous studies, and the cellular mechanisms behind this are still little known. Because of this, we wanted to determine the importance of the role of UR on kainic acid- (KA- induced epilepsy. Oral UR for 6 weeks can successfully attenuate the onset of epileptic seizure in animal tests. Hippocampal mossy fiber sprouting dramatically decreased, while neuronal survival increased with UR treatment in hippocampal CA1 and CA3 areas. Furthermore, oral UR for 6 weeks significantly attenuated the overexpression of astrocyte proliferation and S100B proteins but not γ-aminobutyric acid A (GABAA receptors. These results indicate that oral UR for 6 weeks can successfully attenuate mossy fiber sprouting, astrocyte proliferation, and S100B protein overexpression and increase neuronal survival in KA-induced epileptic rat hippocampus

  17. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Ishizuka, Toshiaki, E-mail: tishizu@ndmc.ac.jp [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan); Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the DNA synthesis via induction of superoxide. Black-Right-Pointing-Pointer Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. Black-Right-Pointing-Pointer Angiotensin II enhanced differentiation into mesodermal progenitor cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT{sub 1}R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression

  18. C/EBPbeta Promotes transition from proliferation to hypertrophic differentiation of chondrocytes through transactivation of p57.

    Directory of Open Access Journals (Sweden)

    Makoto Hirata

    Full Text Available BACKGROUND: Although transition from proliferation to hypertrophic differentiation of chondrocytes is a crucial step for endochondral ossification in physiological skeletal growth and pathological disorders like osteoarthritis, the underlying mechanism remains an enigma. This study investigated the role of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta in chondrocytes during endochondral ossification. METHODOLOGY/PRINCIPAL FINDINGS: Mouse embryos with homozygous deficiency in C/EBPbeta (C/EBPbeta-/- exhibited dwarfism with elongated proliferative zone and delayed chondrocyte hypertrophy in the growth plate cartilage. In the cultures of primary C/EBPbeta-/- chondrocytes, cell proliferation was enhanced while hypertrophic differentiation was suppressed. Contrarily, retroviral overexpression of C/EBPbeta in chondrocytes suppressed the proliferation and enhanced the hypertrophy, suggesting the cell cycle arrest by C/EBPbeta. In fact, a DNA cell cycle histogram revealed that the C/EBPbeta overexpression caused accumulation of cells in the G0/G1 fraction. Among cell cycle factors, microarray and real-time RT-PCR analyses have identified the cyclin-dependent kinase inhibitor p57(Kip2 as the transcriptional target of C/EBPbeta. p57(Kip2 was co-localized with C/EBPbeta in late proliferative and pre-hypertrophic chondrocytes of the mouse growth plate, which was decreased by the C/EBPbeta deficiency. Luciferase-reporter and electrophoretic mobility shift assays identified the core responsive element of C/EBPbeta in the p57(Kip2 promoter between -150 and -130 bp region containing a putative C/EBP motif. The knockdown of p57(Kip2 by the siRNA inhibited the C/EBPbeta-induced chondrocyte hypertrophy. Finally, when we created the experimental osteoarthritis model by inducing instability in the knee joints of adult mice of wild-type and C/EBPbeta+/- littermates, the C/EBPbeta insufficiency caused resistance to joint cartilage destruction

  19. Effects of low-intensity electromagnetic fields on the proliferation and differentiation of cultured mouse bone marrow stromal cells.

    Science.gov (United States)

    Zhong, Cheng; Zhang, Xin; Xu, Zhengjian; He, Rongxin

    2012-09-01

    Electromagnetic fields (EMFs) used in stem-cell tissue engineering can help elucidate their biological principles. The aim of this study was to investigate the effects of low-intensity EMFs on cell proliferation, differentiation, and cycle in mouse bone marrow stromal cells (BMSCs) and the in vivo effects of EMFs on BMSC. Harvested BMSCs were cultured for 3 generations and divided into 4 groups. The methylthiotetrazole (MTT) assay was used to evaluate cell proliferation, and alkaline phosphatase activity was measured via a colorimetric assay on the 3rd, 7th, and 10th days. Changes in cell cycle also were analyzed on the 7th day, and bone nodule formation was analyzed on the 12th day. Additionally, the expression of the collagen I gene was examined by reverse transcription-polymerase chain reaction (RT-PCR) on the 10th day. The BMSCs of the irradiated group and the control group were transplanted into cortical bone of different mice femurs separately, with poly(lactic-co-glycolic acid) (PLGA) serving as a scaffold. After 4 and 8 weeks, bone the bone specimens of mice were sliced and stained by hematoxylin and eosin separately. The results showed that EMFs (0.5 mT, 50 Hz) accelerated cellular proliferation, enhanced cellular differentiation, and increased the percentage of cells in the G(2)/M+S (postsynthetic gap 2 period/mitotic phase + S phase) of the stimulation. The EMF-exposed groups had significantly higher collagen I messenger RNA levels than the control group. The EMF + osteogenic medium-treated group readily formed bone nodules. Hematoxylin and eosin staining showed a clear flaking of bone tissue in the irradiated group. Irradiation of BMSCs with low-intensity EMFs (0.5 mT, 50 Hz) increased cell proliferation and induced cell differentiation. The results of this study did not establish a stricter animal model for studying osteogenesis, and only short-term results were investigated. Further study of the mechanism of EMF is needed.

  20. Effect of nicotine on the proliferation and chondrogenic differentiation of the human Wharton's jelly mesenchymal stem cells.

    Science.gov (United States)

    Yang, Xu; Qi, Yongjian; Avercenc-Leger, Léonore; Vincourt, Jean-Baptiste; Hupont, Sébastien; Huselstein, Céline; Wang, Hui; Chen, Liaobin; Magdalou, Jacques

    2017-01-01

    Osteoarthritis (OA) is a chronic joint disease characterized by a progressive and irreversible degeneration of articular cartilage. Among the environmental risk factors of OA, tobacco consumption features prominently, although, there is a great controversy regarding the role of tobacco smoking in OA development. Among the numerous chemicals present in cigarette smoke, nicotine is one of the most physiologically active molecules. The aim of the study was (i) to measure the impact of nicotine on the proliferation and chondrogenic differentiation of mesenchymal stem cells from the human Wharton's jelly (hWJ-MSCs) into chondrocytes, (ii) to investigate whether the α7 nicotinic acetylcholine receptors (nAChRs) was expressed in hWJ-MSCs and could play a role in the process. The project benefits from the availability of an umbilical cord bank from which hWJ-MSCs were originated. The hWJ-MSCs were cultured and used up to passage 5. The proliferation of hWJ-MSCs with 5 μM nicotine was measured by the MTT assay on the 1st, 2nd, 3rd, and 6th day. Flow cytometry analysis was used to detect cell apoptosis/necrosis by Annexin V/PI double-staining. The chondrogenic differentiation grade of hWJ-MSCs induced by TGFβ3 was assessed by the Sirius red and Alcian blue staining. The expression of markers genes was followed by quantitative real-time PCR. The expression of nAChRs was followed by RT-PCR. The functional activity of α7 nAChR was evaluated by calcium (Ca2+) influx mediated by nicotine using the Fluo-4 NW Calcium assay. The proliferation of hWJ-MSCs was significantly impaired by nicotine (5 μM) from the 3rd day of treatment, but nicotine did not significantly induce modifications on the viability of hWJ-MSCs. Alcian blue staining indicated that the amount of proteoglycan was more abundant in control group than in the nicotine group, but no difference was observed on the total collagen amount using Sirius red staining. The mRNA expression of Sox9, type II collagen (Col2a1

  1. Two Ellagic Acids Isolated from Roots of Sanguisorba officinalis L. Promote Hematopoietic Progenitor Cell Proliferation and Megakaryocyte Differentiation

    Directory of Open Access Journals (Sweden)

    Xiaoping Gao

    2014-04-01

    Full Text Available Using a bioassay-directed chromatographic separation, two ellagic acids were obtained from the ethyl acetate extract of the roots of Sanguisorba officinalis L. On the basis of chemical and spectroscopic methods, the two ellagic acids were identified as 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside and 3,3',4-tri-O-methylellagic acid. Stimulation of cell proliferation was assayed in hematopoietic progenitor cells using the Cell Counting kit-8 method. The megakaryocyte differentiation was determined in human erythroleukemia (HEL cells using Giemsa staining and flow cytometry analysis. The ellagic acids significantly stimulated the proliferation of Baf3/Mpl cells. Morphology analysis and megakaryocyte specific-marker CD41 staining confirmed that the ellagic acids induced megakaryocyte differentiation in HEL cells. This is the first time that 3,3',4-tri-O-methylellagic acid or 3,3',4-tri-O-methylellagic acid-4'-O-β-d-xyloside are reported to induce megakaryopoiesis, suggesting a class of small molecules which differ from others non-peptidyl, and appears to have potential for clinical development as a therapeutic agent for patients with blood platelet disorders.

  2. Neural stem cell proliferation and differentiation in the conductive PEDOT-HA/Cs/Gel scaffold for neural tissue engineering.

