Effects of cellular differentiation, chromosomal integration and 5-aza-2'-deoxycytidine treatment on human papillomavirus-16 DNA methylation in cultured cell lines.

PubMed

Kalantari, Mina; Lee, Denis; Calleja-Macias, Itzel E; Lambert, Paul F; Bernard, Hans-Ulrich

2008-05-10

Human papillomavirus-16 (HPV-16) genomes in cell culture and in situ are affected by polymorphic methylation patterns, which can repress the viral transcription. In order to understand some of the underlying mechanisms, we investigated changes of the methylation of HPV-16 DNA in cell cultures in response to cellular differentiation, to recombination with cellular DNA, and to an inhibitor of methylation. Undifferentiated W12E cells, derived from a precancerous lesion, contained extrachromosomal HPV-16 DNA with a sporadically methylated enhancer-promoter segment. Upon W12E cell differentiation, the viral DNA was demethylated, suggesting a link between differentiation and the epigenetic state of HPV-16 DNA. The viral genomes present in two W12I clones, in which individual copies of the HPV-16 genome have integrated into cellular DNA (type 1 integrants), were unmethylated, akin to that seen in the cervical carcinoma cell line SiHa (also a type 1 integrant). This finding is consistent with hypomethylation being necessary for continued viral gene expression. In contrast, two of three type 2 integrant W12I clones, containing concatemers of HPV-16 genomes integrated into the cellular DNA contained hypermethylated viral DNA, as observed in the cervical carcinoma cell line CaSki (also a type 2 integrant). A third, type 2, W12I clone, interestingly with fewer copies of the viral genome, contained unmethylated HPV-16 genomes. Epithelial differentiation of W12I clones did not lead to demethylation of chromosomally integrated viral genomes as was seen for extrachromosomal HPV-16 DNA in W12E clones. Hypomethylation of CaSki cells in the presence of the DNA methylation inhibitor 5-aza-2'-deoxycytidine reduced the cellular viability, possibly as a consequence of toxic effects of an excess of HPV-16 gene products. Our data support a model wherein (i) the DNA methylation state of extrachromosomal HPV16 replicons and epithelial differentiation are inversely coupled during the viral life cycle, (ii) integration of the viral genome into the host chromosome events leads to an alteration in methylation patterns on the viral genome that is dependent upon the type of integration event and possibly copy number, and (iii) integration universally results in the viral DNA becoming refractory to changes in methylation state upon cellular differentiation that are observed with extrachromosomal HPV-16 genomes.

Chromatin in embryonic stem cell neuronal differentiation.

PubMed

Meshorer, E

2007-03-01

Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny.

Cellular and Molecular Mechanisms of Sexual Differentiation in the Mammalian Nervous System

PubMed Central

Forger, Nancy G.; Strahan, J. Alex; Castillo-Ruiz, Alexandra

2016-01-01

Neuroscientists are likely to discover new sex differences in the coming years, spurred by the National Institutes of Health initiative to include both sexes in preclinical studies. This review summarizes the current state of knowledge of the cellular and molecular mechanisms underlying sex differences in the mammalian nervous system, based primarily on work in rodents. Cellular mechanisms examined include neurogenesis, migration, the differentiation of neurochemical and morphological cell phenotype, and cell death. At the molecular level we discuss evolving roles for epigenetics, sex chromosome complement, the immune system, and newly identified cell signaling pathways. We review recent findings on the role of the environment, as well as genome-wide studies with some surprising results, causing us to rethink often-used models of sexual differentiation. We end by pointing to future directions, including an increased awareness of the important contributions of tissues outside of the nervous system to sexual differentiation of the brain. PMID:26790970

Transcriptomic Profiling Discloses Molecular and Cellular Events Related to Neuronal Differentiation in SH-SY5Y Neuroblastoma Cells.

PubMed

Pezzini, Francesco; Bettinetti, Laura; Di Leva, Francesca; Bianchi, Marzia; Zoratti, Elisa; Carrozzo, Rosalba; Santorelli, Filippo M; Delledonne, Massimo; Lalowski, Maciej; Simonati, Alessandro

2017-05-01

Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.

Aging and differentiation in yeast populations: elders with different properties and functions.

PubMed

Palková, Zdena; Wilkinson, Derek; Váchová, Libuše

2014-02-01

Over the past decade, it has become evident that similarly to cells forming metazoan tissues, yeast cells have the ability to differentiate and form specialized cell types. Examples of yeast cellular differentiation have been identified both in yeast liquid cultures and within multicellular structures occupying solid surfaces. Most current knowledge on different cell types comes from studies of the spatiotemporal internal architecture of colonies developing on various media. With a few exceptions, yeast cell differentiation often concerns nongrowing, stationary-phase cells and leads to the formation of cell subpopulations differing in stress resistance, cell metabolism, respiration, ROS production, and others. These differences can affect longevity of particular subpopulations. In contrast to liquid cultures, where various cell types are dispersed within stationary-phase populations, cellular differentiation depends on the specific position of particular cells within multicellular colonies. Differentiated colonies, thus, resemble primitive multicellular organisms, in which the gradients of certain compounds and the position of cells within the structure affect cellular differentiation. In this review, we summarize and compare the properties of diverse types of differentiated chronologically aging yeast cells that have been identified in colonies growing on different media, as well as of those found in liquid cultures. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

CELLULAR DIFFERENTIATION AND THE AGING PROCESS IN CARTILAGINOUS TISSUES

PubMed Central

Shulman, Herbert J.; Meyer, Karl

1968-01-01

Primary cell cultures of differentiated chondrocytes were shown to produce chondroitin-4-sulfate as the predominant mucopolysaccharide, with suggestive evidence for the synthesis of keratan sulfate and possibly chondroitin-6-sulfate. Chicken embryonic cartilage was shown to be composed mainly of chondroitin-4-sulfate, with a small amount of chondroitin-6-sulfate, but essentially no keratan sulfate. These findings were compared to the data of others, and a hypothesis explaining the aging process in cartilage in terms of cellular differentiation was presented. PMID:5688079

Epstein-Barr virus growth/latency III program alters cellular microRNA expression

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

Cameron, Jennifer E.; Tulane Cancer Center, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL79, New Orleans, LA 70112; Fewell, Claire

The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lowermore » in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.« less

Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

PubMed Central

Sunami, Yoshitaka; Araki, Marito; Hironaka, Yumi; Morishita, Soji; Kobayashi, Masaki; Liew, Ei Leen; Edahiro, Yoko; Tsutsui, Miyuki; Ohsaka, Akimichi; Komatsu, Norio

2013-01-01

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation. PMID:23460888

Biological and molecular characterization of cellular differentiation in Tetrahymena vorax: a potential biocontrol protozoan.

PubMed

Green, M M; LeBoeuf, R D; Churchill, P F

2000-01-01

Tetrahymena vorax (T. vorax) is an indigenous fresh water protozoan with the natural biological potential to maintain a specific aquatic microbial flora by ingesting and eliminating specific microorganism. To investigate the molecular mechanisms controlling Tetrahymena vorax (T. vorax) cellular differentiation from a small-mouth vegetative cell to a voracious large-mouth carnivore capable of ingesting prey ciliates and bacteria from aquatic environments, we use DNA subtraction and gene discovery techniques to identify and isolate T. vorax differentiation-specific genes. The physiological necessity for one newly discovered gene, SUBII-TG, was determined in vivo using an antisense oligonucleotide directed against the 5' SUBII-TG DNA sequence. The barriers to delivering antisense oligonucleotides to the cytoplasm of T. vorax were circumvented by employing a new but simple procedure of processing the oligonucleotide with the differentiation stimulus, stomatin. In these studies, the antisense oligonucleotide down-regulated SUBII-TG mRNA expression, and blocked differentiation and ingestion of prey ciliates. The ability to down-regulate SUBII-TG expression with the antisense oligonucleotide suggests that the molecular mechanisms controlling the natural biological activities of T. vorax can be manipulated to further study its cellular differentiation and potential as a biocontrol microorganism.

Spectral Monitoring of Surfactant Clearance during Alveolar Epithelial Type II Cell Differentiation

PubMed Central

Swain, Robin J.; Kemp, Sarah J.; Goldstraw, Peter; Tetley, Teresa D.; Stevens, Molly M.

2008-01-01

In this study, we report on the noninvasive identification of spectral markers of alveolar type II (ATII) cell differentiation in vitro using Raman microspectroscopy. ATII cells are progenitor cells for alveolar type I (ATI) cells in vivo, and spontaneously differentiate toward an ATI-like phenotype in culture. We analyzed undifferentiated and differentiated primary human ATII cells, and correlated Raman spectral changes to cellular changes in morphology and marker protein synthesis (surfactant protein C, alkaline phosphatase, caveolin-1). Undifferentiated ATII cells demonstrated spectra with strong phospholipid vibrations, arising from alveolar surfactant stored within cytoplasmic lamellar bodies (Lbs). Differentiated ATI-like cells yielded spectra with significantly less lipid content. Factor analysis revealed a phospholipid-dominated spectral component as the main discriminator between the ATII and ATI-like phenotypes. Spectral modeling of the data revealed a significant decrease in the spectral contribution of cellular lipids—specifically phosphatidyl choline, the main constituent of surfactant, as ATII cells differentiate. These observations were consistent with the clearance of surfactant from Lbs as ATII cells differentiate, and were further supported by cytochemical staining for Lbs. These results demonstrate the first spectral characterization of primary human ATII cells, and provide insight into the biochemical properties of alveolar surfactant in its unperturbed cellular environment. PMID:18820234

Oxidative Stress, Redox Regulation and Diseases of Cellular Differentiation

PubMed Central

Ye, Zhi-Wei; Zhang, Jie; Townsend, Danyelle M.; Tew, Kenneth D.

2015-01-01

Background Within cells, there is a narrow concentration threshold that governs whether reactive oxygen species (ROS) induce toxicity or act as second messengers. Scope of review We discuss current understanding of how ROS arise, facilitate cell signaling, cause toxicities and disease related to abnormal cell differentiation and those (primarily) sulfur based pathways that provide nucleophilicity to offset these effects. Primary conclusions Cellular redox homeostasis mediates a plethora of cellular pathways that determine life and death events. For example, ROS intersect with GSH based enzyme pathways to influence cell differentiation, a process integral to normal hematopoiesis, but also affecting a number of diverse cell differentiation related human diseases. Recent attempts to manage such pathologies have focused on intervening in some of these pathways, with the consequence that differentiation therapy targeting redox homeostasis has provided a platform for drug discovery and development. General Significance The balance between electrophilic oxidative stress and protective biomolecular nucleophiles predisposes the evolution of modern life forms. Imbalances of the two can produce aberrant redox homeostasis with resultant pathologies. Understanding the pathways involved provides opportunities to consider interventional strategies. PMID:25445706

Effect of 3D Cultivation Conditions on the Differentiation of Endodermal Cells

PubMed Central

Petrakova, O. S.; Ashapkin, V. V.; Voroteliak, E. A.; Bragin, E. Y.; Shtratnikova, V. Y.; Chernioglo, E. S.; Sukhanov, Y. V.; Terskikh, V. V.; Vasiliev, A. V.

2012-01-01

Cellular therapy of endodermal organs is one of the most important issues in modern cellular biology and biotechnology. One of the most promising directions in this field is the study of the transdifferentiation abilities of cells within the same germ layer. A method for anin vitroinvestigation of the cell differentiation potential (the cell culture in a three-dimensional matrix) is described in this article. Cell cultures of postnatal salivary gland cells and postnatal liver progenitor cells were obtained; their comparative analysis under 2D and 3D cultivation conditions was carried out. Both cell types have high proliferative abilities and can be cultivated for more than 20 passages. Under 2D cultivation conditions, the cells remain in an undifferentiated state. Under 3D conditions, they undergo differentiation, which was confirmed by a lower cell proliferation and by an increase in the differentiation marker expression. Salivary gland cells can undergo hepatic and pancreatic differentiation under 3D cultivation conditions. Liver progenitor cells also acquire a pancreatic differentiation capability under conditions of 3D cultivation. Thus, postnatal salivary gland cells exhibit a considerable differentiation potential within the endodermal germ layer and can be used as a promising source of endodermal cells for the cellular therapy of liver pathologies. Cultivation of cells under 3D conditions is a useful model for thein vitroanalysis of the cell differentiation potential. PMID:23346379

Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus.

PubMed

Abdi, Khadar; Lai, Chun-Hsiang; Paez-Gonzalez, Patricia; Lay, Mark; Pyun, Joon; Kuo, Chay T

2018-04-25

Specialized, differentiated cells often perform unique tasks that require them to maintain a stable phenotype. Multiciliated ependymal cells (ECs) are unique glial cells lining the brain ventricles, important for cerebral spinal fluid circulation. While functional ECs are needed to prevent hydrocephalus, they have also been reported to generate new neurons: whether ECs represent a stable cellular population remains unclear. Via a chemical screen we found that mature ECs are inherently plastic, with their multiciliated state needing constant maintenance by the Foxj1 transcription factor, which paradoxically is rapidly turned over by the ubiquitin-proteasome system leading to cellular de-differentiation. Mechanistic analyses revealed a novel NF-κB-independent IKK2 activity stabilizing Foxj1 in mature ECs, and we found that known IKK2 inhibitors including viruses and growth factors robustly induced Foxj1 degradation, EC de-differentiation, and hydrocephalus. Although mature ECs upon de-differentiation can divide and regenerate multiciliated ECs, we did not detect evidence supporting EC's neurogenic potential.

Induction of CD4 T cell memory by local cellular collectivity.

PubMed

Polonsky, Michal; Rimer, Jacob; Kern-Perets, Amos; Zaretsky, Irina; Miller, Stav; Bornstein, Chamutal; David, Eyal; Kopelman, Naama Meira; Stelzer, Gil; Porat, Ziv; Chain, Benjamin; Friedman, Nir

2018-06-15

Cell differentiation is directed by signals driving progenitors into specialized cell types. This process can involve collective decision-making, when differentiating cells determine their lineage choice by interacting with each other. We used live-cell imaging in microwell arrays to study collective processes affecting differentiation of naïve CD4 + T cells into memory precursors. We found that differentiation of precursor memory T cells sharply increases above a threshold number of locally interacting cells. These homotypic interactions involve the cytokines interleukin-2 (IL-2) and IL-6, which affect memory differentiation orthogonal to their effect on proliferation and survival. Mathematical modeling suggests that the differentiation rate is continuously modulated by the instantaneous number of locally interacting cells. This cellular collectivity can prioritize allocation of immune memory to stronger responses. Copyright © 2018, American Association for the Advancement of Science.

Mitochondrial activity in the regulation of stem cell self-renewal and differentiation.

PubMed

Khacho, Mireille; Slack, Ruth S

2017-12-01

Mitochondria are classically known as the essential energy producers in cells. As such, the activation of mitochondrial metabolism upon cellular differentiation was deemed a necessity to fuel the high metabolic needs of differentiated cells. However, recent studies have revealed a direct role for mitochondrial activity in the regulation of stem cell fate and differentiation. Several components of mitochondrial metabolism and respiration have now been shown to regulate different aspects of stem cell differentiation through signaling, transcriptional, proteomic and epigenetic modulations. In light of these findings mitochondrial metabolism is no longer considered a consequence of cellular differentiation, but rather a key regulatory mechanism of this process. This review will focus on recent progress that defines mitochondria as the epicenters for the regulation of stem cell fate decisions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior.

PubMed

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-24

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an Apc Min/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion.

Cellular context-dependent consequences of Apc mutations on gene regulation and cellular behavior

PubMed Central

Hashimoto, Kyoichi; Yamada, Yosuke; Semi, Katsunori; Yagi, Masaki; Tanaka, Akito; Itakura, Fumiaki; Aoki, Hitomi; Kunisada, Takahiro; Woltjen, Knut; Haga, Hironori; Sakai, Yoshiharu; Yamamoto, Takuya; Yamada, Yasuhiro

2017-01-01

The spectrum of genetic mutations differs among cancers in different organs, implying a cellular context-dependent effect for genetic aberrations. However, the extent to which the cellular context affects the consequences of oncogenic mutations remains to be fully elucidated. We reprogrammed colon tumor cells in an ApcMin/+ (adenomatous polyposis coli) mouse model, in which the loss of the Apc gene plays a critical role in tumor development and subsequently, established reprogrammed tumor cells (RTCs) that exhibit pluripotent stem cell (PSC)-like signatures of gene expression. We show that the majority of the genes in RTCs that were affected by Apc mutations did not overlap with the genes affected in the intestine. RTCs lacked pluripotency but exhibited an increased expression of Cdx2 and a differentiation propensity that was biased toward the trophectoderm cell lineage. Genetic rescue of the mutated Apc allele conferred pluripotency on RTCs and enabled their differentiation into various cell types in vivo. The redisruption of Apc in RTC-derived differentiated cells resulted in neoplastic growth that was exclusive to the intestine, but the majority of the intestinal lesions remained as pretumoral microadenomas. These results highlight the significant influence of cellular context on gene regulation, cellular plasticity, and cellular behavior in response to the loss of the Apc function. Our results also imply that the transition from microadenomas to macroscopic tumors is reprogrammable, which underscores the importance of epigenetic regulation on tumor promotion. PMID:28057861

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation

PubMed Central

Lawag, Abdalla A.; Napper, Jennifer M.; Hunter, Caroline A.; Bacon, Nickolas A.; Deskins, Seth; El-hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C.

2017-01-01

Abstract Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%–90% of the cells die when placed in medium where the major growth factor is granulocyte–macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer. PMID:28910138

HSP90 Inhibition and Cellular Stress Elicits Phenotypic Plasticity in Hematopoietic Differentiation.

