Ap4A induces apoptosis in human cultured cells.

PubMed

Vartanian, A; Alexandrov, I; Prudowski, I; McLennan, A; Kisselev, L

1999-07-30

Diadenosine oligophosphates (Ap(n)A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5',5'''-P1,P3-triphosphate to diadenosine 5',5'''-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of Ap(n)A into cells was achieved by cold shock. Ap4A at 10 microM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.

Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation*

PubMed Central

Hao, Wu; Tashiro, Syoichi; Hasegawa, Tomoka; Sato, Yuiko; Kobayashi, Tami; Tando, Toshimi; Katsuyama, Eri; Fujie, Atsuhiro; Watanabe, Ryuichi; Morita, Mayu; Miyamoto, Kana; Morioka, Hideo; Nakamura, Masaya; Matsumoto, Morio; Amizuka, Norio; Toyama, Yoshiaki; Miyamoto, Takeshi

2015-01-01

Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition. PMID:25998127

Keratin 8/18 regulation of glucose metabolism in normal versus cancerous hepatic cells through differential modulation of hexokinase status and insulin signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathew, Jasmin; Loranger, Anne; Gilbert, Stéphane

2013-02-15

As differentiated cells, hepatocytes primarily metabolize glucose for ATP production through oxidative phosphorylation of glycolytic pyruvate, whereas proliferative hepatocellular carcinoma (HCC) cells undergo a metabolic shift to aerobic glycolysis despite oxygen availability. Keratins, the intermediate filament (IF) proteins of epithelial cells, are expressed as pairs in a lineage/differentiation manner. Hepatocyte and HCC (hepatoma) cell IFs are made solely of keratins 8/18 (K8/K18), thus providing models of choice to address K8/K18 IF functions in normal and cancerous epithelial cells. Here, we demonstrate distinctive increases in glucose uptake, glucose-6-phosphate formation, lactate release, and glycogen formation in K8/K18 IF-lacking hepatocytes and/or hepatoma cellsmore » versus their respective IF-containing counterparts. We also show that the K8/K18-dependent glucose uptake/G6P formation is linked to alterations in hexokinase I/II/IV content and localization at mitochondria, with little effect on GLUT1 status. In addition, we find that the insulin-stimulated glycogen formation in normal hepatocytes involves the main PI-3 kinase-dependent signaling pathway and that the K8/K18 IF loss makes them more efficient glycogen producers. In comparison, the higher insulin-dependent glycogen formation in K8/K18 IF-lacking hepatoma cells is associated with a signaling occurring through a mTOR-dependent pathway, along with an augmentation in cell proliferative activity. Together, the results uncover a key K8/K18 regulation of glucose metabolism in normal and cancerous hepatic cells through differential modulations of mitochondrial HK status and insulin-mediated signaling.« less

Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

PubMed Central

Tan, Kah Yong; Eminli, Sarah; Hettmer, Simone; Hochedlinger, Konrad; Wagers, Amy J.

2011-01-01

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle. PMID:22028872

An RNA tool kit to study the status of mouse ES cells: sex determination and stemness.

PubMed

Jay, F; Ciaudo, C

2013-09-01

Mouse embryonic stem cells (mESCs) are pluripotent stem cells derived from the inner cell mass of the blastocyst. They can be maintained under controlled culture conditions in a pluripotent state, or be induced to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. Several studies have characterised the coding and non-coding (nc) RNA repertoires of mESCs, uncovering highly dynamic variations during the process of differentiation, but also qualitative differences pertaining to sex. For example, up-regulation of the long non-coding RNA Xist on the X chromosome induces gene silencing and X inactivation exclusively during female mESC differentiation. In contrast, specific small RNAs have been shown to be up-regulated during male mESC differentiation. Here, we illustrate how a small set of key coding and ncRNAs can be exploited as dynamic and sensitive markers of the stemness and/or the differentiation status of male or female mESC lines. We describe adapted techniques for the extended characterization and analysis of mESCs from as little material as that cultured in a single 75cm(2) flask. Copyright © 2013 Elsevier Inc. All rights reserved.

Determination of HER2 amplification status in breast cancer cells using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Bi, Xiaohong; Rexer, Brent; Arteaga, Carlos L.; Guo, Mingsheng; Li, Ming; Mahadevan-Jansen, Anita

2010-02-01

The overexpression of HER2 (human epidermal growth factor receptor 2) in breast cancer is associated with increased disease recurrence and worse prognosis. Current diagnosis of HER2 positive breast cancer is time consuming with an estimated 20% inaccuracy. Raman spectroscopy is a proven method for pathological diagnosis based on the molecular composition of tissues. This study aimed to determine the feasibility of Raman spectroscopy to differentially identify the amplification of HER2 in cells. Three cell lines including BT474 (HER2 overexpressing breast cancer cell), MCF-10A (human breast epithelial cell), and MCF-10A with overexpressing HER2, were investigated using a bench top confocal Raman system. A diagnostic algorithm based on generalized linear model (GLM) with elastic-net penalties was established to discriminate 318 spectra collected from the cells, and to identify the spectra regions that differentiate the cell lines. The algorithm was able to differentially identify BT474 breast cancer cells with an overall sensitivity of 100% and specificity of 99%. The results demonstrate the capability of Raman spectroscopy to determine HER2 status in cells. Raman spectroscopy shows promise for application in the diagnosis of HER2 positive breast cancer in clinical practice.

In situ electrochemical detection of embryonic stem cell differentiation.

PubMed

Yea, Cheol-Heon; An, Jeung Hee; Kim, Jungho; Choi, Jeong-Woo

2013-06-20

Stem cell sensors have emerged as a promising technique to electrochemically monitor the functional status and viability of stem cells. However, efficient electrochemical analysis techniques are required for the development of effective electrochemical stem cell sensors. In the current study, we report a newly developed electrochemical cyclic voltammetry (CV) system to determine the status of mouse embryonic stem (ES) cells. 1-Naphthly phosphate (1-NP), which was dephosphorylated by alkaline phosphatase into a 1-naphthol on an undifferentiated mouse ES cell, was used as a substrate to electrochemically monitor the differentiation status of mouse ES cells. The peak current in the cyclic voltammetry of 1-NP increased linearly with the concentration of pure 1-NP (R(2)=0.9623). On the other hand, the peak current in the electrochemical responses of 1-NP decreased as the number of undifferentiated ES cells increased. The increased dephosphorylation of 1-NP to 1-naphthol made a decreased electrochemical signal. Non-toxicity of 1-NP was confirmed. In conclusion, the proposed electrochemical analysis system can be applied to an electrical stem cell chip for diagnosis, drug detection and on-site monitoring. Copyright © 2013 Elsevier B.V. All rights reserved.

Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells.

PubMed

Liu, Zhenguo; Jiang, Yuehua; Hao, Hong; Gupta, Kalpna; Xu, Jian; Chu, Ling; McFalls, Edward; Zweier, Jay; Verfaillie, Catherine; Bache, Robert J

2007-09-01

This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor N(G)-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.

Glycoproteomic Analysis of Glioblastoma Stem Cell Differentiation

PubMed Central

He, Jintang; Liu, Yashu; Zhu, Thant S.; Xie, Xiaolei; Costello, Mark A.; Talsma, Caroline E.; Flack, Callie G.; Crowley, Jessica G.; DiMeco, Francesco; Vescovi, Angelo L.; Fan, Xing; Lubman, David M.

2010-01-01

Cancer stem cells are responsible for tumor formation through self-renewal and differentiation into multiple cell types, and thus represent a new therapeutic target for tumors. Glycoproteins play a critical role in determining the fates of stem cells such as self-renewal, proliferation and differentiation. Here we applied a multi-lectin affinity chromatography and quantitative glycoproteomics approach to analyze alterations of glycoproteins relevant to the differentiation of a glioblastoma-derived stem cell line HSR-GBM1. Three lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and peanut agglutinin (PNA) were used to capture glycoproteins, followed by LC-MS/MS analysis. A total of 73 and 79 high-confidence (FDR < 0.01) glycoproteins were identified from the undifferentiated and differentiated cells, respectively. Label-free quantitation resulted in the discovery of 18 differentially expressed glycoproteins, wherein 9 proteins are localized in the lysosome. All of these lysosomal glycoproteins were up-regulated after differentiation, where their principal function was hydrolysis of glycosyl residues. Protein-protein interaction and functional analyses revealed the active involvement of lysosomes during the process of glioblastoma stem cell differentiation. This work provides glycoprotein markers to characterize differentiation status of glioblastoma stem cells which may be useful in stemcell therapy of glioblastoma. PMID:21110520

Hyperglycemia Promotes Schwann Cell De-differentiation and De-myelination via Sorbitol Accumulation and Igf1 Protein Down-regulation.

PubMed

Hao, Wu; Tashiro, Syoichi; Hasegawa, Tomoka; Sato, Yuiko; Kobayashi, Tami; Tando, Toshimi; Katsuyama, Eri; Fujie, Atsuhiro; Watanabe, Ryuichi; Morita, Mayu; Miyamoto, Kana; Morioka, Hideo; Nakamura, Masaya; Matsumoto, Morio; Amizuka, Norio; Toyama, Yoshiaki; Miyamoto, Takeshi

2015-07-10

Diabetes mellitus (DM) is frequently accompanied by complications, such as peripheral nerve neuropathy. Schwann cells play a pivotal role in regulating peripheral nerve function and conduction velocity; however, changes in Schwann cell differentiation status in DM are not fully understood. Here, we report that Schwann cells de-differentiate into immature cells under hyperglycemic conditions as a result of sorbitol accumulation and decreased Igf1 expression in those cells. We found that de-differentiated Schwann cells could be re-differentiated in vitro into mature cells by treatment with an aldose reductase inhibitor, to reduce sorbitol levels, or with vitamin D3, to elevate Igf1 expression. In vivo DM models exhibited significantly reduced nerve function and conduction, Schwann cell de-differentiation, peripheral nerve de-myelination, and all conditions were significantly rescued by aldose reductase inhibitor or vitamin D3 administration. These findings reveal mechanisms underlying pathological changes in Schwann cells seen in DM and suggest ways to treat neurological conditions associated with this condition. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Donor cell differentiation, reprogramming, and cloning efficiency: elusive or illusive correlation?

PubMed

Oback, B; Wells, D N

2007-05-01

Compared to other assisted reproductive technologies, mammalian nuclear transfer (NT) cloning is inefficient in generating viable offspring. It has been postulated that nuclear reprogramming and cloning efficiency can be increased by choosing less differentiated cell types as nuclear donors. This hypothesis is mainly supported by comparative mouse cloning experiments using early blastomeres, embryonic stem (ES) cells, and terminally differentiated somatic donor cells. We have re-evaluated these comparisons, taking into account different NT procedures, the use of donor cells from different genetic backgrounds, sex, cell cycle stages, and the lack of robust statistical significance when post-blastocyst development is compared. We argue that while the reprogrammability of early blastomeres appears to be much higher than that of somatic cells, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic donor cell lineages. Copyright (c) 2006 Wiley-Liss, Inc.

Melanosome uptake is associated with the proliferation and differentiation of keratinocytes.

PubMed

Choi, Hye-In; Sohn, Kyung-Cheol; Hong, Dong-Kyun; Lee, Young; Kim, Chang Deok; Yoon, Tae-Jin; Park, Jin Woon; Jung, Sunggyun; Lee, Jeung-Hoon; Lee, Young Ho

2014-01-01

Melanosomes are synthesized in melanocytes and transferred to neighboring keratinocytes. However, the associations of melanosome uptake with the proliferation and differentiation of keratinocytes are not fully understood. We examined the associations of melanosome uptake with keratinocyte differentiation and proliferation. SV40T-transformed human epidermal keratinocytes (SV-HEKs) were treated with isolated melanosomes. The effects of melanosome uptake on the proliferation and differentiation of the keratinocytes were analyzed by Western blotting and flow cytometry. The relationship between melanosome uptake and keratinocyte differentiation status was verified by determining the melanin content in the cells. Melanosomes reduced the proliferation of SV-HEKs in a dose-dependent manner, but did not induce differentiation. Melanosome uptake was higher in differentiating keratinocytes compared to non-differentiating keratinocytes, and inhibited significantly by PAR-2 inhibitor. Melanosomes inhibit keratinocyte proliferation. Moreover, melanosome uptake is influenced by keratinocyte differentiation status, being highest in mid-stage differentiating keratinocytes in a PAR-2 dependent manner.

A paired comparison between glioblastoma "stem cells" and differentiated cells.

PubMed

Schneider, Matthias; Ströbele, Stephanie; Nonnenmacher, Lisa; Siegelin, Markus D; Tepper, Melanie; Stroh, Sebastien; Hasslacher, Sebastian; Enzenmüller, Stefanie; Strauss, Gudrun; Baumann, Bernd; Karpel-Massler, Georg; Westhoff, Mike-Andrew; Debatin, Klaus-Michael; Halatsch, Marc-Eric

2016-04-01

Cancer stem cells (CSC) have been postulated to be responsible for the key features of a malignancy and its maintenances, as well as therapy resistance, while differentiated cells are believed to make up the rapidly growing tumour bulk. It is therefore important to understand the characteristics of those two distinct cell populations in order to devise treatment strategies which effectively target both cohorts, in particular with respect to cancers, such as glioblastoma. Glioblastoma is the most common primary brain tumour in adults, with a mean patient survival of 12-15 months. Importantly, therapeutic improvements have not been forthcoming in the last decade. In this study we compare key features of three pairs of glioblastoma cell populations, each pair consisting of stem cell-like and differentiated cells derived from an individual patient. Our data suggest that while growth rates and expression of key survival- and apoptosis-mediating proteins are more similar according to differentiation status than genetic similarity, we found no intrinsic differences in response to standard therapeutic interventions, namely exposure to radiation or the alkylating agent temozolomide. Interestingly, we could demonstrate that both stem cell-like and differentiated cells possess the ability to form stem cell-containing tumours in immunocompromised mice and that differentiated cells could potentially be dedifferentiated to potential stem cells. Taken together our data suggest that the differences between tumour stem cell and differentiated cell are particular fluent in glioblastoma. © 2015 UICC.

Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

PubMed

Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

2018-05-01

Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Transdifferentiation of human periodontal ligament stem cells into pancreatic cell lineage.

PubMed

Lee, Jeong Seok; An, Seong Yeong; Kwon, Il Keun; Heo, Jung Sun

2014-10-01

Human periodontal ligament-derived stem cells (PDLSCs) demonstrate self-renewal capacity and multilineage differentiation potential. In this study, we investigated the transdifferentiation potential of human PDLSCs into pancreatic islet cells. To form three-dimensional (3D) clusters, PDLSCs were cultured in Matrigel with media containing differentiation-inducing agents. We found that after 6 days in culture, PDLSCs underwent morphological changes resembling pancreatic islet-like cell clusters (ICCs). The morphological characteristics of PDLSC-derived ICCs were further assessed using scanning electron microscopy analysis. Using reverse transcription-polymerase chain reaction analysis, we found that pluripotency genes were downregulated, whereas early endoderm and pancreatic differentiation genes were upregulated, in PDLSC-derived ICCs compared with undifferentiated PDLSCs. Furthermore, we found that PDLSC-derived ICCs were capable of secreting insulin in response to high concentrations of glucose, validating their functional differentiation into islet cells. Finally, we also performed dithizone staining, as well as immunofluorescence assays and fluorescence-activated cell sorting analysis for pancreatic differentiation markers, to confirm the differentiation status of PDLSC-derived ICCs. These results demonstrate that PDLSCs can transdifferentiate into functional pancreatic islet-like cells and provide a novel, alternative cell population for pancreatic repair. Copyright © 2014 John Wiley & Sons, Ltd.

Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium.

PubMed

Pelch, Katherine E; Tokar, Erik J; Merrick, B Alex; Waalkes, Michael P

2015-08-01

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. Published by Elsevier Inc.

SELENIUM-DEFICIENCY MODIFIES INFLUENZA INFECTION OF DIFFERENTIATED HUMAN BRONCHIAL EPITHELIAL CELLS

EPA Science Inventory

The nutritional status of the host is important in the defense against invading pathogens. Many studies regarding the effects of host nutritional status on the immune response have demonstrated that suboptimal host nutrition results in impaired host immunity and increased suscept...

Different roles of TGF-β in the multi-lineage differentiation of stem cells

PubMed Central

Wang, Ming-Ke; Sun, Hui-Qin; Xiang, Ying-Chun; Jiang, Fan; Su, Yong-Ping; Zou, Zhong-Min

2012-01-01

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells. Transforming growth factor β (TGF-β) family is a superfamily of growth factors, including TGF-β1, TGF-β2 and TGF-β3, bone morphogenetic proteins, activin/inhibin, and some other cytokines such as nodal, which plays very important roles in regulating a wide variety of biological processes, such as cell growth, differentiation, cell death. TGF-β, a pleiotropic cytokine, has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells, through the Smad pathway, non-Smad pathways including mitogen-activated protein kinase pathways, phosphatidylinositol-3-kinase/AKT pathways and Rho-like GTPase signaling pathways, and their cross-talks. For instance, it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells, immature cardiomyocytes, chondrocytes, neurocytes, hepatic stellate cells, Th17 cells, and dendritic cells. However, TGF-β inhibits the differentiation of stem cells into myotubes, adipocytes, endothelial cells, and natural killer cells. Additionally, TGF-β can provide competence for early stages of osteoblastic differentiation, but at late stages TGF-β acts as an inhibitor. The three mammalian isoforms (TGF-β1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology, and will facilitate both basic research and clinical applications of stem cells. In this article, we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells. PMID:22993659

Simultaneous Fluorescent Gram Staining and Activity Assessment of Activated Sludge Bacteria

PubMed Central

Forster, Scott; Snape, Jason R.; Lappin-Scott, Hilary M.; Porter, Jonathan

2002-01-01

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems. PMID:12324319

Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria.

PubMed

Forster, Scott; Snape, Jason R; Lappin-Scott, Hilary M; Porter, Jonathan

2002-10-01

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.

Pre-differentiation of mesenchymal stromal cells in combination with a microstructured nerve guide supports peripheral nerve regeneration in the rat sciatic nerve model.

PubMed

Boecker, Arne Hendrik; van Neerven, Sabien Geraldine Antonia; Scheffel, Juliane; Tank, Julian; Altinova, Haktan; Seidensticker, Katrin; Deumens, Ronald; Tolba, Rene; Weis, Joachim; Brook, Gary Anthony; Pallua, Norbert; Bozkurt, Ahmet

2016-02-01

Many bioartificial nerve guides have been investigated pre-clinically for their nerve regeneration-supporting function, often in comparison to autologous nerve transplantation, which is still regarded as the current clinical gold standard. Enrichment of these scaffolds with cells intended to support axonal regeneration has been explored as a strategy to boost axonal regeneration across these nerve guides Ansselin et al. (1998). In the present study, 20 mm rat sciatic nerve defects were implanted with a cell-seeded microstructured collagen nerve guide (Perimaix) or an autologous nerve graft. Under the influence of seeded, pre-differentiated mesenchymal stromal cells, axons regenerated well into the Perimaix nerve guide. Myelination-related parameters, like myelin sheath thickness, benefitted from an additional seeding with pre-differentiated mesenchymal stromal cells. Furthermore, both the number of retrogradely labelled sensory neurons and the axon density within the implant were elevated in the cell-seeded scaffold group with pre-differentiated mesenchymal stromal cells. However, a pre-differentiation had no influence on functional recovery. An additional cell seeding of the Perimaix nerve guide with mesenchymal stromal cells led to an extent of functional recovery, independent of the differentiation status, similar to autologous nerve transplantation. These findings encourage further investigations on pre-differentiated mesenchymal stromal cells as a cellular support for peripheral nerve regeneration. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Curcumin and its synthetic analogue dimethoxycurcumin differentially modulates antioxidant status of normal human peripheral blood mononuclear cells.

PubMed

Simon, Emmanuel; Aswini, P; Sameer Kumar, V B; Mankadath, Gokuldas

2018-05-01

Curcumin is a polyphenol derived from the herb Curcuma longa, which has been extensively studied in terms of its antitumour, antioxidant, and chemopreventive activity as well as various other effects. In the present work we compared curcumin with its synthetic analogue dimethoxycurcumin (dimc) in terms of its antioxidant enzyme-modulating effects in human peripheral blood mononuclear cells (PBMC). We found that these compounds modulate antioxidant enzymes differentially. Both curcumin and dimethoxycurcumin effected a decrease in lipid peroxidation status in PBMC, however, curcumin had better activity in this regard. An increase in the activity of catalase was seen in the case of curcumin-treated PBMC, whereas dimc increased catalase activity significantly to almost twofold level. Real time-polymerase chain reaction (RT-PCR) analysis revealed significant up-regulation of catalase at mRNA level post treatment with curcumin as well as dimc, however, dimc had better activity in this regard. Glutathione reductase (GR) activity and reduced glutathione levels increased in the case of peripheral blood mononuclear cells (PBMC) treated with curcumin, however, the trend was reversed with dimethoxycurcumin where, both glutathione reductase activity and reduced glutathione levels were significantly reduced. RT-PCR analysis of glutathione reductase mRNA levels showed decrease in mRNA levels post treatment with dimethoxycurcumin (dimc) further corroborating GR enzyme assay results, however, we could not obtain significant result post curcumin treatment. NFkB reporter assay and western blot analysis of nuclear as well as cytosolic fractions of NFkB revealed that curcumin inhibits NFkB activation whereas inhibition was much less with dimc. It has been reported that curcumin and dimc exerts differential cytotoxicity in normal and tumour cells and the reason for this had been attributed to the differential uptake of these compounds by normal cells and tumour cells. Based on our results we propose that differential modulation of antioxidant enzymes via NFkB pathway could be the reason behind differential cytotoxicity of dimc as well as curcumin in normal cells and tumour cells in addition to differential uptake of these compounds as reported previously.

Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

PubMed

Chao, Zhe; Zheng, Xin-Li; Sun, Rui-Ping; Liu, Hai-Long; Huang, Li-Li; Cao, Zong-Xi; Deng, Chang-Yan; Wang, Feng

2016-07-01

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

Cytokine-Induced Memory-Like Differentiation Enhances Unlicensed Natural Killer Cell Antileukemia and FcγRIIIa-Triggered Responses.

PubMed

Wagner, Julia A; Berrien-Elliott, Melissa M; Rosario, Maximillian; Leong, Jeffrey W; Jewell, Brea A; Schappe, Timothy; Abdel-Latif, Sara; Fehniger, Todd A

2017-03-01

Cytokine-induced memory-like natural killer (NK) cells differentiate after short-term preactivation with IL-12, IL-15, and IL-18 and display enhanced effector function in response to cytokines or tumor targets for weeks after the initial preactivation. Conventional NK cell function depends on a licensing signal, classically delivered by an inhibitory receptor engaging its cognate MHC class I ligand. How licensing status integrates with cytokine-induced memory-like NK cell responses is unknown. We investigated this interaction using killer cell immunoglobulin-like receptor- and HLA-genotyped primary human NK cells. Memory-like differentiation resulted in enhanced IFN-γ production triggered by leukemia targets or FcγRIIIa ligation within licensed NK cells, which exhibited the highest functionality of the NK cell subsets interrogated. IFN-γ production by unlicensed memory-like NK cells was also enhanced to a level comparable with that of licensed control NK cells. Mechanistically, differences in responses to FcγRIIIa-based triggering were not explained by alterations in key signaling intermediates, indicating that the underlying biology of memory-like NK cells is distinct from that of adaptive NK cells in human cytomegalovirus-positive individuals. Additionally, memory-like NK cells responded robustly to cytokine receptor restimulation with no impact of licensing status. These results demonstrate that both licensed and unlicensed memory-like NK cell populations have enhanced functionality, which may be translated to improve leukemia immunotherapy. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Metformin inhibition of neuroblastoma cell proliferation is differently modulated by cell differentiation induced by retinoic acid or overexpression of NDM29 non-coding RNA.

PubMed

Costa, Delfina; Gigoni, Arianna; Würth, Roberto; Cancedda, Ranieri; Florio, Tullio; Pagano, Aldo

2014-01-01

Metformin is a widely used oral hypoglycemizing agent recently proposed as potential anti-cancer drug. In this study we report the antiproliferative effect of metformin treatment in a high risk neuroblastoma cell model, focusing on possible effects associated to different levels of differentiation and/or tumor initiating potential. Antiproliferative and cytotoxic effects of metformin were tested in human SKNBE2 and SH-SY5Y neuroblastoma cell lines and in SKNBE2 cells in which differentiation is induced by retinoic acid treatment or stable overexpression of NDM29 non-coding RNA, both conditions characterized by a neuron-like differentiated phenotype. We found that metformin significantly inhibits the proliferation of NB cells, an effect that correlates with the inhibition of Akt, while AMPK activity resulted unchanged. Notably, metformin effects were modulated in a different ways by differentiating stimuli, being abolished after retinoic acid treatment but potentiated by overexpression of NDM29. These data suggest the efficacy of metformin as neuroblastoma anticancer agent, and support the requirement of further studies on the possible role of the differentiation status on the antiproliferative effects of this drug.

Differentiation-induced skin cancer suppression by FOS, p53, and TACE/ADAM17

PubMed Central

Guinea-Viniegra, Juan; Zenz, Rainer; Scheuch, Harald; Jiménez, María; Bakiri, Latifa; Petzelbauer, Peter; Wagner, Erwin F.

2012-01-01

Squamous cell carcinomas (SCCs) are heterogeneous and aggressive skin tumors for which innovative, targeted therapies are needed. Here, we identify a p53/TACE pathway that is negatively regulated by FOS and show that the FOS/p53/TACE axis suppresses SCC by inducing differentiation. We found that epidermal Fos deletion in mouse tumor models or pharmacological FOS/AP-1 inhibition in human SCC cell lines induced p53 expression. Epidermal cell differentiation and skin tumor suppression were caused by a p53-dependent transcriptional activation of the metalloprotease TACE/ADAM17 (TNF-α–converting enzyme), a previously unknown p53 target gene that was required for NOTCH1 activation. Although half of cutaneous human SCCs display p53-inactivating mutations, restoring p53/TACE activity in mouse and human skin SCCs induced tumor cell differentiation independently of the p53 status. We propose FOS/AP-1 inhibition or p53/TACE reactivating strategies as differentiation-inducing therapies for SCCs. PMID:22772468

TGF-β control of stem cell differentiation genes.

PubMed

Massagué, Joan; Xi, Qiaoran

2012-07-04

The canonical TGF-β/Smad signaling pathway was delineated in the mid 90s and enriched over the past decade with many findings about its specificity, regulation, networking, and malfunctions in disease. However, a growing understanding of the chromatin status of a critical class of TGF-β target genes - the genes controlling differentiation of embryonic stem cells - recently prompted a reexamination of this pathway and its critical role in the regulation of stem cell differentiation. The new findings reveal master regulators of the pluripotent state set the stage for Smad-mediated activation of master regulators of the next differentiation stage. Furthermore, a novel branch of the TGF-β/Smad pathway has been identified in which a chromatin-reading Smad complex makes the master differentiation genes accessible to canonical Smad complexes for transcriptional activation. These findings provide exciting new insights into the global role of TGF-β signaling in the regulators of stem cell fate. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

PubMed

Pandolfini, Luca; Luzi, Ettore; Bressan, Dario; Ucciferri, Nadia; Bertacchi, Michele; Brandi, Rossella; Rocchiccioli, Silvia; D'Onofrio, Mara; Cremisi, Federico

2016-05-06

Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.

Placenta-an alternative source of stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matikainen, Tiina; Laine, Jarmo

2005-09-01

The two most promising practical applications of human stem cells are cellular replacement therapies in human disease and toxicological screening of candidate drug molecules. Both require a source of human stem cells that can be isolated, purified, expanded in number and differentiated into the cell type of choice in a controlled manner. Currently, uses of both embryonic and adult stem cells are investigated. While embryonic stem cells are pluripotent and can differentiate into any specialised cell type, their use requires establishment of embryonic stem cell lines using the inner cell mass of an early pre-implantation embryo. As the blastocyst ismore » destroyed during the process, ethical issues need to be carefully considered. The use of embryonic stem cells is also limited by the difficulties in growing large numbers of the cells without inducing spontaneous differentiation, and the problems in controlling directed differentiation of the cells. The use of adult stem cells, typically derived from bone marrow, but also from other tissues, is ethically non-controversial but their differentiation potential is more limited than that of the embryonic stem cells. Since human cord blood, umbilical cord, placenta and amnion are normally discarded at birth, they provide an easily accessible alternative source of stem cells. We review the potential and current status of the use of adult stem cells derived from the placenta or umbilical cord in therapeutic and toxicological applications.« less

PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes

PubMed Central

Ferguson, Ross; Subramanian, Vasanta

2016-01-01

Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease. PMID:27391595

PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes.

PubMed

Ferguson, Ross; Subramanian, Vasanta

2016-01-01

Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease.

Analysis of E2F factors during epidermal differentiation.

PubMed

Chang, Wing Y; Dagnino, Lina

2005-01-01

The multigene E2F family of transcription factors is central in the control of cell cycle progression. The expression and activity of E2F proteins is tightly regulated transcriptionally and posttranslationally as a function of the proliferation and differentiation status of the cell. In this chapter, we review protocols designed to determine E2F mRNA abundance in tissues by in situ hybridization techniques. The ability to culture primary epidermal keratinocytes and maintain them as either undifferentiated or terminally differentiated cells allows the biochemical and molecular characterization of changes in E2F expression and activity. Thus, we also discuss in detail methods to analyze E2F protein abundance by immunoblot and their ability to bind DNA in cultured cells using electrophoretic mobility shift assays.

Interplay between DNA methylation, histone modification and chromatin remodeling in stem cells and during development.

PubMed

Ikegami, Kohta; Ohgane, Jun; Tanaka, Satoshi; Yagi, Shintaro; Shiota, Kunio

2009-01-01

Genes constitute only a small proportion of the mammalian genome, the majority of which is composed of non-genic repetitive elements including interspersed repeats and satellites. A unique feature of the mammalian genome is that there are numerous tissue-dependent, differentially methylated regions (T-DMRs) in the non-repetitive sequences, which include genes and their regulatory elements. The epigenetic status of T-DMRs varies from that of repetitive elements and constitutes the DNA methylation profile genome-wide. Since the DNA methylation profile is specific to each cell and tissue type, much like a fingerprint, it can be used as a means of identification. The formation of DNA methylation profiles is the basis for cell differentiation and development in mammals. The epigenetic status of each T-DMR is regulated by the interplay between DNA methyltransferases, histone modification enzymes, histone subtypes, non-histone nuclear proteins and non-coding RNAs. In this review, we will discuss how these epigenetic factors cooperate to establish cell- and tissue-specific DNA methylation profiles.

Augmented macrophage differentiation and polarization of tumor-associated macrophages towards M1 subtype in listeria-administered tumor-bearing host.

PubMed

Rai, Rakesh K; Vishvakarma, Naveen K; Mohapatra, Tribhuban M; Singh, Sukh Mahendra

2012-09-01

This study investigates the effect of Listeria administration on differentiation of macrophages from precursor bone marrow cells and functional status of tumor-associated macrophages (TAM). Listeria administration not only resulted in an augmented infiltration of tumor by F4/80 macrophages but also repolarized the functional status of TAM displaying features of some M1 macrophage subtype with upregulated phagocytosis and tumoricidal activity accompanied by altered expression of monocarboxylate transporter-1, toll-like receptor-2, surface markers: CD11c, interleukin-2 receptor, CD62L, and secreted molecules: nitric oxide, interleukin (IL)-1, IL-6, tumor necrosis factor-α, and vascular endothelial growth factor. Declined tumor cell survival and modulated repertoire of cytokines: interferon-γ, IL-6, IL-10, and transforming growth factor-β in tumor microenvironment indicated their role in polarization of TAM towards proinflammatory state. Bone marrow cell of Listeria-administered tumor-bearing mice showed augmented survival, declined expression of p53 upregulated modulator of apoptosis with an upregulated differentiation into activation responsive bone marrow-derived macrophages along with altered expression of macrophage-colony stimulating factor, macrophage-colony stimulating factor receptor, and granulocyte macrophage-colony stimulating factor receptor. These findings indicate that Listeria infection is associated with an augmented differentiation of macrophages accompanied by tumoricidal activation of TAM.

Differentiation and upregulation of heat shock protein 70 induced by a subset of histone deacetylase inhibitors in mouse and human embryonic stem cells.

PubMed

Park, Jeong-A; Kim, Young-Eun; Seok, Hyun-Jeong; Park, Woo-Youn; Kwon, Hyung-Joo; Lee, Younghee

2011-03-01

Inhibiting histone deacetylase (HDAC) activity modulates the epigenetic status of cells, resulting in an alteration of gene expression and cellular function. Here, we investigated the effects of HDAC inhibitors on mouse embryonic stem (ES) cells. The HDAC inhibitors trichostatin A, suberoylanilide hydroxamic acid, sodium butyrate, and valproic acid induced early differentiation of mouse ES cells and triggered induction of heatshock protein (HSP)70. In contrast, class III HDAC inhibitors failed to induce differentiation or HSP70 expression. Transcriptional upregulation of HSP70 was confirmed by mRNA expression analysis, an inhibitor study, and chromatin immunoprecipitation. HSP70 induction was dependent on the SAPK/ JNK, p38, and PI3K/Akt pathways. Differentiation and induction of HSP70 by a subset of HDAC inhibitors was also examined in human ES cells, which suggests that the phenomenon generally occurs in ES cells. A better understanding of the effects of HDAC inhibitors may give more insight into their application in stem cell biology.

Differential response of DU145 and PC3 prostate cancer cells to ionizing radiation: role of reactive oxygen species, GSH and Nrf2 in radiosensitivity.

PubMed

Jayakumar, Sundarraj; Kunwar, Amit; Sandur, Santosh K; Pandey, Badri N; Chaubey, Ramesh C

2014-01-01

Radioresistance is the major impediment in radiotherapy of many cancers including prostate cancer, necessitating the need to understand the factors contributing to radioresistance in tumor cells. In the present study, the role of cellular redox and redox sensitive transcription factor, Nrf2 in the radiosensitivity of prostate cancer cell lines PC3 and DU145, has been investigated. Differential radiosensitivity of PC3 and DU145 cells was assessed using clonogenic assay, flow cytometry, and comet assay. Their redox status was measured using DCFDA and DHR probes. Expression of Nrf2 and its dependent genes was measured by EMSA and real time PCR. Knockdown studies were done using shRNA transfection. PC3 and DU145 cells differed significantly in their radiosensitivity as observed by clonogenic survival, apoptosis and neutral comet assays. Both basal and inducible levels of ROS were higher in PC3 cells than that of DU145 cells. DU145 cells showed higher level of basal GSH content and GSH/GSSG ratio than that of PC3 cells. Further, significant increase in both basal and induced levels of Nrf2 and its dependent genes was observed in DU145 cells. Knock-down experiments and pharmacological intervention studies revealed the involvement of Nrf2 in differential radio-resistance of these cells. Cellular redox status and Nrf2 levels play a causal role in radio-resistance of prostate cancer cells. The pivotal role Nrf2 has been shown in the radioresistance of tumor cells and this study will further help in exploiting this factor in radiosensitization of other tumor cell types. © 2013.

Identification of a role for the nuclear receptor EAR-2 in the maintenance of clonogenic status within the leukemia cell hierarchy

PubMed Central

Ichim, CV; Atkins, HL; Iscove, NN; Wells, RA

2016-01-01

Identification of genes that regulate clonogenicity of acute myelogenous leukemia (AML) cells is hindered by the difficulty of isolating pure populations of cells with defined proliferative abilities. By analyzing the growth of clonal siblings in low passage cultures of the cell line OCI/AML4 we resolved this heterogeneous population into strata of distinct clonogenic potential, permitting analysis of the transcriptional signature of single cells with defined proliferative abilities. By microarray analysis we showed that the expression of the orphan nuclear receptor EAR-2 (NR2F6) is greater in leukemia cells with extensive proliferative capacity than in those that have lost proliferative ability. EAR-2 is expressed highly in long-term hematopoietic stem cells, relative to short-term hematopoietic stem and progenitor cells, and is downregulated in AML cells after induction of differentiation. Exogenous expression of EAR-2 increased the growth of U937 cells and prevented the proliferative arrest associated with terminal differentiation, and blocked differentiation of U937 and 32Dcl3 cells. Conversely, silencing of EAR-2 by short-hairpin RNA initiated terminal differentiation of these cell lines. These data identify EAR-2 as an important factor in the regulation of clonogenicity and differentiation, and establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy. PMID:21637284

Assessment of stem cell differentiation based on genome-wide expression profiles.

PubMed

Godoy, Patricio; Schmidt-Heck, Wolfgang; Hellwig, Birte; Nell, Patrick; Feuerborn, David; Rahnenführer, Jörg; Kattler, Kathrin; Walter, Jörn; Blüthgen, Nils; Hengstler, Jan G

2018-07-05

In recent years, protocols have been established to differentiate stem and precursor cells into more mature cell types. However, progress in this field has been hampered by difficulties to assess the differentiation status of stem cell-derived cells in an unbiased manner. Here, we present an analysis pipeline based on published data and methods to quantify the degree of differentiation and to identify transcriptional control factors explaining differences from the intended target cells or tissues. The pipeline requires RNA-Seq or gene array data of the stem cell starting population, derived 'mature' cells and primary target cells or tissue. It consists of a principal component analysis to represent global expression changes and to identify possible problems of the dataset that require special attention, such as: batch effects; clustering techniques to identify gene groups with similar features; over-representation analysis to characterize biological motifs and transcriptional control factors of the identified gene clusters; and metagenes as well as gene regulatory networks for quantitative cell-type assessment and identification of influential transcription factors. Possibilities and limitations of the analysis pipeline are illustrated using the example of human embryonic stem cell and human induced pluripotent cells to generate 'hepatocyte-like cells'. The pipeline quantifies the degree of incomplete differentiation as well as remaining stemness and identifies unwanted features, such as colon- and fibroblast-associated gene clusters that are absent in real hepatocytes but typically induced by currently available differentiation protocols. Finally, transcription factors responsible for incomplete and unwanted differentiation are identified. The proposed method is widely applicable and allows an unbiased and quantitative assessment of stem cell-derived cells.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Author(s).

Knockdown of SALL4 Protein Enhances All-trans Retinoic Acid-induced Cellular Differentiation in Acute Myeloid Leukemia Cells*

PubMed Central

Liu, Li; Liu, Liang; Leung, Lai-Han; Cooney, Austin J.; Chen, Changyi; Rosengart, Todd K.; Ma, Yupo; Yang, Jianchang

2015-01-01

All-trans retinoic acid (ATRA) is a differentiation agent that revolutionized the treatment of acute promyelocytic leukemia. However, it has not been useful for other types of acute myeloid leukemia (AML). Here we explored the effect of SALL4, a stem cell factor, on ATRA-induced AML differentiation in both ATRA-sensitive and ATRA-resistant AML cells. Aberrant SALL4 expression has been found in nearly all human AML cases, whereas, in normal bone marrow and peripheral blood cells, its expression is only restricted to hematopoietic stem/progenitor cells. We reason that, in AMLs, SALL4 activation may prevent cell differentiation and/or protect self-renewal that is seen in normal hematopoietic stem/progenitor cells. Indeed, our studies show that ATRA-mediated myeloid differentiation can be largely blocked by exogenous expression of SALL4, whereas ATRA plus SALL4 knockdown causes significantly increased AML differentiation and cell death. Mechanistic studies indicate that SALL4 directly associates with retinoic acid receptor α and modulates ATRA target gene expression. SALL4 is shown to recruit lysine-specific histone demethylase 1 (LSD1) to target genes and alter the histone methylation status. Furthermore, coinhibition of LSD1 and SALL4 plus ATRA treatment exhibited the strongest anti-AML effect. These findings suggest that SALL4 plays an unfavorable role in ATRA-based regimes, highlighting an important aspect of leukemia therapy. PMID:25737450

β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

PubMed

Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

2018-01-01

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

Role of Melatonin in the Regulation of Differentiation of T Cells Producing Interleukin-17 (Th17).

PubMed

Kuklina, E M; Glebezdina, N S; Nekrasova, I V

2016-03-01

We studied the ability of melatonin in physiological and pharmacological concentrations to induce and/or regulate differentiation of T cells producing IL-17 (Th17). This hormone produced the opposite effect on CD4+T cells, which depended on their activation status. Melatonin induced the synthesis of IL-17A by intact T cells, but had little effect on activated cells. Melatonin in high (pharmacological) concentration decreased the intracellular expression of this cytokine under conditions of polyclonal activation. Melatonin had a dose-depended effect. Taking into the fact that Th17 cells play an important role in the immune defense, it can be suggested that the regulation of their activity by melatonin contributes to this process.

Loss of end-differentiated β-cell phenotype following pancreatic islet transplantation.

PubMed

Anderson, S J; White, M G; Armour, S L; Maheshwari, R; Tiniakos, D; Muller, Y D; Berishvili, E; Berney, T; Shaw, J A M

2018-03-01

Replacement of pancreatic β-cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life-threatening hypoglycemia in type 1 diabetes. Although near-normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional β-cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress-induced β-cell dysfunction in type 2 diabetes. Here we report loss of end-differentiated β-cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin-positive cells had lost expression of the end-differentiation marker, urocortin-3, or appeared to co-express the α-cell marker, glucagon. In contrast, no insulin + /urocortin-3 - cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end-differentiated phenotype may facilitate β-cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β-cell sources, graft sites, and ultimately fully vascularized bioengineered insulin-secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β-cell phenotype has been maintained. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

The effect of nutritional status and myogenic satellite cell age on turkey satellite cell proliferation, differentiation, and expression of myogenic transcriptional regulatory factors and heparan sulfate proteoglycans syndecan-4 and glypican-1.

PubMed

Harthan, Laura B; McFarland, Douglas C; Velleman, Sandra G

2014-01-01

Posthatch satellite cell mitotic activity is a critical component of muscle development and growth. Satellite cells are myogenic stem cells that can be induced by nutrition to follow other cellular developmental pathways, and whose mitotic activity declines with age. The objective of the current study was to determine the effect of restricting protein synthesis on the proliferation and differentiation, expression of myogenic transcriptional regulatory factors myogenic determination factor 1, myogenin, and myogenic regulatory factor 4, and expression of the heparan sulfate proteoglycans syndecan-4 and glypican-1 in satellite cells isolated from 1-d-, 7-wk-, and 16-wk-old turkey pectoralis major muscle (1 d, 7 wk, and 16 wk cells, respectively) by using variable concentrations of Met and Cys. Four Met concentrations-30 (control), 7.5, 3, or 0 mg/L with 3.2 mg/L of Cys per 1 mg/L of Met-were used for culture of satellite cells to determine the effect of nutrition and age on satellite cell behavior during proliferation and differentiation. Proliferation was reduced by lower Met and Cys concentrations in all ages at 96 h of proliferation. Differentiation was increased in the 1 d Met-restricted cells, whereas the 7 wk cells treated with 3 mg/L of Met had decreased differentiation. Reduced Met and Cys levels from the control did not significantly affect the 16 wk cells at 72 h of differentiation. However, medium with no Met or Cys suppressed differentiation at all ages. The expression of myogenic determination factor 1, myogenin, myogenic regulatory factor 4, syndecan-4, and glypican-1 was differentially affected by age and Met or Cys treatment. These data demonstrate the age-specific manner in which turkey pectoralis major muscle satellite cells respond to nutritional availability and the importance of defining optimal nutrition to maximize satellite cell proliferation and differentiation for subsequent muscle mass accretion.

Neural differentiation of mesenchymal stem cells influences chemotactic responses to HGF.

PubMed

Zheng, Bing; Wang, Chunyan; He, Lihong; Xu, Xiaojing; Qu, Jing; Hu, Jun; Zhang, Huanxiang

2013-01-01

Recently, mesenchymal stem cells (MSCs) have been extensively used for cell-based therapies in neuronal degenerative disease. Although much effort has been devoted to the delineation of factors involved in the migration of MSCs, the relationship between the chemotactic responses and the differentiation status of these cells remains elusive. Here, we report that MSCs in varying neural differentiation states display different chemotactic responses to hepatocyte growth factor (HGF): first, the number of chemotaxing MSCs and the optimal concentrations of HGF that induced the peak migration varied greatly; second, time-lapse video analysis showed that MSCs in certain differentiation state migrated more efficiently toward HGF; third, the phosphorylation levels of Akt, ERK1/2, SAPK/JNK, and p38MAPK were closely related to the differentiation levels of MSCs subjected to HGF; and finally, although inhibition of ERK1/2 signaling significantly attenuated HGF-stimulated transfilter migration of both undifferentiated and differentiating MSCs, abolishment of PI3K/Akt, p38MAPK, or SAPK/JNK signaling only decreased the number of migrated cells in certain differentiation state(s). Blocking of PI3K/Akt or MAPK signaling impaired the migration efficiency and/or speed, the extent of which depends on the cell differentiation states. Meanwhile, F-actin rearrangement, which is essential for MSCs chemotaxis, was induced by HGF, and the time points of cytoskeletal reorganization were different among these cells. Collectively, these results demonstrate that neural differentiation of MSCs influences their chemotactic responses to HGF: MSCs in varying differentiation states possess different migratory capacities, thereby shedding light on optimization of the therapeutic potential of MSCs to be employed for neural regeneration after injury. Copyright © 2012 Wiley Periodicals, Inc.

CCR6+ Th cell distribution differentiates systemic lupus erythematosus patients based on anti-dsDNA antibody status.

PubMed

Zhong, Wei; Jiang, Zhenyu; Wu, Jiang; Jiang, Yanfang; Zhao, Ling

2018-01-01

Systemic lupus erythematosus (SLE) disease has been shown to be associated with the generation of multiple auto-antibodies. Among these, anti-dsDNA antibodies (anti-DNAs) are specific and play a pathogenic role in SLE. Indeed, anti-DNA + SLE patients display a worse disease course. The generation of these pathogenic anti-DNAs has been attributed to the interaction between aberrant T helper (Th) cells and autoimmune B cells. Thus, in this study we have investigated whether CCR6 + Th cells have the ability to differentiate SLE patients based on anti-DNA status, and if their distribution has any correlation with disease activity. We recruited 25 anti-DNA + and 25 anti-DNA - treatment-naive onset SLE patients, matched for various clinical characteristics in our nested matched case-control study. CCR6 + Th cells and their additional subsets were analyzed in each patient by flow cytometry. Anti-DNA + SLE patients specifically had a higher percentage of Th cells expressing CCR6 and CXCR3. Further analysis of CCR6 + Th cell subsets showed that anti-DNA + SLE patients had elevated proportions of Th9, Th17, Th17.1 and CCR4/CXCR3 double-negative (DN) cells. However, the proportions of CCR6 - Th subsets, including Th1 and Th2 cells, did not show any association with anti-DNA status. Finally, we identified a correlation between CCR6 + Th subsets and clinical indicators, specifically in anti-DNA + SLE patients. Our data indicated that CCR6 + Th cells and their subsets were elevated and correlated with disease activity in anti-DNA + SLE patients. We speculated that CCR6 + Th cells may contribute to distinct disease severity in anti-DNA + SLE patients.

Formaldehyde exposure impairs the function and differentiation of NK cells.

PubMed

Kim, Eun-Mi; Lee, Hwa-Youn; Lee, Eun-Hee; Lee, Ki-Mo; Park, Min; Ji, Kon-Young; Jang, Ji-Hun; Jeong, Yun-Hwa; Lee, Kwang-Ho; Yoon, Il-Joo; Kim, Su-Man; Jeong, Moon-Jin; Kim, Kwang Dong; Kang, Hyung-Sik

2013-11-25

We investigated the cytotoxic effects of formaldehyde (FA) on lymphocytes. FA-exposed mice showed a profound reduction not only in the number of natural killer (NK) cells but also in the expression of NK cell-specific receptors, but these mice did not exhibit decreases in the numbers of T or B lymphocytes. FA exposure also induced decreases in NK cytolytic activity and in the expression of NK cell-associated genes, such as IFN-γ, perforin and CD122. To determine the effect of FA on tumorigenicity, C57BL/6 mice were subcutaneously injected with B16F10 melanoma cells after FA exposure. The mass of the B16F10 tumor and the concentration of extravascular polymorphonuclear leukocytes were greater than those in unexposed tumor-bearing control mice. The number and cytolytic activity of NK cells were also reduced in B16F10 tumor-bearing mice exposed to FA. To determine how FA reduces the NK cell number, NK precursor (pNK) cells were treated with FA, and the differentiation status of the NK cells was analyzed. NK cell differentiation was impaired by FA treatment in a concentration-dependent manner. These findings indicate that FA exposure may promote tumor progression by impairing NK cell function and differentiation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Alternative Sources of Adult Stem Cells: Human Amniotic Membrane

NASA Astrophysics Data System (ADS)

Wolbank, Susanne; van Griensven, Martijn; Grillari-Voglauer, Regina; Peterbauer-Scherb, Anja

Human amniotic membrane is a highly promising cell source for tissue engineering. The cells thereof, human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC), may be immunoprivileged, they represent an early developmental status, and their application is ethically uncontroversial. Cell banking strategies may use freshly isolated cells or involve in vitro expansion to increase cell numbers. Therefore, we have thoroughly characterized the effect of in vitro cultivation on both phenotype and differentiation potential of hAEC. Moreover, we present different strategies to improve expansion including replacement of animal-derived supplements by human platelet products or the introduction of the catalytic subunit of human telomerase to extend the in vitro lifespan of amniotic cells. Characterization of the resulting cultures includes phenotype, growth characteristics, and differentiation potential, as well as immunogenic and immunomodulatory properties.

Body mass index and risk of colorectal carcinoma subtypes classified by tumor differentiation status.

PubMed

Hanyuda, Akiko; Cao, Yin; Hamada, Tsuyoshi; Nowak, Jonathan A; Qian, Zhi Rong; Masugi, Yohei; da Silva, Annacarolina; Liu, Li; Kosumi, Keisuke; Soong, Thing Rinda; Jhun, Iny; Wu, Kana; Zhang, Xuehong; Song, Mingyang; Meyerhardt, Jeffrey A; Chan, Andrew T; Fuchs, Charles S; Giovannucci, Edward L; Ogino, Shuji; Nishihara, Reiko

2017-05-01

Previous studies suggest that abnormal energy balance status may dysregulate intestinal epithelial homeostasis and promote colorectal carcinogenesis, yet little is known about how host energy balance and obesity influence enterocyte differentiation during carcinogenesis. We hypothesized that the association between high body mass index (BMI) and colorectal carcinoma incidence might differ according to tumor histopathologic differentiation status. Using databases of the Nurses' Health Study and Health Professionals Follow-up Study, and duplication-method Cox proportional hazards models, we prospectively examined an association between BMI and the incidence of colorectal carcinoma subtypes classified by differentiation features. 120,813 participants were followed for 26 or 32 years and 1528 rectal and colon cancer cases with available tumor pathological data were documented. The association between BMI and colorectal cancer risk significantly differed depending on the presence or absence of poorly-differentiated foci (P heterogeneity  = 0.006). Higher BMI was associated with a higher risk of colorectal carcinoma without poorly-differentiated foci (≥30.0 vs. 18.5-22.4 kg/m 2 : multivariable-adjusted hazard ratio, 1.87; 95% confidence interval, 1.49-2.34; P trend  < 0.001), but not with risk of carcinoma with poorly-differentiated foci (P trend  = 0.56). This differential association appeared to be consistent in strata of tumor microsatellite instability or FASN expression status, although the statistical power was limited. The association between BMI and colorectal carcinoma risk did not significantly differ by overall tumor differentiation, mucinous differentiation, or signet ring cell component (P heterogeneity  > 0.03, with the adjusted α of 0.01). High BMI was associated with risk of colorectal cancer subtype containing no poorly-differentiated focus. Our findings suggest that carcinogenic influence of excess energy balance might be stronger for tumors that retain better intestinal differentiation throughout the tumor areas.

TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

PubMed

Hawse, William F; Boggess, William C; Morel, Penelope A

2017-07-15

The Akt/mTOR pathway is a key driver of murine CD4 + T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. Copyright © 2017 by The American Association of Immunologists, Inc.

Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4+ T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals.

PubMed

Mauro, Claudio; Smith, Joanne; Cucchi, Danilo; Coe, David; Fu, Hongmei; Bonacina, Fabrizia; Baragetti, Andrea; Cermenati, Gaia; Caruso, Donatella; Mitro, Nico; Catapano, Alberico L; Ammirati, Enrico; Longhi, Maria P; Okkenhaug, Klaus; Norata, Giuseppe D; Marelli-Berg, Federica M

2017-03-07

Low-grade systemic inflammation associated to obesity leads to cardiovascular complications, caused partly by infiltration of adipose and vascular tissue by effector T cells. The signals leading to T cell differentiation and tissue infiltration during obesity are poorly understood. We tested whether saturated fatty acid-induced metabolic stress affects differentiation and trafficking patterns of CD4 + T cells. Memory CD4 + T cells primed in high-fat diet-fed donors preferentially migrated to non-lymphoid, inflammatory sites, independent of the metabolic status of the hosts. This was due to biased CD4 + T cell differentiation into CD44 hi -CCR7 lo -CD62L lo -CXCR3 + -LFA1 + effector memory-like T cells upon priming in high-fat diet-fed animals. Similar phenotype was observed in obese subjects in a cohort of free-living people. This developmental bias was independent of any crosstalk between CD4 + T cells and dendritic cells and was mediated via direct exposure of CD4 + T cells to palmitate, leading to increased activation of a PI3K p110δ-Akt-dependent pathway upon priming. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Placental-derived stem cells: Culture, differentiation and challenges

PubMed Central

Oliveira, Maira S; Barreto-Filho, João B

2015-01-01

Stem cell therapy is a promising approach to clinical healing in several diseases. A great variety of tissues (bone marrow, adipose tissue, and placenta) are potentially sources of stem cells. Placenta-derived stem cells (p-SCs) are in between embryonic and mesenchymal stem cells, sharing characteristics with both, such as non-carcinogenic status and property to differentiate in all embryonic germ layers. Moreover, their use is not ethically restricted as fetal membranes are considered medical waste after birth. In this context, the present review will be focused on the biological properties, culture and potential cell therapy uses of placental-derived stem cells. Immunophenotype characterization, mainly for surface marker expression, and basic principles of p-SC isolation and culture (mechanical separation or enzymatic digestion of the tissues, the most used culture media, cell plating conditions) will be presented. In addition, some preclinical studies that were performed in different medical areas will be cited, focusing on neurological, liver, pancreatic, heart, muscle, pulmonary, and bone diseases and also in tissue engineering field. Finally, some challenges for stem cell therapy applications will be highlighted. The understanding of the mechanisms involved in the p-SCs differentiation and the achievement of pure cell populations (after differentiation) are key points that must be clarified before bringing the preclinical studies, performed at the bench, to the medical practice. PMID:26029347

Differential staining of bacteria: gram stain.

PubMed

Moyes, Rita B; Reynolds, Jackie; Breakwell, Donald P

2009-11-01

In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures. (c) 2009 by John Wiley & Sons, Inc.

Stem Cell Metabolism in Cancer and Healthy Tissues: Pyruvate in the Limelight

PubMed Central

Corbet, Cyril

2018-01-01

Normal and cancer stem cells (CSCs) share the remarkable potential to self-renew and differentiate into many distinct cell types. Although most of the stem cells remain under quiescence to maintain their undifferentiated state, they can also undergo cell divisions as required to regulate tissue homeostasis. There is now a growing evidence that cell fate determination from stem cells implies a fine-tuned regulation of their energy balance and metabolic status. Stem cells can shift their metabolic substrate utilization, between glycolysis and mitochondrial oxidative metabolism, during specification and/or differentiation, as well as in order to adapt their microenvironmental niche. Pyruvate appears as a key metabolite since it is at the crossroads of cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. This Review describes how metabolic reprogramming, focusing on pyruvate utilization, drives the fate of normal and CSCs by modulating their capacity for self-renewal, clonal expansion/differentiation, as well as metastatic potential and treatment resistance in cancer. This Review also explores potential therapeutic strategies to restore or manipulate stem cell function through the use of small molecules targeting the pyruvate metabolism. PMID:29403375

DNA Damage Response, Redox Status and Hematopoiesis

PubMed Central

Weiss, Cary N.; Ito, Keisuke

2013-01-01

The ability of hematopoietic stem cells (HSCs) to self-renew and differentiate into progenitors is essential for homeostasis of the hematopoietic system. The longevity of HSCs makes them vulnerable to accumulating DNA damage, which may be leukemogenic or result in senescence and cell death. Additionally, the ability of HSCs to self-renew and differentiate allows DNA damage to spread throughout the hematologic system, leaving the organism vulnerable to disease. In this review we discuss cell fate decisions made in the face of DNA damage and other cellular stresses, and the role of reactive oxygen species in the long-term maintenance of HSCs and their DNA damage response. PMID:24041596

Erythroid differentiation ability of butyric acid analogues: identification of basal chemical structures of new inducers of foetal haemoglobin.

PubMed

Bianchi, Nicoletta; Chiarabelli, Cristiano; Zuccato, Cristina; Lampronti, Ilaria; Borgatti, Monica; Amari, Gabriele; Delcanale, Maurizio; Chiavilli, Francesco; Prus, Eugenia; Fibach, Eitan; Gambari, Roberto

2015-04-05

Several investigations have demonstrated a mild clinical status in patients with β-globin disorders and congenital high persistence of foetal haemoglobin. This can be mimicked by a pharmacological increase of foetal γ-globin genes expression and foetal haemoglobin production. Our goal was to apply a multistep assay including few screening methods (benzidine staining, RT-PCR and HPLC analyses) and erythroid cellular model systems (the K562 cell line and erythroid precursors collected from peripheral blood) to select erythroid differentiation agents with foetal haemoglobin inducing potential. With this methodology, we have identified a butyric acid derivative, namely the 4174 cyclopropanecarboxylic acid compound, able to induce erythroid differentiation without antiproliferative effect in K562 cells and increase of γ-globin gene expression in erythroid precursor cells. The results are relevant for pharmacological treatments of haemoglobinopathies, including β-thalassaemia and sickle cell anaemia. Copyright © 2015 Elsevier B.V. All rights reserved.

Cytotoxicity of Vitex agnus-castus fruit extract and its major component, casticin, correlates with differentiation status in leukemia cell lines.

PubMed

Kikuchi, Hidetomo; Yuan, Bo; Nishimura, Yoshio; Imai, Masahiko; Furutani, Ryota; Kamoi, Saki; Seno, Misako; Fukushima, Shin; Hazama, Shingo; Hirobe, Chieko; Ohyama, Kunio; Hu, Xiao-Mei; Takagi, Norio; Hirano, Toshihiko; Toyoda, Hiroo

2013-12-01

We have demonstrated that an extract from the ripe fruit of Vitex agnus-castus (Vitex) exhibits cytotoxic activities against various types of solid tumor cells, whereas its effects on leukemia cells has not been evaluated to date. In this study, the effects of Vitex and its major component, casticin, on leukemia cell lines, HL-60 and U-937, were investigated by focusing on proliferation, induction of apoptosis and differentiation. Identification and quantitation by NMR spectroscopy showed that casticin accounted for approximate 1% weight of Vitex. Dose-dependent cytotoxicity of Vitex and casticin was observed in both cell lines, and HL-60 cells were more sensitive to the cytotoxicity of Vitex/casticin compared to U-937 cells. Furthermore, compared to unstimulated HL-60 cells, phorbol 12-myristate 13-acetate (PMA)- and 1,25-dihydroxyvitamin D₃ (VD₃)-differentiated HL-60 cells acquired resistance to Vitex/casticin based on the results from cell viability and apoptosis induction analysis. Since the HL-60 cell line is more immature than the U-937 cell line, these results suggested that the levels of cytotoxicity of Vitex/casticin were largely attributed to the degree of differentiation of leukemia cells; that is, cell lines with less differentiated phenotype were more susceptible than the differentiated ones. RT-PCR analysis demonstrated that PMA upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in HL-60 cells, and that anti-ICAM-1 monoclonal antibody not only abrogated PMA-induced aggregation and adhesion of the cells but also restored its sensitivity to Vitex. These results suggested that ICAM-1 plays a crucial role in the acquired resistance in PMA-differentiated HL-60 cells by contributing to cell adhesion. These findings provide fundamental insights into the clinical application of Vitex/casticin for hematopoietic malignancy.

Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy

NASA Astrophysics Data System (ADS)

Stringari, Chiara; Sierra, Robert; Donovan, Peter J.; Gratton, Enrico

2012-04-01

We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic states of differentiating H9 and H1 hESC colonies. This technique is a promising noninvasive tool to monitor hESC metabolism during differentiation, which can have applications in high throughput analysis, drug screening, functional metabolomics and induced pluripotent stem cell generation.

Electrochemical Detection of Human Mesenchymal Stem Cell Differentiation on Fabricated Gold Nano-Dot Cell Chips.

PubMed

An, Jeung Hee; Kim, Seung U; Park, Mi-Kyung; Choi, Jeong Woo

2015-10-01

Human mesenchymal stem cells (MSCs) have the capacity for self-renewal and maintain pluripotency, which is defined by their ability to differentiate into cells such as osteoblasts, neurons, and glial cells. In this study, we report a method for defining the status of human MSCs based on electrochemical detection systems. Gold nano-dot structures were fabricated using a nanoporous alumina mask, and the structural formations were confirmed by scanning electron microscopy (SEM). Human MSCs were allowed to attach to RGD (Arg-Gly-Asp) peptide nanopatterned surfaces, and electrochemical tools were applied to the MSCs attached on the chip surface. The cultured MSCs were shown to differentiate into neural cell types, as indicated by immunocytochemical staining for tyrosine hydroxylase and beta tubulin III. Following treatment with basic fibroblast growth factor (bFGF) for 14 days, most of the B10 cells exhibited bipolar or multipolar morphology with branched processes, and the proportion of B10 cells expressing neuronal cell markers considerably increased. Electrophysiological recordings from MSCs treated with bFGF for 5-14 days were examined with cyclic voltammetry, and the electrochemical signals were shown to increase during differentiation from MSCs to neuronal cells. This human MSC cell line is a useful tool for studying organogenesis, specifically neurogenesis, and in addition, the cell line provides a valuable source of cells for cell therapy. The electrochemical measurement system proposed here could be utilized in electrical cell chips for numerous applications, including cell differentiation, disease diagnosis, drug detection, and on-site monitoring.

Imaging Stem Cells Implanted in Infarcted Myocardium

PubMed Central

Zhou, Rong; Acton, Paul D.; Ferrari, Victor A.

2008-01-01

Stem cell–based cellular cardiomyoplasty represents a promising therapy for myocardial infarction. Noninvasive imaging techniques would allow the evaluation of survival, migration, and differentiation status of implanted stem cells in the same subject over time. This review describes methods for cell visualization using several corresponding noninvasive imaging modalities, including magnetic resonance imaging, positron emission tomography, single-photon emission computed tomography, and bioluminescent imaging. Reporter-based cell visualization is compared with direct cell labeling for short- and long-term cell tracking. PMID:17112999

CpG-STAT3siRNA for Castration-Resistant Prostate Cancer Therapy

DTIC Science & Technology

2015-12-01

RESULTS TLR9 promotes prostate cancer cell engraftment and progression in vivo Previous studies reported expression of the innate immune receptor...cancer cells express innate immune receptors, such as TLR9, normally restricted to the hematopoietic cell lineage [2, 5, 7]. Rather than becoming... innate immune gene family is differentially influenced by DNA stress and p53 status in cancer cells . Cancer Res. 2012; 72:3948–3957. 7. Ilvesaro JM

Clear-cell differentiation and lymphatic invasion, but not the revised TNM classification, predict lymph node metastases in pT1 penile cancer: a clinicopathologic study of 76 patients from a low incidence area.

PubMed

Mannweiler, Sebastian; Sygulla, Stephan; Tsybrovskyy, Oleksiy; Razmara, Yas; Pummer, Karl; Regauer, Sigrid

2013-10-01

Prediction of lymph node (LN) metastases in penile invasive cancer relies on clinical features and histologic characteristics of the primary tumor. Correct prediction, however, is difficult, as only 50% patients undergoing lymphadenectomies will have LN metastases. In 2009, the tumor, nodes, metastases (TNM) classification for staging of early penile cancers was revised. We tested the predictive accuracy of the revised TNM classification in a low incidence area for penile carcinoma. The presence of LN metastases in 76 men with pT1 penile cancers was correlated with the 2009 TNM subclassification, which is based on a combined evaluation of tumor grade and lymphatic invasion, but also with individual parameters, such as histologic grade, lymphatic invasion, perineural invasion, invasion depth, growth pattern and human papilloma virus (HPV) status. 76pT1 penile cancers were reclassified into 31pT1a squamous cell carcinomas (SCC) and 45pT1b (41 SCC; 4 clear-cell carcinomas); 12/22 men (55%; 8 SCC, 4 clear-cell carcinomas) undergoing lymphadenectomy for enlarged inguinal lymph nodes had metastases, 54 patients without enlarged LN and lymphadenectomies had no LN metastases during follow-up of median 47 months. Statistically, clear cell differentiation of the primary carcinoma was highly associated with metastases (100% clear-cell carcinomas vs. 11% SCC) and poor survival (50% vs. 5.5%). Among conventional SCC, only lymphatic invasion showed a highly significant association with metastases with 100% specificity. The 2009 TNM classification, tumor grade alone, perineural invasion, growth pattern, invasion depth or HPV status could not predict LN status. Lymphadenectomy for enlarged LN resulted in 100% sensitivity and 42% predictive probability for identifying metastases and a 16% false positive rate. Statistically, survival correlated significantly with clear-cell differentiation and with lymphatic invasion in both clear-cell carcinomas and conventional SCC. Penile clear-cell carcinomas are more aggressive cancers than SCC. Our observation suggests a benefit of a prophylactic lymphadenectomy for patients with clear-cell carcinomas. Among conventional SCC, only lymphatic invasion predicted LN metastases. Neither tumor grade alone nor perineural invasion, growth pattern, depth of invasion, and subgrouping according to the revised TNM classification correlated with metastases. Clinical evaluation of the LN status was superior to histologic risk stratification. Copyright © 2013 Elsevier Inc. All rights reserved.

Proteomic Characterization of Annexin l (ANX1) and Heat Shock Protein 27 (HSP27) as Biomarkers for Invasive Hepatocellular Carcinoma Cells.

PubMed

Wang, Ruo-Chiau; Huang, Chien-Yu; Pan, Tai-Long; Chen, Wei-Yu; Ho, Chun-Te; Liu, Tsan-Zon; Chang, Yu-Jia

2015-01-01

To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX-2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC-70, protein disulfide isomerase (PDI), and heat shock protein-27 (HSP27), were exclusively up-regulated in SK-Hep-1 cells. Elevated levels of ANX-4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep-1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets.

LDOC1 mRNA is differentially expressed in chronic lymphocytic leukemia and predicts overall survival in untreated patients

PubMed Central

Duzkale, Hatice; Schweighofer, Carmen D.; Coombes, Kevin R.; Barron, Lynn L.; Ferrajoli, Alessandra; O'Brien, Susan; Wierda, William G.; Pfeifer, John; Majewski, Tadeusz; Czerniak, Bogdan A.; Jorgensen, Jeffrey L.; Medeiros, L. Jeffrey; Freireich, Emil J; Keating, Michael J.

2011-01-01

We previously identified LDOC1 as one of the most significantly differentially expressed genes in untreated chronic lymphocytic leukemia (CLL) patients with respect to the somatic mutation status of the immunoglobulin heavy-chain variable region genes. However, little is known about the normal function of LDOC1, its contribution to the pathophysiology of CLL, or its prognostic significance. In this study, we have investigated LDOC1 mRNA expression in a large cohort of untreated CLL patients, as well as in normal peripheral blood B-cell (NBC) subsets and primary B-cell lymphoma samples. We have confirmed that LDOC1 is dramatically down-regulated in mutated CLL cases compared with unmutated cases, and have identified a new splice variant, LDOC1S. We show that LDOC1 is expressed in NBC subsets (naive > memory), suggesting that it may play a role in normal B-cell development. It is also expressed in primary B-cell lymphoma samples, in which its expression is associated with somatic mutation status. In CLL, we show that high levels of LDOC1 correlate with biomarkers of poor prognosis, including cytogenetic markers, unmutated somatic mutation status, and ZAP70 expression. Finally, we demonstrate that LDOC1 mRNA expression is an excellent predictor of overall survival in untreated CLL patients. PMID:21310924

Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Tomonobu M.; World Premier Initiative, iFREC, Osaka University, Osaka 565-0871; Higuchi, Sayaka

Highlights: Black-Right-Pointing-Pointer Change in the epigenetic landscape during myogenesis was optically investigated. Black-Right-Pointing-Pointer Mobility of nuclear proteins was used to state the epigenetic status of the cell. Black-Right-Pointing-Pointer Mobility of nuclear proteins decreased as myogenesis progressed in C2C12. Black-Right-Pointing-Pointer Differentiation state diagram was developed using parameters obtained. -- Abstract: Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extentmore » of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myogenesis using the state diagram developed with the parameters obtained in this study.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pelch, Katherine E.; Tokar, Erik J.; Merrick, B. Alex

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1)more » were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (> 25-fold) and S100P (> 40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (> 15-fold) and NTM (> 1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular transformation.« less

HIV-TB coinfection impairs CD8(+) T-cell differentiation and function while dehydroepiandrosterone improves cytotoxic antitubercular immune responses.

PubMed

Suarez, Guadalupe V; Angerami, Matías T; Vecchione, María B; Laufer, Natalia; Turk, Gabriela; Ruiz, Maria J; Mesch, Viviana; Fabre, Bibiana; Maidana, Patricia; Ameri, Diego; Cahn, Pedro; Sued, Omar; Salomón, Horacio; Bottasso, Oscar A; Quiroga, María F

2015-09-01

Tuberculosis (TB) is the leading cause of death among HIV-positive patients. The decreasing frequencies of terminal effector (TTE ) CD8(+) T cells may increase reactivation risk in persons latently infected with Mycobacterium tuberculosis (Mtb). We have previously shown that dehydroepiandrosterone (DHEA) increases the protective antitubercular immune responses in HIV-TB patients. Here, we aimed to study Mtb-specific cytotoxicity, IFN-γ secretion, memory status of CD8(+) T cells, and their modulation by DHEA during HIV-TB coinfection. CD8(+) T cells from HIV-TB patients showed a more differentiated phenotype with diminished naïve and higher effector memory and TTE T-cell frequencies compared to healthy donors both in total and Mtb-specific CD8(+) T cells. Notably, CD8(+) T cells from HIV-TB patients displayed higher Terminal Effector (TTE ) CD45RA(dim) proportions with lower CD45RA expression levels, suggesting a not fully differentiated phenotype. Also, PD-1 expression levels on CD8(+) T cells from HIV-TB patients increased although restricted to the CD27(+) population. Interestingly, DHEA plasma levels positively correlated with TTE in CD8(+) T cells and in vitro DHEA treatment enhanced Mtb-specific cytotoxic responses and terminal differentiation in CD8(+) T cells from HIV-TB patients. Our data suggest that HIV-TB coinfection promotes a deficient CD8(+) T-cell differentiation, whereas DHEA may contribute to improving antitubercular immunity by enhancing CD8(+) T-cell functions during HIV-TB coinfection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differentiation to adipocytes in accompanied by an increase in the amounts of Gi- and Go-proteins in 3T3-L1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watkins, D.C.; Northup, J.K.; Malbon, C.C.

Treatment of cultures of 3T3-L1 cells with methylisobutyl-xanthine and dexamethasone has been shown to result in accumulation of lipid and conversion to the morphology of adipocytes in more than 90% of the cells. The status of the stimulatory (Gs), inhibitory (Gi) and Go-proteins during the course of 3T3-L1 differentiation was examined. The amount of alpha subunit of Gs (..cap alpha..Gs), assayed by radiolabeling in the presence of cholera toxin and (/sup 32/P)NAD/sup +/, increased upon differentiation as previously described by others. The amounts of ..cap alpha..Gi and ..cap alpha..Go assayed by radiolabeling in the presence of pertussis toxin and (/supmore » 32/P)NAD/sup +/ increased 3-fold upon differentiation. Immunoblots of cell membranes subjected to gel electrophoresis in sodium dodecyl sulfate were probed with two rabbit antisera raised against bovine brain ..cap alpha..Go and with one raised against the..beta..-subunit of the bovine rod-outer-segment G-protein, referred to as transducin. The immunoblotting data confirm the increase upon differentiation of ..cap alpha..Go and also demonstrate an increase in the amount of the ..beta..-subunit. Thus differentiation of 3T3-L1 cells is accompanied by dramatic changes in the complexion of G-proteins in the membranes.« less

Development of hematopoietic stem and progenitor cells from human pluripotent stem cells.

PubMed

Chen, Tong; Wang, Fen; Wu, Mengyao; Wang, Zack Z

2015-07-01

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose. © 2015 Wiley Periodicals, Inc.

[Analysis of tissue-specific differentially methylated genes with differential gene expression in non-small cell lung cancer].

PubMed

Yin, L G; Zou, Z Q; Zhao, H Y; Zhang, C L; Shen, J G; Qi, L; Qi, M; Xue, Z Q

2014-01-01

Adenocarcinoma (ADC) and squamous cell carcinomas (SCC) are two subtypes of non-small cell lung carcinomas which are regarded as the leading cause of cancer-related malignancy worldwide. The aim of this study is to detect the differentially methylated loci (DMLs) and differentially methylated genes (DMGs) of these two tumor sets, and then to illustrate the different expression level of specific methylated genes. Using TCGA database and Illumina HumanMethylation 27 arrays, we first screened the DMGs and DMLs in tumor samples. Then, we explored the BiologicalProcess terms of hypermethylated and hypomethylated genes using Functional Gene Ontology (GO) catalogues. Hypermethylation intensively occurred in CpG-island, whereas hypomethylation was located in non-CpG-island. Most SCC and ADC hypermethylated genes involved GO function of DNA dependenit regulation of transcription, and hypomethylated genes mainly 'enriched in the term of immune responses. Additionally, the expression level of specific differentially methylated genesis distinctbetween ADC and SCC. It is concluded that ADC and SCC have different methylated status that might play an important role in carcinogenesis.