    Science.gov (United States)

    Wang, Shuping; Guan, Shui; Xu, Jianqiang; Li, Wenfang; Ge, Dan; Sun, Changkai; Liu, Tianqing; Ma, Xuehu

    2017-09-26

    Engineering scaffolds with excellent electro-activity is increasingly important in tissue engineering and regenerative medicine. Herein, conductive poly(3,4-ethylenedioxythiophene) doped with hyaluronic acid (PEDOT-HA) nanoparticles were firstly synthesized via chemical oxidant polymerization. A three-dimensional (3D) PEDOT-HA/Cs/Gel scaffold was then developed by introducing PEDOT-HA nanoparticles into a chitosan/gelatin (Cs/Gel) matrix. HA, as a bridge, not only was used as a dopant, but also combined PEDOT into the Cs/Gel via chemical crosslinking. The PEDOT-HA/Cs/Gel scaffold was used as a conductive substrate for neural stem cell (NSC) culture in vitro. The results demonstrated that the PEDOT-HA/Cs/Gel scaffold had excellent biocompatibility for NSC proliferation and differentiation. 3D confocal fluorescence images showed cells attached on the channel surface of Cs/Gel and PEDOT-HA/Cs/Gel scaffolds with a normal neuronal morphology. Compared to the Cs/Gel scaffold, the PEDOT-HA/Cs/Gel scaffold not only promoted NSC proliferation with up-regulated expression of Ki67, but also enhanced NSC differentiation into neurons and astrocytes with up-regulated expression of β tubulin-III and GFAP, respectively. It is expected that this electro-active and bio-active PEDOT-HA/Cs/Gel scaffold will be used as a conductive platform to regulate NSC behavior for neural tissue engineering.

  3. NRG1/ERBB3 signaling in melanocyte development and melanoma: inhibition of differentiation and promotion of proliferation

    Science.gov (United States)

    Buac, Kristina; Xu, Mai; Cronin, Julie; Weeraratna, Ashani T.; Hewitt, Stephen M.; Pavan, William J.

    2010-01-01

    Summary Neuregulin (NRG) signaling through the receptor tyrosine kinase, ERBB3, is required for embryonic development, and dysregulated signaling has been associated with cancer progression. Here we show that NRG1/ERBB3 signaling inhibits melanocyte (MC) maturation and promotes undifferentiated, migratory and proliferative cellular characteristics. Embryonic analyses demonstrated that initial MC specification and distribution were not dependent on ERBB3 signaling. However NRG1/ERBB3 signaling was both necessary and sufficient to inhibit differentiation of later stages of MC development in culture. Analysis of tissue arrays of human melanoma samples suggests that ERBB3 signaling may also contribute to metastatic progression of melanoma as ERBB3 was phosphorylated in primary tumors compared to nevi or metastatic lesions. NRG1-treated MCs demonstrated increased proliferation and invasion and altered morphology concomitant with decreased levels of differentiation genes, increased levels of proliferation genes and altered levels of melanoma progression and metastases genes. ERBB3 activation in primary melanomas suggests that NRG1/ERBB3 signaling may contribute to the progression of melanoma from benign nevi to malignancies. We propose that targeting ERBB3 activation and downstream genes identified in this study may provide novel therapeutic interventions for malignant melanoma. PMID:19659570

  4. Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration.

    Directory of Open Access Journals (Sweden)

    Akihiro Horii

    Full Text Available A class of self-assembling peptide nanofiber scaffolds has been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold, as well as for repairing tissue defects in animals. We report here the development of several peptide nanofiber scaffolds designed specifically for osteoblasts. We designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The motifs included osteogenic growth peptide ALK (ALKRQGRTLYGF bone-cell secreted-signal peptide, osteopontin cell adhesion motif DGR (DGRGDSVAYG and 2-unit RGD binding sequence PGR (PRGDSGYRGDS. We made the new peptide scaffolds by mixing the pure RAD16 and designer-peptide solutions, and we examined the molecular integration of the mixed nanofiber scaffolds using AFM. Compared to pure RAD16 scaffold, we found that these designer peptide scaffolds significantly promoted mouse pre-osteoblast MC3T3-E1 cell proliferation. Moreover, alkaline phosphatase (ALP activity and osteocalcin secretion, which are early and late markers for osteoblastic differentiation, were also significantly increased. We demonstrated that the designer, self-assembling peptide scaffolds promoted the proliferation and osteogenic differentiation of MC3T3-E1. Under the identical culture medium condition, confocal images unequivocally demonstrated that the designer PRG peptide scaffold stimulated cell migration into the 3-D scaffold. Our results suggest that these designer peptide scaffolds may be very useful for promoting bone tissue regeneration.

  5. Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

    Science.gov (United States)

    Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

    2015-07-01

    It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

  6. Inhibition of miR-29c promotes proliferation, and inhibits apoptosis and differentiation in P19 embryonic carcinoma cells.

    Science.gov (United States)

    Chen, Bin; Song, Guixian; Liu, Ming; Qian, Lingmei; Wang, Lihua; Gu, Haitao; Shen, Yahui

    2016-03-01

    In our previous study, the upregulation of microRNA (miR)-29c was identified in the mother of a fetus with a congenital heart defect. However, the functional and regulatory mechanisms of miR‑29c in the development of the heart remain to be elucidated. In the present study, the role and mechanism of miR‑29c inhibition in heart development were investigated in an embryonic carcinoma cell model. Inhibition of miR‑29c promoted proliferation, and suppressed the apoptosis and differentiation of P19 cells. It was also demonstrated that Wingless‑related MMTV integration site 4 (Wnt4) was a target of miR‑29c, determined using bioinformatic analysis combined with luciferase assays. The inhibition of miR‑29c stimulated the WNT4/β‑catenin pathway, promoting proliferation of the P19 cells, but suppressing their differentiation into cardiomyocytes. Furthermore, the inhibition of miR‑29c promoted the expression of B cell lymphoma‑2 and inhibited cell apoptosis. These results demonstrate the significance of miR‑29c in the process of cardiac development and suggest that miR-29c dysregulation may be associated with the occurrence of CHD. Thus, miR-29c may have therapeutic potential in the future.

  7. Proliferation and osteoblastic differentiation of human bone marrow stromal cells on hydroxyapatite/bacterial cellulose nanocomposite scaffolds.

    Science.gov (United States)

    Fang, Bo; Wan, Yi-Zao; Tang, Ting-Tang; Gao, Chuan; Dai, Ke-Rong

    2009-05-01

    In this study, we prepared hydroxyapatite/bacterial cellulose (HAp/BC) nanocomposite scaffolds utilizing the biomimetic technique, and investigated the proliferation and osteoblastic differentiation of stromal cells derived from human bone marrow (hBMSC) on them. Scanning electron microscopy proved that cells could adhere and spread on scaffolds. The hBMSC seeded on the nanocomposites exhibited better adhesion and activity than those seeded upon the pure BC. After 6 days of culture on scaffolds, the cells proliferated faster on the nanocomposites than on the pure BC, as assessed by Alamar Blue assay. Real-time reverse transcription PCR results showed that the alkaline phosphatase (ALP) activity of hBMSC and the expression of osteopontin, osteocalcin, bone sialoprotein, and ALP mRNA were all higher for up to 7 days for hBMSC cultured on the nanocomposites than for those cultured upon the pure BC with and without the presence of osteogenic supplements (L-ascorbic acid, glycerophosphate, and dexamethasone, pproliferation, and differentiation in cultured hBMSC can be modulated by the HAp/BC nanocomposite scaffold properties. In summary, we have developed a scaffold that displays in vitro biocompatibility, which may have potential use for bone tissue engineering.

  8. Role of the amygdala in antidepressant effects on hippocampal cell proliferation and survival and on depression-like behavior in the rat.

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    Jorge E Castro

    Full Text Available The stimulation of adult hippocampal neurogenesis by antidepressants has been associated with multiple molecular pathways, but the potential influence exerted by other brain areas has received much less attention. The basolateral complex of the amygdala (BLA, a region involved in anxiety and a site of action of antidepressants, has been implicated in both basal and stress-induced changes in neural plasticity in the dentate gyrus. We investigated here whether the BLA modulates the effects of the SSRI antidepressant fluoxetine on hippocampal cell proliferation and survival in relation to a behavioral index of depression-like behavior (forced swim test. We used a lesion approach targeting the BLA along with a chronic treatment with fluoxetine, and monitored basal anxiety levels given the important role of this behavioral trait in the progress of depression. Chronic fluoxetine treatment had a positive effect on hippocampal cell survival only when the BLA was lesioned. Anxiety was related to hippocampal cell survival in opposite ways in sham- and BLA-lesioned animals (i.e., negatively in sham- and positively in BLA-lesioned animals. Both BLA lesions and low anxiety were critical factors to enable a negative relationship between cell proliferation and depression-like behavior. Therefore, our study highlights a role for the amygdala on fluoxetine-stimulated cell survival and on the establishment of a link between cell proliferation and depression-like behavior. It also reveals an important modulatory role for anxiety on cell proliferation involving both BLA-dependent and -independent mechanisms. Our findings underscore the amygdala as a potential target to modulate antidepressants' action in hippocampal neurogenesis and in their link to depression-like behaviors.

  9. Differential effects of culture senescence and mechanical stimulation on the proliferation and leiomyogenic differentiation of MSC from different sources: implications for engineering vascular grafts.

    Science.gov (United States)

    Koobatian, Maxwell T; Liang, Mao-Shih; Swartz, Daniel D; Andreadis, Stelios T

    2015-04-01

    We examined the effects of senescence on the proliferation and leiomyogenic differentiation potential of mesenchymal stem cells (MSCs) isolated from bone marrow (BM-MSCs) or hair follicles (HF-MSCs). To this end, we compared ovine HF-MSCs and BM-MSCs in terms of their proliferation and differentiation potential to the smooth muscle cell lineage. We discovered that HF-MSCs are less susceptible to culture senescence compared with BM-MSCs. We hypothesized that application of mechanical forces may enhance the contractility and mechanical properties of vascular constructs prepared from senescent MSCs. Interestingly, HF-MSCs and BM-MSCs responded differently to changes in the mechanical microenvironment, suggesting that despite phenotypic similarities, MSCs from different anatomic locations may activate different pathways in response to the same microenvironmental factors. In turn, this may also suggest that cell-based tissue regeneration approaches may need to be tailored to the stem cell origin, donor age, and culture time for optimal results.