PubMed

Lawag, Abdalla A; Napper, Jennifer M; Hunter, Caroline A; Bacon, Nickolas A; Deskins, Seth; El-Hamdani, Manaf; Govender, Sarah-Leigh; Koc, Emine C; Sollars, Vincent E

2017-10-01

Cancer cells exist in a state of Darwinian selection using mechanisms that produce changes in gene expression through genetic and epigenetic alteration to facilitate their survival. Cellular plasticity, or the ability to alter cellular phenotype, can assist in survival of premalignant cells as they progress to full malignancy by providing another mechanism of adaptation. The connection between cellular stress and the progression of cancer has been established, although the details of the mechanisms have yet to be fully elucidated. The molecular chaperone HSP90 is often upregulated in cancers as they progress, presumably to allow cancer cells to deal with misfolded proteins and cellular stress associated with transformation. The objective of this work is to test the hypothesis that inhibition of HSP90 results in increased cell plasticity in mammalian systems that can confer a greater adaptability to selective pressures. The approach used is a murine in vitro model system of hematopoietic differentiation that utilizes a murine hematopoietic stem cell line, erythroid myeloid lymphoid (EML) clone 1, during their maturation from stem cells to granulocytic progenitors. During the differentiation protocol, 80%-90% of the cells die when placed in medium where the major growth factor is granulocyte-macrophage-colony stimulating factor. Using this selection point model, EML cells exhibit increases in cellular plasticity when they are better able to adapt to this medium and survive. Increases in cellular plasticity were found to occur upon exposure to geldanamycin to inhibit HSP90, when subjected to various forms of cellular stress, or inhibition of histone acetylation. Furthermore, we provide evidence that the cellular plasticity associated with inhibition of HSP90 in this model involves epigenetic mechanisms and is dependent upon high levels of stem cell factor signaling. This work provides evidence for a role of HSP90 and cellular stress in inducing phenotypic plasticity in mammalian systems that has new implications for cellular stress in progression and evolution of cancer.

Complementation of non-tumorigenicity of HPV18-positive cervical carcinoma cells involves differential mRNA expression of cellular genes including potential tumor suppressor genes on chromosome 11q13.

PubMed

Kehrmann, Angela; Truong, Ha; Repenning, Antje; Boger, Regina; Klein-Hitpass, Ludger; Pascheberg, Ulrich; Beckmann, Alf; Opalka, Bertram; Kleine-Lowinski, Kerstin

2013-01-01

The fusion between human tumorigenic cells and normal human diploid fibroblasts results in non-tumorigenic hybrid cells, suggesting a dominant role for tumor suppressor genes in the generated hybrid cells. After long-term cultivation in vitro, tumorigenic segregants may arise. The loss of tumor suppressor genes on chromosome 11q13 has been postulated to be involved in the induction of the tumorigenic phenotype of human papillomavirus (HPV)18-positive cervical carcinoma cells and their derived tumorigenic hybrid cells after subcutaneous injection in immunocompromised mice. The aim of this study was the identification of novel cellular genes that may contribute to the suppression of the tumorigenic phenotype of non-tumorigenic hybrid cells in vivo. We used cDNA microarray technology to identify differentially expressed cellular genes in tumorigenic HPV18-positive hybrid and parental HeLa cells compared to non-tumorigenic HPV18-positive hybrid cells. We detected several as yet unknown cellular genes that play a role in cell differentiation, cell cycle progression, cell-cell communication, metastasis formation, angiogenesis, antigen presentation, and immune response. Apart from the known differentially expressed genes on 11q13 (e.g., phosphofurin acidic cluster sorting protein 1 (PACS1) and FOS ligand 1 (FOSL1 or Fra-1)), we detected novel differentially expressed cellular genes located within the tumor suppressor gene region (e.g., EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) and leucine rich repeat containing 32 (LRRC32) (also known as glycoprotein-A repetitions predominant (GARP)) that may have potential tumor suppressor functions in this model system of non-tumorigenic and tumorigenic HeLa x fibroblast hybrid cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Vitamin D Combined with Aminolevulinate (ALA)-Mediated Photodynamic Therapy (PDT) for Human Psoriasis: A Proof-of-Principle Study

PubMed Central

Maytin, Edward V.; Honari, Golara; Khachemoune, Amor; Taylor, Charles R.; Ortel, Bernhard; Pogue, Brian W.; Sznycer-Taub, Nathaniel; Hasan, Tayyaba

2012-01-01

We previously showed that select agents (methotrexate or Vitamin D), when administered as a preconditioning regimen, are capable of promoting cellular differentiation of epithelial cancer cells while simultaneously enhancing the efficacy of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT). In solid tumors, pretreatment with Vitamin D simultaneously promotes cellular differentiation and leads to selective accumulation of target porphyrins (mainly protoporphyrin IX, PpIX) within diseased tissue. However, questions of whether or not the effects upon cellular differentiation are inexorably linked to PpIX accumulation, and whether these effects might occur in hyperproliferative noncancerous tissues, have remained unanswered. In this paper, we reasoned that psoriasis, a human skin disease in which abnormal cellular proliferation and differentiation plays a major role, could serve as a useful model to test the effects of pro-differentiating agents upon PpIX levels in a non-neoplastic setting. In particular, Vitamin D, a treatment for psoriasis that restores (increases) differentiation, might increase PpIX levels in psoriatic lesions and facilitate their responsiveness to ALA-PDT. This concept was tested in a pilot study of 7 patients with bilaterally-matched psoriatic plaques. A regimen in which calcipotriol 0.005% ointment was applied for 3 days prior to ALA-PDT with blue light, led to preferential increases in PpIX (~130%), and reductions in thickness, redness, scaling, and itching in the pretreated plaques. The results suggest that a larger clinical trial is warranted to confirm a role for combination treatments with Vitamin D and ALA-PDT for psoriasis. PMID:23264699

Physical biology of human brain development.

PubMed

Budday, Silvia; Steinmann, Paul; Kuhl, Ellen

2015-01-01

Neurodevelopment is a complex, dynamic process that involves a precisely orchestrated sequence of genetic, environmental, biochemical, and physical events. Developmental biology and genetics have shaped our understanding of the molecular and cellular mechanisms during neurodevelopment. Recent studies suggest that physical forces play a central role in translating these cellular mechanisms into the complex surface morphology of the human brain. However, the precise impact of neuronal differentiation, migration, and connection on the physical forces during cortical folding remains unknown. Here we review the cellular mechanisms of neurodevelopment with a view toward surface morphogenesis, pattern selection, and evolution of shape. We revisit cortical folding as the instability problem of constrained differential growth in a multi-layered system. To identify the contributing factors of differential growth, we map out the timeline of neurodevelopment in humans and highlight the cellular events associated with extreme radial and tangential expansion. We demonstrate how computational modeling of differential growth can bridge the scales-from phenomena on the cellular level toward form and function on the organ level-to make quantitative, personalized predictions. Physics-based models can quantify cortical stresses, identify critical folding conditions, rationalize pattern selection, and predict gyral wavelengths and gyrification indices. We illustrate that physical forces can explain cortical malformations as emergent properties of developmental disorders. Combining biology and physics holds promise to advance our understanding of human brain development and enable early diagnostics of cortical malformations with the ultimate goal to improve treatment of neurodevelopmental disorders including epilepsy, autism spectrum disorders, and schizophrenia.

Disruption of Testis Cords by Cyclopamine or Forskolin Reveals Independent Cellular Pathways in Testis Organogenesis

PubMed Central

Yao, Humphrey Hung-Chang; Capel, Blanche

2014-01-01

Most studies to date indicate that the formation of testis cords is critical for proper Sertoli cell differentiation, inhibition of germ cell meiosis, and regulation of Leydig cell differentiation. However, the connections between these events are poorly understood. The objective of this study was to dissect the molecular and cellular relationships between these events in testis formation. We took advantage of the different effects of two hedgehog signaling inhibitors, cyclopamine and forskolin, on gonad explant cultures. Both hedgehog inhibitors phenocopied the disruptive effect of Dhh−/− on formation of testis cords without influencing Sertoli cell differentiation. However, they exhibited different effects on other cellular events during testis development. Treatment with cyclopamine did not affect inhibition of germ cell meiosis and mesonephric cell migration but caused defects in Leydig cell differentiation. In contrast, forskolin treatment induced germ cell meiosis, inhibited mesonephric cell migration, and had no effect on Leydig cell differentiation. By carefully contrasting the different effects of these two hedgehog inhibitors, we demonstrate that although formation of testis cords and development of other cell types normally take place in a tightly regulated sequence, each of these events can occur independent of the others. PMID:12051821

A comparative study of metabolic state of stem cells during osteogenic and adipogenic differentiations via fluorescence lifetime imaging microscopy

NASA Astrophysics Data System (ADS)

Chakraborty, Sandeep; Ou, Meng-Hsin; Kuo, Jean-Cheng; Chiou, Arthur

2016-10-01

Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P < 0.001), while the ratio of free to protein-bound NADH ratio decreased significantly in 7- days (P < 0.001) and 14-days (P < 0.001). Thus, our results indicated a higher metabolic rate in both osteogenic and adipogenic differentiation processes when compared with undifferentiated hMSCs. This approach may be further utilized to study proliferation efficiency and differentiation potential of stem cells into other specialized cell lineages.

Differential Cellular Responses to Hedgehog Signalling in Vertebrates—What is the Role of Competence?

PubMed Central

Kiecker, Clemens; Graham, Anthony; Logan, Malcolm

2016-01-01

A surprisingly small number of signalling pathways generate a plethora of cellular responses ranging from the acquisition of multiple cell fates to proliferation, differentiation, morphogenesis and cell death. These diverse responses may be due to the dose-dependent activities of signalling factors, or to intrinsic differences in the response of cells to a given signal—a phenomenon called differential cellular competence. In this review, we focus on temporal and spatial differences in competence for Hedgehog (HH) signalling, a signalling pathway that is reiteratively employed in embryos and adult organisms. We discuss the upstream signals and mechanisms that may establish differential competence for HHs in a range of different tissues. We argue that the changing competence for HH signalling provides a four-dimensional framework for the interpretation of the signal that is essential for the emergence of functional anatomy. A number of diseases—including several types of cancer—are caused by malfunctions of the HH pathway. A better understanding of what provides differential competence for this signal may reveal HH-related disease mechanisms and equip us with more specific tools to manipulate HH signalling in the clinic. PMID:29615599

Metabolic pathways in T cell activation and lineage differentiation.

PubMed

Almeida, Luís; Lochner, Matthias; Berod, Luciana; Sparwasser, Tim

2016-10-01

Recent advances in the field of immunometabolism support the concept that fundamental processes in T cell biology, such as TCR-mediated activation and T helper lineage differentiation, are closely linked to changes in the cellular metabolic programs. Although the major task of the intermediate metabolism is to provide the cell with a constant supply of energy and molecular precursors for the production of biomolecules, the dynamic regulation of metabolic pathways also plays an active role in shaping T cell responses. Key metabolic processes such as glycolysis, fatty acid and mitochondrial metabolism are now recognized as crucial players in T cell activation and differentiation, and their modulation can differentially affect the development of T helper cell lineages. In this review, we describe the diverse metabolic processes that T cells engage during their life cycle from naïve towards effector and memory T cells. We consider in particular how the cellular metabolism may actively support the function of T cells in their different states. Moreover, we discuss how molecular regulators such as mTOR or AMPK link environmental changes to adaptations in the cellular metabolism and elucidate the consequences on T cell differentiation and function. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium.

PubMed

Miyoshi, Hiroyuki; VanDussen, Kelli L; Malvin, Nicole P; Ryu, Stacy H; Wang, Yi; Sonnek, Naomi M; Lai, Chin-Wen; Stappenbeck, Thaddeus S

2017-01-04

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound-associated epithelial (WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 (PGE 2 ) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear β-catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE 2 -Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE 2 and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury. © 2016 The Authors.

Predictive Modeling and Computational Toxicology

EPA Science Inventory

Embryonic development is orchestrated via a complex series of cellular interactions controlling behaviors such as mitosis, migration, differentiation, adhesion, contractility, apoptosis, and extracellular matrix remodeling. Any chemical exposure that perturbs these cellular proce...

Unique Cellular Organization in the Oldest Root Meristem.

PubMed

Hetherington, Alexander J; Dubrovsky, Joseph G; Dolan, Liam

2016-06-20

Roots and shoots of plant bodies develop from meristems-cell populations that self-renew and produce cells that undergo differentiation-located at the apices of axes [1].The oldest preserved root apices in which cellular anatomy can be imaged are found in nodules of permineralized fossil soils called coal balls [2], which formed in the Carboniferous coal swamp forests over 300 million years ago [3-9]. However, no fossil root apices described to date were actively growing at the time of preservation [3-10]. Because the cellular organization of meristems changes when root growth stops, it has been impossible to compare cellular dynamics as stem cells transition to differentiated cells in extinct and extant taxa [11]. We predicted that meristems of actively growing roots would be preserved in coal balls. Here we report the discovery of the first fossilized remains of an actively growing root meristem from permineralized Carboniferous soil with detail of the stem cells and differentiating cells preserved. The cellular organization of the meristem is unique. The position of the Körper-Kappe boundary, discrete root cap, and presence of many anticlinal cell divisions within a broad promeristem distinguish it from all other known root meristems. This discovery is important because it demonstrates that the same general cellular dynamics are conserved between the oldest extinct and extant root meristems. However, its unique cellular organization demonstrates that extant root meristem organization and development represents only a subset of the diversity that has existed since roots first evolved. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cellular Automata for Spatiotemporal Pattern Formation from Reaction-Diffusion Partial Differential Equations

NASA Astrophysics Data System (ADS)

Ohmori, Shousuke; Yamazaki, Yoshihiro

2016-01-01

Ultradiscrete equations are derived from a set of reaction-diffusion partial differential equations, and cellular automaton rules are obtained on the basis of the ultradiscrete equations. Some rules reproduce the dynamical properties of the original reaction-diffusion equations, namely, bistability and pulse annihilation. Furthermore, other rules bring about soliton-like preservation and periodic pulse generation with a pacemaker, which are not obtained from the original reaction-diffusion equations.

Developmental Regulation of Nucleolus Size during Drosophila Eye Differentiation

PubMed Central

Baker, Nicholas E.

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals. PMID:23472166

Role of the Cellular Prion Protein in Oligodendrocyte Precursor Cell Proliferation and Differentiation in the Developing and Adult Mouse CNS

PubMed Central

Bribián, Ana; Gavín, Rosalina; Reina, Manuel; García-Verdugo, José Manuel; Torres, Juan María; de Castro, Fernando; del Río, José Antonio

2012-01-01

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrPc) to this process remains unclear. PrPc is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrPc influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrPc proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrPc knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells. PMID:22529900

CellNet: Network Biology Applied to Stem Cell Engineering

PubMed Central

Cahan, Patrick; Li, Hu; Morris, Samantha A.; da Rocha, Edroaldo Lummertz; Daley, George Q.; Collins, James J.

2014-01-01

SUMMARY Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population, and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering. PMID:25126793

Developmental regulation of nucleolus size during Drosophila eye differentiation.

PubMed

Baker, Nicholas E

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

PubMed Central

Teixeira, Felipe K.; Sanchez, Carlos G.; Hurd, Thomas R.; Seifert, Jessica R. K.; Czech, Benjamin; Preall, Jonathan B.; Hannon, Gregory J.; Lehmann, Ruth

2015-01-01

The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used1,2. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation. PMID:25915123

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.

Classification of C2C12 cells at differentiation by convolutional neural network of deep learning using phase contrast images.

PubMed

Niioka, Hirohiko; Asatani, Satoshi; Yoshimura, Aina; Ohigashi, Hironori; Tagawa, Seiichi; Miyake, Jun

2018-01-01

In the field of regenerative medicine, tremendous numbers of cells are necessary for tissue/organ regeneration. Today automatic cell-culturing system has been developed. The next step is constructing a non-invasive method to monitor the conditions of cells automatically. As an image analysis method, convolutional neural network (CNN), one of the deep learning method, is approaching human recognition level. We constructed and applied the CNN algorithm for automatic cellular differentiation recognition of myogenic C2C12 cell line. Phase-contrast images of cultured C2C12 are prepared as input dataset. In differentiation process from myoblasts to myotubes, cellular morphology changes from round shape to elongated tubular shape due to fusion of the cells. CNN abstract the features of the shape of the cells and classify the cells depending on the culturing days from when differentiation is induced. Changes in cellular shape depending on the number of days of culture (Day 0, Day 3, Day 6) are classified with 91.3% accuracy. Image analysis with CNN has a potential to realize regenerative medicine industry.

Smooth muscle-like tissue constructs with circumferentially oriented cells formed by the cell fiber technology.

PubMed

Hsiao, Amy Y; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

2015-01-01

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.

Smooth Muscle-Like Tissue Constructs with Circumferentially Oriented Cells Formed by the Cell Fiber Technology

PubMed Central

Hsiao, Amy Y.; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

2015-01-01

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments. PMID:25734774

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

PubMed

Amendola, R; Martinez, R; Negroni, A; Venturelli, D; Tanno, B; Calabretta, B; Raschellà, G

1997-09-03

Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect cellular adhesion through regulation of integrin protein expression.

Long-distance communication by specialized cellular projections during pigment pattern development and evolution

PubMed Central

Eom, Dae Seok; Bain, Emily J; Patterson, Larissa B; Grout, Megan E; Parichy, David M

2015-01-01

Changes in gene activity are essential for evolutionary diversification. Yet, elucidating the cellular behaviors that underlie modifications to adult form remains a profound challenge. We use neural crest-derived adult pigmentation of zebrafish and pearl danio to uncover cellular bases for alternative pattern states. We show that stripes in zebrafish require a novel class of thin, fast cellular projection to promote Delta-Notch signaling over long distances from cells of the xanthophore lineage to melanophores. Projections depended on microfilaments and microtubules, exhibited meandering trajectories, and stabilized on target cells to which they delivered membraneous vesicles. By contrast, the uniformly patterned pearl danio lacked such projections, concomitant with Colony stimulating factor 1-dependent changes in xanthophore differentiation that likely curtail signaling available to melanophores. Our study reveals a novel mechanism of cellular communication, roles for differentiation state heterogeneity in pigment cell interactions, and an unanticipated morphogenetic behavior contributing to a striking difference in adult form. DOI: http://dx.doi.org/10.7554/eLife.12401.001 PMID:26701906

Dissecting the Calcium-Induced Differentiation of Human Primary Keratinocytes Stem Cells by Integrative and Structural Network Analyses

PubMed Central

Toufighi, Kiana; Yang, Jae-Seong; Luis, Nuno Miguel; Aznar Benitah, Salvador; Lehner, Ben; Serrano, Luis; Kiel, Christina

2015-01-01

The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation. PMID:25946651

γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake

PubMed Central

Jung, Chang Hwa; Lee, Da-Hye; Ahn, Jiyun; Lee, Hyunjung; Choi, Won Hee; Jang, Young Jin; Ha, Tae-Youl

2015-01-01

Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz), a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhanced binding protein alpha (C/EBPα). Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4) from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1). The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1. PMID:26083118

γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake.

PubMed

Jung, Chang Hwa; Lee, Da-Hye; Ahn, Jiyun; Lee, Hyunjung; Choi, Won Hee; Jang, Young Jin; Ha, Tae-Youl

2015-06-15

Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz), a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhanced binding protein alpha (C/EBPα). Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4) from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1). The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1.