Artificial Extracellular Matrices with Oversulfated Glycosaminoglycan Derivatives Promote the Differentiation of Osteoblast-Precursor Cells and Premature Osteoblasts

PubMed Central

Hempel, Ute; Preissler, Carolin; Möller, Stephanie; Becher, Jana; Rauner, Martina; Hofbauer, Lorenz C.; Dieter, Peter

2014-01-01

Sulfated glycosaminoglycans (GAG) are components of the bone marrow stem cell niche and to a minor extent of mature bone tissue with important functions in regulating stem cell lineage commitment and differentiation. We anticipated that artificial extracellular matrices (aECM) composed of collagen I and synthetically oversulfated GAG derivatives affect preferentially the differentiation of osteoblast-precursor cells and early osteoblasts. A set of gradually sulfated chondroitin sulfate and hyaluronan derivatives was used for the preparation of aECM. All these matrices were analysed with human bone marrow stromal cells to identify the most potent aECM and to determine the influence of the degree and position of sulfate groups and the kind of disaccharide units on the osteogenic differentiation. Oversulfated GAG derivatives with a sulfate group at the C-6 position of the N-acetylglycosamine revealed the most pronounced proosteogenic effect as determined by tissue nonspecific alkaline phosphatase activity and calcium deposition. A subset of the aECM was further analysed with different primary osteoblasts and cell lines reflecting different maturation stages to test whether the effect of sulfated GAG derivatives depends on the maturation status of the cells. It was shown that the proosteogenic effect of aECM was most prominent in early osteoblasts. PMID:24864267

Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation

NASA Astrophysics Data System (ADS)

Meleshina, A. V.; Dudenkova, V. V.; Shirmanova, M. V.; Bystrova, A. S.; Zagaynova, E. V.

2016-03-01

Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

Impact of modeled microgravity on migration, differentiation, and cell cycle control of primitive human hematopoietic progenitor cells.

PubMed

Plett, P Artur; Abonour, Rafat; Frankovitz, Stacy M; Orschell, Christie M

2004-08-01

Migration, proliferation, and differentiation of bone marrow (BM) hematopoietic stem cells (HSC) are important factors in maintaining hematopoietic homeostasis. Homeostatic control of erythrocytes and lymphocytes is perturbed in humans exposed to microgravity (micro-g), resulting in space flight-induced anemia and immunosuppression. We sought to determine whether any of these anomalies can be explained by micro-g-induced changes in migration, proliferation, and differentiation of human BM CD34+ cells, and whether such changes can begin to explain any of the shifts in hematopoietic homeostasis observed in astronauts. BM CD34+ cells were cultured in modeled micro-g (mmicro-g) using NASA's rotating wall vessels (RWV), or in control cultures at earth gravity for 2 to 18 days. Cells were harvested at different times and CD34+ cells assessed for migration potential, cell-cycle kinetics and regulatory proteins, and maturation status. Culture of BM CD34+ cells in RWV for 2 to 3 days resulted in a significant reduction of stromal cell-derived factor 1 (SDF-1alpha)-directed migration, which correlated with decreased expression of F-actin. Modeled micro-g induced alterations in cell-cycle kinetics that were characterized by prolonged S phase and reduced cyclin A expression. Differentiation of primitive CD34+ cells cultured for 14 to 18 days in RWV favored myeloid cell development at the expense of erythroid development, which was significantly reduced compared to controls. These results illustrate that mmicro-g significantly inhibits the migration potential, cell-cycle progression, and differentiation patterns of primitive BM CD34+ cells, which may contribute to some of the hematologic abnormalities observed in humans during space flight.

Transforming growth factor-beta inhibits human antigen-specific CD4+ T cell proliferation without modulating the cytokine response.

PubMed

Tiemessen, Machteld M; Kunzmann, Steffen; Schmidt-Weber, Carsten B; Garssen, Johan; Bruijnzeel-Koomen, Carla A F M; Knol, Edward F; van Hoffen, Els

2003-12-01

Transforming growth factor (TGF)-beta has been demonstrated to play a key role in the regulation of the immune response, mainly by its suppressive function towards cells of the immune system. In humans, the effect of TGF-beta on antigen-specific established memory T cells has not been investigated yet. In this study antigen-specific CD4(+) T cell clones (TCC) were used to determine the effect of TGF-beta on antigen-specific proliferation, the activation status of the T cells and their cytokine production. This study demonstrates that TGF-beta is an adequate suppressor of antigen-specific T cell proliferation, by reducing the cell-cycle rate rather than induction of apoptosis. Addition of TGF-beta resulted in increased CD69 expression and decreased CD25 expression on T cells, indicating that TGF-beta is able to modulate the activation status of in vivo differentiated T cells. On the contrary, the antigen-specific cytokine production was not affected by TGF-beta. Although TGF-beta was suppressive towards the majority of the T cells, insensitivity of a few TCC towards TGF-beta was also observed. This could not be correlated to differential expression of TGF-beta signaling molecules such as Smad3, Smad7, SARA (Smad anchor for receptor activation) and Hgs (hepatocyte growth factor-regulated tyrosine kinase substrate). In summary, TGF-beta has a pronounced inhibitory effect on antigen-specific T cell proliferation without modulating their cytokine production.

Depolarization Alters Phenotype, Maintains Plasticity of Predifferentiated Mesenchymal Stem Cells

PubMed Central

Sundelacruz, Sarah; Levin, Michael

2013-01-01

Although adult stem cell transplantation has been implemented as a therapy for tissue repair, it is limited by the availability of functional adult stem cells. A potential approach to generate stem and progenitor cells may be to modulate the differentiated status of somatic cells. Therefore, there is a need for a better understanding of how the differentiated phenotype of mature cells is regulated. We hypothesize that bioelectric signaling plays an important role in the maintenance of the differentiated state, as it is a functional regulator of the differentiation process in various cells and tissues. In this study, we asked whether the mature phenotype of osteoblasts and adipocytes derived from human mesenchymal stem cells (hMSCs) could be altered by modulation of their membrane potential. hMSC-derived osteoblasts and adipocytes were depolarized by treatment with ouabain, a Na+/K+ ATPase inhibitor, or by treatment with high concentrations of extracellular K+. To characterize the effect of voltage modulation on the differentiated state, the depolarized cells were evaluated for (1) the loss of differentiation markers; (2) the up-regulation of stemness markers and stem properties; and (3) differences in gene expression profiles in response to voltage modulation. hMSC-derived osteoblasts and adipocytes exhibited significant down-regulation of bone and fat tissue markers in response to depolarization, despite the presence of differentiation-inducing soluble factors, suggesting that bioelectric signaling overrides biochemical signaling in the maintenance of cell state. Suppression of the osteoblast or adipocyte phenotype was not accompanied by up-regulation of genes associated with the stem state. Thus, depolarization does not activate the stem cell genetic signature and, therefore, does not induce a full reprogramming event. However, after transdifferentiating the depolarized cells to evaluate for multi-lineage potential, depolarized osteoblasts demonstrated improved ability to achieve correct adipocyte morphology compared with nondepolarized osteoblasts. The present study thus demonstrates that depolarization reduces the differentiated phenotype of hMSC-derived cells and improves their transdifferentiation capacity, but does not restore a stem-like genetic profile. Through global transcript profiling of depolarized osteoblasts, we identified pathways that may mediate the effects of voltage signaling on cell state, which will require a detailed mechanistic inquiry in future studies. PMID:23738690

Human Pluripotent Stem Cells Differentiated in Fully Defined Medium Generate Hematopoietic CD34+ and CD34− Progenitors with Distinct Characteristics

PubMed Central

Chicha, Laurie; Feki, Anis; Boni, Alessandro; Irion, Olivier; Hovatta, Outi; Jaconi, Marisa

2011-01-01

Background Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. Methodology/Principal Findings ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34+ cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34+ cells. ESC-derived CD34+ cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34− cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. Conclusions/Significance Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures. PMID:21364915

Human pluripotent stem cells differentiated in fully defined medium generate hematopoietic CD34- and CD34+ progenitors with distinct characteristics.

PubMed

Chicha, Laurie; Feki, Anis; Boni, Alessandro; Irion, Olivier; Hovatta, Outi; Jaconi, Marisa

2011-02-25

Differentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells. ESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult β-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors. Short-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.

Production of zebrafish cardiospheres and cardiac progenitor cells in vitro and three-dimensional culture of adult zebrafish cardiac tissue in scaffolds.

PubMed

Zeng, Wendy R; Beh, Siew-Joo; Bryson-Richardson, Robert J; Doran, Pauline M

2017-09-01

The hearts of adult zebrafish (Danio rerio) are capable of complete regeneration in vivo even after major injury, making this species of particular interest for understanding the growth and differentiation processes required for cardiac tissue engineering. To date, little research has been carried out on in vitro culture of adult zebrafish cardiac cells. In this work, progenitor-rich cardiospheres suitable for cardiomyocyte differentiation and myocardial regeneration were produced from adult zebrafish hearts. The cardiospheres contained a mixed population of c-kit + and Mef2c + cells; proliferative peripheral cells of possible mesenchymal lineage were also observed. Cellular outgrowth from cardiac explants and cardiospheres was enhanced significantly using conditioned medium harvested from cultures of a rainbow trout cell line, suggesting that fish-specific trophic factors are required for zebrafish cardiac cell expansion. Three-dimensional culture of zebrafish heart cells in fibrous polyglycolic acid (PGA) scaffolds was carried out under dynamic fluid flow conditions. High levels of cell viability and cardiomyocyte differentiation were maintained within the scaffolds. Expression of cardiac troponin T, a marker of differentiated cardiomyocytes, increased during the first 7 days of scaffold culture; after 15 days, premature disintegration of the biodegradable scaffolds led to cell detachment and a decline in differentiation status. This work expands our technical capabilities for three-dimensional zebrafish cardiac cell culture with potential applications in tissue engineering, drug and toxicology screening, and ontogeny research. Biotechnol. Bioeng. 2017;114: 2142-2148. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A gold@polydopamine core-shell nanoprobe for long-term intracellular detection of microRNAs in differentiating stem cells.

PubMed

Choi, Chun Kit K; Li, Jinming; Wei, Kongchang; Xu, Yang J; Ho, Lok Wai C; Zhu, Meiling; To, Kenneth K W; Choi, Chung Hang J; Bian, Liming

2015-06-17

The capability of monitoring the differentiation process in living stem cells is crucial to the understanding of stem cell biology and the practical application of stem-cell-based therapies, yet conventional methods for the analysis of biomarkers related to differentiation require a large number of cells as well as cell lysis. Such requirements lead to the unavoidable loss of cell sources and preclude real-time monitoring of cellular events. In this work, we report the detection of microRNAs (miRNAs) in living human mesenchymal stem cells (hMSCs) by using polydopamine-coated gold nanoparticles (Au@PDA NPs). The PDA shell facilitates the immobilization of fluorescently labeled hairpin DNA strands (hpDNAs) that can recognize specific miRNA targets. The gold core and PDA shell quench the fluorescence of the immobilized hpDNAs, and subsequent binding of the hpDNAs to the target miRNAs leads to their dissociation from Au@PDA NPs and the recovery of fluorescence signals. Remarkably, these Au@PDA-hpDNA nanoprobes can naturally enter stem cells, which are known for their poor transfection efficiency, without the aid of transfection agents. Upon cellular uptake of these nanoprobes, we observe intense and time-dependent fluorescence responses from two important osteogenic marker miRNAs, namely, miR-29b and miR-31, only in hMSCs undergoing osteogenic differentiation and living primary osteoblasts but not in undifferentiated hMSCs and 3T3 fibroblasts. Strikingly, our nanoprobes can afford long-term tracking of miRNAs (5 days) in the differentiating hMSCs without the need of continuously replenishing cell culture medium with fresh nanoprobes. Our results demonstrate the capability of our Au@PDA-hpDNA nanoprobes for monitoring the differentiation status of hMSCs (i.e., differentiating versus undifferentiated) via the detection of specific miRNAs in living stem cells. Our nanoprobes show great promise in the investigation of the long-term dynamics of stem cell differentiation, identification and isolation of specific cell types, and high-throughput drug screening.

Gene expression analysis of whole blood, peripheral blood mononuclear cells, and lymphoblastoid cell lines from the Framingham Heart Study

PubMed Central

Joehanes, Roby; Johnson, Andrew D.; Barb, Jennifer J.; Raghavachari, Nalini; Liu, Poching; Woodhouse, Kimberly A.; O'Donnell, Christopher J.; Munson, Peter J.

2012-01-01

Despite a growing number of reports of gene expression analysis from blood-derived RNA sources, there have been few systematic comparisons of various RNA sources in transcriptomic analysis or for biomarker discovery in the context of cardiovascular disease (CVD). As a pilot study of the Systems Approach to Biomarker Research (SABRe) in CVD Initiative, this investigation used Affymetrix Exon arrays to characterize gene expression of three blood-derived RNA sources: lymphoblastoid cell lines (LCL), whole blood using PAXgene tubes (PAX), and peripheral blood mononuclear cells (PBMC). Their performance was compared in relation to identifying transcript associations with sex and CVD risk factors, such as age, high-density lipoprotein, and smoking status, and the differential blood cell count. We also identified a set of exons that vary substantially between participants, but consistently in each RNA source. Such exons are thus stable phenotypes of the participant and may potentially become useful fingerprinting biomarkers. In agreement with previous studies, we found that each of the RNA sources is distinct. Unlike PAX and PBMC, LCL gene expression showed little association with the differential blood count. LCL, however, was able to detect two genes related to smoking status. PAX and PBMC identified Y-chromosome probe sets similarly and slightly better than LCL. PMID:22045913

The effect of nutritional status on myogenic satellite cell proliferation and differentiation.

PubMed

Powell, D J; McFarland, D C; Cowieson, A J; Muir, W I; Velleman, S G

2013-08-01

Early posthatch satellite cell (SC) mitotic activity is a critical component of muscle development and growth. Satellite cells are stem cells that can be induced by nutrition to follow other cellular developmental pathways. The objective of the current study was to determine the effect of restricting protein synthesis on the proliferation and differentiation of SC, using variable concentrations of Met and Cys to modulate protein synthesis. Broiler pectoralis major SC were cultured and treated with 1 of 6 different Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3/9.6, 1/3.2, or 0/0 mg/L. The effect of Met/Cys concentration on SC proliferation and differentiation was measured, and myonuclear accretion was measured by counting the number of nuclei per myotube during differentiation. The 30/96 mg/L Met/Cys treatment resulted in the highest rate of proliferation compared with all other treatments by 72 h of proliferation (P < 0.05). Differentiation was measured with Met/Cys treatments only during proliferation and the cultures receiving normal differentiation medium (R/N), normal proliferation medium and differentiation medium with variable Met/Cys (N/R), or both proliferation and differentiation receiving variable Met/Cys treatments (R/R). Differentiation responded in a dose-dependent manner to Met/Cys concentration under all 3 of these treatment regimens, with a degree of recovery in the R/N regimen cells following reinstatement of the control medium. Reductions in both proliferation and differentiation were more pronounced as Met/Cys concentrations were further reduced, whereas increased differentiation was observed under the increased Met/Cys concentration treatment when applied during differentiation in the N/R and R/R regimens. The number of nuclei per myotube was significantly decreased in the severely Met/Cys restricted treatments (P < 0.05). These data demonstrate the sensitivity of pectoralis major SC to nutritional availability and the importance of optimal nutrition during both proliferation and differentiation for maximizing SC activity, which will affect subsequent muscle mass accretion.

Wt-p53 action in human leukaemia cell lines corresponding to different stages of differentiation.

PubMed

Rizzo, M G; Zepparoni, A; Cristofanelli, B; Scardigli, R; Crescenzi, M; Blandino, G; Giuliacci, S; Ferrari, S; Soddu, S; Sacchi, A

1998-05-01

Recent studies support the potential application of the wt-p53 gene in cancer therapy. Expression of exogenous wt-p53 suppresses a variety of leukaemia phenotypes by acting on cell survival, proliferation and/or differentiation. As for tumour gene therapy, the final fate of the neoplastic cells is one of the most relevant points. We examined the effects of exogenous wt-p53 gene expression in several leukaemia cell lines to identify p53-responsive leukaemia. The temperature-sensitive p53Val135 mutant or the human wt-p53 cDNA was transduced in leukaemia cell lines representative of different acute leukaemia FAB subtypes, including M1 (KG1), M2 (HL-60), M3 (NB4), M5 (U937) and M6 (HEL 92.1.7), as well as blast crisis of chronic myelogenous leukaemia (BC-CML: K562, BV173) showing diverse differentiation features. By morphological, molecular and biochemical analyses, we have shown that exogenous wt-p53 gene expression induces apoptosis only in cells corresponding to M1, M2 and M3 of the FAB classification and in BC-CML showing morphological and cytochemical features of undifferentiated blast cells. In contrast, it promotes differentiation in the others. Interestingly, cell responsiveness was independent of the vector used and the status of the endogenous p53 gene.

Cell type dependent morphological adaptation in polyelectrolyte hydrogels governs chondrogenic fate.

PubMed

Raghothaman, Deepak; Leong, Meng Fatt; Lim, Tze Chiun; Wan, Andrew C A; Ser, Zheng; Lee, Eng Hin; Yang, Zheng

2016-04-04

Repair of critical-size articular cartilage defects typically involves delivery of cells in biodegradable, 3D matrices. Differences in the developmental status of mesenchymal stem cells (MSCs) and terminally differentiated mature chondrocytes might be a critical factor in engineering appropriate 3D matrices for articular cartilage tissue engineering. This study examined the relationship between material-driven early cell morphological adaptations and chondrogenic outcomes, by studying the influence of aligned collagen type I (Col I) presentation on chondrocytes and MSC in interfacial polyelectrolyte complexation (IPC)-based hydrogels. In the absence of Col I, both chondrocytes and MSCs adopted rounded cell morphology and formed clusters, with chondrocyte clusters favoring the maintenance of hyaline phenotype, while MSC clusters differentiated to fibro-superficial zone-like chondrocytes. Encapsulated chondrocytes in IPC-Col I hydrogel adopted a fibroblastic morphology forming fibro-superficial zone-like phenotype, which could be reversed by inhibiting actin polymerization using cytochalasin D (CytD). In contrast, adoption of fibroblastic morphology by encapsulated MSCs in IPC-Col I facilitated superior chondrogenesis, generating a mature, hyaline neocartilage tissue. CytD treatment abrogated the elongation of MSCs and brought about a single cell-like state, resulting in insignificant chondrogenic differentiation, underscoring the essential requirement of providing matrix environments that are amenable to cell-cell interactions for robust MSC chondrogenic differentiation. Our study demonstrates that MSCs and culture-expanded chondrocytes favour differential microenvironmental niches and emphasizes the importance of designing biomaterials that meet cell type-specific requirements, in adopting chondrocyte or MSC-based approaches for regenerating hyaline, articular cartilage.

Arctigenin exerts anti-colitis efficacy through inhibiting the differentiation of Th1 and Th17 cells via an mTORC1-dependent pathway.

PubMed

Wu, Xin; Dou, Yannong; Yang, Yan; Bian, Difei; Luo, Jinque; Tong, Bei; Xia, Yufeng; Dai, Yue

2015-08-15

Arctigenin, the main effective constituent of Arctium lappa L. fruit, has previously been proven to dramatically attenuate dextran sulfate sodium (DSS)-induced colitis in mice, a frequently used animal model of inflammatory bowel disease (IBD). As Th1 and Th17 cells play a crucial role in the pathogenesis of IBD, the present study addressed whether and how arctigenin exerted anti-colitis efficacy by interfering with the differentiation and activation of Th1/Th17 cells. In vitro, arctigenin was shown to markedly inhibit the differentiation of Th17 cells from naïve T cells, and moderately inhibit the differentiation of Th1 cells, which was accompanied by lowered phosphorylation of STAT3 and STAT4, respectively. In contrast, arctigenin was lack of marked effect on the differentiation of either Th2 or regulatory T cells. Furthermore, arctigenin was shown to suppress the mammalian target of rapamycin complex 1 (mTORC1) pathway in T cells as demonstrated by down-regulated phosphorylation of the downstream target genes p70S6K and RPS6, and it functioned independent of two well-known upstream kinases PI3K/AKT and ERK. Arctigenin was also able to inhibit the activity of mTORC1 by dissociating raptor from mTOR. Interestingly, the inhibitory effect of arctigenin on T cell differentiation disappeared under a status of mTORC1 overactivation via knockdown of tuberous sclerosis complex 2 (TSC2, a negative regulator of mTORC1) or pretreatment of leucine (an agonist of mTOR). In DSS-induced mice, the inhibition of Th1/Th17 responses and anti-colitis effect of arctigenin were abrogated by leucine treatment. In conclusion, arctigenin ameliorates colitis through down-regulating the differentiation of Th1 and Th17 cells via mTORC1 pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

A combination of MTAP and BAP1 immunohistochemistry in pleural effusion cytology for the diagnosis of mesothelioma.

PubMed

Kinoshita, Yoshiaki; Hida, Tomoyuki; Hamasaki, Makoto; Matsumoto, Shinji; Sato, Ayuko; Tsujimura, Tohru; Kawahara, Kunimitsu; Hiroshima, Kenzo; Oda, Yoshinao; Nabeshima, Kazuki

2018-01-01

Homozygous deletion of 9p21 detected by fluorescence in situ hybridization (FISH) and loss of BRCA1-associated protein 1 (BAP1) expression detected by immunohistochemistry (IHC) are useful for the differentiation between malignant pleural mesothelioma (MPM) and reactive mesothelial hyperplasia. The authors previously described that IHC expression of the protein product of the methylthioadenosine phosphorylase (MTAP) gene, which is localized in the 9p21 chromosomal region, was correlated with the deletion status of 9p21 FISH in MPM tissues. In the current study, the authors investigated whether a combination of MTAP and BAP1 IHC could distinguish MPM from reactive mesothelial cells (RMC) in cell blocks obtained from pleural effusions. The authors examined IHC expression of MTAP and BAP1 in cell blocks obtained from pleural effusions of 45 cases of MPM and 21 cases of reactive mesothelial hyperplasia. Furthermore, IHC expression of MTAP was compared with the deletion status of 9p21 FISH. MTAP and BAP1 IHC differentiated MPM from RMC with 100% specificity for both and sensitivities of 42.2% and 60.0%, respectively. The combination of MTAP and BAP1 IHC yielded a sensitivity of 77.8%, which was higher than that of BAP1 IHC alone or 9p21 FISH alone (62.2%). Moreover, a high degree of concordance was observed between the results of MTAP IHC and 9p21 FISH in cell blocks. A combination of MTAP and BAP1 IHC in cell blocks from pleural effusions appears to be a reliable and useful method for differentiating MPM cells from RMC and can be used in the routine diagnosis of MPM. Cancer Cytopathol 2018;126:54-63. © 2017 American Cancer Society. © 2017 American Cancer Society.

Effect of restriction vegan diet's on muscle mass, oxidative status, and myocytes differentiation: A pilot study.

PubMed

Vanacore, Daniela; Messina, Giovanni; Lama, Stefania; Bitti, Giuseppe; Ambrosio, Pasqualina; Tenore, Giancarlo; Messina, Antonietta; Monda, Vincenzo; Zappavigna, Silvia; Boccellino, Mariarosaria; Novellino, Ettore; Monda, Marcellino; Stiuso, Paola

2018-01-10

This study was conceived to evaluate the effects of three different diets on body composition, metabolic parameters, and serum oxidative status. We enrolled three groups of healthy men (omnivores, vegetarians, and vegans) with similar age, weight and BMI, and we observed a significant decrease in muscle mass index and lean body mass in vegan compared to vegetarian and omnivore groups, and higher serum homocysteine levels in vegetarians and vegans compared to omnivores. We studied whether serum from omnivore, vegetarian, and vegan subjects affected oxidative stress, growth and differentiation of both cardiomyoblast cell line H9c2 and H-H9c2 (H9c2 treated with H 2 O 2 to induce oxidative damage). We demonstrated that vegan sera treatment of both H9c2 and H-H9c2 cells induced an increase of TBARS values and cell death and a decrease of free NO 2- compared to vegetarian and omnivorous sera. Afterwards, we investigated the protective effects of vegan, vegetarian, and omnivore sera on the morphological changes induced by H 2 O 2 in H9c2 cell line. We showed that the omnivorous sera had major antioxidant and differentiation properties compared to vegetarian and vegan sera. Finally, we evaluated the influence of the three different groups of sera on MAPKs pathway and our data suggested that ERK expression increased in H-H9c2 cells treated with vegetarian and vegan sera and could promote cell death. The results obtained in this study demonstrated that restrictive vegan diet could not prevent the onset of metabolic and cardiovascular diseases nor protect by oxidative damage. © 2018 Wiley Periodicals, Inc.

Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel.

PubMed

Trojani, Christophe; Weiss, Pierre; Michiels, Jean-François; Vinatier, Claire; Guicheux, Jérôme; Daculsi, Guy; Gaudray, Patrick; Carle, Georges F; Rochet, Nathalie

2005-09-01

The present work evaluates a newly developed silated hydroxypropylmethylcellulose (Si-HPMC)-based hydrogel as a scaffold for 3D culture of osteogenic cells. The pH variation at room temperature catalyzes the reticulation and self-hardening of the viscous polymer solution into a gelatine state. We designed reticulation time, final consistency and pH in order to obtain an easy handling matrice, suitable for in vitro culture and in vivo injection. Three human osteogenic cell lines and normal human osteogenic (HOST) cells were cultured in 3D inside this Si-HPMC hydrogel. We show here that osteosarcoma cells proliferate as clonogenic spheroids and that HOST colonies survive for at least 3 weeks. Mineralization assay and gene expression analysis of osteoblastic markers and cytokines, indicate that all the cells cultured in 3D into this hydrogel, exhibited a more mature differentiation status than cells cultured in monolayer on plastic. This study demonstrates that this Si-HPMC hydrogel is well suited to support osteoblastic survival, proliferation and differentiation when used as a new scaffold for 3D culture and represents also a potential basis for an innovative bone repair material.

Expression and analysis of exogenous proteins in epidermal cells.

PubMed

Dagnino, Lina; Ho, Ernest; Chang, Wing Y

2010-01-01

In this chapter we review protocols for transient transfection of primary keratinocytes. The ability to transfect primary epidermal cells regardless of their differentiation status allows the biochemical and molecular characterization of multiple proteins. We review methods to analyze exogenous protein abundance in transfected keratinocytes by immunoblot and immunoprecipitation. We also present protocols to determine the subcellular distribution of these proteins by indirect immunofluorescence microscopy approaches.

Prevention of Human Mammary Carcinogenesis.

DTIC Science & Technology

1996-07-01

eicosapentaenoic acid (EPA), indole-3-carbinol (13C), 0-carotene (P3-C), (-) epigallocatechin gallate ( EGCG ) and genistein (GEN) effectively down...Alteration in Cell Cycle Progression of 184-B5/HER Cells by (-) Epigallocatechin Gallate ( EGCG ) status % distribution of cellsb, c- ac Sof treatm enta... epigallocatechin gallate ( EGCG ,a green tea polyphenol), indole-3-carbinol (13C,a plant indole) and genistein (GEN,a soy isoflavone) showed differential

The Effect of Chronic Kidney Disease on T Cell Alloimmunity

PubMed Central

Winterberg, Pamela D.; Ford, Mandy L.

2017-01-01

Purpose of review Altered differentiation and activation of T cell subsets occur in patients with CKD, but the impact on graft rejection and protective immunity during transplantation are not fully understood. Recent findings Patients with CKD have decreased frequency of naïve T cells, accumulation of activated, terminally differentiated memory cells, and skewed regulatory versus T helper 17 ratio. Naïve and memory T cell subsets do not appear to improve following kidney transplantation. Retained thymic output is associated with acute rejection, while naïve lymphopenia and accumulation of CD8+TEMRA cells correlate with long-term graft dysfunction. CD28null memory cells accumulate during CKD and appear to confer protection against acute rejection under standard immunosuppression and possibly co-stimulation blockade. T cells bearing CD57 are also increased in patients with CKD and may underlie rejection during co-stimulation blockade. Summary The mechanisms by which CKD alters the differentiation and activation status of T cell subsets is poorly understood. Further research is also needed to understand which cell populations mediate rejection under various immunosuppressive regimens. To date, there is little use of animal models of organ failure in transplant immunology research. CKD mouse models may help identify novel pathways and targets to better control alloimmunity in post-transplant. PMID:27926546

Differentiation‐associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic‐metabolizing activity of “luminal” muscle‐invasive bladder cancers

PubMed Central

Arlt, Volker M.; Indra, Radek; Joel, Madeleine; Stiborová, Marie; Eardley, Ian; Ahmad, Niaz; Otto, Wolfgang; Burger, Maximilian; Rubenwolf, Peter; Phillips, David H.; Southgate, Jennifer

2018-01-01

Extra‐hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self‐defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro‐carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier‐forming differentiated states in vitro. However, ethoxyresorufin O‐deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP‐DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain‐of‐function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle‐invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into “luminal” and “basal” groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over‐expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1‐activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over‐expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP‐function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies. PMID:29323757

In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth

PubMed Central

Bordonaro, Michael; Shirasawa, Senji; Lazarova, Darina L.

2016-01-01

Although neoplastic cells exhibit relatively higher sensitivity to hyperthermia than normal cells, hyperthermia has had variable success as an anti-cancer therapy. This variable outcome might be due to the fact that cancer cells themselves have differential degrees of sensitivity to high temperature. We hypothesized that the varying sensitivity of colorectal cancer (CRC) cells to hyperthermia depends upon the differential induction of survival pathways. Screening of such pathways revealed that Extracellular Signal-Regulated Kinase (ERK) signaling is augmented by hyperthermia, and the extent of this modulation correlates with the mutation status of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). Through clonal growth assays, apoptotic analyses and transcription reporter assays of CRC cells that differ only in KRAS mutation status we established that mutant KRAS cells are more sensitive to hyperthermia, as they exhibit sustained ERK signaling hyperactivation and increased Wingless/Integrated (WNT)/beta-catenin signaling. We propose that whereas increased levels of WNT and ERK signaling and a positive feedback between the two pathways is a major obstacle in anti-cancer therapy today, under hyperthermia the hyperinduction of the pathways and their positive crosstalk contribute to CRC cell death. Ascertaining the causative association between types of mutations and hyperthermia sensitivity may allow for a mutation profile-guided application of hyperthermia as an anti-cancer therapy. Since KRAS and WNT signaling mutations are prevalent in CRC, our results suggest that hyperthermia-based therapy might benefit a significant number, but not all, CRC patients. PMID:27187477

Antagonistic regulation of p57kip2 by Hes/Hey downstream of Notch signaling and muscle regulatory factors regulates skeletal muscle growth arrest.

PubMed

Zalc, Antoine; Hayashi, Shinichiro; Auradé, Frédéric; Bröhl, Dominique; Chang, Ted; Mademtzoglou, Despoina; Mourikis, Philippos; Yao, Zizhen; Cao, Yi; Birchmeier, Carmen; Relaix, Frédéric

2014-07-01

A central question in development is to define how the equilibrium between cell proliferation and differentiation is temporally and spatially regulated during tissue formation. Here, we address how interactions between cyclin-dependent kinase inhibitors essential for myogenic growth arrest (p21(cip1) and p57(kip2)), the Notch pathway and myogenic regulatory factors (MRFs) orchestrate the proliferation, specification and differentiation of muscle progenitor cells. We first show that cell cycle exit and myogenic differentiation can be uncoupled. In addition, we establish that skeletal muscle progenitor cells require Notch signaling to maintain their cycling status. Using several mouse models combined with ex vivo studies, we demonstrate that Notch signaling is required to repress p21(cip1) and p57(kip2) expression in muscle progenitor cells. Finally, we identify a muscle-specific regulatory element of p57(kip2) directly activated by MRFs in myoblasts but repressed by the Notch targets Hes1/Hey1 in progenitor cells. We propose a molecular mechanism whereby information provided by Hes/Hey downstream of Notch as well as MRF activities are integrated at the level of the p57(kip2) enhancer to regulate the decision between progenitor cell maintenance and muscle differentiation. © 2014. Published by The Company of Biologists Ltd.

Magnitude Of Stimulation Dictates The Cannabinoid-Mediated Differential T Cell Response To HIVgp120

EPA Science Inventory

Cannabinoids have immunosuppressive properties, but it is unknown whether cannabinoids further impair the immune status of immunocompromised HIV patients, as approximately 25% of HIV patients smoke marijuana for its putative therapeutic benefit. A surrogate mouse model to induce ...

Wild-type phosphatase and tensin homolog deleted on chromosome 10 improved the sensitivity of cells to rapamycin through regulating phosphorylation of Akt in esophageal squamous cell carcinoma.

PubMed

Lu, Z; Wang, J; Zheng, Y; Yang, S; Liu, M; Chen, X; Wang, C; Hou, G

2017-02-01

Esophageal squamous cell carcinoma (ESCC) is one of the most frequently diagnosed cancers in China, but the etiology and mode of carcinogenesis of this disease remain poorly understood. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), as a negative regulator of Akt/mTOR pathway, frequently mutates or is inactive in many cancers. Although mTOR has been thought a promising cancer therapeutic target, the sensitivity of tumor cells to rapamycin was still to be revaluated. In this study, we measured the effects of rapamycin on cell proliferation and phosphorylation of Akt in ESCC cells with varying degrees of differentiation. And then, the relationship between PTEN status and the sensitivity of cells to rapamycin was investigated in EC9706 cells with or without wild-type PTEN in vitro and in vivo. The results demonstrated ESCC cells with poor differentiation were insensitive to rapamycin of high concentration and rapamycin obviously promoted the phosphorylation of Akt in these cells, but it had no obvious effects on p-Akt in cells with well differentiation. Also, we showed that wild-type PTEN improved the sensitivity of poor differentiation cells to rapamycin through inhibiting phosphorylation of Akt in vitro and in vivo. This study explored the possible molecular mechanism of some ESCC cells insensitive to rapamycin and provided a measure for treating ESCC patients with PTEN inactivation using mTOR inhibitors. © 2015 International Society for Diseases of the Esophagus.

New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

PubMed Central

2013-01-01

Background Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. Results From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Conclusions Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells. PMID:24148232

Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2-dependent regulatory dendritic cell population.

PubMed

Zhang, Bin; Liu, Rui; Shi, Dan; Liu, Xingxia; Chen, Yuan; Dou, Xiaowei; Zhu, Xishan; Lu, Chunhua; Liang, Wei; Liao, Lianming; Zenke, Martin; Zhao, Robert C H

2009-01-01

Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, exert immunomodulatory effects on immune cells, even dendritic cells (DCs). However, whether they influence the destiny of full mature DCs (maDCs) remains controversial. Here we report that MSCs vigorously promote proliferation of maDCs, significantly reduce their expression of Ia, CD11c, CD80, CD86, and CD40 while increasing CD11b expression. Interestingly, though these phenotypes clearly suggest their skew to immature status, bacterial lipopolysaccharide (LPS) stimulation could not reverse this trend. Moreover, high endocytosic capacity, low immunogenicity, and strong immunoregulatory function of MSC-treated maDCs (MSC-DCs) were also observed. Furthermore we found that MSCs, partly via cell-cell contact, drive maDCs to differentiate into a novel Jagged-2-dependent regulatory DC population and escape their apoptotic fate. These results further support the role of MSCs in preventing rejection in organ transplantation and treatment of autoimmune disease.

Inner ear progenitor cells can be generated in vitro from human bone marrow mesenchymal stem cells.

PubMed

Boddy, Sarah L; Chen, Wei; Romero-Guevara, Ricardo; Kottam, Lucksy; Bellantuono, Illaria; Rivolta, Marcelo N

2012-11-01

Mouse mesenchymal stem cells (MSCs) can generate sensory neurons and produce inner ear hair cell-like cells. An equivalent source from humans is highly desirable, given their potential application in patient-specific regenerative therapies for deafness. In this study, we explored the ability of human MSCs (hMSCs) to differentiate into otic lineages. hMSCs were exposed to culture media conditioned by human fetal auditory stem cells. Conditioned media induced the expression of otic progenitor markers PAX8, PAX2, GATA3 and SOX2. After 4 weeks, cells coexpressed ATOH1, MYO7A and POU4F3 (indicators of hair cell lineage) or neuronal markers NEUROG1, POU4F1 and NEFH. Inhibition of WNT signaling prevented differentiation into otic progenitors, while WNT activation partially phenocopied results seen with the conditioned media. This study demonstrates that hMSCs can be driven to express key genes found in the otic lineages and thereby promotes their status as candidates for regenerative therapies for deafness.

Enhanced differentiation potential of human amniotic mesenchymal stromal cells by using three-dimensional culturing.

PubMed

Lin, Xue; Li, Hao Yu; Chen, Lian Feng; Liu, Bo Jiang; Yao, Yian; Zhu, Wen Ling

2013-06-01

The therapeutic potential of human amniotic mesenchymal stromal cells (hAMSCs) remains limited because of their differentiation towards mesenchymal stem cells (MSCs) following adherence. The aim of this study was to develop a three-dimensional (3-D) culture system that would permit hAMSCs to differentiate into cardiomyocyte-like cells. hAMSCs were isolated from human amnions of full-term births collected after Cesarean section. Immunocytochemistry, immunofluorescence and flow cytometry analyses were undertaken to examine hAMSC marker expression for differentiation status after adherence. Membrane currents were determined by patch clamp analysis of hAMSCs grown with or without cardiac lysates. Freshly isolated hAMSCs were positive for human embryonic stem-cell-related markers but their marker profile significantly shifted towards that of MSCs following adherence. hAMSCs cultured in the 3-D culture system in the presence of cardiac lysate expressed cardiomyocyte-specific markers, in contrast to those maintained in standard adherent cultures or those in 3-D cultures without cardiac lysate. hAMSCs cultured in 3-D with cardiac lysate displayed a cardiomyocyte-like phenotype as observed by membrane currents, including a calcium-activated potassium current, a delayed rectifier potassium current and a Ca(2+)-resistant transient outward K(+) current. Thus, although adherence limits the potential of hAMSCs to differentiate into cardiomyocyte-like cells, the 3-D culture of hAMSCs represents a more effective method of their culture for use in regenerative medicine.

EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation

PubMed Central

Béguelin, Wendy; Popovic, Relja; Teater, Matt; Jiang, Yanwen; Bunting, Karen L.; Rosen, Monica; Shen, Hao; Yang, Shao Ning; Wang, Ling; Ezponda, Teresa; Martinez-Garcia, Eva; Zhang, Haikuo; Zhang, Yupeng; Verma, Sharad K.; McCabe, Michael T.; Ott, Heidi M.; Van Aller, Glenn S.; Kruger, Ryan G.; Liu, Yan; McHugh, Charles F.; Scott, David W.; Chung, Young Rock; Kelleher, Neil; Shaknovich, Rita; Creasy, Caretha L.; Gascoyne, Randy D.; Wong, Kwok-Kin; Cerchietti, Leandro C.; Levine, Ross L.; Abdel-Wahab, Omar; Licht, Jonathan D.; Elemento, Olivier; Melnick, Ari M.

2013-01-01

The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B-cells and targeted by somatic mutations in B-cell lymphomas. Here we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions in mice. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B-cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets in B-cells, and in human B-cell lymphomas. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GCB-type DLBCLs are mostly addicted to EZH2, regardless of mutation status, but not the more differentiated ABC-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting. PMID:23680150

Prion potency in stem cells biology.

PubMed

Lopes, Marilene H; Santos, Tiago G

2012-01-01

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.

Expression of different functional isoforms in haematopoiesis.

PubMed

Grech, Godfrey; Pollacco, Joel; Portelli, Mark; Sacco, Keith; Baldacchino, Shawn; Grixti, Justine; Saliba, Christian

2014-01-01

Haematopoiesis is a complex process regulated at various levels facilitating rapid responses to external factors including stress, modulation of lineage commitment and terminal differentiation of progenitors. Although the transcription program determines the RNA pool of a cell, various mRNA strands can be obtained from the same template, giving rise to multiple protein isoforms. The majority of variants and isoforms co-occur in normal haematopoietic cells or are differentially expressed at various maturity stages of progenitor maturation and cellular differentiation within the same lineage or across lineages. Genetic aberrations or specific cellular states result in the predominant expression of abnormal isoforms leading to deregulation and disease. The presence of upstream open reading frames (uORF) in 5' untranslated regions (UTRs) of a transcript, couples the utilization of start codons with the cellular status and availability of translation initiation factors (eIFs). In addition, tissue-specific and cell lineage-specific alternative promoter use, regulates several transcription factors producing transcript variants with variable 5' exons. In this review, we propose to give a detailed account of the differential isoform formation, causing haematological malignancies.

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

PubMed Central

Sun, Yanyan; Zhang, Yanli; Wang, Ziyu; Song, Yang; Wang, Feng

2013-01-01

Background Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism. Methodology/Principal Findings Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats. Conclusions/Significance In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future. PMID:24204972

Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression.

PubMed

Yamashita, Michiko; Inoue, Kazuki; Saeki, Noritaka; Ideta-Otsuka, Maky; Yanagihara, Yuta; Sawada, Yuichiro; Sakakibara, Iori; Lee, Jiwon; Ichikawa, Koichi; Kamei, Yoshiaki; Iimura, Tadahiro; Igarashi, Katsuhide; Takada, Yasutsugu; Imai, Yuuki

2018-01-08

Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice ( Uhrf1 Δ Limb/ Δ Limb ) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from Uhrf1 Δ Limb/ Δ Limb mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including Hspb1 , which is reported to be an IL1-related gene and to affect chondrocyte differentiation. Hspb1 knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as Mmp13 These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation. © 2018. Published by The Company of Biologists Ltd.

Climbing Mount Efficiency--small steps, not giant leaps towards higher cloning success in farm animals.

PubMed

Oback, Björn

2008-07-01

Despite more than a decade of research efforts, farm animal cloning by somatic cell nuclear transfer (SCNT) is still frustratingly inefficient. Inefficiency manifests itself at different levels, which are currently not well integrated. At the molecular level, it leads to widespread genetic, epigenetic and transcriptional aberrations in cloned embryos. At the organismal level, these genome-wide abnormalities compromise development of cloned foetuses and offspring. Specific molecular defects need to be causally linked to specific cloned phenotypes, in order to design specific treatments to correct them. Cloning efficiency depends on the ability of the nuclear donor cell to be fully reprogrammed into an embryonic state and the ability of the enucleated recipient cell to carry out the reprogramming reactions. It has been postulated that reprogrammability of the somatic donor cell epigenome is influenced by its differentiation status. However, direct comparisons between cells of divergent differentiation status within several somatic lineages have found no conclusive evidence for this. Choosing somatic stem cells as donors has not improved cloning efficiency, indicating that donor cell type may be less critical for cloning success. Different recipient cells, on the other hand, vary in their reprogramming ability. In bovine, using zygotes instead of oocytes has increased cloning success. Other improvements in livestock cloning efficiency include better coordinating donor cell type with cell cycle stage and aggregating cloned embryos. In the future, it will be important to demonstrate if these small increases at every step are cumulative, adding up to an integrated cloning protocol with greatly improved efficiency.

Modulation of lens cell adhesion molecules by particle beams

NASA Technical Reports Server (NTRS)

McNamara, M. P.; Bjornstad, K. A.; Chang, P. Y.; Chou, W.; Lockett, S. J.; Blakely, E. A.

2001-01-01

Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast, there was a dose-dependent increase in ICAM-1 immunofluorescence in confluent, but not exponentially-growing cells. These results suggest that proton irradiation downregulates beta 1-integrin and upregulates ICAM-1, potentially contributing to cell death or to aberrant differentiation via modulation of anchorage and/or signal transduction functions. Quantification of the expression levels of the CAMs by Western analysis is in progress.

Stem cell technology for tendon regeneration: current status, challenges, and future research directions

PubMed Central

Lui, Pauline Po Yee

2015-01-01

Tendon injuries are a common cause of physical disability. They present a clinical challenge to orthopedic surgeons because injured tendons respond poorly to current treatments without tissue regeneration and the time required for rehabilitation is long. New treatment options are required. Stem cell-based therapies offer great potential to promote tendon regeneration due to their high proliferative, synthetic, and immunomodulatory activities as well as their potential to differentiate to the target cell types and undergo genetic modification. In this review, I first recapped the challenges of tendon repair by reviewing the anatomy of tendon. Next, I discussed the advantages and limitations of using different types of stem cells compared to terminally differentiated cells for tendon tissue engineering. The safety and efficacy of application of stem cells and their modified counterparts for tendon tissue engineering were then summarized after a systematic literature search in PubMed. The challenges and future research directions to enhance, optimize, and standardize stem cell-based therapies for augmenting tendon repair were then discussed. PMID:26715856

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53

PubMed Central

York, D.; Withers, S. S.; Watson, K. D.; Seo, K. W.; Rebhun, R. B.

2016-01-01

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity. PMID:27333821

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild-type p53.

PubMed

York, D; Withers, S S; Watson, K D; Seo, K W; Rebhun, R B

2017-09-01

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity. © 2016 John Wiley & Sons Ltd.

Zebrafish vitamin K epoxide reductases: expression in vivo, along extracellular matrix mineralization and under phylloquinone and warfarin in vitro exposure.

PubMed

Fernández, Ignacio; Vijayakumar, Parameswaran; Marques, Carlos; Cancela, M Leonor; Gavaia, Paulo J; Laizé, Vincent

2015-06-01

Vitamin K (VK) acts as a cofactor driving the biological activation of VK-dependent proteins and conferring calcium-binding properties to them. As a result, VK is converted into VK epoxide, which must be recycled by VK epoxide reductases (Vkors) before it can be reused. Although VK has been shown to play a central role in fish development, particularly during skeletogenesis, pathways underlying VK actions are poorly understood, while good and reliable molecular markers for VK cycle/homeostasis are still lacking in fish. In the present work, expression of 2 zebrafish vkor genes was characterized along larval development and in adult tissues through qPCR analysis. Zebrafish cell line ZFB1 was used to evaluate in vitro regulation of vkors and other VK cycle-related genes during mineralization and upon 24 h exposure to 0.16 and 0.8 µM phylloquinone (VK1), 0.032 µM warfarin, or a combination of both molecules. Results showed that zebrafish vkors are differentially expressed during larval development, in adult tissues, and during cell differentiation/mineralization processes. Further, several VK cycle intermediates were differentially expressed in ZFB1 cells exposed to VK1 and/or warfarin. Present work provides data identifying different developmental stages and adult tissues where VK recycling is probably highly required, and shows how genes involved in VK cycle respond to VK nutritional status in skeletal cells. Expression of vkor genes can represent a reliable indicator to infer VK nutritional status in fish, while ZFB1 cells could represent a suitable in vitro tool to get insights into the mechanisms underlying VK action on fish bone.

A model for genetic and epigenetic regulatory networks identifies rare pathways for transcription factor induced pluripotency

NASA Astrophysics Data System (ADS)

Artyomov, Maxim; Meissner, Alex; Chakraborty, Arup

2010-03-01

Most cells in an organism have the same DNA. Yet, different cell types express different proteins and carry out different functions. This is because of epigenetic differences; i.e., DNA in different cell types is packaged distinctly, making it hard to express certain genes while facilitating the expression of others. During development, upon receipt of appropriate cues, pluripotent embryonic stem cells differentiate into diverse cell types that make up the organism (e.g., a human). There has long been an effort to make this process go backward -- i.e., reprogram a differentiated cell (e.g., a skin cell) to pluripotent status. Recently, this has been achieved by transfecting certain transcription factors into differentiated cells. This method does not use embryonic material and promises the development of patient-specific regenerative medicine, but it is inefficient. The mechanisms that make reprogramming rare, or even possible, are poorly understood. We have developed the first computational model of transcription factor-induced reprogramming. Results obtained from the model are consistent with diverse observations, and identify the rare pathways that allow reprogramming to occur. If validated, our model could be further developed to design optimal strategies for reprogramming and shed light on basic questions in biology.

Urine biomarkers informative of human kidney allograft rejection and tolerance.

PubMed

Nissaisorakarn, Voravech; Lee, John Richard; Lubetzky, Michelle; Suthanthiran, Manikkam

2018-05-01

We developed urinary cell messenger RNA (mRNA) profiling to monitor in vivo status of human kidney allografts based on our conceptualization that the kidney allograft may function as an in vivo flow cell sorter allowing access of graft infiltrating cells to the glomerular ultrafiltrate and that interrogation of urinary cells is informative of allograft status. For the profiling urinary cells, we developed a two-step preamplification enhanced real-time quantitative PCR (RT-QPCR) assays with a customized amplicon; preamplification compensating for the low RNA yield from urine and the customized amplicon facilitating absolute quantification of mRNA and overcoming the inherent limitations of relative quantification widely used in RT-QPCR assays. Herein, we review our discovery and validation of urinary cell mRNAs as noninvasive biomarkers prognostic and diagnostic of acute cellular rejection (ACR) in kidney allografts. We summarize our results reflecting the utility of urinary cell mRNA profiling for predicting reversal of ACR with anti-rejection therapy; differential diagnosis of kidney allograft dysfunction; and noninvasive diagnosis and prognosis of BK virus nephropathy. Messenger RNA profiles associated with human kidney allograft tolerance are also summarized in this review. Altogether, data supporting the idea that urinary cell mRNA profiles are informative of kidney allograft status and tolerance are reviewed in this report. Copyright © 2018. Published by Elsevier Inc.

Multi-pathway Kinase Signatures of Multipotent Stromal Cells are Predictive for Osteogenic Differentiation

PubMed Central

Platt, Manu O.; Wilder, Catera L.; Wells, Alan; Griffith, Linda G.; Lauffenburger, Douglas A.

2010-01-01

Bone marrow-derived multi-potent stromal cells (MSCs) offer great promise for regenerating tissue. While certain transcription factors have been identified in association with tendency toward particular MSC differentiation phenotypes, the regulatory network of key receptor-mediated signaling pathways activated by extracellular ligands that induce various differentiation responses remain poorly understood. Attempts to predict differentiation fate tendencies from individual pathways in isolation are problematic due to the complex pathway interactions inherent in signaling networks. Accordingly, we have undertaken a multi-variate systems approach integrating experimental measurement of multiple kinase pathway activities and osteogenic differentiation in MSCs, together with computational analysis to elucidate quantitative combinations of kinase signals predictive of cell behavior across diverse contexts. In particular, for culture on polymeric biomaterials surfaces presenting tethered epidermal growth factor (tEGF), type-I collagen, neither, or both, we have found that a partial least-squares regression model yields successful prediction of phenotypic behavior on the basis of two principal components comprising the weighted sums of 8 intracellular phosphoproteins: p-EGFR, p-Akt, p-ERK1/2, p-Hsp27, p-c-jun, p-GSK3α/β, p-p38, and p-STAT3. This combination provides strongest predictive capability for 21-day differentiated phenotype status when calculated from day-7 signal measurements (99%); day-4 (88%) and day-14 (89%) signal measurements are also significantly predictive, indicating a broad time-frame during MSC osteogenesis wherein multiple pathways and states of the kinase signaling network are quantitatively integrated to regulate gene expression, cell processes, and ultimately, cell fate. PMID:19750537

The tyrosine kinase receptor HER2 (erbB-2): from oncogenesis to adipogenesis.

PubMed

Vazquez-Martin, Alejandro; Ortega-Delgado, Francisco Jose; Fernandez-Real, Jose Manuel; Menendez, Javier A

2008-12-01

Recent experimental evidences begin to support the notion that the proto-oncogene HER2 (erbB-2) might unexpectedly function to modulate the adipogenic conversion of preadipocytes. Two opposing scenarios have been proposed, however, to explain the influence of HER2 on adipocyte differentiation. In one hand, down-modulation of HER2 expression and pharmacological reduction of HER2 activity have been related to enhanced adipocyte differentiation. On the contrary, an increased abundance in HER2 has been described in differentiated adipocytes compared with preadipocytes. Considering that expression and activity of the lipogenic enzyme Fatty Acid Synthase (FASN) become up-regulated during adipogenic conversion, we recently hypothesized that a "HER2 --> FASN axis" -a "lipogenic benefit" that has been shown to enhance cancer cell proliferation, survival, chemoresistance and metastasis in biologically aggressive subgroups of breast carcinomas-might also naturally work during the differentiation of preadipocytes. To definitely clarify if the discrepancy between the opposing theories for a role of HER2 during adipocyte differentiation related to the experimental approach utilized to compare the abundance of HER2 in undifferentiated and differentiated adipocytes (i.e., cell lysates containing equivalent protein content versus cell lysates generated from similar cell numbers), we here took advantage of a high content microscopy approach. Using an automated confocal imaging platform, we monitored the expression status of the adipogenic marker FASN and its timing relationship with HER2 not only in individual 3T3-L1 cells but further in whole cultures of 3T3-L1 preadipocytes undergoing adipogenic conversion. Our findings not only confirm a non-oncogenic role for HER2 in the process of adipose differentiation but further suggest that HER2 might represent a previously unrecognized target to manage obesity via the lipogenic enzyme FASN.

Transcriptome study of differential expression in schizophrenia

PubMed Central

Sanders, Alan R.; Göring, Harald H. H.; Duan, Jubao; Drigalenko, Eugene I.; Moy, Winton; Freda, Jessica; He, Deli; Shi, Jianxin; Gejman, Pablo V.

2013-01-01

Schizophrenia genome-wide association studies (GWAS) have identified common SNPs, rare copy number variants (CNVs) and a large polygenic contribution to illness risk, but biological mechanisms remain unclear. Bioinformatic analyses of significantly associated genetic variants point to a large role for regulatory variants. To identify gene expression abnormalities in schizophrenia, we generated whole-genome gene expression profiles using microarrays on lymphoblastoid cell lines (LCLs) from 413 cases and 446 controls. Regression analysis identified 95 transcripts differentially expressed by affection status at a genome-wide false discovery rate (FDR) of 0.05, while simultaneously controlling for confounding effects. These transcripts represented 89 genes with functions such as neurotransmission, gene regulation, cell cycle progression, differentiation, apoptosis, microRNA (miRNA) processing and immunity. This functional diversity is consistent with schizophrenia's likely significant pathophysiological heterogeneity. The overall enrichment of immune-related genes among those differentially expressed by affection status is consistent with hypothesized immune contributions to schizophrenia risk. The observed differential expression of extended major histocompatibility complex (xMHC) region histones (HIST1H2BD, HIST1H2BC, HIST1H2BH, HIST1H2BG and HIST1H4K) converges with the genetic evidence from GWAS, which find the xMHC to be the most significant susceptibility locus. Among the differentially expressed immune-related genes, B3GNT2 is implicated in autoimmune disorders previously tied to schizophrenia risk (rheumatoid arthritis and Graves’ disease), and DICER1 is pivotal in miRNA processing potentially linking to miRNA alterations in schizophrenia (e.g. MIR137, the second strongest GWAS finding). Our analysis provides novel candidate genes for further study to assess their potential contribution to schizophrenia. PMID:23904455

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

PubMed

Vahidi Ferdousi, Leyla; Rocheteau, Pierre; Chayot, Romain; Montagne, Benjamin; Chaker, Zayna; Flamant, Patricia; Tajbakhsh, Shahragim; Ricchetti, Miria

2014-11-01

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs. Copyright © 2014. Published by Elsevier B.V.

Expression of Essential B Cell Development Genes in Horses with Common Variable Immunodeficiency

PubMed Central

Tallmadge, R.L.; Such, K.A.; Miller, K.C.; Matychak, M.B.; Felippe, M.J.B.

2012-01-01

Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p < 0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p < 0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients. PMID:22464097

Physiological oxygen prevents frequent silencing of the DLK1-DIO3 cluster during human embryonic stem cells culture.

PubMed

Xie, Pingyuan; Sun, Yi; Ouyang, Qi; Hu, Liang; Tan, Yueqiu; Zhou, Xiaoying; Xiong, Bo; Zhang, Qianjun; Yuan, Ding; Pan, Yi; Liu, Tiancheng; Liang, Ping; Lu, Guangxiu; Lin, Ge

2014-02-01

Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20-30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs. © AlphaMed Press.

Mechanism of Action of Two Flavone Isomers Targeting Cancer Cells with Varying Cell Differentiation Status

PubMed Central

Parsons, Laura B.; Miller, Gerald E.; Whitted, Crystal; Lynch, Kayla E.; Ramsauer, Robert E.; Patel, Jasmine U.; Wyatt, Jarrett E.; Street, Doris S.; Adams, Carolyn B.; McPherson, Brian; Tsui, Hei Man; Evans, Julie A.; Livesay, Christopher; Torrenegra, Ruben D.; Palau, Victoria E.

2015-01-01

Apoptosis can be triggered in two different ways, through the intrinsic or the extrinsic pathway. The intrinsic pathway is mediated by the mitochondria via the release of cytochrome C while the extrinsic pathway is prompted by death receptor signals and bypasses the mitochondria. These two pathways are closely related to cell proliferation and survival signaling cascades, which thereby constitute possible targets for cancer therapy. In previous studies we introduced two plant derived isomeric flavonoids, flavone A and flavone B which induce apoptosis in highly tumorigenic cancer cells of the breast, colon, pancreas, and the prostate. Flavone A displayed potent cytotoxic activity against more differentiated carcinomas of the colon (CaCo-2) and the pancreas (Panc28), whereas flavone B cytotoxic action is observed on poorly differentiated carcinomas of the colon (HCT 116) and pancreas (MIA PaCa). Apoptosis is induced by flavone A in better differentiated colon cancer CaCo-2 and pancreatic cancer Panc 28 cells via the intrinsic pathway by the inhibition of the activated forms of extracellular signal-regulated kinase (ERK) and pS6, and subsequent loss of phosphorylation of Bcl-2 associated death promoter (BAD) protein, while apoptosis is triggered by flavone B in poorly differentiated colon cancer HCT 116 and MIA PaCa pancreatic cancer cells through the extrinsic pathway with the concomitant upregulation of the phosphorylated forms of ERK and c-JUN at serine 73. These changes in protein levels ultimately lead to activation of apoptosis, without the involvement of AKT. PMID:26606169

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

PubMed

Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

2008-08-25

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

Effect of Cytomegalovirus Co-Infection on Normalization of Selected T-Cell Subsets in Children with Perinatally Acquired HIV Infection Treated with Combination Antiretroviral Therapy

PubMed Central

Kapetanovic, Suad; Aaron, Lisa; Montepiedra, Grace; Anthony, Patricia; Thuvamontolrat, Kasalyn; Pahwa, Savita; Burchett, Sandra; Weinberg, Adriana; Kovacs, Andrea

2015-01-01

Background We examined the effect of cytomegalovirus (CMV) co-infection and viremia on reconstitution of selected CD4+ and CD8+ T-cell subsets in perinatally HIV-infected (PHIV+) children ≥ 1-year old who participated in a partially randomized, open-label, 96-week combination antiretroviral therapy (cART)-algorithm study. Methods Participants were categorized as CMV-naïve, CMV-positive (CMV+) viremic, and CMV+ aviremic, based on blood, urine, or throat culture, CMV IgG and DNA polymerase chain reaction measured at baseline. At weeks 0, 12, 20 and 40, T-cell subsets including naïve (CD62L+CD45RA+; CD95-CD28+), activated (CD38+HLA-DR+) and terminally differentiated (CD62L-CD45RA+; CD95+CD28-) CD4+ and CD8+ T-cells were measured by flow cytometry. Results Of the 107 participants included in the analysis, 14% were CMV+ viremic; 49% CMV+ aviremic; 37% CMV-naïve. In longitudinal adjusted models, compared with CMV+ status, baseline CMV-naïve status was significantly associated with faster recovery of CD8+CD62L+CD45RA+% and CD8+CD95-CD28+% and faster decrease of CD8+CD95+CD28-%, independent of HIV VL response to treatment, cART regimen and baseline CD4%. Surprisingly, CMV status did not have a significant impact on longitudinal trends in CD8+CD38+HLA-DR+%. CMV status did not have a significant impact on any CD4+ T-cell subsets. Conclusions In this cohort of PHIV+ children, the normalization of naïve and terminally differentiated CD8+ T-cell subsets in response to cART was detrimentally affected by the presence of CMV co-infection. These findings may have implications for adjunctive treatment strategies targeting CMV co-infection in PHIV+ children, especially those that are now adults or reaching young adulthood and may have accelerated immunologic aging, increased opportunistic infections and aging diseases of the immune system. PMID:25794163

Differentiated all-trans retinoic acid response of naive CD4+CD25– cells isolated from rats with collagen-induced arthritis and healthy ones under in vitro conditions

PubMed Central

Żyromska, Edyta; Piasecki, Tomasz; Rossowska, Joanna; Kędzierska, Anna; Nowak, Marcin; Żyromski, Marcin; Chełmońska-Soyta, Anna

2017-01-01

Aim o the study To compare the potential of CD4+CD25– cells, isolated from both healthy rats and rats with CIA (Collagen-Induced Arthritis), for differentiation into regulatory T cells in the presence of all-trans retinoic acid in order to learn more about the activation mechanisms and therapeutic potential of regulatory T cells. Material and methods Sorted CD4+CD25– cells were cultured in vitro with/without ATRA, and then the frequency of regulatory T cells and their ability to secrete IL-10 by CD4+ FOXP3+ cells was examined. Gene expression of the foxp3, rarα, rarβ, rxrβ, and ppar β/δ and protein expression of the Rarα, Rarβ, and Rxrβ in cells after stimulation with ATRA were also investigated. Results CD4+CD25– cells isolated from healthy animals or from animals with CIA are characterised by different potential of the differentiation into CD4+CD25+ FOXP3+ cells. Retinoic acid receptor Rxrβ is present in the CD4+CD25– cells isolated from rats with CIA. Conclusions We showed that although ATRA did not increase the frequency of Treg in culture, it significantly increased expression of rarβ and rxrβ only in lymphocytes taken from diseased animals and foxp3 expression only in healthy animals. Moreover, after ATRA stimulation, the frequency of Treg-produced IL-10 tended to be lower in diseased animals than in the healthy group. The results imply that the potential of naïve cell CD4 lymphocytes to differentiate into Tregs and their putative suppressive function is dependent on the donor’s health status. PMID:28680330

Slight changes in the mechanical stimulation affects osteoblast- and osteoclast-like cells in co-culture.

PubMed

Kadow-Romacker, Anke; Duda, Georg N; Bormann, Nicole; Schmidmaier, Gerhard; Wildemann, Britt

2013-12-01

Osteoblast- and osteoclast-like cells are responsible for coordinated bone maintenance, illustrated by a balanced formation and resorption. Both parameters appear to be influenced by mechanical constrains acting on each of these cell types individually. We hypothesized that the interactions between both cell types are also influenced by mechanical stimulation. Co-cultures of osteoblast- and osteoclast-like cells were stimulated with 1,100 µstrain, 0.1 or 0.3 Hz for 1-5 min/day over 5 days. Two different setups depending on the differentiation of the osteoclast-like cells were used: i) differentiation assay for the fusion of pre-osteoclasts to osteoclasts, ii) resorption assay to determine the activity level of osteoclast-like cells. In the differentiation assay (co-culture of osteoblasts with unfused osteoclast precursor cells) the mechanical stimulation resulted in a significant decrease of collagen-1 and osteocalcin produced by osteoblast-like cells. Significantly more TRAP-iso5b was measured after stimulation for 3 min with 0.1 Hz, indicating enhanced osteoclastogenesis. In the resorption assay (co-culture of osteoblasts with fused osteoclasts) the stimulation for 3 min with 0.3 Hz significantly increased the resorption activity of osteoclasts measured by the pit formation and the collagen resorption. The same mechanical stimulation resulted in an increased collagen-1 production by the osteoblast-like cells. The ratio of RANKL/OPG was not different between the groups. These findings demonstrate that already small changes in duration or frequency of mechanical stimulation had significant consequences for the behavior of osteoblast- and osteoclast-like cells in co-culture, which partially depend on the differentiation status of the osteoclast-like cells.

Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

PubMed

Takayama, Kazuo; Mizuguchi, Hiroyuki

2017-02-01

Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Carvacrol promotes angiogenic paracrine potential and endothelial differentiation of human mesenchymal stem cells at low concentrations.

PubMed

Matluobi, Danial; Araghi, Atefeh; Maragheh, Behnaz Faramarzian Azimi; Rezabakhsh, Aysa; Soltani, Sina; Khaksar, Majid; Siavashi, Vahid; Feyzi, Adel; Bagheri, Hesam Saghaei; Rahbarghazi, Reza; Montazersaheb, Soheila

2018-01-01

Phenolic monoterpene compound, named Carvacrol, has been found to exert different biological outcomes. It has been accepted that the angiogenic activity of human mesenchymal stem cells was crucial in the pursuit of appropriate regeneration. In the current experiment, we investigated the contribution of Carvacrol on the angiogenic behavior of primary human mesenchymal stem cells. Mesenchymal stem cells were exposed to Carvacrol in a dose ranging from 25 to 200μM for 48h. We measured cell survival rate by MTT assay and migration rate by a scratch test. The oxidative status was monitored by measuring SOD, GPx activity. The endothelial differentiation was studied by evaluating the level of VE-cadherin and vWF by real-time PCR and ELISA analyses. The content of VEGF and tubulogenesis behavior was monitored in vitro. We also conducted Matrigel plug in vivo CAM assay to assess the angiogenic potential of conditioned media from human mesenchymal stem cells after exposure to Carvacrol. Carvacrol was able to increase mesenchymal stem cell survival and migration rate (p<0.05). An increased activity of SOD was obtained while GPx activity unchanged or reduced. We confirmed the endothelial differentiation of stem cells by detecting vWF and VE-cadherin expression (p<0.05). The VEGF expression was increased and mesenchymal stem cells conditioned media improved angiogenesis tube formation in vitro (p<0.05). Moreover, histological analysis revealed an enhanced microvascular density at the site of Matrigel plug in CAM assay. Our data shed lights on the possibility of a Carvacrol to induce angiogenesis in human mesenchymal stem cells by modulating cell differentiation and paracrine angiogenic response. Copyright © 2017 Elsevier Inc. All rights reserved.

Langerhans cell sarcoma following marginal zone lymphoma: expanding the knowledge on mature B cell plasticity.

PubMed

Ambrosio, Maria Raffaella; De Falco, Giulia; Rocca, Bruno Jim; Barone, Aurora; Amato, Teresa; Bellan, Cristiana; Lazzi, Stefano; Leoncini, Lorenzo

2015-10-01

The concept of unidirectional differentiation of the haematopoietic stem cell has been challenged after recent findings that human B cell progenitors and even mature B cells can be reprogrammed into histiocytic/dendritic cells by altering expression of lineage-associated transcription factors. The conversion of mature B cell lymphomas to Langerhans cell neoplasms is not well documented. Three previous reports have described clonally related follicular lymphoma and Langerhans cell tumours, whereas no case has been published of clonally related marginal zone lymphoma and Langerhans cell sarcoma. We describe the case of a 77-year-old patient who developed a Langerhans cell sarcoma and 6 years later a nodal marginal zone lymphoma. Mutation status examination showed 100 % gene identity to the germline sequence, suggesting direct trans-differentiation or dedifferentiation of the nodal marginal zone lymphoma to the Langerhans cell sarcoma rather than a common progenitor. We found inactivation of paired box 5 (PAX-5) in the lymphoma cells by methylation, along with duplication of part of the long arm of chromosomes 16 and 17 in the sarcoma cells. The absence of PAX-5 could have triggered B cells to differentiate into macrophages and dendritic cells. On the other hand, chromosomal imbalances might have activated genes involved in myeloid lineage maturation, transcription activation and oncogenesis. We hypothesize that this occurred because of previous therapies for nodal marginal zone lymphoma. Better understanding of this phenomenon may help in unravelling the molecular interplay between transcription factors during haematopoietic lineage commitment and may expand the spectrum of clonally related mature B cell neoplasms and Langerhans cell tumours.

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

PubMed

Imai, Hiroyuki; Kano, Kiyoshi; Fujii, Wataru; Takasawa, Ken; Wakitani, Shoichi; Hiyama, Masato; Nishino, Koichiro; Kusakabe, Ken Takeshi; Kiso, Yasuo

2015-01-01

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

Implicit rationing criteria in non-small-cell lung cancer treatment.

PubMed Central

Arndt, K.; Coy, P.; Schaafsma, J.

1996-01-01

Data collected from lung cancer patients attending the Victoria Clinic of the British Columbia Cancer Agency are used to investigate how resources are rationed in the treatment of non-small-cell lung cancer (NSCLC). An ordered logit model is estimated to analyse empirically the relationship between treatment selection and: tumour stage, size and differentiation; the Feinstein index; Karnofsky performance status (KPS); and the patient's age, gender and marital and smoking status. Implicit rationing is found to occur with respect to all of these factors except the Feinstein index, gender and marital status. With respect to age, KPS and smoker status the main empirical results are: (a) an increase in age from 50 to 85 reduces the expected treatment expenditure by 50-70%, depending on the patient's KPS and smoker status; (b) patients with a KPS less than 80 and of 80, receive 30-46% and 75-85%, respectively, of the expected treatment expenditure for patients with a KPS of 90 or 100, depending on age and smoker status; (c) the expected treatment expenditure for active smokers is about 71-86% of the expenditure for non- or former smokers depending on age and KPS. PMID:8611380

Diamine Oxidase from White Pea (Lathyrus sativus) Combined with Catalase Protects the Human Intestinal Caco-2 Cell Line from Histamine Damage.

PubMed

Jumarie, Catherine; Séïde, Marilyne; Marcocci, Lucia; Pietrangeli, Paola; Mateescu, Mircea Alexandru

2017-07-01

Diamine oxidase (DAO) administration has been proposed to treat certain gastrointestinal dysfunctions induced by histamine, an immunomodulator, signaling, and pro-inflammatory factor. However, H 2 O 2 resulting from the oxidative deamination of histamine by DAO may be toxic. The purpose of this study was to investigate to which extent DAO from white pea (Lathyrus sativus), alone or in combination with catalase, may modulate histamine toxicity in the human intestinal Caco-2 cell line. The results show that histamine at concentrations higher than 1 mM is toxic to the Caco-2 cells, independently of the cell differentiation status, with a LC 50 of ≅ 10 mM following a 24-h exposure. Depending on its concentration, DAO increased histamine toxicity to a greater extent in differentiated cells compared to undifferentiated cultures. In the presence of catalase, the DAO-induced increase in histamine toxicity was completely abolished in the undifferentiated cells and only partially decreased in differentiated cells, showing differences in the sensitivity of Caco-2 cells to the products resulting from histamine degradation by DAO (H 2 O 2 , NH 3 , or imidazole aldehyde). It appears that treatment of food histaminosis using a combination of vegetal DAO and catalase would protect against histamine toxicity and prevent H 2 O 2 -induced damage that may occur during histamine oxidative deamination.

p21WAF1 immunohistochemical expression in breast carcinoma: correlations with clinicopathological data, oestrogen receptor status, MIB1 expression, p53 gene and protein alterations and relapse-free survival.

PubMed Central

Barbareschi, M.; Caffo, O.; Doglioni, C.; Fina, P.; Marchetti, A.; Buttitta, F.; Leek, R.; Morelli, L.; Leonardi, E.; Bevilacqua, G.; Dalla Palma, P.; Harris, A. L.

1996-01-01

p21 protein (p21) inhibitor of cyclin-dependent kinases is a critical downstream effector in the p53-specific pathway of growth control. p21 can also be induced by p53-independent pathways in relation to terminal differentiation. We investigated p21 immunoreactivity in normal breast and in 91 breast carcinomas [three in situ ductal carcinomas (DCIS) with microinfiltration and 88 infiltrating carcinomas, 17 of which with an associated DCIS; 57 node negative and 34 node positive] with long-term follow-up (median = 58 months). Seven additional breast carcinomas with known p53 gene mutations were investigated. In normal breast p21 expression was seen in the nuclei of rare luminal cells of acinar structures, and in occasional myoepithelial cells. Poorly differentiated DCIS showed high p21 expression, whereas well-differentiated DCIS tumours showed few p21-reactive cells. p21 was seen in 82 (90%) infiltrating tumours; staining was heterogeneous; the percentage of reactive nuclei ranged from 1% to 35%. High p21 expression (more than 10% of reactive cells) was seen in 24 (26%) cases, and was associated with high tumour grade (P = 0.032); no associations were seen with tumour size, metastases, oestrogen receptor status, MIB1 expression and p53 expression. p21 expression in cases with p53 gene mutations was low in six cases and high in one. High p21 expression was associated with short relapse-free survival (P = 0.003). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8688323

PTEN loss represses glioblastoma tumor initiating cell differentiation via inactivation of Lgl1.

PubMed

Gont, Alexander; Hanson, Jennifer E L; Lavictoire, Sylvie J; Parolin, Doris A; Daneshmand, Manijeh; Restall, Ian J; Soucie, Mathieu; Nicholas, Garth; Woulfe, John; Kassam, Amin; Da Silva, Vasco F; Lorimer, Ian A J

2013-08-01

Glioblastoma multiforme is an aggressive and incurable type of brain tumor. A subset of undifferentiated glioblastoma cells, known as glioblastoma tumor initiating cells (GTICs), has an essential role in the malignancy of this disease and also appears to mediate resistance to radiation therapy and chemotherapy. GTICs retain the ability to differentiate into cells with reduced malignant potential, but the signaling pathways controlling differentiation are not fully understood at this time. PTEN loss is a very common in glioblastoma multiforme and leads to aberrant activation of the phosphoinositide 3-kinase pathway. Increased signalling through this pathway leads to activation of multiple protein kinases, including atypical protein kinase C. In Drosophila, active atypical protein kinase C has been shown to promote the self-renewal of neuroblasts, inhibiting their differentiation along a neuronal lineage. This effect is mediated by atypical protein kinase c-mediated phosphorylation and inactivation of Lgl, a protein that was first characterized as a tumour suppressor in Drosophila. The effects of the atypical protein kinase C/Lgl pathway on the differentiation status of GTICs, and its potential link to PTEN loss, have not been assessed previously. Here we show that PTEN loss leads to the phosphorylation and inactivation of Lgl by atypical protein kinase C in glioblastoma cells. Re-expression of PTEN in GTICs promoted their differentiation along a neuronal lineage. This effect was also seen when atypical protein kinase C was knocked down using RNA interference, and when a non-phosphorylatable, constitutively active form of Lgl was expressed in GTICs. Thus PTEN loss, acting via atypical protein kinase C activation and Lgl inactivation, helps to maintain GTICs in an undifferentiated state.

Murine Mesenchymal Stem Cell Commitment to Differentiation is Regulated by Mitochondrial Dynamics

PubMed Central

Forni, Maria Fernanda; Peloggia, Julia; Trudeau, Kyle; Shirihai, Orian; Kowaltowski, Alicia J.