  10. Habitat Fragmentation Differentially Affects Genetic Variation, Phenotypic Plasticity and Survival in Populations of a Gypsum Endemic.

    Science.gov (United States)

    Matesanz, Silvia; Rubio Teso, María Luisa; García-Fernández, Alfredo; Escudero, Adrián

    2017-01-01

    Habitat fragmentation, i.e., fragment size and isolation, can differentially alter patterns of neutral and quantitative genetic variation, fitness and phenotypic plasticity of plant populations, but their effects have rarely been tested simultaneously. We assessed the combined effects of size and connectivity on these aspects of genetic and phenotypic variation in populations of Centaurea hyssopifolia , a narrow endemic gypsophile that previously showed performance differences associated with fragmentation. We grew 111 maternal families sampled from 10 populations that differed in their fragment size and connectivity in a common garden, and characterized quantitative genetic variation, phenotypic plasticity to drought for key functional traits, and plant survival, as a measure of population fitness. We also assessed neutral genetic variation within and among populations using eight microsatellite markers. Although C. hyssopifolia is a narrow endemic gypsophile, we found substantial neutral genetic variation and quantitative variation for key functional traits. The partition of genetic variance indicated that a higher proportion of variation was found within populations, which is also consistent with low population differentiation in molecular markers, functional traits and their plasticity. This, combined with the generally small effect of habitat fragmentation suggests that gene flow among populations is not restricted, despite large differences in fragment size and isolation. Importantly, population's similarities in genetic variation and plasticity did not reflect the lower survival observed in isolated populations. Overall, our results indicate that, although the species consists of genetically variable populations able to express functional plasticity, such aspects of adaptive potential may not always reflect populations' survival. Given the differential effects of habitat connectivity on functional traits, genetic variation and fitness, our study highlights

  11. Habitat Fragmentation Differentially Affects Genetic Variation, Phenotypic Plasticity and Survival in Populations of a Gypsum Endemic

    Directory of Open Access Journals (Sweden)

    Silvia Matesanz

    2017-05-01

    Full Text Available Habitat fragmentation, i.e., fragment size and isolation, can differentially alter patterns of neutral and quantitative genetic variation, fitness and phenotypic plasticity of plant populations, but their effects have rarely been tested simultaneously. We assessed the combined effects of size and connectivity on these aspects of genetic and phenotypic variation in populations of Centaurea hyssopifolia, a narrow endemic gypsophile that previously showed performance differences associated with fragmentation. We grew 111 maternal families sampled from 10 populations that differed in their fragment size and connectivity in a common garden, and characterized quantitative genetic variation, phenotypic plasticity to drought for key functional traits, and plant survival, as a measure of population fitness. We also assessed neutral genetic variation within and among populations using eight microsatellite markers. Although C. hyssopifolia is a narrow endemic gypsophile, we found substantial neutral genetic variation and quantitative variation for key functional traits. The partition of genetic variance indicated that a higher proportion of variation was found within populations, which is also consistent with low population differentiation in molecular markers, functional traits and their plasticity. This, combined with the generally small effect of habitat fragmentation suggests that gene flow among populations is not restricted, despite large differences in fragment size and isolation. Importantly, population’s similarities in genetic variation and plasticity did not reflect the lower survival observed in isolated populations. Overall, our results indicate that, although the species consists of genetically variable populations able to express functional plasticity, such aspects of adaptive potential may not always reflect populations’ survival. Given the differential effects of habitat connectivity on functional traits, genetic variation and fitness

  12. The effect of insulin, TNFα and DHA on the proliferation, differentiation and lipolysis of preadipocytes isolated from large yellow croaker (Pseudosciaena Crocea R..

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    Xinxia Wang

    Full Text Available Fish final product can be affected by excessive lipid accumulation. Therefore, it is important to develop strategies to control obesity in cultivated fish to strengthen the sustainability of the aquaculture industry. As in mammals, the development of adiposity in fish depends on hormonal, cytokine and dietary factors. In this study, we investigated the proliferation and differentiation of preadipocytes isolated from the large yellow croaker and examined the effects of critical factors such as insulin, TNFα and DHA on the proliferation, differentiation and lipolysis of adipocytes. Preadipocytes were isolated by collagenase digestion, after which their proliferation was evaluated. The differentiation process was optimized by assaying glycerol-3-phosphate dehydrogenase (GPDH activity. Oil red O staining and electron microscopy were performed to visualize the accumulated triacylglycerol. Gene transcript levels were measured using SYBR green quantitative real-time PCR. Insulin promoted preadipocytes proliferation, stimulated cell differentiation and decreased lipolysis of mature adipocytes. TNFα and DHA inhibited cell proliferation and differentiation. While TNFα stimulated mature adipocyte lipolysis, DHA showed no lipolytic effect on adipocytes. The expressions of adipose triglyceride lipase (ATGL, fatty acid synthase (FAS, lipoprotein lipase (LPL and peroxisome proliferator-activated receptor α, γ (PPARα, PPARγ were quantified during preadipocytes differentiation and adipocytes lipolysis to partly explain the regulation mechanisms. In summary, the results of this study indicated that although preadipocytes proliferation and the differentiation process in large yellow croaker are similar to these processes in mammals, the effects of critical factors such as insulin, TNFα and DHA on fish adipocytes development are not exactly the same. Our findings fill in the gaps in the basic data regarding the effects of critical factors on adiposity

  13. Organic cation transporter-mediated ergothioneine uptake in mouse neural progenitor cells suppresses proliferation and promotes differentiation into neurons.

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    Takahiro Ishimoto

    Full Text Available 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 [(3H]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 [(3H]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

  14. Alyssum homolocarpum seeds: phytochemical analysis and effects of the seed oil on neural stem cell proliferation and differentiation.

    Science.gov (United States)

    Hamedi, Azadeh; Ghanbari, Amir; Razavipour, Razieh; Saeidi, Vahid; Zarshenas, Mohammad M; Sohrabpour, Maryam; Azari, Hassan

    2015-07-01

    Pharmacognostic evaluation of medicinal plants may assess their current applications and possibly results in finding new active components. In this study, ash and extractive values and high performance thin layer chromatography fingerprints of Alyssum homolocarpum (Brassicaceae) seed extracts were investigated to elucidate its composition. Differential scanning calorimetry and gas chromatography-mass spectrometry analysis were employed to determine the components of A. homolocarpum seed oil (AHO). Neurosphere assay, in vitro differentiation and immunofluorescence analysis were performed to evaluate the effects of oral administration of AHO (0.5 or 1 g/kg/day for 14 days) on proliferation and differentiation of neural stem cells (NSCs) in adult male BALB/c mice. Total, acid-insoluble and water-soluble ash values were determined as 45.83 ± 5.85, 6.67 ± 2.89 and 28.33 ± 2.89 mg/g, respectively. The extractive values were 4.90, 0.43 and 0.56 % (w/w) for n-hexane, dichloromethane and ethanolic extracts, respectively. Interestingly, AHO was mainly composed of α-linolenic acid (89.71 %), β-sitosterol (3.3 mg/g) and campesterol (0.86 mg/g). Administration of AHO at 1 g/kg/day significantly increased proliferation of NSCs, as evidenced by an increase in mean neurosphere-forming frequency per brain (872.7 ± 15.17) and neurosphere diameter (101 ± 2.48 µm) compared to the control group (424.3 ± 59.29 and 78.63 ± 1.7 µm, respectively; P < 0.05). AHO treatment did not affect in vitro differentiation of the harvested NSCs. Our data show that A. homolocarpum seed oil is a rich source of α-linolenic acid and β-sitosterol with potential therapeutic application to enhance NSC proliferation and recruitment in neurological diseases.

  15. Neutralization of LINGO-1 during in vitro differentiation of neural stem cells results in proliferation of immature neurons.

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    Camilla Lööv

    Full Text Available Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1 antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain.

  16. Neutralization of LINGO-1 during in vitro differentiation of neural stem cells results in proliferation of immature neurons.

    Science.gov (United States)

    Lööv, Camilla; Fernqvist, Maria; Walmsley, Adrian; Marklund, Niklas; Erlandsson, Anna

    2012-01-01

    Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1 antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain.

  17. Differential survival among sSOD-1* genotypes in Chinook Salmon

    Science.gov (United States)

    Hayes, Michael C.; Reisenbichler, Reginald R.; Rubin, Stephen P.; Wetzel, Lisa A.; Marshall , Anne R.