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

Impaired osteogenic differentiation and enhanced cellular receptor of advanced glycation end products sensitivity in patients with type 2 diabetes.

PubMed

Phimphilai, Mattabhorn; Pothacharoen, Peraphan; Kongtawelert, Prachya; Chattipakorn, Nipon

2017-11-01

Preclinical studies have demonstrated impaired osteoblast differentiation in type 2 diabetes (T2DM), which is related to skeletal accumulation of advanced glycation end products (AGEs). However, the role of AGE in osteoblast differentiation in patients with T2DM is unclear. This cross-sectional study was performed to investigate osteoblast differentiation and its association with serum pentosidine and soluble receptor of AGEs (sRAGE). Twenty-seven patients with T2DM and 15 age-matched controls were included to measure sRAGE and osteogenic differentiation in mononuclear cells derived from peripheral blood. The mononuclear cells isolated from patients with T2DM showed a significantly lower rate of osteogenic differentiation (7.4% vs 86.7%, p < 0.0001) with a lower level of ALPL, COL1A1, and BGLAP expression than those of controls by 11-, 44-, and 15-fold respectively, together with nonvisualized mineralization by alizarin red S staining. The levels of pentosidine and sRAGE were comparable in both groups. AGER expression was significantly higher in the T2DM group. BAX expression was also significantly higher in the T2DM group, and showed a strong correlation with AGER expression (r = 0.86, p < 0.0001). Fasting plasma glucose (FPG) level, AGER expression, and BAX expression showed a strong correlation with osteogenic differentiation defects on univariate analysis. However, only FPG showed a correlation with this defect in a multivariate analysis. In conclusion, patients with T2DM showed impairment of osteoblast differentiation, and FPG was an independent risk factor for this impairment. Moreover, T2DM showed a higher cellular sensitivity for activation of receptor of AGEs and higher cellular apoptosis, which may contribute to the defect in osteoblast differentiation.

Harnessing cellular differentiation to improve ALA-based photodynamic therapy in an artificial skin model

NASA Astrophysics Data System (ADS)

Maytin, Edward; Anand, Sanjay; Sato, Nobuyuki; Mack, Judith; Ortel, Bernhard

2005-04-01

During ALA-based photodynamic therapy (PDT), a pro-drug (aminolevulinic acid; ALA) is taken up by tumor cells and metabolically converted to a photosensitizing intermediate (protoporphyrin IX; PpIX). ALA-based PDT, while an emerging treatment modality, remains suboptimal for most cancers (e.g. squamous cell carcinoma of the skin). Many treatment failures may be largely due to insufficient conversion of ALA to PpIX within cells. We discovered a novel way to increase the conversion of ALA to PpIX, by administering agents that can drive terminal differentiation (i.e., accelerate cellular maturation). Terminally-differentiated epithelial cells show higher levels of intracellular PpIX, apparently via increased levels of a rate-limiting enzyme, coproporphyrinogen oxidase (CPO). To study these mechanisms in a three-dimensional tissue, we developed an organotypic model that mimics true epidermal physiology in a majority of respects. A line of rat epidermal keratinocytes (REKs), when grown in raft cultures, displays all the features of a fully-differentiated epidermis. Addition of ALA to the culture medium results in ALA uptake and PpIX synthesis, with subsequent death of keratinocytes upon exposure to blue light. Using this model, we can manipulate cellular differentiation via three different approaches. (1) Vitamin D, a hormone that enhances keratinocyte differentiation; (2) Hoxb13, a nuclear transcription factor that affects the genetically-controlled differentiation program of stratifying cells (3) Hyaluronan, an abundant extracellular matrix molecule that regulates epidermal differentiation. Because the raft cultures contain only a single cell type (no blood, fibroblasts, etc.) the effects of terminal differentiation upon CPO, PpIX, and keratinocyte cell death can be specifically defined.

Determining Regulatory Networks Governing the Differentiation of Embryonic Stem Cells to Pancreatic Lineage

NASA Astrophysics Data System (ADS)

Banerjee, Ipsita

2009-03-01

Knowledge of pathways governing cellular differentiation to specific phenotype will enable generation of desired cell fates by careful alteration of the governing network by adequate manipulation of the cellular environment. With this aim, we have developed a novel method to reconstruct the underlying regulatory architecture of a differentiating cell population from discrete temporal gene expression data. We utilize an inherent feature of biological networks, that of sparsity, in formulating the network reconstruction problem as a bi-level mixed-integer programming problem. The formulation optimizes the network topology at the upper level and the network connectivity strength at the lower level. The method is first validated by in-silico data, before applying it to the complex system of embryonic stem (ES) cell differentiation. This formulation enables efficient identification of the underlying network topology which could accurately predict steps necessary for directing differentiation to subsequent stages. Concurrent experimental verification demonstrated excellent agreement with model prediction.

RNA sequencing supports distinct reactive oxygen species-mediated pathways of apoptosis by high and low size mass fractions of Bay leaf (Lauris nobilis) in HT-29 cells.

PubMed

Rodd, Annabelle L; Ververis, Katherine; Sayakkarage, Dheeshana; Khan, Abdul W; Rafehi, Haloom; Ziemann, Mark; Loveridge, Shanon J; Lazarus, Ross; Kerr, Caroline; Lockett, Trevor; El-Osta, Assam; Karagiannis, Tom C; Bennett, Louise E

2015-08-01

Anti-proliferative and pro-apoptotic effects of Bay leaf (Laurus nobilis) in mammalian cancer and HT-29 adenocarcinoma cells have been previously attributed to effects of polyphenolic and essential oil chemical species. Recently, we demonstrated differentiated growth-regulating effects of high (HFBL) versus low molecular mass (LFBL) aqueous fractions of bay leaf and now confirm by comparative effects on gene expression, that HFBL and LFBL suppress HT-29 growth by distinct mechanisms. Induction of intra-cellular lesions including DNA strand breakage by extra-cellular HFBL, invoked the hypothesis that iron-mediated reactive oxygen species with capacity to penetrate cell membrane, were responsible for HFBL-mediated effects, supported by equivalent effects of HFBL in combination with γ radiation. Activities of HFBL and LFBL were interpreted to reflect differentiated responses to iron-mediated reactive oxygen species (ROS), occurring either outside or inside cells. In the presence of LFBL, apoptotic death was relatively delayed compared with HFBL. ROS production by LFBL mediated p53-dependent apoptosis and recovery was suppressed by promoting G1/S phase arrest and failure of cellular tight junctions. In comparison, intra-cellular anti-oxidant protection exerted by LFBL was absent for extra-cellular HFBL (likely polysaccharide-rich), which potentiated more rapid apoptosis by producing DNA double strand breaks. Differentiated effects on expression of genes regulating ROS defense and chromatic condensation by LFBL versus HFBL, were observed. The results support ferrous iron in cell culture systems and potentially in vivo, can invoke different extra-cellular versus intra-cellular ROS-mediated chemistries, that may be regulated by exogenous, including dietary species.

Establishment of a translational endothelial cell model using directed differentiation of induced pluripotent stem cells from Cynomolgus monkey.

PubMed

Thoma, Eva C; Heckel, Tobias; Keller, David; Giroud, Nicolas; Leonard, Brian; Christensen, Klaus; Roth, Adrian; Bertinetti-Lapatki, Cristina; Graf, Martin; Patsch, Christoph

2016-10-25

Due to their broad differentiation potential, pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced, similar systems for non-human primates (NHPs) are still lacking. However, as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals, the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here, we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs, we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover, we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions, such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species.

Dielectric screening of early differentiation patterns in mesenchymal stem cells induced by steroid hormones.

PubMed

Ron, Amit; Shur, Irena; Daniel, Ramiz; Singh, Ragini Raj; Fishelson, Nick; Croitoru, Nathan; Benayahu, Dafna; Shacham-Diamand, Yosi

2010-06-01

In the framework of this study, target identification and localization of differentiation patterns by means of dielectric spectroscopy is presented. Here, a primary pre-osteoblastic bone marrow-derived MBA-15 cellular system was used to study the variations in the dielectric properties of mesenchymal stem cells while exposed to differentiation regulators. Using the fundamentals of mixed dielectric theories combined with finite numerical tools, the permittivity spectra of MBA-15 cell suspensions have been uniquely analyzed after being activated by steroid hormones to express osteogenic phenotypes. Following the spectral analysis, significant variations were revealed in the dielectric properties of the activated cells in comparison to the untreated populations. Based on the differentiation patterns of MBA-15, the electrical modifications were found to be highly correlated with the activation of specific cellular mechanisms which directly react to the hormonal inductions. In addition, by describing the dielectric dispersion in terms of transfer functions, it is shown that the spectral perturbations are well adapted to variations in the electrical characteristics of the cells. The reported findings vastly emphasize the tight correlation between the cellular and electrical state of the differentiated cells. It therefore emphasizes the vast abilities of impedance-based techniques as potential screening tools for stem cell analysis. Copyright 2009 Elsevier B.V. All rights reserved.

ω-3 polyunsaturated fatty acids direct differentiation of the membrane phenotype in mesenchymal stem cells to potentiate osteogenesis

PubMed Central

Levental, Kandice R.; Surma, Michal A.; Skinkle, Allison D.; Lorent, Joseph H.; Zhou, Yong; Klose, Christian; Chang, Jeffrey T.; Hancock, John F.; Levental, Ilya

2017-01-01

Mammalian cells produce hundreds of dynamically regulated lipid species that are actively turned over and trafficked to produce functional membranes. These lipid repertoires are susceptible to perturbations from dietary sources, with potentially profound physiological consequences. However, neither the lipid repertoires of various cellular membranes, their modulation by dietary fats, nor their effects on cellular phenotypes have been widely explored. We report that differentiation of human mesenchymal stem cells (MSCs) into osteoblasts or adipocytes results in extensive remodeling of the plasma membrane (PM), producing cell-specific membrane compositions and biophysical properties. The distinct features of osteoblast PMs enabled rational engineering of membrane phenotypes to modulate differentiation in MSCs. Specifically, supplementation with docosahexaenoic acid (DHA), a lipid component characteristic of osteoblast membranes, induced broad lipidomic remodeling in MSCs that reproduced compositional and structural aspects of the osteoblastic PM phenotype. The PM changes induced by DHA supplementation potentiated osteogenic differentiation of MSCs concurrent with enhanced Akt activation at the PM. These observations prompt a model wherein the DHA-induced lipidome leads to more stable membrane microdomains, which serve to increase Akt activity and thereby enhance osteogenic differentiation. More broadly, our investigations suggest a general mechanism by which dietary fats affect cellular physiology through remodeling of membrane lipidomes, biophysical properties, and signaling. PMID:29134198

Steap4 Plays a Critical Role in Osteoclastogenesis in Vitro by Regulating Cellular Iron/Reactive Oxygen Species (ROS) Levels and cAMP Response Element-binding Protein (CREB) Activation*

PubMed Central

Zhou, Jian; Ye, Shiqiao; Fujiwara, Toshifumi; Manolagas, Stavros C.; Zhao, Haibo

2013-01-01

Iron is essential for osteoclast differentiation, and iron overload in a variety of hematologic diseases is associated with excessive bone resorption. Iron uptake by osteoclast precursors via the transferrin cycle increases mitochondrial biogenesis, reactive oxygen species production, and activation of cAMP response element-binding protein, a critical transcription factor downstream of receptor activator of NF-κB-ligand-induced calcium signaling. These changes are required for the differentiation of osteoclast precursors to mature bone-resorbing osteoclasts. However, the molecular mechanisms regulating cellular iron metabolism in osteoclasts remain largely unknown. In this report, we provide evidence that Steap4, a member of the six-transmembrane epithelial antigen of prostate (Steap) family proteins, is an endosomal ferrireductase with a critical role in cellular iron utilization in osteoclasts. Specifically, we show that Steap4 is the only Steap family protein that is up-regulated during osteoclast differentiation. Knocking down Steap4 expression in vitro by lentivirus-mediated short hairpin RNAs inhibits osteoclast formation and decreases cellular ferrous iron, reactive oxygen species, and the activation of cAMP response element-binding protein. These results demonstrate that Steap4 is a critical enzyme for cellular iron uptake and utilization in osteoclasts and, thus, indispensable for osteoclast development and function. PMID:23990467

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

PubMed

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

2012-12-07

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

Expression and significance of Ki-67 in lung cancer.

PubMed

Folescu, Roxana; Levai, Codrina Mihaela; Grigoraş, Mirela Loredana; Arghirescu, Teodora Smaranda; Talpoş, Ioana Cristina; Gîndac, Ciprian Mihai; Zamfir, Carmen Lăcrămioara; Poroch, Vladimir; Anghel, Mirella Dorina

2018-01-01

Ki-67 parameter is a proliferation marker in malignant tumors. The increased proliferation activity and the decreased prognosis in lung cancer determined us to investigate different parameters connected to the tumor's aggression, such as cellularity, Ki-67 positivity rate, and proliferating cell nuclear antigen (PCNA). We evaluated the proliferative activity in 62 primary lung tumors by determining the cell's percentage of Ki-67 and immunoreactive PCNA (using MIB-1 and PCNA monoclonal antibodies), classifying Ki-67 and PCNA immunoreactivity into three score groups. The results obtained emphasized a linkage between Ki-67 score with the histological tumor subtype, tumor cellularity and degree of differentiation and with other proliferation immunohistochemistry (IHC) markers, such as p53 cellular tumor antigen. The tumor's cellularity, the Ki-67 positivity rate and PCNA, together with the clinical stage and the histological differentiation bring extra pieces of useful information in order to anticipate the evolution and the prognosis of lung cancer.

Detection and differentiation of coxiella burnetii in biological fluids

DOEpatents

Frazier, Marvin E.; Mallavia, Louis P.; Samuel, James E.; Baca, Oswald G.

1990-01-01

Methods for detecting the presence of Coxiella burenetii in biological samples, as well as a method for differentiating strains of C. burnetii that are capable of causing acute disease from those strains capable of causing chronic disease are disclosed. The methods generally comprise treating cells contained within the biological sample to expose cellular DNA, and hybridizing the cellular DNA (specifically rickettsial DNA) with a C. burnetii-specific labeled DNA probe. Radioisotope and biotin labels are preferred, allowing detection through autoradiography and colorimetric assays, respectively.

Detection and differentiation of coxiella burnetii in biological fluids

DOEpatents

Frazier, Marvin E.; Mallavia, Louis P.; Baca, Oswald G.; Samuel, James E.

1989-01-01

Methods for detecting the presence of Coxiella burnetii in biological samples, as well as a method for differentiating strains of C. burnetii that are capable of causing acute disease from those strains capable of causing chronic disease are disclosed. The methods generally comprise treating cells contained within the biological sample to expose cellular DNA, and hybridizing the cellular DNA (specifically rickettsial DNA) with a C. burnetii-specific labeled DNA probe. Radioisotope and biotin labels are preferred, allowing detection through autoradiography and colorimetric assays, respectively.

Detection and differentiation of coxiella burnetii in biological fluids

DOEpatents

Frazier, Marvin E.; Mallavia, Louis P.; Samuel, James E.; Baca, Oswald G.

1993-01-01

Methods for detecting the presence of Coxiella burnetii in biological samples, as well as a method for differentiating strains of C. burnetii that are capable of causing acute disease from those strains capable of causing chronic disease are disclosed. The methods generally comprise treating cells contained within the biological sample to expose cellular DNA, and hybridizing the cellular DNA with a DNA probe containing DNA sequences that specifically hybridize with C. burnetii DNA of strains associated with the capacity to cause acute or chronic disease.

Discoidin domain receptor 2 (DDR2) regulates proliferation of endochondral cells in mice

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

Kawai, Ikuma; Hisaki, Tomoka; Sugiura, Koji

2012-10-26

Highlights: Black-Right-Pointing-Pointer Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase. Black-Right-Pointing-Pointer DDR2 regulates cell proliferation, cell adhesion, migration, and extracellular matrix remodeling. Black-Right-Pointing-Pointer We produced in vitro and in vivo model to better understand the role of DDR2. Black-Right-Pointing-Pointer DDR2 might play an inhibitory role in the proliferation of chondrocyte. -- Abstract: Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that is activated by fibrillar collagens. DDR2 regulates cell proliferation, cell adhesion, migration, and extracellular matrix remodeling. The decrement of endogenous DDR2 represses osteoblastic marker gene expression and osteogenic differentiation in murine preosteoblastic cells, but themore » functions of DDR2 in chondrogenic cellular proliferation remain unclear. To better understand the role of DDR2 signaling in cellular proliferation in endochondral ossification, we inhibited Ddr2 expression via the inhibitory effect of miRNA on Ddr2 mRNA (miDdr2) and analyzed the cellular proliferation and differentiation in the prechondrocyte ATDC5 cell lines. To investigate DDR2's molecular role in endochondral cellular proliferation in vivo, we also produced transgenic mice in which the expression of truncated, kinase dead (KD) DDR2 protein is induced, and evaluated the DDR2 function in cellular proliferation in chondrocytes. Although the miDdr2-transfected ATDC5 cell lines retained normal differentiation ability, DDR2 reduction finally promoted cellular proliferation in proportion to the decreasing ratio of Ddr2 expression, and it also promoted earlier differentiation to cartilage cells by insulin induction. The layer of hypertrophic chondrocytes in KD Ddr2 transgenic mice was not significantly thicker than that of normal littermates, but the layer of proliferative chondrocytes in KD-Ddr2 transgenic mice was significantly thicker than that of normal littermates. Taken together, our data demonstrated that DDR2 might play a local and essential role in the proliferation of chondrocytes.« less

In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells

PubMed Central

Sella, Sabrina; Adami, Valentina; Amati, Eliana; Bernardi, Martina; Chieregato, Katia; Gatto, Pamela; Menarin, Martina; Pozzato, Alessandro; Pozzato, Gianantonio; Astori, Giuseppe

2018-01-01

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4–64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling. PMID:29293552

Quantitative proteomics analysis highlights the role of redox hemostasis and energy metabolism in human embryonic stem cell differentiation to neural cells.