2015-01-01

Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105+CD90+CD73+CD29+CD34− mesodermal precursors which, after in vitro induction, undergo chondro, adipo and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro and adipocytes and measuring changes in mass, morphology, dynamics and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1 and 2 and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells. PMID:26638184

Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

PubMed

Corneau, Aurélien; Cosma, Antonio; Even, Sophie; Katlama, Christine; Le Grand, Roger; Frachet, Véronique; Blanc, Catherine; Autran, Brigitte

2017-01-01

Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

BaxΔ2 sensitizes colorectal cancer cells to proteasome inhibitor-induced cell death

PubMed Central

Mañas, Adriana; Chen, Wenjing; Nelson, Adam; Yao, Qi; Xiang, Jialing

2018-01-01

Proteasome inhibitors, such as bortezomib and carfilzomib, are FDA approved for the treatment of hemopoietic cancers, but recent studies have shown their great potential for treatment of solid tumors. BaxΔ2, a unique proapoptotic Bax isoform, promotes non-mitochondrial cell death and sensitizes cancer cells to chemotherapy. However, endogenous BaxΔ2 proteins are unstable and susceptible to proteasomal degradation. Here, we screened a panel of proteasome inhibitors in colorectal cancer cells with different Bax statuses. We found that all proteasome inhibitors tested were able to block BaxΔ2 degradation without affecting the level of Baxα or Bcl-2 proteins. Among the inhibitors tested, only bortezomib and carfilzomib were able to induce differential cell death corresponding to the distinct Bax statuses. BaxΔ2-positive cells had a significantly higher level of cell death at low nanomolar concentrations than Baxα-positive or Bax-negative cells. Furthermore, bortezomib-induced cell death in BaxΔ2-positive cells was predominantly dependent on the caspase 8/3 pathway, consistent with our previous studies. These results imply that BaxΔ2 can selectively sensitize cancer cells to proteasome inhibitors, enhancing their potential to treat colon cancer and other solid tumors. PMID:29291406

In vivo microscopy of human leucocytes(Conference Presentation)

NASA Astrophysics Data System (ADS)

Winer, Matan; Yeheskely-Hayon, Daniella; Zeidan, Adel; Yelin, Dvir

2017-02-01

White blood cells (WBC) analysis is an important part of the complete blood count, providing good indication of the patient's immune system status. The most common types of WBCs are the neutrophils and lymphocytes that comprise approximately 60% and 30% of the total WBC count, respectively; differentiating between these cells at the point of care would assist in accurate diagnosis of the possible source of infection (viral or bacterial) and in effective prescription of antibiotics. In this work, we demonstrate the potential of spectrally encoded flow cytometry (SEFC) to non-invasively image WBC in human patients, allowing morphology characterization of the main types of WBCs. The optical setup includes a broadband light that was diffracted and focused onto a single transverse line within the cross section of a small blood vessel at the inner patient lip. Light backscattered from the tissue was measured by a high-speed spectrometer, forming a two-dimensional reflectance confocal image of the flowing cells. By imaging at different depths into vessels of different diameters, we determine optimal imaging conditions (i.e. imaging geometry, speed and depth) for counting the total amount of WBCs and for differentiating between their main types. The presented technology could serve for analyzing the immune system status at the point of care, and for studying the morphological and dynamical characteristics of these cells in vivo.

Anti-Neoplastic Activity of Two Flavone Isomers Derived from Gnaphalium elegans and Achyrocline bogotensis

PubMed Central

Pendleton, Morgan H.; Torrenegra, Ruben D.; Rodriguez, Oscar E.; Harirforoosh, Sam; Ballester, Maria; Lightner, Janet; Krishnan, Koyamangalath; Ramsauer, Victoria P.

2012-01-01

Over 4000 flavonoids have been identified so far and among these, many are known to have antitumor activities. The basis of the relationships between chemical structures, type and position of substituent groups and the effects these compounds exert specifically on cancer cells are not completely elucidated. Here we report the differential cytotoxic effects of two flavone isomers on human cancer cells from breast (MCF7, SK-BR-3), colon (Caco-2, HCT116), pancreas (MIA PaCa, Panc 28), and prostate (PC3, LNCaP) that vary in differentiation status and tumorigenic potential. These flavones are derived from plants of the family Asteraceae, genera Gnaphalium and Achyrocline reputed to have anti-cancer properties. Our studies indicate that 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays potent activity against more differentiated carcinomas of the colon (Caco-2), and pancreas (Panc28), whereas 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) cytototoxic action is observed on poorly differentiated carcinomas of the colon (HCT116), pancreas (Mia PaCa), and breast (SK-BR3). Both flavones induced cell death (>50%) as proven by MTT cell viability assay in these cancer cell lines, all of which are regarded as highly tumorigenic. At the concentrations studied (5–80 µM), neither flavone demonstrated activity against the less tumorigenic cell lines, breast cancer MCF-7 cells, androgen-responsive LNCaP human prostate cancer line, and androgen-unresponsive PC3 prostate cancer cells. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays activity against more differentiated carcinomas of the colon and pancreas, but minimal cytotoxicity on poorly differentiated carcinomas of these organs. On the contrary, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) is highly cytotoxic to poorly differentiated carcinomas of the colon, pancreas, and breast with minimal activity against more differentiated carcinomas of the same organs. These differential effects suggest activation of distinct apoptotic pathways. In conclusion, the specific chemical properties of these two flavone isomers dictate mechanistic properties which may be relevant when evaluating biological responses to flavones. PMID:22768128

Characterization of cell proliferation throughout the brain of the African cichlid fish Astatotilapia burtoni and its regulation by social status.

PubMed

Maruska, Karen P; Carpenter, Russ E; Fernald, Russell D

2012-10-15

New cells are added in the brains of all adult vertebrates, but fishes have some of the greatest potential for neurogenesis and gliogenesis among all taxa, partly due to their indeterminate growth. Little is known, however, about how social interactions influence cell proliferation in the brain of these fishes that comprise the largest group of vertebrates. We used 5-bromo-2'-deoxyuridine (BrdU) to identify and localize proliferation zones in the telencephalon, diencephalon, mesencephalon, and rhombencephalon that were primarily associated with ventricular surfaces in the brain of the African cichlid fish Astatotilapia burtoni. Cell migration was evident in some regions by 1 day post injection, and many newborn cells coexpressed the neuronal marker HuC/D at 30 days, suggesting they had differentiated into neurons. To test the hypothesis that social status and perception of an opportunity to rise in rank influenced cell proliferation, we compared numbers of BrdU-labeled cells in multiple brain nuclei among fish of different social status. Socially suppressed subordinate males had the lowest numbers of proliferating cells in all brain regions examined, but males that were given an opportunity to rise in status had higher cell proliferation rates within 1 day, suggesting rapid upregulation of brain mitotic activity associated with this social transition. Furthermore, socially isolated dominant males had similar numbers of BrdU-labeled cells compared with dominant males that were housed in a socially rich environment, suggesting that isolation has little effect on proliferation and that reduced proliferation in subordinates is a result of the social subordination. These results suggest that A. burtoni will be a useful model to analyze the mechanisms of socially induced neurogenesis in vertebrates. Copyright © 2012 Wiley Periodicals, Inc.

Progenitor Epithelium

PubMed Central

Marty-Santos, Leilani

2015-01-01

Insulin-producing β cells within the vertebrate fetal pancreas acquire their fate in a step-wise manner. Whereas the intrinsic factors dictating the transcriptional or epigenetic status of pancreatic lineages have been intensely examined, less is known about cell–cell interactions that might constitute a niche for the developing β cell lineage. It is becoming increasingly clear that understanding and recapitulating these steps may instruct in vitro differentiation of embryonic stem cells and/or therapeutic regeneration. Indeed, directed differentiation techniques have improved since transitioning from 2D to 3D cultures, suggesting that the 3D microenvironment in which β cells are born is critical. However, to date, it remains unknown whether the changing architecture of the pancreatic epithelium impacts the fate of cells therein. An emerging challenge in the field is to elucidate how progenitors are allocated during key events, such as the stratification and subsequent resolution of the pre-pancreatic epithelium, as well as the formation of lumens and branches. Here, we assess the progenitor epithelium and examine how it might influence the emergence of pancreatic multipotent progenitors (MPCs), which give rise to β cells and other pancreatic lineages. PMID:26216134

Stem Cell Therapy in Bladder Dysfunction: Where Are We? And Where Do We Have to Go?

PubMed Central

Lee, Sang-Rae; Song, Yun Seob; Lee, Hong Jun

2013-01-01

To date, stem cell therapy for the bladder has been conducted mainly on an experimental basis in the areas of bladder dysfunction. The therapeutic efficacy of stem cells was originally thought to be derived from their ability to differentiate into various cell types. Studies about stem cell therapy for bladder dysfunction have been limited to an experimental basis and have been less focused than bladder regeneration. Bladder dysfunction was listed in MESH as “urinary bladder neck obstruction”, “urinary bladder, overactive”, and “urinary bladder, neurogenic”. Using those keywords, several articles were searched and studied. The bladder dysfunction model includes bladder outlet obstruction, cryoinjured, diabetes, ischemia, and spinal cord injury. Adipose derived stem cells (ADSCs), bone marrow stem cells (BMSCs), and skeletal muscle derived stem cells (SkMSCs) are used for transplantation to treat bladder dysfunction. The main mechanisms of stem cells to reconstitute or restore bladder dysfunction are migration, differentiation, and paracrine effects. The aim of this study is to review the stem cell therapy for bladder dysfunction and to provide the status of stem cell therapy for bladder dysfunction. PMID:24151627

Different expression patterns of LGALS1 and LGALS3 in polycythemia vera, essential thrombocythemia and primary myelofibrosis.

PubMed

Moura, L G; Tognon, R; Nunes, N S; Rodrigues, L Cataldi; Ferreira, A F; Kashima, S; Covas, D T; Santana, M; Souto, E X; Perobelli, L; Simões, B P; Dias-Baruffi, M; Castro, F A

2016-10-01

Despite all the knowledge, the cellular and molecular mechanisms involved in myeloproliferative neoplasm (MPN) pathophysiology remain unclear. Authors have shown galectin-1 (Gal-1) and 3 playing roles in tumour angiogenesis and fibrosis, which were correlated with poor prognosis in patients with MPN. In the present study LGALS1 and LGALS3 were differently expressed between polycythemia vera, essential thrombocythemia (ET) and primary myelofibrosis (PMF) diseases. Increased LGALS3 expression was associated with a negative JAK2 V617F status mutation in leucocytes from PMF but not in patients with ET without this mutation. However, a positive Janus kinase 2 (JAK2) V617F cell line established from patients with ET (SET-2 cells) when treated with JAK inhibitor presented high levels of LGALS3. Additionally, high LGALS1 expression was found in CD34(+) cells but not in leucocytes from patients with PMF, in absence of JAK2 V617F mutation, and also in SET-2 cells treated with JAK inhibitor. Thus, our findings indicate that differential expression of LGALS1 and/or LGALS3 in patients with MPN is linked with JAK2 V617F status mutation in these diseases and state of cell differentiation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

p53-dependent and p53-independent anticancer activity of a new indole derivative in human osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cappadone, C., E-mail: concettina.cappadone@unibo.it; Stefanelli, C.; Malucelli, E.

2015-11-13

Osteosarcoma (OS) is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents. The mechanism of formation and development of OS have been studied for a long time. Tumor suppressor pathway governed by p53 gene are known to be involved in the pathogenesis of osteosarcoma. Moreover, loss of wild-type p53 activity is thought to be a major predictor of failure to respond to chemotherapy in various human cancers. In previous studies, we described the activity of a new indole derivative, NSC743420, belonging to the tubulin inhibitors family, capable to induce apoptosis and arrest of themore » cell cycle in the G2/M phase of various cancer cell lines. However, this molecule has never been tested on OS cell line. Here we address the activity of NSC743420 by examine whether differences in the p53 status could influence its effects on cell proliferation and death of OS cells. In particular, we compared the effect of the tested molecule on p53-wild type and p53-silenced U2OS cells, and on SaOS2 cell line, which is null for p53. Our results demonstrated that NSC743420 reduces OS cell proliferation by p53-dependent and p53-independent mechanisms. In particular, the molecule induces proliferative arrest that culminate to apoptosis in SaOS2 p53-null cells, while it brings a cytostatic and differentiating effect in U2OS cells, characterized by the cell cycle arrest in G0/G1 phase and increased alkaline phosphatase activity. - Highlights: • The indole derivative NSC743420 induces antitumor effects on osteosarcoma cells. • p53 status could drive the activity of antitumor agents on osteosarcoma cells. • NSC743420 induces cytostatic and differentiating effects on U2OS cells. • NSC743420 causes apoptosis on p53-null SaOS2 cells.« less

MicroRNA-29a ameliorates glucocorticoid-induced suppression of osteoblast differentiation by regulating β-catenin acetylation.

PubMed

Ko, Jih-Yang; Chuang, Pei-Chin; Chen, Ming-Wen; Ke, Huei-Ching; Wu, Shin-Long; Chang, Yu-Hsuan; Chen, Yu-Shan; Wang, Feng-Sheng

2013-12-01

Excess glucocorticoid treatment induces loss of osteoblast differentiation. Post-translational modification of β-catenin reportedly regulates osteogenic activities in bone cells. This study was undertaken to test whether miR-29a signaling regulates the acetylation status of β-catenin in the glucocorticoid-mediated osteoblast dysfunction. Murine osteoblast cultures were incubated under osteogenic conditions with or without supraphysiological glucocorticoid, miR-29a precursor, antisense oligonucleotides or histone deacetylase 4 (HDAC4) RNA interferences. Osteoblast differentiation was determined by alkaline phosphatase activity, calcium deposition, and von Kossa stain. β-Catenin acetylation and miR-29a transcription were detected by immunoblotting, chromatin immunoprecipitation and quantitative PCR. Protein interaction was detected by fluorescence protein ligation assay. Supraphysiological glucocorticoid treatment repressed osteoblast differentiation and induced loss of miR-29a expression and acetylated β-catenin levels in osteoblast cultures. Gain of miR-29a function attenuated the deleterious effects of glucocorticoid on osteogenic gene expression and mineralized nodule formation, whereas knockdown of miR-29a signaling accelerated loss of osteoblast differentiation capacity. miR-29a reduced HDAC4 signaling and attenuated the glucocorticoid-mediated β-catenin deacetylation and ubiquitination and restored nuclear β-catenin levels. Glucocorticoid-induced loss of miR-29a signaling occurred through transcriptional and translational regulation. Interruption of HDAC4 signaling attenuated the glucocorticoid-induced hypoacetylation of histone H3 at lysine 9 (H3K9Ac) and restored the enrichment of H3K9Ac in miR-29a proximal promoter region and miR-29a transcription in cell cultures. Taken together, excess glucocorticoid-induced loss of miR-29a signaling accelerates β-catenin deacetylation and ubiquitination that impairs osteogenic activities of osteoblast cultures. miR-29a and HDAC4 reciprocal regulation of H3K9 acetylation contributes to the acetylation status of β-catenin and miR-29a expression. Enhancement of miR-29a signaling is an alternative strategy for protecting against the adverse actions of excess glucocorticoid on differentiation capacity of osteogenic cells. © 2013.

[Current status of bone/cartilage tissue engineering towards clinical applications].

PubMed

Ohgushi, Hajime

2014-10-01

Osteo/chondrogenic differentiation capabilities are seen after in vivo implantation of mesenchymal stem cells (MSCs), which are currently used for the patients having bone/cartilage defects. Importantly, the differentiation capabilities are induced by culturing technology, resulting in in vitro bone/cartilage formation. Especially, the in vitro bone tissue is useful for bone tissue regeneration. For cartilage regeneration, culture expanded chondrocytes derived from patient's normal cartilage are also used for the patients having cartilage damages. Recently, the cultured chondrocytes embedded in atelocollagen gel are obtainable as tissue engineered products distributed by Japan Tissue Engineering Co. Ltd. The products are available in the well-regulated hospitals by qualified orthopedic surgeons. The criteria for these hospitals/surgeons have been established. This review paper focuses on current status of bone/cartilage tissue engineering towards clinical applications in Japan.

A systemic evolutionary approach to cancer: Hepatocarcinogenesis as a paradigm.

PubMed

Mazzocca, Antonio; Ferraro, Giovanni; Misciagna, Giovanni; Carr, Brian I

2016-08-01

The systemic evolutionary theory of cancer pathogenesis posits that cancer is generated by the de-emergence of the eukaryotic cell system and by the re-emergence of its archaea (genetic material and cytoplasm) and prokaryotic (mitochondria) subsystems with an uncoordinated behavior. This decreased coordination can be caused by a change in the organization of the eukaryote environment (mainly chronic inflammation), damage to mitochondrial DNA and/or to its membrane composition by many agents (e.g. viruses, chemicals, hydrogenated fatty acids in foods) or damage to nuclear DNA that controls mitochondrial energy production or metabolic pathways, including glycolysis. Here, we postulate that the two subsystems (the evolutionarily inherited archaea and the prokaryote) in a eukaryotic differentiated cell are well integrated, and produce the amount of clean energy that is constantly required to maintain the differentiated status. Conversely, when protracted injuries impair cell or tissue organization, the amount of energy necessary to maintain cell differentiation can be restricted, and this may cause gradual de-differentiation of the eukaryotic cell over time. In cirrhotic liver, for example, this process can be favored by reduced oxygen availability to the organ due to an altered vasculature and the fibrotic barrier caused by the disease. Thus, hepatocarcinogenesis is an ideal example to support our hypothesis. When cancer arises, the pre-eukaryote subsystems become predominant, as shown by the metabolic alterations of cancer cells (anaerobic glycolysis and glutamine utilization), and by their capacity for proliferation and invasion, resembling the primitive symbiotic components of the eukaryotic cell. Copyright © 2016 Elsevier Ltd. All rights reserved.

Improvement of neuronal differentiation by carbon monoxide: Role of pentose phosphate pathway.

PubMed

Almeida, Ana S; Soares, Nuno L; Sequeira, Catarina O; Pereira, Sofia A; Sonnewald, Ursula; Vieira, Helena L A

2018-05-15

Over the last decades, the silent-killer carbon monoxide (CO) has been shown to also be an endogenous cytoprotective molecule able to inhibit cell death and modulate mitochondrial metabolism. Neuronal metabolism is mostly oxidative and neurons also use glucose for maintaining their anti-oxidant status by generation of reduced glutathione (GSH) via the pentose-phosphate pathway (PPP). It is established that neuronal differentiation depends on reactive oxygen species (ROS) generation and signalling, however there is a lack of information about modulation of the PPP during adult neurogenesis. Thus, the main goal of this study was to unravel the role of CO on cell metabolism during neuronal differentiation, particularly by targeting PPP flux and GSH levels as anti-oxidant system. A human neuroblastoma SH-S5Y5 cell line was used, which differentiates into post-mitotic neurons by treatment with retinoic acid (RA), supplemented or not with CO-releasing molecule-A1 (CORM-A1). SH-SY5Y cell differentiation supplemented with CORM-A1 prompted an increase in neuronal yield production. It did, however, not alter glycolytic metabolism, but increased the PPP. In fact, CORM-A1 treatment stimulated (i) mRNA expression of 6-phosphogluconate dehydrogenase (PGDH) and transketolase (TKT), which are enzymes for oxidative and non-oxidative phases of the PPP, respectively and (ii) protein expression and activity of glucose 6-phosphate dehydrogenase (G6PD) the rate-limiting enzyme of the PPP. Likewise, whenever G6PD was knocked-down CO-induced improvement on neuronal differentiation was reverted, while pharmacological inhibition of GSH synthesis did not change CO's effect on the improvement of neuronal differentiation. Both results indicate the key role of PPP in CO-modulation of neuronal differentiation. Furthermore, at the end of SH-SY5Y neuronal differentiation process, CORM-A1 supplementation increased the ratio of reduced and oxidized glutathione (GSH/GSSG) without alteration of GSH metabolism. These data corroborate with PPP stimulation. In conclusion, CO improves neuronal differentiation of SH-S5Y5 cells by stimulating the PPP and modulating the GSH system. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration.

PubMed

Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

2016-11-15

Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk.

Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration

PubMed Central

Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

2016-01-01

Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk. PMID:27705914

Repression of TSC1/TSC2 mediated by MeCP2 regulates human embryo lung fibroblast cell differentiation and proliferation.

PubMed

Wang, Yuanyuan; Chen, Chen; Deng, Ziyu; Bian, Erbao; Huang, Cheng; Lei, Ting; Lv, Xiongwen; Liu, Liping; Li, Jun

2017-03-01

Pulmonary fibrosis (PF) is a severe inflammatory disease with limited effective treatments. It is known that the transdifferentiation of human embryo lung fibroblast (HELF) cells from pulmonary fibroblasts into myofibroblasts, contributes to the progression of pulmonary fibrogenesis. The tuberous sclerosis proteins TSC1 and TSC2 are two key signaling factors which can suppress cell growth and proliferation. However, the roles of TSC1 and TSC2 in lung fibroblast are unclear. Here, we developed a PF model with bleomycin (BLM) in mice and conducted several simulation experiments in HELF cells. Our study shows that the expression of TSC1 and TSC2 in fibrotic mice lung was reduced and stimulation of HELF cells with TGF-β1 resulted in a down-regulation of TSC1 and TSC2. In addition, overexpression of TSC1 or TSC2 decreased cell proliferation and differentiation. Furthermore, we found that reduced expression of TSC1 and TSC2 caused by TGF-β1 is associated with the promoter methylation status of TSC1 and TSC2. MeCP2, controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. We found that expression of TSC1 and TSC2 can be repressed by MeCP2, which regulates HELF cell differentiation and proliferation as myofibroblasts and lead to PF ultimately. Copyright © 2016. Published by Elsevier B.V.

Influence of clinical status and parasite load on erythropoiesis and leucopoiesis in dogs naturally infected with leishmania (Leishmania) chagasi.

PubMed

Trópia de Abreu, Raquel; Carvalho, Maria das Graças; Carneiro, Cláudia Martins; Giunchetti, Rodolfo Cordeiro; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; Coura-Vital, Wendel; Corrêa-Oliveira, Rodrigo; Reis, Alexandre Barbosa

2011-05-10

The bone marrow is considered to be an important storage of parasites in Leishmania-infected dogs, although little is known about cellular genesis in this organ during canine visceral leishmaniasis (CVL). The aim of the present study was to evaluate changes in erythropoiesis and leucopoiesis in bone marrow aspirates from dogs naturally infected with Leishmania chagasi and presenting different clinical statuses and bone marrow parasite densities. The evolution of CVL from asymptomatic to symptomatic status was accompanied by increasing parasite density in the bone marrow. The impact of bone marrow parasite density on cellularity was similar in dogs at different clinical stages, with animals in the high parasite density group. Erythroid and eosinophilic hypoplasia, proliferation of neutrophilic precursor cells and significant increases in lymphocytes and plasma cell numbers were the major alterations observed. Differential bone marrow cell counts revealed increases in the myeloid:erythroid ratio associated to increased numbers of granulopoietic cells in the different clinical groups compared with non-infected dogs. Analysis of the data obtained indicated that the assessment of bone marrow constitutes an additional and useful tool by which to elaborate a prognosis for CVL.

Influence of Clinical Status and Parasite Load on Erythropoiesis and Leucopoiesis in Dogs Naturally Infected with Leishmania (Leishmania) chagasi

PubMed Central

Trópia de Abreu, Raquel; Carvalho, Maria das Graças; Carneiro, Cláudia Martins; Giunchetti, Rodolfo Cordeiro; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; Coura-Vital, Wendel; Corrêa-Oliveira, Rodrigo; Reis, Alexandre Barbosa

2011-01-01

Background The bone marrow is considered to be an important storage of parasites in Leishmania-infected dogs, although little is known about cellular genesis in this organ during canine visceral leishmaniasis (CVL). Methodology/Principal Findings The aim of the present study was to evaluate changes in erythropoiesis and leucopoiesis in bone marrow aspirates from dogs naturally infected with Leishmania chagasi and presenting different clinical statuses and bone marrow parasite densities. The evolution of CVL from asymptomatic to symptomatic status was accompanied by increasing parasite density in the bone marrow. The impact of bone marrow parasite density on cellularity was similar in dogs at different clinical stages, with animals in the high parasite density group. Erythroid and eosinophilic hypoplasia, proliferation of neutrophilic precursor cells and significant increases in lymphocytes and plasma cell numbers were the major alterations observed. Differential bone marrow cell counts revealed increases in the myeloid:erythroid ratio associated to increased numbers of granulopoietic cells in the different clinical groups compared with non-infected dogs. Conclusions Analysis of the data obtained indicated that the assessment of bone marrow constitutes an additional and useful tool by which to elaborate a prognosis for CVL. PMID:21572995

Altered DNA methylation profile in Norwegian patients with Autoimmune Addison's Disease.

PubMed

Bjanesoy, Trine E; Andreassen, Bettina Kulle; Bratland, Eirik; Reiner, Andrew; Islam, Shahinul; Husebye, Eystein S; Bakke, Marit

2014-06-01

Autoimmune Addison's Disease (AAD) is an endocrine and immunological disease of uncertain pathogenesis resulting from the immune system's destruction of the hormone producing cells of the adrenal cortex. The underlying molecular mechanisms are largely unknown, but it is commonly accepted that a combination of genetic susceptibility and environmental impact is critical. In the present study, we identified multiple hypomethylated gene promoter regions in patients with isolated AAD using DNA isolated from CD4+ T cells. The identified differentially methylated regions were distributed evenly across the 10.5-kb-promoter regions covered by the array, and a substantial number localized to promoters of genes involved in immune regulation and autoimmunity. This study reveals a hypomethylated status in CD4+ T cells from AAD patients and indicates differential methylation of promoters of key genes involved in immune responses. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Differential Prognostic Implications of Gastric Signet Ring Cell Carcinoma

PubMed Central

Chon, Hong Jae; Hyung, Woo Jin; Kim, Chan; Park, Sohee; Kim, Jie-Hyun; Park, Chan Hyuk; Ahn, Joong Bae; Kim, Hyunki; Chung, Hyun Cheol; Rha, Sun Young; Noh, Sung Hoon; Jeung, Hei-Cheul

2017-01-01

Objective: The aim of this study was to analyze the clinicopathologic characteristics and prognosis of signet ring cell carcinoma (SRC) according to disease status (early vs advanced gastric cancer) in gastric cancer patients. Background: The prognostic implication of gastric SRC remains a subject of debate. Methods: A retrospective analysis was performed using the clinical records of 7667 patients including 1646 SRC patients who underwent radical gastrectomy between 2001 and 2010. A further analysis was also performed after dividing patients into three groups according to histologic subtype: SRC, well-to-moderately differentiated (WMD), and poorly differentiated adenocarcinoma. Results: SRC patients have younger age distribution and female predominance compared with other histologic subtypes. Notably, the distribution of T stage of SRC patients was distinct, located in extremes (T1: 66.2% and T4: 20%). Moreover, the prognosis of SRC in early gastric cancer and advanced gastric cancer was contrasting. In early gastric cancer, SRC demonstrated more favorable prognosis than WMD after adjusting for age, sex, and stage. In contrast, SRC in advanced gastric cancer displayed worse prognosis than WMD. As stage increased, survival outcomes of SRC continued to worsen compared with WMD. Conclusions: Although conferring favorable prognosis in early stage, SRC has worse prognostic impact as disease progresses. The longstanding controversy of SRC on prognosis may result from disease status at presentation, which leads to differing prognosis compared with tubular adenocarinoma. PMID:27232252

Measles Virus Nucleocapsid (MVNP) Gene Expression and RANK Receptor Signaling in Osteoclast Precursors, Osteoclast Inhibitors Peptide Therapy for Pagets Disease

DTIC Science & Technology

2007-10-01

OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U c . THIS PAGE U UU 27 19b. TELEPHONE NUMBER...and c -Jun kinase activity in osteoclast precursor cells (4). Our hypothesis is that MVNP expression in osteoclast precursors modulates the status...transcription factors such as c - Fos, NFATc1 critical for OCL differentiation were significantly decreased in OIP-1 transgenic mice derived preosteoclast cells

From lymphopoiesis to plasma cells differentiation, the age-related modifications of B cell compartment are influenced by "inflamm-ageing".

PubMed

Bulati, Matteo; Caruso, Calogero; Colonna-Romano, Giuseppina

2017-07-01

Ageing is a complex process characterized by a general decline in physiological functions with increasing morbidity and mortality. The most important aspect of ageing is the chronic inflammatory status, named "inflamm-ageing", strictly associated with the deterioration of the immune function, termed "immunosenescence". Both are causes of increased susceptibility of elderly to infectious diseases, cancer, dementia, cardiovascular diseases and autoimmunity, and of a decreased response to vaccination. It has been widely demonstrated that ageing has a strong impact on the remodelling of the B cell branch of immune system. The first evident effect is the significant decrease in circulating B cells, primarily due to the reduction of new B cell coming from bone marrow (BM) progenitors, as inflammation directly impacts on B lymphopoiesis. Besides, in aged individuals, there is a shift from naïve to memory immunoglobulins production, accompanied by the impaired ability to produce high affinity protective antibodies against newly encountered antigens. This is accompanied by the increase of expanded clones of B cells, which correlates with poor health status. Age-related modifications also occur in naïve/memory B cells subsets. Indeed, in the elderly, there is a reduction of naïve B cells, accompanied by the expansion of memory B cells that show a senescence-associated phenotype. Finally, elderly show the impaired ability of memory B cells to differentiate into plasma cells. It can be concluded that inflammation is the leading cause of the age-related impairment of B cell compartment, which play certainly a key role in the development of age-related diseases. This makes study of B cells in the aged an important tool for monitoring immunosenescence, chronic inflammatory disorders and the effectiveness of vaccines or pharmacological therapies. Copyright © 2017 Elsevier B.V. All rights reserved.

Adult human neural stem cell therapeutics: Current developmental status and prospect.

PubMed

Nam, Hyun; Lee, Kee-Hang; Nam, Do-Hyun; Joo, Kyeung Min

2015-01-26

Over the past two decades, regenerative therapies using stem cell technologies have been developed for various neurological diseases. Although stem cell therapy is an attractive option to reverse neural tissue damage and to recover neurological deficits, it is still under development so as not to show significant treatment effects in clinical settings. In this review, we discuss the scientific and clinical basics of adult neural stem cells (aNSCs), and their current developmental status as cell therapeutics for neurological disease. Compared with other types of stem cells, aNSCs have clinical advantages, such as limited proliferation, inborn differentiation potential into functional neural cells, and no ethical issues. In spite of the merits of aNSCs, difficulties in the isolation from the normal brain, and in the in vitro expansion, have blocked preclinical and clinical study using aNSCs. However, several groups have recently developed novel techniques to isolate and expand aNSCs from normal adult brains, and showed successful applications of aNSCs to neurological diseases. With new technologies for aNSCs and their clinical strengths, previous hurdles in stem cell therapies for neurological diseases could be overcome, to realize clinically efficacious regenerative stem cell therapeutics.

[Cytokine-mediated regulation of expression of Gfi1 and U2afll4 genes activated by T-cells with different differentiation status in vitro].

PubMed

Yurova, K A; Sokhonevich, N A; Khaziakhmatova, O G; Litvinova, L S

2016-01-01

The dose-dependent effects of cytokines (IL-2, IL-7, and IL-15), which have a common g-chain, on mRNA expression of U2afll4 and GFi1 genes involved in regulation of alternative splicing of the Ptprc gene, have been investigated in vitro using T-lymphocyte cultures with different degrees of differentiation. IL-2, IL-7, and IL-15 caused a similar unidirectional inhibitory effect of various severity on restimulated CD45RO+ T-cells exposed to an antigen-independent activation; they caused a dose-dependent decrease of the U2af1l4 gene expression, and an increase of Gfi1 gene expression. This may suggest formation of active forms of the CD45 receptor, and also limitation of the formation of low-molecular short splice variants of the CD45RO receptor. Under conditions of antigen-independent stimulation of naive CD45RA+-cells rIL-7 and IL-15 exhibited opposite effects on U2af1l4 and Gfi1 gene expression. The increase of IL-7 concentrations in the incubation medium of naive cells was accompanied by a decrease in expression of both genes. IL-15 IL-7 exhibited opposite effects. Cytokines possessing a common g-chain (IL-2, IL-7, and IL-15) prevented antigen-independent differentiation of naive T-cells, by preventing the formation of polyclonal "surrogate" cells. In general, the study of the molecular mechanisms of genetic control determining homeostatic processes of T-cells in response to exposure to antigenic or non-antigenic treatments may be important for construction of a general model of self-maintenance and differentiation of immune cells.

Leucine-rich Repeat Neuronal Protein 1 Regulates Differentiation of Embryonic Stem Cells by Posttranslational Modifications of Pluripotency Factors.

PubMed

Liao, Chien Huang; Wang, Ya-Hui; Chang, Wei-Wei; Yang, Bei-Chia; Wu, Tsai-Jung; Liu, Wei-Li; Yu, Alice L; Yu, John

2018-06-11

Stem cell surface markers may facilitate a better understanding of stem cell biology through molecular function studies or serve as tools to monitor the differentiation status and behavior of stem cells in culture or tissue. Thus, it is important to identify additional, novel stem cell markers. We used glycoproteomics to discover surface glycoproteins on human embryonic stem cells (hESCs) that may be useful stem cell markers. We found that a surface glycoprotein, leucine-rich repeat neuronal protein 1 (LRRN1), is expressed abundantly on the surface of hESCs prior to differentiation into embryoid bodies (EBs). Silencing of LRRN1 with short hairpin RNA (shLRRN1) in hESCs resulted in decreased capacity of self-renewal, and skewed differentiation toward endoderm/mesoderm lineages in vitro and in vivo. Meanwhile, the protein expression levels of the pluripotency factors OCT4, NANOG and SOX2 were reduced. Interestingly, the mRNA levels of these pluripotency factors were not affected in LRRN1 silenced cells, but protein half-lives were substantially shortened. Furthermore, we found LRRN1 silencing led to nuclear export and proteasomal degradation of all three pluripotency factors. In addition, the effects on nuclear export were mediated by AKT phosphorylation. These results suggest that LRRN1 plays an important role in maintaining the protein stability of pluripotency factors through AKT phosphorylation, thus maintaining hESC self-renewal capacity and pluripotency. Overall, we found that LRRN1 contributes to pluripotency of hESC by preventing translocation of OCT4, NANOG and SOX2 from nucleus to cytoplasm, thereby lessening their post-translational modification and degradation. This article is protected by copyright. All rights reserved. © 2018 AlphaMed Press.

Deciphering the Role of the Barr Body in Malignancy: An insight into head and neck cancer.

PubMed

Sharma, Deepti; Koshy, George; Gupta, Shruti; Sharma, Bhushan; Grover, Sonal

2017-11-01

X chromosome inactivation is the epitome of epigenetic regulation and long non-coding ribonucleic acid function. The differentiation status of cells has been ascribed to X chromosome activity, with two active X chromosomes generally only observed in undifferentiated or poorly differentiated cells. Recently, several studies have indicated that the reactivation of an inactive X chromosome or X chromosome multiplication correlates with the development of malignancy; however, this concept is still controversial. This review sought to shed light on the role of the X chromosome in cancer development. In particular, there is a need for further exploration of the expression patterns of X-linked genes in cancer cells, especially those in head and neck squamous cell carcinoma (HNSCC), in order to identify different prognostic subpopulations with distinct clinical implications. This article proposes a functional relationship between the loss of the Barr body and the disproportional expression of X-linked genes in HNSCC development.

TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth

PubMed Central

Haricharan, Svasti; Brown, Powel

2015-01-01

Breast cancer is a leading cause of cancer-related death, and it is important to understand pathways that drive the disease to devise effective therapeutic strategies. Our results show that Toll-like receptor 4 (TLR4) drives breast cancer cell growth differentially based on the presence of TP53, a tumor suppressor. TP53 is mutationally inactivated in most types of cancer and is mutated in 30–50% of diagnosed breast tumors. We demonstrate that TLR4 activation inhibits growth of TP53 wild-type cells, but promotes growth of TP53 mutant breast cancer cells by regulating proliferation. This differential effect is mediated by changes in tumor cell cytokine secretion. Whereas TLR4 activation in TP53 mutant breast cancer cells increases secretion of progrowth cytokines, TLR4 activation in TP53 wild-type breast cancer cells increases type I IFN (IFN-γ) secretion, which is both necessary and sufficient for mediating TLR4-induced growth inhibition. This study identifies a novel dichotomous role for TLR4 as a growth regulator and a modulator of tumor microenvironment in breast tumors. These results have translational relevance, demonstrating that TP53 mutant breast tumor growth can be suppressed by pharmacologic TLR4 inhibition, whereas TLR4 inhibitors may in fact promote growth of TP53 wild-type tumors. Furthermore, using data generated by The Cancer Genome Atlas consortium, we demonstrate that the effect of TP53 mutational status on TLR4 activity may extend to ovarian, colon, and lung cancers, among others, suggesting that the viability of TLR4 as a therapeutic target depends on TP53 status in many different tumor types. PMID:26063617

TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth.

PubMed

Haricharan, Svasti; Brown, Powel

2015-06-23

Breast cancer is a leading cause of cancer-related death, and it is important to understand pathways that drive the disease to devise effective therapeutic strategies. Our results show that Toll-like receptor 4 (TLR4) drives breast cancer cell growth differentially based on the presence of TP53, a tumor suppressor. TP53 is mutationally inactivated in most types of cancer and is mutated in 30-50% of diagnosed breast tumors. We demonstrate that TLR4 activation inhibits growth of TP53 wild-type cells, but promotes growth of TP53 mutant breast cancer cells by regulating proliferation. This differential effect is mediated by changes in tumor cell cytokine secretion. Whereas TLR4 activation in TP53 mutant breast cancer cells increases secretion of progrowth cytokines, TLR4 activation in TP53 wild-type breast cancer cells increases type I IFN (IFN-γ) secretion, which is both necessary and sufficient for mediating TLR4-induced growth inhibition. This study identifies a novel dichotomous role for TLR4 as a growth regulator and a modulator of tumor microenvironment in breast tumors. These results have translational relevance, demonstrating that TP53 mutant breast tumor growth can be suppressed by pharmacologic TLR4 inhibition, whereas TLR4 inhibitors may in fact promote growth of TP53 wild-type tumors. Furthermore, using data generated by The Cancer Genome Atlas consortium, we demonstrate that the effect of TP53 mutational status on TLR4 activity may extend to ovarian, colon, and lung cancers, among others, suggesting that the viability of TLR4 as a therapeutic target depends on TP53 status in many different tumor types.

A Novel Approach for the Identification of Pharmacophores Through Differential Toxicity Analysis of Estrogen Receptor Positive and Negative Cell Lines

DTIC Science & Technology

2008-07-31

HSP10, HSP27 , HSP60, and HSP70. HSP27 found to be present at ca. 2-fold higher levels in the antiestrogen-resistant MCF-7/LY2 cell line. The level...of this protein, originally identified in MCF-7 cells, is influenced by estrogens. HSP27 has also been shown to be increased in breast cancer and to...correlate with the ER+ status. The synthesis of HSP27 has also been shown to occur with the development of drug resistance, including that of

CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis.

PubMed

Paulissen, Sandra M J; van Hamburg, Jan Piet; Davelaar, Nadine; Vroman, Heleen; Hazes, Johanna M W; de Jong, Pascal H P; Lubberts, Erik

2015-11-30

Patients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA(+) RA and differences in treatment outcome between these subpopulations suggest that ACPA(+) and ACPA(-) RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA(+) RA. In this context we recently identified a potential pathogenic role for CCR6(+) Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status. We performed a nested matched case-control study including 27 ACPA(+) and 27 ACPA(-) treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4(+)CD45RO(+) (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration. ACPA status was not related to differences in total CD4(+) T cell or memory Th cell proportions. However, ACPA(+) patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4(+) and CCR10(+) Th cells were found. Within the CCR6(+) cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4(+)CCR10(-)), Th17.1 (CXCR3(+)), Th22 (CCR4(+)CCR10(+)) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (p = 0.02), Th17.1 (p = 0.03) and CCR4/CXCR3 DP (p = 0.01) cells were present in ACPA(+) patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6(-)CXCR3(+); p = 0.90), Th2 (CCR6(-)CCR4(+); p = 0.27) and T-regulatory (CD25(hi)FOXP3(+); p = 0.06) cell proportions. Interestingly, CCR6(+) Th cells were inversely correlated with disease duration in ACPA(-) patients (R(2) = -0.35; p < 0.01) but not in ACPA(+) (R(2) < 0.01; p = 0.94) patients. These findings demonstrate that increased peripheral blood CCR6(+) Th cells proportions distinguish ACPA(+) RA from ACPA(-) RA. This suggests that CCR6(+) Th cells are involved in the differences in disease severity and treatment outcome between ACPA(+) and ACPA(-) RA.

Reduced Expression of Siglec-7, NKG2A, and CD57 on Terminally Differentiated CD56-CD16+ Natural Killer Cell Subset Is Associated with Natural Killer Cell Dysfunction in Chronic HIV-1 Clade C Infection.

PubMed

Zulu, Michael Z; Naidoo, Kewreshini K; Mncube, Zenele; Jaggernath, Manjeetha; Goulder, Philip J R; Ndung'u, Thumbi; Altfeld, Marcus; Thobakgale, Christina F

2017-12-01

HIV-1 viremia has been shown to induce several phenotypic and functional abnormalities in natural killer (NK) cells. To assess immune defects associated with HIV viremia, we examined NK cell function, differentiation status, and phenotypic alterations based on expression of inhibitory and activating receptors on NK cells in HIV-1 subtype C chronically infected participants from Durban, South Africa. NK cell phenotypic profiles were characterized by assessing sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7), NKG2A, and NKG2C markers on frozen peripheral blood mononuclear cells from viremic, antiretroviral therapy (ART)-naive HIV-1 chronically infected participants (n = 23), HIV-1 chronically infected participants who had been on combination antiretroviral therapy (cART) for at least 12 months (n = 23) compared with healthy donors (n = 23). NK cell differentiation was assessed by measurement of killer immunoglobulin receptor (KIR) and NKG2A expression; CD57 and CD107a measurements were carried out in HIV viremic and healthy donors. All phenotypic and functional assessments were analyzed by using multicolor flow cytometry. HIV-1-infected participants displayed greater frequencies of the CD56 - CD16 + (CD56negative) NK cell subset compared with healthy donors (p < .0001). Downregulation of Siglec-7 and NKG2A and upregulation of NKG2C were more pronounced in the CD56negative NK cell subset of viremic participants. The CD56negative subset demonstrated a differentiated (KIR + NKG2A - ) phenotype with reduced CD57 expression and lower degranulation capacity in HIV-1-infected participants compared with healthy donors. HIV-1 infection induces the expansion of the CD56negative NK cell subset marked by altered receptor expression profiles that are indicative of impaired function and may explain the overall NK cell dysfunction observed in chronic HIV-1 infection.

MiR-100 regulates cell differentiation and survival by targeting RBSP3, a phosphatase-like tumor suppressor in acute myeloid leukemia

PubMed Central

Zheng, Y-S; Zhang, H; Zhang, X-J; Feng, D-D; Luo, X-Q; Zeng, C-W; Lin, K-Y; Zhou, H; Qu, L-H; Zhang, P; Chen, Y-Q

2012-01-01

Acute myeloblastic leukemia (AML) is characterized by the accumulation of abnormal myeloblasts (mainly granulocyte or monocyte precursors) in the bone marrow and blood. Though great progress has been made for improvement in clinical treatment during the past decades, only minority with AML achieve long-term survival. Therefore, further understanding mechanisms of leukemogenesis and exploring novel therapeutic strategies are still crucial for improving disease outcome. MicroRNA-100 (miR-100), a small non-coding RNA molecule, has been reported as a frequent event aberrantly expressed in patients with AML; however, the molecular basis for this phenotype and the statuses of its downstream targets have not yet been elucidated. In the present study, we found that the expression level of miR-100 in vivo was related to the stage of the maturation block underlying the subtypes of myeloid leukemia. In vitro experiments further demonstrated that miR-100 was required to promote the cell proliferation of promyelocytic blasts and arrest them differentiated to granulocyte/monocyte lineages. Significantly, we identified RBSP3, a phosphatase-like tumor suppressor, as a bona fide target of miR-100 and validated that RBSP3 was involved in cell differentiation and survival in AML. Moreover, we revealed a new pathway that miR-100 regulates G1/S transition and S-phase entry and blocks the terminal differentiation by targeting RBSP3, which partly in turn modulates the cell cycle effectors pRB/E2F1 in AML. These events promoted cell proliferation and blocked granulocyte/monocyte differentiation. Our data highlight an important role of miR-100 in the molecular etiology of AML, and implicate the potential application of miR-100 in cancer therapy. PMID:21643017

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

PubMed

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 will be a new way to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell and patient-derived tumor organoids

PubMed Central

Huang, Ling; Holtzinger, Audrey; Jagan, Ishaan; BeGora, Michael; Lohse, Ines; Ngai, Nicholas; Nostro, Cristina; Wang, Rennian; Muthuswamy, Lakshmi B.; Crawford, Howard C.; Arrowsmith, Cheryl; Kalloger, Steve E.; Renouf, Daniel J.; Connor, Ashton A; Cleary, Sean; Schaeffer, David F.; Roehrl, Michael; Tsao, Ming-Sound; Gallinger, Steven; Keller, Gordon; Muthuswamy, Senthil K.

2016-01-01

There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells (PSCs) into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53R175H induced cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. Culture conditions are also defined for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture, phenotypic heterogeneity of the primary tumor, and retain patient-specific physiologic changes including hypoxia, oxygen consumption, epigenetic marks, and differential sensitivity to EZH2 inhibition. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies. PMID:26501191

Identification of Two Suppressors of CSG2 Calcium Sensitivity, SCS7 and SUR2, as Genes Encoding Hydroxylases of the Sphingolipid Biosynthetic Pathway of Saccharomyces cerevisiae

DTIC Science & Technology

1997-12-10

process can adjusted by experimental alteration of thyroid status. Hyperthyroidism speeds up the time table while hypothyroidism retards it. When 88... canine kidney MDCK cells. The relative amounts ofthe labeled products can be differentially recovered from the basolateral and apical surfaces of these

Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

PubMed

Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

2016-07-01

We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

Expression of genes responsible for cell morphogenesis involved in differentiation in porcine buccal pouch mucosal cells during long-term primary culture and real-time proliferation in vitro.

PubMed

Dyszkiewicz-Konwińska, M; Bryja, A; Jopek, K; Budna, J; Khozmi, R; Jeseta, M; Bukowska, D; Antosik, P; Bruska, M; Nowicki, M; Zabel, M; Kempisty, B

2017-01-01

Recently, using experimental animal model, we demonstrated that porcine buccal pouch mucosal cells reflect increased proliferation capability during primary cultivation in vitro. Although the histological structure and morphogenesis in oral cavity is well recognized, the molecular mechanisms which regulate this process still need further investigation. This study was aimed to analyze the molecular marker expression profile involved in morphogenesis and differentiation capacity of porcine buccal pouch mucosal cells during their long-term primary cultivation in vitro. The experiment was performed on buccal pouch mucosal cells isolated from 80 pubertal crossbred Landrace gilts. After collection, the cells were treated enzymatically and transferred into a primary in vitro culture (IVC) system and cultured for 30 days. The cells were collected for RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using the RTCA system. We found an increased expression of FN1 and SOX9 genes when calculated against ACTB after 7, and 30 days of IVC, (P less than 0.01, P less than 0.001, respectively). The CXCL12 mRNA was down-regulated after 7, 15 and 30 days of IVC, but not statistically significant. Similar expression profile was observed when calculated against HPRT, however, DAB2 was found to be higher expressed at day 15 of IVC, (P less than 0.05). The cell index measured during real-time cell proliferation was substantially increased between 96 h and 147h of IVC and reached the log phase. Since FN1 and SOX9 revealed significant increase of expression after long-term culture in vitro, it is suggested that expression of these differentiation and stemness genes is accompanied by cell proliferation. Moreover, FN1 and SOX9 might be recognized as new markers of buccal pouch mucosal cell proliferation and differentiation in pigs in in vitro primary culture model.

Cigarette smoke condensate induces differential expression and promoter methylation profiles of critical genes involved in lung cancer in NL-20 lung cells in vitro: short-term and chronic exposure.

PubMed

Word, Beverly; Lyn-Cook, Lascelles E; Mwamba, Bibi; Wang, Honggang; Lyn-Cook, Beverly; Hammons, George

2013-01-01

Establishing early diagnostic markers of harm is critical for effective prevention programs and regulation of tobacco products. This study examined effects of cigarette smoke condensate (CSC) on expression and promoter methylation profile of critical genes (DAPK, ECAD, MGMT, and RASSF1A) involved in lung cancer development in different human lung cell lines. NL-20 cells were treated with 0.1-100 μg/ml of CSC for 24 to 72 hrs for short-term exposures. DAPK expression or methylation status was not significantly affected. However, CSC treatment resulted in changes in expression and promoter methylation profile of ECAD, MGMT, and RASSF1A. For chronic studies, cells were exposed to 1 or 10 μg/ml CSC up to 28 days. Cells showed morphological changes associated with transformation and changes in invasion capacities and global methylation status. This study provides critical data suggesting that epigenetic changes could serve as an early biomarker of harm due to exposure to cigarette smoke.

Tumor Trp53 status and genotype affect the bone marrow microenvironment in acute myeloid leukemia

PubMed Central

Jacamo, Rodrigo; Davis, R. Eric; Ling, Xiaoyang; Sonnylal, Sonali; Wang, Zhiqiang; Ma, Wencai; Zhang, Min; Ruvolo, Peter; Ruvolo, Vivian; Wang, Rui-Yu; McQueen, Teresa; Lowe, Scott; Zuber, Johannes; Kornblau, Steven M.; Konopleva, Marina; Andreeff, Michael

2017-01-01

The genetic heterogeneity of acute myeloid leukemia (AML) and the variable responses of individual patients to therapy suggest that different AML genotypes may influence the bone marrow (BM) microenvironment in different ways. We performed gene expression profiling of bone marrow mesenchymal stromal cells (BM-MSC) isolated from normal C57BL/6 mice or mice inoculated with syngeneic murine leukemia cells carrying different human AML genotypes, developed in mice with Trp53 wild-type or nullgenetic backgrounds. We identified a set of genes whose expression in BM-MSC was modulated by all four AML genotypes tested. In addition, there were sets of differentially-expressed genes in AML-exposed BM-MSC that were unique to the particular AML genotype or Trp53 status. Our findings support the hypothesis that leukemia cells alter the transcriptome of surrounding BM stromal cells, in both common and genotype-specific ways. These changes are likely to be advantageous to AML cells, affecting disease progression and response to chemotherapy, and suggest opportunities for stroma-targeting therapy, including those based on AML genotype. PMID:29137349

Glucose Metabolism in T Cells and Monocytes: New Perspectives in HIV Pathogenesis

PubMed Central

Palmer, Clovis S.; Cherry, Catherine L.; Sada-Ovalle, Isabel; Singh, Amit; Crowe, Suzanne M.

2016-01-01

Activation of the immune system occurs in response to the recognition of foreign antigens and receipt of optimal stimulatory signals by immune cells, a process that requires energy. Energy is also needed to support cellular growth, differentiation, proliferation, and effector functions of immune cells. In HIV-infected individuals, persistent viral replication, together with inflammatory stimuli contributes to chronic immune activation and oxidative stress. These conditions remain even in subjects with sustained virologic suppression on antiretroviral therapy. Here we highlight recent studies demonstrating the importance of metabolic pathways, particularly those involving glucose metabolism, in differentiation and maintenance of the activation states of T cells and monocytes. We also discuss how changes in the metabolic status of these cells may contribute to ongoing immune activation and inflammation in HIV- infected persons and how this may contribute to disease progression, establishment and persistence of the HIV reservoir, and the development of co-morbidities. We provide evidence that other viruses such as Epstein–Barr and Flu virus also disrupt the metabolic machinery of their host cells. Finally, we discuss how redox signaling mediated by oxidative stress may regulate metabolic responses in T cells and monocytes during HIV infection. PMID:27211546

Sertoli Cell Wt1 Regulates Peritubular Myoid Cell and Fetal Leydig Cell Differentiation during Fetal Testis Development.

PubMed

Wen, Qing; Wang, Yuqian; Tang, Jixin; Cheng, C Yan; Liu, Yi-Xun

2016-01-01

Sertoli cells play a significant role in regulating fetal testis compartmentalization to generate testis cords and interstitium during development. The Sertoli cell Wilms' tumor 1 (Wt1) gene, which encodes ~24 zinc finger-containing transcription factors, is known to play a crucial role in fetal testis cord assembly and maintenance. However, whether Wt1 regulates fetal testis compartmentalization by modulating the development of peritubular myoid cells (PMCs) and/or fetal Leydig cells (FLCs) remains unknown. Using a Wt1-/flox; Amh-Cre mouse model by deleting Wt1 in Sertoli cells (Wt1SC-cKO) at embryonic day 14.5 (E14.5), Wt1 was found to regulate PMC and FLC development. Wt1 deletion in fetal testis Sertoli cells caused aberrant differentiation and proliferation of PMCs, FLCs and interstitial progenitor cells from embryo to newborn, leading to abnormal fetal testis interstitial development. Specifically, the expression of PMC marker genes α-Sma, Myh11 and Des, and interstitial progenitor cell marker gene Vcam1 were down-regulated, whereas FLC marker genes StAR, Cyp11a1, Cyp17a1 and Hsd3b1 were up-regulated, in neonatal Wt1SC-cKO testes. The ratio of PMC:FLC were also reduced in Wt1SC-cKO testes, concomitant with a down-regulation of Notch signaling molecules Jag 1, Notch 2, Notch 3, and Hes1 in neonatal Wt1SC-cKO testes, illustrating changes in the differentiation status of FLC from their interstitial progenitor cells during fetal testis development. In summary, Wt1 regulates the development of FLC and interstitial progenitor cell lineages through Notch signaling, and it also plays a role in PMC development. Collectively, these effects confer fetal testis compartmentalization.

Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

PubMed

Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

2015-08-01

Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

Glandular differentiation in dedifferentiated chondrosarcoma: molecular evidence of a rare phenomenon.

PubMed

Jour, George; Liu, Yajuan; Ricciotti, Robert; Pritchard, Colin; Hoch, Benjamin L

2015-09-01

Epithelial glandular differentiation in dedifferentiated chondrosarcoma has not been described. Our patient was a 64-year-old man with a history of prostate cancer status post-radiation and hormonal therapy. On screening bone scan, he was found to have increased uptake in his right femoral shaft. Biopsy revealed intermediate-grade conventional chondrosarcoma. Subsequent femoral resection was remarkable for an intermediate-grade chondrosarcomatous component juxtaposed to an area composed of anastomosing nests and cords of malignant epithelial cells showing nuclear atypia and increased mitotic activity. A fibroblastic-appearing spindle cell population was intimately associated with the epithelial cells. The epithelial cells labeled with 34bE12, AE1/AE3, EMA, and Vimentin (both spindled and epithelial components) while being negative for prostate-specific antigen, prostate specific acid phosphatase, cytokeratin 20, thyroid transcription factor-1, and CDX2. The patient developed local recurrence 9 months after the initial resection but has had no metastatic disease and consistently undetectable prostate-specific antigen levels. Deep parallel sequencing of the dedifferentiated component showed a nonsynonymous mutation at exon 4 of IDH1 gene at codon R132 leading to a substitution of arginine, with serine confirming glandular differentiation in dedifferentiated chondrosarcoma. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative Studies of the Proteome, Glycoproteome, and N-Glycome of Clear Cell Renal Cell Carcinoma Plasma before and after Curative Nephrectomy

PubMed Central

2015-01-01

Clear cell renal cell carcinoma is the most prevalent of all reported kidney cancer cases, and currently there are no markers for early diagnosis. This has stimulated great research interest recently because early detection of the disease can significantly improve the low survival rate. Combining the proteome, glycoproteome, and N-glycome data from clear cell renal cell carcinoma plasma has the potential of identifying candidate markers for early diagnosis and prognosis and/or to monitor disease recurrence. Here, we report on the utilization of a multi-dimensional fractionation approach (12P-M-LAC) and LC–MS/MS to comprehensively investigate clear cell renal cell carcinoma plasma collected before (disease) and after (non-disease) curative nephrectomy (n = 40). Proteins detected in the subproteomes were investigated via label-free quantification. Protein abundance analysis revealed a number of low-level proteins with significant differential expression levels in disease samples, including HSPG2, CD146, ECM1, SELL, SYNE1, and VCAM1. Importantly, we observed a strong correlation between differentially expressed proteins and clinical status of the patient. Investigation of the glycoproteome returned 13 candidate glycoproteins with significant differential M-LAC column binding. Qualitative analysis indicated that 62% of selected candidate glycoproteins showed higher levels (upregulation) in M-LAC bound fraction of disease samples. This observation was further confirmed by released N-glycans data in which 53% of identified N-glycans were present at different levels in plasma in the disease vs non-disease samples. This striking result demonstrates the potential for significant protein glycosylation alterations in clear cell renal cell carcinoma cancer plasma. With future validation in a larger cohort, information derived from this study may lead to the development of clear cell renal cell carcinoma candidate biomarkers. PMID:25184692

Glomerular parietal epithelial cells of adult murine kidney undergo EMT to generate cells with traits of renal progenitors

PubMed Central

G, Swetha; Chandra, Vikash; Phadnis, Smruti; Bhonde, Ramesh

2011-01-01

Abstract Glomerular parietal epithelial cells (GPECs) are known to revert to embryonic phenotype in response to renal injury. However, the mechanism of de-differentiation in GPECs and the underlying cellular processes are not fully understood. In the present study, we show that cultured GPECs of adult murine kidney undergo epithelial-mesenchymal transition (EMT) to generate cells, which express CD24, CD44 and CD29 surface antigens. Characterization by qRT-PCR and immunostaining of these clonogenic cells demonstrate that they exhibit metastable phenotype with co-expression of both epithelial (cytokeratin-18) and mesenchymal (vimentin) markers. Transcript analysis by qRT-PCR revealed high expression of metanephric mesenchymal (Pax-2, WT-1, Six-1, Eya-1, GDNF) and uteric bud (Hoxb-7, C-Ret) genes in these cells, indicating their bipotent progenitor status. Incubation of GPECs with EMT blocker Prostaglandin E2, resulted in low expression of renal progenitor markers reflecting the correlation between EMT and acquired stemness in these cells. Additional in vitro renal commitment assays confirmed their functional staminality. When injected into E13.5 kidney rudiments, the cells incorporated into the developing kidney primordia and co-culture with E13.5 spinal cord resulted in branching and tubulogenesis in these cells. When implanted under renal capsule of unilaterally nephrectomized mice, these cells differentiated into immature glomeruli and vascular ducts. Our study demonstrates that EMT plays a major role in imparting plasticity to terminally differentiated GPECs by producing metastable cells with traits of kidney progenitors. The present study would improve our understanding on epithelial cell plasticity, furthering our knowledge of its role in renal repair and regeneration. PMID:19840197

Induced Pluripotent Stem Cells and Periodontal Regeneration.

PubMed

Du, Mi; Duan, Xuejing; Yang, Pishan

Periodontitis is a chronic inflammatory disease which leads to destruction of both the soft and hard tissues of the periodontium. Tissue engineering is a therapeutic approach in regenerative medicine that aims to induce new functional tissue regeneration via the synergistic combination of cells, biomaterials, and/or growth factors. Advances in our understanding of the biology of stem cells, including embryonic stem cells and mesenchymal stem cells, have provided opportunities for periodontal tissue engineering. However, there remain a number of limitations affecting their therapeutic efficiency. Due to the considerable proliferation and differentiation capacities, recently described induced pluripotent stem cells (iPSCs) provide a new way for cell-based therapies for periodontal regeneration. This review outlines the latest status of periodontal tissue engineering and highlights the potential use of iPSCs in periodontal tissue regeneration.

Evaluation of lipid oxidative stress status and inflammation in atopic ocular surface disease

PubMed Central

Wakamatsu, Tais H.; Ayako, Igarashi; Takano, Yoji; Matsumoto, Yukihiro; Ibrahim, Osama M.A.; Okada, Naoko; Satake, Yoshiyuki; Fukagawa, Kazumi; Shimazaki, Jun; Tsubota, Kazuo; Fujishima, Hiroshi

2010-01-01

Background Although the oxidative stress status in atopic skin disease has been reported to be elevated, there are still no studies related to the status of oxidative stress in atopic ocular surface disease. The purpose of this study was to evaluate the ocular surface lipid oxidative stress status and inflammation in atopic keratoconjunctivitis (AKC) patients and normal subjects. Methods Twenty eight eyes of 14 patients (9 males, 5 females) with AKC and 18 eyes of 9 age and sex matched (4 males and 5 females) normal healthy controls were examined in this prospective study. The severity of atopic dermatitis (AD) was scored by the SCORing Atopic Dermatitis (SCORAD) index. All subjects underwent Schirmer test, tear film break up time (BUT), fluorescein/Rose Bengal stainings, tear collection, and brush cytology from the upper palpebral conjunctiva. The brush cytology samples were stained with Diff-Quik for differentiation of inflammatory cells and immunohistochemistry (IHC) staining with HEL (hexanoyl-lysine) and 4-HNE (4-hydroxy-2-nonenal) to study lipid oxidation. HEL and cytokine (interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ)) levels were measured by enzyme-linked immunosorbent assay (ELISA) from tear samples of AKC patients and control subjects. Toluidine Blue and IHC staining with HEL, 4-HNE and cluster of differentiation 45 (CD45) were performed on papillary samples of AKC patients. This study was conducted in compliance with the “Declaration of Helsinki.” Results The tear stability and vital staining scores were significantly worse in eyes of AKC patients (p<0.05) compared to the controls. Inflammatory cells and positively stained conjunctival epithelial cells for HEL and 4-HNE showed a significant elevation in brush cytology samples of AKC patients. Significantly higher levels of HEL and cytokines were detected in tears of AKC patients compared to controls. Papillary specimens also revealed many CD45 inflammatory cells as well as many cells positively stained with HEL and 4-HNE in IHC. A strong significant linear positive correlation between conjunctival inflammation and epithelial lipid oxidative stress status was observed. Conjunctival lipid oxidative stress also correlated strongly with tear HEL levels and epithelial damage scores. Conclusions The ocular surface disease in AKC was characterized by marked tear instability, ocular surface epithelial damage, increase in inflammatory infiltrates and presence of increased lipid oxidation. PMID:21139696

Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity

NASA Astrophysics Data System (ADS)

van de Pavert, Serge A.; Ferreira, Manuela; Domingues, Rita G.; Ribeiro, Hélder; Molenaar, Rosalie; Moreira-Santos, Lara; Almeida, Francisca F.; Ibiza, Sales; Barbosa, Inês; Goverse, Gera; Labão-Almeida, Carlos; Godinho-Silva, Cristina; Konijn, Tanja; Schooneman, Dennis; O'Toole, Tom; Mizee, Mark R.; Habani, Yasmin; Haak, Esther; Santori, Fabio R.; Littman, Dan R.; Schulte-Merker, Stefan; Dzierzak, Elaine; Simas, J. Pedro; Mebius, Reina E.; Veiga-Fernandes, Henrique

2014-04-01

The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity.

PubMed

van de Pavert, Serge A; Ferreira, Manuela; Domingues, Rita G; Ribeiro, Hélder; Molenaar, Rosalie; Moreira-Santos, Lara; Almeida, Francisca F; Ibiza, Sales; Barbosa, Inês; Goverse, Gera; Labão-Almeida, Carlos; Godinho-Silva, Cristina; Konijn, Tanja; Schooneman, Dennis; O'Toole, Tom; Mizee, Mark R; Habani, Yasmin; Haak, Esther; Santori, Fabio R; Littman, Dan R; Schulte-Merker, Stefan; Dzierzak, Elaine; Simas, J Pedro; Mebius, Reina E; Veiga-Fernandes, Henrique

2014-04-03

The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

Influence of the PDE5 inhibitor tadalafil on redox status and antioxidant defense system in C2C12 skeletal muscle cells.

PubMed

Duranti, Guglielmo; Ceci, Roberta; Sgrò, Paolo; Sabatini, Stefania; Di Luigi, Luigi

2017-05-01

Phosphodiesterase type 5 inhibitors (PDE5Is), widely known for their beneficial effects onto male erectile dysfunction, seem to exert favorable effects onto metabolism as well. Tadalafil exposure increases oxidative metabolism of C2C12 skeletal muscle cells. A rise in fatty acid (FA) metabolism, requiring more oxygen, could induce a larger reactive oxygen species (ROS) release as a byproduct thus leading to a redox imbalance. The aim of this study was to determine how PDE5I tadalafil influences redox status in skeletal muscle cells to match the increasing oxidative metabolism. To this purpose, differentiated C2C12 skeletal muscle cells were treated with tadalafil and analyzed for total antioxidant capacity (TAC) and glutathione levels as marker of redox status; enzyme activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) engaged in antioxidant defense; and lipid peroxidation (TBARS) and protein carbonyls (PrCar) as markers of oxidative damage. Tadalafil increased total intracellular glutathione (tGSH), CAT, SOD, and GPx enzymatic activities while no changes were found in TAC. A perturbation of redox status, as showed by the decrease in the ratio between reduced/oxidized glutathione (GSH/GSSG), was observed. Nevertheless, it did not cause any change in TBARS and PrCar levels probably due to the enhancement in the antioxidant enzymatic network. Taken together, these data indicate that tadalafil, besides improving oxidative metabolism, may be beneficial to skeletal muscle cells by enhancing the enzymatic antioxidant system capacity.

Genome Wide DNA Methylation Profiles Provide Clues to the Origin and Pathogenesis of Germ Cell Tumors

PubMed Central

Rijlaarsdam, Martin A.; Tax, David M. J.; Gillis, Ad J. M.; Dorssers, Lambert C. J.; Koestler, Devin C.; de Ridder, Jeroen; Looijenga, Leendert H. J.

2015-01-01

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809). PMID:25859847

Driver or passenger effects of augmented c-Myc and Cdc20 in gliomagenesis.

PubMed

Ji, Ping; Zhou, Xinhui; Liu, Qun; Fuller, Gregory N; Phillips, Lynette M; Zhang, Wei

2016-04-26

Cdc20 and c-Myc are commonly overexpressed in a broad spectrum of cancers, including glioblastoma (GBM). Despite this clear association, whether c-Myc and Cdc20 overexpression is a driver or passenger event in gliomagenesis remains unclear. Both c-Myc and Cdc20 induced the proliferation of primary glial progenitor cells. c-Myc also promoted the formation of soft agar anchorage-independent colonies. In the RCAS/Ntv-a glia-specific transgenic mouse model, c-Myc increased the GBM incidence from 19.1% to 47.4% by 12 weeks of age when combined with kRas and Akt3 in Ntv-a INK4a-ARF (also known as CDKN2A)-null mice. In contrast, Cdc20 decreased the GBM incidence from 19.1% to 9.1%. Moreover, cell differentiation was modulated by c-Myc in kRas/Akt3-induced GBM on the basis of Nestin/GFAP expression (glial progenitor cell differentiation), while Cdc20 had no effect on primary glial progenitor cell differentiation. We used glial progenitor cells from Ntv-a newborn mice to evaluate the role of c-Myc and Cdc20 in the proliferation and transformation of GBM in vitro and in vivo. We further determined whether c-Myc and Cdc20 have a driver or passenger role in GBM development using kRas/Akt3 signals in a RCAS/Ntv-a mouse model. These results suggest that the driver or passenger of oncogene signaling is dependent on cellular status. c-Myc is a driver when combined with kRas/Akt3 oncogenic signals in gliomagenesis, whereas Cdc20 overexpression is a passenger. Inhibition of cell differentiation of c-Myc may be a target for anti-glioma therapy.

Regulation of callus status and cell-suspending culture in naked seed oat (Avena nuda).

PubMed

Cui, L; Fan, Y

1998-01-01

The original calli were obtained by inducing culture of mature embryos of naked seed oat on N6 medium. The original calli were white-colored tumor forms, soft outside and hard inside. These kinds of calli are easy to differentiate into plantlets, and they are not the friable type. Friable embryogenic calli could be obtained by cycled regulated culture on IM1-IM4 medium for 7-8 months from the original calli. They became vigorous, lightish yellow in color, with small grainy forms. Well-separated and fast-growing suspending cell lines have been obtained from the above-mentioned embryogenic calli in the liquid medium. Regenerated plants have been obtained for this kind of suspension line by culturing on the medium for differentiation. The surviving percentage for such plantlets was over 95% after planting in the soil.

Effects of Recombinant Human Lactoferrin on Osteoblast Growth and Bone Status in Piglets.

PubMed

Li, Qiuling; Zhao, Jie; Hu, Wenping; Wang, Jianwu; Yu, Tian; Dai, Yunping; Li, Ning

2018-04-03

Lactoferrin (LF), an ~80 kDa iron-binding glycoprotein, modulates many biological effects, including antimicrobial and immunomodulatory activities. Recently, it was shown that LF also regulates bone cell activity, suggesting its therapeutic effect on postmenopausal bone loss. However, a minimal amount is known regarding the effects of recombinant human LF (rhLF) supplementation on bone status in young healthy infants. We found osteoblast cell differentiation was significantly promoted in vitro. Furthermore, treatment of human osteoblast cells with rhLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein kinase (p44/p42 MAPK, ERK1/2). In order to investigate the effects of rhLF on bone status in vivo, we used a piglet model, which is a useful model for human infants. Piglets were supplemented with rhLF milk for 30 days. Bone formation markers, Serum calcium concentration, bone mineral density (BMD), bone mineral content (BMC), tibia bone strength, and the overall metabolite profile analysis showed that rhLF was advantageous to the bone growth in piglets. These findings suggest that rhLF supplementation benefits neonate bone health by modulating bone formation.

Two-Stage, In Silico Deconvolution of the Lymphocyte Compartment of the Peripheral Whole Blood Transcriptome in the Context of Acute Kidney Allograft Rejection

PubMed Central

Shannon, Casey P.; Balshaw, Robert; Ng, Raymond T.; Wilson-McManus, Janet E.; Keown, Paul; McMaster, Robert; McManus, Bruce M.; Landsberg, David; Isbel, Nicole M.; Knoll, Greg; Tebbutt, Scott J.

2014-01-01

Acute rejection is a major complication of solid organ transplantation that prevents the long-term assimilation of the allograft. Various populations of lymphocytes are principal mediators of this process, infiltrating graft tissues and driving cell-mediated cytotoxicity. Understanding the lymphocyte-specific biology associated with rejection is therefore critical. Measuring genome-wide changes in transcript abundance in peripheral whole blood cells can deliver a comprehensive view of the status of the immune system. The heterogeneous nature of the tissue significantly affects the sensitivity and interpretability of traditional analyses, however. Experimental separation of cell types is an obvious solution, but is often impractical and, more worrying, may affect expression, leading to spurious results. Statistical deconvolution of the cell type-specific signal is an attractive alternative, but existing approaches still present some challenges, particularly in a clinical research setting. Obtaining time-matched sample composition to biologically interesting, phenotypically homogeneous cell sub-populations is costly and adds significant complexity to study design. We used a two-stage, in silico deconvolution approach that first predicts sample composition to biologically meaningful and homogeneous leukocyte sub-populations, and then performs cell type-specific differential expression analysis in these same sub-populations, from peripheral whole blood expression data. We applied this approach to a peripheral whole blood expression study of kidney allograft rejection. The patterns of differential composition uncovered are consistent with previous studies carried out using flow cytometry and provide a relevant biological context when interpreting cell type-specific differential expression results. We identified cell type-specific differential expression in a variety of leukocyte sub-populations at the time of rejection. The tissue-specificity of these differentially expressed probe-set lists is consistent with the originating tissue and their functional enrichment consistent with allograft rejection. Finally, we demonstrate that the strategy described here can be used to derive useful hypotheses by validating a cell type-specific ratio in an independent cohort using the nanoString nCounter assay. PMID:24733377

Annexin A1 Down-Regulation in Head and Neck Cancer Is Associated with Epithelial Differentiation Status

PubMed Central

Pedrero, Juana Maria Garcia; Fernandez, M. Pilar; Morgan, Reginald O.; Zapatero, Agustin Herrero; Gonzalez, Maria Victoria; Nieto, Carlos Suarez; Rodrigo, Juan Pablo

2004-01-01

Annexin A1 (ANXA1) protein expression was evaluated by Western blot in a series of 32 head and neck squamous cell carcinomas (HNSCCs) in a search for molecular alterations that could serve as useful diagnostic/prognostic markers. ANXA1 down-regulation was observed in 24 cases (75%) compared with patient-matched normal epithelium. In relation to clinicopathological variables, ANXA1 down-regulation was significantly associated with advanced T stages (P = 0.029), locoregional lymph node metastases (P = 0.038), advanced disease stage (P = 0.006), hypopharyngeal localization (P = 0.038), and poor histological differentiation (P = 0.005). ANXA1 expression was also analyzed by immunohistochemistry in paraffin-embedded sections from 22 of 32 HNSCCs and 8 premalignant lesions. All dysplastic tissues showed significantly reduced ANXA1 expression compared to a strong positive signal observed in adjacent normal epithelia (except basal and suprabasal cells). A close association was observed between ANXA1 expression and the histological grade in HNSCC. Well-differentiated tumors presented a positive ANXA1 signal in highly keratinized areas whereas moderately and poorly differentiated tumors exhibited very weak or negative staining. Our findings clearly identify ANXA1 as an effective differentiation marker for the histopathological grading of HNSCCs and for the detection of epithelial dysplasia. PMID:14695321

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santamaria-Martinez, Albert; Universitat de Barcelona, Barcelona; Barquinero, Jordi

2009-10-15

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture andmore » sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45{sup -}, CD81{sup +} and Sca-1{sup +}). We also demonstrated that SP clonal cells secrete transforming growth factor {beta}1 (TGF-{beta}1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-{beta}1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.« less

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis.

PubMed

Santamaria-Martínez, Albert; Barquinero, Jordi; Barbosa-Desongles, Anna; Hurtado, Antoni; Pinós, Tomàs; Seoane, Joan; Poupon, Marie-France; Morote, Joan; Reventós, Jaume; Munell, Francina

2009-10-15

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45(-), CD81(+) and Sca-1(+)). We also demonstrated that SP clonal cells secrete transforming growth factor beta1 (TGF-beta1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-beta1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

Metabolic reprogramming: a cancer hallmark even warburg did not anticipate.

PubMed

Ward, Patrick S; Thompson, Craig B

2012-03-20

Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee

2011-11-04

Highlights: Black-Right-Pointing-Pointer Genome-wide DNA methylation pattern in Alzheimer's disease model cell line. Black-Right-Pointing-Pointer Integrated analysis of CpG methylation and mRNA expression profiles. Black-Right-Pointing-Pointer Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. Black-Right-Pointing-Pointer The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterationsmore » in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2 Prime -deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.« less

Adjuvant Effect of Killed Propionibacterium acnes on Mouse Peritoneal B-1 Lymphocytes and Their Early Phagocyte Differentiation

PubMed Central

Mussalem, Juliana Sekeres; Squaiella-Baptistão, Carla Cristina; Teixeira, Daniela; Yendo, Tatiana Mina; Thies, Felipe Garutti; Popi, Ana Flavia; Mariano, Mario; Longo-Maugéri, Ieda

2012-01-01

B-1 lymphocytes are the predominant cells in mouse peritoneal cavity. They express macrophage and lymphocyte markers and are divided into B-1a, B-1b and B-1c subtypes. The role of B-1 cells is not completely clear, but they are responsible for natural IgM production and seem to play a regulatory role. An enriched B-1b cell population can be obtained from non-adherent peritoneal cell cultures, and we have previously demonstrated that these cells undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to the substrate in vitro. Nevertheless, the B-1 cell response to antigens or adjuvants has been poorly investigated. Because killed Propionibacterium acnes exhibits immunomodulatory effects on both macrophages and B-2 lymphocytes, we analyzed whether a killed bacterial suspension or its soluble polysaccharide (PS) could modulate the absolute number of peritoneal B-1 cells in BALB/c mice, the activation status of these cells and their ability to differentiate into phagocytes in vitro. In vivo, P. acnes treatment elevated the absolute number of all B-1 subsets, whereas PS only increased B-1c. Moreover, the bacterium increased the number of B-1b cells that were positive for MHC II, TLR2, TLR4, TLR9, IL-4, IL-5 and IL-12, in addition to up-regulating TLR9, CD80 and CD86 expression. PS increased B-1b cell expression of TLR4, TLR9, CD40 and CD86, as well as IL-10 and IL-12 synthesis. Both of the treatments decreased the absolute number of B-1b cells in vitro, suggesting their early differentiation into B-1 cell-derived phagocytes (B-1CDP). We also observed a higher phagocytic activity from the phagocytes that were derived from B-1b cells after P. acnes and PS treatment. The adjuvant effect that P. acnes has on B-1 cells, mainly the B-1b subtype, reinforces the importance of B-1 cells in the innate and adaptive immune responses. PMID:22448280

Adjuvant effect of killed Propionibacterium acnes on mouse peritoneal B-1 lymphocytes and their early phagocyte differentiation.