    2011-01-01

    Differential survival and growth were tested in Chinook salmon Oncorhynchus tshawytscha expressing two common alleles, *–100 and *–260, at the superoxide dismutase locus (sSOD-1*). These tests were necessary to support separate studies in which the two alleles were used as genetic marks under the assumption of mark neutrality. Heterozygous adults were used to produce progeny with –100/–100, –100/–260, and –260/–260 genotypes that were reared in two natural streams and two hatcheries in the states of Washington and Oregon. The latter also were evaluated as returning adults. In general, the genotype ratios of juveniles reared at hatcheries were consistent with high survival and little or no differential survival in the hatchery. Adult returns at one hatchery were significantly different from the expected proportions, and the survival of the –260/–260 genotype was 0.56–0.89 times that of the –100/–100 genotype over four year-classes. Adult returns at a second hatchery (one year-class) were similar but not statistically significant: survival of the –260/–260genotype relative to the –100/–100 genotype was 0.76. The performance of the heterozygote group was intermediate at both hatcheries. Significant differences in growth were rarely observed among hatchery fish (one year-class of juveniles and one age-class of adult males) but were consistent with greater performance for the –100/–100 genotype. Results from two groups of juveniles reared in streams (one year-class from each stream) suggested few differences in growth, but the observed genotype ratios were significantly different from the expected ratios in one stream. Those differences were consistent with the adult data; survival for the –260/–260 genotype was 76% of that of the –100/–100 genotype. These results, which indicate nonneutrality among sSOD-1* genotypes, caused us to modify our related studies and suggest caution in the interpretation of results and analyses in

  18. Effect of low-level diode laser on proliferation and osteogenic differentiation of dental pulp stem cells

    Science.gov (United States)

    Tabatabaei, Fahimeh S.; Torshabi, Maryam; Mojahedi Nasab, Masoud; Khosraviani, Keikhosro; Khojasteh, Arash

    2015-09-01

    This study assessed the effect of low-level laser irradiation (LLLI) on the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs). DPSCs were exposed to 810 nm laser light (0.1, 0.2, or 0.3 J cm-2) for 7 d (60 s daily). The negative control group (cells in regular medium) and positive control group (cells in osteogenic medium (OM)) were not lased. One group of cells in OM was irradiated with laser operated at 0.2 J cm-2. Cell viability was evaluated at 24 h and one week after the last day of laser irradiation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Osteogenic differentiation was assessed using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and alizarin Red S staining. Cell proliferation was not affected by laser irradiation at 24 h except in one group (cells in OM exposed to laser at 0.2 J cm-2). However, one week after the last day of laser irradiation, it was significantly increased in groups exposed to laser at 0.1 or 0.2 J cm-2 and decreased in groups containing OM (P  cell differentiation. Laser at 0.3 J cm-2 increased bone sialoprotein (BSP) and decreased alkaline phosphatase (ALP). Mineralized nodules were only observed in groups containing OM. Considering these findings, LLLI may be used as a novel approach for preconditioning of DPSCs in vitro prior to bone tissue engineering.

  19. Differential Survival for Men and Women with HIV/AIDS-Related Neurologic Diagnoses.

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    Martha L Carvour

    Full Text Available Neurologic complications of human immunodeficiency virus (HIV infection and acquired immune deficiency syndrome (AIDS frequently lead to disability or death in affected patients. The aim of this study was to determine whether survival patterns differ between men and women with HIV/AIDS-related neurologic disease (neuro-AIDS.Retrospective cohort data from a statewide surveillance database for HIV/AIDS were used to characterize survival following an HIV/AIDS-related neurologic diagnosis for men and women with one or more of the following conditions: cryptococcosis, toxoplasmosis, primary central nervous system lymphoma, progressive multifocal leukoencephalopathy, and HIV-associated dementia. A second, non-independent cohort was formed using university-based cases to confirm and extend the findings from the statewide data. Kaplan-Meier analysis was used to compare the survival experiences for men and women in the cohorts. Cox regression was employed to characterize survival while controlling for potential confounders in the study population.Women (n=27 had significantly poorer outcomes than men (n=198 in the statewide cohort (adjusted hazard ratio=2.31, 95% CI: 1.22 to 4.35, and a similar, non-significant trend was observed among university-based cases (n=17 women, 154 men. Secondary analyses suggested that this difference persisted over the course of the AIDS epidemic and was not attributable to differential antiretroviral therapy responses among men and women.The survival disadvantage of women compared to men should be confirmed and the mechanisms underlying this disparity elucidated. If this relationship is confirmed, targeted clinical and public health efforts might be directed towards screening, treatment, and support for women affected by neuro-AIDS.

  20. Differential Survival for Men and Women with HIV/AIDS-Related Neurologic Diagnoses.

    Science.gov (United States)

    Carvour, Martha L; Harms, Jerald P; Lynch, Charles F; Mayer, Randall R; Meier, Jeffery L; Liu, Dawei; Torner, James C

    2015-01-01

    Neurologic complications of human immunodeficiency virus (HIV) infection and acquired immune deficiency syndrome (AIDS) frequently lead to disability or death in affected patients. The aim of this study was to determine whether survival patterns differ between men and women with HIV/AIDS-related neurologic disease (neuro-AIDS). Retrospective cohort data from a statewide surveillance database for HIV/AIDS were used to characterize survival following an HIV/AIDS-related neurologic diagnosis for men and women with one or more of the following conditions: cryptococcosis, toxoplasmosis, primary central nervous system lymphoma, progressive multifocal leukoencephalopathy, and HIV-associated dementia. A second, non-independent cohort was formed using university-based cases to confirm and extend the findings from the statewide data. Kaplan-Meier analysis was used to compare the survival experiences for men and women in the cohorts. Cox regression was employed to characterize survival while controlling for potential confounders in the study population. Women (n=27) had significantly poorer outcomes than men (n=198) in the statewide cohort (adjusted hazard ratio=2.31, 95% CI: 1.22 to 4.35), and a similar, non-significant trend was observed among university-based cases (n=17 women, 154 men). Secondary analyses suggested that this difference persisted over the course of the AIDS epidemic and was not attributable to differential antiretroviral therapy responses among men and women. The survival disadvantage of women compared to men should be confirmed and the mechanisms underlying this disparity elucidated. If this relationship is confirmed, targeted clinical and public health efforts might be directed towards screening, treatment, and support for women affected by neuro-AIDS.

  1. Simulation of fracture healing incorporating mechanoregulation of tissue differentiation and dispersal/proliferation of cells

    NARCIS (Netherlands)

    Andreykiv, A.; Van Keulen, F.; Prendergast, P.J.

    2007-01-01

    Modelling the course of healing of a long bone subjected to loading has been the subject of several investigations. These have succeeded in predicting the differentiation of tissues in the callus in response to a static mechanical load and the diffusion of biological factors. In this paper an

  2. The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline

    NARCIS (Netherlands)

    Bailey, Alexis S.; Batista, Pedro J.; Gold, Rebecca S.; Grace Chen, Y.; de Rooij, Dirk G.|info:eu-repo/dai/nl/153112336; Chang, Howard Y.; Fuller, Margaret T.

    2017-01-01

    The switch from mitosis to meiosis is the key event marking onset of differentiation in the germline stem cell lineage. In Drosophila, the translational repressor Bgcn is required for spermatogonia to stop mitosis and transition to meiotic prophase and the spermatocyte state. Here we show that the

  3. Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells

    Science.gov (United States)

    Dreyer-Andersen, Nanna; Almeida, Ana Sofia; Jensen, Pia; Kamand, Morad; Okarmus, Justyna; Rosenberg, Tine; Friis, Stig Düring; Martínez Serrano, Alberto; Blaabjerg, Morten; Kristensen, Bjarne Winther; Skrydstrup, Troels; Gramsbergen, Jan Bert; Vieira, Helena L. A.

    2018-01-01

    Exploratory studies using human fetal tissue have suggested that intrastriatal transplantation of dopaminergic neurons may become a future treatment for patients with Parkinson’s disease. However, the use of human fetal tissue is compromised by ethical, regulatory and practical concerns. Human stem cells constitute an alternative source of cells for transplantation in Parkinson’s disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting in both protection and generation of reactive oxygen species. The present study investigated the effect of CO produced by a novel CO-releasing molecule on dopaminergic differentiation of human neural stem cells. Short-term exposure to 25 ppm CO at days 0 and 4 significantly increased the relative content of β-tubulin III-immunoreactive immature neurons and tyrosine hydroxylase expressing catecholaminergic neurons, as assessed 6 days after differentiation. Also the number of microtubule associated protein 2-positive mature neurons had increased significantly. Moreover, the content of apoptotic cells (Caspase3) was reduced, whereas the expression of a cell proliferation marker (Ki67) was left unchanged. Increased expression of hypoxia inducible factor-1α and production of reactive oxygen species (ROS) in cultures exposed to CO may suggest a mechanism involving mitochondrial alterations and generation of ROS. In conclusion, the present procedure using controlled, short-term CO exposure allows efficient dopaminergic differentiation of human neural stem cells at low cost and may as such be useful for derivation of cells for experimental studies and future development of donor cells for transplantation in Parkinson’s disease. PMID:29338033

  4. Comparative Analysis of Proliferation and Differentiation Potentials of Stem Cells from Inflamed Pulp of Deciduous Teeth and Stem Cells from Exfoliated Deciduous Teeth

    Directory of Open Access Journals (Sweden)

    Shi Yu

    2014-01-01

    Full Text Available Stem cells isolated from exfoliated deciduous teeth (SHEDs are highly capable of proliferation and differentiation, and they represent good cell sources for mesenchymal stem cell- (MSC- mediated dental tissue regeneration, but the supply of SHEDs is limited. A previous study found that stem cells could be isolated from inflamed tissues, but it is unknown whether primary dental pulp diagnosed with irreversible pulpitis might contain stem cells with appropriate tissue regeneration capacity. In this study, we aimed to isolate stem cells from both inflamed pulps of deciduous teeth (SCIDs and SHEDs from Chinese children and to compare their proliferation and differentiation potentials. Our results showed that SCIDs were positive for cell surface markers, including CD105, CD90, and CD146, and they had high proliferation ability and osteogenic, adipogenic, and chondrogenic differentiation potentials. There was no significant difference in proliferation and differentiation potentials between SCIDs and SHEDs. The mRNA of inflammatory factors, including IL-1β, IL-6, and TNF-α, was expressed at similar levels in SCIDs and SHEDs, but SCIDs secreted more TNF-α protein. In conclusion, our in vitro results showed that SCIDs have proliferation and differentiation potentials similar to those of SHEDs. Thus, SCIDs represent a new potentially applicable source for MSC mediated tissue regeneration.