PubMed

Fathi, Ali; Hatami, Maryam; Vakilian, Haghighat; Han, Chia-Li; Chen, Yu-Ju; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

2014-04-14

Neural differentiation of human embryonic stem cells (hESCs) is a unique opportunity for in vitro analyses of neurogenesis in humans. Extrinsic cues through neural plate formation are well described in the hESCs although intracellular mechanisms underlying neural development are largely unknown. Proteome analysis of hESC differentiation to neural cells will help to further define molecular mechanisms involved in neurogenesis in humans. Using a two-dimensional differential gel electrophoresis (2D-DIGE) system, we analyzed the proteome of hESC differentiation to neurons at three stages, early neural differentiation, neural ectoderm and mature neurons. Out of 137 differentially accumulated protein spots, 118 spots were identified using MALDI-TOF/TOF and LC MS/MS. We observed that proteins involved in redox hemostasis, vitamin and energy metabolism and ubiquitin dependent proteolysis were more abundant in differentiated cells, whereas the abundance of proteins associated with RNA processing and protein folding was higher in hESCs. Higher abundance of proteins involved in maintaining cellular redox state suggests the importance of redox hemostasis in neural differentiation. Furthermore, our results support the concept of a coupling mechanism between neuronal activity and glucose utilization. The protein network analysis showed that the majority of the interacting proteins were associated with the cell cycle and cellular proliferation. These results enhanced our understanding of the molecular dynamics that underlie neural commitment and differentiation. In highlighting the role of redox and unique metabolic properties of neuronal cells, the present findings add insight to our understanding of hESC differentiation to neurons. The abundance of fourteen proteins involved in maintaining cellular redox state, including 10 members of peroxiredoxin (Prdx) family, mainly increased during differentiation, thus highlighting a link of neural differentiation to redox. Our results revealed markedly higher expression of genes encoding enzymes involved in the glycolysis and amino acid synthesis during differentiation. Protein network analysis predicted a number of critical mediators in hESC differentiation. These proteins included TP53, CTNNB1, SMARCA4, TNF, TERT, E2F1, MYC, RB1, and AR. Copyright © 2014 Elsevier B.V. All rights reserved.

Determination of tenogenic differentiation in human mesenchymal stem cells by terahertz waves for measurement of the optical property of cellular suspensions

NASA Astrophysics Data System (ADS)

Morita, Yasuyuki; Azuchi, Kosuke; Ju, Yang; Suzuki, Satoshi; Xu, Baiyao; Yamamoto, Shuhei

2014-06-01

Technology for identifying stem cell-to-tenocyte differentiation that is non-contact and non-destructive in vitro is essential in tissue engineering. It has been found that expression of various RNA and proteins produced by differentiated cells is elevated when human bone marrow mesenchymal stem cells (hBMSCs) differentiate into tenocytes. Also, such biomolecules have absorption bands in the terahertz range. Thus, we attempted to evaluate whether terahertz waves could be used to distinguish hBMSC-to-tenocyte differentiation. Terahertz time-domain spectroscopy (THz-TDS) using femtosecond laser pulses was used for terahertz measurements. HBMSCs differentiated into tenocytes with mechanical stimulation: 10% cyclical uniaxial stretching at 1 Hz for 24 or 48 h. Cellular suspensions before and after differentiation were measured with terahertz waves. Complex refractive index, consisting of a refractive index (real) and an extinction coefficient (imaginary) obtained from the transmitted terahertz signals, was evaluated before and after differentiation at 1.0 THz. As a result, the THz-TDS system enabled discrimination of hBMSC-to-tenocyte differentiation due to the marked contrast in optical parameter before and after differentiation. This is the first report of the potential of a THz-TDS system for the detection of tenogenic differentiation using a non-contact and non-destructive in vitro technique.

The Faceted Discrete Growth and Phase Differentiation During the Directional Solidification of 20SiMnMo5 Steel

NASA Astrophysics Data System (ADS)

Ma, Xiaoping; Li, Dianzhong

2018-07-01

The microstructures, segregation and cooling curve were investigated in the directional solidification of 20SiMnMo5 steel. The typical characteristic of faceted growth is identified. The microstructures within the single cellular and within the single dendritic arm, together with the contradictive segregation distribution against the cooling curve, verify the discrete crystal growth in multi-scales. Not only the single cellular/dendritic arm but also the single martensite zone within the single cellular/dendritic arm is produced by the discrete growth. In the viewpoint of segregation, the basic domain following continuous growth has not been revealed. Along with the multi-scale faceted discrete growth, the phase differentiation happens for both the solid and liquid. The differentiated liquid phases appear and evolve with different sizes, positions, compositions and durations. The physical mechanism for the faceted discrete growth is qualitatively established based on the nucleation of new faceted steps induced by the composition gradient and temperature gradient.

CellNet: network biology applied to stem cell engineering.

PubMed

Cahan, Patrick; Li, Hu; Morris, Samantha A; Lummertz da Rocha, Edroaldo; Daley, George Q; Collins, James J

2014-08-14

Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering. Copyright © 2014 Elsevier Inc. All rights reserved.

The Faceted Discrete Growth and Phase Differentiation During the Directional Solidification of 20SiMnMo5 Steel

NASA Astrophysics Data System (ADS)

Ma, Xiaoping; Li, Dianzhong

2018-03-01

The microstructures, segregation and cooling curve were investigated in the directional solidification of 20SiMnMo5 steel. The typical characteristic of faceted growth is identified. The microstructures within the single cellular and within the single dendritic arm, together with the contradictive segregation distribution against the cooling curve, verify the discrete crystal growth in multi-scales. Not only the single cellular/dendritic arm but also the single martensite zone within the single cellular/dendritic arm is produced by the discrete growth. In the viewpoint of segregation, the basic domain following continuous growth has not been revealed. Along with the multi-scale faceted discrete growth, the phase differentiation happens for both the solid and liquid. The differentiated liquid phases appear and evolve with different sizes, positions, compositions and durations. The physical mechanism for the faceted discrete growth is qualitatively established based on the nucleation of new faceted steps induced by the composition gradient and temperature gradient.

Cellular stress associated with the differentiation of Plasmodium berghei ookinetes.

PubMed

Duran-Bedolla, Josefina; Téllez-Sosa, Juan; Valdovinos-Torres, Humberto; Pavón, Natalia; Buelna-Chontal, Mabel; Tello-López, Angel T; Argotte-Ramos, Rocio; Rodríguez, Mario Henry; Rodríguez, María Carmen

2017-04-01

For malaria transmission, Plasmodium parasites must develop in the mosquito vector. Oxidative stress in the insect midgut, triggered by environmental changes (e.g., pH and temperature), influences the cellular signaling involved in differentiation from gametocytes to mobile ookinetes for the purpose of parasite survival. Oxidative stress activates the homeostatic response to stress characterized by the phosphorylation eIF2α, the attenuation of protein synthesis, and the transcription of genes participating in the unfolded protein response and antioxidant processes, forming a part of an integrated stress response (ISR). We hypothesized that ISR operates during the differentiation of gametocytes to ookinetes to assure Plasmodium survival. Using in-vitro conditions resembling the mosquito midgut conditions, we cultured Plasmodium berghei gametocytes to ookinetes and evaluated the redox balance by detecting reactive oxygen species and superoxide dismutase activity. Additionally, we evaluated the phosphorylation of eIF2α, the attenuation of the global protein synthesis, and the gene expression of cellular stress markers (e.g., endoplasmic reticulum chaperones and antioxidant molecules, measured by reverse-transcription quantitative polymerase chain reaction), finding that these processes were all taking place, probably to improve survival during the differentiation of Plasmodium berghei ookinetes.

Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

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

Eom, Young Woo; Biomedical Research Institute, Lifeliver Co., Ltd., Suwon; Lee, Jong Eun

2011-04-29

Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of humanmore » adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.« less

Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

PubMed

Movahednia, Mohammad Mehdi; Kidwai, Fahad Karim; Zou, Yu; Tong, Huei Jinn; Liu, Xiaochen; Islam, Intekhab; Toh, Wei Seong; Raghunath, Michael; Cao, Tong

2015-04-01

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-β1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.

Cryopreservation has no effect on function of natural killer cells differentiated in vitro from umbilical cord blood CD34(+) cells.

PubMed

Domogala, Anna; Madrigal, J Alejandro; Saudemont, Aurore

2016-06-01

Natural killer (NK) cells offer the potential for a powerful cellular immunotherapy because they can target malignant cells without being direct effectors of graft-versus-host disease. We have previously shown that high numbers of functional NK cells can be differentiated in vitro from umbilical cord blood (CB) CD34(+) cells. To develop a readily available, off-the-shelf cellular product, it is essential that NK cells differentiated in vitro can be frozen and thawed while maintaining the same phenotype and functions. We evaluated the phenotype and function of fresh and frozen NK cells differentiated in vitro. We also assessed whether the concentration of NK cells at the time of freezing had an impact on cell viability. We found that cell concentration of NK cells at the time of freezing did not have an impact on their viability and on cell recovery post-thaw. Moreover, freezing of differentiated NK cells in vitro did not affect their phenotype, cytotoxicity and degranulation capacity toward K562 cells, cytokine production and proliferation. We are therefore able to generate large numbers of functional NK cells from CB CD34(+) cells that maintain the same phenotype and function post-cryopreservation, which will allow for multiple infusions of a highly cytotoxic NK cell product. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Role of H1 Linker Histones in Mammalian Development and Stem Cell Differentiation

PubMed Central

Pan, Chenyi; Fan, Yuhong

2016-01-01

H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome. PMID:26689747

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation

PubMed Central

Song, Juhyun; Oh, Yumi; Kim, Jong Youl; Cho, Kyoung Joo

2016-01-01

Purpose Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. Materials and Methods This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. Results Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. Conclusion Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders. PMID:27593875

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation.

PubMed

Song, Juhyun; Oh, Yumi; Kim, Jong Youl; Cho, Kyoung Joo; Lee, Jong Eun

2016-11-01

Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders.

Mechanisms by which HPV Induces a Replication Competent Environment in Differentiating Keratinocytes

PubMed Central

Moody, Cary A.

2017-01-01

Human papillomaviruses (HPV) are the causative agents of cervical cancer and are also associated with other genital malignancies, as well as an increasing number of head and neck cancers. HPVs have evolved their life cycle to contend with the different cell states found in the stratified epithelium. Initial infection and viral genome maintenance occurs in the proliferating basal cells of the stratified epithelium, where cellular replication machinery is abundant. However, the productive phase of the viral life cycle, including productive replication, late gene expression and virion production, occurs upon epithelial differentiation, in cells that normally exit the cell cycle. This review outlines how HPV interfaces with specific cellular signaling pathways and factors to provide a replication-competent environment in differentiating cells. PMID:28925973

Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

EPA Science Inventory

An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

Cellular and multicellular form and function.

PubMed

Liu, Wendy F; Chen, Christopher S

2007-11-10

Engineering artificial tissue constructs requires the appropriate spatial arrangement of cells within scaffolds. The introduction of microengineering tools to the biological community has provided a valuable set of techniques to manipulate the cellular environment, and to examine how cell structure affects cellular function. Using micropatterning techniques, investigators have found that the geometric presentation of cell-matrix adhesions are important regulators of various cell behaviors including cell growth, proliferation, differentiation, polarity and migration. Furthermore, the presence of neighboring cells in multicellular aggregates has a significant impact on the proliferative and differentiated state of cells. Using microengineering tools, it will now be possible to manipulate the various environmental factors for practical applications such as engineering tissue constructs with greater control over the physical structure and spatial arrangement of cells within their surrounding microenvironment.

Optimization of differentiation time of mesenchymal-stem-cell to tenocyte under a cyclic stretching with a microgrooved culture membrane and selected measurement cells.

PubMed

Morita, Yasuyuki; Yamashita, Takahiro; Toku, Toku; Ju, Yang

2018-01-01

There is a need for efficient stem cell-to-tenocyte differentiation techniques for tendon tissue engineering. More than 1 week is required for tenogenic differentiation with chemical stimuli, including co-culturing. Research has begun to examine the utility of mechanical stimuli, which reduces the differentiation time to several days. However, the precise length of time required to differentiate human bone marrow-derived mesenchymal stem cells (hBMSCs) into tenocytes has not been clarified. Understanding the precise time required is important for future tissue engineering projects. Therefore, in this study, a method was developed to more precisely determine the length of time required to differentiate hBMSCs into tenocytes with cyclic stretching stimulus. First, it had to be determined how stretching stimulation affected the cells. Microgrooved culture membranes were used to suppress cell orientation behavior. Then, only cells oriented parallel to the microgrooves were selected and evaluated for protein synthesis levels for differentiation. The results revealed that growing cells on the microgrooved membrane and selecting optimally-oriented cells for measurement improved the accuracy of the differentiation evaluation, and that hBMSCs differentiated into tenocytes in approximately 10 h. The differentiation time corresponded to the time required for cellular cytoskeleton reorganization and cellular morphology alterations. This suggests that cells, when subjected to mechanical stimulus, secrete mRNAs and proteins for both cytoskeleton reorganization and differentiation.

The effect of myotonic dystrophy transcript levels and location on muscle differentiation

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

Mastroyiannopoulos, Nikolaos P.; Chrysanthou, Elina; Kyriakides, Tassos C.

2008-12-12

In myotonic dystrophy type I (DM1), nuclear retention of mutant DMPK transcripts compromises muscle cell differentiation. Although several reports have identified molecular defects in myogenesis, it remains still unclear how exactly the retention of the mutant transcripts induces this defect. We have recently created a novel cellular model in which the mutant DMPK 3' UTR transcripts were released to the cytoplasm of myoblasts by using the WPRE genetic element. As a result, muscle cell differentiation was repaired. In this paper, this cellular model was further exploited to investigate the effect of the levels and location of the mutant transcripts onmore » muscle differentiation. Results show that the levels of these transcripts were proportional to the inhibition of both the initial fusion of myoblasts and the maturity of myotubes. Moreover, the cytoplasmic export of the mutant RNAs to the cytoplasm caused less inhibition only in the initial fusion of myoblasts.« less

Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum During Myocyte DIfferentiation

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

Stenoien, David L.; Knyushko, Tatyana V.; Londono, Monica P.

2007-06-01

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) family members are transmembrane proteins that play an essential role in regulating intracellular calcium levels. Phospholamban (PLB), a 52 amino acid phosphoprotein, regulates SERCA activity in adult heart and skeletal muscle. Using the C2C12 myocyte cell line, we find endogenous PLB constitutively expressed in both myoblasts and myotubes, whereas SERCA expression coincides with activation of the differentiation program. PLB has a punctuate distribution in myoblasts changing to a reticular distribution in myotubes where it colocalizes with SERCAs. To examine the distribution and dynamics of PLB and SERCA, we expressed fluorescent fusion proteins (GFP, CFP, andmore » YFP) of PLB and SERCA in myoblasts. Coexpressed PLB and SERCA localize to distinct cellular compartments in myoblasts but begin to colocalize as cells differentiate. Fluorescence Recovery After Photobleaching (FRAP) studies show different recovery patterns for each protein in myoblasts confirming their localization to distinct compartments. To extend these studies, we created stable cell lines expressing O6-alkylguanine-DNA alkyltransferase (AGT) fusions with PLB or SERCA to track their localization as myocytes differentiate. These experiments demonstrate that PLB localizes to punctate vesicles in myoblasts and adopts a reticular distribution that coincides with SERCA distribution after differentiation. Colocalization experiments indicate that a subset of PLB in myoblasts colocalizes with endosomes, Golgi, and the plasma membrane however PLB also localizes to other, as yet unidentified vesicles. Our results indicate that differentiation plays a critical role in regulating PLB distribution to ensure its colocalization within the same cellular compartment as SERCA in differentiated cells. The presence and altered distribution of PLB in undifferentiated myoblasts raises the possibility that this protein has additional functions distinct from SERCA regulation.« less

Abiotic Stress Tolerance in Plants: Myriad Roles of Ascorbate Peroxidase

PubMed Central

Pandey, Saurabh; Fartyal, Dhirendra; Agarwal, Aakrati; Shukla, Tushita; James, Donald; Kaul, Tanushri; Negi, Yogesh K.; Arora, Sandeep; Reddy, Malireddy K.

2017-01-01

One of the most significant manifestations of environmental stress in plants is the increased production of Reactive Oxygen Species (ROS). These ROS, if allowed to accumulate unchecked, can lead to cellular toxicity. A battery of antioxidant molecules is present in plants for keeping ROS levels under check and to maintain the cellular homeostasis under stress. Ascorbate peroxidase (APX) is a key antioxidant enzyme of such scavenging systems. It catalyses the conversion of H2O2 into H2O, employing ascorbate as an electron donor. The expression of APX is differentially regulated in response to environmental stresses and during normal plant growth and development as well. Different isoforms of APX show differential response to environmental stresses, depending upon their sub-cellular localization, and the presence of specific regulatory elements in the upstream regions of the respective genes. The present review delineates role of APX isoforms with respect to different types of abiotic stresses and its importance as a key antioxidant enzyme in maintaining cellular homeostasis. PMID:28473838

Reprogramming cellular identity for regenerative medicine

PubMed Central

Cherry, Anne B.C.; Daley, George Q.

2012-01-01

The choreographed development of over 200 distinct differentiated cell types from a single zygote is a complex and poorly understood process. Whereas development leads unidirectionally towards more restricted cell fates, recent work in cellular reprogramming has proven that striking conversions of one cellular identity into another can be engineered, promising countless applications in biomedical research and paving the way for modeling disease with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. We here review evidence demonstrating that because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. However, manipulating in vitro culture conditions may ultimately enable cell-based models to recapitulate gene-environment interactions. Here, we discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration. PMID:22424223

454 Transcriptome sequencing suggests a role for two-component signalling in cellularization and differentiation of barley endosperm transfer cells.

PubMed

Thiel, Johannes; Hollmann, Julien; Rutten, Twan; Weber, Hans; Scholz, Uwe; Weschke, Winfriede

2012-01-01

Cell specification and differentiation in the endosperm of cereals starts at the maternal-filial boundary and generates the endosperm transfer cells (ETCs). Besides the importance in assimilate transfer, ETCs are proposed to play an essential role in the regulation of endosperm differentiation by affecting development of proximate endosperm tissues. We attempted to identify signalling elements involved in early endosperm differentiation by using a combination of laser-assisted microdissection and 454 transcriptome sequencing. 454 sequencing of the differentiating ETC region from the syncytial state until functionality in transfer processes captured a high proportion of novel transcripts which are not available in existing barley EST databases. Intriguingly, the ETC-transcriptome showed a high abundance of elements of the two-component signalling (TCS) system suggesting an outstanding role in ETC differentiation. All components and subfamilies of the TCS, including distinct kinds of membrane-bound receptors, have been identified to be expressed in ETCs. The TCS system represents an ancient signal transduction system firstly discovered in bacteria and has previously been shown to be co-opted by eukaryotes, like fungi and plants, whereas in animals and humans this signalling route does not exist. Transcript profiling of TCS elements by qRT-PCR suggested pivotal roles for specific phosphorelays activated in a coordinated time flow during ETC cellularization and differentiation. ETC-specificity of transcriptionally activated TCS phosphorelays was assessed for early differentiation and cellularization contrasting to an extension of expression to other grain tissues at the beginning of ETC maturation. Features of candidate genes of distinct phosphorelays and transcriptional activation of genes putatively implicated in hormone signalling pathways hint at a crosstalk of hormonal influences, putatively ABA and ethylene, and TCS signalling. Our findings suggest an integral function for the TCS in ETC differentiation possibly coupled to sequent hormonal regulation by ABA and ethylene.