PubMed

Mussalem, Juliana Sekeres; Squaiella-Baptistão, Carla Cristina; Teixeira, Daniela; Yendo, Tatiana Mina; Thies, Felipe Garutti; Popi, Ana Flavia; Mariano, Mario; Longo-Maugéri, Ieda

2012-01-01

B-1 lymphocytes are the predominant cells in mouse peritoneal cavity. They express macrophage and lymphocyte markers and are divided into B-1a, B-1b and B-1c subtypes. The role of B-1 cells is not completely clear, but they are responsible for natural IgM production and seem to play a regulatory role. An enriched B-1b cell population can be obtained from non-adherent peritoneal cell cultures, and we have previously demonstrated that these cells undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to the substrate in vitro. Nevertheless, the B-1 cell response to antigens or adjuvants has been poorly investigated. Because killed Propionibacterium acnes exhibits immunomodulatory effects on both macrophages and B-2 lymphocytes, we analyzed whether a killed bacterial suspension or its soluble polysaccharide (PS) could modulate the absolute number of peritoneal B-1 cells in BALB/c mice, the activation status of these cells and their ability to differentiate into phagocytes in vitro. In vivo, P. acnes treatment elevated the absolute number of all B-1 subsets, whereas PS only increased B-1c. Moreover, the bacterium increased the number of B-1b cells that were positive for MHC II, TLR2, TLR4, TLR9, IL-4, IL-5 and IL-12, in addition to up-regulating TLR9, CD80 and CD86 expression. PS increased B-1b cell expression of TLR4, TLR9, CD40 and CD86, as well as IL-10 and IL-12 synthesis. Both of the treatments decreased the absolute number of B-1b cells in vitro, suggesting their early differentiation into B-1 cell-derived phagocytes (B-1CDP). We also observed a higher phagocytic activity from the phagocytes that were derived from B-1b cells after P. acnes and PS treatment. The adjuvant effect that P. acnes has on B-1 cells, mainly the B-1b subtype, reinforces the importance of B-1 cells in the innate and adaptive immune responses.

Paradoxical Role of DNA Methylation in Activation of FoxA2 Gene Expression during Endoderm Development*

PubMed Central

Bahar Halpern, Keren; Vana, Tal; Walker, Michael D.

2014-01-01

The transcription factor FoxA2 is a master regulator of endoderm development and pancreatic beta cell gene expression. To elucidate the mechanisms underlying the activation of the FoxA2 gene during differentiation, we have compared the epigenetic status of undifferentiated human embryonic stem cells (hESCs), hESC-derived early endoderm stage cells (CXCR4+ cells), and pancreatic islet cells. Unexpectedly, a CpG island in the promoter region of the FoxA2 gene displayed paradoxically high levels of DNA methylation in expressing tissues (CXCR4+, islets) and low levels in nonexpressing tissues. This CpG island region was found to repress reporter gene expression and bind the Polycomb group protein SUZ12 and the DNA methyltransferase (DNMT)3b preferentially in undifferentiated hESCs as compared with CXCR4+ or islets cells. Consistent with this, activation of FoxA2 gene expression, but not CXCR4 or SOX17, was strongly inhibited by 5-aza-2′-deoxycytidine and by knockdown of DNMT3b. We hypothesize that in nonexpressing tissues, the lack of DNA methylation allows the binding of DNA methyltransferases and repressing proteins, such as Polycomb group proteins; upon differentiation, DNMT activation leads to CpG island methylation, causing loss of repressor protein binding. These results suggest a novel and unexpected role for DNA methylation in the activation of FoxA2 gene expression during differentiation. PMID:25016019

Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Se Jeong; Gu, Dong Ryun; Center for Metabolic Function Regulation

2016-06-17

Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reducedmore » following the activation of adenosine 5′ monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.« less

Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Gia-Ming; Present address: The University of Chicago, Section of Hematology/Oncology, 900 E. 57th Street, Room 7134, Chicago, IL 60637; Bain, Lisa J., E-mail: lbain@clemson.edu

2012-05-01

Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site ofmore » myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (∼ 2-fold) to the myogenin promoter at the transcription start site (− 40 to + 42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (− 40 to + 42). -- Highlights: ► Igf-1 expression is decreased in C2C12 cells after 20 nM arsenite exposure. ► Arsenic exposure alters histone remodeling on the myogenin promoter. ► Glp expression, a H3–K9 methyltransferase, was increased in arsenic-exposed cells. ► Ezh2 and Dnmt3a localization to the myogenin promoter is induced by arsenic.« less

Impact of copper oxide nanomaterials on differentiated and undifferentiated Caco-2 intestinal epithelial cells; assessment of cytotoxicity, barrier integrity, cytokine production and nanomaterial penetration.

PubMed

Ude, Victor C; Brown, David M; Viale, Luca; Kanase, Nilesh; Stone, Vicki; Johnston, Helinor J

2017-08-23

Copper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact. Undifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 μg/cm 2 Cu (equivalent to 1.95 to 250 μg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 μg/cm 2 for CuO NMs, and 4.25 μg/cm 2 for copper sulphate (CuSO 4 ), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO 4 on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO 4 on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO 4 was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (P app ); a measure of barrier permeability to CuO NMs. For all experiments, CuSO 4 was used as an ionic control. CuO NMs and CuSO 4 caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO 4 translocated across the differentiated Caco-2 cell monolayer. CuO NM mediated IL-8 production was over 2-fold higher in undifferentiated cells. A reduction in cell viability in differentiated cells was not responsible for the lower level of cytokine production observed. Both CuO NMs and CuSO 4 decreased TEER values to a similar extent, and caused tight junction dysfunction (ZO-1 staining), suggesting that barrier integrity was disrupted. CuO NMs and CuSO 4 stimulated IL-8 production by Caco-2 cells, decreased barrier integrity and thereby increased the P app and translocation of Cu. There was no significant enhancement in potency of the CuO NMs compared to CuSO 4 . Differentiated Caco-2 cells were identified as a powerful model to assess the impacts of ingested NMs on the GI tract.

A post-transcriptional mechanism pacing expression of neural genes with precursor cell differentiation status.

PubMed

Dai, Weijun; Li, Wencheng; Hoque, Mainul; Li, Zhuyun; Tian, Bin; Makeyev, Eugene V

2015-07-06

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3' UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly, TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand, inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons.

Multiple Cellular Responses to Serotonin Contribute to Epithelial Homeostasis

PubMed Central

Pai, Vaibhav P.; Horseman, Nelson D.

2011-01-01

Epithelial homeostasis incorporates the paradoxical concept of internal change (epithelial turnover) enabling the maintenance of anatomical status quo. Epithelial cell differentiation and cell loss (cell shedding and apoptosis) form important components of epithelial turnover. Although the mechanisms of cell loss are being uncovered the crucial triggers that modulate epithelial turnover through regulation of cell loss remain undetermined. Serotonin is emerging as a common autocrine-paracine regulator in epithelia of multiple organs, including the breast. Here we address whether serotonin affects epithelial turnover. Specifically, serotonin's roles in regulating cell shedding, apoptosis and barrier function of the epithelium. Using in vivo studies in mouse and a robust model of differentiated human mammary duct epithelium (MCF10A), we show that serotonin induces mammary epithelial cell shedding and disrupts tight junctions in a reversible manner. However, upon sustained exposure, serotonin induces apoptosis in the replenishing cell population, causing irreversible changes to the epithelial membrane. The staggered nature of these events induced by serotonin slowly shifts the balance in the epithelium from reversible to irreversible. These finding have very important implications towards our ability to control epithelial regeneration and thus address pathologies of aberrant epithelial turnover, which range from degenerative disorders (e.g.; pancreatitis and thyrioditis) to proliferative disorders (e.g.; mastitis, ductal ectasia, cholangiopathies and epithelial cancers). PMID:21390323

Chick derived induced pluripotent stem cells by the poly-cistronic transposon with enhanced transcriptional activity.

PubMed

Katayama, Masafumi; Hirayama, Takashi; Tani, Tetsuya; Nishimori, Katsuhiko; Onuma, Manabu; Fukuda, Tomokazu

2018-02-01

Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species. © 2017 Wiley Periodicals, Inc.

Effect of Antibiotics against Mycoplasma sp. on Human Embryonic Stem Cells Undifferentiated Status, Pluripotency, Cell Viability and Growth

PubMed Central

Romorini, Leonardo; Riva, Diego Ariel; Blüguermann, Carolina; Videla Richardson, Guillermo Agustin; Scassa, Maria Elida; Sevlever, Gustavo Emilio; Miriuka, Santiago Gabriel

2013-01-01

Human embryonic stem cells (hESCs) are self-renewing pluripotent cells that can differentiate into specialized cells and hold great promise as models for human development and disease studies, cell-replacement therapies, drug discovery and in vitro cytotoxicity tests. The culture and differentiation of these cells are both complex and expensive, so it is essential to extreme aseptic conditions. hESCs are susceptible to Mycoplasma sp. infection, which is hard to detect and alters stem cell-associated properties. The purpose of this work was to evaluate the efficacy and cytotoxic effect of PlasmocinTM and ciprofloxacin (specific antibiotics used for Mycoplasma sp. eradication) on hESCs. Mycoplasma sp. infected HUES-5 884 (H5 884, stable hESCs H5-brachyury promoter-GFP line) cells were effectively cured with a 14 days PlasmocinTM 25 µg/ml treatment (curative treatment) while maintaining stemness characteristic features. Furthermore, cured H5 884 cells exhibit the same karyotype as the parental H5 line and expressed GFP, through up-regulation of brachyury promoter, at day 4 of differentiation onset. Moreover, H5 cells treated with ciprofloxacin 10 µg/ml for 14 days (mimic of curative treatment) and H5 and WA09 (H9) hESCs treated with PlasmocinTM 5 µg/ml (prophylactic treatment) for 5 passages retained hESCs features, as judged by the expression of stemness-related genes (TRA1-60, TRA1-81, SSEA-4, Oct-4, Nanog) at mRNA and protein levels. In addition, the presence of specific markers of the three germ layers (brachyury, Nkx2.5 and cTnT: mesoderm; AFP: endoderm; nestin and Pax-6: ectoderm) was verified in in vitro differentiated antibiotic-treated hESCs. In conclusion, we found that PlasmocinTM and ciprofloxacin do not affect hESCs stemness and pluripotency nor cell viability. However, curative treatments slightly diminished cell growth rate. This cytotoxic effect was reversible as cells regained normal growth rate upon antibiotic withdrawal. PMID:23936178

Signed weighted gene co-expression network analysis of transcriptional regulation in murine embryonic stem cells

PubMed Central

Mason, Mike J; Fan, Guoping; Plath, Kathrin; Zhou, Qing; Horvath, Steve

2009-01-01

Background Recent work has revealed that a core group of transcription factors (TFs) regulates the key characteristics of embryonic stem (ES) cells: pluripotency and self-renewal. Current efforts focus on identifying genes that play important roles in maintaining pluripotency and self-renewal in ES cells and aim to understand the interactions among these genes. To that end, we investigated the use of unsigned and signed network analysis to identify pluripotency and differentiation related genes. Results We show that signed networks provide a better systems level understanding of the regulatory mechanisms of ES cells than unsigned networks, using two independent murine ES cell expression data sets. Specifically, using signed weighted gene co-expression network analysis (WGCNA), we found a pluripotency module and a differentiation module, which are not identified in unsigned networks. We confirmed the importance of these modules by incorporating genome-wide TF binding data for key ES cell regulators. Interestingly, we find that the pluripotency module is enriched with genes related to DNA damage repair and mitochondrial function in addition to transcriptional regulation. Using a connectivity measure of module membership, we not only identify known regulators of ES cells but also show that Mrpl15, Msh6, Nrf1, Nup133, Ppif, Rbpj, Sh3gl2, and Zfp39, among other genes, have important roles in maintaining ES cell pluripotency and self-renewal. We also report highly significant relationships between module membership and epigenetic modifications (histone modifications and promoter CpG methylation status), which are known to play a role in controlling gene expression during ES cell self-renewal and differentiation. Conclusion Our systems biologic re-analysis of gene expression, transcription factor binding, epigenetic and gene ontology data provides a novel integrative view of ES cell biology. PMID:19619308

Distinct Histopathologic and Molecular Alterations in Inflammatory Bowel Disease-Associated Intestinal Adenocarcinoma: c-MYC Amplification is Common and Associated with Mucinous/Signet Ring Cell Differentiation.

PubMed

Hartman, Douglas J; Binion, David G; Regueiro, Miguel D; Miller, Caitlyn; Herbst, Cameron; Pai, Reetesh K

2018-05-17

Chronic idiopathic inflammatory bowel disease (IBD) is a significant risk factor for the development of intestinal adenocarcinoma. The underlying molecular alterations in IBD-associated intestinal adenocarcinoma remain largely unknown. We compared the clinicopathologic and molecular features of 35 patients with 47 IBD-associated intestinal adenocarcinomas with a consecutive series of 451 patients with sporadic colorectal carcinoma identified at our institution and published data on sporadic colorectal carcinoma. c-MYC amplification was the most frequent molecular alteration identified in 33% of IBD-associated intestinal adenocarcinoma that is a significantly higher frequency than in sporadic colorectal carcinoma (8%) (P = 0.0001). Compared to sporadic colorectal carcinoma, IBD-associated intestinal adenocarcinomas more frequently demonstrated mucinous differentiation (60% vs 25%, P < 0.001) and signet ring cell differentiation (28% vs 4%, P < 0.001). Mucinous and signet ring cell differentiation were significantly associated with the presence of c-MYC amplification (both with P < 0.05). HER2 positivity (11%), KRAS exon 2 or 3 mutation (10%), and IDH1 mutation (7%) were less commonly observed in IBD-associated intestinal adenocarcinoma. There was an association between poor survival and HER2 status with 3 of 4 patients having HER2-positive adenocarcinoma dead of disease at last clinical follow-up; however, no statistically significant survival effect was identified for any of the molecular alterations identified. We demonstrate that IBD-associated intestinal adenocarcinomas have a high frequency of c-MYC amplification that is associated with mucinous and signet ring cell differentiation. Many of the identified molecular alterations have potential therapeutic relevance, including HER2 amplification, IDH1 mutation, and low frequency KRAS mutation.

Combined effects of dentin sialoprotein and bone morphogenetic protein-2 on differentiation in human cementoblasts.

PubMed

Lee, So-Youn; Auh, Q-Schick; Kang, Soo-Kyung; Kim, Hyung-Joon; Lee, Jung-Woo; Noh, Kwantae; Jang, Jun-Hyeog; Kim, Eun-Cheol

2014-07-01

The aim of this study is to determine the effects of the combination of recombinant human BMP-2 (rh-BMP-2) and dentin sialoprotein (rh-DSP) on growth and differentiation in human cementoblasts and determine the underlying signal transduction mechanism. Compared to treatment of cementoblasts with either rh-BMP-2 or rh-DSP alone, the combination of rh-BMP-2 and rh-DSP synergistically increased cell growth, ALP activity, nodule formation and expression of differentiation markers. The differentiation-promoting effect was also observed in periodontal ligament cells and an osteoblastic cell line. Likewise, combination of rh-DSP and rh-BMP-2 increased BMP-2 mRNA expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. The expression levels of α2β1 integrin and RhoA, as well as the phosphorylation status of FAK and Akt, were increased by the combination of rh-BMP-2 and rh-DSP in a time-dependent manner. In addition, rh-BMP-2 and rh-DSP enhanced expression of Wnt ligands, β-catenin activation and GSK-3β phosphorylation, all of which were inhibited by the Wnt receptor antagonist DKK1. Furthermore, treatment with rh-DSP plus rh-BMP-2 resulted in phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 and also induced the nuclear translocation of the NF-κB p65 subunit, which was blocked by noggin. This study demonstrates for the first time that rh-DSP and rh-BMP-2 act synergistically, enhancing each other's ability to stimulate cementoblastic cell growth and differentiation in vitro via autocrine BMP, integrin, Wnt/β-catenin, MAP kinase and NF-κB pathways. These results support the therapeutic potential of a combination strategy for aiding periodontal regeneration.

High-throughput testing in head and neck squamous cell carcinoma identifies agents with preferential activity in human papillomavirus-positive or negative cell lines.

PubMed

Ghasemi, Farhad; Black, Morgan; Sun, Ren X; Vizeacoumar, Frederick; Pinto, Nicole; Ruicci, Kara M; Yoo, John; Fung, Kevin; MacNeil, Danielle; Palma, David A; Winquist, Eric; Mymryk, Joe S; Ailles, Laurie A; Datti, Alessandro; Barrett, John W; Boutros, Paul C; Nichols, Anthony C

2018-05-25

Head and neck squamous cell carcinoma (HNSCC) is a common cancer diagnosis worldwide. Despite advances in treatment, HNSCC has very poor survival outcomes, emphasizing an ongoing need for development of improved therapeutic options. The distinct tumor characteristics of human papillomavirus (HPV)-positive vs . HPV-negative disease necessitate development of treatment strategies tailored to tumor HPV-status. High-throughput robotic screening of 1,433 biologically and pharmacologically relevant compounds at a single dose (4 μM) was carried out against 6 HPV-positive and 20 HPV-negative HNSCC cell lines for preliminary identification of therapeutically relevant compounds. Statistical analysis was further carried out to differentiate compounds with preferential activity against cell lines stratified by the HPV-status. These analyses yielded 57 compounds with higher activity in HPV-negative cell lines, and 34 with higher-activity in HPV-positive ones. Multi-point dose-response curves were generated for six of these compounds (Ryuvidine, MK-1775, SNS-032, Flavopiridol, AZD-7762 and ARP-101), confirming Ryuvidine to have preferential potency against HPV-negative cell lines, and MK-1775 to have preferential potency against HPV-positive cell lines. These data comprise a valuable resource for further investigation of compounds with therapeutic potential in the HNSCC.

ZEB1 promotes the progression and metastasis of cervical squamous cell carcinoma via the promotion of epithelial-mesenchymal transition

PubMed Central

Ma, Yihui; Zheng, Xiangyu; Zhou, Jun; Zhang, Ying; Chen, Kuisheng

2015-01-01

Objective: The process of epithelial-mesenchymal transition (EMT) clearly contributes to cancer metastasis. The aim of this study was to investigate the expression of the EMT-related transcription repressor ZEB1 and the expression of EMT-associated markers (E-cadherin, β-catenin and N-cadherin) in cervical squamous cell carcinoma. In addition, the role of ZEB1 and these EMT-associated markers in the progression and metastasis of cervical squamous cell carcinoma was explored. Methods: The expression of ZEB1, E-cadherin, β-catenin and N-cadherin was evaluated in 81 specimens of cervical squamous cell carcinoma by immunohistochemistry; the clinicopathological significance of these markers was then analyzed. Results: 1) Of the 81 samples, 37 cases (45.7%) were positive for ZEB1, and nuclear expression of ZEB1 in tumor cells was positively associated with the differentiation status of the tumor tissue (P < 0.05), vascular invasion (P < 0.05) and lymph node metastasis (P < 0.05). 2) The loss of E-cadherin and β-catenin expression in tumor cells and the acquisition of N-cadherin expression were positively associated with the differentiation status of the tumor tissue (P < 0.05) and with the occurrence of vascular invasion (P < 0.05). 3) A significant negative correlation was observed between ZEB1 and E-cadherin expression (Spearman = -0.636, P < 0.05) and between ZEB1 and β-catenin expression (Spearman = -0.417, P < 0.05). Moreover, a significant positive correlation was observed between ZEB1 and N-cadherin expression (Spearman = 0.557, P < 0.05). Conclusions: These results emphasize the role of EMT in cervical squamous cell carcinoma. The upregulation of ZEB1 is associated with the abnormal expression of E-cadherin, β-catenin and N-cadherin, which might promote the progression and metastasis of cervical squamous cell carcinoma. PMID:26617850

Macrophage Migration Inhibitory Factor Stimulates Angiogenic Factor Expression and Correlates With Differentiation and Lymph Node Status in Patients With Esophageal Squamous Cell Carcinoma

PubMed Central

Ren, Yi; Law, Simon; Huang, Xin; Lee, Ping Yin; Bacher, Michael; Srivastava, Gopesh; Wong, John

2005-01-01

Objective: The objectives of this study were: 1) to examine the expression of macrophage migration inhibitory factor (MIF) in esophageal squamous cell carcinoma (ESCC); 2) to see if a relationship exists between MIF expression, clinicopathologic features, and long-term prognosis; and 3) to ascertain the possible biologic function of MIF in angiogenesis. Summary Background Data: MIF has been linked to fundamental processes such as those controlling cell proliferation, cell survival, angiogenesis, and tumor progression. Its role in ESCC, and the correlation of MIF expression and tumor pathologic features in patients, has not been elucidated. Methods: The expression of MIF in tumor and nontumor tissues was examined by immunohistochemical staining. Concentrations of MIF, vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) in patients’ sera and in the supernatant of tumor cells culture were examined by ELISA. Correlations with clinicopathologic factors were made. Results: In 72 patients with ESCC, intracellular MIF was overexpressed in esophagectomy specimens. The expression of MIF correlated with both tumor differentiation and lymph node status. The median survival in the low-MIF expression group (<50% positively stained cancer cells on immunohistochemistry) and high expression group (≥50% positively stained cancer cells) was 28.3 months and 15.8 months, respectively (P = 0.03). The 3-year survival rates for the 2 groups were 37.7% and 12.1%, respectively. MIF expression was related to microvessel density; increased MIF serum levels also correlated with higher serum levels of VEGF. In addition, in vitro MIF stimulation of esophageal cancer cell lines induced a dose-dependent increase in VEGF and IL-8 secretion. Conclusions: These results demonstrate, for the first time, that human esophageal carcinomas express and secrete large amounts of MIF. Through its effects on VEGF and IL-8, MIF may serve as an autocrine factor in angiogenesis and thus play an important role in the pathogenesis of ESCC. PMID:15973102

[Methylation Status of the SOCS3 Gene Promoter in H2228 Cells and 
EML4-ALK-positive Lung Cancer Tissues].

PubMed

Liu, Chunlai; Li, Yongwen; Dong, Yunlong; Zhang, Hongbing; Li, Ying; Liu, Hongyu; Chen, Jun

2016-09-20

The EML4-ALK fusion gene is a newly discovered driver gene of non-small cell lung cancer and exhibits special clinical and pathological features. The JAK-STAT signaling pathway, an important downstream signaling pathway of EML4-ALK, is aberrantly sustained and activated in EML4-ALK-positive lung cancer cells fusion gene, but the underlying reason remains unknown. The suppressor of cytokine signaling (SOCS) is a negative regulatory factor that mainly inhibits the proliferation, differentiation, and induction of apoptotic cells by inhibiting the JAK-STAT signaling pathway. The aberrant methylation of the SOCS gene leads to inactivation of tumors and abnormal activation of the JAK2-STAT signaling pathway. The aim of this study is to investigate the methylation status of the SOCS3 promoter in EML4-ALK-positive H2228 cells and lung cancer tissues. The methylation status of the SOCS3 promoter in EML4-ALK-positive H2228 lung cancer cells and lung cancer tissues was detected by methylation-specific PCR (MSP) analysis and verified by DNA sequencing. The expression levels of SOCS3 in H2228 cells were detected by Western blot and Real-time PCR analyses after treatment with the DNA methyltransferase inhibitor 5'-Aza-dC. MSP and DNA sequencing assay results indicated the presence of SOCS3 promoter methylation in H2228 cells as well as in three cases of seven EML4-ALK-positive lung cancer tissues. The expression level of SOCS3 significantly increased in H2228 cells after 5'-Aza-dC treatment. The aerrant methylation of the SOCS3 promoter region in EML4-ALK (+) H2228 cells and lung cancer tissues may be significantly involved in the pathogenesis of EML4-ALK-positive lung cancer.

Remyelinating Oligodendrocyte Precursor Cell miRNAs from the Sfmbt2 Cluster Promote Cell Cycle Arrest and Differentiation

PubMed Central

Kuypers, Nicholas J.; Bankston, Andrew N.; Howard, Russell M.; Beare, Jason E.

2016-01-01

Oligodendrocyte (OL) loss contributes to the functional deficits underlying diseases with a demyelinating component. Remyelination by oligodendrocyte progenitor cells (OPCs) can restore these deficits. To understand the role that microRNAs (miRNAs) play in remyelination, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase-EGFP+ mice were treated with cuprizone, and OPCs were sorted from the corpus callosum. Microarray analysis revealed that Sfmbt2 family miRNAs decreased during cuprizone treatment. One particular Sfmbt2 miRNA, miR-297c-5p, increased during mouse OPC differentiation in vitro and during callosal development in vivo. When overexpressed in both mouse embryonic fibroblasts and rat OPCs (rOPCs), cell cycle analysis revealed that miR-297c-5p promoted G1/G0 arrest. Additionally, miR-297c-5p transduction increased the number of O1+ rOPCs during differentiation. Luciferase reporter assays confirmed that miR-297c-5p targets cyclin T2 (CCNT2), the regulatory subunit of positive transcription elongation factor b, a complex that inhibits OL maturation. Furthermore, CCNT2-specific knockdown promoted rOPC differentiation while not affecting cell cycle status. Together, these data support a dual role for miR-297c-5p as both a negative regulator of OPC proliferation and a positive regulator of OL maturation via its interaction with CCNT2. SIGNIFICANCE STATEMENT This work describes the role of oligodendrocyte progenitor cell (OPC) microRNAs (miRNAs) during remyelination and development in vivo and differentiation in vitro. This work highlights the importance of miRNAs to OPC biology and describes miR-297c-5p, a novel regulator of OPC function. In addition, we identified CCNT2 as a functional target, thus providing a mechanism by which miR-297c-5p imparts its effects on differentiation. These data are important, given our lack of understanding of OPC miRNA regulatory networks and their potential clinical value. Therefore, efforts to understand the role of miR-297c-5p in pathological conditions and its potential for facilitating repair may provide future therapeutic strategies to treat demyelination. PMID:26843650

Down-regulation of CD73 on B cells of patients with viremic HIV correlates with B cell activation and disease progression.

PubMed

Kim, Eun-Seong; Ackermann, Christin; Tóth, Ilona; Dierks, Patrick; Eberhard, Johanna M; Wroblewski, Raluca; Scherg, Felix; Geyer, Matthias; Schmidt, Reinhold E; Beisel, Claudia; Bockhorn, Maximilian; Haag, Friedrich; van Lunzen, Jan; Schulze Zur Wiesch, Julian

2017-05-01

Recently, alterations of the T cell expression of the ectonucleotidases, CD39 and CD73, during HIV infection have been described. Here, peripheral ( n = 70) and lymph nodal B cells ( n = 10) of patients with HIV at different stages of disease as well as uninfected individuals were analyzed via multicolor flow cytometry with regard to expression of CD39 and CD73 and differentiation, proliferation, and exhaustion status. Patients with chronic, untreated HIV showed a significantly decreased frequency of CD73-expressing B cells ( P < 0.001) compared with healthy controls. Decreased frequencies of CD39 + CD73 + B cells in patients with HIV correlated with low CD4 + counts ( P < 0.0256) as well as increased proliferation and exhaustion status as determined by Ki-67 and programmed death-1 expression. Down-regulation of CD73 was observed in naive and memory B cells as determined by CD27 and CD21. Neither HIV elite controller patients nor antiretroviral therapy-treated patients had significantly lower CD39 and CD73 expression on B cells compared with healthy controls. Of importance, low CD73 + expression on B cells was associated with modulated in vitro B cell function. Further in vivo studies are warranted to evaluate the in vivo role of phenotypic loss of CD73 in B cell dysregulation in HIV. © Society for Leukocyte Biology.

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1→S transition.

PubMed

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay; Sarvepalli, Kavitha; Sadhale, Parag P; Nath, Utpal

2011-07-01

The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1→S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1→S arrest is discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Understanding the reconstitution of the B-cell compartment in bone marrow and blood after treatment for B-cell precursor acute lymphoblastic leukaemia.

PubMed

Theunissen, Prisca M J; van den Branden, Anouk; Van Der Sluijs-Gelling, Alita; De Haas, Valerie; Beishuizen, Auke; van Dongen, Jacques J M; Van Der Velden, Vincent H J

2017-07-01

A better understanding of the reconstitution of the B-cell compartment during and after treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) will help to assess the immunological status and needs of post-treatment BCP-ALL patients. Using 8-colour flow cytometry and proliferation-assays, we studied the composition and proliferation of both the B-cell precursor (BCP) population in the bone marrow (BM) and mature B-cell population in peripheral blood (PB) during and after BCP-ALL therapy. We found a normal BCP differentiation pattern and a delayed formation of classical CD38 dim -naive mature B-cells, natural effector B-cells and memory B-cells in patients after chemotherapy. This B-cell differentiation/maturation pattern was strikingly similar to that during initial B-cell development in healthy infants. Tissue-resident plasma cells appeared to be partly protected from chemotherapy. Also, we found that the fast recovery of naive mature B-cell numbers after chemotherapy was the result of increased de novo BCP generation, rather than enhanced B-cell proliferation in BM or PB. These results indicate that post-treatment BCP-ALL patients will eventually re-establish a B-cell compartment with a composition and B-cell receptor repertoire similar to that in healthy children. Additionally, the formation of a new memory B-cell compartment suggests that revaccination might be beneficial after BCP-ALL therapy. © 2017 John Wiley & Sons Ltd.

Recloned dogs derived from adipose stem cells of a transgenic cloned beagle.

PubMed

Oh, Hyun Ju; Park, Jung Eun; Kim, Min Jung; Hong, So Gun; Ra, Jeong Chan; Jo, Jung Youn; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2011-04-15

A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning. Copyright © 2011 Elsevier Inc. All rights reserved.

In vitro regeneration of kidney from pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osafune, Kenji, E-mail: osafu@cira.kyoto-u.ac.jp; PRESTO, Japan Science and Technology Agency; JST Yamanaka iPS Cell Special Project, Japan Science and Technology Agency

2010-10-01

Although renal transplantation has proved a successful treatment for the patients with end-stage renal failure, the therapy is hampered by the problem of serious shortage of donor organs. Regenerative medicine using stem cells, including cell transplantation therapy, needs to be developed to solve the problem. We previously identified the multipotent progenitor cells in the embryonic mouse kidney that can give rise to several kinds of epithelial cells found in adult kidney, such as glomerular podocytes and renal tubular epithelia. Establishing the method to generate the progenitors from human pluripotent stem cells that have the capacity to indefinitely proliferate in vitromore » is required for the development of kidney regeneration strategy. We review the current status of the research on the differentiation of pluripotent stem cells into renal lineages and describe cues to promote this research field.« less

NUMB does not impair growth and differentiation status of experimental gliomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Euskirchen, Philipp, E-mail: philipp.euskirchen@charite.de; Laboratory for Gene Therapy and Molecular Imaging, Max-Planck-Institute for Neurological Research, Cologne; Skaftnesmo, Kai-Ove

2011-12-10

The cell fate determinant NUMB orchestrates asymmetric cell division in flies and mammals and has lately been suggested to have a tumor suppressor function in breast and lung cancer. Here, we studied NUMB in the context of malignant gliomas. We used ectopic expression of NUMB in order to inhibit proliferation and induce differentiation in glioma cells by alteration of Notch, Hedgehog and p53 signaling. We found that NUMB is consistently expressed in glioma biopsies with predominance of NUMB2/4 isoforms as determined by isoform-specific real-time PCR and Western blotting. Upon lentiviral overexpression, in vitro proliferation rate and the grade of differentiationmore » as assessed by morphology and expression of neural and glial markers remained unchanged. Orthotopic xenografts of NUMB-transduced human U87 glioma cells could be established in nude rats without impairing engraftment or causing significant changes in morphology based on magnetic resonance imaging (MRI). The previously reported alteration of Hedgehog and p53 signaling by NUMB could not be recapitulated in glioma cells. We thus show that in experimental gliomas, NUMB overexpression most likely does not exert a tumor suppressor function such as seen in epithelial cancers.« less

Status of epigenetic chromatin modification enzymes and esophageal squamous cell carcinoma risk in northeast Indian population.

PubMed

Singh, Virendra; Singh, Laishram C; Singh, Avninder P; Sharma, Jagannath; Borthakur, Bibhuti B; Debnath, Arundhati; Rai, Avdhesh K; Phukan, Rup K; Mahanta, Jagadish; Kataki, Amal C; Kapur, Sujala; Saxena, Sunita

2015-01-01

Esophageal cancer incidence is reported in high frequency in northeast India. The etiology is different from other population at India due to wide variations in dietary habits or nutritional factors, tobacco/betel quid chewing and alcohol habits. Since DNA methylation, histone modification and miRNA-mediated epigenetic processes alter the gene expression, the involvement of these processes might be useful to find out epigenetic markers of esophageal cancer risk in northeast Indian population. The present investigation was aimed to carryout differential expression profiling of chromatin modification enzymes in tumor and normal tissue collected from esophageal squamous cell carcinoma (ESCC) patients. Differential mRNA expression profiling and their validation was done by quantitative real time PCR and tissue microarray respectively. Univariate and multiple logistic regression analysis were used to analyze the epidemiological data. mRNA expression data was analyzed by Student t-test. Fisher exact test was used for tissue microarray data analysis. Higher expression of enzymes regulating methylation (DOT1L and PRMT1) and acetylation (KAT7, KAT8, KAT2A and KAT6A) of histone was found associated with ESCC risk. Tissue microarray done in independent cohort of 75 patients revealed higher nuclear protein expression of KAT8 and PRMT1 in tumor similar to mRNA expression. Expression status of PRMT1 and KAT8 was found declined as we move from low grade to high grade tumor. Betel nut chewing, alcohol drinking and dried fish intake were significantly associated with increased risk of esophageal cancer among the study subject. Study suggests the association of PRMT1 and KAT8 with esophageal cancer risk and its involvement in the transition process of low to high grade tumor formation. The study exposes the differential status of chromatin modification enzymes between tumor and normal tissue and points out that relaxed state of chromatin facilitates more transcriptionally active genome in esophageal carcinogenesis.

Chronic endurance exercise affects paracrine action of CD31+ and CD34+ cells on endothelial tube formation

PubMed Central

Landers-Ramos, Rian Q.; Sapp, Ryan M.; Jenkins, Nathan T.; Murphy, Anna E.; Cancre, Lucile; Chin, Eva R.; Spangenburg, Espen E.

2015-01-01

We aimed to determine if chronic endurance-exercise habits affected redox status and paracrine function of CD34+ and CD34−/CD31+ circulating angiogenic cells (CACs). Subjects were healthy, nonsmoking men and women aged 18–35 yr and categorized by chronic physical activity habits. Blood was drawn from each subject for isolation and culture of CD34+ and CD34−/CD31+ CACs. No differences in redox status were found in any group across either cell type. Conditioned media (CM) was generated from the cultured CACs and used in an in vitro human umbilical vein endothelial cell-based tube assay. CM from CD34+ cells from inactive individuals resulted in tube structures that were 29% shorter in length (P < 0.05) and 45% less complex (P < 0.05) than the endurance-trained group. CD34−/CD31+ CM from inactive subjects resulted in tube structures that were 26% shorter in length (P < 0.05) and 42% less complex (P < 0.05) than endurance-trained individuals. Proteomics analyses identified S100A8 and S100A9 in the CM. S100A9 levels were 103% higher (P < 0.05) and S100A8 was 97% higher in the CD34−/CD31+ CM of inactive subjects compared with their endurance-trained counterparts with no significant differences in either protein in the CM of CD34+ CACs as a function of training status. Recombinant S100A8/A9 treatment at concentrations detected in inactive subjects' CD34−/CD31+ CAC CM also reduced tube formation (P < 0.05). These findings are the first, to our knowledge, to demonstrate a differential paracrine role in CD34+ and CD34−/CD31+ CACs on tube formation as a function of chronic physical activity habits and identifies a differential secretion of S100A9 by CD34−/CD31+ CACs due to habitual exercise. PMID:26055789

DOE Office of Scientific and Technical Information (OSTI.GOV)

Becerikli, Mustafa; Jacobsen, Frank; Rittig, Andrea

Soft tissue sarcomas (STS) are characterized by co-participation of several epigenetic and genetic events during tumorigenesis. Having bypassed cellular senescence barriers during oncogenic transformation, the factors further affecting growth rate of STS cells remain poorly understood. Therefore, we investigated the role of gene silencing (DNA promoter methylation of LINE-1, PTEN), genetic aberrations (karyotype, KRAS and BRAF mutations) as well as their contribution to the proliferation rate and migratory potential that underlies “initial” and “final” passage sarcoma cells. Three different cell lines were used, SW982 (synovial sarcoma), U2197 (malignant fibrous histiocytoma (MFH)) and HT1080 (fibrosarcoma). Increased proliferative potential of final passagemore » STS cells was not associated with significant differences in methylation (LINE-1, PTEN) and mutation status (KRAS, BRAF), but it was dependent on the amount of chromosomal aberrations. Collectively, our data demonstrate that these fairly differentiated/advanced cancer cell lines have still the potential to gain an additional spontaneous growth benefit without external influences and that maintenance of increased proliferative potential towards longevity of STS cells (having crossed senescence barriers) may be independent of overt epigenetic alterations. -- Highlights: Increased proliferative potential of late passage STS cells was: • Not associated with epigenetic changes (methylation changes at LINE-1, PTEN). • Not associated with mutation status of KRAS, BRAF. • Dependent on presence/absence of chromosomal aberrations.« less

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: an improved approach in cartilage tissue engineering.

PubMed

Moroz, Andrei; Bittencourt, Renata Aparecida Camargo; Almeida, Renan Padron; Felisbino, Sérgio Luis; Deffune, Elenice

2013-01-01

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

A Comprehensive Proteomics Analysis Reveals a Secretory Path- and Status-Dependent Signature of Exosomes Released from Tumor-Associated Macrophages.

PubMed

Zhu, Yinghui; Chen, Xianwei; Pan, Qingfei; Wang, Yang; Su, Siyuan; Jiang, Cuicui; Li, Yang; Xu, Ningzhi; Wu, Lin; Lou, Xiaomin; Liu, Siqi

2015-10-02

Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Tumor-associated macrophages (TAMs) are important constituents of the tumor microenvironment; thus, it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAM status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1- and TAM-derived exosomes have different RNA profiles, which may result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding the complicated roles of macrophage-derived exosomes in the tumor microenvironment.

Differential effect of grape seed extract against human non-small-cell lung cancer cells: the role of reactive oxygen species and apoptosis induction.

PubMed

Tyagi, Alpna; Raina, Komal; Gangar, Subhash; Kaur, Manjinder; Agarwal, Rajesh; Agarwal, Chapla

2013-01-01

The present study examines grape seed extract (GSE) efficacy against a series of non-small-cell lung cancer (NSCLC) cell lines that differ in their Kras and p53 status to establish GSE potential as a cytotoxic agent against a wide range of lung cancer cells. GSE suppressed growth and induced apoptotic death in NSCLC cells irrespective of their k-Ras status, with more sensitivity toward H460 and H322 (wt k-Ras) than A549 and H1299 cells (mutated k-Ras). Mechanistic studies in A549 and H460 cells, selected, based on comparative efficacy of GSE at higher and lower doses, respectively, showed that apoptotic death involves cytochrome c release associated caspases 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, strong phosphorylation of ERK1/2 and JNK1/2, downregulation of cell survival proteins, and upregulated proapoptotic Bak expression. Importantly, GSE treatment caused a strong superoxide radical-associated oxidative stress, significantly decreased intracellular reduced glutathione levels, suggesting, for the first time, the involvement of GSE-caused oxidative stress in its apoptotic inducing activity in these cells. Because GSE is a widely-consumed dietary agent with no known untoward effects, our results support future studies to establish GSE efficacy and usefulness against NSCLC control.

Characterization of keratinocyte differentiation induced by ascorbic acid: protein kinase C involvement and vitamin C homeostasis.

PubMed

Savini, Isabella; Catani, Maria Valeria; Rossi, Antonello; Duranti, Guglielmo; Melino, Gerry; Avigliano, Luciana

2002-02-01

Epidermal keratinocytes undergo differentiation in response to several stimuli to form the cornified envelope, a structure that contributes to the barrier function of skin. Although differentiation has been extensively analyzed, the precise role of vitamin C during this process is still not defined. Ascorbic acid, besides acting as a radical scavenger, has been shown to promote mesenchymal differentiation. In this study, we found that keratinocytes grown in ascorbate-supplemented medium developed a differentiated phenotype, as demonstrated by enhanced expression of marker genes and increase in cornified envelope content. The pro-differentiating effects of ascorbate were mediated by the protein-kinase-C-dependent induction of activating protein 1 DNA binding activity; indeed, down-modulation of protein kinase C activity abolished differentiation triggered by ascorbic acid. Although vitamin C appeared to regulate the same signaling pathway modulated by calcium, a classical in vitro inducer of epidermal differentiation, nonetheless terminally differentiated keratinocytes exhibited different ascorbate homeostasis and cellular antioxidant status. Indeed, we found that, unlike calcium, differentiation promoted by ascorbate was accompanied by (i) an enhanced ascorbate transport, due to overexpression of specific transporters, (ii) a great efficiency of dehydroascorbate uptake, and (iii) an increase in glutathione content with respect to proliferating cells. Ascorbic acid may be useful to promote epidermal differentiation, avoiding depletion of hydrophilic antioxidant stores.

OGEE v2: an update of the online gene essentiality database with special focus on differentially essential genes in human cancer cell lines.

PubMed

Chen, Wei-Hua; Lu, Guanting; Chen, Xiao; Zhao, Xing-Ming; Bork, Peer

2017-01-04

OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased numbers of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives.

PubMed

Cima, Igor; Wen Yee, Chay; Iliescu, Florina S; Phyo, Wai Min; Lim, Kiat Hon; Iliescu, Ciprian; Tan, Min Han

2013-01-01

This review will cover the recent advances in label-free approaches to isolate and manipulate circulating tumor cells (CTCs). In essence, label-free approaches do not rely on antibodies or biological markers for labeling the cells of interest, but enrich them using the differential physical properties intrinsic to cancer and blood cells. We will discuss technologies that isolate cells based on their biomechanical and electrical properties. Label-free approaches to analyze CTCs have been recently invoked as a valid alternative to "marker-based" techniques, because classical epithelial and tumor markers are lost on some CTC populations and there is no comprehensive phenotypic definition for CTCs. We will highlight the advantages and drawbacks of these technologies and the status on their implementation in the clinics.

Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A

PubMed Central

Kelly, Richard D. W.; Mahmud, Arsalan; McKenzie, Matthew; Trounce, Ian A.; St John, Justin C.

2012-01-01

DNA methylation is an essential mechanism controlling gene expression during differentiation and development. We investigated the epigenetic regulation of the nuclear-encoded, mitochondrial DNA (mtDNA) polymerase γ catalytic subunit (PolgA) by examining the methylation status of a CpG island within exon 2 of PolgA. Bisulphite sequencing identified low methylation levels (<10%) within exon 2 of mouse oocytes, blastocysts and embryonic stem cells (ESCs), while somatic tissues contained significantly higher levels (>40%). In contrast, induced pluripotent stem (iPS) cells and somatic nuclear transfer ESCs were hypermethylated (>20%), indicating abnormal epigenetic reprogramming. Real time PCR analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) immunoprecipitated DNA suggests active DNA methylation and demethylation within exon 2 of PolgA. Moreover, neural differentiation of ESCs promoted de novo methylation and demethylation at the exon 2 locus. Regression analysis demonstrates that cell-specific PolgA expression levels were negatively correlated with DNA methylation within exon 2 and mtDNA copy number. Finally, using chromatin immunoprecipitation (ChIP) against RNA polymerase II (RNApII) phosphorylated on serine 2, we show increased DNA methylation levels are associated with reduced RNApII transcriptional elongation. This is the first study linking nuclear DNA epigenetic regulation with mtDNA regulation during differentiation and cell specialization. PMID:22941637

[The relationship between human papillomavirus (HPV) infection and penile cancer].

PubMed

Yumura, Yasushi; Hattori, Yusuke; Noda, Hideyuki; Kondo, Kei-Ichi; Noguchi, Kazumi; Sasaki, Takeshi; Kasuga, Jun; Kubota, Yoshinobu

2009-11-01

Human papillomavirus (HPV) may be carcinogenic effectors in a variety of human lower genital tract malignancies. We evaluated HPV status with respect to clinical and pathological features and prognosis of penile carcinoma. We searched for HPV infected cells (Koilocytosis) within the primary lesion of cancer tissue from 78 patients with penile squamous cell carcinoma. The following variables were recorded : age, tumor size, clinical stage, lymphatic and venous invasion, histologic and nuclear grade, Broders grade, infiltration status, and lymph node and distant metastasis. Koilocytosis were detected 55.1% (43 of 78) of patients. Tumors with Koilocytosis had better differentiation (p=0.0443) and lower grade (better keratinized) in Broders grading system (p=0.0116) than HPV negative tumors. No difference was found in the 5-year survival rate (p=0.5693). Our data suggest that the presence of Koilocytosis does not influence prognosis in penile cancer.

Advances in translational research in neuro-oncology.

PubMed

Fueyo, Juan; Gomez-Manzano, Candelaria; Yung, W K Alfred

2011-03-01

During the last decade, we have witnessed several key advances in the field of neuro-oncology. First, there were conceptual advances in the molecular and cell biology of malignant gliomas including the discovery in 2004 of brain tumor stem cells. Second, the Cancer Genome Atlas project has been extremely useful in the discovery of new molecular markers, including mutations in the IDH1 gene, and has led to a new classification of gliomas based on the differentiation status and mesenchymal transformation. In addition, use of the 1p/19q marker and O6-methylguanine-DNA methyltransferase methylation status have been identified as guides for patient selection for therapies and represent the first steps toward personalized medicine for treating gliomas. Finally, progress has been made in treatment strategies including the establishment of temozolomide as the criterion standard for treating gliomas, the adoption of bevacizumab in the clinical setting, and developments in experimental biological therapies including cancer vaccines and oncolytic adenoviruses.

Relating tumor score to hematology in green turtles with fibropapillomatosis in Hawaii

USGS Publications Warehouse

Work, Thierry M.; Balazs, George H.

1999-01-01

The relationship between hematologic status and severity of tumor affliction in green turtles (Chelonia mydas) with fibropapillomatosis (FP) was examined. During 1 wk periods in July 1997 and July 1998, we bled 108 free-ranging green turtles from Pala'au (Molokai, Hawaii, USA) where FP is endemic. Blood was analyzed for hematocrit, estimated total solids, total white blood cell (WBC) count and differential WBC count. Each turtle was assigned a subjective tumor score ranging from 0 (no visible external tumors) to 3 (heavily tumored) that indicated the severity of FP. There was a progressive increase in monocytes and a decrease in all other hematologic parameters except heterophils and total numbers of white blood cells as tumor score increased. These data indicate that tumor score can relate to physiologic status of green turtles afflicted with FP, and that tumor score is a useful field monitor of severity of FP in this species.

Transcriptomics and proteomics show that selenium affects inflammation, cytoskeleton, and cancer pathways in human rectal biopsies.

PubMed

Méplan, Catherine; Johnson, Ian T; Polley, Abigael C J; Cockell, Simon; Bradburn, David M; Commane, Daniel M; Arasaradnam, Ramesh P; Mulholland, Francis; Zupanic, Anze; Mathers, John C; Hesketh, John

2016-08-01

Epidemiologic studies highlight the potential role of dietary selenium (Se) in colorectal cancer prevention. Our goal was to elucidate whether expression of factors crucial for colorectal homoeostasis is affected by physiologic differences in Se status. Using transcriptomics and proteomics followed by pathway analysis, we identified pathways affected by Se status in rectal biopsies from 22 healthy adults, including 11 controls with optimal status (mean plasma Se = 1.43 μM) and 11 subjects with suboptimal status (mean plasma Se = 0.86 μM). We observed that 254 genes and 26 proteins implicated in cancer (80%), immune function and inflammatory response (40%), cell growth and proliferation (70%), cellular movement, and cell death (50%) were differentially expressed between the 2 groups. Expression of 69 genes, including selenoproteins W1 and K, which are genes involved in cytoskeleton remodelling and transcription factor NFκB signaling, correlated significantly with Se status. Integrating proteomics and transcriptomics datasets revealed reduced inflammatory and immune responses and cytoskeleton remodelling in the suboptimal Se status group. This is the first study combining omics technologies to describe the impact of differences in Se status on colorectal expression patterns, revealing that suboptimal Se status could alter inflammatory signaling and cytoskeleton in human rectal mucosa and so influence cancer risk.-Méplan, C., Johnson, I. T., Polley, A. C. J., Cockell, S., Bradburn, D. M., Commane, D. M., Arasaradnam, R. P., Mulholland, F., Zupanic, A., Mathers, J. C., Hesketh, J. Transcriptomics and proteomics show that selenium affects inflammation, cytoskeleton, and cancer pathways in human rectal biopsies. © The Author(s).

Flow cytometry analysis of T-cell subsets in cerebrospinal fluid of narcolepsy type 1 patients with long-lasting disease.

PubMed

Moresco, Monica; Lecciso, Mariangela; Ocadlikova, Darina; Filardi, Marco; Melzi, Silvia; Kornum, Birgitte Rahbek; Antelmi, Elena; Pizza, Fabio; Mignot, Emmanuel; Curti, Antonio; Plazzi, Giuseppe

2018-04-01

Type 1 narcolepsy (NT1) is a central hypersomnia linked to the destruction of hypocretin-producing neurons. A great body of genetic and epidemiological data points to likely autoimmune disease aetiology. Recent reports have characterized peripheral blood T-cell subsets in NT1, whereas data regarding the cerebrospinal fluid (CSF) immune cell composition are lacking. The current study aimed to characterize the T-cell and natural killer (NK) cell subsets in NT1 patients with long disease course. Immune cell subsets from CSF and peripheral blood mononuclear cell (PBMC) samples were analysed by flow cytometry in two age-balanced and sex-balanced groups of 14 NT1 patients versus 14 healthy controls. The frequency of CSF cell groups was compared with PBMCs. Non-parametric tests were used for statistical analyses. The NT1 patients did not show significant differences of CSF immune cell subsets compared to controls, despite a trend towards higher CD4 + terminally differentiated effector memory T cells. T cells preferentially displayed a memory phenotype in the CSF compared to PBMCs. Furthermore, a reduced frequency of CD4 + terminally differentiated effector memory T cells and an increased frequency of NK CD56 bright cells was observed in PBMCs from patients compared to controls. Finally, the ratio between CSF and peripheral CD4 + terminally differentiated effector memory T cells was two-fold increased in NT1 patients versus controls. Significant differences in PBMCs and in CSF/PBMC ratios of immune cell profile were found in NT1 patients compared to healthy controls. These differences might have arisen from the different HLA status, or be primary or secondary to hypocretin deficiency. Further functional studies in patients close to disease onset are required to understand NT1 pathophysiology. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative Gene Expression Analyses Reveal Distinct Molecular Signatures between Differentially Reprogrammed Cardiomyocytes.

PubMed

Zhou, Yang; Wang, Li; Liu, Ziqing; Alimohamadi, Sahar; Yin, Chaoying; Liu, Jiandong; Qian, Li

2017-09-26

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) or directly reprogrammed from non-myocytes (induced cardiomyocytes [iCMs]) are promising sources for heart regeneration or disease modeling. However, the similarities and differences between iPSC-CMs and iCMs are still unknown. Here, we performed transcriptome analyses of beating iPSC-CMs and iCMs generated from cardiac fibroblasts (CFs) of the same origin. Although both iPSC-CMs and iCMs establish CM-like molecular features globally, iPSC-CMs exhibit a relatively hyperdynamic epigenetic status, whereas iCMs exhibit a maturation status that more closely resembles that of adult CMs. Based on gene expression of metabolic enzymes, iPSC-CMs primarily employ glycolysis, whereas iCMs utilize fatty acid oxidation as the main pathway. Importantly, iPSC-CMs and iCMs exhibit different cell-cycle status, alteration of which influenced their maturation. Therefore, our study provides a foundation for understanding the pros and cons of different reprogramming approaches. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Vitamin D3 contributes to enhanced osteogenic differentiation of MSCs under oxidative stress condition via activating the endogenous antioxidant system.

PubMed

Zhou, J; Wang, F; Ma, Y; Wei, F

2018-06-02

The anti-oxidative effects of vitamin D3 (Vd3) on mesenchymal stem cells (MSCs) have not been studied before. The present study suggested that Vd3 could not only promote the osteogenic differentiation of MSCs under normal condition but also partly protect it from oxidative stress damage by activating the endogenous antioxidant system. Evolving evidence proved that oxidative stress caused by reactive oxygen species (ROS) overproduction might lead to bone loss. Vd3, a commonly used osteogenic induction drug, was proved to exhibit potent anti-oxidative effects on other cell types. The present study aims to investigate the protective effects of Vd3 on oxidative stress-induced dysfunctions of MSCs, as well as its underlying mechanisms. The H 2 O 2 was used as exogenous reactive oxygen species (ROS). The influence of ROS and anti-oxidative protection of Vd3 on MSCs were analyzed too. Multi-techniques were used to assess the beneficial effects of Vd3 on MSCs under oxidative stress condition. The results demonstrated that Vd3 could significantly attenuate the H 2 O 2 -induced cell injury of MSCs via Sirt1/FoxO1 signaling pathway, and reduced the H 2 O 2 exposure-induced intracellular oxidative stress status of MSCs. What's more, the H 2 O 2 exposure resulted in the decreased osteogenic differentiation of MSCs, as evidenced by decreased alkaline phosphatase activity, calcium deposition level, and osteogenic differentiation gene mRNA levels, but the injury was restored via Vd3 administration. The results suggested that Vd3 could not only promote the osteogenic differentiation of osteoblastic cells under normal condition but also partly protect the cell from oxidative stress damage by activating endogenous antioxidant system. The study shed light on the new roles of Vd3 in bone modeling and remodeling regulation.

Downregulation of monocytic differentiation via modulation of CD147 by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

PubMed

Sasidhar, Manda V; Chevooru, Sai Krishnaveni; Eickelberg, Oliver; Hartung, Hans-Peter; Neuhaus, Oliver

2017-01-01

CD147 is an activation induced glycoprotein that promotes the secretion and activation of matrix metalloproteinases (MMPs) and is upregulated during the differentiation of macrophages. Interestingly, some of the molecular functions of CD147 rely on its glycosylation status: the highly glycosylated forms of CD147 induce MMPs whereas the lowly glycosylated forms inhibit MMP activation. Statins are hydroxy-methylglutaryl coenzyme A reductase inhibitors that block the synthesis of mevalonate, thereby inhibiting all mevalonate-dependent pathways, including isoprenylation, N-glycosylation and cholesterol synthesis. In this study, we investigated the role of statins in the inhibition of macrophage differentiation and the associated process of MMP secretion through modulation of CD147. We observed that differentiation of the human monocytic cell line THP-1 to a macrophage phenotype led to upregulation of CD147 and CD14 and that this effect was inhibited by statins. At the molecular level, statins altered CD147 expression, structure and function by inhibiting isoprenylation and N-glycosylation. In addition, statins induced a shift of CD147 from its highly glycosylated form to its lowly glycosylated form. This shift in N-glycosylation status was accompanied by a decrease in the production and functional activity of MMP-2 and MMP-9. In conclusion, these findings describe a novel molecular mechanism of immune regulation by statins, making them interesting candidates for autoimmune disease therapy.

Concise Review: Multifaceted Characterization of Human Mesenchymal Stem Cells for Use in Regenerative Medicine

PubMed Central

Samsonraj, Rebekah M.; Raghunath, Michael; Nurcombe, Victor; Hui, James H.

2017-01-01

Abstract Mesenchymal stem cells (MSC) hold great potential for regenerative medicine because of their ability for self‐renewal and differentiation into tissue‐specific cells such as osteoblasts, chondrocytes, and adipocytes. MSCs orchestrate tissue development, maintenance and repair, and are useful for musculoskeletal regenerative therapies to treat age‐related orthopedic degenerative diseases and other clinical conditions. Importantly, MSCs produce secretory factors that play critical roles in tissue repair that support both engraftment and trophic functions (autocrine and paracrine). The development of uniform protocols for both preparation and characterization of MSCs, including standardized functional assays for evaluation of their biological potential, are critical factors contributing to their clinical utility. Quality control and release criteria for MSCs should include cell surface markers, differentiation potential, and other essential cell parameters. For example, cell surface marker profiles (surfactome), bone‐forming capacities in ectopic and orthotopic models, as well as cell size and granularity, telomere length, senescence status, trophic factor secretion (secretome), and immunomodulation, should be thoroughly assessed to predict MSC utility for regenerative medicine. We propose that these and other functionalities of MSCs should be characterized prior to use in clinical applications as part of comprehensive and uniform guidelines and release criteria for their clinical‐grade production to achieve predictably favorable treatment outcomes for stem cell therapy. Stem Cells Translational Medicine 2017;6:2173–2185 PMID:29076267

Gilz-Activin A as a Novel Signaling Axis Orchestrating Mesenchymal Stem Cell and Th17 Cell Interplay

PubMed Central

Luz-Crawford, Patricia; Espinosa-Carrasco, Gabriel; Ipseiz, Natacha; Contreras, Rafael; Tejedor, Gautier; Medina, Daniel A.; Vega-Letter, Ana-Maria; Ngo, Devi; Morand, Eric F.; Pène, Jérôme; Hernandez, Javier; Jorgensen, Christian; Djouad, Farida

2018-01-01

Mesenchymal stem cells (MSC) are highly immunosuppressive cells able to reduce chronic inflammation through the active release of mediators. Recently, we showed that glucocorticoid-induced leucine zipper (Gilz) expression by MSC is involved in their therapeutic effect by promoting the generation of regulatory T cells. However, the mechanisms underlying this pivotal role of Gilz remain elusive. Methods and Results In this study, we have uncovered evidence that Gilz modulates the phenotype and function of Th1 and Th17 cells likely by upregulating the level of Activin A and NO2 secreted by MSC. Adoptive transfer experiments sustained this Gilz-dependent suppressive effect of MSC on Th1 and Th17 cell functions. In immunoregulatory MSC, obtained by priming with IFN-γ and TNF-α, Gilz was translocated to the nucleus and bound to the promoters of inos and Activin βA to induce their expression. The increased expression of Activin A directly impacted on Th17 cells fate by repressing their differentiation program through the activation of Smad3/2 and enhancing IL-10 production. Conclusion Our results reveal how Gilz controls inos and Activin βA gene expression to ultimately assign immunoregulatory status to MSC able to repress the pathogenic Th17 cell differentiation program and uncover Activin A as a novel mediator of MSC in this process. PMID:29344311

Gilz-Activin A as a Novel Signaling Axis Orchestrating Mesenchymal Stem Cell and Th17 Cell Interplay.

PubMed

Luz-Crawford, Patricia; Espinosa-Carrasco, Gabriel; Ipseiz, Natacha; Contreras, Rafael; Tejedor, Gautier; Medina, Daniel A; Vega-Letter, Ana-Maria; Ngo, Devi; Morand, Eric F; Pène, Jérôme; Hernandez, Javier; Jorgensen, Christian; Djouad, Farida

2018-01-01

Mesenchymal stem cells (MSC) are highly immunosuppressive cells able to reduce chronic inflammation through the active release of mediators. Recently, we showed that glucocorticoid-induced leucine zipper (Gilz) expression by MSC is involved in their therapeutic effect by promoting the generation of regulatory T cells. However, the mechanisms underlying this pivotal role of Gilz remain elusive. Methods and Results In this study, we have uncovered evidence that Gilz modulates the phenotype and function of Th1 and Th17 cells likely by upregulating the level of Activin A and NO 2 secreted by MSC. Adoptive transfer experiments sustained this Gilz-dependent suppressive effect of MSC on Th1 and Th17 cell functions. In immunoregulatory MSC, obtained by priming with IFN-γ and TNF-α, Gilz was translocated to the nucleus and bound to the promoters of inos and Activin βA to induce their expression. The increased expression of Activin A directly impacted on Th17 cells fate by repressing their differentiation program through the activation of Smad3/2 and enhancing IL-10 production. Conclusion Our results reveal how Gilz controls inos and Activin βA gene expression to ultimately assign immunoregulatory status to MSC able to repress the pathogenic Th17 cell differentiation program and uncover Activin A as a novel mediator of MSC in this process.

Cell fusion phenomena detected after in utero transplantation of Ds-red-harboring porcine amniotic fluid stem cells into EGFP transgenic mice.

PubMed

Peng, Shao-Yu; Chen, Yu-Hsu; Chou, Chih-Jen; Wang, Yao-Horng; Lee, Hung-Maan; Cheng, Winston Teng-Kui; Shaw, S W Steven; Wu, Shinn-Chih

2014-05-01

Amniotic fluid stem cells (AFSCs) are derived from the amniotic fluid of the developing fetus and can give rise to diverse differentiated cells of ectoderm, mesoderm, and endoderm lineages. Intrauterine transplantation is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant dams. Certain disease such as osteogenesis imperfecta was successfully treated in affected fetal mice using this method. However, the donor cell destiny remains uncertain. The purpose of this study was to investigate the biodistribution and cell fate of Ds-red-harboring porcine AFSCs (Ds-red pAFSCs) after intrauterine transplantation into enhanced green fluorescent protein-harboring fetuses of pregnant mice. Pregnant mice (12.5 days) underwent open laparotomy with intrauterine pAFSC transplantation (5 × 10(4) cells per pup) into fetal peritoneal cavity. Three weeks after birth, the mice were sacrificed. Several samples from different organs were obtained for histological examination and flow cytometric analysis. Ds-red pAFSCs migrated most frequently into the intestines. Furthermore, enhanced green fluorescent protein and red fluorescent protein signals were co-expressed in the intestine and liver cells via immunohistochemistry studies. In utero xenotransplantation of pAFSCs fused with recipient intestinal cells instead of differentiating or maintaining the undifferentiated status in the tissue. © 2014 John Wiley & Sons, Ltd.

Level of PAX5 in differential diagnosis of non-Hodgkin's lymphoma

PubMed Central

Bharti, Brij; Shukla, Sachin; Tripathi, Ratnakar; Mishra, Suman; Kumar, Mohan; Pandey, Manoj; Mishra, Rajnikant

2016-01-01

Background & objectives: The PAX5, a paired box transcription factor and B-cell activator protein (BSAP), activates B-cell commitment genes and represses non-B-cell lineage genes. About 14 transcript variants of PAX5 have been observed in human. Any alteration in its expression pattern leads to lymphogenesis or associated diseases and carcinogenesis in non-lymphoid tissues. Its mechanisms of function in pathophysiology of non-Hodgkin's lymphoma (NHL) are unclear. This study was intended to explore influence of PAX5 in cascade of NHL pathogenesis and diagnosis. Methods: Samples of 65 patients were evaluated by immunohistochemical staining for cellular localization of PAX5, CD19, CD3, cABL, p53, Ras and Raf and by TUNEL assay, RNA-isolation and reverse transcriptase (RT)-PCR, Western blot analysis, and lactate dehydrogenase (LDH) specific staining. Results: B-cell type NHL patients were positive for PAX5, p53, Ras, CD19, Raf and CD3. All of them showed TUNEL-positive cells. The differential expression pattern of PAX5, CD19, p53, CD3, ZAP70, HIF1α, Ras, Raf and MAPK (mitogen-activated protein kinase) at the levels of transcripts and proteins was observed. The LDH assay showed modulation of LDH4 and LDH5 isoforms in the lymph nodes of NHL patients. Interpretation & conclusions: The histological observations suggested that the patients represent diverse cases of NHL like mature B-cell type, mature T-cell type and high grade diffuse B-cell type NHL. The findings indicate that patients with NHL may also be analyzed for status of PAX5, CD19 and ZAP70, and their transcriptional and post-translational variants for the differential diagnosis of NHL and therapy. PMID:27748274

Differential Lectin Agglutination of Fetal, Dividing-Postnatal, and Malignant Hepatocytes

PubMed Central

Becker, F. F.

1974-01-01

Numerous studies have reported the capacity of the lectin, concanavalin A, to agglutinate selected cell-types. The finding that cells transformed in culture, embryonic cells, and malignant cells are all agglutinated by this substance, may contribute to our understanding of the oncogenic process. The present study compared the response to concanavalin A of rat hepatocytes derived from livers of differing developmental and mitotic-status as well as those derived from malignant liver tumors (hepatomas). Fetal hepatocytes and hepatoma cells were highly susceptible to agglutination while hepatocytes from post-natal livers, whether dividing or quiescent, were not. Treatment with protease(s) did not make the interphase hepatocyte agglutinable. These data emphasize the importance of examining a wide variety of cells in attempting to understand the interaction of lectins on cell surfaces, and further, demonstrate the value of obtaining cells directly from tissue(s) during differing physiologic and pathologic states. Images PMID:4373708

Cardiac Biomarkers: a Focus on Cardiac Regeneration

PubMed Central

Forough, Reza; Scarcello, Catherine; Perkins, Matthew

2011-01-01

Historically, biomarkers have been used in two major ways to maintain and improve better health status: first, for diagnostic purposes, and second, as specific targets to treat various diseases. A new era in treatment and even cure for the some diseases using reprograming of somatic cells is about to be born. In this approach, scientists are successfully taking human skin cells (previously considered terminally-differentiated cells) and re-programming them into functional cardiac myocytes and other cell types in vitro. A cell reprograming approach for treatment of cardiovascular diseases will revolutionize the field of medicine and significantly expand the human lifetime. Availability of a comprehensive catalogue for cardiac biomarkers is necessary for developing cell reprograming modalities to treat cardiac diseases, as well as for determining the progress of reprogrammed cells as they become cardiac cells. In this review, we present a comprehensive survey of the cardiac biomarkers currently known. PMID:23074366

Calorie Restriction Attenuates Terminal Differentiation of Immune Cells.

PubMed

White, Matthew J; Beaver, Charlotte M; Goodier, Martin R; Bottomley, Christian; Nielsen, Carolyn M; Wolf, Asia-Sophia F M; Boldrin, Luisa; Whitmore, Charlotte; Morgan, Jennifer; Pearce, Daniel J; Riley, Eleanor M

2016-01-01

Immune senescence is a natural consequence of aging and may contribute to frailty and loss of homeostasis in later life. Calorie restriction increases healthy life-span in C57BL/6J (but not DBA/2J) mice, but whether this is related to preservation of immune function, and how it interacts with aging, is unclear. We compared phenotypic and functional characteristics of natural killer (NK) cells and T cells, across the lifespan, of calorie-restricted (CR) and control C57BL/6 and DBA/2 mice. Calorie restriction preserves a naïve T cell phenotype and an immature NK cell phenotype as mice age. The splenic T cell populations of CR mice had higher proportions of CD11a - CD44 lo cells, lower expression of TRAIL, KLRG1, and CXCR3, and higher expression of CD127, compared to control mice. Similarly, splenic NK cells from CR mice had higher proportions of less differentiated CD11b - CD27 + cells and correspondingly lower proportions of highly differentiated CD11b + CD27 - NK cells. Within each of these subsets, cells from CR mice had higher expression of CD127, CD25, TRAIL, NKG2A/C/E, and CXCR3 and lower expression of KLRG1 and Ly49 receptors compared to controls. The effects of calorie restriction on lymphoid cell populations in lung, liver, and lymph nodes were identical to those seen in the spleen, indicating that this is a system-wide effect. The impact of calorie restriction on NK cell and T cell maturation is much more profound than the effect of aging and, indeed, calorie restriction attenuates these age-associated changes. Importantly, the effects of calorie restriction on lymphocyte maturation were more marked in C57BL/6 than in DBA/2J mice indicating that delayed lymphocyte maturation correlates with extended lifespan. These findings have implications for understanding the interaction between nutritional status, immunity, and healthy lifespan in aging populations.

Epigenetic regulation of Atoh1 guides hair cell development in the mammalian cochlea.

PubMed

Stojanova, Zlatka P; Kwan, Tao; Segil, Neil

2015-10-15

In the developing cochlea, sensory hair cell differentiation depends on the regulated expression of the bHLH transcription factor Atoh1. In mammals, if hair cells die they do not regenerate, leading to permanent deafness. By contrast, in non-mammalian vertebrates robust regeneration occurs through upregulation of Atoh1 in the surviving supporting cells that surround hair cells, leading to functional recovery. Investigation of crucial transcriptional events in the developing organ of Corti, including those involving Atoh1, has been hampered by limited accessibility to purified populations of the small number of cells present in the inner ear. We used µChIP and qPCR assays of FACS-purified cells to track changes in the epigenetic status of the Atoh1 locus during sensory epithelia development in the mouse. Dynamic changes in the histone modifications H3K4me3/H3K27me3, H3K9ac and H3K9me3 reveal a progression from poised, to active, to repressive marks, correlating with the onset of Atoh1 expression and its subsequent silencing during the perinatal (P1 to P6) period. Inhibition of acetylation blocked the increase in Atoh1 mRNA in nascent hair cells, as well as ongoing hair cell differentiation during embryonic organ of Corti development ex vivo. These results reveal an epigenetic mechanism of Atoh1 regulation underlying hair cell differentiation and subsequent maturation. Interestingly, the H3K4me3/H3K27me3 bivalent chromatin structure observed in progenitors persists at the Atoh1 locus in perinatal supporting cells, suggesting an explanation for the latent capacity of these cells to transdifferentiate into hair cells, and highlighting their potential as therapeutic targets in hair cell regeneration. © 2015. Published by The Company of Biologists Ltd.

Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor.

PubMed

Vernardis, Spyros I; Terzoudis, Konstantinos; Panoskaltsis, Nicki; Mantalaris, Athanasios

2017-02-06

Human pluripotent stem cells (hPSCs) are adhesion-dependent cells that require cultivation in colonies to maintain growth and pluripotency. Robust differentiation protocols necessitate single cell cultures that are achieved by use of ROCK (Rho kinase) inhibitors. ROCK inhibition enables maintenance of stem cell phenotype; its effects on metabolism are unknown. hPSCs were exposed to 10 μM ROCK inhibitor for varying exposure times. Pluripotency (TRA-1-81, SSEA3, OCT4, NANOG, SOX2) remained unaffected, until after prolonged exposure (96 hrs). Gas chromatography-mass spectrometry metabolomics analysis identified differences between ROCK-treated and untreated cells as early as 12 hrs. Exposure for 48 hours resulted in reduction in glycolysis, glutaminolysis, the citric acid (TCA) cycle as well as the amino acids pools, suggesting the adaptation of the cells to the new culture conditions, which was also reflected by the expression of the metabolic regulators, mTORC1 and tp53 and correlated with cellular proliferation status. While gene expression and protein levels did not reveal any changes in the physiology of the cells, metabolomics revealed the fluctuating state of the metabolism. The above highlight the usefulness of metabolomics in providing accurate and sensitive information on cellular physiological status, which could lead to the development of robust and optimal stem cell bioprocesses.

Fructose-bisphosphate aldolase a is a potential metastasis-associated marker of lung squamous cell carcinoma and promotes lung cell tumorigenesis and migration.

PubMed

Du, Sha; Guan, Zhuzhu; Hao, Lihong; Song, Yang; Wang, Lan; Gong, Linlin; Liu, Lu; Qi, Xiaoyu; Hou, Zhaoyuan; Shao, Shujuan

2014-01-01

Fructose-bisphosphate aldolase A (ALDOA) is a key enzyme in glycolysis and is responsible for catalyzing the reversible conversion of fructose-1,6-bisphosphate to glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. ALDOA contributes to various cellular functions such as muscle maintenance, regulation of cell shape and mobility, striated muscle contraction, actin filament organization and ATP biosynthetic process. Here, we reported that ALDOA is a highly expressed in lung squamous cell carcinoma (LSCC) and its expression level is correlated with LSCC metastasis, grades, differentiation status and poor prognosis. Depletion of ALDOA expression in the lung squamous carcinoma NCI-H520 cells reduces the capabilities of cell motility and tumorigenesis. These data suggest that ALDOA could be a potential marker for LSCC metastasis and a therapeutic target for drug development.

Autologous Pluripotent Stem Cell-Derived β-Like Cells for Diabetes Cellular Therapy.

PubMed

Millman, Jeffrey R; Pagliuca, Felicia W

2017-05-01

Development of stem cell technologies for cell replacement therapy has progressed rapidly in recent years. Diabetes has long been seen as one of the first applications for stem cell-derived cells because of the loss of only a single cell type-the insulin-producing β-cell. Recent reports have detailed strategies that overcome prior hurdles to generate functional β-like cells from human pluripotent stem cells in vitro, including from human induced pluripotent stem cells (hiPSCs). Even with this accomplishment, addressing immunological barriers to transplantation remains a major challenge for the field. The development of clinically relevant hiPSC derivation methods from patients and demonstration that these cells can be differentiated into β-like cells presents a new opportunity to treat diabetes without immunosuppression or immunoprotective encapsulation or with only targeted protection from autoimmunity. This review focuses on the current status in generating and transplanting autologous β-cells for diabetes cell therapy, highlighting the unique advantages and challenges of this approach. © 2017 by the American Diabetes Association.