  5. Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo.

    Directory of Open Access Journals (Sweden)

    Andrea Caceres

    Full Text Available BACKGROUND: The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub. Of the 7 lysines (K least is known about effects of modification of K6. METHODOLOGY AND PRINCIPAL FINDINGS: We replaced K6 with tryptophan (W because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP. Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27(kip, a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues. CONCLUSIONS AND SIGNIFICANCE: K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues

  6. Expression of GLUT-1 in epithelial ovarian carcinoma: correlation with tumor cell proliferation, angiogenesis, survival and ability to predict optimal cytoreduction.

    Science.gov (United States)

    Semaan, Assaad; Munkarah, Adnan R; Arabi, Haitham; Bandyopadhyay, Sudeshna; Seward, Shelly; Kumar, Sanjeev; Qazi, Aamer; Hussein, Yasser; Morris, Robert T; Ali-Fehmi, Rouba

    2011-04-01

    GLUT-1 is involved at various steps in the processes of tumor progression. The objective of this study was to examine the relationship between GLUT-1 expression and tumor proliferation and angiogenesis in epithelial ovarian carcinoma. Specimens from 213 patients with epithelial ovarian carcinoma were evaluated by immunohistochemistry for GLUT-1, Ki-67, and vascular endothelial growth factor. Tumor microvessel density was assessed with CD34 immunostaining. We investigated the relationships between GLUT-1 expression and clinicopathologic characteristics, tumor angiogenesis (tumor MVD and vascular endothelial growth factor expression), and tumor proliferation (Ki-67). The effect of GLUT-1 expression on patient survival and on the volume of residual disease after cytoreduction was determined. There was a significant positive correlation between expression of GLUT-1, Ki-67, and microvessel density. In univariate survival analysis, high GLUT-1 expression, high Ki-67 expression and high tumor microvessel density showed a significant impact on patient survival (p=0.0001). In multivariate analysis including patients with all tumor stages, after controlling for age, race, stage, grade, MVD, and the 3 markers (GLUT-1, Ki-67 and VEGF), only age (HR 1.5; 95% CI 1-2.3), stage (HR 3.6; 95% CI 1.8-7.5) and grade (HR 2.3; 95% CI 1.2-4.5) retained their significance as independent poor prognostic factors. Tumors simultaneously overexpressing GLUT-1 and Ki-67 were less likely to be optimally cytoreduced as compared to tumors overexpressing only one or neither of those two markers (OR: 3.8, p=0.01). Expression of GLUT-1 correlates with tumor proliferation and microvessel density in epithelial ovarian carcinoma. In addition, patients with rapidly proliferating advanced stage tumors overexpressing GLUT-1 have a lesser chance for optimal cytoreduction. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. Survival

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — These data provide information on the survival of California red-legged frogs in a unique ecosystem to better conserve this threatened species while restoring...

  8. Chondroregulatory action of prolactin on proliferation and differentiation of mouse chondrogenic ATDC5 cells in 3-dimensional micromass cultures

    Energy Technology Data Exchange (ETDEWEB)

    Seriwatanachai, Dutmanee [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Krishnamra, Nateetip [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand); Charoenphandhu, Narattaphol, E-mail: naratt@narattsys.com [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Mouse chondrogenic ATDC5 cells expressed PRL receptor mRNAs and proteins. Black-Right-Pointing-Pointer Low PRL concentration (10 ng/mL) increased chondrocyte viability and differentiation. Black-Right-Pointing-Pointer Higher PRL concentrations ( Greater-Than-Or-Slanted-Equal-To 100 ng/mL) decreased viability and increased apoptosis. -- Abstract: A recent investigation in lactating rats has provided evidence that the lactogenic hormone prolactin (PRL) increases endochondral bone growth and bone elongation, presumably by accelerating apoptosis of hypertrophic chondrocytes in the growth plate and/or subsequent chondrogenic matrix mineralization. Herein, we demonstrated the direct chondroregulatory action of PRL on proliferation, differentiation and apoptosis of chondrocytes in 3-dimensional micromass culture of mouse chondrogenic ATDC5 cell line. The results showed that ATDC5 cells expressed PRL receptor (PRLR) transcripts, and responded typically to PRL by downregulating PRLR expression. Exposure to a low PRL concentration of 10 ng/mL, comparable to the normal levels in male and non-pregnant female rats, increased chondrocyte viability, differentiation, proteoglycan accumulation, and mRNA expression of several chondrogenic differentiation markers, such as Sox9, ALP and Hspg2. In contrast, high PRL concentrations of Greater-Than-Or-Slanted-Equal-To 100 ng/mL, comparable to the levels in pregnancy or lactation, decreased chondrocyte viability by inducing apoptosis, with no effect on chondrogenic marker expression. It could be concluded that chondrocytes directly but differentially responded to non-pregnant and pregnant/lactating levels of PRL, thus suggesting the stimulatory effect of PRL on chondrogenesis in young growing individuals, and supporting the hypothesis of hypertrophic chondrocyte apoptosis in the growth plate of lactating rats.

  9. HCMV Reprogramming of Infected Monocyte Survival and Differentiation: A Goldilocks Phenomenon

    Directory of Open Access Journals (Sweden)

    Emily V. Stevenson

    2014-02-01

    Full Text Available The wide range of disease pathologies seen in multiple organ sites associated with human cytomegalovirus (HCMV infection results from the systemic hematogenous dissemination of the virus, which is mediated predominately by infected monocytes. In addition to their role in viral spread, infected monocytes are also known to play a key role in viral latency and life-long persistence. However, in order to utilize infected monocytes for viral spread and persistence, HCMV must overcome a number of monocyte biological hurdles, including their naturally short lifespan and their inability to support viral gene expression and replication. Our laboratory has shown that HCMV is able to manipulate the biology of infected monocytes in order to overcome these biological hurdles by inducing the survival and differentiation of infected monocytes into long-lived macrophages capable of supporting viral gene expression and replication. In this current review, we describe the unique aspects of how HCMV promotes monocyte survival and differentiation by inducing a “finely-tuned” macrophage cell type following infection. Specifically, we describe the induction of a uniquely polarized macrophage subset from infected monocytes, which we argue is the ideal cellular environment for the initiation of viral gene expression and replication and, ultimately, viral spread and persistence within the infected host.

  10. NKAP Regulates Invariant NKT Cell Proliferation and Differentiation into ROR-γt-Expressing NKT17 Cells.

    Science.gov (United States)

    Thapa, Puspa; Chen, Meibo W; McWilliams, Douglas C; Belmonte, Paul; Constans, Megan; Sant'Angelo, Derek B; Shapiro, Virginia Smith

    2016-06-15

    Invariant NKT (iNKT) cells are a unique lineage with characteristics of both adaptive and innate lymphocytes, and they recognize glycolipids presented by an MHC class I-like CD1d molecule. During thymic development, iNKT cells also differentiate into NKT1, NKT2, and NKT17 functional subsets that preferentially produce cytokines IFN-γ, IL-4, and IL-17, respectively, upon activation. Newly selected iNKT cells undergo a burst of proliferation, which is defective in mice with a specific deletion of NKAP in the iNKT cell lineage, leading to severe reductions in thymic and peripheral iNKT cell numbers. The decreased cell number is not due to defective homeostasis or increased apoptosis, and it is not rescued by Bcl-xL overexpression. NKAP is also required for differentiation into NKT17 cells, but NKT1 and NKT2 cell development and function are unaffected. This failure in NKT17 development is rescued by transgenic expression of promyelocytic leukemia zinc finger; however, the promyelocytic leukemia zinc finger transgene does not restore iNKT cell numbers or the block in positive selection into the iNKT cell lineage in CD4-cre NKAP conditional knockout mice. Therefore, NKAP regulates multiple steps in iNKT cell development and differentiation. Copyright © 2016 by The American Association of Immunologists, Inc.

  11. Engineered Biomaterials Control Differentiation and Proliferation of Human-Embryonic-Stem-Cell-Derived Cardiomyocytes via Timed Notch Activation

    Directory of Open Access Journals (Sweden)

    Jason C. Tung

    2014-03-01

    Full Text Available For cell-based treatments of myocardial infarction, a better understanding of key developmental signaling pathways and more robust techniques for producing cardiomyocytes are required. Manipulation of Notch signaling has promise as it plays an important role during cardiovascular development, but previous studies presented conflicting results that Notch activation both positively and negatively regulates cardiogenesis. We developed surface- and microparticle-based Notch-signaling biomaterials that function in a time-specific activation-tunable manner, enabling precise investigation of Notch activation at specific developmental stages. Using our technologies, a biphasic effect of Notch activation on cardiac differentiation was found: early activation in undifferentiated human embryonic stem cells (hESCs promotes ectodermal differentiation, activation in specified cardiovascular progenitor cells increases cardiac differentiation. Signaling also induces cardiomyocyte proliferation, and repeated doses of Notch-signaling microparticles further enhance cardiomyocyte population size. These results highlight the diverse effects of Notch activation during cardiac development and provide approaches for generating large quantities of cardiomyocytes.