454 Transcriptome Sequencing Suggests a Role for Two-Component Signalling in Cellularization and Differentiation of Barley Endosperm Transfer Cells

PubMed Central

Thiel, Johannes; Hollmann, Julien; Rutten, Twan; Weber, Hans; Scholz, Uwe; Weschke, Winfriede

2012-01-01

Background Cell specification and differentiation in the endosperm of cereals starts at the maternal-filial boundary and generates the endosperm transfer cells (ETCs). Besides the importance in assimilate transfer, ETCs are proposed to play an essential role in the regulation of endosperm differentiation by affecting development of proximate endosperm tissues. We attempted to identify signalling elements involved in early endosperm differentiation by using a combination of laser-assisted microdissection and 454 transcriptome sequencing. Principal Findings 454 sequencing of the differentiating ETC region from the syncytial state until functionality in transfer processes captured a high proportion of novel transcripts which are not available in existing barley EST databases. Intriguingly, the ETC-transcriptome showed a high abundance of elements of the two-component signalling (TCS) system suggesting an outstanding role in ETC differentiation. All components and subfamilies of the TCS, including distinct kinds of membrane-bound receptors, have been identified to be expressed in ETCs. The TCS system represents an ancient signal transduction system firstly discovered in bacteria and has previously been shown to be co-opted by eukaryotes, like fungi and plants, whereas in animals and humans this signalling route does not exist. Transcript profiling of TCS elements by qRT-PCR suggested pivotal roles for specific phosphorelays activated in a coordinated time flow during ETC cellularization and differentiation. ETC-specificity of transcriptionally activated TCS phosphorelays was assessed for early differentiation and cellularization contrasting to an extension of expression to other grain tissues at the beginning of ETC maturation. Features of candidate genes of distinct phosphorelays and transcriptional activation of genes putatively implicated in hormone signalling pathways hint at a crosstalk of hormonal influences, putatively ABA and ethylene, and TCS signalling. Significance Our findings suggest an integral function for the TCS in ETC differentiation possibly coupled to sequent hormonal regulation by ABA and ethylene. PMID:22848641

Emended descriptions of Prevotella denticola, Prevotella loescheii, Prevotella veroralis, and Prevotella melaninogenica.

PubMed

Wu, C C; Johnson, J L; Moore, W E; Moore, L V

1992-10-01

During studies of human periodontal disease, a number of bacterial strains were encountered that, on the basis of results of standard biochemical tests, appeared to be Prevotella buccalis, Prevotella denticola, Prevotella melaninogenica, or Prevotella loescheii. However, use of the standard biochemical tests, cellular fatty acid analyses, and the polyacrylamide gel electrophoresis patterns of soluble proteins resulted in conflicting identifications of these strains. The results of tests for cellobiose fermentation, inulin fermentation, and pigment production were responsible for most of the discordant results. Cellular fatty acid analyses in which the Microbial Identification System was used did not differentiate these strains from validly described species, even though separate library entries were created for them. DNA reassociation determinations in which the S1 nuclease procedure was used showed that cellobiose fermentation and pigment production are variable among strains of P. melaninogenica and P. denticola and that fermentation of xylan is not a reliable characteristic for differentiating P. buccalis from Prevotella veroralis. In contrast to previous indications, most strains of P. veroralis do not ferment xylan. These species can be differentiated by DNA-DNA reassociation and by cellular fatty acid analysis, using the Microbial Identification System, but differentiation by currently described phenotypic characteristics is not reliable. Similarly, P. loescheii and the genetically distinct (but closely related) D1C-20 group cannot be distinguished reliably from each other or from P. veroralis, P. denticola, and P. melaninogenica on the basis of currently described phenotypic tests other than cellular fatty acid composition or, for some species, electrophoretic patterns of soluble whole-cell proteins.

Prenatal Alcohol Exposure and Cellular Differentiation

PubMed Central

Veazey, Kylee J.; Muller, Daria; Golding, Michael C.

2013-01-01

Exposure to alcohol significantly alters the developmental trajectory of progenitor cells and fundamentally compromises tissue formation (i.e., histogenesis). Emerging research suggests that ethanol can impair mammalian development by interfering with the execution of molecular programs governing differentiation. For example, ethanol exposure disrupts cellular migration, changes cell–cell interactions, and alters growth factor signaling pathways. Additionally, ethanol can alter epigenetic mechanisms controlling gene expression. Normally, lineage-specific regulatory factors (i.e., transcription factors) establish the transcriptional networks of each new cell type; the cell’s identity then is maintained through epigenetic alterations in the way in which the DNA encoding each gene becomes packaged within the chromatin. Ethanol exposure can induce epigenetic changes that do not induce genetic mutations but nonetheless alter the course of fetal development and result in a large array of patterning defects. Two crucial enzyme complexes—the Polycomb and Trithorax proteins—are central to the epigenetic programs controlling the intricate balance between self-renewal and the execution of cellular differentiation, with diametrically opposed functions. Prenatal ethanol exposure may disrupt the functions of these two enzyme complexes, altering a crucial aspect of mammalian differentiation. Characterizing the involvement of Polycomb and Trithorax group complexes in the etiology of fetal alcohol spectrum disorders will undoubtedly enhance understanding of the role that epigenetic programming plays in this complex disorder. PMID:24313167

Positive and Negative Regulatory Mechanisms for Fine-Tuning Cellularity and Functions of Medullary Thymic Epithelial Cells.

PubMed

Akiyama, Taishin; Tateishi, Ryosuke; Akiyama, Nobuko; Yoshinaga, Riko; Kobayashi, Tetsuya J

2015-01-01

Self-tolerant T cells and regulatory T cells develop in the thymus. A wide variety of cell-cell interactions in the thymus is required for the differentiation, proliferation, and repertoire selection of T cells. Various secreted and cell surface molecules expressed in thymic epithelial cells (TECs) mediate these processes. Moreover, cytokines expressed by cells of hematopoietic origin regulate the cellularity of TECs. Tumor necrosis factor (TNF) family RANK ligand, lymphotoxin, and CD40 ligand, expressed in T cells and innate lymphoid cells (ILCs), promote the differentiation and proliferation of medullary TECs (mTECs) that play critical roles in the induction of immune tolerance. A recent study suggests that interleukin-22 (IL-22) produced by ILCs promotes regeneration of TECs after irradiation. Intriguingly, tumor growth factor-β and osteoprotegerin limit cellularity of mTECs, thereby attenuating regulatory T cell generation. We will review recent insights into the molecular basis for cell-cell interactions regulating differentiation and proliferation of mTECs and also discuss about a perspective on use of mathematical models for understanding this complicated system.

Quantifying Cell Fate Decisions for Differentiation and Reprogramming of a Human Stem Cell Network: Landscape and Biological Paths

PubMed Central

Li, Chunhe; Wang, Jin

2013-01-01

Cellular reprogramming has been recently intensively studied experimentally. We developed a global potential landscape and kinetic path framework to explore a human stem cell developmental network composed of 52 genes. We uncovered the underlying landscape for the stem cell network with two basins of attractions representing stem and differentiated cell states, quantified and exhibited the high dimensional biological paths for the differentiation and reprogramming process, connecting the stem cell state and differentiated cell state. Both the landscape and non-equilibrium curl flux determine the dynamics of cell differentiation jointly. Flux leads the kinetic paths to be deviated from the steepest descent gradient path, and the corresponding differentiation and reprogramming paths are irreversible. Quantification of paths allows us to find out how the differentiation and reprogramming occur and which important states they go through. We show the developmental process proceeds as moving from the stem cell basin of attraction to the differentiation basin of attraction. The landscape topography characterized by the barrier heights and transition rates quantitatively determine the global stability and kinetic speed of cell fate decision process for development. Through the global sensitivity analysis, we provided some specific predictions for the effects of key genes and regulation connections on the cellular differentiation or reprogramming process. Key links from sensitivity analysis and biological paths can be used to guide the differentiation designs or reprogramming tactics. PMID:23935477

The effect of differentiation agents on inflammatory and oxidative responses of the human neuroblastoma cell line SK-N-SH.

PubMed

Niewiarowska-Sendo, Anna; Patrzalek, Katarzyna; Kozik, Andrzej; Guevara-Lora, Ibeth

2015-01-01

Obtaining a suitable experimental cellular model is a major problem for neuroscience studies. Neuroblastoma cell lines have been often applied in studies related to pathological disorders of nervous system. However, in the search for an ideal model, these cells must be differentiated to cancel their tumor character. The subsequent reactions that are caused by differentiation are not always indifferent to the same model. We evaluated the effect of two well known substances, used for SH-N-SK cell line differentiation, retinoic acid (RA) and phorbol-12-myristate-13-acetate (PMA), on the induction of pro-inflammatory and pro-oxidative reactions in these cells. Cells differentiated with PMA were able to produce significantly higher amounts of pro-inflammatory cytokines whereas the release of nitric oxide radicals was similar to that in undifferentiated cells. On the contrary, in RA-differentiated cells no significant changes in cytokine production were observed and the nitric oxide release was decreased. Additionally, the RA-differentiated neuronal model was more sensible to lipopolysaccharide stimulation, producing pro-inflammatory cytokines abundantly. These results suggest that RA-differentiated SH-N-SK cells provide a more suitable experimental model for the study of molecular and cellular mechanisms of the inflammation and oxidative stress in neuronal cells.

Usefulness of Cellular Analysis of Bronchoalveolar Lavage Fluid for Predicting the Etiology of Pneumonia in Critically Ill Patients

PubMed Central

Hong, Hyo-Lim; Kim, Sung-Han; Huh, Jin Won; Sung, Heungsup; Lee, Sang-Oh; Kim, Mi-Na; Jeong, Jin-Yong; Lim, Chae-Man; Kim, Yang Soo; Woo, Jun Hee; Koh, Younsuck

2014-01-01

Background The usefulness of bronchoalveolar lavage (BAL) fluid cellular analysis in pneumonia has not been adequately evaluated. This study investigated the ability of cellular analysis of BAL fluid to differentially diagnose bacterial pneumonia from viral pneumonia in adult patients who are admitted to intensive care unit. Methods BAL fluid cellular analysis was evaluated in 47 adult patients who underwent bronchoscopic BAL following less than 24 hours of antimicrobial agent exposure. The abilities of BAL fluid total white blood cell (WBC) counts and differential cell counts to differentiate between bacterial and viral pneumonia were evaluated using receiver operating characteristic (ROC) curve analysis. Results Bacterial pneumonia (n = 24) and viral pneumonia (n = 23) were frequently associated with neutrophilic pleocytosis in BAL fluid. BAL fluid median total WBC count (2,815/µL vs. 300/µL, P<0.001) and percentage of neutrophils (80.5% vs. 54.0%, P = 0.02) were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. In ROC curve analysis, BAL fluid total WBC count showed the best discrimination, with an area under the curve of 0.855 (95% CI, 0.750–0.960). BAL fluid total WBC count ≥510/µL had a sensitivity of 83.3%, specificity of 78.3%, positive likelihood ratio (PLR) of 3.83, and negative likelihood ratio (NLR) of 0.21. When analyzed in combination with serum procalcitonin or C-reactive protein, sensitivity was 95.8%, specificity was 95.7%, PLR was 8.63, and NLR was 0.07. BAL fluid total WBC count ≥510/µL was an independent predictor of bacterial pneumonia with an adjusted odds ratio of 13.5 in multiple logistic regression analysis. Conclusions Cellular analysis of BAL fluid can aid early differential diagnosis of bacterial pneumonia from viral pneumonia in critically ill patients. PMID:24824328

Use of Lightweight Cellular Mats to Reduce the Settlement of Structure on Soft Soil

NASA Astrophysics Data System (ADS)

Ganasan, R.; Lim, A. J. M. S.; Wijeyesekera, D. C.

2016-07-01

Construction of structures on soft soils gives rise to some difficulties in Malaysia and other country especially in settlement both in short and long term. The focus of this research is to minimize the differential and non-uniform settlement on peat soil with the use of an innovative cellular mat. The behaviour and performance of the lightweight geo-material (in block form) is critically investigated and in particular the use as a fill in embankment on soft ground. Hemic peat soil, sponge and innovative cellular mat will be used as the main material in this study. The monitoring in settlement behavior from this part of research will be done as laboratory testing only. The uneven settlement in this problem was uniquely monitored photographically using spot markers. In the end of the research, it is seen that the innovative cellular mat has reduce the excessive and differential settlement up to 50% compare to flexible and rigid foundations. This had improve the stiffness of soils as well as the porous contain in cellular structure which help in allowing water/moisture to flow through in or out thus resulting in prevent the condition of floating.

Cellular genetic therapy.

PubMed

Del Vecchio, F; Filareto, A; Spitalieri, P; Sangiuolo, F; Novelli, G

2005-01-01

Cellular genetic therapy is the ultimate frontier for those pathologies that are consequent to a specific nonfunctional cellular type. A viable cure for there kinds of diseases is the replacement of sick cells with healthy ones, which can be obtained from the same patient or a different donor. In fact, structures can be corrected and strengthened with the introduction of undifferentiated cells within specific target tissues, where they will specialize into the desired cellular types. Furthermore, consequent to the recent results obtained with the transdifferentiation experiments, a process that allows the in vitro differentiation of embryonic and adult stem cells, it has also became clear that many advantages may be obtained from the use of stem cells to produce drugs, vaccines, and therapeutic molecules. Since stem cells can sustain lineage potentials, the capacity for differentiation, and better tolerance for the introduction of exogenous genes, they are also considered as feasible therapeutic vehicles for gene therapy. In fact, it is strongly believed that the combination of cellular genetic and gene therapy approaches will definitely allow the development of new therapeutic strategies as well as the production of totipotent cell lines to be used as experimental models for the cure of genetic disorders.

Modeling of coupled differential equations for cellular chemical signaling pathways: Implications for assay protocols utilized in cellular engineering.

PubMed

O'Clock, George D

2016-08-01

Cellular engineering involves modification and control of cell properties, and requires an understanding of fundamentals and mechanisms of action for cellular derived product development. One of the keys to success in cellular engineering involves the quality and validity of results obtained from cell chemical signaling pathway assays. The accuracy of the assay data cannot be verified or assured if the effect of positive feedback, nonlinearities, and interrelationships between cell chemical signaling pathway elements are not understood, modeled, and simulated. Nonlinearities and positive feedback in the cell chemical signaling pathway can produce significant aberrations in assay data collection. Simulating the pathway can reveal potential instability problems that will affect assay results. A simulation, using an electrical analog for the coupled differential equations representing each segment of the pathway, provides an excellent tool for assay validation purposes. With this approach, voltages represent pathway enzyme concentrations and operational amplifier feedback resistance and input resistance values determine pathway gain and rate constants. The understanding provided by pathway modeling and simulation is strategically important in order to establish experimental controls for assay protocol structure, time frames specified between assays, and assay concentration variation limits; to ensure accuracy and reproducibility of results.

Erythroid cell growth and differentiation in vitro in the simulated microgravity environment of the NASA rotating wall vessel bioreactor

NASA Technical Reports Server (NTRS)

Sytkowski, A. J.; Davis, K. L.

2001-01-01

Prolonged exposure of humans and experimental animals to the altered gravitational conditions of space flight has adverse effects on the lymphoid and erythroid hematopoietic systems. Although some information is available regarding the cellular and molecular changes in lymphocytes exposed to microgravity, little is known about the erythroid cellular changes that may underlie the reduction in erythropoiesis and resultant anemia. We now report a reduction in erythroid growth and a profound inhibition of erythropoietin (Epo)-induced differentiation in a ground-based simulated microgravity model system. Rauscher murine erythroleukemia cells were grown either in tissue culture vessels at 1 x g or in the simulated microgravity environment of the NASA-designed rotating wall vessel (RWV) bioreactor. Logarithmic growth was observed under both conditions; however, the doubling time in simulated microgravity was only one-half of that seen at 1 x g. No difference in apoptosis was detected. Induction with Epo at the initiation of the culture resulted in differentiation of approximately 25% of the cells at 1 x g, consistent with our previous observations. In contrast, induction with Epo at the initiation of simulated microgravity resulted in only one-half of this degree of differentiation. Significantly, the growth of cells in simulated microgravity for 24 h prior to Epo induction inhibited the differentiation almost completely. The results suggest that the NASA RWV bioreactor may serve as a suitable ground-based microgravity simulator to model the cellular and molecular changes in erythroid cells observed in true microgravity.

Mesenchymal Stem Cells Retain Their Defining Stem Cell Characteristics After Exposure to Ionizing Radiation

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

Nicolay, Nils H., E-mail: n.nicolay@dkfz.de; Department of Molecular and Radiation Oncology, German Cancer Research Center, Heidelberg; Sommer, Eva

2013-12-01

Purpose: Mesenchymal stem cells (MSCs) have the ability to migrate to lesion sites and undergo differentiation into functional tissues. Although this function may be important for tissue regeneration after radiation therapy, the influence of ionizing radiation (IR) on cellular survival and the functional aspects of differentiation and stem cell characteristics of MSCs have remained largely unknown. Methods and Materials: Radiation sensitivity of human primary MSCs from healthy volunteers and primary human fibroblast cells was examined, and cellular morphology, cell cycle effects, apoptosis, and differentiation potential after exposure to IR were assessed. Stem cell gene expression patterns after exposure to IRmore » were studied using gene arrays. Results: MSCs were not more radiosensitive than human primary fibroblasts, whereas there were considerable differences regarding radiation sensitivity within individual MSCs. Cellular morphology, cytoskeletal architecture, and cell motility were not markedly altered by IR. Even after high radiation doses up to 10 Gy, MSCs maintained their differentiation potential. Compared to primary fibroblast cells, MSCs did not show an increase in irradiation-induced apoptosis. Gene expression analyses revealed an upregulation of various genes involved in DNA damage response and DNA repair, but expression of established MSC surface markers appeared only marginally influenced by IR. Conclusions: These data suggest that human MSCs are not more radiosensitive than differentiated primary fibroblasts. In addition, upon photon irradiation, MSCs were able to retain their defining stem cell characteristics both on a functional level and regarding stem cell marker expression.« less

Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation

PubMed Central

Bermudez, Yira; Benavente, Claudia A.; Meyer, Ralph G.; Coyle, W. Russell; Jacobson, Myron K.; Jacobson, Elaine L.