A BAP1 Mutation-specific MicroRNA Signature Predicts Clinical Outcomes in Clear Cell Renal Cell Carcinoma Patients with Wild-type BAP1

PubMed Central

Ge, Yu-Zheng; Xu, Lu-Wei; Zhou, Chang-Cheng; Lu, Tian-Ze; Yao, Wen-Tao; Wu, Ran; Zhao, You-Cai; Xu, Xiao; Hu, Zhi-Kai; Wang, Min; Yang, Xiao-Bing; Zhou, Liu-Hua; Zhong, Bing; Xu, Zheng; Li, Wen-Cheng; Zhu, Jia-Geng; Jia, Rui-Peng

2017-01-01

Background: Clear cell renal cell carcinoma (ccRCC) is the most prevalent histologic subtype of kidney cancers in adults, which could be divided into two distinct subgroups according to the BRCA1 associated protein-1 (BAP1) mutation status. In the current study, we comprehensively analyzed the genome-wide microRNA (miRNA) expression profiles in ccRCC, with the aim to identify the differentially expressed miRNAs between BAP1 mutant and wild-type tumors, and generate a BAP1 mutation-specific miRNA signature for ccRCC patients with wild-type BAP1. Methods: The BAP1 mutation status and miRNA profiles in BAP1 mutant and wild-type tumors were analyzed. Subsequently, the association of the differentially expressed miRNAs with patient survival was examined, and a BAP1 mutation-specific miRNA signature was generated and examined with Kaplan-Meier survival, univariate and multivariate Cox regression analyses. Finally, the bioinformatics methods were adopted for the target prediction of selected miRNAs and functional annotation analyses. Results: A total of 350 treatment-naïve primary ccRCC patients were selected from The Cancer Genome Atlas project, among which 35 (10.0%) subjects carried mutant BAP1 and had a shorter overall survival (OS) time. Furthermore, 33 miRNAs were found to be differentially expressed between BAP1 mutant and wild-type tumors, among which 11 (miR-149, miR-29b-2, miR-182, miR-183, miR-21, miR-365-2, miR-671, miR-365-1, miR-10b, miR-139, and miR-181a-2) were significantly associated with OS in ccRCC patients with wild-type BAP1. Finally, a BAP1 mutation-specific miRNA signature consisting of 11 miRNAs was generated and validated as an independent prognostic parameter. Conclusions: In summary, our study identified a total of 33 miRNAs differentially expressed between BAP1 mutant and wild-type tumors, and generated a BAP1 mutation-specific miRNA signature including eleven miRNAs, which could serve as a novel prognostic biomarker for ccRCC patients with wild-type BAP1. PMID:28900502

Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance.

PubMed

Wermann, Hendrik; Stoop, Hans; Gillis, Ad J M; Honecker, Friedemann; van Gurp, Ruud J H L M; Ammerpohl, Ole; Richter, Julia; Oosterhuis, J Wolter; Bokemeyer, Carsten; Looijenga, Leendert H J

2010-08-01

Differences in the global methylation pattern, ie hyper- as well as hypo-methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5-(m)cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam-2 before and after demethylation using 5-azacytidine. Exposure to 5-azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell-specific marker VASA, showed increased expression. Following treatment with 5-azacytidine, TCam-2 cells were analysed using a high-throughput methylation screen for changes in the methylation sites of 14,000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs.

Human papillomavirus reduces the prognostic value of nodal involvement in tonsillar squamous cell carcinomas.

PubMed

Straetmans, Jos M J A A; Olthof, Nadine; Mooren, Jeroen J; de Jong, Jos; Speel, Ernst-Jan M; Kremer, Bernd

2009-10-01

Assessment of the prognostic value of nodal status in relation to human papillomavirus (HPV) status and the various treatment modalities in tonsillar squamous cell carcinomas (TSCC). Retrospective 5-year survival analysis. A 5-year follow-up of disease-free, disease-specific, and overall survival in a group of 81 patients with TSCC was conducted. The nodal status and integration of HPV-DNA in the genome (detected with fluorescence in situ hybridization) as prognostic indicators were examined while correcting for other clinical parameters (smoking habits, alcohol consumption, treatment modality, differentiation, TNM classification). Of TSCCs, 41% were positive for HPV type 16. In these TSCCs, the primary tumor was significantly smaller when compared to HVP-negative TSCCs (P = .04), whereas the percentage of cases with cervical metastases was identical. In the total population, it was not nodal involvement, but rather HPV manifestation, which was related to patient prognosis. Within the treatment modalities (surgery combined with radiotherapy and radiotherapy alone), neither nodal status nor HPV were prognostic indicators. Since a substantial percentage of TSCCs are HPV-positive and metastasizes to cervical lymph nodes in less advanced primary tumors, the N status is an unreliable prognostic indicator in TSCCs. HPV is only prognostically relevant in the total tumor population, but loses its value within patient groups receiving a single treatment modality. The value of HPV for prognosis of patients with TSCC requires further study.

Population balance modeling: current status and future prospects.

PubMed

Ramkrishna, Doraiswami; Singh, Meenesh R

2014-01-01

Population balance modeling is undergoing phenomenal growth in its applications, and this growth is accompanied by multifarious reviews. This review aims to fortify the model's fundamental base, as well as point to a variety of new applications, including modeling of crystal morphology, cell growth and differentiation, gene regulatory processes, and transfer of drug resistance. This is accomplished by presenting the many faces of population balance equations that arise in the foregoing applications.

Human papillomavirus molecular biology.

PubMed

Harden, Mallory E; Munger, Karl

Human papillomaviruses are small DNA viruses with a tropism for squamous epithelia. A unique aspect of human papillomavirus molecular biology involves dependence on the differentiation status of the host epithelial cell to complete the viral lifecycle. A small group of these viruses are the etiologic agents of several types of human cancers, including oral and anogenital tract carcinomas. This review focuses on the basic molecular biology of human papillomaviruses. Copyright © 2016 Elsevier B.V. All rights reserved.

A candidate anti-HIV reservoir compound, auranofin, exerts a selective ‘anti-memory' effect by exploiting the baseline oxidative status of lymphocytes

PubMed Central

Chirullo, B; Sgarbanti, R; Limongi, D; Shytaj, I L; Alvarez, D; Das, B; Boe, A; DaFonseca, S; Chomont, N; Liotta, L; III Petricoin, E; Norelli, S; Pelosi, E; Garaci, E; Savarino, A; Palamara, A T

2013-01-01

Central memory (TCM) and transitional memory (TTM) CD4+ T cells are known to be the major cellular reservoirs for HIV, as these cells can harbor a transcriptionally silent form of viral DNA that is not targeted by either the immune system or current antiretroviral drug regimens. In the present study, we explored the molecular bases of the anti-HIV reservoir effects of auranofin (AF), a pro-oxidant gold-based drug and a candidate compound for a cure of AIDS. We here show that TCM and TTM lymphocytes have lower baseline antioxidant defenses as compared with their naive counterpart. These differences are mirrored by the effects exerted by AF on T-lymphocytes: AF was able to exert a pro-differentiating and pro-apoptotic effect, which was more pronounced in the memory subsets. AF induced an early activation of the p38 mitogen-activated protein kinase (p38 MAPK) followed by mitochondrial depolarization and a final burst in intracellular peroxides. The pro-differentiating effect was characterized by a downregulation of the CD27 marker expression. Interestingly, AF-induced apoptosis was inhibited by pyruvate, a well-known peroxide scavenger, but pyruvate did not inhibit the pro-differentiating effect of AF, indicating that the pro-apoptotic and pro-differentiating effects involve different pathways. In conclusion, our results demonstrate that AF selectively targets the TCM/TTM lymphocyte subsets, which encompass the HIV reservoir, by affecting redox-sensitive cell death pathways. PMID:24309931

Mammary stem cells and the differentiation hierarchy: current status and perspectives

PubMed Central

Visvader, Jane E.; Stingl, John

2014-01-01

The mammary epithelium is highly responsive to local and systemic signals, which orchestrate morphogenesis of the ductal tree during puberty and pregnancy. Based on transplantation and lineage tracing studies, a hierarchy of stem and progenitor cells has been shown to exist among the mammary epithelium. Lineage tracing has highlighted the existence of bipotent mammary stem cells (MaSCs) in situ as well as long-lived unipotent cells that drive morphogenesis and homeostasis of the ductal tree. Moreover, there is accumulating evidence for a heterogeneous MaSC compartment comprising fetal MaSCs, slow-cycling cells, and both long-term and short-term repopulating cells. In parallel, diverse luminal progenitor subtypes have been identified in mouse and human mammary tissue. Elucidation of the normal cellular hierarchy is an important step toward understanding the “cells of origin” and molecular perturbations that drive breast cancer. PMID:24888586

A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate

PubMed Central

Lozano-Velasco, Estefanía; Vallejo, Daniel; Esteban, Francisco J.; Doherty, Chris; Hernández-Torres, Francisco; Franco, Diego

2015-01-01

The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the transcription factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5+ satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine. PMID:26055324

Involvement of thiol-based mechanisms in plant development.

PubMed

Rouhier, Nicolas; Cerveau, Delphine; Couturier, Jérémy; Reichheld, Jean-Philippe; Rey, Pascal

2015-08-01

Increasing knowledge has been recently gained regarding the redox regulation of plant developmental stages. The current state of knowledge concerning the involvement of glutathione, glutaredoxins and thioredoxins in plant development is reviewed. The control of the thiol redox status is mainly ensured by glutathione (GSH), a cysteine-containing tripeptide and by reductases sharing redox-active cysteines, glutaredoxins (GRXs) and thioredoxins (TRXs). Indeed, thiol groups present in many regulatory proteins and metabolic enzymes are prone to oxidation, ultimately leading to post-translational modifications such as disulfide bond formation or glutathionylation. This review focuses on the involvement of GSH, GRXs and TRXs in plant development. Recent studies showed that the proper functioning of root and shoot apical meristems depends on glutathione content and redox status, which regulate, among others, cell cycle and hormone-related processes. A critical role of GRXs in the formation of floral organs has been uncovered, likely through the redox regulation of TGA transcription factor activity. TRXs fulfill many functions in plant development via the regulation of embryo formation, the control of cell-to-cell communication, the mobilization of seed reserves, the biogenesis of chloroplastic structures, the metabolism of carbon and the maintenance of cell redox homeostasis. This review also highlights the tight relationships between thiols, hormones and carbon metabolism, allowing a proper development of plants in relation with the varying environment and the energy availability. GSH, GRXs and TRXs play key roles during the whole plant developmental cycle via their antioxidant functions and the redox-regulation of signaling pathways. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

Mitochondrial Respiratory Function Induces Endogenous Hypoxia

PubMed Central

Prior, Sara; Kim, Ara; Yoshihara, Toshitada; Tobita, Seiji; Takeuchi, Toshiyuki; Higuchi, Masahiro

2014-01-01

Hypoxia influences many key biological functions. In cancer, it is generally believed that hypoxic condition is generated deep inside the tumor because of the lack of oxygen supply. However, consumption of oxygen by cancer should be one of the key means of regulating oxygen concentration to induce hypoxia but has not been well studied. Here, we provide direct evidence of the mitochondrial role in the induction of intracellular hypoxia. We used Acetylacetonatobis [2-(2′-benzothienyl) pyridinato-kN, kC3’] iridium (III) (BTP), a novel oxygen sensor, to detect intracellular hypoxia in living cells via microscopy. The well-differentiated cancer cell lines, LNCaP and MCF-7, showed intracellular hypoxia without exogenous hypoxia in an open environment. This may be caused by high oxygen consumption, low oxygen diffusion in water, and low oxygen incorporation to the cells. In contrast, the poorly-differentiated cancer cell lines: PC-3 and MDAMB231 exhibited intracellular normoxia by low oxygen consumption. The specific complex I inhibitor, rotenone, and the reduction of mitochondrial DNA (mtDNA) content reduced intracellular hypoxia, indicating that intracellular oxygen concentration is regulated by the consumption of oxygen by mitochondria. HIF-1α was activated in endogenously hypoxic LNCaP and the activation was dependent on mitochondrial respiratory function. Intracellular hypoxic status is regulated by glucose by parabolic dose response. The low concentration of glucose (0.045 mg/ml) induced strongest intracellular hypoxia possibly because of the Crabtree effect. Addition of FCS to the media induced intracellular hypoxia in LNCaP, and this effect was partially mimicked by an androgen analog, R1881, and inhibited by the anti-androgen, flutamide. These results indicate that mitochondrial respiratory function determines intracellular hypoxic status and may regulate oxygen-dependent biological functions. PMID:24586439

Isolation and characterization of 2 new human rotator cuff and long head of biceps tendon cells possessing stem cell-like self-renewal and multipotential differentiation capacity.

PubMed

Randelli, Pietro; Conforti, Erika; Piccoli, Marco; Ragone, Vincenza; Creo, Pasquale; Cirillo, Federica; Masuzzo, Pamela; Tringali, Cristina; Cabitza, Paolo; Tettamanti, Guido; Gagliano, Nicoletta; Anastasia, Luigi

2013-07-01

Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside. Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration. Controlled laboratory study. Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties. Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types--namely, osteoblasts, adipocytes, and skeletal muscle cells. This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status. Rotator cuff and long head of the biceps tendons contain a stem cell population that can proliferate in vitro and could constitute an easily accessible stem cell source to develop novel therapies for tendon regeneration.

Lower expression of CADM1 and higher expression of MAL in Merkel cell carcinomas are associated with Merkel cell polyomavirus infection and better prognosis.

PubMed

Iwasaki, Takeshi; Matsushita, Michiko; Nonaka, Daisuke; Nagata, Keiko; Kato, Masako; Kuwamoto, Satoshi; Murakami, Ichiro; Hayashi, Kazuhiko

2016-02-01

Merkel cell carcinoma (MCC) is a clinically aggressive neuroendocrine skin cancer; 80% of the cases are associated with the Merkel cell polyomavirus (MCPyV). We previously reported that MCPyV-negative MCCs have more irregular nuclei with abundant cytoplasm and significantly unfavorable outcomes than do MCPyV-positive MCCs. These results suggest that some cell adhesion or structural stabilization molecules are differently expressed depending on MCPyV infection status. Thus, we investigated the association of prognosis or MCPyV infection status in MCCs with cell adhesion molecule 1 (CADM1)/differentially expressed in adenocarcinoma of the lung protein 1 (DAL-1)/membrane protein, palmitoylated 3 (MPP3) tripartite complex and mal T-cell differentiation protein (MAL) expression, which play important roles in cell adhesion and oncogenesis and are related to cancer outcomes in various malignancies, to elucidate the role of these molecules. We analyzed the pathological and molecular characteristics of 26 MCPyV-positive and 15 MCPyV-negative MCCs. Univariate Cox regression analysis showed that advanced age (hazard ratio [HR], 8.249; P = .007) and high CADM1 expression (HR, 5.214; P = .012) were significantly unfavorable overall survival parameters, whereas MCPyV infection (HR, 0.043, P < .001) and lower MAL expression (HR, 0.273; P = .018) were significantly favorable. On multivariate analysis, only MCPyV infection was significantly favorable for overall survival (HR, 0.04; P = .005). Hypermethylation of CADM1, DAL-1, and MAL promoters was detected in 1 of 18, 15 of 27, and 1 of 13 cases, respectively. Double immunostaining for cytokeratin 20 and CADM1, DAL-1, or MAL showed that nonneoplastic Merkel cells expressed DAL-1 and MAL but not CADM1. This study revealed that MCPyV-negative MCCs significantly expressed higher CADM1 and lower MAL than MCPyV-positive MCCs; these expression levels were markedly related to unfavorable outcomes. These data will give us important insights to develop novel molecular target therapies for MCCs. Copyright © 2015 Elsevier Inc. All rights reserved.

Real-time estimation of paracellular permeability of cerebral endothelial cells by capacitance sensor array

NASA Astrophysics Data System (ADS)

Hyun Jo, Dong; Lee, Rimi; Hyoung Kim, Jin; Oh Jun, Hyoung; Geol Lee, Tae; Hun Kim, Jeong

2015-06-01

Vascular integrity is important in maintaining homeostasis of brain microenvironments. In various brain diseases including Alzheimer’s disease, stroke, and multiple sclerosis, increased paracellular permeability due to breakdown of blood-brain barrier is linked with initiation and progression of pathological conditions. We developed a capacitance sensor array to monitor dielectric responses of cerebral endothelial cell monolayer, which could be utilized to evaluate the integrity of brain microvasculature. Our system measured real-time capacitance values which demonstrated frequency- and time-dependent variations. With the measurement of capacitance at the frequency of 100 Hz, we could differentiate the effects of vascular endothelial growth factor (VEGF), a representative permeability-inducing factor, on endothelial cells and quantitatively analyse the normalized values. Interestingly, we showed differential capacitance values according to the status of endothelial cell monolayer, confluent or sparse, evidencing that the integrity of monolayer was associated with capacitance values. Another notable feature was that we could evaluate the expression of molecules in samples in our system with the reference of real-time capacitance values. We suggest that this dielectric spectroscopy system could be successfully implanted as a novel in vitro assay in the investigation of the roles of paracellular permeability in various brain diseases.

The DEAD-box RNA helicase Ddx39ab is essential for myocyte and lens development in zebrafish.

PubMed

Zhang, Linlin; Yang, Yuxi; Li, Beibei; Scott, Ian C; Lou, Xin

2018-04-23

RNA helicases from the DEAD-box family are found in almost all organisms and have important roles in RNA metabolism, including RNA synthesis, processing and degradation. The function and mechanism of action of most of these helicases in animal development and human disease remain largely unexplored. In a zebrafish mutagenesis screen to identify genes essential for heart development we identified a mutant that disrupts the gene encoding the RNA helicase DEAD-box 39ab ( ddx39ab ). Homozygous ddx39ab mutant embryos exhibit profound cardiac and trunk muscle dystrophy, along with lens abnormalities, caused by abrupt terminal differentiation of cardiomyocyte, myoblast and lens fiber cells. Loss of ddx39ab hindered splicing of mRNAs encoding epigenetic regulatory factors, including members of the KMT2 gene family, leading to misregulation of structural gene expression in cardiomyocyte, myoblast and lens fiber cells. Taken together, these results show that Ddx39ab plays an essential role in establishment of the proper epigenetic status during differentiation of multiple cell lineages. © 2018. Published by The Company of Biologists Ltd.

A post-transcriptional mechanism pacing expression of neural genes with precursor cell differentiation status

PubMed Central

Dai, Weijun; Li, Wencheng; Hoque, Mainul; Li, Zhuyun; Tian, Bin; Makeyev, Eugene V.

2015-01-01

Nervous system (NS) development relies on coherent upregulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3′-untranslated regions (3′ UTRs) of multiple neural transcripts contain AU-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously implicated in regulation of mRNA stability. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines in the neural lineage because of a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Importantly, TTP downregulation in this context is essential for proper neuronal differentiation. On the other hand, inactivation of TTP in non-neuronal cells leads to dramatic upregulation of multiple NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated upregulation of these transcripts in neurons. PMID:26144867

Splenic marginal zone lymphoma.

PubMed

Piris, Miguel A; Onaindía, Arantza; Mollejo, Manuela

Splenic marginal zone lymphoma (SMZL) is an indolent small B-cell lymphoma involving the spleen and bone marrow characterized by a micronodular tumoral infiltration that replaces the preexisting lymphoid follicles and shows marginal zone differentiation as a distinctive finding. SMZL cases are characterized by prominent splenomegaly and bone marrow and peripheral blood infiltration. Cells in peripheral blood show a villous cytology. Bone marrow and peripheral blood characteristic features usually allow a diagnosis of SMZL to be performed. Mutational spectrum of SMZL identifies specific findings, such as 7q loss and NOTCH2 and KLF2 mutations, both genes related with marginal zone differentiation. There is a striking clinical variability in SMZL cases, dependent of the tumoral load and performance status. Specific molecular markers such as 7q loss, p53 loss/mutation, NOTCH2 and KLF2 mutations have been found to be associated with the clinical variability. Distinction from Monoclonal B-cell lymphocytosis with marginal zone phenotype is still an open issue that requires identification of precise and specific thresholds with clinical meaning. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tissue Inhibitor of Metalloproteinases–3 Moderates the Proinflammatory Status of Macrophages

PubMed Central

Gharib, Sina A.; Bench, Eli M.; Sussman, Samuel W.; Wang, Roy T.; Rims, Cliff; Birkland, Timothy P.; Wang, Ying; Manicone, Anne M.; McGuire, John K.; Parks, William C.

2013-01-01

Tissue inhibitor of metalloproteinases–3 (TIMP-3) has emerged as a key mediator of inflammation. Recently, we reported that the resolution of inflammation is impaired in Timp3−/− mice after bleomycin-induced lung injury. Here, we demonstrate that after LPS instillation (another model of acute lung injury), Timp3−/− mice demonstrate enhanced and persistent neutrophilia, increased numbers of infiltrated macrophages, and delayed weight gain, compared with wild-type (WT) mice. Because macrophages possess broad immune functions and can differentiate into cells that either stimulate inflammation (M1 macrophages) or are immunosuppressive (M2 macrophages), we examined whether TIMP-3 influences macrophage polarization. Comparisons of the global gene expression of unstimulated or LPS-stimulated bone marrow–derived macrophages (BMDMs) from WT and Timp3−/− mice revealed that Timp3−/− BMDMs exhibited an increased expression of genes associated with proinflammatory (M1) macrophages, including Il6, Il12, Nos2, and Ccl2. Microarray analyses also revealed a baseline difference in gene expression between WT and Timp3−/− BMDMs, suggesting altered macrophage differentiation. Furthermore, the treatment of Timp3−/− BMDMs with recombinant TIMP-3 rescued this altered gene expression. We also examined macrophage function, and found that Timp3−/− M1 cells exhibit significantly more neutrophil chemotactic activity and significantly less soluble Fas ligand–induced caspase-3/7 activity, a marker of apoptosis, compared with WT M1 cells. Macrophage differentiation into immunosuppressive M2 cells is mediated by exposure to IL-4/IL-13, and we found that Timp3−/− M2 macrophages demonstrated a lower expression of genes associated with an anti-inflammatory phenotype, compared with WT M2 cells. Collectively, these findings indicate that TIMP-3 functions to moderate the differentiation of macrophages into proinflammatory (M1) cells. PMID:23742180

Squamous Cell Carcinoma Antigen-encoding Genes SERPINB3/B4 as Potentially Useful Markers for the Stratification of HNSCC Tumours.

PubMed

Saidak, Zuzana; Morisse, Mony Chenda; Chatelain, Denis; Sauzay, Chloé; Houessinon, Aline; Guilain, Nelly; Soyez, Marion; Chauffert, Bruno; Dakpé, Stéphanie; Galmiche, Antoine

2018-03-01

The squamous cell carcinoma antigen (SCCA), encoded by the genes SERPINB3/B4, is a tumour marker produced by head and neck squamous cell carcinoma (HNSCC). We aimed to examine SERPINB3/B4 mRNA levels and its clinical significance in the therapeutic context. We retrieved mRNA expression levels, clinical, pathological and genomic data for 520 HNSCC from The Cancer Genome Atlas (TCGA). HNSCC tumours express high levels of SERPINB3/B4 mRNA. SERPINB3 expression differs depending on Human papillomavirus (HPV) infection status, primary tumour location, grade and differentiation, extension to lymph nodes and extracapsular spread. Interestingly, we observed an association between SERPINB3/B4 and the presence of tumour immune infiltrate as well as the expression of the immune checkpoint regulators PD-L1/PD-L2 that depended on HPV status. Our findings point to potential interest of SERPINB3/B4 for the stratification of HNSCC patients in the therapeutic context. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Concise Review: Multifaceted Characterization of Human Mesenchymal Stem Cells for Use in Regenerative Medicine.

PubMed

Samsonraj, Rebekah M; Raghunath, Michael; Nurcombe, Victor; Hui, James H; van Wijnen, Andre J; Cool, Simon M

2017-12-01

Mesenchymal stem cells (MSC) hold great potential for regenerative medicine because of their ability for self-renewal and differentiation into tissue-specific cells such as osteoblasts, chondrocytes, and adipocytes. MSCs orchestrate tissue development, maintenance and repair, and are useful for musculoskeletal regenerative therapies to treat age-related orthopedic degenerative diseases and other clinical conditions. Importantly, MSCs produce secretory factors that play critical roles in tissue repair that support both engraftment and trophic functions (autocrine and paracrine). The development of uniform protocols for both preparation and characterization of MSCs, including standardized functional assays for evaluation of their biological potential, are critical factors contributing to their clinical utility. Quality control and release criteria for MSCs should include cell surface markers, differentiation potential, and other essential cell parameters. For example, cell surface marker profiles (surfactome), bone-forming capacities in ectopic and orthotopic models, as well as cell size and granularity, telomere length, senescence status, trophic factor secretion (secretome), and immunomodulation, should be thoroughly assessed to predict MSC utility for regenerative medicine. We propose that these and other functionalities of MSCs should be characterized prior to use in clinical applications as part of comprehensive and uniform guidelines and release criteria for their clinical-grade production to achieve predictably favorable treatment outcomes for stem cell therapy. Stem Cells Translational Medicine 2017;6:2173-2185. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Phenotypic analysis of perennial airborne allergen-specific CD4+ T cells in atopic and non-atopic individuals.

PubMed

Crack, L R; Chan, H W; McPherson, T; Ogg, G S

2011-11-01

Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4+ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4+ T cells in adult individuals with severe persistent AD in comparison with healthy controls. Using HLA class II tetrameric complexes based on a HLA-DPB1*0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. Ex vivo Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. Circulating Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals. © 2011 Blackwell Publishing Ltd.

Hyperglycemia impedes definitive endoderm differentiation of human embryonic stem cells by modulating histone methylation patterns.

PubMed

Chen, A C H; Lee, Y L; Fong, S W; Wong, C C Y; Ng, E H Y; Yeung, W S B

2017-06-01

Exposure to maternal diabetes during fetal growth is a risk factor for the development of type II diabetes (T2D) in later life. Discovery of the mechanisms involved in this association should provide valuable background for therapeutic treatments. Early embryogenesis involves epigenetic changes including histone modifications. The bivalent histone methylation marks H3K4me3 and H3K27me3 are important for regulating key developmental genes during early fetal pancreas specification. We hypothesized that maternal hyperglycemia disrupted early pancreas development through changes in histone bivalency. A human embryonic stem cell line (VAL3) was used as the cell model for studying the effects of hyperglycemia upon differentiation into definitive endoderm (DE), an early stage of the pancreatic lineage. Hyperglycemic conditions significantly down-regulated the expression levels of DE markers SOX17, FOXA2, CXCR4 and EOMES during differentiation. This was associated with retention of the repressive histone methylation mark H3K27me3 on their promoters under hyperglycemic conditions. The disruption of histone methylation patterns was observed as early as the mesendoderm stage, with Wnt/β-catenin signaling being suppressed during hyperglycemia. Treatment with Wnt/β-catenin signaling activator CHIR-99021 restored the expression levels and chromatin methylation status of DE markers, even in a hyperglycemic environment. The disruption of DE development was also found in mouse embryos at day 7.5 post coitum from diabetic mothers. Furthermore, disruption of DE differentiation in VAL3 cells led to subsequent impairment in pancreatic progenitor formation. Thus, early exposure to hyperglycemic conditions hinders DE development with a possible relationship to the later impairment of pancreas specification.

Fructose-Bisphosphate Aldolase A Is a Potential Metastasis-Associated Marker of Lung Squamous Cell Carcinoma and Promotes Lung Cell Tumorigenesis and Migration

PubMed Central

Hao, Lihong; Song, Yang; Wang, Lan; Gong, Linlin; Liu, Lu; Qi, Xiaoyu; Hou, Zhaoyuan; Shao, Shujuan

2014-01-01

Fructose-bisphosphate aldolase A (ALDOA) is a key enzyme in glycolysis and is responsible for catalyzing the reversible conversion of fructose-1,6-bisphosphate to glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. ALDOA contributes to various cellular functions such as muscle maintenance, regulation of cell shape and mobility, striated muscle contraction, actin filament organization and ATP biosynthetic process. Here, we reported that ALDOA is a highly expressed in lung squamous cell carcinoma (LSCC) and its expression level is correlated with LSCC metastasis, grades, differentiation status and poor prognosis. Depletion of ALDOA expression in the lung squamous carcinoma NCI-H520 cells reduces the capabilities of cell motility and tumorigenesis. These data suggest that ALDOA could be a potential marker for LSCC metastasis and a therapeutic target for drug development. PMID:24465716

SCAPs Regulate Differentiation of DFSCs During Tooth Root Development in Swine

PubMed Central

Wu, Xiaoshan; Hu, Lei; Li, Yan; Li, Yang; Wang, Fu; Ma, Ping; Wang, Jinsong; Zhang, Chunmei; Jiang, Canhua; Wang, Songlin

2018-01-01

The tooth root transmits and balances occlusal forces through the periodontium to the alveolar bone. The periodontium, including the gingiva, the periodontal ligament, the cementum and the partial alveolar bone, derives from the dental follicle (DF), except for the gingiva. In the early developmental stages, the DF surrounds the tooth germ as a sphere and functions to promote tooth eruption. However, the morphological dynamics and factors regulating the differentiation of the DF during root elongation remain largely unknown. Miniature pigs are regarded as a useful experimental animal for modeling in craniofacial research because they are similar to humans with respect to dentition and mandible anatomy. In the present study, we used the third deciduous incisor of miniature pig as the model to investigate the factors influencing DF differentiation during root development. We found that the DF was shaped like a crescent and was located between the root apical and the alveolar bone. The expression levels of WNT5a, β-Catenin, and COL-I gradually increased from the center of the DF (beneath the apical foramen) to the lateral coronal corner, where the DF differentiates into the periodontium. To determine the potential regulatory role of the apical papilla on DF cell differentiation, we co-cultured dental follicle stem cells (DFSCs) with stem cells of the apical papilla (SCAPs). The osteogenesis and fibrogenesis abilities of DFSCs were inhibited when being co-cultured with SCAPs, suggesting that the fate of the DF can be regulated by signals from the apical papilla. The apical papilla may sustain the undifferentiated status of DFSCs before root development finishes. These data yield insight into the interaction between the root apex and surrounding DF tissues in root and periodontium development and shed light on the future study of root regeneration in large mammals. PMID:29511365

Differential Reprogramming of Isogenic Colorectal Cancer Cells by Distinct Activating KRAS Mutations

PubMed Central

2015-01-01

Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS. PMID:25599653

Physical Forces Modulate Oxidative Status and Stress Defense Meditated Metabolic Adaptation of Yeast Colonies: Spaceflight and Microgravity Simulations

NASA Astrophysics Data System (ADS)

Hammond, Timothy G.; Allen, Patricia L.; Gunter, Margaret A.; Chiang, Jennifer; Giaever, Guri; Nislow, Corey; Birdsall, Holly H.

2018-05-01

Baker's yeast ( Saccharomyces cerevisiae) has broad genetic homology to human cells. Although typically grown as 1-2mm diameter colonies under certain conditions yeast can form very large (10 + mm in diameter) or `giant' colonies on agar. Giant yeast colonies have been used to study diverse biomedical processes such as cell survival, aging, and the response to cancer pharmacogenomics. Such colonies evolve dynamically into complex stratified structures that respond differentially to environmental cues. Ammonia production, gravity driven ammonia convection, and shear defense responses are key differentiation signals for cell death and reactive oxygen system pathways in these colonies. The response to these signals can be modulated by experimental interventions such as agar composition, gene deletion and application of pharmaceuticals. In this study we used physical factors including colony rotation and microgravity to modify ammonia convection and shear stress as environmental cues and observed differences in the responses of both ammonia dependent and stress response dependent pathways We found that the effects of random positioning are distinct from rotation. Furthermore, both true and simulated microgravity exacerbated both cellular redox responses and apoptosis. These changes were largely shear-response dependent but each model had a unique response signature as measured by shear stress genes and the promoter set which regulates them These physical techniques permitted a graded manipulation of both convection and ammonia signaling and are primed to substantially contribute to our understanding of the mechanisms of drug action, cell aging, and colony differentiation.

Discrimination of Stem Cell Status after Subjecting Cynomolgus Monkey Pluripotent Stem Cells to Naïve Conversion

PubMed Central

Honda, Arata; Kawano, Yoshihiro; Izu, Haruna; Choijookhuu, Narantsog; Honsho, Kimiko; Nakamura, Tomonori; Yabuta, Yukihiro; Yamamoto, Takuya; Takashima, Yasuhiro; Hirose, Michiko; Sankai, Tadashi; Hishikawa, Yoshitaka; Ogura, Atsuo; Saitou, Mitinori

2017-01-01

Experimental animal models have played an indispensable role in the development of human induced pluripotent stem cell (iPSC) research. The derivation of high-quality (so-called “true naïve state”) iPSCs of non-human primates enhances their application and safety for human regenerative medicine. Although several attempts have been made to convert human and non-human primate PSCs into a truly naïve state, it is unclear which evaluation methods can discriminate them as being truly naïve. Here we attempted to derive naïve cynomolgus monkey (Cm) (Macaca fascicularis) embryonic stem cells (ESCs) and iPSCs. Several characteristics of naïve Cm ESCs including colony morphology, appearance of naïve-related mRNAs and proteins, leukaemia inhibitory factor dependency, and mitochondrial respiration were confirmed. Next, we generated Cm iPSCs and converted them to a naïve state. Transcriptomic comparison of PSCs with early Cm embryos elucidated the partial achievement (termed naïve-like) of their conversion. When these were subjected to in vitro neural differentiation, enhanced differentiating capacities were observed after naïve-like conversion, but some lines exhibited heterogeneity. The difficulty of achieving contribution to chimeric mouse embryos was also demonstrated. These results suggest that Cm PSCs could ameliorate their in vitro neural differentiation potential even though they could not display true naïve characteristics. PMID:28349944

Physical Forces Modulate Oxidative Status and Stress Defense Meditated Metabolic Adaptation of Yeast Colonies: Spaceflight and Microgravity Simulations

NASA Astrophysics Data System (ADS)

Hammond, Timothy G.; Allen, Patricia L.; Gunter, Margaret A.; Chiang, Jennifer; Giaever, Guri; Nislow, Corey; Birdsall, Holly H.

2017-12-01

Baker's yeast (Saccharomyces cerevisiae) has broad genetic homology to human cells. Although typically grown as 1-2mm diameter colonies under certain conditions yeast can form very large (10 + mm in diameter) or `giant' colonies on agar. Giant yeast colonies have been used to study diverse biomedical processes such as cell survival, aging, and the response to cancer pharmacogenomics. Such colonies evolve dynamically into complex stratified structures that respond differentially to environmental cues. Ammonia production, gravity driven ammonia convection, and shear defense responses are key differentiation signals for cell death and reactive oxygen system pathways in these colonies. The response to these signals can be modulated by experimental interventions such as agar composition, gene deletion and application of pharmaceuticals. In this study we used physical factors including colony rotation and microgravity to modify ammonia convection and shear stress as environmental cues and observed differences in the responses of both ammonia dependent and stress response dependent pathways We found that the effects of random positioning are distinct from rotation. Furthermore, both true and simulated microgravity exacerbated both cellular redox responses and apoptosis. These changes were largely shear-response dependent but each model had a unique response signature as measured by shear stress genes and the promoter set which regulates them These physical techniques permitted a graded manipulation of both convection and ammonia signaling and are primed to substantially contribute to our understanding of the mechanisms of drug action, cell aging, and colony differentiation.

Development of the terminally differentiated state sensitizes epiphyseal chondrocytes to apoptosis through caspase-3 activation.

PubMed

Pucci, Bruna; Adams, Christopher S; Fertala, Jolanta; Snyder, Bradley C; Mansfield, Kyle D; Tafani, Marco; Freeman, Theresa; Shapiro, Irving M

2007-03-01

The maturation of epiphyseal chondrocytes is accompanied by dramatic changes in energy metabolism and shifts in proteins concerned with the induction of apoptosis. We evaluated the role of mitochondria in this process by evaluating the membrane potential (Delta psi m) of chondrocytes of embryonic tibia and the epiphyseal growth plate. We observed that there was a maturation-dependent change in fluorescence, indicating a fall in the Delta psi m. The level of mitochondrial Bcl-2 was decreased during maturation, while in the same time period there was an obvious increase in Bax levels in the mitochondrial fraction of the terminally differentiated chondrocytes. Bcl(xL), another anti-apoptotic protein, was also robustly expressed in the mitochondrial fraction, but its expression was not dependent on the maturation status of the chondrocytes. We found that caspase-3 was present throughout the growth plate and in hypertrophic cells in culture. We blocked caspase-3 activity and found that alkaline phosphatase staining and mineral formation was decreased, and the cells had lost their characteristic shape. Moreover, we noted that the undifferentiated cells were insensitive to elevated concentrations of inorganic phosphate (Pi). It is concluded that during hypertrophy, the change in membrane potential, the increased binding of a pro-apoptotic protein to mitochondria, and the activation of caspase-3 serve to prime cells for apoptosis. Only when the terminally differentiated chondrocytes are challenged with low levels of apoptogens there is activation of apoptosis. Copyright 2006 Wiley-Liss, Inc.

Complete response in 5 out of 38 patients with advanced hepatocellular carcinoma treated with stem cell differentiation stage factors: case reports from a single centre.

PubMed

Livraghi, Tito; Ceriani, R; Palmisano, A; Pedicini, V; Pich, M G; Tommasini, M A; Torzilli, G

2011-02-01

Hepatocellular carcinoma (HCC) represents the third cause of cancer-related death. Because HCC is multi-centric with time, excluding the few transplanted patients, sooner or later it becomes untreatable with loco-regional therapies and, until some years ago, it was not responsive to systemic therapies. In 2005 a randomized trial indicated the efficacy of a product containing stem cell differentiation stage factors (SCDSF) taken from zebra fish embryos during the stage in which the totipotent stem cells are differentiating into the pluripotent adult stem cells. In such a trial the patients, with "intermediate" and "advanced" HCC according to BCLC/AASLD guidelines, presented benefit in terms of performance status (PS) and objective tumoral response, with some cases (2.4%) of complete response (CR). The aim of this cohort study is to report the experience of a tertiary referral center on the evidence of cases of CR in patients with "advanced" stage HCC treated with SCDSF as supportive care. CR was regarded as sustained disappearance of the neoplastic areas or blood supply therein, accompanied by normalization of AFP levels. Out of 49 patients consecutively recruited and retrospectively evaluated, 38 had "advanced" stage and 11 "terminal" stage. In 5 patients with "advanced" stage a sustained CR was reported (13.1%). Improvement on PS was obtained in 17 patients (34.6%). No side effects occurred. SCDSF treatment confirmed its efficacy in patients with "advanced" HCC, in terms of PS and tumoral response.

Immune status of free-ranging green turtles with fibropapillomatosis from Hawaii

USGS Publications Warehouse

Work, Thierry M.; Rameyer, Robert; Balazs, George H.; Cray, Carolyn; Chang, Sandra P.

2001-01-01

Cell-mediated and humoral immune status of free-ranging green turtles (Chelonia mydas) in Hawaii (USA) with and without fibropapillornatosis (FP) were assessed. Tumored and non-tumored turtles from Kaneohe Bay (KB) on the island of Oahu and from FP-free areas on the west (Kona/Kohala) coast of the island of Hawaii were sampled from April 1998 through February 1999. Turtles on Oahu were grouped (0-3) for severity of tumors with 0 for absence of tumors, 1 for light, 2 for moderate, and 3 for most severe. Turtles were weighed, straight carapace length measured and the regression slope of weight to straight carapace length compared between groups (KB0, KB1, KB2, KB3, Kona). Blood was assayed for differential white blood cell count, hematocrit, in vitro peripheral blood mononuclear cell (PBMC) proliferation in the presence of concanavalin A (ConA) and phytohaemagglutinin (PHA), and protein electrophoresis. On Oahu, heterophil/lymphocyte ratio increased while eosinophil/monocyte ratio decreased with increasing tumors score. Peripheral blood mononuclear cell proliferation indices for ConA and PHA were significantly lower for turtles with tumor scores 2 and 3. Tumor score 3 turtles (KB3) had significantly lower hematocrit, total protein, alpha 1, alpha 2, and gamma globulins than the other four groups. No significant differences in immune status were seen between non-tumored (or KB1) turtles from Oahu and Hawaii. There was no significant difference between groups in regression slopes of body condition to carapace length. We conclude that turtles with severe FP are imunosuppressed. Furthermore, the lack of significant difference in immune status between non-tumored (and KB1) turtles from Oahu and Kona/Kohala indicates that immunosuppression may not be a prerequisite for development of FP.

Periodontal Ligament Stem Cells: Current Status, Concerns, and Future Prospects

PubMed Central

Zhu, Wenjun; Liang, Min

2015-01-01

Periodontal ligament stem cells (PDLSCs), which reside in the perivascular space of the periodontium, possess characteristics of mesenchymal stem cells and are a promising tool for periodontal regeneration. Recently, great progress has been made in PDLSC transplantation. Investigators are attempting to maximize the proliferation and differentiation potential of PDLSCs by modifying culture conditions and applying growth factors. Nevertheless, problems remain. First, incomparability among different studies must be minimized by establishing standard guidelines for culture and identification of PDLSCs. Notably, attention should be paid to the biological safety of PDLSC transplantation. The present review updates the latest findings regarding PDLSCs and discusses standard criteria for culture and identification of PDLSCs. Finally, the review calls for careful consideration of PDLSC transplantation safety. PMID:25861283

Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma.

PubMed

Dumble, Melissa L; Croager, Emma J; Yeoh, George C T; Quail, Elizabeth A

2002-03-01

Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.

Functional Definition of Progenitors Versus Mature Endothelial Cells Reveals Key SoxF-Dependent Differentiation Process.

PubMed

Patel, Jatin; Seppanen, Elke J; Rodero, Mathieu P; Wong, Ho Yi; Donovan, Prudence; Neufeld, Zoltan; Fisk, Nicholas M; Francois, Mathias; Khosrotehrani, Kiarash

2017-02-21

During adult life, blood vessel formation is thought to occur via angiogenic processes involving branching from existing vessels. An alternate proposal suggests that neovessels form from endothelial progenitors able to assemble the intimal layers. We here aimed to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological and pathological situations such as normal aorta, lungs, and wound healing, tumors, and placenta, as well. Based on protein expression levels of common endothelial markers using flow cytometry, 3 subpopulations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells. Lineage tracing by using Cdh5cre ERt2 /Rosa-YFP reporter strategy demonstrated that the CD31-/loVEGFR2lo/intracellular endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2lo/intracellular transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi/extracellular population. EVP cells arose from vascular-resident beds that could not be transferred by bone marrow transplantation. Furthermore, EVP displayed progenitor-like status with a high proportion of cells in a quiescent cell cycle phase as assessed in wounds, tumors, and aorta. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by colony-forming capacity in limiting dilution and by transplantation in Matrigel plugs in recipient mice. RNA sequencing revealed prominent gene expression differences between EVP and D cells. In particular, EVP cells highly expressed genes related to progenitor function including Sox9 , Il33 , Egfr , and Pdfgrα. Conversely, D cells highly expressed genes related to differentiated endothelium including Ets1&2 , Gata2 , Cd31 , Vwf , and Notch . The RNA sequencing also pointed to an essential role of the Sox18 transcription factor. The role of SOX18 in the differentiation process was validated by using lineage-tracing experiments based on S ox18Cre ERt2 /Rosa-YFP mice. Besides, in the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced. Our findings support an entirely novel endothelial hierarchy, from EVP to TA to D, as defined by self-renewal, differentiation, and molecular profiling of an endothelial progenitor. This paradigm shift in our understanding of vascular-resident endothelial progenitors in tissue regeneration opens new avenues for better understanding of cardiovascular biology. © 2016 American Heart Association, Inc.

Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability.

PubMed

Lovelock, Paul K; Wong, Ee Ming; Sprung, Carl N; Marsh, Anna; Hobson, Karen; French, Juliet D; Southey, Melissa; Sculley, Tom; Pandeya, Nirmala; Brown, Melissa A; Chenevix-Trench, Georgia; Spurdle, Amanda B; McKay, Michael J

2007-09-01

Assays to determine the pathogenicity of unclassified sequence variants in disease-associated genes include the analysis of lymphoblastoid cell lines (LCLs). We assessed the ability of several assays of LCLs to distinguish carriers of germline BRCA1 and BRCA2 gene mutations from mutation-negative controls to determine their utility for use in a diagnostic setting. Post-ionising radiation cell viability and micronucleus formation, and telomere length were assayed in LCLs carrying BRCA1 or BRCA2 mutations, and in unaffected mutation-negative controls. Post-irradiation cell viability and micronucleus induction assays of LCLs from individuals carrying pathogenic BRCA1 mutations, unclassified BRCA1 sequence variants or wildtype BRCA1 sequence showed significant phenotypic heterogeneity within each group. Responses were not consistent with predicted functional consequences of known pathogenic or normal sequences. Telomere length was also highly heterogeneous within groups of LCLs carrying pathogenic BRCA1 or BRCA2 mutations, and normal BRCA1 sequences, and was not predictive of mutation status. Given the significant degree of phenotypic heterogeneity of LCLs after gamma-irradiation, and the lack of association with BRCA1 or BRCA2 mutation status, we conclude that the assays evaluated in this study should not be used as a means of differentiating pathogenic and non-pathogenic sequence variants for clinical application. We suggest that a range of normal controls must be included in any functional assays of LCLs to ensure that any observed differences between samples reflect the genotype under investigation rather than generic inter-individual variation.

Circulating lymphocyte levels and relationship with infection status in patients with relapsing-remitting multiple sclerosis treated with daclizumab beta.

PubMed

Giovannoni, Gavin; Wiendl, Heinz; Turner, Benjamin; Umans, Kimberly; Mokliatchouk, Oksana; Castro-Borrero, Wanda; Greenberg, Steven J; McCroskery, Peter; Giannattasio, Giorgio

2017-09-01

Reversible lymphocyte count reductions have occurred following daclizumab beta treatment for relapsing-remitting multiple sclerosis. To analyse total and differential lymphocyte levels and relationship with infection status. In DECIDE, blood samples were collected at 12-week intervals from daclizumab beta- ( n = 919) or intramuscular interferon beta-1a-treated ( n = 922) patients. Infections/serious infections were assessed proximate to grade 2/3 lymphopenia or low CD4 + /CD8 + T-cell counts. Total safety population (TSP) data were additionally analysed from the entire clinical development programme ( n = 2236). Over 96 weeks in DECIDE, mean absolute lymphocyte count (ALC), CD4 + and CD8 + T-cell counts decreased <10% (7.1% vs 1.6%, 9.7% vs 2.0%, 9.3% vs 5.9%: daclizumab beta vs interferon beta-1a, respectively); shifts to ALC below lower limit of normal occurred in 13% versus 15%, respectively. Grade 3 lymphopenia was uncommon (TSP: <1%) and transient. Lymphocyte changes generally occurred within 24 weeks after treatment initiation and were reversible within 12 weeks of discontinuation. In DECIDE, mean CD4 + /CD8 + T-cell counts were similar regardless of infection status. TSP data were consistent with DECIDE. When observed, ALC and CD4 + /CD8 + T-cell count decreases in daclizumab beta-treated patients were generally mild-to-modest, reversible upon treatment discontinuation and not associated with increased risk of infections, including opportunistic infections.

On the role of transforming growth factor-beta in the growth inhibitory effects of retinoic acid in human pancreatic cancer cells.

PubMed

Singh, Brahmchetna; Murphy, Richard F; Ding, Xian-Zhong; Roginsky, Alexandra B; Bell, Richard H; Adrian, Thomas E

2007-12-24

Retinoids are potent growth inhibitory and differentiating agents in a variety of cancer cell types. We have shown that retinoids induce growth arrest in all pancreatic cancer cell lines studied, regardless of their p53 and differentiation status. However, the mechanism of growth inhibition is not known. Since TGF-beta2 is markedly induced by retinoids in other cancers and mediates MUC4 expression in pancreatic cancer cells, we investigated the role of TGF-beta in retinoic acid-mediated growth inhibition in pancreatic cancer cells. Retinoic acid markedly inhibited proliferation of two cell lines (Capan-2 and Hs766T) in a concentration and time-dependent manner. Retinoic acid increased TGF-beta2 mRNA content and secretion of the active and latent forms of TGF-beta2 (measured by ELISA and bioassay). The concentrations of active and TGF-beta2 secreted in response to 0.1 - 10 muM retinoic acid were between 1-5 pM. TGF-beta2 concentrations within this range also inhibited proliferation. A TGF-beta neutralizing antibody blocked the growth inhibitory effects of retinoic acid in Capan-2 cells and partially inhibitory the effects in Hs766T cells. These findings indicate that TGF-beta can cause growth inhibition of pancreatic cancer cells, in a p53-independent manner. Furthermore, it demonstrates the fundamental role of TGF-beta in growth inhibition in response to retinoic acid treatment is preserved in vitro.

Pregnancy persistently affects memory T cell populations.

PubMed

Kieffer, Tom E C; Faas, Marijke M; Scherjon, Sicco A; Prins, Jelmer R

2017-02-01

Pregnancy is an immune challenge to the maternal immune system. The effects of pregnancy on maternal immunity and particularly on memory T cells during and after pregnancy are not fully known. This observational study aims to show the short term and the long term effects of pregnancy on the constitution, size and activation status of peripheral human memory T-lymphocyte populations. Effector memory (EM) and central memory (CM) T-lymphocytes were analyzed using flow cytometry of peripheral blood from 14 nulligravid, 12 primigravid and 15 parous women that were on average 18 months postpartum. The short term effects were shown by the significantly higher CD4+ EM cell and activated CD4+ memory cell proportions in primigravid women compared to nulligravid women. The persistent effects found in this study were the significantly higher proportions of CD4+ EM, CD4+ CM and activated memory T cells in parous women compared to nulligravid women. In contrast to CD4+ cells, activation status of CD8+ memory cells did not differ between the groups. This study shows that pregnancy persistently affects the pre-pregnancy CD4+ memory cell pool in human peripheral blood. During pregnancy, CD4+ T-lymphocytes might differentiate into EM cells followed by persistent higher proportions of CD4+ CM and EM cells postpartum. The persistent effects of pregnancy on memory T cells found in this study support the hypothesis that memory T cells are generated during pregnancy and that these cells could be involved in the lower complication risks in multiparous pregnancies in humans. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Human Decidua-Derived Mesenchymal Cells Are a Promising Source for the Generation and Cell Banking of Human Induced Pluripotent Stem Cells

PubMed Central

Shofuda, Tomoko; Kanematsu, Daisuke; Fukusumi, Hayato; Yamamoto, Atsuyo; Bamba, Yohei; Yoshitatsu, Sumiko; Suemizu, Hiroshi; Nakamura, Masato; Sugimoto, Yoshikazu; Furue, Miho Kusuda; Kohara, Arihiro; Akamatsu, Wado; Okada, Yohei; Okano, Hideyuki; Yamasaki, Mami; Kanemura, Yonehiro

2013-01-01

Placental tissue is a biomaterial with remarkable potential for use in regenerative medicine. It has a three-layer structure derived from the fetus (amnion and chorion) and the mother (decidua), and it contains huge numbers of cells. Moreover, placental tissue can be collected without any physical danger to the donor and can be matched with a variety of HLA types. The decidua-derived mesenchymal cells (DMCs) are highly proliferative fibroblast-like cells that express a similar pattern of CD antigens as bone marrow-derived mesenchymal cells (BM-MSCs). Here we demonstrated that induced pluripotent stem (iPS) cells could be efficiently generated from DMCs by retroviral transfer of reprogramming factor genes. DMC-hiPS cells showed equivalent characteristics to human embryonic stem cells (hESCs) in colony morphology, global gene expression profile (including human pluripotent stem cell markers), DNA methylation status of the OCT3/4 and NANOG promoters, and ability to differentiate into components of the three germ layers in vitro and in vivo. The RNA expression of XIST and the methylation status of its promoter region suggested that DMC-iPSCs, when maintained undifferentiated and pluripotent, had three distinct states: (1) complete X-chromosome reactivation, (2) one inactive X-chromosome, or (3) an epigenetic aberration. Because DMCs are derived from the maternal portion of the placenta, they can be collected with the full consent of the adult donor and have considerable ethical advantages for cell banking and the subsequent generation of human iPS cells for regenerative applications. PMID:26858858

Malat1 as an evolutionarily conserved lncRNA, plays a positive role in regulating proliferation and maintaining undifferentiated status of early-stage hematopoietic cells.

PubMed

Ma, Xian-Yong; Wang, Jian-Hui; Wang, Jing-Lan; Ma, Charles X; Wang, Xiao-Chun; Liu, Feng-Song

2015-09-03

The metastasis-associated lung adenocarcinoma transcription 1 (Malat1) is a highly conserved long non-coding RNA (lncRNA) gene. Previous studies showed that Malat1 is abundantly expressed in many tissues and involves in promoting tumor growth and metastasis by modulating gene expression and target protein activities. However, little is known about the biological function and regulation mechanism of Malat1 in normal cell proliferation. In this study we conformed that Malat1 is highly conserved across vast evolutionary distances amongst 20 species of mammals in terms of sequence, and found that mouse Malat1 expresses in tissues of liver, kidney, lung, heart, testis, spleen and brain, but not in skeletal muscle. After treating erythroid myeloid lymphoid (EML) cells with All-trans Retinoic Acid (ATRA), we investigated the expression and regulation of Malat1 during hematopoietic differentiation, the results showed that ATRA significantly down regulates Malat1 expression during the differentiation of EML cells. Mouse LRH (Lin-Rhodamine(low) Hoechst(low)) cells that represent the early-stage progenitor cells show a high level of Malat1 expression, while LRB (Lin - Hoechst(Low) Rhodamine(Bright)) cells that represent the late-stage progenitor cells had no detectable expression of Malat1. Knockdown experiment showed that depletion of Malat1 inhibits the EML cell proliferation. Along with the down regulation of Malat1, the tumor suppressor gene p53 was up regulated during the differentiation. Interestingly, we found two p53 binding motifs with help of bioinformatic tools, and the following chromatin immunoprecipitation (ChIP) test conformed that p53 acts as a transcription repressor that binds to Malat1's promoter. Furthermore, we testified that p53 over expression in EML cells causes down regulation of Malat1. In summary, this study indicates Malat1 plays a critical role in maintaining the proliferation potential of early-stage hematopoietic cells. In addition to its biological function, the study also uncovers the regulation pattern of Malat1 expression mediated by p53 in hematopoietic differentiation. Our research shed a light on exploring the Malat1 biological role including therapeutic significance to inhibit the proliferation potential of malignant cells.

The polymethoxy flavonoid sudachitin suppresses inflammatory bone destruction by directly inhibiting osteoclastogenesis due to reduced ROS production and MAPK activation in osteoclast precursors

PubMed Central

Kitano, Victor J.; Shimada, Jun

2018-01-01

Inflammatory bone diseases, including rheumatoid arthritis, periodontitis and peri-implantitis, are associated not only with the production of inflammatory cytokines but also with local oxidative status, which is defined by intracellular reactive oxygen species (ROS). Osteoclast differentiation has been reported to be related to increased intracellular ROS levels in osteoclast lineage cells. Sudachitin, which is a polymethoxyflavone derived from Citrus sudachi, possesses antioxidant properties and regulates various functions in mammalian cells. However, the effects of sudachitin on inflammatory bone destruction and osteoclastogenesis remain unknown. In calvaria inflamed by a local lipopolysaccharide (LPS) injection, inflammation-induced bone destruction and the accompanying elevated expression of osteoclastogenesis-related genes were reduced by the co-administration of sudachitin and LPS. Moreover, sudachitin inhibited osteoclast formation in cultures of isolated osteoblasts and osteoclast precursors. However, sudachitin rather increased the expression of receptor activator of NF-κB ligand (RANKL), which is an important molecule triggering osteoclast differentiation, and the mRNA ratio of RANKL/osteoprotegerin that is a decoy receptor for RANKL, in the isolated osteoblasts, suggesting the presence of additional target cells. When osteoclast formation was induced from osteoclast precursors derived from bone marrow cells in the presence of soluble RANKL and macrophage colony-stimulating factor, sudachitin inhibited osteoclastogenesis without influencing cell viability. Consistently, the expression of osteoclast differentiation-related molecules including c-fos, NFATc1, cathepsin K and osteoclast fusion proteins such as DC-STAMP and Atp6v0d2 was reduced by sudachitin. In addition, sudachitin decreased activation of MAPKs such as Erk and JNK and the ROS production evoked by RANKL in osteoclast lineage cells. Our findings suggest that sudachitin is a useful agent for the treatment of anti-inflammatory bone destruction. PMID:29342179

The polymethoxy flavonoid sudachitin suppresses inflammatory bone destruction by directly inhibiting osteoclastogenesis due to reduced ROS production and MAPK activation in osteoclast precursors.

PubMed

Ohyama, Yoko; Ito, Junta; Kitano, Victor J; Shimada, Jun; Hakeda, Yoshiyuki

2018-01-01

Inflammatory bone diseases, including rheumatoid arthritis, periodontitis and peri-implantitis, are associated not only with the production of inflammatory cytokines but also with local oxidative status, which is defined by intracellular reactive oxygen species (ROS). Osteoclast differentiation has been reported to be related to increased intracellular ROS levels in osteoclast lineage cells. Sudachitin, which is a polymethoxyflavone derived from Citrus sudachi, possesses antioxidant properties and regulates various functions in mammalian cells. However, the effects of sudachitin on inflammatory bone destruction and osteoclastogenesis remain unknown. In calvaria inflamed by a local lipopolysaccharide (LPS) injection, inflammation-induced bone destruction and the accompanying elevated expression of osteoclastogenesis-related genes were reduced by the co-administration of sudachitin and LPS. Moreover, sudachitin inhibited osteoclast formation in cultures of isolated osteoblasts and osteoclast precursors. However, sudachitin rather increased the expression of receptor activator of NF-κB ligand (RANKL), which is an important molecule triggering osteoclast differentiation, and the mRNA ratio of RANKL/osteoprotegerin that is a decoy receptor for RANKL, in the isolated osteoblasts, suggesting the presence of additional target cells. When osteoclast formation was induced from osteoclast precursors derived from bone marrow cells in the presence of soluble RANKL and macrophage colony-stimulating factor, sudachitin inhibited osteoclastogenesis without influencing cell viability. Consistently, the expression of osteoclast differentiation-related molecules including c-fos, NFATc1, cathepsin K and osteoclast fusion proteins such as DC-STAMP and Atp6v0d2 was reduced by sudachitin. In addition, sudachitin decreased activation of MAPKs such as Erk and JNK and the ROS production evoked by RANKL in osteoclast lineage cells. Our findings suggest that sudachitin is a useful agent for the treatment of anti-inflammatory bone destruction.

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

PubMed

Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

2006-04-01

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

Heterogeneity, histological features and DNA ploidy in oral carcinoma by image-based analysis.

PubMed

Diwakar, N; Sperandio, M; Sherriff, M; Brown, A; Odell, E W

2005-04-01

Oral squamous carcinomas appear heterogeneous on DNA ploidy analysis. However, this may be partly a result of sample dilution or the detection limit of techniques. The aim of this study was to determine whether oral squamous carcinomas are heterogeneous for ploidy status using image-based ploidy analysis and to determine whether ploidy status correlates with histological parameters. Multiple samples from 42 oral squamous carcinomas were analysed for DNA ploidy using an image-based system and scored for histological parameters. 22 were uniformly aneuploid, 1 uniformly tetraploid and 3 uniformly diploid. 16 appeared heterogeneous but only 8 appeared to be genuinely heterogeneous when minor ploidy histogram peaks were taken into account. Ploidy was closely related to nuclear pleomorphism but not differentiation. Sample variation, detection limits and diagnostic criteria account for much of the ploidy heterogeneity observed. Confident diagnosis of diploid status in an oral squamous cell carcinoma requires a minimum of 5 samples.

miR-300 mediates Bmi1 function and regulates differentiation in primitive cardiac progenitors

PubMed Central

Cruz, F M; Tomé, M; Bernal, J A; Bernad, A

2015-01-01

B lymphoma Mo-MLV insertion region 1 (Bmi1) is a polycomb-family transcriptional factor critical for self-renewal in many adult stem cells and human neoplasia. We sought to identify microRNAs regulated by Bmi1 that could play a role in multipotent cardiac progenitor cell (CPC) decisions. We found that miR-300, a poorly characterized microRNA mapping in the Dlk1-Dio3 microRNA cluster, was positively regulated by Bmi1 in CPCs. Forced expression of miR-300 in CPCs promoted an improved stemness signature with a significant increase in Oct4 levels, a reduction in senescence progression and an enhanced proliferative status via p19 activation and inhibition of p16 accumulation. Endothelial and cardiogenic differentiation were clearly compromised by sustained miR-300 expression. Additionally, RNA and protein analysis revealed a significant reduction in key cardiac transcription factors, including Nkx2.5 and Tbx5. Collectively, these results suggest that some functions attributed to Bmi1 are due to induction of miR-300, which decreases the cardiogenic differentiation potential of multipotent CPCs in vitro and promotes self-renewal. PMID:26512961

Generation of a TALEN-mediated, p63 knock-in in human induced pluripotent stem cells.

PubMed

Kobayashi, Yuki; Hayashi, Ryuhei; Quantock, Andrew J; Nishida, Kohji

2017-12-01

The expression of p63 in surface ectodermal cells during development of the cornea, skin, oral mucosa and olfactory placodes is integral to the process of cellular self-renewal and the maintenance of the epithelial stem cell status. Here, we used TALEN technology to generate a p63 knock-in (KI) human induced pluripotent stem (hiPS) cell line in which p63 expression can be visualized via enhanced green fluorescent protein (EGFP) expression. The KI-hiPS cells maintained pluripotency and expressed the stem cell marker gene, ΔNp63α. They were also able to successfully differentiate into functional corneal epithelial cells as assessed by p63 expression in reconstructed corneal epithelium. This approach enables the tracing of p63-expressing cell lineages throughout epithelial development, and represents a promising application in the field of stem cell research. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Looking Out From the Top: Differential Effects of Status and Power on Perspective Taking.

PubMed

Blader, Steven L; Shirako, Aiwa; Chen, Ya-Ru

2016-06-01

The impact of hierarchical rank on perspective taking is both practically and theoretically important, prompting considerable research attention to this issue. However, prior research has primarily examined how power affects perspective taking, and has neglected to investigate the impact of status (i.e., the respect and esteem that an individual holds in the eyes of others). Yet status represents a distinct and ubiquitous basis of hierarchical differentiation, one that may profoundly affect perspective taking. The current research addresses this gap, theorizing and testing the prediction that high status enhances perspective taking, in contrast to prior research that has generally found that high power diminishes perspective taking. Five studies, examining various forms of perspective taking across diverse paradigms, provide converging evidence that status and power exert differential effects on perspective taking. Moreover, these studies provide insight regarding the distinction between status and power, as well as the distinct psychology associated with status. © 2016 by the Society for Personality and Social Psychology, Inc.

Identification of genuine primary pulmonary NK cell lymphoma via clinicopathologic observation and clonality assay.

PubMed

Gong, Li; Wei, Long-Xiao; Huang, Gao-Sheng; Zhang, Wen-Dong; Wang, Lu; Zhu, Shao-Jun; Han, Xiu-Juan; Yao, Li; Lan, Miao; Li, Yan-Hong; Zhang, Wei

2013-08-19

Extranodal natural killer (NK)/T-cell lymphoma, nasal type, is an uncommon lymphoma associated with the Epstein-Barr virus (EBV). It most commonly involves the nasal cavity and upper respiratory tract. Primary pulmonary NK/T cell lymphoma is extremely rare. If a patient with a NK or T-cell tumor has an unusual reaction to treatment or an unusual prognosis, it is wise to differentiate NK from T-cell tumors. The clinicopathologic characteristics, immunophenotype, EBV in situ hybridization, and T cell receptor (TCR) gene rearrangement of primary pulmonary NK cell lymphoma from a 73-year-old Chinese woman were investigated and the clonal status was determined using female X-chromosomal inactivation mosaicism and polymorphisms at the phosphoglycerate kinase (PGK) gene. The lesion showed the typical histopathologic characteristics and immunohistochemical features of NK/T cell lymphoma. However, the sample was negative for TCR gene rearrangement. A clonality assay demonstrated that the lesion was monoclonal. It is concluded that this is the first recorded case of genuine primary pulmonary NK cell lymphoma. The purpose of the present work is to recommend that pathologists carefully investigate the whole lesion to reduce the likelihood that primary pulmonary NK cell lymphoma will be misdiagnosed as an infectious lesion. In addition, TCR gene rearrangement and clonal analysis, which is based on female X-chromosomal inactivation mosaicism and polymorphisms at PGK and androgen receptor (AR) loci, were found to play important roles in differentiating NK cell lymphoma from T cell lymphoma. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5205300349457729.

Loss of CD28 on Peripheral T Cells Decreases the Risk for Early Acute Rejection after Kidney Transplantation

PubMed Central

Dedeoglu, Burç; Meijers, Ruud W. J.; Klepper, Mariska; Hesselink, Dennis A.; Baan, Carla C.; Litjens, Nicolle H. R.; Betjes, Michiel G. H.

2016-01-01

Background End-stage renal disease patients have a dysfunctional, prematurely aged peripheral T-cell system. Here we hypothesized that the degree of premature T-cell ageing before kidney transplantation predicts the risk for early acute allograft rejection (EAR). Methods 222 living donor kidney transplant recipients were prospectively analyzed. EAR was defined as biopsy proven acute allograft rejection within 3 months after kidney transplantation. The differentiation status of circulating T cells, the relative telomere length and the number of CD31+ naive T cells were determined as T-cell ageing parameters. Results Of the 222 patients analyzed, 30 (14%) developed an EAR. The donor age and the historical panel reactive antibody score were significantly higher (p = 0.024 and p = 0.039 respectively) and the number of related donor kidney transplantation was significantly lower (p = 0.018) in the EAR group. EAR-patients showed lower CD4+CD28null T-cell numbers (p<0.01) and the same trend was observed for CD8+CD28null T-cell numbers (p = 0.08). No differences regarding the other ageing parameters were found. A multivariate Cox regression analysis showed that higher CD4+CD28null T-cell numbers was associated with a lower risk for EAR (HR: 0.65, p = 0.028). In vitro, a significant lower percentage of alloreactive T cells was observed within CD28null T cells (p<0.001). Conclusion Immunological ageing-related expansion of highly differentiated CD28null T cells is associated with a lower risk for EAR. PMID:26950734

[A European discussion about stem cells for therapeutic use].

PubMed

Boer, G J

2002-06-29

Stem cells as a source material for growing cellular transplants to repair dysfunctional organs appear to be a new challenge for medical science. Though stem cells are also present in foetal and adult organs, embryonic stem cells from the pre-implantation embryo in particular have the potency to proliferate easily in vitro and the capacity to differentiate into all the body's organ-specific cells. Therefore, these are the ideal cells for developing new cell transplantation therapies for diseases such as Parkinson's disease, diabetes mellitus and heart failure. The use of spare in vitro fertilization (IVF) embryos or pre-implantation embryos specially created to harvest human embryonic stem cells is, however, controversial and an ethical problem. In a European discussion platform organised by the European Commission Research Directorate-General, the status quo of the progress was presented and subsequently commented upon and discussed in terms of medical-ethical, social, industrial and patient interests. The expectations of this new medical technology were high, but clinical trials seem only acceptable once the in vitro differentiation of stem cells can be adequately controlled and once it is known how in vitro prepared stem cells behave after implantation. The ethical justification of the use of in vitro pre-implantation embryos remains controversial. The prevailing view is that the interests of severely ill patients for whom no adequate therapy exists, surmounts the interest of protection of a human in vitro pre-implantation embryo, regardless of whether it was the result of IVF or of transplantation of a somatic cell nucleus of the patient in an enucleated donor egg cell (therapeutic cloning).

Periodontal ligament cellular structures engineered with electrospun poly(DL-lactide-co-glycolide) nanofibrous membrane scaffolds.

PubMed

Inanç, Bülend; Arslan, Y Emre; Seker, Sükran; Elçin, A Eser; Elçin, Y Murat

2009-07-01

Periodontal tissue engineering is expected to overcome the limitations associated with the existing regenerative techniques for the treatment of periodontal defects involving alveolar bone, cementum, and periodontal ligament. Cell-based tissue engineering approaches involve the utilization of in vitro expanded cells with regenerative capacity and their delivery to the appropriate sites via biomaterial scaffolds. The aim of this study was to establish living periodontal ligament cell-containing structures on electrospun poly(DL-lactic-co-glycolic acid) (PLGA) nanofiber membrane scaffolds, assess their viability and characteristics, and engineer multilayered structures amenable to easy handling. Human periodontal ligament (hPDL) cells were expanded in explant culture and then characterized morphologically and immunohistochemically. PLGA nanofiber membranes were prepared by the electrospinning process; mechanical tensile properties were determined, surface topography, nanofiber size, and porosity status were investigated with SEM. Cells were seeded on the membranes at approximately 50,000 cell/cm(2) and cultured for 21 days either in expansion or in osteogenic induction medium. Cell adhesion and viability were demonstrated using SEM and MTT, respectively, and osteogenic differentiation was determined with IHC and immunohistomorphometric evaluation of osteopontin, osteocalcin, and bone sialoprotein marker expression. At days 3, 6, 9, and 12 additional cell/membrane layers were deposited on the existing ones and multilayered hybrid structures were established. Results indicate the feasibility of periodontal ligament cell-containing tissue-like structures engineering with PDL cells and electrospun nanofiber PLGA scaffolds supporting cell adhesion, viability and osteogenic differentiation properties of cells in hybrid structures amenable to macroscopic handling.

Chronic Lymphocytic Leukemia B-Cell Normal Cellular Counterpart: Clues From a Functional Perspective

PubMed Central

Darwiche, Walaa; Gubler, Brigitte; Marolleau, Jean-Pierre; Ghamlouch, Hussein

2018-01-01

Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of small mature-looking CD19+ CD23+ CD5+ B-cells that accumulate in the blood, bone marrow, and lymphoid organs. To date, no consensus has been reached concerning the normal cellular counterpart of CLL B-cells and several B-cell types have been proposed. CLL B-cells have remarkable phenotypic and gene expression profile homogeneity. In recent years, the molecular and cellular biology of CLL has been enriched by seminal insights that are leading to a better understanding of the natural history of the disease. Immunophenotypic and molecular approaches (including immunoglobulin heavy-chain variable gene mutational status, transcriptional and epigenetic profiling) comparing the normal B-cell subset and CLL B-cells provide some new insights into the normal cellular counterpart. Functional characteristics (including activation requirements and propensity for plasma cell differentiation) of CLL B-cells have now been investigated for 50 years. B-cell subsets differ substantially in terms of their functional features. Analysis of shared functional characteristics may reveal similarities between normal B-cell subsets and CLL B-cells, allowing speculative assignment of a normal cellular counterpart for CLL B-cells. In this review, we summarize current data regarding peripheral B-cell differentiation and human B-cell subsets and suggest possibilities for a normal cellular counterpart based on the functional characteristics of CLL B-cells. However, a definitive normal cellular counterpart cannot be attributed on the basis of the available data. We discuss the functional characteristics required for a cell to be logically considered to be the normal counterpart of CLL B-cells. PMID:29670635

Analysis of chronic lymphotic leukemia transcriptomic profile: differences between molecular subgroups.

PubMed

Jantus Lewintre, Eloisa; Reinoso Martín, Cristina; Montaner, David; Marín, Miguel; José Terol, María; Farrás, Rosa; Benet, Isabel; Calvete, Juan J; Dopazo, Joaquín; García-Conde, Javier

2009-01-01

B cell chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder with a variable clinical course. Patients with unmutated IgV(H) gene show a shorter progression-free and overall survival than patients with immunoglobulin heavy chain variable regions (IgV(H)) gene mutated. In addition, BCL6 mutations identify a subgroup of patients with high risk of progression. Gene expression was analysed in 36 early-stage patients using high-density microarrays. Around 150 genes differentially expressed were found according to IgV(H) mutations, whereas no difference was found according to BCL6 mutations. Functional profiling methods allowed us to distinguish KEGG and gene ontology terms showing coordinated gene expression changes across subgroups of CLL. We validated a set of differentially expressed genes according to IgV(H) status, scoring them as putative prognostic markers in CLL. Among them, CRY1, LPL, CD82 and DUSP22 are the ones with at least equal or superior performance to ZAP70 which is actually the most used surrogate marker of IgV(H) status.

Flow cytometric characterization of freshwater crayfish hemocytes for the examination of physiological status in wild and captive animals.

PubMed

Taylor, Sean; Landman, Michael J; Ling, Nicholas

2009-09-01

Enumeration of invertebrate hemocytes is a potentially powerful tool for the determination of physiological effects of extrinsic stressors, such as hypoxia, disease, and toxicant exposure. A detailed flow cytometric method of broad application was developed for the objective characterization and enumeration of the hemocytes of New Zealand freshwater crayfish Paranephrops planifrons for the purpose of physiological health assessment. Hemocyte populations were isolated by flow cytometric sorting based on differential light scatter properties followed by morphological characterization via light microscopy and software image analysis. Cells were identified as hyaline, semigranular, and granular hemocytes based on established invertebrate hemocyte classification. A characteristic decrease in nuclear size, an increase in granularity between the hyaline and granular cells, and the eccentric location of nuclei in granular cells were also observed. The granulocyte subpopulations were observed to possess varying degrees of granularity. The developed methodology was used to perform total and differential hemocyte counts from three lake populations and between wild and captive crayfish specimens. Differences in total and differential hemocyte counts were not observed among the wild populations. However, specimens held in captivity for 14 d exhibited a significant 63% reduction in total hemocyte count, whereas the relative hemocyte proportions remained the same. These results demonstrate the utility of this method for the investigation of subacute stressor effects in selected decapod crustaceans.

Longitudinal trends of total white blood cell and differential white blood cell counts of atomic bomb survivors.

PubMed

Hsu, Wan-Ling; Tatsukawa, Yoshimi; Neriishi, Kazuo; Yamada, Michiko; Cologne, John; Fujiwara, Saeko

2010-01-01

In studying the late health effects of atomic-bomb (A-bomb) survivors, earlier findings were that white blood cell (WBC) count increased with radiation dose in cross-sectional studies. However, a persistent effect of radiation on WBC count and other risk factors has yet to be confirmed. The objectives of the present study were 1) to examine the longitudinal relationship between A-bomb radiation dose and WBC and differential WBC counts among A-bomb survivors and 2) to investigate the potential confounding risk factors (such as age at exposure and smoking status) as well as modification of the radiation dose-response. A total of 7,562 A-bomb survivors in Hiroshima and Nagasaki were included in this study from 1964-2004. A linear mixed model was applied using the repeated WBC measurements. During the study period, a secular downward trend of WBC count was observed. Radiation exposure was a significant risk factor for elevated WBC and differential WBC counts over time. A significant increase of WBC counts among survivors with high radiation dose (> 2 Gy) was detected in men exposed below the age of 20 and in women regardless of age at exposure. Effects on WBC of low dose radiation remain unclear, however. Cigarette smoking produced the most pronounced effect on WBC counts and its impact was much larger than that of radiation exposure.

[Humans or material? Three levels of the discourse about the stem cell research from theological-ethical view].

PubMed

Ernst, Stephan

2004-01-01

In the debate on the ethical evaluation of the stem cell research three levels can be differentiated. The first level of argumentation is that of weighing up goods: The possible therapeutical success for thousands of humans seems to justify the consumption of a few embryos. It is show, that this, however, already presupposes - on a second argumentation level - a judgement on the moral status of the embryo. Different moments of time, when human dignity and life protection are ascribe to the embryo, have already been discussed, but in spite of all rationality of the arguments a consensus has not been reached. On this third level of argumentation two fundamental meanings of reality can be differentiated. The empirical-observing and the communicative-participating view. These lead to a different moral evaluation of the embryo. This contribution votes for the priority of the communicative-participating view. It receives addition support by theology and Christian faith.