  12. Effect of the alcoholic extract of Ashwagandha leaves and its components on proliferation, migration, and differentiation of glioblastoma cells: combinational approach for enhanced differentiation.

    Science.gov (United States)

    Shah, Navjot; Kataria, Hardeep; Kaul, Sunil C; Ishii, Tetsuro; Kaur, Gurcharan; Wadhwa, Renu

    2009-09-01

    Ashwagandha (Withania somnifera) is widely used in the Indian traditional system of medicine, Ayurveda. Although it is claimed to have a large variety of health-promoting effects, including therapeutic effects on stress and disease, the mechanisms of action have not yet been determined. In the present study, we aimed to investigate the growth inhibition and differentiation potential of the alcoholic extract of Ashwagandha leaves (i-Extract), its different constituents (Withaferin A, Withanone, Withanolide A) and their combinations on glioma (C6 and YKG1) cell lines. Withaferin A, Withanone, Withanolide A and i-Extract markedly inhibited the proliferation of glioma cells in a dose-dependent manner and changed their morphology toward the astrocytic type. Molecular analysis revealed that the i-Extract and some of its components caused enhanced expression of glial fibrillary acidic protein, change in the immunostaining pattern of mortalin from perinuclear to pancytoplasmic, delay in cell migration, and increased expression of neuronal cell adhesion molecules. The data suggest that the i-Extract and its components have the potential to induce senescence-like growth arrest and differentiation in glioma cells. These assays led us to formulate a unique combination formula of i-Extract components that caused enhanced differentiation of glial cells.

  13. Association of Glioblastoma Multiforme Stem Cell Characteristics, Differentiation, and Microglia Marker Genes with Patient Survival

    Directory of Open Access Journals (Sweden)

    Sandra Bien-Möller

    2018-01-01

    Full Text Available Patients with glioblastoma multiforme (GBM are at high risk to develop a relapse despite multimodal therapy. Assumedly, glioma stem cells (GSCs are responsible for treatment resistance of GBM. Identification of specific GSC markers may help to develop targeted therapies. Here, we performed expression analyses of stem cell (ABCG2, CD44, CD95, CD133, ELF4, Nanog, and Nestin as well as differentiation and microglia markers (GFAP, Iba1, and Sparc in GBM compared to nonmalignant brain. Furthermore, the role of these proteins for patient survival and their expression in LN18 stem-like neurospheres was analyzed. At mRNA level, ABCG2 and CD95 were reduced, GFAP was unchanged; all other investigated markers were increased in GBM. At protein level, CD44, ELF4, Nanog, Nestin, and Sparc were elevated in GBM, but only CD133 and Nestin were strongly associated with survival time. In addition, ABCG2 and GFAP expression was decreased in LN18 neurospheres whereas CD44, CD95, CD133, ELF4, Nanog, Nestin, and Sparc were upregulated. Altogether only CD133 and Nestin were associated with survival rates. This raises concerns regarding the suitability of the other target structures as prognostic markers, but makes both CD133 and Nestin candidates for GBM therapy. Nevertheless, a search for more specific marker proteins is urgently needed.

  14. Ciliary neurotrophic factor reduces proliferation and promotes differentiation of TH-MYCN transformed sympathoadrenal progenitors

    Science.gov (United States)

    DeWitt, John; Pappas, Anthony; Nishi, Rae

    2014-01-01

    Neuroblastoma is a childhood cancer caused by transformation of sympathoadrenal progenitors. By following the formation of tumors in homozygous TH-MYCN mice, an established mouse model of neuroblastoma, we were able to capture transformed cells prior to the formation of large, vascularized tumors in order to determine the responsiveness of cells to neurotrophic factors. We discovered that the CNTF receptor is abundantly expressed in tumor cells from these mice. Furthermore, CNTF, but not nerve growth factor, brain-derived nerve growth factor, NT-3, or glial cell line derived neurotrophic factor, promoted neuronal differentiation and withdrawal from the cell cycle. Thus, transformation of sympathoadrenal progenitors by MYCN overexpression differentially affects responsiveness to neurotrophic molecules. PMID:25171250

  15. Ciliary neurotrophic factor reduces the proliferation and promotes the differentiation of TH- MYCN transformed sympathoadrenal progenitors.

    Science.gov (United States)

    DeWitt, John; Pappas, Anthony; Nishi, Rae

    2014-01-01

    Neuroblastoma is a childhood cancer caused by the transformation of sympathoadrenal progenitors. By following the formation of tumors in homozygous TH-MYCN mice, an established mouse model of neuroblastoma, we were able to capture transformed cells prior to the formation of large, vascularized tumors in order to determine the responsiveness of cells to neurotrophic factors. We discovered that the ciliary neurotrophic factor (CNTF) receptor is abundantly expressed in tumor cells from these mice. Furthermore, CNTF - but not nerve growth factor, brain-derived nerve growth factor, neurotrophin 3, or glial cell line-derived neurotrophic factor - promoted neuronal differentiation and withdrawal from the cell cycle. Thus, the transformation of sympathoadrenal progenitors by MYCN overexpression differentially affects responsiveness to neurotrophic molecules. © 2014 S. Karger AG, Basel.

  16. Terminal Differentiation of Adult Hippocampal Progenitor Cells Is a Step Functionally Dissociable from Proliferation and Is Controlled by Tis21, Id3 and NeuroD2.

    Science.gov (United States)

    Micheli, Laura; Ceccarelli, Manuela; Gioia, Roberta; D'Andrea, Giorgio; Farioli-Vecchioli, Stefano; Costanzi, Marco; Saraulli, Daniele; Cestari, Vincenzo; Tirone, Felice

    2017-01-01

    Cell proliferation and differentiation are interdependent processes. Here, we have asked to what extent the two processes of neural progenitor cell amplification and differentiation are functionally separated. Thus, we analyzed whether it is possible to rescue a defect of terminal differentiation in progenitor cells of the dentate gyrus, where new neurons are generated throughout life, by inducing their proliferation and/or their differentiation with different stimuli appropriately timed. As a model we used the Tis21 knockout mouse, whose dentate gyrus neurons, as demonstrated by us and others, have an intrinsic defect of terminal differentiation. We first tested the effect of two proliferative as well as differentiative neurogenic stimuli, one pharmacological (fluoxetine), the other cognitive (the Morris water maze (MWM) training). Both effectively enhanced the number of new dentate gyrus neurons produced, and fluoxetine also reduced the S-phase length of Tis21 knockout dentate gyrus progenitor cells and increased the rate of differentiation of control cells, but neither factor enhanced the defective rate of differentiation. In contrast, the defect of terminal differentiation was fully rescued by in vivo infection of proliferating dentate gyrus progenitor cells with retroviruses either silencing Id3, an inhibitor of neural differentiation, or expressing NeuroD2, a proneural gene expressed in terminally differentiated dentate gyrus neurons. This is the first demonstration that NeuroD2 or the silencing of Id3 can activate the differentiation of dentate gyrus neurons, complementing a defect of differentiation. It also highlights how the rate of differentiation of dentate gyrus neurons is regulated genetically at several levels and that a neurogenic stimulus for amplification of neural stem/progenitor cells may not be sufficient in itself to modify this rate.

  17. Role of ciliary neurotrophic factor in the proliferation and differentiation of neural stem cells.

    Science.gov (United States)

    Ding, Jun; He, Zhili; Ruan, Juan; Ma, Zilong; Liu, Ying; Gong, Chengxin; Iqbal, Khalid; Sun, Shenggang; Chen, Honghui

    2013-01-01

    Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine that has been fully studied for its structure, receptor, and signaling pathways and its multiplex effects on neural system, skeletal muscle, and weight control. Recent research demonstrates that CNTF also plays an important role in neurogenesis and the differentiation of neural stem cells. In this article, we summarize the general characteristics of CNTF and its function on neural stem cells, which could be a valuable therapeutic strategy in treating neurological disorders.

  18. Sox2 Activates Cell Proliferation and Differentiation in the Respiratory Epithelium

    OpenAIRE

    Tompkins, David H.; Besnard, Valérie; Lange, Alexander W.; Keiser, Angela R.; Wert, Susan E.; Bruno, Michael D.; Whitsett, Jeffrey A.

    2010-01-01

    Sox2, a transcription factor critical for the maintenance of embryonic stem cells and induction of pluripotent stem cells, is expressed exclusively in the conducting airway epithelium of the lung, where it is required for differentiation of nonciliated, goblet, and ciliated cells. To determine the role of Sox2 in respiratory epithelial cells, Sox2 was selectively and conditionally expressed in nonciliated airway epithelial cells and in alveolar type II cells in the adult mouse. Sox2 induced e...

  19. The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline

    OpenAIRE

    Bailey, Alexis S; Batista, Pedro J; Gold, Rebecca S; Chen, Y Grace; de Rooij, Dirk G; Chang, Howard Y; Fuller, Margaret T

    2017-01-01

    The switch from mitosis to meiosis is the key event marking onset of differentiation in the germline stem cell lineage. In Drosophila, the translational repressor Bgcn is required for spermatogonia to stop mitosis and transition to meiotic prophase and the spermatocyte state. Here we show that the mammalian Bgcn homolog YTHDC2 facilitates a clean switch from mitosis to meiosis in mouse germ cells, revealing a conserved role for YTHDC2 in this critical cell fate transition. YTHDC2-deficient ma...