2011-01-01

Background Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through Gi-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. Results Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional Gi-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. Conclusions The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s) of nicotinic acid receptors in human skin homeostasis. PMID:21655214

Immunohistochemical analysis of human milk fat globulin expression in extramammary Paget's disease.

PubMed

Ohnishi, T; Watanabe, S

2001-03-01

Primary extramammary Paget's disease is thought to be an intraepidermal carcinoma indicating apocrine secretory differentiation. In addition to expression in breast tissue, human milk fat globulin (HMFG) is expressed in the normal apocrine glands and tumours with apocrine differentiation. In this study HMFG expression in extramammary Paget's disease was analysed immunohistochemically in 18 cases of primary extramammary Paget's disease and two cases of secondary extramammary Paget's disease. The proportion and staining pattern of positive tumour cells with the anti-HMFG antibody was variable in each case. Cytoplasmic staining was observed frequently in dermal invasion and metastasis of Paget cells. The variabilities were thought to be due to modulation of the cellular localization of the cell surface component, HMFG, according to changes in cellular differentiation or malignant potency.

Simulation of elastic wave propagation using cellular automata and peridynamics, and comparison with experiments

DOE PAGES

Nishawala, Vinesh V.; Ostoja-Starzewski, Martin; Leamy, Michael J.; ...

2015-09-10

Peridynamics is a non-local continuum mechanics formulation that can handle spatial discontinuities as the governing equations are integro-differential equations which do not involve gradients such as strains and deformation rates. This paper employs bond-based peridynamics. Cellular Automata is a local computational method which, in its rectangular variant on interior domains, is mathematically equivalent to the central difference finite difference method. However, cellular automata does not require the derivation of the governing partial differential equations and provides for common boundary conditions based on physical reasoning. Both methodologies are used to solve a half-space subjected to a normal load, known as Lamb’smore » Problem. The results are compared with theoretical solution from classical elasticity and experimental results. Furthermore, this paper is used to validate our implementation of these methods.« less

Correlation of cellular factors and differential scrapie prion permissiveness in ovine microglia

USDA-ARS?s Scientific Manuscript database

Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrP-C) is misfolded into an accumulating, disease-associated isoform (PrP-D). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors con...

A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

PubMed

Bohnenpoll, Tobias; Wittern, Anna B; Mamo, Tamrat M; Weiss, Anna-Carina; Rudat, Carsten; Kleppa, Marc-Jens; Schuster-Gossler, Karin; Wojahn, Irina; Lüdtke, Timo H-W; Trowe, Mark-Oliver; Kispert, Andreas

2017-08-01

The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

Effects of differentiation on the phospholipid and phospholipid fatty acid composition of N1E-115 neuroblastoma cells.

PubMed

Murphy, E J; Horrocks, L A

1993-04-07

The effects of differentiation on the phospholipid and phospholipid fatty acid composition of N1E-115 neuroblastoma cells were determined. The cellular lipids were extracted on days 0, 3 and 7, following the addition of 1.2% dimethylsulfoxide to induce cellular differentiation. Proportions of ethanolamine glycerophospholipids (EtnGpl), phosphatidylinositol (PtdIns) and sphingomyelin (CerPCho) were significantly elevated following differentiation. The mole percentage of choline glycerophospholipids (ChoGpl) decreased with differentiation. The plasmalogens, both choline and ethanolamine, increased by 1.3- and 2.3-fold, respectively, during differentiation. The fatty acid composition of the phospholipid classes was also altered. PtdIns and ChoGpl had decreased proportions of polyenoic fatty acids, while these proportions were increased in EtnGpl. Both ChoGpl and EtnGpl had increased n-3/n-6 series fatty acid ratios, but this ratio was decreased in PtdIns. The mole percentage of arachidonic acid was significantly decreased in both PtdIns and ChoGpl, but elevated in EtnGpl and may be a result of the increase in ethanolamine plasmalogen. Thus, differentiation did not increase the overall mole percentage of polyenoic FA in the cells nor increase the n-6 series fatty acid proportions. We speculate plasmalogens may have a role in the differentiation process or in maintaining the cell in the differentiated state.

TRIENNIAL GROWTH AND DEVELOPMENT SYMPOSIUM: Factors influencing bovine intramuscular adipose tissue development and cellularity.

PubMed

Albrecht, E; Schering, L; Liu, Y; Komolka, K; Kühn, C; Wimmers, K; Gotoh, T; Maak, S

2017-05-01

Appearance, distribution, and amount of intramuscular fat (IMF), often referred to as marbling, are highly variable and depend on environmental and genetic factors. On the molecular level, the concerted action of several drivers, including hormones, receptors, transcription factors, etc., determines where clusters of adipocytes arise. Therefore, the aim of future studies remains to identify such factors as biological markers of IMF to increase the ability to identify animals that deposit IMF early in age to increase efficiency of high-quality meat production. In an attempt to unravel the cellular development of marbling, we investigated the abundance of markers for adipogenic differentiation during fattening of cattle and the transcriptome of muscle and dissected IMF. Markers of different stages of adipogenic differentiation are well known from cell culture experiments. They are usually transiently expressed, such as delta-like homolog 1 (DLK1) that is abundant in preadipocytes and absent during differentiation to mature adipocytes. It is even a greater challenge to detect those markers in live animals. Within skeletal muscles, hyperplasia and hypertrophy of adipocytes can be observed throughout life. Therefore, development of marbling requires, on the cellular level, recruitment, proliferation, and differentiation of adipogenic cells to store excess energy in the form of lipids in new cells. In a recent study, we investigated the localization and abundance of early markers of adipogenic differentiation, such as DLK1, in bovine muscle tissue. An inverse relationship between IMF content and number of DLK1-positive cells in bovine muscle was demonstrated. Considering the cellular environment of differentiating adipocytes in muscle and the secretory action of adipocytes and myocytes, it becomes obvious that cross talk between cells via adipokines and myokines may be important for IMF development. Secreted proteins can act on other cells, inhibiting or stimulating their function via autocrine and paracrine actions. Such factors with potential influence on IMF, among them, agouti signaling protein and thrombospondin 4, were identified in transcriptome analyses and further investigated. Furthermore, results from transcriptome analysis indicate involvement of genes that are not directly related to adipogenesis and lipid metabolism, providing new candidates for future research.

HOXC9-Induced Differentiation in Neuroblastoma Development

DTIC Science & Technology

2014-10-01

Neuroblastoma Development PRINCIPAL INVESTIGATOR: Han-Fei Ding RECIPIENT: Georgia Health Sciences University Research Institute, Inc... Neuroblastoma  Development   5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-12-1-0613 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...role in determining the differentiation states of neuroblastoma tumors, with higher levels of HOXC9 promoting differentiation. At the cellular level

Physico-electrochemical Characterization of Pluripotent Stem Cells during Self-Renewal or Differentiation by a Multi-modal Monitoring System.

PubMed

Low, Karen; Wong, Lauren Y; Maldonado, Maricela; Manjunath, Chetas; Horner, Christopher B; Perez, Mark; Myung, Nosang V; Nam, Jin

2017-05-09

Monitoring pluripotent stem cell behaviors (self-renewal and differentiation to specific lineages/phenotypes) is critical for a fundamental understanding of stem cell biology and their translational applications. In this study, a multi-modal stem cell monitoring system was developed to quantitatively characterize physico-electrochemical changes of the cells in real time, in relation to cellular activities during self-renewal or lineage-specific differentiation, in a non-destructive, label-free manner. The system was validated by measuring physical (mass) and electrochemical (impedance) changes in human induced pluripotent stem cells undergoing self-renewal, or subjected to mesendodermal or ectodermal differentiation, and correlating them to morphological (size, shape) and biochemical changes (gene/protein expression). An equivalent circuit model was used to further dissect the electrochemical (resistive and capacitive) contributions of distinctive cellular features. Overall, the combination of the physico-electrochemical measurements and electrical circuit modeling collectively offers a means to longitudinally quantify the states of stem cell self-renewal and differentiation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Major transcriptome re-organisation and abrupt changes in signalling, cell cycle and chromatin regulation at neural differentiation in vivo.

PubMed

Olivera-Martinez, Isabel; Schurch, Nick; Li, Roman A; Song, Junfang; Halley, Pamela A; Das, Raman M; Burt, Dave W; Barton, Geoffrey J; Storey, Kate G

2014-08-01

Here, we exploit the spatial separation of temporal events of neural differentiation in the elongating chick body axis to provide the first analysis of transcriptome change in progressively more differentiated neural cell populations in vivo. Microarray data, validated against direct RNA sequencing, identified: (1) a gene cohort characteristic of the multi-potent stem zone epiblast, which contains neuro-mesodermal progenitors that progressively generate the spinal cord; (2) a major transcriptome re-organisation as cells then adopt a neural fate; and (3) increasing diversity as neural patterning and neuron production begin. Focussing on the transition from multi-potent to neural state cells, we capture changes in major signalling pathways, uncover novel Wnt and Notch signalling dynamics, and implicate new pathways (mevalonate pathway/steroid biogenesis and TGFβ). This analysis further predicts changes in cellular processes, cell cycle, RNA-processing and protein turnover as cells acquire neural fate. We show that these changes are conserved across species and provide biological evidence for reduced proteasome efficiency and a novel lengthening of S phase. This latter step may provide time for epigenetic events to mediate large-scale transcriptome re-organisation; consistent with this, we uncover simultaneous downregulation of major chromatin modifiers as the neural programme is established. We further demonstrate that transcription of one such gene, HDAC1, is dependent on FGF signalling, making a novel link between signals that control neural differentiation and transcription of a core regulator of chromatin organisation. Our work implicates new signalling pathways and dynamics, cellular processes and epigenetic modifiers in neural differentiation in vivo, identifying multiple new potential cellular and molecular mechanisms that direct differentiation. © 2014. Published by The Company of Biologists Ltd.

Proteomic analysis of the response of α-ketoglutarate-producer Yarrowia lipolytica WSH-Z06 to environmental pH stimuli.

PubMed

Guo, Hongwei; Wan, Hui; Chen, Hongwen; Fang, Fang; Liu, Song; Zhou, Jingwen

2016-10-01

During bioproduction of short-chain carboxylates, a shift in pH is a common strategy for enhancing the biosynthesis of target products. Based on two-dimensional gel electrophoresis, comparative proteomics analysis of general and mitochondrial protein samples was used to investigate the cellular responses to environmental pH stimuli in the α-ketoglutarate overproducer Yarrowia lipolytica WSH-Z06. The lower environmental pH stimuli tensioned intracellular acidification and increased the level of reactive oxygen species (ROS). A total of 54 differentially expressed protein spots were detected, and 11 main cellular processes were identified to be involved in the cellular response to environmental pH stimuli. Slight decrease in cytoplasmic pH enhanced the cellular acidogenicity by elevating expression level of key enzymes in tricarboxylic acid cycle (TCA cycle). Enhanced energy biosynthesis, ROS elimination, and membrane potential homeostasis processes were also employed as cellular defense strategies to compete with environmental pH stimuli. Owing to its antioxidant role of α-ketoglutarate, metabolic flux shifted to α-ketoglutarate under lower pH by Y. lipolytica in response to acidic pH stimuli. The identified differentially expressed proteins provide clues for understanding the mechanisms of the cellular responses and for enhancing short-chain carboxylate production through metabolic engineering or process optimization strategies in combination with manipulation of environmental conditions.

Vaccine induced differential expressions of miRNAs at cytolytic stage in chickens resistant or susceptible to Marek’s disease

USDA-ARS?s Scientific Manuscript database

Gene expression regulation is critical for all cellular processes since dysregulation of it often results in elevated disease risk and compromised cellular immunity. MicroRNAs (miRNAs) directly regulate gene expression post-transcriptionally through base-pairing with regions in the 3’-untranslated s...

Differential programming of p53-deficient embryonic cells during rotenone block

EPA Science Inventory

Mitochondrial dysfunction has been implicated in chemical toxicities. The present study used an in vitro model to investigate the differential expression of metabolic pathways during cellular stress in p53- efficient embryonic fibroblasts compared to p53-deficient cells. These c...

Biological effects of weightlessness and clinostatic conditions registered in cells of root meristem and cap of higher plants

NASA Astrophysics Data System (ADS)

Sytnik, K. M.; Kordyum, E. L.; Belyavskaya, N. A.; Nedukha, E. M.; Tarasenko, V. A.

Research in cellular reproduction, differentiation and vital activity, i.e. processes underlying the development and functioning of organisms, plants included, is essential for solving fundamental and applied problems of space biology. Detailed anatomical analysis of roots of higher plants grown on board the Salyut 6 orbital research station show that under conditions of weightlessness for defined duration mitosis, cytokinesis and tissue differentiation in plant vegetative organs occur essentially normally. At the same time, certain rearrangements in the structural organization of cellular organelles - mainly the plastid apparatus, mitochondria, Golgi apparatus and nucleus - are established in the root meristem and cap of the experimental plants. This is evidence for considerable changes in cellular metabolism. The structural changes in the subcellular level arising under spaceflight conditions are partially absent in clinostat experiments designed to simulate weightlessness. Various clinostatic conditions have different influences on the cell structural and functional organization than does space flight. It is suggested that alterations of cellular metabolism under weightlessness and clinostatic conditions occur within existing genetic programs.

Cancer Risk-Assessment of Radiation Damage in Ataxia Telangiectasia Heterozygous Human Breast Epithelial Cell Cultures

NASA Technical Reports Server (NTRS)

Applewhite, Lisa C.

2002-01-01

This paper describes the study of the markers of cellular changes that are found during the onset of carcinogenesis. Several of the biological factors are markers of stress response, oncoprotein expression, and differentiation factors. Oxidative stress response agents such as heat shock proteins (HSPs) protect cells from oxidative stresses such as ionizing radiation. The onocoprotein HER-2/neu, a specific breast cancer marker, indicates early onset of cancer. Additional structural and morphogenetic markers of differentiation were considered in order to determine initial cellular changes at the initial onset of cancer. As an additional consideration, all-trans retinoic acid (RA), a differentiation agent, was considered because of its known role in regulating normal differentiation and inhibiting tumor proliferation via specific nuclear receptors. This paper discusses study and results of the preliminary analyses of gamma irradiation of AT heterozygous human breast epithelial cells (WH). Comparisons are also made of the effects various RA concentrations post-irradiation.

The Effect of Spaceflight on Cartilage Cell Cycle and Differentiation

NASA Technical Reports Server (NTRS)

Doty, Stephen B.; Stiner, Dalina; Telford, William G.

2000-01-01

In vivo studies have shown that spaceflight results in loss of bone and muscle. In an effort to understand the mechanisms of these changes, cell cultures of cartilage, bone and muscle have been subjected to spaceflight to study the microgravity effects on differentiated cells. However it now seems possible that the cell differentiation process itself may be the event(s) most affected by spaceflight. For example, osteoblast-like cells have been shown to have reduced cellular activity in microgravity due to an underdifferentiated state (Carmeliet, et al, 1997). And reduced human lymphocyte growth in spaceflight was related to increased apoptosis (Lewis, et al, 1998). Which brings us to the question of whether reduced cellular activity in space is due to an effect on the differentiated cell, an effect on the cell cycle and cell proliferation, or an effect on cell death. This question has not been specifically addressed on previous flights and was the question behind die present study.

Abnormal differentiation, hyperplasia and embryonic/perinatal lethality in BK5-T/t transgenic mice

PubMed Central

Chen, Xin; Schneider-Broussard, Robin; Hollowell, Debra; McArthur, Mark; Jeter, Collene R.; Benavides, Fernando; DiGiovanni, John; Tang, Dean G.

2009-01-01

The cell-of-origin has a great impact on the types of tumors that develop and the stem/progenitor cells have long been considered main targets of malignant transformation. The SV40 large T and small t antigens (T/t), have been targeted to multiple differentiated cellular compartments in transgenic mice. In most of these studies, transgenic animals develop tumors without apparent defects in animal development. In this study, we used the bovine keratin 5 (BK5) promoter to target the T/t antigens to stem/progenitor cell-containing cytokeratin 5 (CK5) cellular compartment. A transgene construct, BK5-T/t, was made and microinjected into the male pronucleus of FVB/N mouse oocytes. After implanting ∼1700 embryos, only 7 transgenics were obtained, including 4 embryos (E9.5, E13, E15, and E20) and 3 postnatal animals, which died at P1, P2, and P18, respectively. Immunohistological analysis revealed aberrant differentiation and prominent hyperplasia in several transgenic CK5 tissues, especially the upper digestive organs (tongue, oral mucosa, esophagus, and forestomach) and epidermis, the latter of which also showed focal dysplasia. Altogether, these results indicate that constitutive expression of the T/t antigens in CK5 cellular compartment results in abnormal epithelial differentiation and leads to embryonic/perinatal animal lethality. PMID:19272531

Investigating the interplay between substrate stiffness and ligand chemistry in directing mesenchymal stem cell differentiation within 3D macro-porous substrates.

PubMed

Haugh, Matthew G; Vaughan, Ted J; Madl, Christopher M; Raftery, Rosanne M; McNamara, Laoise M; O'Brien, Fergal J; Heilshorn, Sarah C

2018-07-01

Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior. Copyright © 2018 Elsevier Ltd. All rights reserved.

Prenatal alcohol exposure and cellular differentiation: a role for Polycomb and Trithorax group proteins in FAS phenotypes?

PubMed

Veazey, Kylee J; Muller, Daria; Golding, Michael C

2013-01-01

Exposure to alcohol significantly alters the developmental trajectory of progenitor cells and fundamentally compromises tissue formation (i.e., histogenesis). Emerging research suggests that ethanol can impair mammalian development by interfering with the execution of molecular programs governing differentiation. For example, ethanol exposure disrupts cellular migration, changes cell-cell interactions, and alters growth factor signaling pathways. Additionally, ethanol can alter epigenetic mechanisms controlling gene expression. Normally, lineage-specific regulatory factors (i.e., transcription factors) establish the transcriptional networks of each new cell type; the cell's identity then is maintained through epigenetic alterations in the way in which the DNA encoding each gene becomes packaged within the chromatin. Ethanol exposure can induce epigenetic changes that do not induce genetic mutations but nonetheless alter the course of fetal development and result in a large array of patterning defects. Two crucial enzyme complexes--the Polycomb and Trithorax proteins--are central to the epigenetic programs controlling the intricate balance between self-renewal and the execution of cellular differentiation, with diametrically opposed functions. Prenatal ethanol exposure may disrupt the functions of these two enzyme complexes, altering a crucial aspect of mammalian differentiation. Characterizing the involvement of Polycomb and Trithorax group complexes in the etiology of fetal alcohol spectrum disorders will undoubtedly enhance understanding of the role that epigenetic programming plays in this complex disorder.