Thinking Differentially: A Response to Issues in Differential Response

ERIC Educational Resources Information Center

Fluke, John D.; Merkel-Holguin, Lisa; Schene, Patricia

2013-01-01

This is a response to the document by Hughes et al. in this issue that offers a critique of the status of differential response (DR). We find the document to be helpful in intent, but do not find that it reflects scientifically sound methods, and contains many mischaracterizations of the status, impetus, research, and evaluation of DR to date. We…

Versican-Derived Matrikines Regulate Batf3-Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer.

PubMed

Hope, Chelsea; Emmerich, Philip B; Papadas, Athanasios; Pagenkopf, Adam; Matkowskyj, Kristina A; Van De Hey, Dana R; Payne, Susan N; Clipson, Linda; Callander, Natalie S; Hematti, Peiman; Miyamoto, Shigeki; Johnson, Michael G; Deming, Dustin A; Asimakopoulos, Fotis

2017-09-01

Colorectal cancer originates within immunologically complex microenvironments. To date, the benefits of immunotherapy have been modest, except in neoantigen-laden mismatch repair-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. In this article, we report that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) strongly correlated with CD8 + T cell infiltration in colorectal cancer, regardless of mismatch repair status. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8 + T cell infiltration. Tumor-intrinsic WNT pathway activation was associated with CD8 + T cell exclusion and VCAN accumulation. In addition to regulating VCAN levels at the tumor site, VCAN proteolysis results in the generation of bioactive fragments with novel functions (VCAN-derived matrikines). Versikine, a VCAN-derived matrikine, enhanced the generation of CD103 + CD11c hi MHCII hi conventional dendritic cells (cDCs) from Flt3L-mobilized primary bone marrow-derived progenitors, suggesting that VCAN proteolysis may promote differentiation of tumor-seeding DC precursors toward IRF8- and BATF3-expressing cDCs. Intratumoral BATF3-dependent DCs are critical determinants for T cell antitumor immunity, effector T cell trafficking to the tumor site, and response to immunotherapies. Our findings provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker and VCAN-derived matrikines as novel immunotherapy agents. Copyright © 2017 by The American Association of Immunologists, Inc.

Epigenetic repression of HOXB cluster in oral cancer cell lines.

PubMed

Xavier, Flávia Caló Aquino; Destro, Maria Fernanda de Souza Setubal; Duarte, Carina Magalhães Esteves; Nunes, Fabio Daumas

2014-08-01

Aberrant DNA methylation is a fundamental transcriptional control mechanism in carcinogenesis. The expression of homeobox genes is usually controlled by an epigenetic mechanism, such as the methylation of CpG islands in the promoter region. The aim of this study was to describe the differential methylation pattern of HOX genes in oral squamous cell carcinoma (OSCC) cell lines and transcript status in a group of hypermethylated and hypomethylated genes. Quantitative analysis of DNA methylation was performed on two OSCC cell lines (SCC4 and SCC9) using a method denominated Human Homeobox Genes EpiTect Methyl qPCR Arrays, which allowed fast, precise methylation detection of 24 HOX specific genes without bisulfite conversion. Methylation greater than 50% was detected in HOXA11, HOXA6, HOXA7, HOXA9, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXC8 and HOXD10. Both cell lines demonstrated similar hypermethylation status for eight HOX genes. A similar pattern of promoter hypermethylation and hypomethylation was demonstrated for the HOXB cluster and HOXA cluster, respectively. Moreover, the hypermethylation profile of the HOXB cluster, especially HOXB4, was correlated with decreased transcript expression, which was restored following treatment with 5-aza-2'-deoxycytidine. The homeobox methylation profile in OSCC cell lines is consistent with an epigenetic biomarker. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Effect of Inflammatory Status on Butyrate and Folate Uptake by Tumoral (Caco-2) and Non-Tumoral (IEC-6) Intestinal Epithelial Cells

PubMed Central

Couto, Mafalda R.; Gonçalves, Pedro; Catarino, Telmo A.; Martel, Fátima

2017-01-01

Objective Colorectal cancer (CRC) is the second leading cause of cancer death in occidental countries. Chronic inflammatory bowel disease (crohn’s disease and ulcerative colitis) is associated with an increased risk for CRC development. The aim of this work was to investigate the relationship between inflammatory status and absorption of nutrients with a role in CRC pathogenesis. Materials and Methods In this experimental study, we evaluated the in vitro effect of tumour necrosis factor-alpha (TNF-α), interferon-γ (IF-γ), and acetylsalicylic acid on 14C-butyrate (14C- BT), 3H-folic acid (3H-FA) uptake, and on proliferation, viability and differentiation of Caco-2 and IEC-6 cells in culture. Results The proinflammatory cytokines TNF-α and INF-γ were found to decrease uptake of a low concentration of 14C-BT (10 µM) by Caco-2 (tumoral) and IEC-6 (normal) intestinal epithelial cell lines. However, the effect of TNF-α and INF-γ in IEC-6 cells is most probably related to a cytotoxic and antiproliferative impact. In contrast, INF-γ increases uptake of a high concentration (10 mM) of 14C-BT in Caco-2 cells. The anticarcinogenic effect of BT (10 mM) in these cells is not affected by the presence of this cytokine. On the other hand, acetylsalicylic acid stimulates 14C-BT uptake by Caco-2 cells and potentiates its antiproliferative effect. Finally, both TNF-α and INF-γ cause a significant decrease in 3H-FA uptake by Caco-2 cells. Conclusion The inflammatory status has an impact upon cellular uptake of BT and FA, two nutrients with a role in CRC pathogenesis. Moreover, the anti-inflammatory acetylsalicylic acid potentiates the anticarcinogenic effect of BT in Caco-2 cells by increasing its cellular uptake. PMID:28580313

Advances in Translational Research in Neuro-oncology

PubMed Central

Fueyo, Juan; Gomez-Manzano, Candelaria; Yung, W. K. Alfred

2011-01-01

During the last decade, we have witnessed several key advances in the field of neurooncology. First, there were conceptual advances in the molecular and cell biology of malignant gliomas including the discovery in 2004 of brain tumor stem cells. Second, the Cancer Genome Atlas project has been extremely useful in the discovery of new molecular markers, including mutations in the IDH1 gene, and has led to a new classification of gliomas based on the differentiation status and mesenchymal transformation. In addition, use of the 1p/19q marker and O6-methylguanine-DNA methyltransferase methylation status have been identified as guides for patient selection for therapies and represent the first steps toward personalized medicine for treating gliomas. Finally, progress has been made in treatment strategies including the establishment of temozolomide as the criterion standard for treating gliomas, the adoption of bevacizumab in the clinical setting, and developments in experimental biological therapies including cancer vaccines and oncolytic adenoviruses. PMID:21059986

Proteomic Comparison and MRM-Based Comparative Analysis of Metabolites Reveal Metabolic Shift in Human Prostate Cancer Cell Lines.

PubMed

Shu, Qingbo; Cai, Tanxi; Chen, Xiulan; Zhu, Helen He; Xue, Peng; Zhu, Nali; Xie, Zhensheng; Wei, Shasha; Zhang, Qing; Niu, Lili; Gao, Wei-Qiang; Yang, Fuquan

2015-08-07

One of the major challenges in prostate cancer therapy remains the development of effective treatments for castration-resistant prostate cancer (CRPC), as the underlying mechanisms for its progression remain elusive. Previous studies showed that androgen receptor (AR) is crucially involved in regulation of metabolism in prostate cancer (PCa) cells throughout the transition from early stage, androgen-sensitive PCa to androgen-independent CRPC. AR achieves such metabolic rewiring directively either via its transcriptional activity or via interactions with AMP-activated protein kinase (AMPK). However, due to the heterogeneous expression and activity status of AR in PCa cells, it remains a challenge to investigate the links between AR status and metabolic alterations. To this end, we compared the proteomes of three pairs of androgen-sensitive (AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR(+)/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling approach. Our results revealed that most of the differentially expressed proteins between each pair function in metabolism, indicating a metabolic shift between AS and AI cells, as further validated by multiple reaction monitoring (MRM)-based quantification of nucleotides and relative comparison of fatty acids between these cell lines. Furthermore, increased adenylate kinase isoenzyme 1 (AK1) in AS relative to AI cells may result in activation of AMPK, representing a major regulatory factor involved in the observed metabolic shift in PCa cells.

Cellular and Molecular effects of Vitamin D on Carcinogenesis

PubMed Central

Welsh, JoEllen

2011-01-01

Epidemiologic data suggest that the incidence and severity of many types of cancer inversely correlates with indices of vitamin D status. The vitamin D receptor (VDR) is highly expressed in epithelial cells at risk for carcinogenesis including those resident in skin, breast, prostate and colon, providing a direct molecular link by which vitamin D status impacts on carcinogenesis. Consistent with this concept, activation of VDR by its ligand 1,25-dihydroxyvitamin D (1,25D) triggers comprehensive genomic changes in epithelial cells that contribute to maintenance of the differentiated phenotype, resistance to cellular stresses and protection of the genome. Many epithelial cells also express the vitamin D metabolizing enzyme CYP27B1 which enables autocrine generation of 1,25D from the circulating vitamin D metabolite 25-hydroxyvitamin D (25D), critically linking overall vitamin D status with cellular anti-tumor actions. Furthermore, pre-clinical studies in animal models has demonstrated that dietary supplementation with vitamin D or chronic treatment with VDR agonists decreases tumor development in skin, colon, prostate and breast. Conversely, deletion of the VDR gene in mice alters the balance between proliferation and apoptosis, increases oxidative DNA damage, and enhances susceptibility to carcinogenesis in these tissues. Because VDR expression is retained in many human tumors, vitamin D status may be an important modulator of cancer progression in persons living with cancer. Collectively, these observations have reinforced the need to further define the molecular actions of the VDR and the human requirement for vitamin D in relation to cancer development and progression. PMID:22085499

Differentiating the effects of status and power: a justice perspective.

PubMed

Blader, Steven L; Chen, Ya-Ru

2012-05-01

Few empirical efforts have been devoted to differentiating status and power, and thus significant questions remain about differences in how status and power impact social encounters. We conducted 5 studies to address this gap. In particular, these studies tested the prediction that status and power would have opposing effects on justice enacted toward others. In the first 3 studies, we directly compared the effects of status and power on people's enactment of distributive (Study 1) and procedural (Studies 2 and 3) justice. In the last 2 studies, we orthogonally manipulated status and power and examined their main and interactive effects on people's enactment of distributive (Study 4) and procedural (Study 5) justice. As predicted, all 5 studies showed consistent evidence that status is positively associated with justice toward others, while power is negatively associated with justice toward others. The effects of power are moderated, however, by an individual's other orientation (Studies 2, 3, 4, and 5), and the effects of status are moderated by an individual's dispositional concern about status (Study 5). Furthermore, Studies 4 and 5 also demonstrated that status and power interact, such that the positive effect of status on justice emerges when power is low and not when power is high, providing further evidence for differential effects between power and status. Theoretical implications for the literatures on status, power, and distributive/procedural justice are discussed.

Autocrine stimulation of human hepatocytes triggers late DNA synthesis and stabilizes long-term differentiation in vitro.

PubMed

Leckel, Kerstin; Strey, Christoph; Bechstein, Wolf O; Boost, Kim A; Auth, Marcus K H; El Makhfi, Amal; Juengel, Eva; Wedel, Steffen; Jones, Jon; Jonas, Dietger; Blaheta, Roman A

2008-05-01

Isolated human hepatocytes are of great value in investigating cell transplantation, liver physiology, pathology, and drug metabolism. Though hepatocytes possess a tremendous proliferative capacity in vivo, their ability to grow in culture is severely limited. We postulated that repeated medium change, common to most in vitro systems, may prevent long-term maintenance of hepato-specific functions and growth capacity. To verify our hypotheses we compared the DNA synthesis and differentiation status of isolated human hepatocytes, cultured in medium which was renewed every day or was not changed for 3 weeks ('autocrine' setting). Daily medium change led to rapid hepatocellular de-differentiation without any signs of DNA replication. In contrast, the autocrine setting allowed hepatocytes to become highly differentiated, demonstrated by an elevated ASGPr expression level, and increased albumin and fibrinogen synthesis and release. Cytokeratin 18 filaments were stably expressed, whereas cytokeratin 19 remained undetectable. Hepatocytes growing in an autocrine fashion were activated in the presence of hepatocyte growth factor (HGF), evidenced by c-Met phosphorylation. However, HGF response was not achieved when the culture medium was renewed daily. Furthermore, the autocrine setting evoked a late but strong interleukin 6 release into the culture supernatant, reaching maximum values after a 10-day cultivation period, and intense BrdU incorporation after a further 5-day period. Our data suggest that preservation of the same medium creates environmental conditions which allow hepatocytes to control their differentiation status and DNA synthesis in an autocrine fashion. Further studies are necessary to identify the key mediators involved in autocrine communication and to design the optimal culture configuration for clinical application.

Characterization of a family of structurally related glycoproteins expressing beta 1-6-branched asparagine-linked oligosaccharides in human colon carcinoma cells.

PubMed

Laferté, S; Loh, L C

1992-04-01

Previous studies have established that metastatic tumour cells express high levels of beta 1-6-branched Asn-linked oligosaccharides which can be detected with the lectin leucoagglutinin (L-PHA) [Dennis, Laferté, Waghorne, Breitman & Kerbel (1987) Science 236, 582-585]. In order to identify L-PHA-binding glycoproteins which may play a role specifically in colon cancer, we have prepared monoclonal antibodies (MAbs) to the moderately well-differentiated human colon carcinoma cell line HT29. In this paper we present the initial characterization of a family of structurally related L-PHA-binding glycoproteins detected by MAb 1H9 which are differentially expressed and processed by HT29 cells and by two other human colon carcinoma cell lines, SW480 and SW620. In contrast to HT29, the SW480 and SW620 cell lines were established from a poorly differentiated grade III/IV primary tumour and one of its lymph node metastases respectively. MAb 1H9 detects in HT29 cells a conformational determinant present on three L-PHA-binding glycoproteins of 100, 70 and 25kDa, as well as a 74 kDa glycoprotein with high-mannose-type Asn-linked oligosaccharides. Pulse-chase experiments and peptide mapping analyses revealed that the 74 kDa and 100 kDa species are related by carbohydrate processing and are probably derived from a common 76 kDa precursor. On the other hand, the 70 kDa glycoprotein is synthesized from an endoglycosidase H-sensitive precursor of 56 kDa which is structurally related to, but distinct from, the aforementioned 76 kDa precursor. In addition, the 100 kDa species is secreted into the culture medium, whereas the 70 kDa glycoprotein is retained intracellularly. SW480 and SW620 cells showed qualitative and quantitative differences from HT29 cells, including increased secretion of a smaller L-PHA-binding glycoprotein of 92 kDa into the culture medium, as well as apparent differences in glycosylation of the intracellular 66 kDa glycoprotein. These results suggested that the expression, glycosylation and subcellular localization of this family of L-PHA-binding glycoproteins may correlate with the differentiation status of colon cancer cells and/or reflect biochemical changes. characteristic of more progressive metastatic tumours.

PTSD is associated with an increase in aged T cell phenotypes in adults living in Detroit

PubMed Central

Aiello, Allison E.; Dowd, Jennifer B.; Jayabalasingham, Bamini; Feinstein, Lydia; Uddin, Monica; Simanek, Amanda M.; Cheng, Caroline K.; Galea, Sandro; Wildman, Derek E.; Koenen, Karestan; Pawelec, Graham

2016-01-01

Background Psychosocial stress is thought to play a key role in the acceleration of immunological aging. This study investigated the relationship between lifetime and past-year history of post-traumatic stress disorder (PTSD) and the distribution of T cell phenotypes thought to be characteristic of immunological aging. Methods Data were from 85 individuals who participated in the community-based Detroit Neighborhood Health Study. Immune markers assessed included the CD4:CD8 ratio, the ratio of late-differentiated effector (CCR7-CD45RA+CD27-CD28-) to naïve (CCR7+CD45RA+CD27+CD28+) T cells, the percentage of KLRG1-expressing cells, and the percentage of CD57-expressing cells. Results In models adjusted for age, gender, race/ethnicity, education, smoking status, and medication use, we found that past-year PTSD was associated with statistically significant differences in the CD8+ T cell population, including a higher ratio of late-differentiated effector to naïve T cells, a higher percentage of KLRG1+ cells, and a higher percentage of CD57+ cells. The percentage of CD57+ cells in the CD4 subset was also significantly higher and the CD4:CD8 ratio significantly lower among individuals who had experienced past-year PTSD. Lifetime PTSD was also associated with differences in several parameters of immune aging. Conclusions PTSD is associated with an aged immune phenotype and should be evaluated as a potential catalyzer of accelerated immunological aging in future studies. PMID:26894484

Effect of aqueous and lipophilic mullet (Mugil cephalus) Bottarga extracts on the growth and lipid profile of intestinal Caco-2 cells.

PubMed

Rosa, Antonella; Atzeri, Angela; Deiana, Monica; Melis, M Paola; Loru, Debora; Incani, Alessandra; Cabboi, Barbara; Dessì, M Assunta

2011-03-09

The importance of n-3 polyunsaturated fatty acid (n-3 PUFA) intake has long been recognized in human nutrition. Although health benefits, n-3 PUFA are subject to rapid and/or extensive oxidation during processing and storage, resulting in potential alteration in nutritional composition and quality of food. Bottarga, a salted and semi-dried mullet ( Mugil cephalus ) ovary product, is proposed as an important source of n-3 PUFA, having high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we investigated the extent of lipid oxidation of grated bottarga samples during 7 months of storage at -20 °C and room temperature under light exposure. Cell viability, lipid composition, and lipid peroxidation were measured in intestinal differentiated Caco-2 cell monolayers after 6-48 h of incubation with lipid and hydrophilic extracts obtained from bottarga samples at different storage conditions. The storage of bottarga did not affect the n-3 PUFA level, but differences were observed in hydroperoxide levels in samples from different storage conditions. All tested bottarga extracts did not show a toxic effect on cell viability of differentiated Caco-2 cells. Epithelial cells incubated with bottarga oil had significant changes in fatty acid composition but not in cholesterol levels with an accumulation of EPA, DHA, and 22:5. Cell hydroperoxides were higher in treated cells, in relation to the oxidative status of bottarga oil. Moreover, the bottarga lipid extract showed an in vitro inhibitory effect on the growth of a colon cancer cell line (undifferentiated Caco-2 cells).

Effect of Storage Temperature on the Phenotype of Cultured Epidermal Cells Stored in Xenobiotic-Free Medium.

PubMed

Jackson, Catherine; Eidet, Jon R; Reppe, Sjur; Aass, Hans Christian D; Tønseth, Kim A; Roald, Borghild; Lyberg, Torstein; Utheim, Tor P

2016-06-01

Cultured epidermal cell sheets (CECS) are used in the treatment of large area burns to the body and have potential to treat limbal stem cell deficiency (LSCD) as shown in animal studies. Despite widespread use, storage options for CECS are limited. Short-term storage allows flexibility in scheduling surgery, quality control and improved transportation to clinics worldwide. Recent evidence points to the phenotype of cultured epithelial cells as a critical predictor of post-operative success following transplantation of CECS in burns and in transplantation of cultured epithelial cells in patients with LSCD. This study, therefore assessed the effect of a range of temperatures, spanning 4-37 °C, on the phenotype of CECS stored over a 2-week period in a xenobiotic-free system. Progenitor cell (p63, ΔNp63α and ABCG2) and differentiation (C/EBPδ and CK10) associated marker expression was assessed using immunocytochemistry. Immunohistochemistry staining of normal skin for the markers p63, ABCG2 and C/EBPδ was also carried out. Assessment of progenitor cell side population (SP) was performed using JC1 dye by flow cytometry. P63 expression remained relatively constant throughout the temperature range but was significantly lower compared to control between 20 and 28 °C (p < 0.05). High C/EBPδ together with low p63 suggested more differentiation beginning at 20 °C and above. Lower CK10 and C/EBPδ expression most similar to control was seen at 12 °C. The percentage of ABCG2 positive cells was most similar to control between 8 and 24 °C. Between 4 and 24 °C, the SP fluctuated, but was not significantly different compared to control. Results were supported by staining patterns indicating differentiation status associated with markers in normal skin sections. Lower storage temperatures, and in particular 12 °C, merit further investigation as optimal storage temperature for maintenance of undifferentiated phenotype in CECS.

Mesenchymal stem cells reside in anterior cruciate ligament remnants in situ.

PubMed

Fu, Weili; Li, Qi; Tang, Xin; Chen, Gang; Zhang, Chenghao; Li, Jian

2016-07-01

It has been reported that the anterior cruciate ligament (ACL) has certain self-healing ability after acute injury or with primary suture repair. Many studies have confirmed that a remnant preservation technique with ACL reconstruction contributes to biological augmentation for ACL healing. However, it remains unclear whether mesenchymal stem cells (MSC) reside in ACL remnants in situ. The aim of this study was to investigate the methods of culture and identification of MSC derived from the remnants of ACL rupture patients and to analyse these MSC's properties. The cells of ACL remnants from the ACL rupture patients were isolated by the methods of enzymatic digestion and cultured in vitro to the third passage under the microscope to observe their morphology and growth status. The third passage of isolated cells was analysed for the identification of immunophenotype, osteogenic, adipogenic and chondrogenic differentiation. On the third to fifth days of in vitro culture, a few cells of long fusiform shape appeared and were adherent to the plastic walls. On the sixth to ninth days, cells clustered and colonies were observed. The third passage cells showed uniform cell morphology and good proliferation, with appearance of the typical surface markers of MSC, CD29, CD44, CD90 and CD105. The surface markers of CD34 and CD45 of haematopoietic stem cells were not expressed. Under appropriate conditions of in vitro culture, isolated cells could be differentiated into osteoblasts that deposit mineralised matrix and express early osteogenic markers, adipocytes that accumulate lipid droplets in cytoplasm and chondrocytes that secrete chondrogenic-specific matrix aggrecan and collagen II. Real-time polymerase chain reaction (PCR) analysis demonstrated that the specific mRNA expression of osteogenesis, adipogenesis and chondrogenesis increased significantly compared with the control groups at day zero. Stem cells derived in situ from the human ACL stump were successfully isolated and characterised. Those isolated cells were identified as MSC according to their adherent ability, morphology, surface markers and multilineage differentiation potential. MSC derived from ACL remnants could be a potential source of seeding cells for ligament regeneration.

Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals

PubMed Central

Kumar, Dharmendra; Talluri, Thirumala R; Anand, Taruna; Kues, Wilfried A

2015-01-01

Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals. PMID:25815117

NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

NASA Technical Reports Server (NTRS)

Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

2005-01-01

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

Engineering Stem Cells for Biomedical Applications

PubMed Central

Yin, Perry T.; Han, Edward

2018-01-01

Stem cells are characterized by a number of useful properties, including their ability to migrate, differentiate, and secrete a variety of therapeutic molecules such as immunomodulatory factors. As such, numerous pre-clinical and clinical studies have utilized stem cell-based therapies and demonstrated their tremendous potential for the treatment of various human diseases and disorders. Recently, efforts have focused on engineering stem cells in order to further enhance their innate abilities as well as to confer them with new functionalities, which can then be used in various biomedical applications. These engineered stem cells can take on a number of forms. For instance, engineered stem cells encompass the genetic modification of stem cells as well as the use of stem cells for gene delivery, nanoparticle loading and delivery, and even small molecule drug delivery. The present Review gives an in-depth account of the current status of engineered stem cells, including potential cell sources, the most common methods used to engineer stem cells, and the utilization of engineered stem cells in various biomedical applications, with a particular focus on tissue regeneration, the treatment of immunodeficiency diseases, and cancer. PMID:25772134

Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood.

PubMed

Toepfner, Nicole; Herold, Christoph; Otto, Oliver; Rosendahl, Philipp; Jacobi, Angela; Kräter, Martin; Stächele, Julia; Menschner, Leonhard; Herbig, Maik; Ciuffreda, Laura; Ranford-Cartwright, Lisa; Grzybek, Michal; Coskun, Ünal; Reithuber, Elisabeth; Garriss, Geneviève; Mellroth, Peter; Henriques-Normark, Birgitta; Tregay, Nicola; Suttorp, Meinolf; Bornhäuser, Martin; Chilvers, Edwin R; Berner, Reinhard; Guck, Jochen

2018-01-13

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis. © 2018, Toepfner et al.

Long non-coding RNAs in B-cell malignancies: a comprehensive overview

PubMed Central

Taiana, Elisa; Neri, Antonino

2017-01-01

B-cell malignancies constitute a large part of hematological neoplasias. They represent a heterogeneous group of diseases, including Hodgkin's lymphoma, most non-Hodgkin's lymphomas (NHL), some leukemias and myelomas. B-cell malignancies reflect defined stages of normal B-cell differentiation and this represents the major basis for their classification. Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nucleotides, for which many recent studies have demonstrated a function in regulating gene expression, cell biology and carcinogenesis. Deregulated expression levels of lncRNAs have been observed in various types of cancers including hematological malignancies. The involvement of lncRNAs in cancer initiation and progression and their attractive features both as biomarker and for therapeutic research are becoming increasingly evident. In this review, we summarize the recent literature to highlight the status of the knowledge of lncRNAs role in normal B-cell development and in the pathogenesis of B-cell tumors. PMID:28947998

Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood

PubMed Central

Toepfner, Nicole; Herold, Christoph; Otto, Oliver; Rosendahl, Philipp; Jacobi, Angela; Kräter, Martin; Stächele, Julia; Menschner, Leonhard; Herbig, Maik; Ciuffreda, Laura; Ranford-Cartwright, Lisa; Grzybek, Michal; Coskun, Ünal; Reithuber, Elisabeth; Garriss, Geneviève; Mellroth, Peter; Henriques-Normark, Birgitta; Tregay, Nicola; Suttorp, Meinolf; Bornhäuser, Martin; Chilvers, Edwin R; Berner, Reinhard

2018-01-01

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis. PMID:29331015

The effect of women's status and community on the gender differential in children's nutrition in India.

PubMed

Bose, Sunita

2011-09-01

This study uses the third National Family Health Survey (2005-06) in India to investigate whether differences in women's status, both at the individual and community levels, can explain the persistent gender differential in nutritional allocation among children. The results show that girls are less likely than boys to receive supplemental food and more likely to be malnourished. In general it appears that higher women's status within a community, as well as higher maternal status, have beneficial effects on a daughter's nutritional status. Further, the moderating effects of community appear to be more consistent and stronger than the individual-level characteristics. A positive relationship between the percentage of literate women in a community and the gender differential in malnutrition appears to be an exception to the general findings regarding the beneficial nature of women's status on a daughter's well-being, showing the need for more than just basic adult literacy drives in communities to overcome the problem of daughter neglect.

Social status and the pursuit of positive social identity: Systematic domains of intergroup differentiation and discrimination for high- and low- status groups

PubMed Central

Oldmeadow, Julian A.; Fiske, Susan T.

2013-01-01

Research on intergroup discrimination has focused on the cognitive and motivational mechanisms involved, but the role of stereotype content has been neglected. Drawing on social identity theory and stereotype content research, the current studies investigated the role of stereotype content in intergroup differentiation and discrimination. Across two studies, students from high- and low-status groups differentiated themselves positively on stereotypes of competence and warmth respectively, and in allocations of resources in domains relevant to competence (academics, research) and warmth (sports, community outreach). Furthermore, there was evidence that discrimination by high- and low-status groups was driven by their respective stereotypes of competence and warmth. It is argued that stereotypes of competence and warmth, derived from status and power relations between groups, define the domains in which groups pursue positively distinct identities. PMID:24319344

MALDI imaging reveals NCOA7 as a potential biomarker in oral squamous cell carcinoma arising from oral submucous fibrosis.

PubMed

Xie, Xiaoyan; Jiang, Yuchen; Yuan, Yao; Wang, Peiqi; Li, Xinyi; Chen, Fangman; Sun, Chongkui; Zhao, Hang; Zeng, Xin; Jiang, Lu; Zhou, Yu; Dan, Hongxia; Feng, Mingye; Liu, Rui; Chen, Qianming

2016-09-13

Oral squamous cell carcinoma (OSCC) ranks among the most common cancer worldwide, and is associated with severe morbidity and high mortality. Oral submucous fibrosis (OSF), characterized by fibrosis of the mucosa of the upper digestive tract, is a pre-malignant lesion, but the molecular mechanisms underlying this malignant transformation remains to be elucidated. In this study, matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS)-based proteomic strategy was employed to profile the differentially expressed peptides/proteins between OSCC tissues and the corresponding adjacent non-cancerous OSF tissues. Sixty-five unique peptide peaks and nine proteins were identified with altered expression levels. Of them, expression of NCOA7 was found to be up-regulated in OSCC tissues by immunohistochemistry staining and western blotting, and correlated with a pan of clinicopathologic parameters, including lesion site, tumor differentiation status and lymph node metastasis. Further, we show that overexpression of NCOA7 promotes OSCC cell proliferation in either in vitro or in vivo models. Mechanistic study demonstrates that NCOA7 induces OSCC cell proliferation probably by activating aryl hydrocarbon receptor (AHR). The present study suggests that NCOA7 is a potential biomarker for early diagnosis of OSF malignant transformation, and leads to a better understanding of the molecular mechanisms responsible for OSCC development.

Neuroprotective Effects of Transplanted Mesenchymal Stromal Cells-derived Human Umbilical Cord Blood Neural Progenitor Cells in EAE.

PubMed

Rafieemehr, Hassan; Kheyrandish, Maryam; Soleimani, Masoud

2015-12-01

Multiple Sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. The aim of this study was to investigate the neuroprotective effects of transplanted human umbilical cord blood mesenchymal stromal cells (UCB-MSC) derived neural progenitor cell (MDNPC) in EAE, an experimental model of MS. To initiate neuronal differentiation of UCB-MSCs, the pre-induction medium was removed and replaced with induction media containing retinoic acid, b FGF, h EGF, NGF, IBMX and ascorbic acid for one week. The expression of neural genes was examined in comparison to control group by real-time PCR assay. Then, experimental autoimmune encephalitis (EAE) was induced using myelin oligodendrocyte glycoprotein (MOG, 35-55 peptides) in 24 C57BL/6 mice. After induction, the mice were divided in four groups (n=6) as follows: healthy, PBS, UCB-MSCs and MDNPC, respectively. At the end of the study, disease status in all the groups was analyzed using hematoxylin-eosin (H&E) staining of brain sections. We found that UCB-MSCs exhibit neuronal differentiation potential in vitro and transplanted MDNPC lowered clinical score and reduced CNS leukocyte infiltration compared to untreated mice. Our results showed that MDNPC from UCB may be a proper candidate for regenerative therapy in MS and other neurodegenerative diseases.

Relationship Between Clinicopathological Characteristics and PET/CT Uptake in Esophageal Squamous Cell Carcinoma: [18F]Alfatide versus [18F]FDG.

PubMed

Dong, Yinjun; Wei, Yuchun; Chen, Guanxuan; Huang, Yong; Song, Pingping; Liu, Shuguang; Zheng, Jinsong; Cheng, Monica; Yuan, Shuanghu

2018-06-04

To assess a novel radiotracer aluminum [ 18 F]fluoride-1,4,7-triazacyclononane-triacetic acid-pegylated dimeric RGD ([ 18 F]ALF-NOTA-PRGD 2 , denoted as [ 18 F]Alfatide) for positron emission tomography (PET)/X-ray computed tomography (CT) and explore the relationships between clinicopathological characteristics and maximum standard uptake values in primary (SUV P ) and metastatic lymph nodes (SUV LN ) of patients with esophageal squamous cell carcinoma (ESCC), as verified by pathologic examination and compared with those obtained with 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]DG) PET. We prospectively enrolled patients with newly diagnosed ESCC who agreed to undergo [ 18 F]Alfatide PET/CT or [ 18 F]FDG PET/CT scans before surgery at Shandong Cancer Hospital from May 2011 to July 2017. SUVs and the pathological tumor-node-metastasis (pTNM) stages of primary tumors and metastatic lymph nodes (LNs) were measured and confirmed pathologically. Immunohistochemical (IHC) staining for integrin αvβ3 was performed on tumor samples (both primary tumors and metastatic LNs) collected from nine patients. Of 61 patients who underwent PET/CT scans, 46 then underwent curative surgery and were included in our analysis (n = 21 for [ 18 F]Alfatide PET/CT and n = 25 for [ 18 F]FDG PET/CT). No significant differences in the SUV P on [ 18 F]Alfatide PET/CT or [ 18 F]FDG PET/CT were observed among the cohorts according to gender, pathological stage, T stage, status of LNs, and differentiation (all P > 0.05). The SUV LN differed significantly between the pathological stages and status of LNs both on [ 18 F]Alfatide PET/CT (P = 0.03, 0.003) and [ 18 F]FDG PET/CT (P = 0.001. < 0.001), but not according to gender (P = 0.128, 0.129), T stage (P = 0.791, 0.727), or tumor differentiation (P = 0.049, 0.053). Significant positive correlations were observed between the SUV LN on [ 18 F]Alfatide PET/CT and [ 18 F]FDG PET/CT, and pathological stage (r = 0.52, P = 0.016; r = 0.503, P = 0.01), LN status (r = 0.73, P < 0.001; r = 0.649, P < 0.001), and differentiation (r = 0.509, P < 0.019; r = 0.459, P = 0.021) were observed. No significant differences were found between the relationships of SUV P with SUV LN , length, age, gender, pathological stage, T stage, status of LN, or differentiation, or of SUV LN with length, age, gender, or T stage both on [ 18 F]Alfatide PET/CT and [ 18 F]FDG PET/CT (all P > 0.05). The quantitated expression levels of αvβ3 in primary tumors and metastatic LNs were 1.67 ± 1.12 and 3.42 ± 2.93, respectively (P = 0.031). Our results suggest that SUV LN is influenced by pathological stage, LN status, and differentiation. SUV LN may therefore serve as a new parameter for risk stratification of with ESCC patients. Moreover, [ 18 F]Alfatide PET can provide complementary molecular information about ESCC metastasis.

Zoledronic acid has differential antitumor activity in the pre- and postmenopausal bone microenvironment in vivo.

PubMed

Ottewell, Penelope D; Wang, Ning; Brown, Hannah K; Reeves, Kimberly J; Fowles, C Anne; Croucher, Peter I; Eaton, Colby L; Holen, Ingunn

2014-06-01

Clinical trials in early breast cancer have suggested that benefits of adjuvant bone-targeted treatments are restricted to women with established menopause. We developed models that mimic pre- and postmenopausal status to investigate effects of altered bone turnover on growth of disseminated breast tumor cells. Here, we report a differential antitumor effect of zoledronic acid (ZOL) in these two settings. Twleve-week-old female Balb/c-nude mice with disseminated MDA-MB-231 breast tumor cells in bone underwent sham operation or ovariectomy (OVX), mimicking the pre- and postmenopausal bone microenvironment, respectively. To determine the effects of bone-targeted therapy, sham/OVX animals received saline or 100 μg/kg ZOL weekly. Tumor growth was assessed by in vivo imaging and effects on bone by real-time PCR, micro-CT, histomorphometry, and measurements of bone markers. Disseminated tumor cells were detected by two-photon microscopy. OVX increased bone resorption and induced growth of disseminated tumor cells in bone. Tumors were detected in 83% of animals following OVX (postmenopausal model) compared with 17% following sham operation (premenopausal model). OVX had no effect on tumors outside of bone. OVX-induced tumor growth was completely prevented by ZOL, despite the presence of disseminated tumor cells. ZOL did not affect tumor growth in bone in the sham-operated animals. ZOL increased bone volume in both groups. This is the first demonstration that tumor growth is driven by osteoclast-mediated mechanisms in models that mimic post- but not premenopausal bone, providing a biologic rationale for the differential antitumor effects of ZOL reported in these settings. Clin Cancer Res; 20(11); 2922-32. ©2014 AACR. ©2014 American Association for Cancer Research.

Growth-inhibitory and antiangiogenic activity of the MEK inhibitor PD0325901 in malignant melanoma with or without BRAF mutations.

PubMed

Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

2009-08-01

The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC(50) in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G(1)-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27(KIP1)) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.

Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations12

PubMed Central

Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

2009-01-01

The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma. PMID:19649202

Modeling Alzheimer’s disease with human induced pluripotent stem (iPS) cells

PubMed Central

Mungenast, Alison E.; Siegert, Sandra; Tsai, Li-Huei

2018-01-01

In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer’s disease (AD) have been often failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD. PMID:26657644

Modeling Alzheimer's disease with human induced pluripotent stem (iPS) cells.

PubMed

Mungenast, Alison E; Siegert, Sandra; Tsai, Li-Huei

2016-06-01

In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer's disease (AD) have been failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas9 will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD. Copyright © 2015 Elsevier Inc. All rights reserved.

Redox homeostasis: the linchpin in stem cell self-renewal and differentiation.

PubMed

Wang, Kui; Zhang, Tao; Dong, Qiang; Nice, Edouard Collins; Huang, Canhua; Wei, Yuquan

2013-03-14

Stem cells are characterized by their unique ability of self-renewal to maintain the so-called stem cell pool. Over the past decades, reactive oxygen species (ROS) have been recognized as toxic aerobic metabolism byproducts that are harmful to stem cells, leading to DNA damage, senescence or cell death. Recently, a growing body of literature has shown that stem cells reside in redox niches with low ROS levels. The balance of Redox homeostasis facilitates stem cell self-renewal by an intricate network. Thus, to fully decipher the underlying molecular mechanisms involved in the maintenance of stem cell self-renewal, it is critical to address the important role of redox homeostasis in the regulation of self-renewal and differentiation of stem cells. In this regard, we will discuss the regulatory mechanisms involved in the subtly orchestrated balance of redox status in stem cells by scavenger antioxidant enzyme systems that are well monitored by the hypoxia niches and crucial redox regulators including forkhead homeobox type O family (FoxOs), apurinic/apyrimidinic (AP) endonuclease1/redox factor-1 (APE1/Ref-1), nuclear factor erythroid-2-related factor 2 (Nrf2) and ataxia telangiectasia mutated (ATM). We will also introduce several pivotal ROS-sensitive molecules, such as hypoxia-inducible factors, p38 mitogen-activated protein kinase (p38) and p53, involved in the redox-regulated stem cell self-renewal. Specifically, all the aforementioned molecules can act as 'redox sensors' by virtue of redox modifications of their cysteine residues, which are critically important in the control of protein function. Given the importance of redox homeostasis in the regulation of stem cell self-renewal, understanding the underlying molecular mechanisms involved will provide important new insights into stem cell biology.

Redox homeostasis: the linchpin in stem cell self-renewal and differentiation

PubMed Central

Wang, Kui; Zhang, Tao; Dong, Qiang; Nice, Edouard Collins; Huang, Canhua; Wei, Yuquan

2013-01-01

Stem cells are characterized by their unique ability of self-renewal to maintain the so-called stem cell pool. Over the past decades, reactive oxygen species (ROS) have been recognized as toxic aerobic metabolism byproducts that are harmful to stem cells, leading to DNA damage, senescence or cell death. Recently, a growing body of literature has shown that stem cells reside in redox niches with low ROS levels. The balance of Redox homeostasis facilitates stem cell self-renewal by an intricate network. Thus, to fully decipher the underlying molecular mechanisms involved in the maintenance of stem cell self-renewal, it is critical to address the important role of redox homeostasis in the regulation of self-renewal and differentiation of stem cells. In this regard, we will discuss the regulatory mechanisms involved in the subtly orchestrated balance of redox status in stem cells by scavenger antioxidant enzyme systems that are well monitored by the hypoxia niches and crucial redox regulators including forkhead homeobox type O family (FoxOs), apurinic/apyrimidinic (AP) endonuclease1/redox factor-1 (APE1/Ref-1), nuclear factor erythroid-2-related factor 2 (Nrf2) and ataxia telangiectasia mutated (ATM). We will also introduce several pivotal ROS-sensitive molecules, such as hypoxia-inducible factors, p38 mitogen-activated protein kinase (p38) and p53, involved in the redox-regulated stem cell self-renewal. Specifically, all the aforementioned molecules can act as ‘redox sensors' by virtue of redox modifications of their cysteine residues, which are critically important in the control of protein function. Given the importance of redox homeostasis in the regulation of stem cell self-renewal, understanding the underlying molecular mechanisms involved will provide important new insights into stem cell biology. PMID:23492768

Induced PTF1a expression in pancreatic ductal adenocarcinoma cells activates acinar gene networks, reduces tumorigenic properties, and sensitizes cells to gemcitabine treatment.

PubMed

Jakubison, Brad L; Schweickert, Patrick G; Moser, Sarah E; Yang, Yi; Gao, Hongyu; Scully, Kathleen; Itkin-Ansari, Pamela; Liu, Yunlong; Konieczny, Stephen F

2018-05-02

Pancreatic acinar cells synthesize, package, and secrete digestive enzymes into the duodenum to aid in nutrient absorption and meet metabolic demands. When exposed to cellular stresses and insults, acinar cells undergo a dedifferentiation process termed acinar-ductal metaplasia (ADM). ADM lesions with oncogenic mutations eventually give rise to pancreatic ductal adenocarcinoma (PDAC). In healthy pancreata, the basic helix-loop-helix (bHLH) factors MIST1 and PTF1a coordinate an acinar-specific transcription network that maintains the highly developed differentiation status of the cells, protecting the pancreas from undergoing a transformative process. However, when MIST1 and PTF1a gene expression is silenced, cells are more prone to progress to PDAC. In this study, we tested whether induced MIST1 or PTF1a expression in PDAC cells could (i) re-establish the transcriptional program of differentiated acinar cells and (ii) simultaneously reduce tumor cell properties. As predicted, PTF1a induced gene expression of digestive enzymes and acinar-specific transcription factors, while MIST1 induced gene expression of vesicle trafficking molecules as well as activation of unfolded protein response components, all of which are essential to handle the high protein production load that is characteristic of acinar cells. Importantly, induction of PTF1a in PDAC also influenced cancer-associated properties, leading to a decrease in cell proliferation, cancer stem cell numbers, and repression of key ATP-binding cassette efflux transporters resulting in heightened sensitivity to gemcitabine. Thus, activation of pancreatic bHLH transcription factors rescues the acinar gene program and decreases tumorigenic properties in pancreatic cancer cells, offering unique opportunities to develop novel therapeutic intervention strategies for this deadly disease. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Value of p16(INK)⁴(a) in the pathology of invasive penile squamous cell carcinomas: A report of 202 cases.

PubMed

Cubilla, Antonio L; Lloveras, Belén; Alejo, Maria; Clavero, Omar; Chaux, Alcides; Kasamatsu, Elena; Monfulleda, Núria; Tous, Sara; Alemany, Laia; Klaustermeier, Joellen; Muñoz, Nubia; Quint, Wim; de Sanjose, Silvia; Bosch, Francisco Xavier

2011-02-01

One third to one half of penile squamous cell carcinomas (SCCs) are related to human papillomavirus (HPV) infection. Viral detection is usually carried out by polymerase chain reaction (PCR) or other molecular methods. In this study, we evaluated p16(INK)⁴(a) immunohistochemical expression, which is simpler and less costly, as a potential marker of high-risk HPV (HR-HPV) infection in penile SCC. We pathologically classified 202 invasive penile carcinomas and performed HPV genotyping by short PCR fragment (SPF)₁₀ PCR and p16(INK)⁴(a) immunohistochemistry. We also evaluated HPV and p16(INK)⁴(a) according to the histologic subtypes of penile SCC. Tumors depicting continuous p16(INK)⁴(a) immunostain in all neoplastic cells were considered positive. HPV and p16(INK)⁴(a) status were compared using classifier performances and concordance indexes. Evidence of HPV (low-risk and high-risk genotypes) was found in 63 cases (31%) by PCR. Fifty-three p16(INK)⁴(a)-positive cases were identified (26%). Overexpression of p16(INK)⁴(a) had a sensitivity of 67% and a specificity of 91% for defining the HPV status. Concordance indexes between p16(INK)⁴(a) and HPV status were high (≥78%) in general cases and in all histologic subtypes of penile SCC. The stain was useful in the differential diagnosis of basaloid and low-grade warty carcinomas. Low-risk HPV genotypes were found in 5 tumors, 4 of which were p16(INK)⁴(a) negative. Basaloid and nonbasaloid high-grade (grade 3) SCCs were more likely to be HR-HPV positive when compared with grades 1 to 2 tumors (P<0.000001 and 0.0417, respectively). p16(INK)⁴(a) overexpression was found to be a reliable marker for HR-HPV and a helpful tool in the differential diagnosis of low-grade verruciform and high-grade solid penile tumors. SCC variants depicting basaloid features were more likely to be HPV and p16(INK)⁴(a) positive than low-grade, keratinizing lesions. We also observed a tendency toward HPV positivity in high-grade nonbasaloid tumors. Our results indicated a concordance between HPV and p16(INK)⁴(a) status and this observation may have diagnostic and prognostic implications.

Current status of mucins in the diagnosis and therapy of cancer

PubMed Central

Rachagani, Satyanarayana; Torres, Maria P.; Moniaux, Nicolas; Batra, Surinder K.

2010-01-01

Mucins are the most abundant high molecular weight glycoproteins in mucus. Their nature and glycosylation content dictates the biochemical and biophysical properties of viscoelastic secretions, pointing out an important role in diverse biological functions, such as differentiation, cell adhesions, immune responses, and cell signaling. Mucins are expressed in tubular organs by specialized epithelial cells in the body. Their aberrant expression is well documented in a variety of inflammatory or malignant diseases. From a prognosis point of view, their expression and alterations in glycosylation are associated with the development and progression of malignant diseases. Therefore, mucins can be used as valuable markers to distinguish between normal and disease conditions. Indeed, this alteration in glycosylation patterns generates several epitopes in the oligosaccharide side chains that can be used as diagnostic and/or prognostic markers. Furthermore, these characteristic tumor-associated epitopes are extensively used as appropriate immunotargets of malignant epithelial cells. Therefore, in an effort to detect and treat cancer at the earliest stage possible, mucins are analyzed as potential markers of disease for diagnosis, progression, and for therapeutic purposes. In this review, we focused on the current status of the distribution of mucins in normal and pathologic conditions and their clinical use both in cancer diagnosis and therapeutics treatments. PMID:19904814

Imaging Mitochondrial Redox Potential and Its Possible Link to Tumor Metastatic Potential

PubMed Central

Li, Lin Z.

2012-01-01

Cellular redox states can regulate cell metabolism, growth, differentiation, motility, apoptosis, signaling pathways, and gene expressions etc. Growing body of literature suggest importance of redox status for cancer progression. While most studies on redox state were done on cells and tissue lysates, it is important to understand the role of redox state in tissue in vivo/ex vivo and image its heterogeneity. Redox scanning is a clinically-translatable method for imaging tissue mitochondrial redox potential with a submillimeter resolution. Redox scanning data in mouse models of human cancers demonstrate a correlation between mitochondrial redox state and tumor metastatic potential. I will discuss the significance of this correlation and possible directions for future research. PMID:22895837

Effect of lipoic acid and α-glyceryl-phosphoryl-choline on astroglial cell proliferation and differentiation in primary culture.

PubMed

Grasso, S; Bramanti, V; Tomassoni, D; Bronzi, D; Malfa, G; Traini, E; Napoli, M; Renis, M; Amenta, F; Avola, R

2014-01-01

Lipoic acid plays a crucial role as antioxidant and metabolic component of enzymes involved in glucose metabolism of different cell types. Choline alphoscerate (α-glyceryl-phosphoryl-choline [αGPC]) is a semisynthetic derivative of phosphatidylcholines representing, among acetilcholine precursors, a cholinergic drug. In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 or 21 DIV astrocyte cultures treated with 50 μM (+)lipoic acid or (+/-)lipoic acid and/or 10 mM αGPC for 24 hr. In addition, we evaluated the possible genoprotective effect by analysis of DNA status detected by alkaline comet assay. The addition of single drugs [(+)lipoic acid, (+/-)lipoic acid, or αGPC] induced an "upward modulation" of the expression of biomarkers used in our study. On the contrary, the cotreatment with either (+)lipoic acid + αGPC or (+/-)lipoic + αGPC surprisingly showed no significant modification or even a downregulation of the above-mentioned biomarkers. This latter finding demonstrated no additional effect after the cotreatment with both drugs with respect to the single treatments alone. Further studies are necessary to clarify the specific mechanism evoked by the processing of these neuroprotective agents in our in vitro models. Finally, these preliminary findings may represent a good tool with which to clarify the antioxidant and metabolic roles played by lipoic acid in proliferating and differentiating astroglial cell cultures, during an interactive cross-talk between glial and neuronal cells, after brain lesions or damage correlated with oxidative stress that may occur in some degenerative diseases. Copyright © 2013 Wiley Periodicals, Inc.

Nutritional status and CD4 cell counts in patients with HIV/AIDS receiving antiretroviral therapy.

PubMed

Santos, Ana Célia Oliveira dos; Almeida, Ana Maria Rampeloti

2013-01-01

Even with current highly active antiretroviral therapy, individuals with AIDS continue to exhibit important nutritional deficits and reduced levels of albumin and hemoglobin, which may be directly related to their cluster of differentiation 4 (CD4) cell counts. The aim of this study was to characterize the nutritional status of individuals with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and relate the findings to the albumin level, hemoglobin level and CD4 cell count. Patients over 20 years of age with AIDS who were hospitalized in a university hospital and were receiving antiretroviral therapy were studied with regard to clinical, anthropometric, biochemical and sociodemographic characteristics. Body mass index, percentage of weight loss, arm circumference, triceps skinfold and arm muscle circumference were analyzed. Data on albumin, hemoglobin, hematocrit and CD4 cell count were obtained from patient charts. Statistical analysis was performed using Fisher's exact test, Student's t-test for independent variables and the Mann-Whitney U-test. The level of significance was set to 0.05 (α = 5%). Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) 17.0 software for Windows. Of the 50 patients evaluated, 70% were male. The prevalence of malnutrition was higher when the definition was based on arm circumference and triceps skinfold measurement. The concentrations of all biochemical variables were significantly lower among patients with a body mass index of less than 18.5kg/m2. The CD4 cell count, albumin, hemoglobin and hematocrit anthropometric measures were directly related to each other. These findings underscore the importance of nutritional follow-up for underweight patients with AIDS, as nutritional status proved to be related to important biochemical alterations.

Stem cells in nephrology: present status and future.

PubMed

Watorek, Ewa; Klinger, Marian

2006-01-01

Stem cell biology is currently developing rapidly because of the potential therapeutic utility of stem cells. The ability to acquire any desired phenotype raises hope for regenerative therapies. Manipulation of these cells is a potentially valuable tool; however, the mechanisms of stem cell differentiation and plasticity are currently beyond our control. In the field of nephrology, the presence of adult kidney stem cells has been debated. Renal adult stem cells may be descendants of some early kidney progenitors, or may be derived from bone marrow. Evidence of a hematopoietic stem-cell contribution to renal repair encourages the possibility of bone marrow or stem cell transplantation as a means of treating autoimmune glomerulopathies. The transplantation of fetal kidney tissue containing renal progenitors, which then develop into functional nephrons, is a step towards renal regeneration. According to recent reports, the development of functional nephrons from human mesenchymal stem cells in rodent whole-embryo culture is possible. Establishing in vitro self organs from autologous stem cells would be a promising therapeutic solution in light of the shortage of allogenic organs and the unresolved problem of chronic allograft rejection.

Control of Aβ release from human neurons by differentiation status and RET signaling.

PubMed

Scholz, Diana; Chernyshova, Yana; Leist, Marcel

2013-01-01

Few studies have compared the processing of endogenous human amyloid precursor protein (APP) in younger and older neurons. Here, we characterized LUHMES cells as a human model to study Alzheimer's disease-related processes during neuronal maturation and aging. Differentiated LUHMES expressed and spontaneously processed APP via the secretase pathways, and they secreted amyloid β (Aβ) peptide. This was inhibited by cholesterol depletion or secretase inhibition, but not by block of tau phosphorylation. In vitro aged cells increased Aβ secretion without upregulation of APP or secretases. We identified the medium constituent glial cell line-derived neurotrophic factor (GDNF) as responsible for this effect. GDNF-triggered Aβ release was associated with rapid upregulation of the GDNF coreceptor "rearranged during transfection" (RET). Other direct (neurturin) or indirect (nerve growth factor) RET activators also increased Aβ, whereas different neurotrophins were ineffective. Downstream of RET, we found activation of protein kinase B (AKT) to be involved. Accordingly, inhibitors of the AKT regulator phosphatidylinositol-3-kinase completely blocked GDNF-triggered AKT phosphorylation and Aβ increase. This suggests that RET signaling affects Aβ release from aging neurons. Copyright © 2013 Elsevier Inc. All rights reserved.

Phytochemicals for breast cancer therapy: current status and future implications.

PubMed

Siddiqui, Jawed Akhtar; Singh, Aru; Chagtoo, Megha; Singh, Nidhi; Godbole, Madan Madhav; Chakravarti, Bandana

2015-01-01

Breast cancer is one of the most common malignancies among women, representing nearly 30% of newly diagnosed cancers every year. Till date, various therapeutic interventions, including surgery, chemotherapy, hormonal therapy, and radiotherapy are available and are known to cause a significant decline in the overall mortality rate. However, therapeutic resistance, recurrence and lack of treatment in metastasis are the major challenges that need to be addressed. Increasing evidence suggests the presence of cancer stem cells (CSCs) in heterogeneous population of breast tumors capable of selfrenewal and differentiation and is considered to be responsible for drug resistance and recurrence. Therefore, compound that can target both differentiated cancer cells, as well as CSCs, may provide a better treatment strategy. Due to safe nature of dietary agents and health products, investigators are introducing them into clinical trials in place of chemotherapeutic agents.This current review focuses on phytochemicals, mainly flavonoids that are in use for breast cancer therapy in preclinical phase. As phytochemicals have several advantages in breast cancer and cancer stem cells, new synthetic series for breast cancer therapy from analogues of most potent natural molecule can be developed via rational drug design approach.

Differential methylation of the oxytocin receptor gene in patients with anorexia nervosa: a pilot study.

PubMed

Kim, Youl-Ri; Kim, Jeong-Hyun; Kim, Mi Jeong; Treasure, Janet

2014-01-01

Recent studies in patients with anorexia nervosa suggest that oxytocin may be involved in the pathophysiology of anorexia nervosa. We examined whether there was evidence of variation in methylation status of the oxytocin receptor (OXTR) gene in patients with anorexia nervosa that might account for these findings. We analyzed the methylation status of the CpG sites in a region from the exon 1 to the MT2 regions of the OXTR gene in buccal cells from 15 patients and 36 healthy women using bisulfite sequencing. We further examined whether methylation status was associated with markers of illness severity or form. We identified six CpG sites with significant differences in average methylation levels between the patient and control groups. Among the six differentially methylated CpG sites, five showed higher than average methylation levels in patients than those in the control group (64.9-88.8% vs. 6.6-45.0%). The methylation levels of these five CpG sites were negatively associated with body mass index (BMI). BMI, eating disorders psychopathology, and anxiety were identified in a regression analysis as factors affecting the methylation levels of these CpG sites with more variation accounted for by BMI. Epigenetic misregulation of the OXTR gene may be implicated in anorexia nervosa, which may either be a mechanism linking environmental adversity to risk or may be a secondary consequence of the illness.

Differential Expression Profile of ZFX Variants Discriminates Breast Cancer Subtypes

PubMed

Pourkeramati, Fatemeh; Asadi, Malek Hossein; Shakeri, Shahryar; Farsinejad, Alireza

2018-05-13

ZFX is a transcriptional regulator in embryonic stem cells that plays an important role in pluripotency and self-renewal. ZFX is widely expressed in pluripotent stem cells and is down-regulated during differentiation of embryonic stem cells. ZFX has five different variants that encode three different protein isoforms. While several reports have determined the overexpression of ZFX in a variety of somatic cancers, the expression of ZFX-spliced variants in cancer cells is not well-understood. We investigated the expression of ZFX variants in a series of breast cancer tissues and cell lines using quantitative PCR. The expression of ZFX variant 1/3 was higher in tumor tissue compared to marginal tissue. In contrast, the ZFX variant 5 was down-regulated in tumor tissues. While the ZFX variant 1/3 and ZFX variant 5 expression significantly increased in low-grade tumors, ZFX variant 4 was strongly expressed in high-grade tumors and demonstrating lymphatic invasion. In addition, our result revealed a significant association between the HER2 status and the expression of ZFX-spliced variants. Our data suggest that the expression of ZFX-spliced transcripts varies between different types of breast cancer and may contribute to their tumorigenesis process. Hence, ZFX-spliced transcripts could be considered as novel tumor markers with a probable value in diagnosis, prognosis, and therapy of breast cancer.

Does Simulated Spaceflight Modify Epigenetic Status During Bone Remodeling?

NASA Technical Reports Server (NTRS)

Thomas, Nicholas J.; Stevick, Rebecca J.; Tran, Luan H.; Nalavadi, Mohit O.; Almeida, Eduardo A.C.; Globus, Ruth K.; Alwood, Joshua S.

2015-01-01

Little is known about the effects of spaceflight conditions on epigenetics. The term epigenetics describes changes to the genome that can affect expression of a gene without changes to the sequence of DNA. Epigenetic processes are thought to underlie cellular differentiation, where transcription of specific genes occurs in response to key stimuli, and may be heritable - passing from one cell to its daughter cell. We hypothesize that the mechanical environment during spaceflight, namely microgravity-induced weightlessness or exercise regulate gene expression in the osteoblast-lineage cells both to control bone formation by osteoblasts and bone resorption by osteoclasts, which continually shapes bone structure throughout life. Similarly we intend to evaluate how radiation regulates these same bone cell activity and differentiation related genes. We further hypothesize that the regulation in bone cell gene expression is at least partially controlled through epigenetic mechanisms of methylation or small non-coding RNA (microRNAs). We have acquired preliminary data suggesting that global genome methylation is modified in response to axial compression of the tibia - a model of exercise. We intend to pursue these hypotheses wherein we will evaluate changes in gene expression and, congruently, changes in epigenetic state in bones from mice subjected to the aforementioned conditions: hindlimb unloading to simulate weightlessness, axial compression of the tibia, or radiation exposure in order to gain insight into the role of epigenetics in spaceflight-induced bone loss.

ATR inhibition broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status

PubMed Central

Huntoon, Catherine J.; Flatten, Karen S.; Wahner Hendrickson, Andrea E.; Huehls, Amelia M.; Sutor, Shari L.; Kaufmann, Scott H.; Karnitz, Larry M.

2013-01-01

Replication stress and DNA damage activate the ATR-CHK1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and CHK1 kinases in ovarian cancer cells using genetic and pharmacological inhibitors of in combination with cisplatin, topotecan, gemcitabine and the poly(ADP-ribose)-polymerase (PARP) inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNAi-mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan and gemcitabine each activated CHK1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 or the CHK1 inhibitor MK-8776 blocked ATR-mediated CHK1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-CHK1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. Additionally, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and CHK1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents, and that CHK1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-CHK1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells. PMID:23548269

Differences in Cell Division Rates Drive the Evolution of Terminal Differentiation in Microbes

PubMed Central

Matias Rodrigues, João F.; Rankin, Daniel J.; Rossetti, Valentina; Wagner, Andreas; Bagheri, Homayoun C.

2012-01-01

Multicellular differentiated organisms are composed of cells that begin by developing from a single pluripotent germ cell. In many organisms, a proportion of cells differentiate into specialized somatic cells. Whether these cells lose their pluripotency or are able to reverse their differentiated state has important consequences. Reversibly differentiated cells can potentially regenerate parts of an organism and allow reproduction through fragmentation. In many organisms, however, somatic differentiation is terminal, thereby restricting the developmental paths to reproduction. The reason why terminal differentiation is a common developmental strategy remains unexplored. To understand the conditions that affect the evolution of terminal versus reversible differentiation, we developed a computational model inspired by differentiating cyanobacteria. We simulated the evolution of a population of two cell types –nitrogen fixing or photosynthetic– that exchange resources. The traits that control differentiation rates between cell types are allowed to evolve in the model. Although the topology of cell interactions and differentiation costs play a role in the evolution of terminal and reversible differentiation, the most important factor is the difference in division rates between cell types. Faster dividing cells always evolve to become the germ line. Our results explain why most multicellular differentiated cyanobacteria have terminally differentiated cells, while some have reversibly differentiated cells. We further observed that symbioses involving two cooperating lineages can evolve under conditions where aggregate size, connectivity, and differentiation costs are high. This may explain why plants engage in symbiotic interactions with diazotrophic bacteria. PMID:22511858

Genes Related to Antiviral Activity, Cell Migration, and Lysis Are Differentially Expressed in CD4+ T Cells in Human T Cell Leukemia Virus Type 1-Associated Myelopathy/Tropical Spastic Paraparesis Patients

PubMed Central

Pinto, Mariana Tomazini; Malta, Tathiane Maistro; Rodrigues, Evandra Strazza; Pinheiro, Daniel Guariz; Panepucci, Rodrigo Alexandre; Malmegrim de Farias, Kelen Cristina Ribeiro; Sousa, Alessandra De Paula; Takayanagui, Osvaldo Massaiti; Tanaka, Yuetsu; Covas, Dimas Tadeu

2014-01-01

Abstract Human T cell leukemia virus type 1 (HTLV-1) preferentially infects CD4+ T cells and these cells play a central role in HTLV-1 infection. In this study, we investigated the global gene expression profile of circulating CD4+ T cells from the distinct clinical status of HTLV-1-infected individuals in regard to TAX expression levels. CD4+ T cells were isolated from asymptomatic HTLV-1 carrier (HAC) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients in order to identify genes involved in HAM/TSP development using a microarray technique. Hierarchical clustering analysis showed that healthy control (CT) and HTLV-1-infected samples clustered separately. We also observed that the HAC and HAM/TSP groups clustered separately regardless of TAX expression. The gene expression profile of CD4+ T cells was compared among the CT, HAC, and HAM/TSP groups. The paxillin (Pxn), chemokine (C-X-C motif ) receptor 4 (Cxcr4), interleukin 27 (IL27), and granzyme A (Gzma) genes were differentially expressed between the HAC and HAM/TSP groups, regardless of TAX expression. The perforin 1 (Prf1) and forkhead box P3 (Foxp3) genes were increased in the HAM/TSP group and presented a positive correlation to the expression of TAX and the proviral load (PVL). The frequency of CD4+FOXP3+ regulatory T cells (Treg) was higher in HTLV-1-infected individuals. Foxp3 gene expression was positively correlated with cell lysis-related genes (Gzma, Gzmb, and Prf1). These findings suggest that CD4+ T cell activity is distinct between the HAC and HAM/TSP groups. PMID:24041428

Use of Hes1-GFP reporter mice to assess activity of the Hes1 promoter in bone cells under chronic inflammation

PubMed Central

Zhang, Hengwei; Sun, Wen; Li, Xing; Wang, Mengmeng; Boyce, Brendan F; Hilton, Matthew J; Xing, Lianping

2016-01-01

Notch signaling plays a critical role in maintaining bone homeostasis partially by controlling the formation of osteoblasts from mesenchymal stem cells (MSCs). We reported that TNF activates Notch signaling in MSCs which inhibits osteoblast differentiation in TNF transgenic (TNF-Tg) mice, a mouse model of chronic inflammatory arthritis. In the current study, we used Hes1-GFP and Hes1-GFP/TNF-Tg mice to study the distribution and dynamic change of Notch active cells in normal and inflammatory bone loss and mechanisms mediating their enhanced proliferation. We found that Hes1-GFP+ cells are composed of cells expressing mesenchymal, hematopoietic and endothelial surface markers. CD45−/Hes1-GFP+ cells express high levels of mesenchymal markers and form CFU-F and CFU-ALP colonies. Expansion of CFU-F colonies is associated with a rapid increase in Hes1-GFP+ cell numbers and their GFP intensity. The GFP signal is lost when a CFU-F colony differentiates into an ALP+ osteoblast colony. TNF increases the numbers of CD45−/Hes1-GFP+ cells, which are stained negatively for osteoblast marker osteocalcin and localized adjacent to endosteal and trabecular bone surfaces. CD45−/Hes1-GFP+ cells in Hes1-GFP/TNF-Tg mice have increased BrdU incorporation and PDGFRβ levels. TNF increases the number of proliferating Hes1-GFP+ cells, which is prevented by a specific PDGFRβ inhibitor. Notch inhibition blocks TNF-mediated PDGFRβ expression and cell proliferation. Thus, TNF-induced MSC proliferation is mediated by PDGFRβ signal, which works at downstream of Notch. Hes1-GFP mice can be used to assess the activation status of Notch in bone cells. PMID:27269414

Use of Hes1-GFP reporter mice to assess activity of the Hes1 promoter in bone cells under chronic inflammation.

PubMed

Zhang, Hengwei; Sun, Wen; Li, Xing; Wang, Mengmeng; Boyce, Brendan F; Hilton, Matthew J; Xing, Lianping

2016-09-01

Notch signaling plays a critical role in maintaining bone homeostasis partially by controlling the formation of osteoblasts from mesenchymal stem cells (MSCs). We reported that TNF activates Notch signaling in MSCs which inhibits osteoblast differentiation in TNF transgenic (TNF-Tg) mice, a mouse model of chronic inflammatory arthritis. In the current study, we used Hes1-GFP and Hes1-GFP/TNF-Tg mice to study the distribution and dynamic change of Notch active cells in normal and inflammatory bone loss and mechanisms mediating their enhanced proliferation. We found that Hes1-GFP+ cells are composed of cells expressing mesenchymal, hematopoietic and endothelial surface markers. CD45-/Hes1-GFP+ cells express high levels of mesenchymal markers and form CFU-F and CFU-ALP colonies. Expansion of CFU-F colonies is associated with a rapid increase in Hes1-GFP+ cell numbers and their GFP intensity. The GFP signal is lost when a CFU-F colony differentiates into an ALP+ osteoblast colony. TNF increases the numbers of CD45-/Hes1-GFP+ cells, which are stained negatively for osteoblast marker osteocalcin and localized adjacent to endosteal and trabecular bone surfaces. CD45-/Hes1-GFP+ cells in Hes1-GFP/TNF-Tg mice have increased BrdU incorporation and PDGFRβ levels. TNF increases the number of proliferating Hes1-GFP+ cells, which is prevented by a specific PDGFRβ inhibitor. Notch inhibition blocks TNF-mediated PDGFRβ expression and cell proliferation. Thus, TNF-induced MSC proliferation is mediated by PDGFRβ signal, which works at downstream of Notch. Hes1-GFP mice can be used to assess the activation status of Notch in bone cells. Copyright © 2016 Elsevier Inc. All rights reserved.

High Expression of FAM83B Predicts Poor Prognosis in Patients with Pancreatic Ductal Adenocarcinoma and Correlates with Cell Cycle and Cell Proliferation

PubMed Central

Shen, Chao-qin; Yan, Ting-Ting; Liu, Wei; Zhu, Xiao-qiang; Tian, Xiang-long; Fu, Xue-liang; Hua, Rong; Zhang, Jun-feng; Huo, Yan-miao; Liu, De-jun; Yang, Jian-yu; Sun, Yong-Wei; Fang, Jing-Yuan; Chen, Hao-Yan; Hong, Jie

2017-01-01

FAM83B (family with sequence similarity 83, member B) seems to emerge as a new class of players involved in the development of a variety of malignant tumors. Yet the molecular mechanisms are not well understood. The present study is intended to investigate the expression and function of FAM83B in pancreatic ductal adenocarcinoma (PDAC). In this study, we found that the expression of FAM83B was significantly increased both in PDAC cell lines and PDAC tumor tissues. FAM83B expression was positively related with advanced clinical stage and poor vital status. Higher FAM83B expression predicted shorter overall survival in PDAC patients, regardless of lymphatic metastasis status and histological differentiation. Actually, FAM83B may act as an independent prognostic indicator as well. What's more, down-regulation of FAM83B in PDAC cells contributed to G0/G1 phase arrest and inhibition of cell proliferation. Finally, a subcutaneous xenograft model indicated that knockdown of FAM83B significantly reduced the tumor volume in vivo. Our findings have provided supporting evidence for the potential molecular biomarker role of FAM83B in PDAC. It's of great interest and broad significance to target FAM83B in PDAC, which may conduce to develop a meaningful and effective strategy in the diagnosis and treatment of PDAC. PMID:29158787

Rice pectin methylesterase inhibitor28 (OsPMEI28) encodes a functional PMEI and its overexpression results in a dwarf phenotype through increased pectin methylesterification levels.

PubMed

Nguyen, Hong Phuong; Jeong, Ho Young; Jeon, Seung Ho; Kim, Donghyuk; Lee, Chanhui

2017-01-01

Pectin methylesterases (PMEs, EC 3.1.1.11) belonging to carbohydrate esterase family 8 cleave the ester bond between a galacturonic acid and an methyl group and the resulting change in methylesterification level plays an important role during the growth and development of plants. Optimal pectin methylesterification status in each cell type is determined by the balance between PME activity and post-translational PME inhibition by PME inhibitors (PMEIs). Rice contains 49 PMEIs and none of them are functionally characterized. Genomic sequence analysis led to the identification of rice PMEI28 (OsPMEI28). Recombinant OsPMEI28 exhibited inhibitory activity against commercial PME protein with the highest activities detected at pH 8.5. Overexpression of OsPMEI28 in rice resulted in an increased level of cell wall bound methylester groups and differential changes in the composition of cell wall neutral monosaccharides and lignin content in culm tissues. Consequently, transgenic plants overexpressing OsPMEI28 exhibited dwarf phenotypes and reduced culm diameter. Our data indicate that OsPMEI28 functions as a critical structural modulator by regulating the degree of pectin methylesterification and that an impaired status of pectin methylesterification affects physiochemical properties of the cell wall components and causes abnormal cell extensibility in rice culm tissues. Copyright © 2016 Elsevier GmbH. All rights reserved.

Genome-wide gene expression effects in B6C3F1 mouse intestinal epithelia following 7 and 90days of exposure to hexavalent chromium in drinking water.

PubMed

Kopec, Anna K; Kim, Suntae; Forgacs, Agnes L; Zacharewski, Timothy R; Proctor, Deborah M; Harris, Mark A; Haws, Laurie C; Thompson, Chad M

2012-02-15

Chronic administration of high doses of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) elicits alimentary cancers in mice. To further elucidate key events underlying tumor formation, a 90-day drinking water study was conducted in B6C3F1 mice. Differential gene expression was examined in duodenal and jejunal epithelial samples following 7 or 90days of exposure to 0, 0.3, 4, 14, 60, 170 or 520mg/L SDD in drinking water. Genome-wide microarray analyses identified 6562 duodenal and 4448 jejunal unique differentially expressed genes at day 8, and 4630 and 4845 unique changes, respectively, in the duodenum and jejunum at day 91. Comparative analysis identified significant overlap in duodenal and jejunal differential gene expression. Automated dose-response modeling identified >80% of the differentially expressed genes exhibited sigmoidal dose-response curves with EC(50) values ranging from 10 to 100mg/L SDD. Only 16 genes satisfying the dose-dependent differential expression criteria had EC(50) values <10mg/L SDD, 3 of which were regulated by Nrf2, suggesting oxidative stress in response to SDD at low concentrations. Analyses of differentially expressed genes identified over-represented functions associated with oxidative stress, cell cycle, lipid metabolism, and immune responses consistent with the reported effects on redox status and histopathology at corresponding SDD drinking water concentrations. Collectively, these data are consistent with a mode of action involving oxidative stress and cytotoxicity as early key events. This suggests that the tumorigenic effects of chronic Cr(VI) oral exposure likely require chronic tissue damage and compensatory epithelial cell proliferation. Copyright © 2011 Elsevier Inc. All rights reserved.

Genome-wide gene expression effects in B6C3F1 mouse intestinal epithelia following 7 and 90 days of exposure to hexavalent chromium in drinking water

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kopec, Anna K.; Kim, Suntae; Forgacs, Agnes L.

2012-02-15

Chronic administration of high doses of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) elicits alimentary cancers in mice. To further elucidate key events underlying tumor formation, a 90-day drinking water study was conducted in B6C3F1 mice. Differential gene expression was examined in duodenal and jejunal epithelial samples following 7 or 90 days of exposure to 0, 0.3, 4, 14, 60, 170 or 520 mg/L SDD in drinking water. Genome-wide microarray analyses identified 6562 duodenal and 4448 jejunal unique differentially expressed genes at day 8, and 4630 and 4845 unique changes, respectively, in the duodenum and jejunum at day 91.more » Comparative analysis identified significant overlap in duodenal and jejunal differential gene expression. Automated dose–response modeling identified > 80% of the differentially expressed genes exhibited sigmoidal dose–response curves with EC{sub 50} values ranging from 10 to 100 mg/L SDD. Only 16 genes satisfying the dose-dependent differential expression criteria had EC{sub 50} values < 10 mg/L SDD, 3 of which were regulated by Nrf2, suggesting oxidative stress in response to SDD at low concentrations. Analyses of differentially expressed genes identified over-represented functions associated with oxidative stress, cell cycle, lipid metabolism, and immune responses consistent with the reported effects on redox status and histopathology at corresponding SDD drinking water concentrations. Collectively, these data are consistent with a mode of action involving oxidative stress and cytotoxicity as early key events. This suggests that the tumorigenic effects of chronic Cr(VI) oral exposure likely require chronic tissue damage and compensatory epithelial cell proliferation. Highlights: ► Mouse small intestine gene expression is highly responsive to hexavalent chromium [Cr(VI)]. ► Cr(VI) elicits more differential gene expression after 7 days of exposure than 90 days of exposure. ► Oral exposure to Cr(VI) leads to oxidative stress, cell cycle, lipid and immune dysregulation. ► Cr(VI) elicits dose-dependent changes in gene expression with an overall median EC{sub 50} of 47 mg/L SDD.« less

Adaptive Regulation of Osteopontin Production by Dendritic Cells Through the Bidirectional Interaction With Mesenchymal Stromal Cells.

PubMed

Scutera, Sara; Salvi, Valentina; Lorenzi, Luisa; Piersigilli, Giorgia; Lonardi, Silvia; Alotto, Daniela; Casarin, Stefania; Castagnoli, Carlotta; Dander, Erica; D'Amico, Giovanna; Sozzani, Silvano; Musso, Tiziana

2018-01-01

Mesenchymal stromal cells (MSCs) exert immunosuppressive effects on immune cells including dendritic cells (DCs). However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN), a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β). OPN induction required cell-to-cell contact, mediated at least in part, by β1 integrin (CD29). Conversely, activated MSCs inhibited the release of OPN via the production of soluble factors with a major role played by Prostaglandin E 2 (PGE 2 ). Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE 2 inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM) hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c + ) are a small percentage of BM cells, we demonstrated colocalization of CD271 + MSCs with CD1c + DCs in normal and myelodysplastic BM. OPN reactivity was observed in occasional CD1c + cells in the proximity of CD271 + MSCs. Altogether, these results candidate OPN as a signal modulated by MSCs according to their activation status and involved in DC regulation of MSC differentiation.

The promising potential of menstrual stem cells for antenatal diagnosis and cell therapy.