  20. Water Extract of Ashwagandha Leaves Limits Proliferation and Migration, and Induces Differentiation in Glioma Cells

    Directory of Open Access Journals (Sweden)

    Hardeep Kataria

    2011-01-01

    Full Text Available Root extracts of Withania somnifera (Ashwagandha are commonly used as a remedy for a variety of ailments and a general tonic for overall health and longevity in the Indian traditional medicine system, Ayurveda. We undertook a study to investigate the anti-proliferative and differentiation-inducing activities in the water extract of Ashwagandha leaves (ASH-WEX by examining in glioma cells. Preliminary detection for phytochemicals was performed by thin-layer chromatography. Cytotoxicity was determined using trypan blue and MTT assays. Expression level of an hsp70 family protein (mortalin, glial cell differentiation marker [glial fibrillary acidic protein (GFAP] and neural cell adhesion molecule (NCAM were analyzed by immunocytochemistry and immunoblotting. Anti-migratory assay was also done using wound-scratch assay. Expression levels of mortalin, GFAP and NCAM showed changes, subsequent to the treatment with ASH-WEX. The data support the existence of anti-proliferative, differentiation-inducing and anti-migratory/anti-metastasis activities in ASH-WEX that could be used as potentially safe and complimentary therapy for glioma.

  1. Water Extract of Ashwagandha Leaves Limits Proliferation and Migration, and Induces Differentiation in Glioma Cells

    Science.gov (United States)

    Kataria, Hardeep; Shah, Navjot; Kaul, Sunil C.; Wadhwa, Renu; Kaur, Gurcharan

    2011-01-01

    Root extracts of Withania somnifera (Ashwagandha) are commonly used as a remedy for a variety of ailments and a general tonic for overall health and longevity in the Indian traditional medicine system, Ayurveda. We undertook a study to investigate the anti-proliferative and differentiation-inducing activities in the water extract of Ashwagandha leaves (ASH-WEX) by examining in glioma cells. Preliminary detection for phytochemicals was performed by thin-layer chromatography. Cytotoxicity was determined using trypan blue and MTT assays. Expression level of an hsp70 family protein (mortalin), glial cell differentiation marker [glial fibrillary acidic protein (GFAP)] and neural cell adhesion molecule (NCAM) were analyzed by immunocytochemistry and immunoblotting. Anti-migratory assay was also done using wound-scratch assay. Expression levels of mortalin, GFAP and NCAM showed changes, subsequent to the treatment with ASH-WEX. The data support the existence of anti-proliferative, differentiation-inducing and anti-migratory/anti-metastasis activities in ASH-WEX that could be used as potentially safe and complimentary therapy for glioma. PMID:20007262

  2. Peroxisome proliferator-activated receptorα agonists differentially regulate inhibitor of DNA binding expression in rodents and human cells.

    Science.gov (United States)

    González, María Del Carmen; Corton, J Christopher; Acero, Nuria; Muñoz-Mingarro, Dolores; Quirós, Yolanda; Alvarez-Millán, Juan José; Herrera, Emilio; Bocos, Carlos

    2012-01-01

    Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

  3. Short Stat5-interacting peptide derived from phospholipase C-β3 inhibits hematopoietic cell proliferation and myeloid differentiation.

    Directory of Open Access Journals (Sweden)

    Hiroki Yasudo

    Full Text Available Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN. Our recent study found that phospholipase C (PLC-β3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-β3-dependent manner. PLC-β3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-β3 (PLC-β3-CT accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-β3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998 suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-β3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-β3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies.

  4. Preconditioning Human Mesenchymal Stem Cells with a Low Concentration of BMP2 Stimulates Proliferation and Osteogenic Differentiation In Vitro

    DEFF Research Database (Denmark)

    Lysdahl, Helle; Baatrup, Anette; Foldager, Casper Bindzus

    2014-01-01

    treatment strategy in which human bone marrow-derived mesenchymal stem cells (hMSCs) are preconditioned with low concentrations of BMP2 for a short time in vitro. hMSCs in suspension were stimulated for 15 min with 10 and 20 ng/mL of BMP2. After the BMP2 was removed, the cells were seeded and cultured......MSCs. This implies that preconditioning with BMP2 might be more effective at inducing proliferation and osteogenic differentiation of hMSCs than continuous stimulation. Preconditioning with BMP2 could benefit the clinical application of BMP2 since side effects from high-dose treatments could be avoided....

  5. Effects of Naringin on Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Lihua Yin

    2015-01-01

    Full Text Available This study is to explore the osteogenesis potential of the human periodontal ligament stem cells (hPDLSCs induced by naringin in vitro and in vitro. The results confirmed that 1 μM naringin performs the best effect and a collection of bone-related genes (RUNX2, COL1A2, OPN, and OCN had significantly higher expression levels compared to the control group. Furthermore, a typical trabecular structure was observed in vivo, surrounded by a large amount of osteoblasts. These results demonstrated that naringin, at a concentration of 1 μM, can efficiently promote the proliferation and differentiation of hPDLSCs both in vitro and in vivo.

  6. Effects of Glucocorticoid Receptor Small Interfering RNA Delivered Using Poly Lactic-Co-Glycolic Acid Microparticles on Proliferation and Differentiation Capabilities of Human Mesenchymal Stromal Cells

    Science.gov (United States)

    Wei, Na; Joshi, Vijaya; Yu, Yang; Kim, NaJung; Krishnamachari, Yogita; Zhang, Qiang

    2012-01-01

    Bone marrow–derived mesenchymal stem cells (MSC) are a potential attractive source of cells for stem cell–based tissue regeneration, but the small number and reduced capabilities of MSC proliferation and differentiation due to in vitro replicative senescence and donor-associated pathophysiological factors, including age and estrogen depletion, severely restrict their potential usefulness in clinical applications. Glucocorticoids (GC) are well-known steroid hormones that regulate MSC proliferation and differentiation, but the defined effects and underlying mechanisms of endogenous glucocorticoids on MSC characteristics are not understood. This study investigated the effects of the blockage of endogenous GC using glucocorticoid receptor (GR) small interfering RNA (siRNA) delivered using biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles on proliferation and differentiation capabilities of human MSC in vitro. The results show that we can prepare PLGA microparticles as a delivery system for GR siRNA and maintain release of siRNA up to 40 days in vitro. Transfection of GR siRNA significantly downregulates GR and upregulates the expression of fibroblast growth factor-2 and Sox-11 of human MSC. MSC that have proliferated with endogenous GC blocked in vitro have greater proliferation rates and exhibit upregulated expression of osteogenic markers (alkaline phosphatase and core binding factor alpha 1) under differentiation stimulation after 1 week. Under adipogenic differentiation, MSC proliferated in vitro with siRNA transfection, resulting in significantly lower adipogenic markers (peroxisome proliferator-activated receptor and lipoprotein lipase) than controls. In conclusion, PLGA particles can serve as a tool for delivery of GR siRNA to effectively block the effects of endogenous GC on MSC, which has the potential to improve the capabilities of human MSC for clinical application by preventing replicative senescence. PMID:21988716

  7. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Favi, Pelagie M.; Benson, Roberto S. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Neilsen, Nancy R. [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Hammonds, Ryan L. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Bates, Cassandra C. [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Stephens, Christopher P. [Department of Surgery, Graduate School of Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Center for Materials Processing, University of Tennessee, Knoxville, TN 37996 (United States); Dhar, Madhu S., E-mail: mdhar@utk.edu [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States)

    2013-05-01

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications. - Highlights: ► Critical point drying induces fibre bundling unlike lyophilisation. ► Cells positively expressed undifferentiated pluripotent stem cell markers. ► BCs were cytocompatible, supported cell adhesion, proliferation and differentiation ► Cells seeded on BC scaffolds were viable and metabolically active. ► Findings demonstrate that BC and EqMSCs are promising tissue engineered constructs.

  8. [Effect of electroacupuncture on differentiation and proliferation of hippocampal nerve stem cells in splenic asthenia pedo-rats].

    Science.gov (United States)

    Zhuo, Yuan-yuan; Yang, Zhuo-xin; Wu, Jia-man

    2011-10-01

    To observe the effect of electroacupuncture (EA) on the differentiation and proliferation of nerve stem cells in the hippocampal dentate gyrus (DG) in splenic asthenia pedo-rats so as to study its central mechanism. A total of 72 SD male rats were randomly assigned to normal control group (n=24), model group (n=24) and EA group (n=24) which were further divided into 7 d, 14 d, 28 d and 49 d time-points (n=6). Splenic asthenia model was established by intraperitoneal injection of reserpine and gavage of Dahuang (Radix et Rhizoma Rhei) fluid. EA was applied to bilateral "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) for 20 min, once daily for 7, 14, 28 and 49 days respectively. Brdu, Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE) expression in the DG of hippocampus were detected by immunohistochemistry double staining. Compared with the normal control group, the numbers of Brdu, Brdu/GFAP, Brdu/NSE Immunoreactive (IR) positive cells in the DG of hippocampus on day 7 and 14, and that of Brdu/Nestin IR-positive cells on day 7 were decreased considerably in the model group (P 0.05). EA of ST 36 and SP 6 can effectively suppress splenic asthenia syndrome-induced decrease of the numbers of Brdu, Brdu/GFAP, Brdu/Nestin and Brdu/NSE IR-positive cells in the DG of hippocampus at the early stage in the splenic asthenia rats, which may contribute to its effect in improving splenic asthenia symptoms in clinic by promoting the proliferation and differentiation of some nerve stem cells in the hippocampus.