The effect of noncoherent red light irradiation on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

PubMed

Peng, Fei; Wu, Hua; Zheng, Yadong; Xu, Xiqiang; Yu, Jizhe

2012-05-01

Mesenchymal stem cells (MSCs) are promising for use in regenerative medicine. Low-level light irradiation (LLLI) has been shown to modulate various processes in different biological systems. The aim of our study was to investigate the effect of red light emitted from a light-emitting diode (LED) on bone marrow MSCs with or without osteogenic supplements. MSCs both with and without osteogenic supplements were divided into four groups, and each group was irradiated at doses of 0, 1, 2 and 4 J/cm(2). Cellular proliferation was evaluated using WST-8 and 5-ethynyl-2'-deoxyuridine (EdU) fluorescence staining. The alkaline phosphatase activity, mineralization, and expression of osteoblast master genes (Col1α1, Alpl, Bglap and Runx2) were monitored as indicators of MSC differentiation towards osteoblasts. In groups without osteogenic supplements, red light at all doses significantly stimulated cellular proliferation, whereas the osteogenic phenotype of the MSCs was not enhanced. In groups with osteogenic supplements, red light increased alkaline phosphatase activity and mineralized nodule formation, and stimulated the expression of Bglap and Runx2, but decreased cellular proliferation. In conclusion, nonconherent red light can promote proliferation but cannot induce osteogenic differentiation of MSCs in normal media, while it enhances osteogenic differentiation and decreases proliferation of MSCs in media with osteogenic supplements.

Osteoblast dysfunctions in bone diseases: from cellular and molecular mechanisms to therapeutic strategies.

PubMed

Marie, Pierre J

2015-04-01

Several metabolic, genetic and oncogenic bone diseases are characterized by defective or excessive bone formation. These abnormalities are caused by dysfunctions in the commitment, differentiation or survival of cells of the osteoblast lineage. During the recent years, significant advances have been made in our understanding of the cellular and molecular mechanisms underlying the osteoblast dysfunctions in osteoporosis, skeletal dysplasias and primary bone tumors. This led to suggest novel therapeutic approaches to correct these abnormalities such as the modulation of WNT signaling, the pharmacological modulation of proteasome-mediated protein degradation, the induction of osteoprogenitor cell differentiation, the repression of cancer cell proliferation and the manipulation of epigenetic mechanisms. This article reviews our current understanding of the major cellular and molecular mechanisms inducing osteoblastic cell abnormalities in age-related bone loss, genetic skeletal dysplasias and primary bone tumors, and discusses emerging therapeutic strategies to counteract the osteoblast abnormalities in these disorders of bone formation.

Developmental Progression in the Coral Acropora digitifera Is Controlled by Differential Expression of Distinct Regulatory Gene Networks

PubMed Central

Reyes-Bermudez, Alejandro; Villar-Briones, Alejandro; Ramirez-Portilla, Catalina; Hidaka, Michio; Mikheyev, Alexander S.

2016-01-01

Corals belong to the most basal class of the Phylum Cnidaria, which is considered the sister group of bilaterian animals, and thus have become an emerging model to study the evolution of developmental mechanisms. Although cell renewal, differentiation, and maintenance of pluripotency are cellular events shared by multicellular animals, the cellular basis of these fundamental biological processes are still poorly understood. To understand how changes in gene expression regulate morphogenetic transitions at the base of the eumetazoa, we performed quantitative RNA-seq analysis during Acropora digitifera’s development. We collected embryonic, larval, and adult samples to characterize stage-specific transcription profiles, as well as broad expression patterns. Transcription profiles reconstructed development revealing two main expression clusters. The first cluster grouped blastula and gastrula and the second grouped subsequent developmental time points. Consistently, we observed clear differences in gene expression between early and late developmental transitions, with higher numbers of differentially expressed genes and fold changes around gastrulation. Furthermore, we identified three coexpression clusters that represented discrete gene expression patterns. During early transitions, transcriptional networks seemed to regulate cellular fate and morphogenesis of the larval body. In late transitions, these networks seemed to play important roles preparing planulae for switch in lifestyle and regulation of adult processes. Although developmental progression in A. digitifera is regulated to some extent by differential coexpression of well-defined gene networks, stage-specific transcription profiles appear to be independent entities. While negative regulation of transcription is predominant in early development, cell differentiation was upregulated in larval and adult stages. PMID:26941230

Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous system.

PubMed

Metzger, Marco; Bareiss, Petra M; Nikolov, Ivan; Skutella, Thomas; Just, Lothar

2007-01-01

Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell-cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement.

Sordaria macrospora, a model organism to study fungal cellular development.

PubMed

Engh, Ines; Nowrousian, Minou; Kück, Ulrich

2010-12-01

During the development of multicellular eukaryotes, the processes of cellular growth and organogenesis are tightly coordinated. Since the 1940s, filamentous fungi have served as genetic model organisms to decipher basic mechanisms underlying eukaryotic cell differentiation. Here, we focus on Sordaria macrospora, a homothallic ascomycete and important model organism for developmental biology. During its sexual life cycle, S. macrospora forms three-dimensional fruiting bodies, a complex process involving the formation of different cell types. S. macrospora can be used for genetic, biochemical and cellular experimental approaches since diverse tools, including fluorescence microscopy, a marker recycling system and gene libraries, are available. Moreover, the genome of S. macrospora has been sequenced and allows functional genomics analyses. Over the past years, our group has generated and analysed a number of developmental mutants which has greatly enhanced our fundamental understanding about fungal morphogenesis. In addition, our recent research activities have established a link between developmental proteins and conserved signalling cascades, ultimately leading to a regulatory network controlling differentiation processes in a eukaryotic model organism. This review summarizes the results of our recent findings, thus advancing current knowledge of the general principles and paradigms underpinning eukaryotic cell differentiation and development. Copyright © 2010 Elsevier GmbH. All rights reserved.

Early changes in staurosporine-induced differentiated RGC-5 cells indicate cellular injury response to nonlethal blue light exposure.

PubMed

Zhang, Pei; Huang, Chen; Wang, Wei; Wang, Minshu

2015-06-01

Blue light has been previously demonstrated to induce injury of retinal cells. The cellular responses to nonlethal blue light exposure for each type of retinal cell are of particular interest but remain undetermined. Based on the doses of blue light reported in previous research to be nonlethal to retinal pigment epithelial cells, here we investigated whether and to what extent such doses of blue light are cytotoxic to staurosporine-differentiated RGC-5 cells. RGC-5 cells were differentiated for 24 hours using 200 nM staurosporine. The resulting cells were cultured and exposed to blue light at three different energy levels (1, 10, and 50 J cm(-2)). Cellular morphologies were investigated with an inverted microscope and cell viability was assessed with a Cell Counting Kit-8 (CCK-8) assay. The generation of intracellular reactive oxygen species (ROS) was evaluated by H2DCFDA. After loading of MitoTracker Green FM dye, the mitochondrial contents were analyzed using flow cytometry. The lactate dehydrogenase (LDH) activities in the media were also measured. The level of lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA). Treatment of the cells for 24 hours with 200 nM staurosporine successfully induced the differentiation of RGC-5 cells. No morphological changes were observed in the ssdRGC-5 cells exposed to blue light at 50 J cm(-2), which was the highest energy level tested. Exposure of the ssdRGC-5 cells to this energy level of blue light did, however, decrease their numbers by approximately 72.1% compared to the numbers of such cells found after being left in the dark. Remarkably, the levels of ROS generation and mitochondrial contents were, respectively, increased to 142% and 118% of those of the control by a 10 J cm(-2) exposure of blue light. The LDH activities and MDA levels exhibited no obvious changes in the blue light-exposed ssdRGC-5 cells compared to the control cells. In vitro nonlethal blue light exposure led to cellular damage of staurosporine-differentiated RGC-5 cells. These increases in oxidative stress and mitochondrial content were the early steps of the cellular response to the exposure of relatively low doses (10 J cm(-2)) of blue light.

Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

PubMed Central

2010-01-01

Introduction Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues. PMID:20964822

Evolution of Microbial Quorum Sensing to Human Global Quorum Sensing: An Insight into How Gap Junctional Intercellular Communication Might Be Linked to the Global Metabolic Disease Crisis.

PubMed

Trosko, James E

2016-06-15

The first anaerobic organism extracted energy for survival and reproduction from its source of nutrients, with the genetic means to ensure protection of its individual genome but also its species survival. While it had a means to communicate with its community via simple secreted molecules ("quorum sensing"), the eventual shift to an aerobic environment led to multi-cellular metazoan organisms, with evolutionary-selected genes to form extracellular matrices, stem cells, stem cell niches, and a family of gap junction or "connexin" genes. These germinal and somatic stem cells responded to extracellular signals that triggered intra-cellular signaling to regulate specific genes out of the total genome. These extra-cellular induced intra-cellular signals also modulated gap junctional intercellular communication (GJIC) in order to regulate the new cellular functions of symmetrical and asymmetrical cell division, cell differentiation, modes of cell death, and senescence. Within the hierarchical and cybernetic concepts, differentiated by neurons organized in the brain of the Homo sapiens, the conscious mind led to language, abstract ideas, technology, myth-making, scientific reasoning, and moral decision-making, i.e., the creation of culture. Over thousands of years, this has created the current collision between biological and cultural evolution, leading to the global "metabolic disease" crisis.

Folding, But Not Surface Area Expansion, Is Associated with Cellular Morphological Maturation in the Fetal Cerebral Cortex

PubMed Central

Studholme, Colin; Frias, Antonio E.

2017-01-01

Altered macroscopic anatomical characteristics of the cerebral cortex have been identified in individuals affected by various neurodevelopmental disorders. However, the cellular developmental mechanisms that give rise to these abnormalities are not understood. Previously, advances in image reconstruction of diffusion magnetic resonance imaging (MRI) have made possible high-resolution in utero measurements of water diffusion anisotropy in the fetal brain. Here, diffusion anisotropy within the developing fetal cerebral cortex is longitudinally characterized in the rhesus macaque, focusing on gestation day (G85) through G135 of the 165 d term. Additionally, for subsets of animals characterized at G90 and G135, immunohistochemical staining was performed, and 3D structure tensor analyses were used to identify the cellular processes that most closely parallel changes in water diffusion anisotropy with cerebral cortical maturation. Strong correlations were found between maturation of dendritic arbors on the cellular level and the loss of diffusion anisotropy with cortical development. In turn, diffusion anisotropy changes were strongly associated both regionally and temporally with cortical folding. Notably, the regional and temporal dependence of diffusion anisotropy and folding were distinct from the patterns observed for cerebral cortical surface area expansion. These findings strengthen the link proposed in previous studies between cellular-level changes in dendrite morphology and noninvasive diffusion MRI measurements of the developing cerebral cortex and support the possibility that, in gyroencephalic species, structural differentiation within the cortex is coupled to the formation of gyri and sulci. SIGNIFICANCE STATEMENT Abnormal brain morphology has been found in populations with neurodevelopmental disorders. However, the mechanisms linking cellular level and macroscopic maturation are poorly understood, even in normal brains. This study contributes new understanding to this subject using serial in utero MRI measurements of rhesus macaque fetuses, from which macroscopic and cellular information can be derived. We found that morphological differentiation of dendrites was strongly associated both regionally and temporally with folding of the cerebral cortex. Interestingly, parallel associations were not observed with cortical surface area expansion. These findings support the possibility that perturbed morphological differentiation of cells within the cortex may underlie abnormal macroscopic characteristics of individuals affected by neurodevelopmental disorders. PMID:28069920

Histomorphometric analysis of nuclear and cellular volumetric alterations in oral lichen planus, lichenoid lesions and normal oral mucosa using image analysis software.

PubMed

Venkatesiah, Sowmya S; Kale, Alka D; Hallikeremath, Seema R; Kotrashetti, Vijayalakshmi S

2013-01-01

Lichen planus is a chronic inflammatory mucocutaneous disease that clinically and histologically resembles lichenoid lesions, although the latter has a different etiology. Though criteria have been suggested for differentiating oral lichen planus from lichenoid lesions, confusion still prevails. To study the cellular and nuclear volumetric features in the epithelium of normal mucosa, lichen planus, and lichenoid lesions to determine variations if any. A retrospective study was done on 25 histologically diagnosed cases each of oral lichen planus, oral lichenoid lesions, and normal oral mucosa. Cellular and nuclear morphometric measurements were assessed on hematoxylin and eosin sections using image analysis software. Analysis of variance test (ANOVA) and Tukey's post-hoc test. The basal cells of oral lichen planus showed a significant increase in the mean nuclear and cellular areas, and in nuclear volume; there was a significant decrease in the nuclear-cytoplasmic ratio as compared to normal mucosa. The suprabasal cells showed a significant increase in nuclear and cellular areas, nuclear diameter, and nuclear and cellular volumes as compared to normal mucosa. The basal cells of oral lichenoid lesions showed significant difference in the mean cellular area and the mean nuclear-cytoplasmic ratio as compared to normal mucosa, whereas the suprabasal cells differed significantly from normal mucosa in the mean nuclear area and the nuclear and cellular volumes. Morphometry can differentiate lesions of oral lichen planus and oral lichenoid lesions from normal oral mucosa. Thus, morphometry may serve to discriminate between normal and premalignant lichen planus and lichenoid lesions. These lesions might have a high risk for malignant transformation and may behave in a similar manner with respect to malignant transformation.

[The role of telomerase activity in non-invasive diagnostics of bladder cancer].

PubMed

Glybochko, P V; Alyaev, J G; Potoldykova, N V; Polyakovsky, K A; Vinarov, A Z; Glukhov, A I; Gordeev, S A

2016-08-01

To evaluate the potentials of determining the telomerase activity (TA) in the cellular material of the urine for noninvasive diagnosis of bladder cancer (BC). Evaluation of TA was performed in the urine of 48 patients with bladder cancer (study group) before and after transurethral resection of the bladder wall (n=38), an open resection of the bladder (n=4), and cystectomy (n=6). TA was also evaluated in 48 tumor tissue samples obtained from these patients during removal of the bladder tumor. Each sample of the tumor tissue was separated into two parts, one of which was subjected to histological examination, and the latter was used to determine the telomerase activity. In all cases, the diagnosis of bladder cancer was confirmed morphologically. Determination of TA in the samples was performed by the modified TRAP-method (telomerase repeat amplification protocol), RT-PCR, PCR, and electrophoresis. As a control, cell material of the urine and tissue in 12 patients with chronic cystitis was investigated. TA before surgery was found in 45 (93.75%) of 48 samples of cellular material of the urine from patients with suspected bladder cancer. BC was histologically verified in all patients in this group. In the postoperative period, TA was not observed in the 48 samples of cellular material of the urine from patients with BC. In the control group of patients with histologically verified cystitis, weak TA was determined only in one sample of cellular material of the urine. The analysis indicates statistically significant predominance of patients with bladder cancer in case of TA in the urine (P=0.001). TA was detected in all samples of tumor tissue. We also analyzed the dependence of TA levels in urine and tissue on the degree of BC differentiation. In patients with highly differentiated BC, mean AT in the cellular materials of the urine was 0,61% (n=15), in patients with moderately differentiated BC - 0.95% (n=23), in patients with low-grade bladder cancer - 1.33% (n=10); in other words, increase in the TA levels with decreasing the degree of differentiation was observed. This finding can be used in the prognosis of the course of disease based on determining the TA level in these patients. Preliminary data indicate the possibility of use of determining the TA in cellular material of the urine for the diagnosis and monitoring of bladder cancer recurrence.

Stimulation of neural differentiation in human bone marrow mesenchymal stem cells by extremely low-frequency electromagnetic fields incorporated with MNPs.

PubMed

Choi, Yun-Kyong; Lee, Dong Heon; Seo, Young-Kwon; Jung, Hyun; Park, Jung-Keug; Cho, Hyunjin

2014-10-01

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have been investigated as a new cell-therapeutic solution due to their capacity that could differentiate into neural-like cells. Extremely low-frequency electromagnetic fields (ELF-EMFs) therapy has emerged as a novel technique, using mechanical stimulus to differentiate hBM-MSCs and significantly enhance neuronal differentiation to affect cellular and molecular reactions. Magnetic iron oxide (Fe3O4) nanoparticles (MNPs) have recently achieved widespread use for biomedical applications and polyethylene glycol (PEG)-labeled nanoparticles are used to increase their circulation time, aqueous solubility, biocompatibility, and nonspecific cellular uptake as well as to decrease immunogenicity. Many studies have used MNP-labeled cells for differentiation, but there have been no reports of MNP-labeled neural differentiation combined with EMFs. In this study, synthesized PEG-phospholipid encapsulated magnetite (Fe3O4) nanoparticles are used on hBM-MSCs to improve their intracellular uptake. The PEGylated nanoparticles were exposed to the cells under 50 Hz of EMFs to improve neural differentiation. First, we measured cell viability and intracellular iron content in hBM-MSCs after treatment with MNPs. Analysis was conducted by RT-PCR, and immunohistological analysis using neural cell type-specific genes and antibodies after exposure to 50 Hz electromagnetic fields. These results suggest that electromagnetic fields enhance neural differentiation in hBM-MSCs incorporated with MNPs and would be an effective method for differentiating neural cells.

Metabostemness: a new cancer hallmark.

PubMed

Menendez, Javier A; Alarcón, Tomás

2014-01-01

The acquisition of and departure from stemness in cancer tissues might not only be hardwired by genetic controllers, but also by the pivotal regulatory role of the cellular metabotype, which may act as a "starter dough" for cancer stemness traits. We have coined the term metabostemness to refer to the metabolic parameters causally controlling or functionally substituting the epitranscriptional orchestration of the genetic reprograming that redirects normal and tumor cells toward less-differentiated cancer stem cell (CSC) cellular states. Certain metabotypic alterations might operate as pivotal molecular events rendering a cell of origin susceptible to epigenetic rewiring required for the acquisition of aberrant stemness and, concurrently, of refractoriness to differentiation. The metabostemness attribute can remove, diminish, or modify the nature of molecular barriers present in Waddington's epigenetic landscapes, thus allowing differentiated cells to more easily (re)-enter into CSC cellular macrostates. Activation of the metabostemness trait can poise cells with chromatin states competent for rapid dedifferentiation while concomitantly setting the idoneous metabolic stage for later reprograming stimuli to finish the journey from non-cancerous into tumor-initiating cells. Because only a few permitted metabotypes will be compatible with the operational properties owned by CSC cellular states, the metabostemness property provides a new framework through which to pharmacologically resolve the apparently impossible problem of discovering drugs aimed to target the molecular biology of the cancer stemness itself. The metabostemness cancer hallmark generates a shifting oncology theory that should guide a new era of metabolo-epigenetic cancer precision medicine.