PubMed

Khoury, Maroun; Alcayaga-Miranda, Francisca; Illanes, Sebastián E; Figueroa, Fernando E

2014-01-01

Menstrual-derived stem cells (MenSCs) are a new source of mesenchymal stem cells isolated from the menstrual fluid. Currently, there is a growing interest in their clinical potential due to fact that they are multipotent, highly proliferative, and easy to obtain in a non-invasive manner. Sampling can be repeated periodically in a simplified and reproducible manner devoid of complications that no existing cell source can match. MenSCs are also free of ethical dilemmas, and display novel properties with regard to presently known adult derived stem cells. This review details their distinctive biological properties regarding immunophenotype and function, proliferation rate, differentiation potential, and paracrine effects mediated by secreted factors. Their possible role in antenatal diagnosis is also discussed. While more insight on their immunomodulatory and diagnostic properties is needed, the impact of clinical and epidemiological factors, such as age, use of contraceptives, or hormonal status still requires further investigations to properly assess their current and future use in clinical application and diagnosis.

Research progress on the proliferation and differentiation of

NASA Astrophysics Data System (ADS)

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

Cancer stem cell markers in patterning differentiation and in prognosis of oral squamous cell carcinoma.

PubMed

Mohanta, Simple; Siddappa, Gangotri; Valiyaveedan, Sindhu Govindan; Dodda Thimmasandra Ramanjanappa, Ravindra; Das, Debashish; Pandian, Ramanan; Khora, Samanta Sekhar; Kuriakose, Moni Abraham; Suresh, Amritha

2017-06-01

Differentiation is a major histological parameter determining tumor aggressiveness and prognosis of the patient; cancer stem cells with their slow dividing and undifferentiated nature might be one of the factors determining the same. This study aims to correlate cancer stem cell markers (CD44 and CD147) with tumor differentiation and evaluate their subsequent effect on prognosis. Immunohistochemical analysis in treatment naïve oral cancer patients (n = 53) indicated that the expression of CD147 was associated with poorly differentiated squamous cell carcinoma and moderately differentiated squamous cell carcinoma (p < 0.01). Furthermore, co-expression analysis showed that 45% each of moderately differentiated squamous cell carcinoma and poorly differentiated squamous cell carcinoma patients were CD44 high /CD147 high as compared to only 10% of patients with well-differentiated squamous cell carcinoma. A three-way analysis indicated that differentiation correlated with recurrence and survival (p < 0.05) in only the patients with CD44 high /CD147 high cohort. Subsequently, relevance of these cancer stem cell markers in patterning the differentiation characteristics was evaluated in oral squamous cell carcinoma cell lines originating from different grades of oral cancer. Flowcytometry-based analysis indicated an increase in CD44 + /CD147 + cells in cell lines of poorly differentiated squamous cell carcinoma (94.35 ± 1.14%, p < 0.001) and moderately differentiated squamous cell carcinoma origin (93.49 ± 0.47%, p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin (23.12% ± 0.49%). Expression profiling indicated higher expression of cancer stem cell and epithelial-mesenchymal transition markers in SCC029B (poorly differentiated squamous cell carcinoma originated; p ≤ 0.001), which was further translated into increased spheroid formation, migration, and invasion (p < 0.001) as compared to cell line of well-differentiated squamous cell carcinoma origin. This study suggests that CD44 and CD147 together improve the prognostic efficacy of tumor differentiation; in vitro results further point out that these markers might be determinant of differentiation characteristics, imparting properties of increased self-renewal, migration, and invasion.

In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

PubMed Central

Yamauchi, Kaori; Hasegawa, Kouichi; Chuma, Shinichiro; Nakatsuji, Norio; Suemori, Hirofumi

2009-01-01

Background Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. Methods and Findings To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. Conclusion VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates. PMID:19399191

Differential neurogenic effects of casein-derived opioid peptides on neuronal stem cells: implications for redox-based epigenetic changes.

PubMed

Trivedi, Malav; Zhang, Yiting; Lopez-Toledano, Miguel; Clarke, Andrew; Deth, Richard

2016-11-01

Food-derived peptides, such as β-casomorphin BCM7, have potential to cross the gastrointestinal tract and blood-brain barrier and are associated with neurological disorders and neurodevelopmental disorders. We previously established a novel mechanism through which BCM7 affects the antioxidant levels in neuronal cells leading to inflammatory consequences. In the current study, we elucidated the effects of casein-derived peptides on neuronal development by using the neurogenesis of neural stem cells (NSCs) as an experimental model. First, the transient changes in intracellular thiol metabolites during NSC differentiation (neurogenesis) were investigated. Next, the neurogenic effects of food-derived opioid peptides were measured, along with changes in intracellular thiol metabolites, redox status and global DNA methylation levels. We observed that the neurogenesis of NSCs was promoted by human BCM7 to a greater extent, followed by A2-derived BCM9 in contrast to bovine BCM7, which induced increased astrocyte formation. The effect was most apparent when human BCM7 was administered for 1day starting on 3days postplating, consistent with immunocytochemistry. Furthermore, neurogenic changes regulated by bovine BCM7 and morphine were associated with an increase in the glutathione/glutathione disulfide ratio and a decrease in the S-adenosylmethionine/S-adenosylhomocysteine ratio, indicative of changes in the redox and the methylation states. Finally, bovine BCM7 and morphine decreased DNA methylation in differentiating NSCs. In conclusion, these results suggest that food-derived opioid peptides and morphine regulated neurogenesis and differentiation of NSCs through changes in the redox state and epigenetic regulation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The differentiation directions of the bone marrow stromal cells under modeling microgravity

NASA Astrophysics Data System (ADS)

Nesterenko, Olga; Rodionova, Natalia; Katkova, Olena

Within experiments on rats simulating microgravity by base load remove from back limbs (duration of the experiment 1,5 months) on marrow stromal cells cultures (ex vivo, in vitro) comprising osteogenic cells-predecessors, extracted from femurs, studied their peculiarities of the colony formation ablity, the cell structure, some cytological and ultra-structural characteristics and differentiation direction. It was found that that under microgravity conditions there is a decline of the stromal cells colony formation intensity, decrease of the colonies size and cells mitotic activity that indicates decrease of their growth potential. Both in control and in experiment the colonies were presented by population of low-differentiated cells, differentiated cells and mature cells. The comparative cytological and morphometric analysis have shown that the studied stromal cells in colonies have the smaller sizes, more elongated shape, and higher nucleocytoplasmic ratio. Cells composition in the experiment colonies is reliably different by the ratio of the low-differentiating to being differentiated cells; a ratio of low-differentiated to already differentiated cells; ratio of differentiated cells to total number of all cells. In comparison with control group, amount of the cells passed trough a differentiation stage and mature cells in colonies is decreased by 3 to 4 times. Among the differentiated stromal cells in colonies increasing amount of adipocytes was revealed. The analysis of electron microscope microphotographs showed that in osteogenic cells differentiated under microgravity conditions, there is a reduction of the specific volume of a granular endoplasmic reticulum, Golgi's complex and quantity of nuclei reduction that indicates depression of the specific biosyntheses process intensity in cells. The increase of lysosomes and myelinic structures quantity is linked to organelles partial reduction. Consolidation of mitochondrias is an evidence of the cells’ energy metabolism disorder. In differentiated cells, disorganization and a cytoskeleton destruction was observed. Results showed that under microgravity conditions proliferative and differentiation (including osteogenic) potentialities of low-differentiated marrow stromal cells decreased, induction of their adipocytic differentiation was observes as well. Obtained results make a new contribution into gravitation sensitivity mechanisms understanding for stromal cells of the bone marrow which contain osteogenic cells- predecessors, features of the osteoporosis development.

HIV-specific CD8+ T cells: serial killers condemned to die?

PubMed

Petrovas, Constantinos; Mueller, Yvonne M; Katsikis, Peter D

2004-04-01

An increasing body of evidence supports a key role for cytotoxic CD8+ T cells (CTL) in controlling HIV infection. Although a vigorous HIV-specific CD8+ T cell response is raised during the primary infection, these cells ultimately fail to control virus and prevent disease progression. The failure of CTL to control HIV infection has been attributed to a number of strategies HIV employs to evade the immune system. Recently, intrinsic defects in the CTL themselves have been proposed to contribute to the failure of CTL to control HIV. HIV-specific CD8+ T cells differ in their effector/memory phenotype from other virus-specific CD8+ T cells indicating that their differentiation status differs. This altered differentiation may affect effector functions as well as homing properties of these cells. Other studies have indicated that activation of HIV-specific CTL may be impaired and this contributes to their dysfunction. The effector function of these CTL may also be affected. There are conflicting reports about their ability to kill, whereas IFNgamma production does not appear to be impaired in these cells. In this review we focus on recent work indicating that apoptosis may be an important mechanism through which HIV evades the CTL response. In particular, HIV-specific CD8+ T cells are highly susceptible to CD95/Fas-induced apoptosis. This leads to the hypothesis that virus-specific cytotoxic T cells can be eliminated upon binding CD95L/FasL on HIV-infected cells. Understanding the intrinsic defects of CTL in HIV infection could lead to new therapeutic strategies and optimized vaccination protocols that enhance the HIV-specific cytotoxic response.

Comparative marker analysis after isolation and culture of testicular cells from the immature marmoset.

PubMed

Albert, Silvia; Wistuba, Joachim; Eildermann, Katja; Ehmcke, Jens; Schlatt, Stefan; Gromoll, Joerg; Kossack, Nina

2012-01-01

The marmoset monkey is a valuable model in reproductive medicine. While previous studies have evaluated germ cell dynamics in the postnatal marmoset, the features of testicular somatic cells remain largely unknown. Therefore, the aim of this study was to establish marmoset-specific markers for Sertoli and peritubular cells (PTCs) and to compare protocols for the enrichment and culture of testicular cell types. Immunohistochemistry of Sertoli and PTC-specific markers - anti-müllerian hormone (AMH), vimentin (VIM), α-smooth muscle actin (SMA) - was performed and corresponding RNA expression profiles were established by quantitative PCR analysis (SOX9,AMH, FSHR,VIM, and SMA). For these analyses, testicular tissue from newborn (n = 4), 8-week-old (n = 4) and adult (n = 3) marmoset monkeys was used. Protocols for the enrichment and culture of testicular cell fractions from the 8-week-old marmoset monkeys (n = 3) were evaluated and cells were analyzed using germ cell- and somatic cell-specific markers. The expression of AMH, VIM and SMA reflects the proportion and differentiation status of Sertoli and PTCs at the RNA and the protein levels. While applied protocols did not support the propagation of germ cells in vitro, our analyses revealed that PTCs maintain their proliferative potential and constitute the dominant cell type after short- and long-term culture. Expression of functionally meaningful testicular somatic markers is similar in the human and the marmoset monkey, indicating that this primate can indeed be used as model for human testicular development. The PTC culture system established in this study will facilitate the identification of factors influencing male sex differentiation and spermatogenesis. Copyright © 2012 S. Karger AG, Basel.

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

PubMed

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.

TCR-pMHC encounter differentially regulates transcriptomes of tissue-resident CD8 T cells.

PubMed

Yoshizawa, Akihiro; Bi, Kevin; Keskin, Derin B; Zhang, Guanglan; Reinhold, Bruce; Reinherz, Ellis L

2018-01-01

To investigate the role of TCR-pMHC interaction in regulating lung CD8 tissue-resident T cell (T R ) differentiation, polyclonal responses were compared against NP 366-374 /D b and PA 224-233 /D b , two immunodominant epitopes that arise during influenza A infection in mice. Memory niches distinct from iBALTs develop within the lamina propria, supporting CD103 + and CD103 - CD8 T R generation and intraepithelial translocation. Gene set enrichment analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) identify dominant TCR, adherens junction, RIG-I-like and NOD-like pattern recognition receptor as well as TGF-β signaling pathways and memory signatures among PA 224-233 /D b T cells consistent with T resident memory (T RM ) status. In contrast, NP 366-374 /D b T cells exhibit enrichment of effector signatures, upregulating pro-inflammatory mediators even among T RM . While NP 366-374 /D b T cells manifest transcripts linked to canonical exhaustion pathways, PA 224-233 /D b T cells exploit P2rx7 purinoreceptor attenuation. The NP 366-374 /D b CD103 + subset expresses the antimicrobial lactotransferrin whereas PA 224-233 /D b CD103 + utilizes pore-forming mpeg-1, with <22% of genes correspondingly upregulated in CD103 + (or CD103 - ) subsets of both specificities. Thus, TCR-pMHC interactions among T R and antigen presenting cells in a tissue milieu strongly impact CD8 T cell biology. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

PubMed Central

Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Park, Ji-Sung; Lee, Seung-Chan; Baregundi Subbarao, Raghavendra; Lee, Sung-Lim; Park, Bong-Wook; King, William Allan; Rho, Gyu-Jin

2015-01-01

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson's correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson's correlation revealed high correlation (r > 0.9) between NF3 and NFopt. Overall, the present study showed that HMBS, YWHAZ, SDHA, and TBP are suitable reference genes for qRT-PCR in porcine MSCs. PMID:25972899

EMMPRIN expression in oral squamous cell carcinomas: correlation with tumor proliferation and patient survival.

PubMed

Monteiro, Luís Silva; Delgado, Maria Leonor; Ricardo, Sara; Garcez, Fernanda; do Amaral, Barbas; Pacheco, José Júlio; Lopes, Carlos; Bousbaa, Hassan

2014-01-01

The aim of our study was to explore the clinicopathological and prognostic significance of extracellular matrix metalloproteinase inducer (EMMPRIN) expression in oral squamous cell carcinomas (OSCC), and its relation with the proliferative tumor status of OSCC. We examined EMMPRIN and Ki-67 proteins expression by immunohistochemistry in 74 cases with OSCC. Statistical analysis was conducted to examine their clinicopathological and prognostic significance in OSCC. EMMPRIN membrane expression was observed in all cases, with both membrane and cytoplasmic tumor expression in 61 cases (82.4%). EMMPRIN overexpression was observed in 56 cases (75.7%). Moderately or poorly differentiated tumors showed EMMPRIN overexpression more frequently than well-differentiated tumors (P = 0.002). Overexpression of EMMPRIN was correlated with high Ki-67 expression (P = 0.004). In the multivariate analysis, EMMPRIN overexpression reveals an adverse independent prognostic value for cancer-specific survival (CSS) (P = 0.034). Our results reveal that EMMPRIN protein is overexpressed in more than two-thirds of OSCC cases, especially in high proliferative and less differentiated tumors. The independent value of EMMPRIN overexpression in CSS suggests that this protein could be used as an important biological prognostic marker for patients with OSCC. Moreover, the high expression of EMMPRIN makes it a possible therapeutic target in OSCC patients.

Thyroid hormone actions on male reproductive system of teleost fish.

PubMed

Tovo-Neto, Aldo; da Silva Rodrigues, Maira; Habibi, Hamid R; Nóbrega, Rafael Henrique

2018-04-17

Thyroid hormones (THs) play important roles in the regulation of many biological processes of vertebrates, such as growth, metabolism, morphogenesis and reproduction. An increasing number of studies have been focused on the involvement of THs in the male reproductive system of vertebrates, in particular of fish. Therefore, this mini-review aims to summarize the main findings on THs role in male reproductive system of fish, focusing on sex differentiation, testicular development and spermatogenesis. The existing data in the literature have demonstrated that THs exert their roles at the different levels of the hypothalamic-pituitary-gonadal (HPG) axis. In general a positive correlation has been shown between THs and fish reproductive status; where THs are associated with testicular development, growth and maturation. Recently, the molecular mechanisms underlying the role of THs in spermatogenesis have been unraveled in zebrafish testis. THs promote germ cell proliferation and differentiation by increasing a stimulatory growth factor of spermatogenesis produced by Sertoli cells. In addition, THs enhanced the gonadotropin-induced androgen release in zebrafish testis. Next to their functions in the adult testis, THs are involved in the gonadal sex differentiation through modulating sex-related gene expression, and testicular development via regulation of Sertoli cell proliferation. In conclusion, this mini-review showed that THs modulate the male reproductive system during the different life stages of fish. The physiological and molecular mechanisms showed a link between the thyroid and reproduction, suggesting a possibly co-evolution and interdependence of these two systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Microfluidic engineering of neural stem cell niches for fate determination

PubMed Central

Ma, Jingyun; Li, Na; Wang, Liang; Shen, Liming; Sun, Yu; Wang, Yajun; Zhao, Jingyuan; Wei, Wenjuan; Ren, Yan; Liu, Jing

2017-01-01

Neural stem cell (NSC) transplantation has great therapeutic potential for neurodegenerative diseases and central nervous system injuries. Successful NSC replacement therapy requires precise control over the cellular behaviors. However, the regulation of NSC fate is largely unclear, which severely restricts the potential clinical applications. To develop an effective model, we designed an assembled microfluidic system to engineer NSC niches and assessed the effects of various culture conditions on NSC fate determination. Five types of NSC microenvironments, including two-dimensional (2D) cellular monolayer culture, 2D cellular monolayer culture on the extracellular matrix (ECM), dispersed cells in the ECM, three-dimensional (3D) spheroid aggregates, and 3D spheroids cultured in the ECM, were constructed within an integrated microfluidic chip simultaneously. In addition, we evaluated the influence of static and perfusion culture on NSCs. The efficiency of this approach was evaluated comprehensively by characterization of NSC viability, self-renewal, proliferation, and differentiation into neurons, astrocytes, or oligodendrocytes. Differences in the status and fate of NSCs governed by the culture modes and micro-niches were analyzed. NSCs in the microfluidic device demonstrated good viability, the 3D culture in the ECM facilitated NSC self-renewal and proliferation, and 2D culture in the static state and spheroid culture under perfusion conditions benefited NSC differentiation. Regulation of NSC self-renewal and differentiation on this microfluidic device could provide NSC-based medicinal products and references for distinct nerve disease therapy. PMID:28798841

Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4+ T-Cells

PubMed Central

Jensen, Helle; Folkersen, Lasse; Skov, Søren

2012-01-01

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4+ T-cells, however recently a subset of NKG2D+ CD4+ T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D+ CD4+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D+ CD4+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D+ CD4+ T-cells, generated from HCMV-primed CD4+ T-cells. We show that the HCMV-primed NKG2D+ CD4+ T-cells possess a higher differentiated phenotype than the NKG2D– CD4+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4+ T-cells, whereas it is produced de novo in resting CD4+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D+ CD4+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression. PMID:22870231

Regulation and gene expression profiling of NKG2D positive human cytomegalovirus-primed CD4+ T-cells.

PubMed

Jensen, Helle; Folkersen, Lasse; Skov, Søren

2012-01-01

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8(+) T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4(+) T-cells, however recently a subset of NKG2D(+) CD4(+) T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D(+) CD4(+) T-cells possesses effector-like functions, thus resembling the subsets of NKG2D(+) CD4(+) T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4(+) T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D(+) CD4(+) T-cells, generated from HCMV-primed CD4(+) T-cells. We show that the HCMV-primed NKG2D(+) CD4(+) T-cells possess a higher differentiated phenotype than the NKG2D(-) CD4(+) T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+) T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4(+) T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4(+) T-cells, whereas it is produced de novo in resting CD4(+) T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+) CD4(+) T-cells, as well as the mechanisms regulating NKG2D cell surface expression.

Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines.

PubMed

Hearst, Scoty M; Gilder, Andrew S; Negi, Sandeep S; Davis, Misty D; George, Eric M; Whittom, Angela A; Toyota, Cory G; Husedzinovic, Alma; Gruss, Oliver J; Hebert, Michael D

2009-06-01

Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells.

High-molecular-weight fibronectin synthesized by adenoid cystic carcinoma cells of salivary gland origin.

PubMed

Toyoshima, K; Kimura, S; Cheng, J; Oda, Y; Mori, K J; Saku, T

1999-03-01

To understand the morphogenesis of characteristic cribriform structures and the frequent invasion of salivary adenoid cystic carcinomas (ACC) along such basement membrane-rich structures as peripheral nerves, we have isolated fibronectin (FN) from the culture media of ACC3 cells established from a parotid ACC and characterized its glycosylation and alternative splicing status. FN isolated from ACC3 cells (ACC-FN) showed a molecular mass of 315 kDa in SDS-PAGE and was less heterogeneous and larger than plasma FN (pFN) or FNs from other cell sources. Differential enzymatic treatments of immunoprecipitated ACC-FN with neuraminidase, peptide-N-glycosidase F and endo-alpha-N-acetylgalactosaminidase revealed that ACC-FN was composed of a polypeptide chain of 270 kDa, with 10 kDa each of N-linked and O-linked oligosaccharide chains. Reverse transcription polymerase chain reaction (RT-PCR), in-situ hybridization, and immunofluorescence studies showed that most ACC-FNs contained ED-A, ED-B and IIICS regions in the molecules. This alternative splicing status of ACC-FN seemed to contribute to its less heterogeneous and larger molecular form. Cell attachment assay demonstrated that ACC-FN was more potent than pFN in adhesion of ACC3 cells. The results indicated that ACC-FN may function as a substrate for attachment of ACC3 cells, or that ACC3 cells trap and retain ACC-FN in their pericellular space. This isoform of FN may play an important role in the mode of invasion of ACC and the formation of stromal pseudocysts in the characteristic cribriform structure of ACC.

Prebiotics effect on immune and hepatic oxidative status and gut morphology of white sea bream (Diplodus sargus).

PubMed

Guerreiro, Inês; Couto, Ana; Machado, Marina; Castro, Carolina; Pousão-Ferreira, Pedro; Oliva-Teles, Aires; Enes, Paula

2016-03-01

The aim of this study was to evaluate the effects of short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS) and galactooligosaccharides (GOS) on immune and hepatic oxidative status, and gut morphology of white sea bream juveniles. Four diets were formulated: a control diet with fish meal (FM) and plant feedstuffs (PF) (30FM:70PF) and three test diets similar to the control but supplemented with 1% of scFOS, XOS or GOS. Dietary prebiotic incorporation did not affect total blood cell counts, hematocrit, hemoglobin, red blood indices or differential white blood cell counts. Fish fed GOS had lower ACH50 and nitric oxide than fish fed control diet. XOS enhanced immune status through the increase in alternative complement pathway (ACH50), lysozyme and total immunoglobulin. The higher activity of glucose 6-phosphate dehydrogenase in fish fed FOS compared to the other dietary groups was the only related antioxidant enzyme affected by prebiotics in the liver. GOS ameliorated the precocious adverse effects of PF based diet on gut histomorphology, as denoted by the lower incidence of histological alterations in fish fed GOS for 15 days. In conclusion, XOS and GOS at 1% might have potential to be used as prebiotics in white sea bream juveniles. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Diagnostic value of MYB protein expression in adenoid cystic carcinoma and status of MYB gene copy number].

PubMed

Huo, Zhen; Zeng, Xuan; Wu, Shafei; Wu, Huanwen; Meng, Yunxiao; Liu, Yuanyuan; Luo, Yufeng; Cao, Jinling; Liang, Zhiyong

2015-08-01

To explore the diagnostic value of MYB protein expression for adenoid cystic carcinoma and its differential diagnosis from other salivary gland tumors, and to further investigate the status of MYB gene copy number. MYB expression was studied by immunohistochemistry in 34 adenoid cystic carcinomas, 55 non-adenoid cystic carcinomas (other salivary gland tumors) including 10 pleomorphic adenomas, 10 basal cell adenomas, 10 epithelial-myoepithelial carcinomas, 9 basal cell adenocarcinomas, 8 mucoepidermoid carcinomas, 4 carcinoma in pleomorphic adenomas, and 4 polymorphous low-grade adenocarcinoma. MYB gene copy number status was detected by FISH in MYB protein-positive cases. 82.4% (28/34) of adenoid cystic carcinomas were MYB protein-positive, compared with 9.1% (5/55) of non-adenoid cystic carcinomas, and the difference between the two groups was statistically significant (P < 0.01). 2/18 of adenoid cystic carcinomas had duplication of MYB gene by FISH, and all non-adenoid cystic carcinomas were negative although the difference was not statistically significant (P = 0.435). MYB protein expression is a useful diagnostic marker for adenoid cystic carcinomas in its separation from other salivary gland tumors. In addition, duplication of MYB gene is no a major mechanism for the MYB protein overexpression.

How important are hemoparasites to migratory songbirds? Evaluating physiological measures and infection status in three neotropical migrants during stopover.

PubMed

Cornelius, E A; Davis, A K; Altizer, S A

2014-01-01

Long-distance migrations are energetically expensive for many animals, including migratory songbirds. During these demanding journeys, birds likely face limitations in allocating resources to different physiological functions, including lipid reserves needed to fuel the migration and costly immune defense against pathogens. We sampled three species of long-distance migratory songbirds during their fall migration through coastal Georgia and quantified their body condition, subcutaneous fat reserves, and infection status with blood parasites (Hemoproteus and Plasmodium). We also quantified cellular immunity, on the basis of total and differential white blood cell counts, and estimated individual stress levels, using the heterophil∶lymphocyte (H∶L) ratio. We tested whether birds infected with blood parasites had decreased fat measures, poorer body condition, or increased stress levels (as reflected by H∶L ratios). We also examined relationships between immune cell profiles and the following variables: body condition, subcutaneous fat, infection status, age, and species. Infected birds did not show greater H∶L ratios, poorer body condition, or lower fat measures, but in one species infected individuals showed significantly elevated leukocyte counts. Although we found little evidence for negative relationships between immune cell counts and body condition or fat measures, as might reflect underlying trade-offs in resource allocation, our results concerning hemoparasites are consistent with past work and suggest that chronic hemoparasite infections might have minimal effects on the outcome of long-distance migratory flight.

Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

PubMed

Barbakadze, Tamar; Goloshvili, Galina; Narmania, Nana; Zhuravliova, Elene; Mikeladze, David

2017-10-01

Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. According to the results, it may be proposed that hypoxia can decrease S-nitrosylation of soluble-H-Ras in D PC12 cells and abolish the inhibitory effect of NO-HRas in mitochondrial oxidative metabolism. Copyright© by Royan Institute. All rights reserved.

Histone H3K9 Trimethylase Eggless Controls Germline Stem Cell Maintenance and Differentiation

PubMed Central

Zhou, Jian; McDowell, William; Park, Jungeun; Haug, Jeff; Staehling, Karen; Tang, Hong; Xie, Ting

2011-01-01

Epigenetic regulation plays critical roles in the regulation of cell proliferation, fate determination, and survival. It has been shown to control self-renewal and lineage differentiation of embryonic stem cells. However, epigenetic regulation of adult stem cell function remains poorly defined. Drosophila ovarian germline stem cells (GSCs) are a productive adult stem cell system for revealing regulatory mechanisms controlling self-renewal and differentiation. In this study, we show that Eggless (Egg), a H3K9 methyltransferase in Drosophila, is required in GSCs for controlling self-renewal and in escort cells for regulating germ cell differentiation. egg mutant ovaries primarily exhibit germ cell differentiation defects in young females and gradually lose GSCs with time, indicating that Egg regulates both germ cell maintenance and differentiation. Marked mutant egg GSCs lack expression of trimethylated H3K9 (H3k9me3) and are rapidly lost from the niche, but their mutant progeny can still differentiate into 16-cell cysts, indicating that Egg is required intrinsically to control GSC self-renewal but not differentiation. Interestingly, BMP-mediated transcriptional repression of differentiation factor bam in marked egg mutant GSCs remains normal, indicating that Egg is dispensable for BMP signaling in GSCs. Normally, Bam and Bgcn interact with each other to promote GSC differentiation. Interestingly, marked double mutant egg bgcn GSCs are still lost, but their progeny are able to differentiate into 16-cell cysts though bgcn mutant GSCs normally do not differentiate, indicating that Egg intrinsically controls GSC self-renewal through repressing a Bam/Bgcn-independent pathway. Surprisingly, RNAi-mediated egg knockdown in escort cells leads to their gradual loss and a germ cell differentiation defect. The germ cell differentiation defect is at least in part attributed to an increase in BMP signaling in the germ cell differentiation niche. Therefore, this study has revealed the essential roles of histone H3K9 trimethylation in controlling stem cell maintenance and differentiation through distinct mechanisms. PMID:22216012

Expression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblast.

PubMed

Haider, S; Kunihs, V; Fiala, C; Pollheimer, J; Knöfler, M

2017-09-01

TGF-β superfamily members are thought to play a pivotal role in placental development and differentiation. However, their downstream effectors, the Smad transcription factors, have been poorly investigated in human trophoblasts. Expression and localisation of the canonical TGF-β targets Smad2/3 and their regulators (Smad4 and Smad7) were investigated in first trimester placenta and purified cytotrophoblast (CTB) subtypes using immunofluorescence, western blotting and qPCR. Canonical and non-canonical activation was analysed in nuclear/cytoplasmic extracts of trophoblast subtypes as well as in tissue sections using antibodies against Smad2/3, phosphorylated either at the C-terminus (pSmad2C/3C) or in their linker regions (pSmad2L/3L). Smad phosphorylation was also examined in differentiating extravillous trophoblasts (EVTs) in the absence or presence of decidual stromal cell (DSC)-conditioned medium. Smad2, Smad4 and Smad7 protein were uniformly expressed between 6th and 12th week placentae and the different isolated CTB subtypes. Activated pSmad2L was mainly detected in nuclei and cytoplasm of villous CTBs, whereas pSmad2C was absent from these cells. In contrast, pSmad2C could be detected in the cytoplasm of cell column trophoblasts and in the cytoplasm/nuclei of EVTs. Smad3 and its phosphorylated forms pSmad3C and pSmad3L specifically localised to EVT nuclei. During EVT differentiation autocrine activation of pSmad2C/3C and pSmad3L was observed. DSC-conditioned medium further increased Smad2/3 phosphorylation in EVTs. The lack of pSmad2C in villous CTBs suggests that other mitogens than TGF-β could promote Smad2 linker phosphorylation under homeostatic conditions. Whereas autocrine signalling activates Smad2/3 in differentiating EVTs, paracrine factors contribute to Smad phosphorylation in these cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cancer (stem) cell differentiation: An inherent or acquired property?

PubMed

Mohr, Marieke; Zänker, Kurt S; Dittmar, Thomas

2015-12-01

There is a growing list of data indicating that cancer (stem) cells could functionally adapt foreign tissue features, such as endothelial-like cells or neuroendocrine cells, express lineage markers or could differentiate into various lineages in response to appropriate differentiation criteria. The finding that cancer (stem) cells may possess some kind of differentiation capacity poses the question whether this might be an inherent or acquired property. Cancer stem cells share stem cell characteristics and may thus possess an inherent differentiation capacity enabling the cells to respond to various differentiation stimuli. Considering the plasticity of cancer (stem) cells, even non-tumorigenic (and putatively non-differentiable) tumor cells could give rise to tumorigenic tumor stem cells, exhibiting stem cell characteristics including an inherent differentiation capacity. On the contrary, cancer (stem) cells may have acquired differentiation capacity as a consequence of a previous cell fusion event with cell types exhibiting differentiation potential and being fusogenic, such as macrophages or stem cells. Of pivotal interest in a tumor context are macrophages, which chiefly foster the chronically inflamed tumor microenvironment. Because chronically inflamed tissue is a well-known trigger for cell fusion and both macrophages and stem cells are highly fusogenic we conclude that cell fusion events between these cell types and cancer (stem) cells should frequently occur, thereby giving rise to hybrid cells exhibiting not only novel properties, like an enhanced metastatogenic phenotype, but also parental characteristics, such as differentiation capacity. Conceivably, the combination of both properties might be advantageous for metastasizing cancer (stem) cells to adapt better and faster to a foreign organ tissue environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Differential targeting of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1, by chelators with anti-proliferative activity in a range of tumor cell-types

PubMed Central

Moussa, Rayan S.; Kovacevic, Zaklina; Richardson, Des R.

2015-01-01

Chelators such as 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) target tumor cell iron pools and inhibit proliferation. These agents also modulate multiple targets, one of which is the cyclin-dependent kinase inhibitor, p21. Hence, this investigation examined the mechanism of action of these compounds in targeting p21. All the chelators up-regulated p21 mRNA in the five tumor cell-types assessed. In contrast, examining their effect on total p21 protein levels, these agents induced either: (1) down-regulation in MCF-7 cells; (2) up-regulation in SK-MEL-28 and CFPAC-1 cells; or (3) had no effect in LNCaP and SK-N-MC cells. The nuclear localization of p21 was also differentially affected by the ligands depending upon the cell-type, with it being decreased in MCF-7 cells, but increased in SK-MEL-28 and CFPAC-1 cells. Further studies assessing the mechanisms responsible for these effects demonstrated that p21 expression was not correlated with p53 status, suggesting a p53-independent mechanism. Considering this, we examined proteins that modulate p21 independently of p53, namely NDRG1, MDM2 and ΔNp63. These studies demonstrated that a dominant negative MDM2 isoform (p75MDM2) closely resembled p21 expression in response to chelation in three cell lines. These data suggest MDM2 may be involved in the regulation of p21 by chelators. PMID:26335183

Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

PubMed

Behringer, Richard; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, Andras

2016-12-01

Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size. © 2016 Cold Spring Harbor Laboratory Press.

Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells.

PubMed

Kondo, Yuki; Nurani, Alif Meem; Saito, Chieko; Ichihashi, Yasunori; Saito, Masato; Yamazaki, Kyoko; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Fukuda, Hiroo

2016-06-01

Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells. © 2016 American Society of Plant Biologists. All rights reserved.

EMODIN DOWNREGULATES CELL PROLIFERATION MARKERS DURING DMBA INDUCED ORAL CARCINOGENESIS IN GOLDEN SYRIAN HAMSTERS.

PubMed

Manimaran, Asokan; Buddhan, Rajamanickam; Manoharan, Shanmugam

2017-01-01

Cell-cycle disruption is the major characteristic features of neoplastic transformation and the status of cell-cycle regulators can thus be utilized to assess the prognostic significance in patients with cancer. The PCNA, cyclin D1, CDK4, CDK6 and survivin expression in the buccal mucosa was utilized to evaluate the Emodin efficacy on abnormal cell proliferation during 7,12-dimethylbenz(a)anthracene (DMBA) induced oral carcinogenesis in golden Syrian hamsters. Topical application of DMBA, three times a week for 14 weeks, on the hamsters' buccal pouches developed well differentiated squamous cell carcinoma. Cyclin D1 and PCNA over-expression and up-regulation of CDK4, CDK6 and survivin were noticed in the buccal mucosa of hamsters treated with DMBA alone. Emodin administration (50mg/kg b.w) orally to hamsters treated with DMBA down-regulated the expression of cell proliferation markers in the buccal mucosa. The anti-cell proliferative role of Emodin is owing to its modulating efficacy on cell-cycle markers towards the tumor suppression during DMBA induced oral carcinogenesis.

JAG1-Mediated Notch Signaling Regulates Secretory Cell Differentiation of the Human Airway Epithelium.

PubMed

Gomi, Kazunori; Staudt, Michelle R; Salit, Jacqueline; Kaner, Robert J; Heldrich, Jonna; Rogalski, Allison M; Arbelaez, Vanessa; Crystal, Ronald G; Walters, Matthew S

2016-08-01

Basal cells (BC) are the stem/progenitor cells of the human airway epithelium capable of differentiating into secretory and ciliated cells. Notch signaling activation increases BC differentiation into secretory cells, but the role of individual Notch ligands in regulating this process in the human airway epithelium is largely unknown. The objective of this study was to define the role of the Notch ligand JAG1 in regulating human BC differentiation. JAG1 over-expression in BC increased secretory cell differentiation, with no effect on ciliated cell differentiation. Conversely, knockdown of JAG1 decreased expression of secretory cell genes. These data demonstrate JAG1-mediated Notch signaling regulates differentiation of BC into secretory cells.

Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Fei; Kishida, Tsunao; Ejima, Akika

Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblastsmore » from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.« less

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation

PubMed Central

Azimova, Dinara R.

2016-01-01

Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+–H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. Our findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms. PMID:27821494

Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids.

PubMed

Huang, Ling; Holtzinger, Audrey; Jagan, Ishaan; BeGora, Michael; Lohse, Ines; Ngai, Nicholas; Nostro, Cristina; Wang, Rennian; Muthuswamy, Lakshmi B; Crawford, Howard C; Arrowsmith, Cheryl; Kalloger, Steve E; Renouf, Daniel J; Connor, Ashton A; Cleary, Sean; Schaeffer, David F; Roehrl, Michael; Tsao, Ming-Sound; Gallinger, Steven; Keller, Gordon; Muthuswamy, Senthil K

2015-11-01

There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53(R175H) induces cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. We also define culture conditions for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture and phenotypic heterogeneity of the primary tumor and retain patient-specific physiological changes, including hypoxia, oxygen consumption, epigenetic marks and differences in sensitivity to inhibition of the histone methyltransferase EZH2. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies.

Soft matrix supports osteogenic differentiation of human dental follicle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Viale-Bouroncle, Sandra; Voellner, Florian; Moehl, Christoph

Highlights: {yields} Rigid stiffness supports osteogenic differentiation in mesenchymal stem cells (MSCs). {yields} Our study examined stiffness and differentiation of dental follicle cells (DFCs). {yields} Soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs. {yields} DFCs and MSCs react contrarily to soft and rigid surface stiffness. -- Abstract: The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness andmore » cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.« less

The expression of hematopoietic progenitor cell antigen CD34 is regulated by DNA methylation in a site-dependent manner in gastrointestinal stromal tumours.

PubMed

Bure, Irina; Braun, Alexander; Kayser, Claudia; Geddert, Helene; Schaefer, Inga-Marie; Cameron, Silke; Ghadimi, Michael B; Ströbel, Philipp; Werner, Martin; Hartmann, Arndt; Wiemann, Stefan; Agaimy, Abbas; Haller, Florian; Moskalev, Evgeny A

2017-12-01

The anatomic site-dependent expression of hematopoietic progenitor cell antigen CD34 is a feature of gastrointestinal stromal tumours (GISTs). The basis for the differential CD34 expression is only incompletely understood. This study aimed at understanding the regulation of CD34 in GISTs and clarification of its site-dependent expression. Two sample sets of primary GISTs were interrogated including 52 fresh-frozen and 134 paraffin-embedded and formalin-fixed specimens. DNA methylation analysis was performed by HumanMethylation450 BeadChip array in three cell lines derived from gastric and intestinal GISTs, and differentially methylated CpG sites were established upstream of CD34. The methylation degree was further quantified by pyrosequencing, and inverse correlation with CD34 mRNA and protein abundance was revealed. The gene's expression could be activated upon induction of DNA hypomethylation with 5-aza-2'-deoxycytidine in GIST-T1 cells. In patient samples, a strong inverse correlation of DNA methylation degree with immunohistochemically evaluated CD34 expression was documented. Both CD34 expression and DNA methylation levels were specific to the tumours' anatomic location and mutation status. A constant decrease in methylation levels was observed ranging from almost 100% hypermethylation in intestinal GISTs from duodenum to hypomethylation in rectum. CD34 was heavily methylated in gastric PDGFRA-mutant GISTs in comparison to hypomethylated KIT-mutant counterparts. Next to CD34 hypermethylation, miR-665 was predicted and experimentally confirmed to target CD34 mRNA in GIST-T1 cells. Our results suggest that CD34 expression in GISTs may undergo a complex control by DNA methylation and miR-665. Differential methylation and expression of CD34 in GISTs along the gastrointestinal tract axis and in tumours that harbour different gain-of-function mutations suggest the origin from different cell populations in the gastrointestinal tract. © 2017 UICC.

Spermatogonial stem cell transplantation and male infertility: Current status and future directions.

PubMed

Forbes, Connor M; Flannigan, Ryan; Schlegel, Peter N

2018-03-01

To summarise the current state of research into spermatogonial stem cell (SSC) therapies with a focus on future directions, as SSCs show promise as a source for preserving or initiating fertility in otherwise infertile men. We performed a search for publications addressing spermatogonial stem cell transplantation in the treatment of male infertility. The search engines PubMed and Google Scholar were used from 1990 to 2017. Search terms were relevant for spermatogonial stem cell therapies. Titles of publications were screened for relevance; abstracts were read, if related and full papers were reviewed for directly pertinent original research. In all, 58 papers were found to be relevant to this review, and were included in appropriate subheadings. This review discusses the various techniques that SSCs are being investigated to treat forms of male infertility. Evidence does not yet support clinical application of SSCs in humans. However, significant progress in the in vitro and in vivo development of SSCs, including differentiation into functional germ cells, gives reason for cautious optimism for future research.

GENE EXPRESSION CHANGES AFTER SEIZURE PRECONDITIONING IN THE THREE MAJOR HIPPOCAMPAL CELL LAYERS

PubMed Central

Borges, Karin; Shaw, Renee; Dingledine, Raymond

2008-01-01

Rodents experience hippocampal damage after status epilepticus (SE) mainly in pyramidal cells while sparing the dentate granule cell layer (DGCL). Hippocampal damage was prevented in rats that had been preconditioned by brief seizures on two consecutive days before SE. To identify neuroprotective genes and biochemical pathways changed after preconditioning we compared the effect of preconditioning on gene expression in the CA1 and CA3 pyramidal and DGCLs, harvested by laser capture microscopy. In the DGCL the expression of 632 genes was altered, compared to only 151 and 58 genes in CA1 and CA3 pyramidal cell layers. Most of the differentially expressed genes regulate tissue structure and intra- and extracellular signaling, including neurotransmission. A selective upregulation of energy metabolism transcripts occurred in CA1 pyramidal cells relative to the DGCL. These results reveal a broad transcriptional response of the DGCL to preconditioning, and suggest several mechanisms underlying the neuroprotective effect of preconditioning seizures. PMID:17239605

Quantitative multiplex immunohistochemistry reveals myeloid-inflamed tumor-immune complexity associated with poor prognosis

PubMed Central

Tsujikawa, Takahiro; Kumar, Sushil; Borkar, Rohan N.; Azimi, Vahid; Thibault, Guillaume; Chang, Young Hwan; Balter, Ariel; Kawashima, Rie; Choe, Gina; Sauer, David; El Rassi, Edward; Clayburgh, Daniel R.; Kulesz-Martin, Molly F.; Lutz, Eric R.; Zheng, Lei; Jaffee, Elizabeth M.; Leyshock, Patrick; Margolin, Adam A.; Mori, Motomi; Gray, Joe W.; Flint, Paul W.; Coussens, Lisa M.

2017-01-01

SUMMARY Here we describe a multiplexed immunohistochemical platform, with computational image processing workflows including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas, and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination, and revealed that response to therapy correlated with degree of mono-myelocytic cell density, and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification, and provide digital image processing pipelines (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment. PMID:28380359

Conditionally reprogrammed primary airway epithelial cells maintain morphology, lineage and disease specific functional characteristics.

PubMed

Martinovich, Kelly M; Iosifidis, Thomas; Buckley, Alysia G; Looi, Kevin; Ling, Kak-Ming; Sutanto, Erika N; Kicic-Starcevich, Elizabeth; Garratt, Luke W; Shaw, Nicole C; Montgomery, Samuel; Lannigan, Francis J; Knight, Darryl A; Kicic, Anthony; Stick, Stephen M

2017-12-21

Current limitations to primary cell expansion led us to test whether airway epithelial cells derived from healthy children and those with asthma and cystic fibrosis (CF), co-cultured with an irradiated fibroblast feeder cell in F-medium containing 10 µM ROCK inhibitor could maintain their lineage during expansion and whether this is influenced by underlying disease status. Here, we show that conditionally reprogrammed airway epithelial cells (CRAECs) can be established from both healthy and diseased phenotypes. CRAECs can be expanded, cryopreserved and maintain phenotypes over at least 5 passages. Population doublings of CRAEC cultures were significantly greater than standard cultures, but maintained their lineage characteristics. CRAECs from all phenotypes were also capable of fully differentiating at air-liquid interface (ALI) and maintained disease specific characteristics including; defective CFTR channel function cultures and the inability to repair wounds. Our findings indicate that CRAECs derived from children maintain lineage, phenotypic and importantly disease-specific functional characteristics over a specified passage range.

Comparative expression profile of NOD1/2 and certain acute inflammatory cytokines in thermal-stressed cell culture model of native and crossbred cattle

NASA Astrophysics Data System (ADS)

Bhanuprakash, V.; Singh, Umesh; Sengar, Gyanendra Singh; Raja, T. V.; Sajjanar, Basavraj; Alex, Rani; Kumar, Sushil; Alyethodi, R. R.; Kumar, Ashish; Sharma, Ankur; Kumar, Suresh; Bhusan, Bharat; Deb, Rajib

2017-05-01

Thermotolerance depends mainly on the health and immune status of the animals. The variation in the immune status of the animals may alter the level of tolerance of animals exposed to heat or cold stress. The present study was conducted to investigate the expression profile of two important nucleotide binding and oligomerization domain receptors (NLRs) (NOD1 and NOD2) and their central signalling molecule RIP2 gene during in vitro thermal-stressed bovine peripheral blood mononuclear cells (PBMCs) of native (Sahiwal) and crossbred (Sahiwal X HF) cattle. We also examined the differential expression profile of certain acute inflammatory cytokines in in vitro thermal-stressed PBMC culture among native and its crossbred counterparts. Results revealed that the expression profile of NOD1/2 positively correlates with the thermal stress, signalling molecule and cytokines. Present findings also highlighted that the expression patterns during thermal stress were comparatively superior among indigenous compared to crossbred cattle which may add references regarding the better immune adaptability of Zebu cattle.

Molecular Mechanisms of Stem/Progenitor Cell Maintenance in the Adrenal Cortex

PubMed Central

Lerario, Antonio Marcondes; Finco, Isabella; LaPensee, Christopher; Hammer, Gary Douglas

2017-01-01

The adrenal cortex is characterized by three histologically and functionally distinct zones: the outermost zona glomerulosa (zG), the intermediate zona fasciculata, and the innermost zona reticularis. Important aspects of the physiology and maintenance of the adrenocortical stem/progenitor cells have emerged in the last few years. Studies have shown that the adrenocortical cells descend from a pool of progenitors that are localized in the subcapsular region of the zG. These cells continually undergo a process of centripetal displacement and differentiation, which is orchestrated by several paracrine and endocrine cues, including the pituitary-derived adrenocorticotrophic hormone, and angiotensin II. However, while several roles of the endocrine axes on adrenocortical function are well established, the mechanisms coordinating the maintenance of an undifferentiated progenitor cell pool with self-renewal capacity are poorly understood. Local factors, such as the composition of the extracellular matrix (ECM) with embedded signaling molecules, and the activity of major paracrine effectors, including ligands of the sonic hedgehog and Wnt signaling pathways, are thought to play a major role. Particularly, the composition of the ECM, which exhibits substantial differences within each of the three histologically distinct concentric zones, has been shown to influence the differentiation status of adrenocortical cells. New data from other organ systems and different experimental paradigms strongly support the conclusion that the interactions of ECM components with cell-surface receptors and secreted factors are key determinants of cell fate. In this review, we summarize established and emerging data on the paracrine and autocrine regulatory loops that regulate the biology of the progenitor cell niche and propose a role for bioengineered ECM models in further elucidating this biology in the adrenal. PMID:28386245

The role of morphine on rat neural stem cells viability, neuro-angiogenesis and neuro-steroidgenesis properties.

PubMed

Abdyazdani, Nima; Nourazarian, Alireza; Nozad Charoudeh, Hojjatollah; Kazemi, Masoumeh; Feizy, Navid; Akbarzade, Maryam; Mehdizadeh, Amir; Rezaie, Jafar; Rahbarghazi, Reza

2017-01-01

A lack of comprehensive data exists on the effect of morphine on neural stem cell neuro-steroidogenesis and neuro-angiogenesis properties. We, herein, investigated the effects of morphine (100μM), naloxone (100μM) and their combination on rat neural stem cells viability, clonogenicity and Ki-67 expression over a period of 72h. Any alterations in the total fatty acids profile under treatment protocols were elucidated by direct transesterification method. We also monitored the expression of p53, aromatase and 5-alpha reductase by real-time PCR assay. To examine angiogenic capacity, in vitro tubulogenesis and the level of VE-cadherin transcript were investigated during neural to endothelial differentiation under the experimental procedure. Cells supplemented with morphine displayed reduced survival (p<0.01) and clonogenicity (p<0.001). Flow cytometric analysis showed a decrease in Ki-67 during 72h. Naloxone potentially blunted morphine-induced all effects. The normal levels of fatty acids, including saturated and unsaturated were altered by naloxone and morphine supplements. Following 48h, the up-regulation of p53, aromatase and 5-alpha reductase genes occurred in morphine-primed cells. Using three-dimensional culture models of angiogenesis and real time PCR assay, we showed morphine impaired the tubulogenesis properties of neural stem cells (p<0.001) by the inhibition of trans-differentiation into vascular cells and led to decrease of in VE-cadherin expression. Collectively, morphine strongly impaired the healthy status of neural stem cells by inducing p53 and concurrent elevation of aromatase and 5-alpha reductase activities especially during early 48h. Also, neural stem cells-being exposed to morphine lost their potency to elicit angiogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Phytochemical Ginkgolide B Attenuates Amyloid-β1-42 Induced Oxidative Damage and Altered Cellular Responses in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Gill, Iqbal; Kaur, Sukhchain; Kaur, Navrattan; Dhiman, Monisha; Mantha, Anil K

2017-01-01

Oxidative stress is an upsurge in reactive oxygen/nitrogen species (ROS/RNS), which aggravates damage to cellular components viz. lipids, proteins, and nucleic acids resulting in impaired cellular functions and neurological pathologies including Alzheimer's disease (AD). In the present study, we have examined amyloid-β (Aβ)-induced oxidative stress responses, a major cause for AD, in the undifferentiated and differentiated human neuroblastoma SH-SY5Y cells. Aβ1-42-induced oxidative damage was evaluated on lipids by lipid peroxidation; proteins by protein carbonyls; antioxidant status by SOD and GSH enzyme activities; and DNA and RNA damage levels by evaluating the number of AP sites and 8-OHG base damages produced. In addition, the neuro-protective role of the phytochemical ginkgolide B (GB) in countering Aβ1-42-induced oxidative stress was assessed. We report that the differentiated cells are highly vulnerable to Aβ1-42-induced oxidative stress events as exerted by the deposition of Aβ in AD. Results of the current study suggest that the pre-treatment of GB, followed by Aβ1-42 treatment for 24 h, displayed neuro-protective potential, which countered Aβ1-42-induced oxidative stress responses in both undifferentiated and differentiated SH-SY5Y neuronal cells by: 1) hampering production of ROS and RNS; 2) reducing lipid peroxidation; 3) decreasing protein carbonyl content; 4) restoring antioxidant activities of SOD and GSH enzymes; and 5) maintaining genome integrity by reducing the oxidative DNA and RNA base damages. In conclusion, Aβ1-42 induces oxidative damage to the cellular biomolecules, which are associated with AD pathology, and are protected by the pre-treatment of GB against Aβ-toxicity. Taken together, this study advocates for phytochemical-based therapeutic interventions against AD.

Materials for Neural Differentiation, Trans-Differentiation, and Modeling of Neurological Disease.

PubMed

Gong, Lulu; Cao, Lining; Shen, Zhenmin; Shao, Li; Gao, Shaorong; Zhang, Chao; Lu, Jianfeng; Li, Weida

2018-04-01

Neuron regeneration from pluripotent stem cells (PSCs) differentiation or somatic cells trans-differentiation is a promising approach for cell replacement in neurodegenerative diseases and provides a powerful tool for investigating neural development, modeling neurological diseases, and uncovering the mechanisms that underlie diseases. Advancing the materials that are applied in neural differentiation and trans-differentiation promotes the safety, efficiency, and efficacy of neuron regeneration. In the neural differentiation process, matrix materials, either natural or synthetic, not only provide a structural and biochemical support for the monolayer or three-dimensional (3D) cultured cells but also assist in cell adhesion and cell-to-cell communication. They play important roles in directing the differentiation of PSCs into neural cells and modeling neurological diseases. For the trans-differentiation of neural cells, several materials have been used to make the conversion feasible for future therapy. Here, the most current applications of materials for neural differentiation for PSCs, neuronal trans-differentiation, and neurological disease modeling is summarized and discussed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improvement of Cell Survival During Human Pluripotent Stem Cell Definitive Endoderm Differentiation

PubMed Central

Wang, Han; Luo, Xie; Yao, Li; Lehman, Donna M.

2015-01-01

Definitive endoderm (DE) is a vital precursor for internal organs such as liver and pancreas. Efficient protocol to differentiate human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) to DE is essential for regenerative medicine and for modeling diseases; yet, poor cell survival during DE differentiation remains unsolved. In this study, our use of B27 supplement in modified differentiation protocols has led to a substantial improvement. We used an SOX17-enhanced green fluorescent protein (eGFP) reporter hESC line to compare and modify established DE differentiation protocols. Both total live cell numbers and the percentages of eGFP-positive cells were used to assess differentiation efficiency. Among tested protocols, three modified protocols with serum-free B27 supplement were developed to generate a high number of DE cells. Massive cell death was avoided during DE differentiation and the percentage of DE cells remained high. When the resulting DE cells were further differentiated toward the pancreatic lineage, the expression of pancreatic-specific markers was significantly increased. Similar high DE differentiation efficiency was observed in H1 hESCs and iPSCs through the modified protocols. In B27 components, bovine serum albumin was found to facilitate DE differentiation and cell survival. Using our modified DE differentiation protocols, satisfactory quantities of quality DE can be produced as primary material for further endoderm lineage differentiation. PMID:26132288

Engineering Stem Cells for Biomedical Applications.

PubMed

Yin, Perry T; Han, Edward; Lee, Ki-Bum

2016-01-07

Stem cells are characterized by a number of useful properties, including their ability to migrate, differentiate, and secrete a variety of therapeutic molecules such as immunomodulatory factors. As such, numerous pre-clinical and clinical studies have utilized stem cell-based therapies and demonstrated their tremendous potential for the treatment of various human diseases and disorders. Recently, efforts have focused on engineering stem cells in order to further enhance their innate abilities as well as to confer them with new functionalities, which can then be used in various biomedical applications. These engineered stem cells can take on a number of forms. For instance, engineered stem cells encompass the genetic modification of stem cells as well as the use of stem cells for gene delivery, nanoparticle loading and delivery, and even small molecule drug delivery. The present Review gives an in-depth account of the current status of engineered stem cells, including potential cell sources, the most common methods used to engineer stem cells, and the utilization of engineered stem cells in various biomedical applications, with a particular focus on tissue regeneration, the treatment of immunodeficiency diseases, and cancer. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of a family of structurally related glycoproteins expressing beta 1-6-branched asparagine-linked oligosaccharides in human colon carcinoma cells.

PubMed Central

Laferté, S; Loh, L C

1992-01-01

Previous studies have established that metastatic tumour cells express high levels of beta 1-6-branched Asn-linked oligosaccharides which can be detected with the lectin leucoagglutinin (L-PHA) [Dennis, Laferté, Waghorne, Breitman & Kerbel (1987) Science 236, 582-585]. In order to identify L-PHA-binding glycoproteins which may play a role specifically in colon cancer, we have prepared monoclonal antibodies (MAbs) to the moderately well-differentiated human colon carcinoma cell line HT29. In this paper we present the initial characterization of a family of structurally related L-PHA-binding glycoproteins detected by MAb 1H9 which are differentially expressed and processed by HT29 cells and by two other human colon carcinoma cell lines, SW480 and SW620. In contrast to HT29, the SW480 and SW620 cell lines were established from a poorly differentiated grade III/IV primary tumour and one of its lymph node metastases respectively. MAb 1H9 detects in HT29 cells a conformational determinant present on three L-PHA-binding glycoproteins of 100, 70 and 25kDa, as well as a 74 kDa glycoprotein with high-mannose-type Asn-linked oligosaccharides. Pulse-chase experiments and peptide mapping analyses revealed that the 74 kDa and 100 kDa species are related by carbohydrate processing and are probably derived from a common 76 kDa precursor. On the other hand, the 70 kDa glycoprotein is synthesized from an endoglycosidase H-sensitive precursor of 56 kDa which is structurally related to, but distinct from, the aforementioned 76 kDa precursor. In addition, the 100 kDa species is secreted into the culture medium, whereas the 70 kDa glycoprotein is retained intracellularly. SW480 and SW620 cells showed qualitative and quantitative differences from HT29 cells, including increased secretion of a smaller L-PHA-binding glycoprotein of 92 kDa into the culture medium, as well as apparent differences in glycosylation of the intracellular 66 kDa glycoprotein. These results suggested that the expression, glycosylation and subcellular localization of this family of L-PHA-binding glycoproteins may correlate with the differentiation status of colon cancer cells and/or reflect biochemical changes. characteristic of more progressive metastatic tumours. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 10. PMID:1567368

Uric acid disrupts hypochlorous acid production and the bactericidal activity of HL-60 cells.

PubMed

Carvalho, Larissa A C; Lopes, João P P B; Kaihami, Gilberto H; Silva, Railmara P; Bruni-Cardoso, Alexandre; Baldini, Regina L; Meotti, Flavia C

2018-06-01

Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl - /H 2 O 2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Comparative toxicogenomic analysis of oral Cr(VI) exposure effects in rat and mouse small intestinal epithelia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kopec, Anna K.; Thompson, Chad M.; Kim, Suntae

2012-07-15

Continuous exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal tumors in mice but not rats. Concentration-dependent gene expression effects were evaluated in female F344 rat duodenal and jejunal epithelia following 7 and 90 days of exposure to 0.3–520 mg/L (as sodium dichromate dihydrate, SDD) in drinking water. Whole-genome microarrays identified 3269 and 1815 duodenal, and 4557 and 1534 jejunal differentially expressed genes at 8 and 91 days, respectively, with significant overlaps between the intestinal segments. Functional annotation identified gene expression changes associated with oxidative stress, cell cycle, cell death, and immune response that weremore » consistent with reported changes in redox status and histopathology. Comparative analysis with B6C3F1 mouse data from a similarly designed study identified 2790 differentially expressed rat orthologs in the duodenum compared to 5013 mouse orthologs at day 8, and only 1504 rat and 3484 mouse orthologs at day 91. Automated dose–response modeling resulted in similar median EC{sub 50}s in the rodent duodenal and jejunal mucosae. Comparative examination of differentially expressed genes also identified divergently regulated orthologs. Comparable numbers of differentially expressed genes were observed at equivalent Cr concentrations (μg Cr/g duodenum). However, mice accumulated higher Cr levels than rats at ≥ 170 mg/L SDD, resulting in a ∼ 2-fold increase in the number of differentially expressed genes. These qualitative and quantitative differences in differential gene expression, which correlate with differences in tissue dose, likely contribute to the disparate intestinal tumor outcomes. -- Highlights: ► Cr(VI) elicits dose-dependent changes in gene expression in rat intestine. ► Cr(VI) elicits less differential gene expression in rats compared to mice. ► Cr(VI) gene expression can be phenotypically anchored to intestinal changes. ► Species-specific and divergent changes are consistent with species-specific tumors.« less

ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter

PubMed Central

Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

2014-01-01

One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance. PMID:25089713

ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter.

PubMed

Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

2014-08-01

One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance.

Real-time monitoring of inflammation status in 3T3-L1 adipocytes possessing a secretory Gaussia luciferase gene under the control of nuclear factor-kappa B response element

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagasaki, Haruka; Yoshimura, Takeshi; Aoki, Naohito, E-mail: n-aoki@bio.mie-u.ac.jp

2012-04-13

Highlights: Black-Right-Pointing-Pointer Inflammation status in adipocytes can be monitored by the new assay system. Black-Right-Pointing-Pointer Only an aliquot of conditioned medium is required without cell lysis. Black-Right-Pointing-Pointer Inflammation-attenuating compounds can be screened more conveniently. -- Abstract: We have established 3T3-L1 cells possessing a secretory Gaussia luciferase (GLuc) gene under the control of nuclear factor-kappa B (NF-{kappa}B) response element. The 3T3-L1 cells named 3T3-L1-NF-{kappa}B-RE-GLuc could differentiate into adipocyte as comparably as parental 3T3-L1 cells. Inflammatory cytokines such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1{beta} induced GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes in a concentration- and time-dependent manner. GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLucmore » adipocytes was also induced when cultured with RAW264.7 macrophages and was dramatically enhanced by lipopolysaccharide (LPS)-activated macrophages. An NF-{kappa}B activation inhibitor BAY-11-7085 and an antioxidant N-acetyl cysteine significantly suppressed GLuc secretion induced by macrophages. Finally, we found that rosemary-derived carnosic acid strongly suppressed GLuc secretion induced by macrophages and on the contrary up-regulated adiponectin secretion. Collectively, by using 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes, inflammation status can be monitored in real time and inflammation-attenuating compounds can be screened more conveniently.« less

Incidence of lung cancer histologic cell-types according to neighborhood factors: A population based study in California

PubMed Central

McKinley, Meg; Gali, Kathleen; Patel, Manali; Clarke, Christina; Wakelee, Heather; Haile, Robert; Gomez, Scarlett Lin; Cheng, Iona

2018-01-01

Background The relationships between neighborhood factors (i.e., neighborhood socioeconomic status (nSES) and ethnic enclave) and histologic subtypes of lung cancer for racial/ethnic groups, particularly Hispanics and Asian American/Pacific Islanders (AAPIs), are poorly understood. Methods We conducted a population-based study of 75,631 Californians diagnosed with lung cancer from 2008 through2012. We report incidence rate ratios (IRRs) for lung cancer histologic cell-types by nSES among racial/ethnic groups (non-Hispanic (NH) Whites, NH Blacks, Hispanics and AAPIs) and according to Hispanic or Asian neighborhood ethnic enclave status among Hispanics and AAPIs, respectively. In addition, we examined incidence jointly by nSES and ethnic enclave. Results Patterns of lung cancer incidence by nSES and ethnic enclave differed across race/ethnicity, sex, and histologic cell-type. For adenocarcinoma, Hispanic males and females, residing in both low nSES and high nSES neighborhoods that were low enclave, had higher incidence rates compared to those residing in low nSES, high enclave neighborhoods; males (IRR, 1.17 [95% CI, 1.04–1.32] and IRR, 1.15 [95% CI, 1.02–1.29], respectively) and females (IRR, 1.29 [95% CI, 1.15–1.44] and IRR, 1.51 [95% CI, 1.36–1.67], respectively). However, AAPI males residing in both low and high SES neighborhoods that were also low enclave had lower adenocarcinoma incidence. Conclusions Neighborhood factors differentially influence the incidence of lung cancer histologic cell-types with heterogeneity in these associations by race/ethnicity and sex. For Hispanic males and females and AAPI males, neighborhood ethnic enclave status is strongly associated with lung adenocarcinoma incidence. PMID:29791458

Toxicity of aged gasoline exhaust particles to normal and diseased airway epithelia

NASA Astrophysics Data System (ADS)

Künzi, Lisa; Krapf, Manuel; Daher, Nancy; Dommen, Josef; Jeannet, Natalie; Schneider, Sarah; Platt, Stephen; Slowik, Jay G.; Baumlin, Nathalie; Salathe, Matthias; Prévôt, André S. H.; Kalberer, Markus; Strähl, Christof; Dümbgen, Lutz; Sioutas, Constantinos; Baltensperger, Urs; Geiser, Marianne

2015-06-01

Particulate matter (PM) pollution is a leading cause of premature death, particularly in those with pre-existing lung disease. A causative link between particle properties and adverse health effects remains unestablished mainly due to complex and variable physico-chemical PM parameters. Controlled laboratory experiments are required. Generating atmospherically realistic aerosols and performing cell-exposure studies at relevant particle-doses are challenging. Here we examine gasoline-exhaust particle toxicity from a Euro-5 passenger car in a uniquely realistic exposure scenario, combining a smog chamber simulating atmospheric ageing, an aerosol enrichment system varying particle number concentration independent of particle chemistry, and an aerosol deposition chamber physiologically delivering particles on air-liquid interface (ALI) cultures reproducing normal and susceptible health status. Gasoline-exhaust is an important PM source with largely unknown health effects. We investigated acute responses of fully-differentiated normal, distressed (antibiotics-treated) normal, and cystic fibrosis human bronchial epithelia (HBE), and a proliferating, single-cell type bronchial epithelial cell-line (BEAS-2B). We show that a single, short-term exposure to realistic doses of atmospherically-aged gasoline-exhaust particles impairs epithelial key-defence mechanisms, rendering it more vulnerable to subsequent hazards. We establish dose-response curves at realistic particle-concentration levels. Significant differences between cell models suggest the use of fully-differentiated HBE is most appropriate in future toxicity studies.

Toxicity of aged gasoline exhaust particles to normal and diseased airway epithelia

PubMed Central

Künzi, Lisa; Krapf, Manuel; Daher, Nancy; Dommen, Josef; Jeannet, Natalie; Schneider, Sarah; Platt, Stephen; Slowik, Jay G.; Baumlin, Nathalie; Salathe, Matthias; Prévôt, André S. H.; Kalberer, Markus; Strähl, Christof; Dümbgen, Lutz; Sioutas, Constantinos; Baltensperger, Urs; Geiser, Marianne

2015-01-01

Particulate matter (PM) pollution is a leading cause of premature death, particularly in those with pre-existing lung disease. A causative link between particle properties and adverse health effects remains unestablished mainly due to complex and variable physico-chemical PM parameters. Controlled laboratory experiments are required. Generating atmospherically realistic aerosols and performing cell-exposure studies at relevant particle-doses are challenging. Here we examine gasoline-exhaust particle toxicity from a Euro-5 passenger car in a uniquely realistic exposure scenario, combining a smog chamber simulating atmospheric ageing, an aerosol enrichment system varying particle number concentration independent of particle chemistry, and an aerosol deposition chamber physiologically delivering particles on air-liquid interface (ALI) cultures reproducing normal and susceptible health status. Gasoline-exhaust is an important PM source with largely unknown health effects. We investigated acute responses of fully-differentiated normal, distressed (antibiotics-treated) normal, and cystic fibrosis human bronchial epithelia (HBE), and a proliferating, single-cell type bronchial epithelial cell-line (BEAS-2B). We show that a single, short-term exposure to realistic doses of atmospherically-aged gasoline-exhaust particles impairs epithelial key-defence mechanisms, rendering it more vulnerable to subsequent hazards. We establish dose-response curves at realistic particle-concentration levels. Significant differences between cell models suggest the use of fully-differentiated HBE is most appropriate in future toxicity studies. PMID:26119831

Toxicity of aged gasoline exhaust particles to normal and diseased airway epithelia.

PubMed

Künzi, Lisa; Krapf, Manuel; Daher, Nancy; Dommen, Josef; Jeannet, Natalie; Schneider, Sarah; Platt, Stephen; Slowik, Jay G; Baumlin, Nathalie; Salathe, Matthias; Prévôt, André S H; Kalberer, Markus; Strähl, Christof; Dümbgen, Lutz; Sioutas, Constantinos; Baltensperger, Urs; Geiser, Marianne

2015-06-29

Particulate matter (PM) pollution is a leading cause of premature death, particularly in those with pre-existing lung disease. A causative link between particle properties and adverse health effects remains unestablished mainly due to complex and variable physico-chemical PM parameters. Controlled laboratory experiments are required. Generating atmospherically realistic aerosols and performing cell-exposure studies at relevant particle-doses are challenging. Here we examine gasoline-exhaust particle toxicity from a Euro-5 passenger car in a uniquely realistic exposure scenario, combining a smog chamber simulating atmospheric ageing, an aerosol enrichment system varying particle number concentration independent of particle chemistry, and an aerosol deposition chamber physiologically delivering particles on air-liquid interface (ALI) cultures reproducing normal and susceptible health status. Gasoline-exhaust is an important PM source with largely unknown health effects. We investigated acute responses of fully-differentiated normal, distressed (antibiotics-treated) normal, and cystic fibrosis human bronchial epithelia (HBE), and a proliferating, single-cell type bronchial epithelial cell-line (BEAS-2B). We show that a single, short-term exposure to realistic doses of atmospherically-aged gasoline-exhaust particles impairs epithelial key-defence mechanisms, rendering it more vulnerable to subsequent hazards. We establish dose-response curves at realistic particle-concentration levels. Significant differences between cell models suggest the use of fully-differentiated HBE is most appropriate in future toxicity studies.

Voltage-gated sodium channel expression and action potential generation in differentiated NG108-15 cells.

PubMed

Liu, Jinxu; Tu, Huiyin; Zhang, Dongze; Zheng, Hong; Li, Yu-Long

2012-10-25

The generation of action potential is required for stimulus-evoked neurotransmitter release in most neurons. Although various voltage-gated ion channels are involved in action potential production, the initiation of the action potential is mainly mediated by voltage-gated Na+ channels. In the present study, differentiation-induced changes of mRNA and protein expression of Na+ channels, Na+ currents, and cell membrane excitability were investigated in NG108-15 cells. Whole-cell patch-clamp results showed that differentiation (9 days) didn't change cell membrane excitability, compared to undifferentiated state. But differentiation (21 days) induced the action potential generation in 45.5% of NG108-15 cells (25/55 cells). In 9-day-differentiated cells, Na+ currents were mildly increased, which was also found in 21-day differentiated cells without action potential. In 21-day differentiated cells with action potential, Na+ currents were significantly enhanced. Western blot data showed that the expression of Na+ channels was increased with differentiated-time dependent manner. Single-cell real-time PCR data demonstrated that the expression of Na+ channel mRNA was increased by 21 days of differentiation in NG108-15 cells. More importantly, the mRNA level of Na+ channels in cells with action potential was higher than that in cells without action potential. Differentiation induces expression of voltage-gated Na+ channels and action potential generation in NG108-15 cells. A high level of the Na+ channel density is required for differentiation-triggered action potential generation.

Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines.

PubMed

Karanam, Narasimha Kumar; Srinivasan, Kalayarasan; Ding, Lianghao; Sishc, Brock; Saha, Debabrata; Story, Michael D

2017-03-30

The use of tumor-treating fields (TTFields) has revolutionized the treatment of recurrent and newly diagnosed glioblastoma (GBM). TTFields are low-intensity, intermediate frequency, alternating electric fields that are applied to tumor regions and cells using non-invasive arrays. The predominant mechanism by which TTFields are thought to kill tumor cells is the disruption of mitosis. Using five non-small cell lung cancer (NSCLC) cell lines we found that there is a variable response in cell proliferation and cell killing between these NSCLC cell lines that was independent of p53 status. TTFields treatment increased the G2/M population, with a concomitant reduction in S-phase cells followed by the appearance of a sub-G1 population indicative of apoptosis. Temporal changes in gene expression during TTFields exposure was evaluated to identify molecular signaling changes underlying the differential TTFields response. The most differentially expressed genes were associated with the cell cycle and cell proliferation pathways. However, the expression of genes found within the BRCA1 DNA-damage response were significantly downregulated (P<0.05) during TTFields treatment. DNA double-strand break (DSB) repair foci increased when cells were exposed to TTFields as did the appearance of chromatid-type aberrations, suggesting an interphase mechanism responsible for cell death involving DNA repair. Exposing cells to TTFields immediately following ionizing radiation resulted in increased chromatid aberrations and a reduced capacity to repair DNA DSBs, which were likely responsible for at least a portion of the enhanced cell killing seen with the combination. These findings suggest that TTFields induce a state of 'BRCAness' leading to a conditional susceptibility resulting in enhanced sensitivity to ionizing radiation and provides a strong rationale for the use of TTFields as a combined modality therapy with radiation or other DNA-damaging agents.

Modeling human neurological disorders with induced pluripotent stem cells.

PubMed

Imaizumi, Yoichi; Okano, Hideyuki

2014-05-01

Human induced pluripotent stem (iPS) cells obtained by reprogramming technology are a source of great hope, not only in terms of applications in regenerative medicine, such as cell transplantation therapy, but also for modeling human diseases and new drug development. In particular, the production of iPS cells from the somatic cells of patients with intractable diseases and their subsequent differentiation into cells at affected sites (e.g., neurons, cardiomyocytes, hepatocytes, and myocytes) has permitted the in vitro construction of disease models that contain patient-specific genetic information. For example, disease-specific iPS cells have been established from patients with neuropsychiatric disorders, including schizophrenia and autism, as well as from those with neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. A multi-omics analysis of neural cells originating from patient-derived iPS cells may thus enable investigators to elucidate the pathogenic mechanisms of neurological diseases that have heretofore been unknown. In addition, large-scale screening of chemical libraries with disease-specific iPS cells is currently underway and is expected to lead to new drug discovery. Accordingly, this review outlines the progress made via the use of patient-derived iPS cells toward the modeling of neurological disorders, the testing of existing drugs, and the discovery of new drugs. The production of human induced pluripotent stem (iPS) cells from the patients' somatic cells and their subsequent differentiation into specific cells have permitted the in vitro construction of disease models that contain patient-specific genetic information. Furthermore, innovations of gene-editing technologies on iPS cells are enabling new approaches for illuminating the pathogenic mechanisms of human diseases. In this review article, we outlined the current status of neurological diseases-specific iPS cell research and described recently obtained knowledge in the form of actual examples. © 2013 International Society for Neurochemistry.

Thiol redox transitions in cell signaling: a lesson from N-acetylcysteine.

PubMed

Parasassi, Tiziana; Brunelli, Roberto; Costa, Graziella; De Spirito, Marco; Krasnowska, Ewa; Lundeberg, Thomas; Pittaluga, Eugenia; Ursini, Fulvio

2010-06-29

The functional status of cells is under the control of external stimuli affecting the function of critical proteins and eventually gene expression. Signal sensing and transduction by messengers to specific effectors operate by post-translational modification of proteins, among which thiol redox switches play a fundamental role that is just beginning to be understood. The maintenance of the redox status is, indeed, crucial for cellular homeostasis and its dysregulation towards a more oxidized intracellular environment is associated with aberrant proliferation, ultimately related to diseases such as cancer, cardiovascular disease, and diabetes. Redox transitions occur in sensitive cysteine residues of regulatory proteins relevant to signaling, their evolution to metastable disulfides accounting for the functional redox switch. N-acetylcysteine (NAC) is a thiol-containing compound that is able to interfere with redox transitions of thiols and, thus, in principle, able to modulate redox signaling. We here review the redox chemistry of NAC, then screen possible mechanisms to explain the effects observed in NAC-treated normal and cancer cells; such effects involve a modification of global gene expression, thus of functions and morphology, with a leitmotif of a switch from proliferation to terminal differentiation. The regulation of thiol redox transitions in cell signaling is, therefore, proposed as a new tool, holding promise not only for a deeper explanation of mechanisms, but indeed for innovative pharmacological interventions.