  9. Emerging role of LRRK2 in human neural progenitor cell cycle progression, survival and differentiation.

    Science.gov (United States)

    Milosevic, Javorina; Schwarz, Sigrid C; Ogunlade, Vera; Meyer, Anne K; Storch, Alexander; Schwarz, Johannes

    2009-06-15

    Despite a comprehensive mapping of the Parkinson's disease (PD)-related mRNA and protein leucine-rich repeat kinase 2 (LRRK2) in the mammalian brain, its physiological function in healthy individuals remains enigmatic. Based on its structural features and kinase properties, LRRK2 may interact with other proteins involved in signalling pathways. Here, we show a widespread LRRK2 mRNA and/or protein expression in expanded or differentiated human mesencephalic neural progenitor cells (hmNPCs) and in post-mortem substantia nigra PD patients. Using small interfering RNA duplexes targeting LRRK2 in hmNPCs following their differentiation into glia and neurons, we observed a reduced number of dopaminergic neurons due to apoptosis in LRRK2 knockdown samples. LRRK2-deficient hmNPCs exhibited elevated cell cycle- and cell death-related markers. In conclusion, a reduction of LRRK2 expression in hmNPCs severely impaired dopaminergic differentiation and/or survival of dopaminergic neurons most likely via preserving or reactivating the cell cycle.

  10. Emerging role of LRRK2 in human neural progenitor cell cycle progression, survival and differentiation

    Directory of Open Access Journals (Sweden)

    Meyer Anne K

    2009-06-01

    Full Text Available Abstract Despite a comprehensive mapping of the Parkinson's disease (PD-related mRNA and protein leucine-rich repeat kinase 2 (LRRK2 in the mammalian brain, its physiological function in healthy individuals remains enigmatic. Based on its structural features and kinase properties, LRRK2 may interact with other proteins involved in signalling pathways. Here, we show a widespread LRRK2 mRNA and/or protein expression in expanded or differentiated human mesencephalic neural progenitor cells (hmNPCs and in post-mortem substantia nigra PD patients. Using small interfering RNA duplexes targeting LRRK2 in hmNPCs following their differentiation into glia and neurons, we observed a reduced number of dopaminergic neurons due to apoptosis in LRRK2 knockdown samples. LRRK2-deficient hmNPCs exhibited elevated cell cycle- and cell death-related markers. In conclusion, a reduction of LRRK2 expression in hmNPCs severely impaired dopaminergic differentiation and/or survival of dopaminergic neurons most likely via preserving or reactivating the cell cycle.

  11. The Role of Nephronectin on Proliferation and Differentiation in Human Dental Pulp Stem Cells

    Directory of Open Access Journals (Sweden)

    Jia Tang

    2017-01-01

    Full Text Available Aim. The purpose of the current study was to investigate the effects of nephronectin (Npnt in human dental pulp stem cells (hDPSCs. Methodology. Npnt was coated to nontissue culture-treated polystyrene (non-PS plates. The presence of immobilized protein on the surface was detected by polyclonal rabbit primary anti-Npnt antibody. Then the cell number was counted and compared with PBS-, bovine serum albumin- (BSA-, fish scale type I collagen- (FCOL1-, and human fibronectin- (Fn- coated wells. Cell proliferation was assessed using CCK-8 assay. Cell morphology was observed under light microscopy and fluorescence microscopy. Lastly, the mRNA expression profiles of integrins, dentin sialophosphoprotein (DSPP, bone sialoprotein (BSP, and mineralization capacity of hDPSCs were investigated by real time RT-PCR and alizarin red staining, respectively. Results. Npnt mediates hDPSC adhesion and spreading partially via the Arg-Gly-Asp (RGD motif. Npnt enhanced the mRNA expression of ITGA1, ITGA4, ITGA7, and ITGB1 on day five. Npnt downregulated DSPP but significantly upregulated BSP mRNA expression at day 28. Further, Npnt and FCOL1 accelerated the matrix mineralization in hDPSCs. Conclusions. The current findings implicate that Npnt would be favorable to recruit hDPSCs and conducive to mineralization in hDPSCs. The combination of Npnt with hDPSCs may offer a promising approach for hard tissue regeneration.

  12. Fragile x mental retardation protein regulates proliferation and differentiation of adult neural stem/progenitor cells.

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    Yuping Luo

    2010-04-01

    Full Text Available Fragile X syndrome (FXS, the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP. FMRP is an RNA-binding protein that can regulate the translation of specific mRNAs. Adult neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. In this study, we investigated whether Fmrp deficiency affects adult neurogenesis. We show that in a mouse model of fragile X syndrome, adult neurogenesis is indeed altered. The loss of Fmrp increases the proliferation and alters the fate specification of adult neural progenitor/stem cells (aNPCs. We demonstrate that Fmrp regulates the protein expression of several components critical for aNPC function, including CDK4 and GSK3beta. Dysregulation of GSK3beta led to reduced Wnt signaling pathway activity, which altered the expression of neurogenin1 and the fate specification of aNPCs. These data unveil a novel regulatory role for Fmrp and translational regulation in adult neurogenesis.

  13. The Drosophila gonads: models for stem cell proliferation, self-renewal, and differentiation

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    John E. La Marca

    2014-12-01

    Full Text Available The male and female gonads of Drosophila melanogaster have developed into powerful model systems for both the study of stem cell behaviours, and for understanding how stem cell misregulation can lead to cancers. Using these systems, one is able to observe and manipulate the resident stem cell populations in vivo with a great deal of licence. The tractability of the testis and ovary also allow researchers to explore a range of cellular mechanisms, such as proliferation and polarity, as well as the influence exerted by the local environment through a host of highly-conserved signalling pathways. Importantly, many of the cellular behaviours and processes studied in the Drosophila testis and ovary are known to be disrupted, or otherwise misregulated, in human tumourigenic cells. Here, we review the mechanisms relating to stem cell behaviour, though we acknowledge there are many other fascinating aspects of gametogenesis, including the invasive behaviour of migratory border cells in the Drosophila ovary that, though relevant to the study of tumourigenesis, will unfortunately not be covered.

  14. Effects of Syzygium aromaticum, Cinnamomum zeylanicum, and Salvia triloba extracts on proliferation and differentiation of dental pulp stem cells.

    Science.gov (United States)

    Mendi, Ayşegül; Yağci, Beyza Gökçınar; Kiziloğlu, Mustafa; Saraç, Nurdan; Yilmaz, Derviş; Uğur, Aysel; Uçkan, Duygu

    2017-01-01

    Hypersensitivity, local irritative and cytotoxic effects are known for the chemical components of Syzygium aromaticum and Cinnamomum zeylanicum contained in dental materials. However, there is no intimate data in dentistry using the whole extracts of these plants and introducing new ones. Salvia triloba is a well-known anti-inflammatory plant that correspondingly could be used in several dental traumas. We aimed to show and compare the effect of S. aromaticum, C. zeylanicum, and S. triloba extracts on dental pulp stem cells (DPSCs) proliferation, differentiation, and immune responses. Using xCELLigence, a real time monitoring system, we obtained a growth curve of DPSCs with different concentrations of the Extracts. A dose of 10 μg/mL was the most efficient concentration for vitality. Osteogenic differentiation and anti-inflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. The level of osteonectin (ON, early osteogenic marker) decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation) differed among the extracts, in which S. aromaticum presented the highest value, followed by S. triloba and C. zeylanicum. Surprisingly, the determined calcium granules were reduced in S. aromaticum and S. triloba. In response to tumor necrosis factor alpha (TNF-α), S. triloba-treated DPSCs showed the most reduced level of IL-6 cytokine level. We suggest C. zeylanicum as a promising osteogenic inducer and S. triloba as a potent anti-inflammatory agent, which could be used safely in biocomposite or scaffold fabrications for dentistry. Because calcium granule formation and cell viability play a critical role in hard tissue formation, S. aromaticum in dentistry should be strictly controlled, and the mechanism leading to reduced calcium granule formation should be identified.

  15. Effects of Syzygium aromaticum, Cinnamomum zeylanicum, and Salvia triloba extracts on proliferation and differentiation of dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Ayşegül Mendi

    Full Text Available Abstract Hypersensitivity, local irritative and cytotoxic effects are known for the chemical components of Syzygium aromaticum and Cinnamomum zeylanicum contained in dental materials. However, there is no intimate data in dentistry using the whole extracts of these plants and introducing new ones. Salvia triloba is a well-known anti-inflammatory plant that correspondingly could be used in several dental traumas. Objectives: We aimed to show and compare the effect of S. aromaticum, C. zeylanicum, and S. triloba extracts on dental pulp stem cells (DPSCs proliferation, differentiation, and immune responses. Material and Methods: Using xCELLigence, a real time monitoring system, we obtained a growth curve of DPSCs with different concentrations of the Extracts. A dose of 10 μg/mL was the most efficient concentration for vitality. Osteogenic differentiation and anti-inflammatory activities were determined by using an ELISA Kit to detect early and late markers of differentiation. Results: The level of osteonectin (ON, early osteogenic marker decreased, which indicated that the osteogenic differentiation may be accelerated with addition of extracts. However, the level of osteocalcin (OCN, late osteogenic marker and sign of calcium granulation differed among the extracts, in which S. aromaticum presented the highest value, followed by S. triloba and C. zeylanicum. Surprisingly, the determined calcium granules were reduced in S. aromaticum and S. triloba. In response to tumor necrosis factor a