Differentially expressed microRNAs in lung adenocarcinoma invert effects of copy number aberrations of prognostic genes

PubMed Central

Tokar, Tomas; Pastrello, Chiara; Ramnarine, Varune R.; Zhu, Chang-Qi; Craddock, Kenneth J.; Pikor, Larrisa A.; Vucic, Emily A.; Vary, Simon; Shepherd, Frances A.; Tsao, Ming-Sound; Lam, Wan L.; Jurisica, Igor

2018-01-01

In many cancers, significantly down- or upregulated genes are found within chromosomal regions with DNA copy number alteration opposite to the expression changes. Generally, this paradox has been overlooked as noise, but can potentially be a consequence of interference of epigenetic regulatory mechanisms, including microRNA-mediated control of mRNA levels. To explore potential associations between microRNAs and paradoxes in non-small-cell lung cancer (NSCLC) we curated and analyzed lung adenocarcinoma (LUAD) data, comprising gene expressions, copy number aberrations (CNAs) and microRNA expressions. We integrated data from 1,062 tumor samples and 241 normal lung samples, including newly-generated array comparative genomic hybridization (aCGH) data from 63 LUAD samples. We identified 85 “paradoxical” genes whose differential expression consistently contrasted with aberrations of their copy numbers. Paradoxical status of 70 out of 85 genes was validated on sample-wise basis using The Cancer Genome Atlas (TCGA) LUAD data. Of these, 41 genes are prognostic and form a clinically relevant signature, which we validated on three independent datasets. By meta-analysis of results from 9 LUAD microRNA expression studies we identified 24 consistently-deregulated microRNAs. Using TCGA-LUAD data we showed that deregulation of 19 of these microRNAs explains differential expression of the paradoxical genes. Our results show that deregulation of paradoxical genes is crucial in LUAD and their expression pattern is maintained epigenetically, defying gene copy number status. PMID:29507679

Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.

PubMed

Morsczeck, C

2006-02-01

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.

LET-418/Mi2 and SPR-5/LSD1 cooperatively prevent somatic reprogramming of C. elegans germline stem cells.

PubMed

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-04-08

Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, "sensitization" of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis.

LET-418/Mi2 and SPR-5/LSD1 Cooperatively Prevent Somatic Reprogramming of C. elegans Germline Stem Cells

PubMed Central

Käser-Pébernard, Stéphanie; Müller, Fritz; Wicky, Chantal

2014-01-01

Summary Throughout their journey to forming new individuals, germline stem cells must remain totipotent, particularly by maintaining a specific chromatin structure. However, the place epigenetic factors occupy in this process remains elusive. So far, “sensitization” of chromatin by modulation of histone arrangement and/or content was believed to facilitate transcription-factor-induced germ cell reprogramming. Here, we demonstrate that the combined reduction of two epigenetic factors suffices to reprogram C. elegans germ cells. The histone H3K4 demethylase SPR-5/LSD1 and the chromatin remodeler LET-418/Mi2 function together in an early process to maintain germ cell status and act as a barrier to block precocious differentiation. This epigenetic barrier is capable of limiting COMPASS-mediated H3K4 methylation, because elevated H3K4me3 levels correlate with germ cell reprogramming in spr-5; let-418 mutants. Interestingly, germ cells deficient for spr-5 and let-418 mainly reprogram as neurons, suggesting that neuronal fate might be the first to be derepressed in early embryogenesis. PMID:24749077

Mapping human pluripotent stem cell differentiation pathways using high throughput single-cell RNA-sequencing.

PubMed

Han, Xiaoping; Chen, Haide; Huang, Daosheng; Chen, Huidong; Fei, Lijiang; Cheng, Chen; Huang, He; Yuan, Guo-Cheng; Guo, Guoji

2018-04-05

Human pluripotent stem cells (hPSCs) provide powerful models for studying cellular differentiations and unlimited sources of cells for regenerative medicine. However, a comprehensive single-cell level differentiation roadmap for hPSCs has not been achieved. We use high throughput single-cell RNA-sequencing (scRNA-seq), based on optimized microfluidic circuits, to profile early differentiation lineages in the human embryoid body system. We present a cellular-state landscape for hPSC early differentiation that covers multiple cellular lineages, including neural, muscle, endothelial, stromal, liver, and epithelial cells. Through pseudotime analysis, we construct the developmental trajectories of these progenitor cells and reveal the gene expression dynamics in the process of cell differentiation. We further reprogram primed H9 cells into naïve-like H9 cells to study the cellular-state transition process. We find that genes related to hemogenic endothelium development are enriched in naïve-like H9. Functionally, naïve-like H9 show higher potency for differentiation into hematopoietic lineages than primed cells. Our single-cell analysis reveals the cellular-state landscape of hPSC early differentiation, offering new insights that can be harnessed for optimization of differentiation protocols.

A clinicopathologic prediction model for postoperative recurrence in stage Ia non-small cell lung cancer.

PubMed

Zhang, Yang; Sun, Yihua; Xiang, Jiaqing; Zhang, Yawei; Hu, Hong; Chen, Haiquan

2014-10-01

Controversy remains over the appropriate postoperative management for patients with stage Ia non-small cell lung cancer who underwent complete surgical resection as a result of a heterogeneous prognosis. We aimed to identify the predictive factors for recurrence in these patients to aid in the decision making. We reviewed 344 patients with stage Ia non-small cell lung cancer to analyze the associations between recurrence-free survival and the following clinicopathologic variables: age, gender, smoking history, family history, preoperative serum carcinoembryonic antigen level, type of surgical resection, tumor location, tumor histology, lymphovascular invasion, tumor differentiation, and pathologic T status. Cox multivariate survival analysis revealed that central tumor location (P=.019), stage T1b (P=.006), high histologic grade (including large cell carcinoma, solid predominant, micropapillary predominant, and invasive mucinous adenocarcinoma, P=.007), poor differentiation (P=.022), and lymphovascular invasion (P=.035) were independently associated with recurrence-free survival. A nomogram for predicting the probability of 3-year recurrence-free survival was developed using the 5 variables. This model shows good calibration, reasonable discrimination (concordance index=0.733), and small overfitting (2.6%) demonstrated by bootstrapping. We developed a clinicopathologic prediction model for postoperative recurrence in stage Ia non-small cell lung cancer. This model can help with the selection of appropriate postoperative therapeutic strategies for these patients. Copyright © 2014 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

Differential clinicopathological features in microsatellite instability-positive colorectal cancers depending on CIMP status.

PubMed

Bae, Jeong Mo; Kim, Mi Jung; Kim, Jung Ho; Koh, Jae Moon; Cho, Nam-Yun; Kim, Tae-You; Kang, Gyeong Hoon

2011-07-01

Microsatellite instability-positive (MSI+) colorectal cancers (CRCs) are divided into CpG island methylator phenotype-positive (CIMP+) and CpG island methylator phenotype-negative (CIMP-) tumors. The repertoire of inactivated genes in CIMP+/MSI+ CRCs overlaps with but is likely to differ from that of CIMP-/MSI+ CRCs. Because epigenotypic differences are likely to be manifested as phenotypic differences, CIMP+/MSI+ CRCs are expected to differ from CIMP-/MSI+ CRCs in some clinicopathological features. This study aimed to characterize both common and different features between the two subtypes. A total of 72 MSI+ CRCs were analyzed for their methylation status in eight CIMP panel markers using MethyLight assay. CIMP+/MSI+ and CIMP-/MSI+ CRCs were compared regarding clinicopathologic features and mutation in KRAS/BRAF. An independent set of MSI+ CRCs (n = 97) was analyzed for their relationship of CIMP+ status with clinical outcome. Eighteen cases (25%) were CIMP+, and this CIMP+ subtype was highly correlated with older age (P < 0.001). Polypoid gross appearance without ulceration was observed only in CIMP-/MSI+ CRCs (18.5%, P = 0.057). CIMP+/MSI+ CRCs were closely associated with poor differentiation, medullary appearance, signet ring cell appearance, and acinar-form appearance, whereas the CIMP-/MSI+ subtype was closely associated with intraglandular eosinophilic mucin and stratified nuclei (all P values <0.05). Patients with CIMP+/MSI+ CRCs showed worse overall survival than patients with CIMP-/MSI+ CRCs. Our results demonstrate heterogeneity in the clinicopathological features of MSI+ CRCs depending on CIMP status. The observation that CIMP+ and CIMP- subtypes showed different clinical behaviors may provide a clue for establishing subtype-specific therapeutic strategies for these two subtypes.

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

PubMed Central

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

BMP signaling balances proliferation and differentiation of muscle satellite cell descendants

PubMed Central

2011-01-01

Background The capacity of muscle to grow or to regenerate after damage is provided by adult stem cells, so called satellite cells, which are located under the basement lamina of each myofiber. Upon activation satellite cells enter the cell cycle, proliferate and differentiate into myoblasts, which fuse to injured myofibers or form new fibers. These processes are tightly controlled by many growth factors. Results Here we investigate the role of bone morphogenetic proteins (BMPs) during satellite cell differentiation. Unlike the myogenic C2C12 cell line, primary satellite cells do not differentiate into osteoblasts upon BMP signaling. Instead BMP signaling inhibits myogenic differentiation of primary satellite cells ex vivo. In contrast, inhibition of BMP signaling results in cell cycle exit, followed by enhanced myoblast differentiation and myotube formation. Using an in vivo trauma model we demonstrate that satellite cells respond to BMP signals during the regeneration process. Interestingly, we found the BMP inhibitor Chordin upregulated in primary satellite cell cultures and in regenerating muscles. In both systems Chordin expression follows that of Myogenin, a marker for cells committed to differentiation. Conclusion Our data indicate that BMP signaling plays a critical role in balancing proliferation and differentiation of activated satellite cells and their descendants. Initially, BMP signals maintain satellite cells descendants in a proliferating state thereby expanding cell numbers. After cells are committed to differentiate they upregulate the expression of the BMP inhibitor Chordin thereby supporting terminal differentiation and myotube formation in a negative feedback mechanism. PMID:21645366

Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

PubMed

Kim, So-Yeon; Kim, Ye-Ryung; Park, Woo-Jae; Kim, Han Su; Jung, Sung-Chul; Woo, So-Youn; Jo, Inho; Ryu, Kyung-Ha; Park, Joo-Won

2015-01-01

Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on β-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Inhibition of phosphatidylinositol 3-kinase causes apoptosis in retinoic acid differentiated hl-60 leukemia cells.

PubMed

Ma, Jin; Liu, Qiang; Zeng, Yi-Xin

2004-01-01

Phosphatidylinositol 3-kinase (PI3-K) signaling may inhibit apoptosis in neoplastic cells. The PI-3K inhibitor wortmannin renders cells apoptosis-prone. Inducers of differentiation may also cause apoptosis. To detect the effect of wortmannin on the survival of differentiated human acute promyeloid leukemia cells, HL-60 cells were induced to differentiation with treatment of all trans-retinoic acid (ATRA) followed by treatment with wortmannin. Results showed that apoptosis occurred in cells that underwent differentiation, but not in undifferentiated HL-60 cells. The pro-apoptotic molecule, Bad, played a role in this apoptotic mechanism. Thus, the survival of differentiated HL-60 cells induced by ATRA depends on the ability of the PI3-K pathway to transduce survival signals; the PI3-K inhibitor, wortmannin, can induce apoptosis of differentiated HL-60 cells. These results may indicate a novel method for treating cancer with differentiation induction and signal pathway regulation.

Detection of Methylated Circulating DNA as Noninvasive Biomarkers for Breast Cancer Diagnosis

PubMed Central

Cheuk, Isabella Wai Yin; Shin, Vivian Yvonne

2017-01-01

Internationally, breast cancer is the most common female cancer, and is induced by a combination of environmental, genetic, and epigenetic risk factors. Despite the advancement of imaging techniques, invasive sampling of breast epithelial cells is the only definitive diagnostic procedure for patients with breast cancer. To date, molecular biomarkers with high sensitivity and specificity for the screening and early detection of breast cancer are lacking. Recent evidence suggests that the detection of methylated circulating cell-free DNA in the peripheral blood of patients with cancer may be a promising quantitative and noninvasive method for cancer diagnosis. Methylation detection based on a multi-gene panel, rather than on the methylation status of a single gene, may be used to increase the sensitivity and specificity of breast cancer screening. In this review, the results of 14 relevant studies, investigating the efficacy of cell-free DNA methylation screening for breast cancer diagnosis, have been summarized. The genetic risk factors for breast cancer, the methods used for breast cancer detection, and the techniques and limitations related to the detection of cell-free DNA methylation status, have also been reviewed and discussed. From this review, we conclude that the analysis of peripheral blood or other samples to detect differentially methylated cell-free DNA is a promising technique for use in clinical settings, and may improve the sensitivity of screening for both, early detection and disease relapse, and thus improve the future prognosis of patients with breast cancer. PMID:28382090

High Glucose Inhibits Neural Stem Cell Differentiation Through Oxidative Stress and Endoplasmic Reticulum Stress.

PubMed

Chen, Xi; Shen, Wei-Bin; Yang, Penghua; Dong, Daoyin; Sun, Winny; Yang, Peixin

2018-06-01

Maternal diabetes induces neural tube defects by suppressing neurogenesis in the developing neuroepithelium. Our recent study further revealed that high glucose inhibited embryonic stem cell differentiation into neural lineage cells. However, the mechanism whereby high glucose suppresses neural differentiation is unclear. To investigate whether high glucose-induced oxidative stress and endoplasmic reticulum (ER) stress lead to the inhibition of neural differentiation, the effect of high glucose on neural stem cell (the C17.2 cell line) differentiation was examined. Neural stem cells were cultured in normal glucose (5 mM) or high glucose (25 mM) differentiation medium for 3, 5, and 7 days. High glucose suppressed neural stem cell differentiation by significantly decreasing the expression of the neuron marker Tuj1 and the glial cell marker GFAP and the numbers of Tuj1 + and GFAP + cells. The antioxidant enzyme superoxide dismutase mimetic Tempol reversed high glucose-decreased Tuj1 and GFAP expression and restored the numbers of neurons and glial cells differentiated from neural stem cells. Hydrogen peroxide treatment imitated the inhibitory effect of high glucose on neural stem cell differentiation. Both high glucose and hydrogen peroxide triggered ER stress, whereas Tempol blocked high glucose-induced ER stress. The ER stress inhibitor, 4-phenylbutyrate, abolished the inhibition of high glucose or hydrogen peroxide on neural stem cell differentiation. Thus, oxidative stress and its resultant ER stress mediate the inhibitory effect of high glucose on neural stem cell differentiation.

Differential Susceptibility to Effects of the Home Environment on Motor Developmental Outcomes of Preschool Children: Low Birthweight Status as a Susceptibility Factor

ERIC Educational Resources Information Center

Wu, Jennifer Chun-Li; Chiang, Tung-liang

2016-01-01

Low birthweight (LBW) children tend to have higher risks of developmental problems. According to differential susceptibility hypothesis, these putatively vulnerable children may also disproportionately benefit from positive environmental exposure. This study aimed to examine whether LBW status moderates home environmental influences on…

Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

PubMed

Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

2012-03-01

Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. Copyright © 2011 AlphaMed Press.

c-Myc-Induced Survivin Is Essential for Promoting the Notch-Dependent T Cell Differentiation from Hematopoietic Stem Cells

PubMed Central

Haque, Rizwanul; Song, Jianyong; Haque, Mohammad; Lei, Fengyang; Sandhu, Praneet; Ni, Bing; Zheng, Songguo; Fang, Deyu; Yang, Jin-Ming; Song, Jianxun

2017-01-01

Notch is indispensable for T cell lineage commitment, and is needed for thymocyte differentiation at early phases. During early stages of T cell development, active Notch prevents other lineage potentials including B cell lineage and myeloid cell (e.g., dendritic cell) lineage. Nevertheless, the precise intracellular signaling pathways by which Notch promotes T cell differentiation remain unclear. Here we report that the transcription factor c-Myc is a key mediator of the Notch signaling–regulated T cell differentiation. In a well-established in vitro differentiation model of T lymphocytes from hematopoietic stem cells, we showed that Notch1 and 4 directly promoted c-Myc expression; dominant-negative (DN) c-Myc inhibited early T cell differentiation. Moreover, the c-Myc expression activated by Notch signaling increased the expression of survivin, an inhibitor of apoptosis (IAP) protein. We further demonstrated that over-expression of c-Myc increased the abundance of survivin and the T cell differentiation thereof, whereas dn c-Myc reduced survivin levels and concomitantly retarded the differentiation. The c-Myc–dependent survivin induction is functionally germane, because Notch-dependent T cell differentiation was canceled by the depletion of survivin. These results identify both c-Myc and survivin as important mediators of the Notch signaling–regulated differentiation of T lymphocytes from hematopoietic stem cells. PMID:28272325

Tumor differentiation as related to sentinel lymph node status in gastric cancer

PubMed Central

Lavy, Ron; Kapiev, Andronik; Hershkovitz, Yehuda; Poluksht, Natan; Rabin, Igor; Chikman, Bar; Shapira, Zahar; Wasserman, Ilan; Sandbank, Judith; Halevy, Ariel

2014-01-01

AIM: To investigate the influence of tumor grade on sentinel lymph node (SLN) status in patients with gastric cancer (GC). METHODS: We retrospectively studied 71 patients with GC who underwent SLN mapping during gastric surgery to evaluate the relationship between SLN status and tumor grade. RESULTS: Poorly differentiated tumors were detected in 50/71 patients, while the other 21 patients had moderately differentiated tumors. SLNs were identified in 58/71 patients (82%). In 41 of the 58 patients that were found to have stained nodes (70.7%), the tumor was of the poorly differentiated type (group I), while in the remaining patients with stained nodes 17/58 (29.3%), the tumor was of the moderately differentiated type (group II). Positive SLNs were found in 22/41 patients in group I (53.7%) and in 7/17 patients in group II (41.2%) (P = 0.325). The rate of positivity for the SLNs in the two groups (53.7% vs 41.2%) was not statistically significant (P = 0.514). CONCLUSION: Most of our patients were found to have poorly differentiated adenocarcinoma of the stomach and there was no correlation between tumor grade and SLN involvement. PMID:24627734

Enrichment of short interspersed transposable elements to embryonic stem cell-specific hypomethylated gene regions.

PubMed

Muramoto, Hiroki; Yagi, Shintaro; Hirabayashi, Keiji; Sato, Shinya; Ohgane, Jun; Tanaka, Satoshi; Shiota, Kunio

2010-08-01

Embryonic stem cells (ESCs) have a distinctive epigenome, which includes their genome-wide DNA methylation modification status, as represented by the ESC-specific hypomethylation of tissue-dependent and differentially methylated regions (T-DMRs) of Pou5f1 and Nanog. Here, we conducted a genome-wide investigation of sequence characteristics associated with T-DMRs that were differentially methylated between ESCs and somatic cells, by focusing on transposable elements including short interspersed elements (SINEs), long interspersed elements (LINEs) and long terminal repeats (LTRs). We found that hypomethylated T-DMRs were predominantly present in SINE-rich/LINE-poor genomic loci. The enrichment for SINEs spread over 300 kb in cis and there existed SINE-rich genomic domains spreading continuously over 1 Mb, which contained multiple hypomethylated T-DMRs. The characterization of sequence information showed that the enriched SINEs were relatively CpG rich and belonged to specific subfamilies. A subset of the enriched SINEs were hypomethylated T-DMRs in ESCs at Dppa3 gene locus, although SINEs are overall methylated in both ESCs and the liver. In conclusion, we propose that SINE enrichment is the genomic property of regions harboring hypomethylated T-DMRs in ESCs, which is a novel aspect of the ESC-specific epigenomic information.

Effects of arbuscular mycorrhizal colonization and phosphorus application on nuclear ploidy in Allium porrum plants.

PubMed

Fusconi, Anna; Lingua, Guido; Trotta, Antonio; Berta, Graziella

2005-07-01

Arbuscular mycorrhizal (AM) colonization can strongly affect the plant cell nucleus, causing displacement from the periphery to the center of the cell, hypertrophy and polyploidization. The hypertrophy response has been shown in a variety of AM plants whilst polyploidization has been reported only in Lycopersicon esculentum, a multiploid species with a small genome. In order to determine whether polyploidization is a general plant response to AM colonization, analyses were performed on Allium porrum, a plant with a large genome, which is much less subject to polyploidization than L. esculentum. The ploidy status of leaves, complete root systems and four zones of the adventitious roots was investigated in relation to phosphorus content, AM colonization and root differentiation in A. porrum plants grown under two different regimes of phosphate nutrition in order to distinguish direct effects of the fungus from those of improved nutrition. Results showed the presence of two nuclear populations (2C and 4C) in all treatments and samples. Linear regression analyses suggested a general negative correlation between phosphorus content and the proportion of 2C nuclei. The percentage of 2C nuclei (and consequently that of 4C nuclei), was also influenced by AM colonization, differentiation and ageing of the root cells, which resulted in earlier occurrence, in time and space, of polyploid nuclei.

Transcriptomic analysis and mutational status of IDH1 in paired primary-recurrent intrahepatic cholangiocarcinoma.

PubMed

Peraldo-Neia, C; Ostano, P; Cavalloni, G; Pignochino, Y; Sangiolo, D; De Cecco, L; Marchesi, E; Ribero, D; Scarpa, A; De Rose, A M; Giuliani, A; Calise, F; Raggi, C; Invernizzi, P; Aglietta, M; Chiorino, G; Leone, F

2018-06-05

Effective target therapies for intrahepatic cholangiocarcinoma (ICC) have not been identified so far. One of the reasons may be the genetic evolution from primary (PR) to recurrent (REC) tumors. We aim to identify peculiar characteristics and to select potential targets specific for recurrent tumors. Eighteen ICC paired PR and REC tumors were collected from 5 Italian Centers. Eleven pairs were analyzed for gene expression profiling and 16 for mutational status of IDH1. For one pair, deep mutational analysis by Next Generation Sequencing was also carried out. An independent cohort of patients was used for validation. Two class-paired comparison yielded 315 differentially expressed genes between REC and PR tumors. Up-regulated genes in RECs are involved in RNA/DNA processing, cell cycle, epithelial to mesenchymal transition (EMT), resistance to apoptosis, and cytoskeleton remodeling. Down-regulated genes participate to epithelial cell differentiation, proteolysis, apoptotic, immune response, and inflammatory processes. A 24 gene signature is able to discriminate RECs from PRs in an independent cohort; FANCG is statistically associated with survival in the chol-TCGA dataset. IDH1 was mutated in the RECs of five patients; 4 of them displayed the mutation only in RECs. Deep sequencing performed in one patient confirmed the IDH1 mutation in REC. RECs are enriched for genes involved in EMT, resistance to apoptosis, and cytoskeleton remodeling. Key players of these pathways might be considered druggable targets in RECs. IDH1 is mutated in 30% of RECs, becoming both a marker of progression and a target for therapy.

Bestatin Inhibits Cell Growth, Cell Division, and Spore Cell Differentiation in Dictyostelium discoideum

PubMed Central

Poloz, Yekaterina; Catalano, Andrew

2012-01-01

Bestatin methyl ester (BME) is an inhibitor of Zn2+-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have used Dictyostelium as a model organism to study the effects of BME. Only two Zn2+-binding aminopeptidases have been identified in Dictyostelium to date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development of Dictyostelium, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation in Dictyostelium likely through inhibition of PsaA. PMID:22345351

Differential chemosensitivity to antifolate drugs between RAS and BRAF melanoma cells

PubMed Central

2014-01-01

Background The importance of the genetic background of cancer cells for the individual susceptibility to cancer treatments is increasingly apparent. In melanoma, the existence of a BRAF mutation is a main predictor for successful BRAF-targeted therapy. However, despite initial successes with these therapies, patients relapse within a year and have to move on to other therapies. Moreover, patients harbouring a wild type BRAF gene (including 25% with NRAS mutations) still require alternative treatment such as chemotherapy. Multiple genetic parameters have been associated with response to chemotherapy, but despite their high frequency in melanoma nothing is known about the impact of BRAF or NRAS mutations on the response to chemotherapeutic agents. Methods Using cell proliferation and DNA methylation assays, FACS analysis and quantitative-RT-PCR we have characterised the response of a panel of NRAS and BRAF mutant melanoma cell lines to various chemotherapy drugs, amongst them dacarbazine (DTIC) and temozolomide (TMZ) and DNA synthesis inhibitors. Results Although both, DTIC and TMZ act as alkylating agents through the same intermediate, NRAS and BRAF mutant cells responded differentially only to DTIC. Further analysis revealed that the growth-inhibitory effects mediated by DTIC were rather due to interference with nucleotide salvaging, and that NRAS mutant melanoma cells exhibit higher activity of the nucleotide synthesis enzymes IMPDH and TK1. Importantly, the enhanced ability of RAS mutant cells to use nucleotide salvaging resulted in resistance to DHFR inhibitors. Conclusion In summary, our data suggest that the genetic background in melanoma cells influences the response to inhibitors blocking de novo DNA synthesis, and that defining the RAS mutation status could be used to stratify patients for the use of antifolate drugs. PMID:24941944

Osteopontin expression is correlated with differentiation and good prognosis in medullary thyroid carcinoma.

PubMed

Ferreira, Luciana Bueno; Eloy, Catarina; Pestana, Ana; Lyra, Joana; Moura, Margarida; Prazeres, Hugo; Tavares, Catarina; Sobrinho-Simões, Manuel; Gimba, Etel; Soares, Paula

2016-04-01

Osteopontin (OPN) or secreted phosphoprotein 1 (SPP1) is a matricellular glycoprotein whose expression is elevated in various types of cancer and has been shown to be involved in tumourigenesis and metastasis in many malignancies, including follicular cell-derived thyroid carcinomas. Its role in C-cell-derived thyroid lesions and tumours remains to be established. The objective of this study is to clarify the role of OPN expression in the development of medullary thyroid carcinoma (MTC). OPN expression was analysed in a series of 116 MTCs by immunohistochemistry and by qPCR mRNA quantification of the 3 OPN isoforms (OPNa, OPNb and OPNc) in six cases from which fresh frozen tissue was available. Statistical tests were used to evaluate the relationship of OPN expression and the clinicopathological and molecular characteristics of patients and tumours. OPN expression was detected in 91 of 116 (78.4%) of the MTC. We also observed high OPN expression in C-cell hyperplasia as well as in C-cells scattered in the thyroid parenchyma adjacent to the tumours. OPN expression was significantly associated with smaller tumour size, PTEN nuclear expression and RAS status, and suggestively associated with non-invasive tumours. OPNa isoform was expressed significantly at higher levels in tumours than in non-tumour samples. OPNb and OPNc presented similar levels of expression in all samples. Furthermore, OPNa isoform overexpression was significantly associated with reduced growth and viability in the MTC-derived cell line (TT). The expression of OPN in normal C-cells and C-cell hyperplasia suggests that OPN is a differentiation marker of C-cells, rather than a marker of biological aggressiveness in this setting. At variance with other cancers, OPN expression is associated with good prognostic features in MTC. © 2016 European Society of Endocrinology.

Characterization of SLCO5A1/OATP5A1, a Solute Carrier Transport Protein with Non-Classical Function

PubMed Central

Sebastian, Katrin; Detro-Dassen, Silvia; Rinis, Natalie; Fahrenkamp, Dirk; Müller-Newen, Gerhard; Merk, Hans F.; Schmalzing, Günther

2013-01-01

Organic anion transporting polypeptides (OATP/SLCO) have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (∼6.5-fold) and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of ∼105 kDa and ∼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of ∼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20) and genes implicated in developmental processes (e.g. TGM2). A single nucleotide polymorphism leading to the exchange of amino acid 33 (L→F) revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration. PMID:24376674

E2A-positive gastric MALT lymphoma has weaker plasmacytoid infiltrates and stronger expression of the memory B-cell-associated miR-223: possible correlation with stage and treatment response.

PubMed

Liu, Ting-Yun; Chen, Shee-Uan; Kuo, Sung-Hsin; Cheng, Ann-Lii; Lin, Chung-Wu

2010-11-01

Extranodal marginal-zone lymphoma of mucosa-associated lymphoid tissue of the stomach (gastric MALT lymphoma) is derived from memory B cells of the marginal zone. Normal memory B cells do not express markers of germinal-center B cells, such as E2A (immunoglobulin enhancer-binding factor E12/E47), B-cell chronic lymphocytic leukemia/lymphoma 6 (BCL6), or activation-induced cytidine deaminase (AID). E2A is a transcription factor that induces somatic hypermutations and blocks plasma cell differentiation. In 50 stage-I(E)/II(E1) gastric MALT lymphomas, we confirmed that all cases were BCL6(-)/AID(-), but a subset (50%, 25/50) was E2A(+). As E2A(-) and E2A(+) gastric MALT lymphomas had similar numbers of somatic hypermutations without intraclonal variations, which implied an origin from memory B cells, the expression of E2A was best regarded as a marker of aberrant follicular differentiation. Although the status of somatic hypermutation was not affected by E2A, E2A(+) gastric MALT lymphoma showed less plasmacytoid infiltrates and higher expressions of miRNA-223, a microRNA associated with memory B cells. Clinically, E2A(+) gastric MALT lymphomas were more likely to spread to perigastric lymph nodes and were less responsive to Helicobacter eradication therapy than were E2A(-) gastric MALT lymphomas. Taken together, aberrant E2A expression is a diagnostic feature of a subtype of gastric MALT lymphoma with weaker plasmacytoid infiltrates and stronger miR-223 expression. A prospective study would be necessary to verify the association between E2A expression and a poor response to Helicobacter eradication therapy.

Defective interleukin-4/Stat6 activity correlates with increased constitutive expression of negative regulators SOCS-3, SOCS-7, and CISH in colon cancer cells.

PubMed

Liu, Xiao Hong; Xu, Shuang Bing; Yuan, Jia; Li, Ben Hui; Zhang, Yan; Yuan, Qin; Li, Pin Dong; Li, Feng; Zhang, Wen Jie

2009-12-01

Interleukin-4 (IL-4)-induced Stat6 activities (phenotypes) vary among human cancer cells, of which the HT-29 cell line carries an active Stat6(high) phenotype, while Caco-2 carries a defective Stat6(null) phenotype, respectively. Cancer cells with Stat6(high) show resistance to apoptosis and exaggerated metastasis, suggesting the clinical significance of Stat6 phenotypes. We previously showed that Stat6(high) HT-29 cells exhibited low constitutive expression of Stat6-negative regulators SOCS-1 and SHP-1 because of gene hypermethylation. This study further examined the constitutive expression of other closely related SOCS family numbers including SOCS-3, SOCS-5, SOCS-7, and CISH using RT-PCR. Similar to SOCS-1 and SHP-1, Stat6(high) HT-29 cells expressed low constitutive mRNA of SOCS-3, SOCS-7, and CISH than Stat6(null) Caco-2 cells. Interestingly, DNA demethylation using 5-aza-2'-deoxycytidine in HT-29 cells up-regulated mRNA expression of the above genes, indicating a hypermethylation status, which was confirmed by methylation-specific sequencing in selected SOCS-3 gene. Furthermore, defective Stat6(null) Caco-2 exhibited impaired phosphorylation of Stat6 after IL-4 stimulation by flow cytometry, in keeping with the notion of an over-performed negative regulation. The findings that IL-4/Stat6 phenotypes show differential expression of multiple negative regulators suggest a model that a collective force of powerful negative regulators, directly and indirectly, acts on Stat6 activation, which may result in differential Stat6 phenotypes.

Transcription factor Mohawk and the pathogenesis of human anterior cruciate ligament degradation

PubMed Central

Nakahara, Hiroyuki; Hasegawa, Akihiko; Otabe, Koji; Ayabe, Fumiaki; Matsukawa, Tetsuya; Onizuka, Naoko; Ito, Yoshiaki; Ozaki, Toshifumi; Lotz, Martin K.; Asahara, Hiroshi

2013-01-01

Objective To investigate the expression and function of Mohawk (MKX) in human adult anterior cruciate ligament (ACL) tissues and ligament cells from normal and osteoarthritis-affected knees. Methods Knee joints were obtained at autopsy within 24-48 hours postmortem from 13 normal donors (age 36.9±11.0 years), 16 OA donors (age 79.7±11.4 years) and 8 old donors without OA (age 76.9±12.9 years). All cartilage surfaces were graded macroscopically. MKX expression was analyzed by immunohistochemistry and quantitative PCR. ACL-derived cells were used to study regulation of MKX expression by IL-1β. MKX was knocked down by siRNA to analyze function of MKX in extracellular matrix (ECM) production and differentiation in ACL-derived cells. Results The expression of MKX was significantly decreased in ACL-derived cells from OA knees compared with normal knees. Consistent with this finding, immunohistochemistry showed that MKX positive cells were significantly reduced in ACL tissues from OA donors in particular in cells located in disorientated fibers. In ACL-derived cells, IL-1β strongly suppressed MKX gene expression and reduced ligament ECM genes, COL1A1 and TNXB. On the other hand, SOX9, chondrocyte master transcription factor, was up regulated by IL-1β treatment. Importantly, knock down of MKX expression by siRNA upregulated SOX9 expression in ACL-derived cells, whereas the expression of COL1A1 and TNXB were decreased. Conclusion Reduced expression of MKX is a feature of degenerated ACL in OA-affected joints and this may be in part mediated by IL-1β. MKX appears necessary to maintain the tissue specific cellular differentiation status and ECM production in adult human tendons and ligaments. PMID:23686683

The breast cancer antigen 5T4 interacts with Rab11, and is a target and regulator of Rab11 mediated trafficking.

PubMed

Harris, Janelle L; Dave, Keyur; Gorman, Jeffrey; Khanna, Kum Kum

2018-06-01

5T4 is a transmembrane glycoprotein with limited expression in normal adult tissues and expression in some solid tumours. It is unclear whether 5T4 is preferentially expressed by stem or differentiated cell types. Modes of 5T4 regulation are unknown despite its ongoing development as a cancer immunotherapy target. Our aims were to clarify the differentiation status of 5T4 expressing cells in breast cancer and to understand the mechanism underlying 5T4 membrane presentation. We analysed 5T4 expression in breast cancer cell populations by flow cytometery and found that 5T4 is highly expressed on differentiated cells, where it localizes to focal adhesions. Using immunoprecipitation and mass spectrometry, we identified interactions between 5T4 and the membrane trafficking proteins Rab11, Rab18 and ARF6. Mechanistically we found that Rab11 and Rab18 have oppositional roles in controlling expression and surface presentation of 5T4. 5T4 depletion stabilizes Rab11 protein expression with a consequent stimulation transferrin surface labelling, indicating that 5T4 represses endocytic activity. Successful immunotherapeutic targeting of 5T4 requires surface presentation and different immunotherapy strategies require surface presentation versus endocytosis. While breast cancer cells with high 5T4 surface expression and rapid cell surface turnover would be susceptible to antibody-drug conjugates that rely on intracellular release, 5T4 positive cells with lower expression or lower turnover may still be responsive to T-cell mediated approaches. We find that endocytosis of 5T4 is strongly Rab11 dependent and as such Rab11 activity could affect the success or failure of 5T4-targetted immunotherapy, particularly for antibody-drug conjugate approaches. In fact, 5T4 itself represses Rab11 expression. This newly uncovered relationship between Rab11 and 5T4 suggests that breast tumours with high 5T4 expression may not have efficient endocytic uptake of 5T4-targetted immunotherapeutics. This should be considered when selecting amongst the different types of immunotherapies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Wnt/β-Catenin Expression Does Not Correlate with Serum Alkaline Phosphatase Concentration in Canine Osteosarcoma Patients

PubMed Central

Piskun, Caroline M.; Muthuswamy, Anantharaman; Huelsmeyer, Michael K.; Thompson, Victoria; Stein, Timothy J.

2011-01-01

Osteosarcoma is an aggressive malignancy of the bone and an increase in serum alkaline phosphatase concentration has clinical prognostic value in both humans and canines. Increased serum alkaline phosphatase concentration at the time of diagnosis has been associated with poorer outcomes for osteosarcoma patients. The biology underlying this negative prognostic factor is poorly understood. Given that activation of the Wnt signaling pathway has been associated with alkaline phosphatase expression in osteoblasts, we hypothesized that the Wnt/β-catenin signaling pathway would be differentially activated in osteosarcoma tissue based on serum ALP status. Archived canine osteosarcoma samples and primary canine osteosarcoma cell lines were used to evaluate the status of Wnt/β-catenin signaling pathway activity through immunohistochemical staining, western immunoblot analyses, quantitative reverse-transcription polymerase chain reaction, and a Wnt-responsive promoter activity assay. We found no significant difference in β-catenin expression or activation between OSA populations differing in serum ALP concentration. Pathway activity was mildly increased in the primary OSA cell line generated from a patient with increased serum ALP compared to the normal serum ALP OSA cell line. Further investigation into the mechanisms underlying differences in serum ALP concentration is necessary to improve our understanding of the biological implications of this negative prognostic indicator. PMID:22022527

Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest

PubMed Central

Carén, Helena; Stricker, Stefan H.; Bulstrode, Harry; Gagrica, Sladjana; Johnstone, Ewan; Bartlett, Thomas E.; Feber, Andrew; Wilson, Gareth; Teschendorff, Andrew E.; Bertone, Paul; Beck, Stephan; Pollard, Steven M.

2015-01-01

Summary Glioblastoma (GBM) is an aggressive brain tumor whose growth is driven by stem cell-like cells. BMP signaling triggers cell-cycle exit and differentiation of GBM stem cells (GSCs) and, therefore, might have therapeutic value. However, the epigenetic mechanisms that accompany differentiation remain poorly defined. It is also unclear whether cell-cycle arrest is terminal. Here we find only a subset of GSC cultures exhibit astrocyte differentiation in response to BMP. Although overtly differentiated non-cycling astrocytes are generated, they remain vulnerable to cell-cycle re-entry and fail to appropriately reconfigure DNA methylation patterns. Chromatin accessibility mapping identified loci that failed to alter in response to BMP and these were enriched in SOX transcription factor-binding motifs. SOX transcription factors, therefore, may limit differentiation commitment. A similar propensity for cell-cycle re-entry and de-differentiation was observed in GSC-derived oligodendrocyte-like cells. These findings highlight significant obstacles to BMP-induced differentiation as therapy for GBM. PMID:26607953

Oscillatory Protein Expression Dynamics Endows Stem Cells with Robust Differentiation Potential

PubMed Central

Kaneko, Kunihiko

2011-01-01

The lack of understanding of stem cell differentiation and proliferation is a fundamental problem in developmental biology. Although gene regulatory networks (GRNs) for stem cell differentiation have been partially identified, the nature of differentiation dynamics and their regulation leading to robust development remain unclear. Herein, using a dynamical system modeling cell approach, we performed simulations of the developmental process using all possible GRNs with a few genes, and screened GRNs that could generate cell type diversity through cell-cell interactions. We found that model stem cells that both proliferated and differentiated always exhibited oscillatory expression dynamics, and the differentiation frequency of such stem cells was regulated, resulting in a robust number distribution. Moreover, we uncovered the common regulatory motifs for stem cell differentiation, in which a combination of regulatory motifs that generated oscillatory expression dynamics and stabilized distinct cellular states played an essential role. These findings may explain the recently observed heterogeneity and dynamic equilibrium in cellular states of stem cells, and can be used to predict regulatory networks responsible for differentiation in stem cell systems. PMID:22073296

Harnessing the potential of lung stem cells for regenerative medicine.

PubMed

McQualter, Jonathan L; Anthony, Desiree; Bozinovski, Steven; Prêle, Cecilia M; Laurent, Geoffrey J

2014-11-01

In response to recurrent exposure to environmental insults such as allergens, pollution, irritants, smoke and viral/bacterial infection, the epithelium of the lung is continually damaged. Homeostasis of the lung requires a balance between immune regulation and promotion of tissue regeneration, which requires the co-ordinated proliferation and differentiation of stem and progenitor cells. In this review we reflect on the current understanding of lung epithelial stem and progenitor cells and advocate a model hierarchy in which self-renewing multipotent lung epithelial stem cells give rise to lineage restricted progenitor cells that repopulate airway and alveolar epithelial cell lineages during homeostasis and repair. We also discuss the role of mesenchymal progenitor cells in maintaining the structural integrity of the lung and propose a model in which mesenchymal cells act as the quintessential architects of lung regeneration by providing molecular signals, such as FGF-10, to regulate the fate and specificity of epithelial stem and progenitor cells. Moreover, we discuss the current status and future prospects for translating lung stem cell therapies to the clinic to replace, repair, or regenerate diseased lung tissue. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation. Copyright © 2014 Elsevier Ltd. All rights reserved.

miR-21 promotes the differentiation of hair follicle-derived neural crest stem cells into Schwann cells

PubMed Central

Ni, Yuxin; Zhang, Kaizhi; Liu, Xuejuan; Yang, Tingting; Wang, Baixiang; Fu, Li; A, Lan; Zhou, Yanmin

2014-01-01

Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair follicles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA (miR)-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist (agomir-21), the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist (antagomir-21), the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regulating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA. PMID:25206896

All-Trans-Retinoic Acid Stimulates Overexpression of Tumor Protein D52 (TPD52, Isoform 3) and Neuronal Differentiation of IMR-32 Cells.

PubMed

Kotapalli, Sudha Sravanti; Dasari, Chandrashekhar; Duscharla, Divya; Kami Reddy, Karthik Reddy; Kasula, Manjula; Ummanni, Ramesh

2017-12-01

Tumor protein D52 (TPD52), a proto-oncogene is overexpressed in a variety of epithelial carcinomas and plays an important role in cell proliferation, migration, and cell death. In the present study we found that the treatment of IMR-32 neuroblastoma (NB) cells with retinoic acid (RA) stimulates an increase in expression of TPD52. TPD52 expression is detectable after 72 h, can be maintained till differentiation of NB cells suggesting that TPD52 is involved in differentiation. Here, we demonstrate that TPD52 is essential for RA to promote differentiation of NB cells. Our results show that exogenous expression of EGFP-TPD52 in IMR-32 cells resulted cell differentiation even without RA. RA by itself and with overexpression of TPD52 can increase the ability of NB cells differentiation. Interestingly, transfection of IMR-32 cells with a specific small hairpin RNA for efficient knockdown of TPD52 attenuated RA induced NB cells differentiation. Transcriptional and translational level expression of neurotropic (BDNF, NGF, Nestin) and differentiation (β III tubulin, NSE, TH) factors in NB cells with altered TPD52 expression and/or RA treatment confirmed essential function of TPD52 in cellular differentiation. Furthermore, we show that TPD52 protects cells from apoptosis and arrest cell proliferation by varying expression of p27Kip1, activation of Akt and ERK1/2 thus promoting cell differentiation. Additionally, inhibition of STAT3 activation by its specific inhibitor arrested NB cells differentiation by EGFP-TPD52 overexpression with or without RA. Taken together, our data reveal that TPD52 act through activation of JAK/STAT signaling pathway to undertake NB cells differentiation induced by RA. J. Cell. Biochem. 118: 4358-4369, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Ion Channel Gene Expression in Lung Adenocarcinoma: Potential Role in Prognosis and Diagnosis

PubMed Central

Ko, Jae-Hong; Gu, Wanjun; Lim, Inja; Bang, Hyoweon; Ko, Eun A.; Zhou, Tong

2014-01-01

Ion channels are known to regulate cancer processes at all stages. The roles of ion channels in cancer pathology are extremely diverse. We systematically analyzed the expression patterns of ion channel genes in lung adenocarcinoma. First, we compared the expression of ion channel genes between normal and tumor tissues in patients with lung adenocarcinoma. Thirty-seven ion channel genes were identified as being differentially expressed between the two groups. Next, we investigated the prognostic power of ion channel genes in lung adenocarcinoma. We assigned a risk score to each lung adenocarcinoma patient based on the expression of the differentially expressed ion channel genes. We demonstrated that the risk score effectively predicted overall survival and recurrence-free survival in lung adenocarcinoma. We also found that the risk scores for ever-smokers were higher than those for never-smokers. Multivariate analysis indicated that the risk score was a significant prognostic factor for survival, which is independent of patient age, gender, stage, smoking history, Myc level, and EGFR/KRAS/ALK gene mutation status. Finally, we investigated the difference in ion channel gene expression between the two major subtypes of non-small cell lung cancer: adenocarcinoma and squamous-cell carcinoma. Thirty ion channel genes were identified as being differentially expressed between the two groups. We suggest that ion channel gene expression can be used to improve the subtype classification in non-small cell lung cancer at the molecular level. The findings in this study have been validated in several independent lung cancer cohorts. PMID:24466154

Correlation between ploidy status using flow cytometry and nucleolar organizer regions in benign and malignant epithelial odontogenic tumors.

PubMed

Mohamed Mahmoud, Sarah Ahmed; El-Rouby, Dalia Hussein; El-Ghani, Safa Fathy Abd; Badawy, Omnia Mohamed

2017-06-01

Differentiation between the aggressive benign odontogenic tumors and their malignant counterparts is controversial and difficult. While flow cytometry (FCM) allowed DNA analysis in neoplasia, argyrophilic organizer regions (AgNORs) number and/or size in a nucleus are correlated with the ribosomal gene activity and therefore with cellular proliferation. The aim of this research was to study the diagnostic accuracy of FCM and AgNORs staining in differentiating between benign and malignant epithelial odontogenic tumors and to correlate between these two interventions. Sixteen benign cases [8 cases of ameloblastoma (AB) and 8 cases of keratocystic odontogenic tumor (KCOT)] and 13 malignant epithelial odontogenic tumors [8 cases of ameloblastic carcinoma (ABC) and 5 cases of clear cell odontogenic carcinoma(CCOC)] were included in the current study. For FCM analysis, a single cell suspension from Formalin fixed paraffin-embedded (FFPE) tumors was prepared according to a modified method described by Hedley (1989) and AgNORs staining were performed in accordance to the Ploton protocol (1986). Analysis of AgNORs was performed using both quantitative and qualitative methods. The work revealed that all the examined tumors were diploid, except for 40% of CCOC cases. The S-phase fraction (SPF) value, AgNORs count and AgNORs area/cell showed statistically significant difference on comparing benign and malignant groups. A weak positive correlation was observed between SPF and AgNORs count. The SPF value was considered to be more sensitive and specific in differentiation between aggressive benign and malignant epithelial odontogenic tumors in comparison to AgNORs counting. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-Wide Assessment of Differential DNA Methylation Associated with Autoantibody Production in Systemic Lupus Erythematosus.

PubMed

Chung, Sharon A; Nititham, Joanne; Elboudwarej, Emon; Quach, Hong L; Taylor, Kimberly E; Barcellos, Lisa F; Criswell, Lindsey A

2015-01-01

Systemic lupus erythematosus (SLE) is characterized by the development of autoantibodies associated with specific clinical manifestations. Previous studies have shown an association between differential DNA methylation and SLE susceptibility, but have not investigated SLE-related autoantibodies. Our goal was to determine whether DNA methylation is associated with production of clinically relevant SLE-related autoantibodies, with an emphasis on the anti-dsDNA autoantibody. In this study, we characterized the methylation status of 467,314 CpG sites in 326 women with SLE. Using a discovery and replication study design, we identified and replicated significant associations between anti-dsDNA autoantibody production and the methylation status of 16 CpG sites (pdiscovery<1.07E-07 and preplication<0.0029) in 11 genes. Associations were further investigated using multivariable regression to adjust for estimated leukocyte cell proportions and population substructure. The adjusted mean DNA methylation difference between anti-dsDNA positive and negative cases ranged from 1.2% to 19%, and the adjusted odds ratio for anti-dsDNA autoantibody production comparing the lowest and highest methylation tertiles ranged from 6.8 to 18.2. Differential methylation for these CpG sites was also associated with anti-SSA, anti-Sm, and anti-RNP autoantibody production. Overall, associated CpG sites were hypomethylated in autoantibody positive compared to autoantibody negative cases. Differential methylation of CpG sites within the major histocompatibility region was not strongly associated with autoantibody production. Genes with differentially methylated CpG sites represent multiple biologic pathways, and have not been associated with autoantibody production in genetic association studies. In conclusion, hypomethylation of CpG sites within genes from different pathways is associated with anti-dsDNA, anti-SSA, anti-Sm, and anti-RNP production in SLE, and these associations are not explained by genetic variation. Thus, studies of epigenetic mechanisms such as DNA methylation represent a complementary method to genetic association studies to identify biologic pathways that may contribute to the clinical heterogeneity of autoimmune diseases.

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

PubMed Central

Kutleša, Snježana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B.; Jurecic, Roland

2011-01-01

Objective Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. Materials and Methods EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. Results The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Tα, RAG-1, and T-cell receptor – Vβ genes; and 3) produced interferon-γ in response to T-cell receptor stimulation. Conclusions These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation. PMID:19447159

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

PubMed

Kutlesa, Snjezana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland

2009-08-01

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

Arrested neural and advanced mesenchymal differentiation of glioblastoma cells-comparative study with neural progenitors

PubMed Central

2009-01-01

Background Although features of variable differentiation in glioblastoma cell cultures have been reported, a comparative analysis of differentiation properties of normal neural GFAP positive progenitors, and those shown by glioblastoma cells, has not been performed. Methods Following methods were used to compare glioblastoma cells and GFAP+NNP (NHA): exposure to neural differentiation medium, exposure to adipogenic and osteogenic medium, western blot analysis, immunocytochemistry, single cell assay, BrdU incorporation assay. To characterize glioblastoma cells EGFR amplification analysis, LOH/MSI analysis, and P53 nucleotide sequence analysis were performed. Results In vitro differentiation of cancer cells derived from eight glioblastomas was compared with GFAP-positive normal neural progenitors (GFAP+NNP). Prior to exposure to differentiation medium, both types of cells showed similar multilineage phenotype (CD44+/MAP2+/GFAP+/Vimentin+/Beta III-tubulin+/Fibronectin+) and were positive for SOX-2 and Nestin. In contrast to GFAP+NNP, an efficient differentiation arrest was observed in all cell lines isolated from glioblastomas. Nevertheless, a subpopulation of cells isolated from four glioblastomas differentiated after serum-starvation with varying efficiency into derivatives indistinguishable from the neural derivatives of GFAP+NNP. Moreover, the cells derived from a majority of glioblastomas (7 out of 8), as well as GFAP+NNP, showed features of mesenchymal differentiation when exposed to medium with serum. Conclusion Our results showed that stable co-expression of multilineage markers by glioblastoma cells resulted from differentiation arrest. According to our data up to 95% of glioblastoma cells can present in vitro multilineage phenotype. The mesenchymal differentiation of glioblastoma cells is advanced and similar to mesenchymal differentiation of normal neural progenitors GFAP+NNP. PMID:19216795

Tissue Engineered Strategies for Skeletal Muscle Injury

PubMed Central

Longo, Umile Giuseppe; Loppini, Mattia; Berton, Alessandra; Spiezia, Filippo; Maffulli, Nicola; Denaro, Vincenzo

2012-01-01

Skeletal muscle injuries are common in athletes, occurring with direct and indirect mechanisms and marked residual effects, such as severe long-term pain and physical disability. Current therapy consists of conservative management including RICE protocol (rest, ice, compression and elevation), nonsteroidal anti-inflammatory drugs, and intramuscular corticosteroids. However, current management of muscle injuries often does not provide optimal restoration to preinjury status. New biological therapies, such as injection of platelet-rich plasma and stem-cell-based therapy, are appealing. Although some studies support PRP application in muscle-injury management, reasons for concern persist, and further research is required for a standardized and safe use of PRP in clinical practice. The role of stem cells needs to be confirmed, as studies are still limited and inconsistent. Further research is needed to identify mechanisms involved in muscle regeneration and in survival, proliferation, and differentiation of stem cells. PMID:25098362

No Interactive Effects of Sex and Persistent Cytomegalovirus on Immune Phenotypes in Young Children: The Generation R Study.

PubMed

Jansen, Michelle A E; van den Heuvel, Diana; Jaddoe, Vincent W V; Moll, Henriette A; van Zelm, Menno C

2017-03-15

Persistent infections with cytomegalovirus (CMV) differentially affect the host immune phenotype in middle-aged males and females. Because CMV already impacts on T-cell memory at a young age, we studied whether these effects were modified by sex in 1,079 children with an average age of 6 years. Sex and CMV independently impacted on multiple B-cell and T-cell subsets. However, there was no significant effect of their interaction. Importantly, the effects of sex and CMV were in part explained by age and infection with other herpesviruses. Thus, immune aging is likely to be more complex, with involvement of hormonal changes with age, socioeconomic status, birth characteristics, and pathogen exposure. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

21 CFR 864.5220 - Automated differential cell counter.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated differential cell counter. 864.5220... § 864.5220 Automated differential cell counter. (a) Identification. An automated differential cell... have the capability to flag, count, or classify immature or abnormal hematopoietic cells of the blood...

21 CFR 864.5220 - Automated differential cell counter.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated differential cell counter. 864.5220... § 864.5220 Automated differential cell counter. (a) Identification. An automated differential cell... have the capability to flag, count, or classify immature or abnormal hematopoietic cells of the blood...

21 CFR 864.5220 - Automated differential cell counter.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated differential cell counter. 864.5220... § 864.5220 Automated differential cell counter. (a) Identification. An automated differential cell... have the capability to flag, count, or classify immature or abnormal hematopoietic cells of the blood...

21 CFR 864.5220 - Automated differential cell counter.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated differential cell counter. 864.5220... § 864.5220 Automated differential cell counter. (a) Identification. An automated differential cell... have the capability to flag, count, or classify immature or abnormal hematopoietic cells of the blood...

21 CFR 864.5220 - Automated differential cell counter.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated differential cell counter. 864.5220... § 864.5220 Automated differential cell counter. (a) Identification. An automated differential cell... have the capability to flag, count, or classify immature or abnormal hematopoietic cells of the blood...

Papillary renal cell carcinoma: a clinicopathological and whole-genome exon sequencing study

PubMed Central

Liu, Kunpeng; Ren, Yuan; Pang, Lijuan; Qi, Yan; Jia, Wei; Tao, Lin; Hu, Zhengyan; Zhao, Jin; Zhang, Haijun; Li, Li; Yue, Haifeng; Han, Juan; Liang, Weihua; Hu, Jianming; Zou, Hong; Yuan, Xianglin; Li, Feng

2015-01-01

Papillary renal cell carcinoma (PRCC) represents the second most common histological subtype of RCC, and comprises 2 subtypes. Prognosis for type 1 PRCC is relatively good, whereas type 2 PRCC is associated with poor clinical outcomes. The aim of the present study was to evaluate the clinicopathological and mutations characteristics of PRCC. Hence, we reported on 13 cases of PRCC analyzed using whole-exome sequencing. Histologically, type 2 PRCC showed a higher nuclear grade and lymphovascular invasion rate versus type 1 PRCC (P < 0.05). Immunostaining revealed type 1 PRCC had higher CK7 and lower Top IIα expression rates (P < 0.05). Whole-exome sequencing data analysis revealed that the mutational statuses of 373 genes (287 missense, 69 silent, 6 nonsense, and 11 synonymous mutations) differed significantly between PRCC and normal renal tissues (P < 0.05). Functional enrichment analysis was used to classify the 287 missense-mutated genes into 11 biological process clusters (comprised of 61 biological processes) and 5 pathways, involved in cell adhesion, microtubule-based movement, the cell cycle, polysaccharide biosynthesis, muscle cell development and differentiation, cell death, and negative regulation. Associated pathways included the ATP-binding cassette transporter, extracellular matrix-receptor interaction, lysosome, complement and coagulation cascades, and glyoxylate and dicarboxylate metabolism pathways. The missense mutation status of 19 genes differed significantly between the groups (P < 0.05), and alterations in the EEF1D, RFNG, GPR142, and RAB37 genes were located in different chromosomal regions in type 1 and 2 PRCC. These mutations may contribute to future studies on pathogenic mechanisms and targeted therapy of PRCC. PMID:26339402

Phosphohistone-H3 (PHH3) is prognostic relevant in Merkel cell carcinomas but Merkel cell polyomavirus is a more powerful prognostic factor than AJCC clinical stage, PHH3, Ki-67 or mitotic indices.

PubMed

Iwasaki, Takeshi; Matsushita, Michiko; Nonaka, Daisuke; Kato, Masako; Nagata, Keiko; Murakami, Ichiro; Hayashi, Kazuhiko

2015-08-01

Merkel cell carcinomas (MCCs) associated with Merkel cell polyomavirus (MCPyV) have better prognosis than those without MCPyV. The relationship between mitotic index (MI) and MCC outcome has remained elusive because of the difficulty in differentiating mitotic cells from apoptotic ones. We evaluated the role of phosphohistone-H3 (PHH3) (Ser10), a new mitotic count biomarker, in MCPyV-positive or -negative MCC patients, and assessed its prognostic value in comparison to Ki-67 labeling index or MI using hematoxylin and eosin (HE) staining. We compared the prognostic value of PHH3 mitotic index with that of MI by HE in 19 MCPyV-positive and 9 MCPyV-negative MCC patients. PHH3-positive immunoreactivity was mostly observed in mitotic figures. Multivariate analysis significantly showed that MCPyV status (HR, 0.004; 95% CI 0.0003-0.058) and the American Joint Committee of Cancer (AJCC) stage (HR, 5.02; 95% CI 1.23-20.51) were observed as significantly independent prognostic factors for OS. PHH3-positive cell counts/10 HPF was a slightly significant independent prognostic factor for OS (HR, 4.96; 95% CI 0.93-26.55). PHH3-positive MI and MCPyV status in MCC patients are useful in prognostication, although MCPyV-infection is a more powerful prognostic factor in MCCs than the AJCC scheme on proliferation or mitotic indices. © 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

Down-regulated RPS3a/nbl expression during retinoid-induced differentiation of HL-60 cells: a close association with diminished susceptibility to actinomycin D-stimulated apoptosis.

PubMed

Russell, L; Naora, H; Naora, H

2000-04-01

The efficacy of anticancer agents significantly depends on the differential susceptibility of undifferentiated cancer cells and differentiated normal cells to undergo apoptosis. We previously found that enhanced expression of RPS3a/nbl, which apparently encodes a ribosomal protein, seems to prime cells for apoptosis, while suppressing such enhanced expression triggers cell death. The present study found that HL-60 cells induced to differentiate by all-trans retinoic acid did not undergo apoptosis following treatment with actinomycin D whereas undifferentiated HL-60 cells were highly apoptosis-susceptible, confirming earlier suggestions that differentiated cells have diminished apoptosis-susceptibility. Undifferentiated HL-60 cells highly expressed RPS3a/nbl whereas all-trans retinoic acid -induced differentiated cells exhibited markedly reduced levels, suggesting that apoptosis-resistance of differentiated cells could be due to low RPS3a/nbl expression. Down-regulation of enhanced RPS3a/nbl expression was also observed in cells induced to differentiate with the retinoid 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1- propenyl]benzoic acid without any significant induction of cell death. While down-regulation of RPS3a/nbl expression during differentiation did not apparently induce apoptosis, RPS3a/nbl antisense oligomers triggered death of undifferentiated HL-60 cells, but not of retinoid-induced differentiated cells. It therefore seems that while down-regulation of enhanced RPS3a/nbl expression can induce apoptosis in undifferentiated cells, down-regulation of enhanced RPS3a/nbl expression during differentiation occurs independently of apoptosis, and could be regarded as reverting the primed condition to the unprimed (low RPS3a/nbl) state.

Induction of temporally dissociated morphological and physiological differentiation of N1E-115 cells.

PubMed

Cosgrove, C; Cobbett, P

1991-07-01

Clonal cells derived from neural tumors have been widely used to study the processes of neuronal differentiation in vitro. The murine neuroblastoma clone N1E-115 has recently been shown to differentiate morphologically in response to removal of serum from the culture medium. In the present study, the nature and time course of electrophysiological differentiation of N1E-115 cells maintained in serum-free medium was examined. Differentiated cells had a higher resting potential and lower input conductance than nondifferentiated cells. Differentiated but not nondifferentiated cells generated current evoked action potentials, and differentiated cells fired spontaneous, repetitive action potentials after 13 days in serum-free medium. The rate of potential change during the depolarizing and repolarizing phases of the action potential became faster as the duration of maintenance of cells in serum-free medium increased. Remarkably, morphological differentiation appeared to be complete after exposure to serum-free medium for 5 days but electrophysiological differentiation was not complete until 13 days in this medium.

Consequences of metal exposure on retinoid metabolism in vertebrates: a review.

PubMed

Defo, M A; Spear, P A; Couture, P

2014-02-10

What we generally refer to as 'vitamin A' is a group of naturally-occurring molecules structurally similar to retinol that are capable of exerting biological activity. These retinoids are essential to diverse physiological functions including vision, immune response, bone mineralization, reproduction, cell differentiation, and growth. As well, some retinoids have antioxidant properties. Independent studies published over the last few decades have revealed that many fish and wildlife populations living in highly polluted environments have altered retinoid status possibly associated with retinoid metabolic or homeostatic mechanisms. Substantial evidence links organic contaminant exposure with changes in retinoid status in animal populations, but only a few detailed studies have been published implicating inorganic contaminants such as metals. This mini-review selectively deals with field and laboratory studies reporting associations between environmental contaminants, especially trace metals, and alterations in retinoid status. Both essential and non-essential trace metals have been reported to affect retinoid status. This review focuses on metabolic imbalances of retinoids in relation to metal contamination and illustrates possible modes of action. The role of retinoids as antioxidants and their potential as biomarkers of metal contamination are discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Rheb may complex with RASSF1A to coordinate Hippo and TOR signaling.

PubMed

Nelson, Nicholas; Clark, Geoffrey J

2016-06-07

The TOR pathway is a vital component of cellular homeostasis that controls the synthesis of proteins, nucleic acids and lipids. Its core is the TOR kinase. Activation of the TOR pathway suppresses autophagy, which plays a vital but complex role in tumorigenesis. The TOR pathway is regulated by activation of the Ras-related protein Rheb, which can bind mTOR. The Hippo pathway is a major growth control module that regulates cell growth, differentiation and apoptosis. Its core consists of an MST/LATS kinase cascade that can be activated by the RASSF1A tumor suppressor. The TOR and Hippo pathways may be coordinately regulated to promote cellular homeostasis. However, the links between the pathways remain only partially understood. We now demonstrate that in addition to mTOR regulation, Rheb also impacts the Hippo pathway by forming a complex with RASSF1A. Using stable clones of two human lung tumor cell lines (NCI-H1792 and NCI-H1299) with shRNA-mediated silencing or ectopic overexpression of RASSF1A, we show that activated Rheb stimulates the Hippo pathway, but is suppressed in its ability to stimulate the TOR pathway. Moreover, by selectively labeling autophagic vacuoles we show that RASSF1A inhibits the ability of Rheb to suppress autophagy and enhance cell growth. Thus, we identify a new connection that impacts coordination of Hippo and TOR signaling. As RASSF1A expression is frequently lost in human tumors, the RASSF1A status of a tumor may impact not just its Hippo pathway status, but also its TOR pathway status.

Modulation of human osteoclastogenesis and osteoblastogenesis by lycopene.

PubMed

Costa-Rodrigues, João; Fernandes, Maria Helena; Pinho, Olívia; Monteiro, Pedro Ribeiro Rocha

2018-03-15

Lycopene is a lipid-soluble pigment that is mainly found in tomato. It is the carotenoid that presents the highest antioxidant potential, and due to that, it has been implicated in a decrease of the risk of several oxidative-stress-related disorders, such as cancer, inflammatory diseases and osteoporosis. Nevertheless, at the present, there is no detailed information about how lycopene affects bone metabolism. The aim of the present work was to characterize the cellular and molecular effects of lycopene on human osteoclast and osteoblast differentiation and function. It was observed that lycopene, at levels found in plasma after the ingestion of lycopene-containing products, decreased osteoclast differentiation but did not affect cell density/survival; calcium-phosphate resorbing ability was also decreased. On the other hand, osteoblast proliferation (via a decrease on apoptosis) and differentiation were increased in the presence of lycopene. The observed effects in both cell types appeared to be related to significant changes in MEK signaling pathway, but also in protein kinase C pathway in osteoclasts and NFkB signaling in osteoblasts. In conclusion, lycopene appears to promote an anabolic state of bone metabolism, stimulating osteoblastogenesis and inhibiting osteoclastogenesis, which may contribute to the promotion of a proper health status of bone tissue. This information might be relevant for the prevention and delay in the progression of osteolytic bone conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

NASA Technical Reports Server (NTRS)

Chen, Silvia S.; Revoltella, Roberto P.; Papini, Sandra; Michelini, Monica; Fitzgerald, Wendy; Zimmerberg, Joshua; Margolis, Leonid

2003-01-01

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- and Biliary-Like Cells

PubMed Central

Woods, Alisa G.; Lazar, Catalin; Radu, Gabriel L.; Darie, Costel C.; Branza-Nichita, Norica

2013-01-01

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells. PMID:23977166

Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells.

PubMed

Shu, Sai-Nan; Wei, Lai; Wang, Jiang-Hua; Zhan, Yu-Tao; Chen, Hong-Song; Wang, Yu

2004-10-01

To investigate the different effects of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) on hepatic differentiation. MSCs from rat bone marrow were isolated and cultured by standard methods. HSCs from rat bone marrow were isolated and purified by magnetic activated cell sorting. Both cell subsets were induced. Morphology, RT-PCR and immunocytochemistry were used to identify the hepatic differentiation grade. MSCs exhibited round in shape after differentiation, instead of fibroblast-like morphology before differentiation. Albumin mRNA and protein were expressed positively in MSCs, without detection of alpha-fetoprotein (AFP). HSCs were polygonal in shape after differentiation. The expression of albumin signal decreased and AFP signal increased. The expression of CK18 was continuous in MSCs and HSCs both before and after induction. Both MSCs and HSCs have hepatic differentiation capabilities. However, their capabilities are not the same. MSCs can differentiate into mature hepatocyte-like cells, never expressing early hepatic specific genes, while Thy-1.1(+) cells are inclined to differentiate into hepatic stem cell-like cells, with an increasing AFP expression and a decreasing albumin signal. CK18 mRNA is positive in Thy-1.1(+) cells and MSCs, negative in Thy-1.1(-) cells. It seems that CK18 has some relationship with Thy-1.1 antigen, and CK18 may be a predictive marker of hepatic differentiation capability.

Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells.

PubMed

Petrareanu, Catalina; Macovei, Alina; Sokolowska, Izabela; Woods, Alisa G; Lazar, Catalin; Radu, Gabriel L; Darie, Costel C; Branza-Nichita, Norica

2013-01-01

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.

Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiotherapy response prediction

PubMed Central

Jeong, Youngtae; Hoang, Ngoc T.; Lovejoy, Alexander; Stehr, Henning; Newman, Aaron M.; Gentles, Andrew J.; Kong, William; Truong, Diana; Martin, Shanique; Chaudhuri, Aadel; Heiser, Diane; Zhou, Li; Say, Carmen; Carter, Justin N.; Hiniker, Susan M.; Loo, Billy W.; West, Robert B.; Beachy, Philip; Alizadeh, Ash A.; Diehn, Maximilian

2016-01-01

Lung squamous cell carcinomas (LSCC) pathogenesis remains incompletely understood and biomarkers predicting treatment response remain lacking. Here we describe novel murine LSCC models driven by loss of Trp53 and Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal, suggesting that mutations in these genes lead to expansion of mutant stem cell clones. Deletion of Trp53 and Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histological and molecular features of human LSCCs, indicating that they represent the likely cell of origin in this model. Deletion of Keap1 promoted tumor aggressiveness, metastasis, and resistance to oxidative stress and radiotherapy (RT). KEAP1/NRF2 mutation status predicted risk of local recurrence after RT in non-small lung cancer (NSCLC) patients and could be non-invasively identified in circulating tumor DNA. Thus, KEAP1/NRF2 mutations could serve as predictive biomarkers for personalization of therapeutic strategies for NSCLCs. PMID:27663899

Student Perceptions of Self-Disclosure in the Classroom Based on Perceived Status Differentials.

ERIC Educational Resources Information Center

Klinger-Vartabedian, Laurel; O'Flaherty, Kathleen

A study examined the extent to which perceived teacher status differentials influenced students' perceptions of teacher self-disclosure. This study involved a two-step procedure. Initially 30 students in a basic speech section were asked to recall examples of teacher self-disclosure that had occurred in classes in which they were enrolled. Then 13…

Two stages of enteropathogenic Escherichia coli intestinal pathogenicity are up and down-regulated by the epithelial cell differentiation.

PubMed

Gabastou, J M; Kernéis, S; Bernet-Camard, M F; Barbat, A; Coconnier, M H; Kaper, J B; Servin, A L

1995-09-01

Pathogens and eucaryotic cells are active partners during the process of pathogenicity. To gain access to enterocytes and to cross the epithelial membrane, many enterovirulent microorganisms interact with the brush border membrane-associated components as receptors. Recent reports provide evidence that intestinal cell differentiation plays a role in microbial pathogenesis. Human enteropathogenic Escherichia coli (EPEC) develop their pathogenicity upon infecting enterocytes. To determine if intestinal epithelial cell differentiation influences EPEC pathogenicity, we examined the infection of human intestinal epithelial cells by JPN 15 (pMAR7) [EAF+ eae+] EPEC strain as a function of the cell differentiation. The human embryonic intestinal INT407 cells, the human colonic T84 cells, the human undifferentiated HT-29 cells (HT-29 Std) and two enterocytic cell lines, HT-29 glc-/+ and Caco-2 cells, were used as cellular models. Cells were infected apically with the EPEC strain and the cell-association and cell-entry were examined by quantitative determination using metabolically radiolabeled bacteria, as well as by light, scanning and transmission electron microscopy. [EAF+ eae+] EPEC bacteria efficiently colonized the cultured human intestinal cells. Diffuse bacterial adhesion occurred to undifferentiated HT-29 Std and INT407 cells, whereas characteristic EPEC cell clusters were observed on fully differentiated enterocytic HT-29 glc-/+ cells and on colonic crypt T84 cells. As shown using the Caco-2 cell line, which spontaneously differentiates in culture, the formation of EPEC clusters increased as a function of the epithelial cell differentiation. In contrast, efficient cell-entry of [EAF+ eae+] EPEC bacteria occurred in recently differentiated Caco-2 cells and decreased when the cells were fully differentiated.(ABSTRACT TRUNCATED AT 250 WORDS)

YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Yi-Ting; Ding, Jing-Ya; Li, Ming-Yang

2012-09-10

Tight regulation of cell numbers by controlling cell proliferation and apoptosis is important during development. Recently, the Hippo pathway has been shown to regulate tissue growth and organ size in Drosophila. In mammalian cells, it also affects cell proliferation and differentiation in various tissues, including the nervous system. Interplay of several signaling cascades, such as Notch, Wnt, and Sonic Hedgehog (Shh) pathways, control cell proliferation during neuronal differentiation. However, it remains unclear whether the Hippo pathway coordinates with other signaling cascades in regulating neuronal differentiation. Here, we used P19 cells, a mouse embryonic carcinoma cell line, as a model tomore » study roles of YAP, a core component of the Hippo pathway, in neuronal differentiation. P19 cells can be induced to differentiate into neurons by expressing a neural bHLH transcription factor gene Ascl1. Our results showed that YAP promoted cell proliferation and inhibited neuronal differentiation. Expression of Yap activated Shh but not Wnt or Notch signaling activity during neuronal differentiation. Furthermore, expression of Yap increased the expression of Patched homolog 1 (Ptch1), a downstream target of the Shh signaling. Knockdown of Gli2, a transcription factor of the Shh pathway, promoted neuronal differentiation even when Yap was over-expressed. We further demonstrated that over-expression of Yap inhibited neuronal differentiation in primary mouse cortical progenitors and Gli2 knockdown rescued the differentiation defect in Yap over-expressing cells. In conclusion, our study reveals that Shh signaling acts downstream of YAP in regulating neuronal differentiation. -- Highlights: Black-Right-Pointing-Pointer YAP promotes cell proliferation and inhibits neuronal differentiation in P19 cells. Black-Right-Pointing-Pointer YAP promotes Sonic hedgehog signaling activity during neuronal differentiation. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap-overexpression phenotype in P19 cells. Black-Right-Pointing-Pointer Knockdown of Gli2 rescues the Yap-overexpression phenotype in cortical progenitors.« less

Hematopoietic stem cells: can old cells learn new tricks?

PubMed

Ho, Anthony D; Punzel, Michael

2003-05-01

Since the establishment of cell lines derived from human embryonic stem (ES) cells, it has been speculated that out of such "raw material," we could some day produce all sorts of replacement parts for the human body. Human pluripotent stem cells can be isolated from embryonic, fetal, or adult tissues. Enormous self-renewal capacity and developmental potential are the characteristics of ES cells. Somatic stem cells, especially those derived from hematopoietic tissues, have also been reported to exhibit developmental potential heretofore not considered possible. The initial evidences for the plasticity potential of somatic stem cells were so encouraging that the opponents of ES cell research used them as arguments for restricting ES cell research. In the past months, however, critical issues have been raised challenging the validity and the interpretation of the initial data. Whereas hematopoietic stem-cell therapy has been a clinical reality for almost 40 years, there is still a long way to go in basic research before novel therapy strategies with stem cells as replacement for other organ systems can be established. Given the present status, we should keep all options open for research in ES cells and adult stem cells to appreciate the complexity of their differentiation pathways and the relative merits of various types of stem cells for regenerative medicine.

The effect of syndecan-4 and glypican-1 knockdown on the proliferation and differentiation of turkey satellite cells differing in age and growth rates.

PubMed

Velleman, Sandra G; Clark, Daniel L; Tonniges, Jeffrey R

2018-09-01

Posthatch skeletal muscle growth requires myogenic satellite cells and the dynamic expression of cell membrane-associated proteins. The membrane associated heparan sulfate proteoglycans, syndecan-4 and glypican-1, link the satellite cell niche to the intracellular environment. Sydnecan-4 and glypican-1 are differentially expressed with age in turkey satellite cells and their over-expression impacts both satellite cell proliferation and differentiation, but their effect on satellite cells from lines with different growth potentials is not known. The objective of the current study was to determine if syndecan-4 and glypican-1 regulation of satellite cell proliferation and differentiation is affected by age and growth selection. Pectoralis major satellite cells isolated at 1 d, 7 and 16-wk of age from a Randombred Control 2 (RBC2) line and a 16-wk body weight (F) line selected from the RBC2 line turkeys were studied. Syndecan-4 and glypican-1 expression was knocked down in both lines. The F-line cells proliferated faster than RBC2 line cells regardless of age, while differentiation tended to be greater in RBC2 line cells than F-line cells at each age. Syndecan-4 knockdown decreased proliferation at 7- and 16-wk but not 1 d cells, and increased differentiation at 1 d and 7 wk but not 16 wk cells. Glypican-1 knockdown differentially affected proliferation depending on cell age, whereas differentiation was decreased for 7- and 16-wk but not 1 d cells. These data suggest syndecan-4 and glypican-1 differentially affected satellite cell function in an age-dependent manner, but had little impact on differences in proliferation and differentiation due to growth selection. Copyright © 2018. Published by Elsevier Inc.

Chondrogenic Differentiation Increases Antidonor Immune Response to Allogeneic Mesenchymal Stem Cell Transplantation

PubMed Central

Ryan, Aideen E; Lohan, Paul; O'Flynn, Lisa; Treacy, Oliver; Chen, Xizhe; Coleman, Cynthia; Shaw, Georgina; Murphy, Mary; Barry, Frank; Griffin, Matthew D; Ritter, Thomas

2014-01-01

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3+ and CD68+ infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use. PMID:24184966

Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines

PubMed Central

Hearst, Scoty M.; Gilder, Andrew S.; Negi, Sandeep S.; Davis, Misty D.; George, Eric M.; Whittom, Angela A.; Toyota, Cory G.; Husedzinovic, Alma; Gruss, Oliver J.; Hebert, Michael D.

2009-01-01

Summary Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells. PMID:19435804