Metabostemness: A New Cancer Hallmark

PubMed Central

Menendez, Javier A.; Alarcón, Tomás

2014-01-01

The acquisition of and departure from stemness in cancer tissues might not only be hardwired by genetic controllers, but also by the pivotal regulatory role of the cellular metabotype, which may act as a “starter dough” for cancer stemness traits. We have coined the term metabostemness to refer to the metabolic parameters causally controlling or functionally substituting the epitranscriptional orchestration of the genetic reprograming that redirects normal and tumor cells toward less-differentiated cancer stem cell (CSC) cellular states. Certain metabotypic alterations might operate as pivotal molecular events rendering a cell of origin susceptible to epigenetic rewiring required for the acquisition of aberrant stemness and, concurrently, of refractoriness to differentiation. The metabostemness attribute can remove, diminish, or modify the nature of molecular barriers present in Waddington’s epigenetic landscapes, thus allowing differentiated cells to more easily (re)-enter into CSC cellular macrostates. Activation of the metabostemness trait can poise cells with chromatin states competent for rapid dedifferentiation while concomitantly setting the idoneous metabolic stage for later reprograming stimuli to finish the journey from non-cancerous into tumor-initiating cells. Because only a few permitted metabotypes will be compatible with the operational properties owned by CSC cellular states, the metabostemness property provides a new framework through which to pharmacologically resolve the apparently impossible problem of discovering drugs aimed to target the molecular biology of the cancer stemness itself. The metabostemness cancer hallmark generates a shifting oncology theory that should guide a new era of metabolo-epigenetic cancer precision medicine. PMID:25325014

Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line

PubMed Central

2011-01-01

Background When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. Results A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. Conclusions The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells. PMID:22111699

Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

PubMed Central

Harden, Mallory E.; Prasad, Nripesh; Griffiths, Anthony

2017-01-01

ABSTRACT The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. PMID:28049151

Maize early endosperm growth and development: from fertilization through cell type differentiation.

PubMed

Leroux, Brian M; Goodyke, Austin J; Schumacher, Katelyn I; Abbott, Chelsi P; Clore, Amy M; Yadegari, Ramin; Larkins, Brian A; Dannenhoffer, Joanne M

2014-08-01

• Given the worldwide economic importance of maize endosperm, it is surprising that its development is not the most comprehensively studied of the cereals. We present detailed morphometric and cytological descriptions of endosperm development in the maize inbred line B73, for which the genome has been sequenced, and compare its growth with four diverse Nested Association Mapping (NAM) founder lines.• The first 12 d of B73 endosperm development were described using semithin sections of plastic-embedded kernels and confocal microscopy. Longitudinal sections were used to compare endosperm length, thickness, and area.• Morphometric comparison between Arizona- and Michigan-grown B73 showed a common pattern. Early endosperm development was divided into four stages: coenocytic, cellularization through alveolation, cellularization through partitioning, and differentiation. We observed tightly synchronous nuclear divisions in the coenocyte, elucidated that the onset of cellularization was coincident with endosperm size, and identified a previously undefined cell type (basal intermediate zone, BIZ). NAM founders with small mature kernels had larger endosperms (0-6 d after pollination) than lines with large mature kernels.• Our B73-specific model of early endosperm growth links developmental events to relative endosperm size, while accounting for diverse growing conditions. Maize endosperm cellularizes through alveolation, then random partitioning of the central vacuole. This unique cellularization feature of maize contrasts with the smaller endosperms of Arabidopsis, barley, and rice that strictly cellularize through repeated alveolation. NAM analysis revealed differences in endosperm size during early development, which potentially relates to differences in timing of cellularization across diverse lines of maize. © 2014 Botanical Society of America, Inc.

IN UTERO EXPOSURE TO THE FUNGICIDE PROCYMIDONE AND DIBUTYL PHTHALATE PRODUCE DOSE ADDITIVE DISRUPTIONS OF MALE RAT SEXUAL DIFFERENTIATION

EPA Science Inventory

Procymidone (PRO) and dibutyl phthalate (DBP) alter male rat sexual differentiation by disrupting the androgen-signaling pathway via distinctly different cellular mechanisms of toxicity. DBP inhibits fetal Leydig cell androgen production whereas PRO binds AR and blocks androgen a...

Modems for emerging digital cellular-mobile radio system

NASA Technical Reports Server (NTRS)

Feher, Kamilo

1991-01-01

Digital modem techniques for emerging digital cellular telecommunications-mobile radio system applications are described and analyzed. In particular, theoretical performance, experimental results, principles of operation, and various architectures of pi/4-QPSK (pi/4-shifted coherent or differential QPSK) modems for second-generation US digital cellular radio system applications are presented. The spectral/power efficiency and performance of the pi/4-QPSK modems (American and Japanese digital cellular emerging standards) are studied and briefly compared to GMSK (Gaussian minimum-shift keying) modems (proposed for European DECT and GSM cellular standards). Improved filtering strategies and digital pilot-aided (digital channel sounding) techniques are also considered for pi/4-QPSK and other digital modems. These techniques could significantly improve the performance of digital cellular and other digital land mobile and satellite mobile radio systems. More spectrally efficient modem trends for future cellular/mobile (land mobile) and satellite communication systems applications are also highlighted.

Dynamic regulation of nuclear architecture and mechanics—a rheostatic role for the nucleus in tailoring cellular mechanosensitivity

PubMed Central

Lee, David A.

2017-01-01

ABSTRACT Nuclear architecture, a function of both chromatin and nucleoskeleton structure, is known to change with stem cell differentiation and differs between various somatic cell types. These changes in nuclear architecture are associated with the regulation of gene expression and genome function in a cell-type specific manner. Biophysical stimuli are known effectors of differentiation and also elicit stimuli-specific changes in nuclear architecture. This occurs via the process of mechanotransduction whereby extracellular mechanical forces activate several well characterized signaling cascades of cytoplasmic origin, and potentially some recently elucidated signaling cascades originating in the nucleus. Recent work has demonstrated changes in nuclear mechanics both with pluripotency state in embryonic stem cells, and with differentiation progression in adult mesenchymal stem cells. This review explores the interplay between cytoplasmic and nuclear mechanosensitivity, highlighting a role for the nucleus as a rheostat in tuning the cellular mechano-response. PMID:28152338

Dynamic regulation of nuclear architecture and mechanics-a rheostatic role for the nucleus in tailoring cellular mechanosensitivity.

PubMed

Thorpe, Stephen D; Lee, David A

2017-05-04

Nuclear architecture, a function of both chromatin and nucleoskeleton structure, is known to change with stem cell differentiation and differs between various somatic cell types. These changes in nuclear architecture are associated with the regulation of gene expression and genome function in a cell-type specific manner. Biophysical stimuli are known effectors of differentiation and also elicit stimuli-specific changes in nuclear architecture. This occurs via the process of mechanotransduction whereby extracellular mechanical forces activate several well characterized signaling cascades of cytoplasmic origin, and potentially some recently elucidated signaling cascades originating in the nucleus. Recent work has demonstrated changes in nuclear mechanics both with pluripotency state in embryonic stem cells, and with differentiation progression in adult mesenchymal stem cells. This review explores the interplay between cytoplasmic and nuclear mechanosensitivity, highlighting a role for the nucleus as a rheostat in tuning the cellular mechano-response.

Hepatocyte nuclear factor-4alpha induces transdifferentiation of hematopoietic cells into hepatocytes.

PubMed

Khurana, Satish; Jaiswal, Amit K; Mukhopadhyay, Asok

2010-02-12

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Restoration of promyelocytic leukemia protein-nuclear bodies in neuroblastoma cells enhances retinoic acid responsiveness.

PubMed

Yu, Jiang Hong; Nakajima, Ayako; Nakajima, Hiroshi; Diller, Lisa R; Bloch, Kenneth D; Bloch, Donald B

2004-02-01

Neuroblastoma is the most common solid tumor of infancy and is believed to result from impaired differentiation of neuronal crest embryonal cells. The promyelocytic leukemia protein (PML)-nuclear body is a cellular structure that is disrupted during the pathogenesis of acute promyelocytic leukemia, a disease characterized by impaired myeloid cell differentiation. During the course of studies to examine the composition and function of PML-nuclear bodies, we observed that the human neuroblastoma cell line SH-SY5Y lacked these structures and that the absence of PML-nuclear bodies was a feature of N- and I-type, but not S-type, neuroblastoma cell lines. Induction of neuroblastoma cell differentiation with 5-bromo-2'deoxyuridine, all-trans-retinoic acid, or IFN-gamma induced PML-nuclear body formation. PML-nuclear bodies were not detected in tissue sections prepared from undifferentiated neuroblastomas but were present in neuroblasts in differentiating tumors. Expression of PML in neuroblastoma cells restored PML-nuclear bodies, enhanced responsiveness to all-trans-retinoic acid, and induced cellular differentiation. Pharmacological therapies that increase PML expression may prove to be important components of combined modalities for the treatment of neuroblastoma.

In Vitro Hepatic Trans-Differentiation of Human Mesenchymal Stem Cells Using Sera from Congestive/Ischemic Liver during Cardiac Failure

PubMed Central

Bishi, Dillip Kumar; Mathapati, Santosh; Cherian, Kotturathu Mammen; Guhathakurta, Soma; Verma, Rama Shanker

2014-01-01

Cellular therapy for end-stage liver failures using human mesenchymal stem cells (hMSCs)-derived hepatocytes is a potential alternative to liver transplantation. Hepatic trans-differentiation of hMSCs is routinely accomplished by induction with commercially available recombinant growth factors, which is of limited clinical applications. In the present study, we have evaluated the potential of sera from cardiac-failure-associated congestive/ischemic liver patients for hepatic trans-differentiation of hMSCs. Results from such experiments were confirmed through morphological changes and expression of hepatocyte-specific markers at molecular and cellular level. Furthermore, the process of mesenchymal-to-epithelial transition during hepatic trans-differentiation of hMSCs was confirmed by elevated expression of E-Cadherin and down-regulation of Snail. The functionality of hMSCs-derived hepatocytes was validated by various liver function tests such as albumin synthesis, urea release, glycogen accumulation and presence of a drug inducible cytochrome P450 system. Based on these findings, we conclude that sera from congestive/ischemic liver during cardiac failure support a liver specific microenvironment for effective hepatic trans-differentiation of hMSCs in vitro. PMID:24642599

The altered expression of perineuronal net elements during neural differentiation.

PubMed

Eskici, Nazli F; Erdem-Ozdamar, Sevim; Dayangac-Erden, Didem

2018-01-01

Perineuronal nets (PNNs), which are localized around neurons during development, are specialized forms of neural extracellular matrix with neuroprotective and plasticity-regulating roles. Hyaluronan and proteoglycan link protein 1 (HAPLN1), tenascin-R (TNR) and aggrecan (ACAN) are key elements of PNNs. In diseases characterized by neuritogenesis defects, the expression of these proteins is known to be downregulated, suggesting that PNNs may have a role in neural differentiation. In this study, the mRNA and protein levels of HAPLN1, TNR and ACAN were determined and compared at specific time points of neural differentiation. We used PC12 cells as the in vitro model because they reflect this developmental process. On day 7, the HAPLN1 mRNA level showed a 2.9-fold increase compared to the non-differentiated state. However, the cellular HAPLN1 protein level showed a decrease, indicating that the protein may have roles in neural differentiation, and may be secreted during the early period of differentiation. By contrast, TNR mRNA and protein levels remained unchanged, and the amount of cellular ACAN protein showed a 3.7-fold increase at day 7. These results suggest that ACAN may be secreted after day 7, possibly due to its large amount of post-translational modifications. Our results provide preliminary data on the expression of PNN elements during neural differentiation. Further investigations will be performed on the role of these elements in neurological disease models.

Synergetic effect of topological cue and periodic mechanical tension-stress on osteogenic differentiation of rat bone mesenchymal stem cells.

PubMed

Liu, Yao; Yang, Guang; Ji, Huanzhong; Xiang, Tao; Luo, En; Zhou, Shaobing

2017-06-01

Mesenchymal stem cells (MSCs) are able to self-renew and differentiate into tissues of mesenchymal origin, making them to be significant for cell-based therapies, such as metabolic bone diseases and bone repair. Regulating the differentiation of MSCs is significant for bone regeneration. Electrospun fibers mimicking natural extracellular matrix (ECM), is an effective artificial ECM to regulate the behaviors and fates of MSCs. The aligned electrospun fibers can modulate polar cell pattern of bone mesenchymal stem cells, which leads to more obvious osteogenic differentiation. Apart from the topographic effect of electrospun fibers, mechanical cues can also intervene the cell behaviors. In this study, the osteogenic differentiation of rat bone mesenchymal stem cells was evaluated, which were cultured on aligned/random electrospun fiber mats materials under mechanical tension intervention. Scanning electron microscope and immune-fluorescent staining were used to directly observe the polarity changing of cellular morphology and cytoskeleton. The results proved that aligned electrospun fibers could be more conducive to promote osteogenic differentiation of rat bone mesenchymal stem cells and this promotion of osteogenic differentiation was enhanced by tension intervention. These results were correlated to the quantitative real-time PCR assay. In general, culturing rat bone mesenchymal stem cells on electrospun fibers under the intervention of mechanical tension is an effective way to mimic a more real cellular microenvironment. Copyright © 2017 Elsevier B.V. All rights reserved.

Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

PubMed

Tang, Bolin; Zhang, Bo; Zhuang, Junjun; Wang, Qi; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

2018-07-01

Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding state of integrin α5β1 with fibronectin induces effective activation of integrin-mediated FAK/ERK signaling pathway to upregulate cellular osteogenic differentiation. This work provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components

PubMed Central

García-Cruz, Karla V.; García-Ponce, Berenice; Garay-Arroyo, Adriana; Sanchez, María De La Paz; Ugartechea-Chirino, Yamel; Desvoyes, Bénédicte; Pacheco-Escobedo, Mario A.; Tapia-López, Rosalinda; Ransom-Rodríguez, Ivan; Gutierrez, Crisanto; Alvarez-Buylla, Elena R.

2016-01-01

Background Morphogenesis depends on the concerted modulation of cell proliferation and differentiation. Such modulation is dynamically adjusted in response to various external and internal signals via complex transcriptional regulatory networks that mediate between such signals and regulation of cell-cycle and cellular responses (proliferation, growth, differentiation). In plants, which are sessile, the proliferation/differentiation balance is plastically adjusted during their life cycle and transcriptional networks are important in this process. MADS-box genes are key developmental regulators in eukaryotes, but their role in cell proliferation and differentiation modulation in plants remains poorly studied. Methods We characterize the XAL1 loss-of-function xal1-2 allele and overexpression lines using quantitative cellular and cytometry analyses to explore its role in cell cycle, proliferation, stem-cell patterning and transition to differentiation. We used quantitative PCR and cellular markers to explore if XAL1 regulates cell-cycle components and PLETHORA1 (PLT1) gene expression, as well as confocal microscopy to analyse stem-cell niche organization. Key Results We previously showed that XAANTAL1 (XAL1/AGL12) is necessary for Arabidopsis root development as a promoter of cell proliferation in the root apical meristem. Here, we demonstrate that XAL1 positively regulates the expression of PLT1 and important components of the cell cycle: CYCD3;1, CYCA2;3, CYCB1;1, CDKB1;1 and CDT1a. In addition, we show that xal1-2 mutant plants have a premature transition to differentiation with root hairs appearing closer to the root tip, while endoreplication in these plants is partially compromised. Coincidently, the final size of cortex cells in the mutant is shorter than wild-type cells. Finally, XAL1 overexpression-lines corroborate that this transcription factor is able to promote cell proliferation at the stem-cell niche. Conclusion XAL1 seems to be an important component of the networks that modulate cell proliferation/differentiation transition and stem-cell proliferation during Arabidopsis root development; it also regulates several cell-cycle components. PMID:27474508

How and where to build a root hair.

PubMed

Dolan, L

2001-12-01

The root hair of Arabidopsis has become a model system for investigations of the patterning and morphogenesis of cells in plants. A cascade of transcriptional regulators controls the pattern of cellular differentiation. Recently, one of the genes that plays a specific role in cellular differentiation in roots, WEREWOLF, has been shown to be functionally equivalent to GLABRA1, which functions only in the shoot. The cloning of genes defined by mutants with defective root-hair growth has provided insights into the roles of the cell wall, ion transport and the cytoskeleton during hair growth. Genetic analyses continue to identify mutants that will be instructive in furthering our understanding of the growth and development of root-hair cells.

The developmental origin of brain tumours: a cellular and molecular framework.

PubMed

Azzarelli, Roberta; Simons, Benjamin D; Philpott, Anna

2018-05-14

The development of the nervous system relies on the coordinated regulation of stem cell self-renewal and differentiation. The discovery that brain tumours contain a subpopulation of cells with stem/progenitor characteristics that are capable of sustaining tumour growth has emphasized the importance of understanding the cellular dynamics and the molecular pathways regulating neural stem cell behaviour. By focusing on recent work on glioma and medulloblastoma, we review how lineage tracing contributed to dissecting the embryonic origin of brain tumours and how lineage-specific mechanisms that regulate stem cell behaviour in the embryo may be subverted in cancer to achieve uncontrolled proliferation and suppression of differentiation. © 2018. Published by The Company of Biologists Ltd.

Furanodictine A and B: amino sugar analogues produced by cellular slime mold Dictyostelium discoideum showing neuronal differentiation activity.

PubMed

Kikuchi, H; Saito, Y; Komiya, J; Takaya, Y; Honma, S; Nakahata, N; Ito, A; Oshima, Y

2001-10-19

We investigated the constituents of Dictyostelium discoideum to clarify the diversity of secondary metabolites of Dictyostelium cellular slime molds and to explore biologically active substances that could be useful in the development of novel drugs. From a methanol extract of the multicellular fruit body of D. discoideum, we isolated two novel amino sugar analogues, furanodictine A (1) and B (2). They are the first 3,6-anhydrosugars to be isolated from natural sources. Their relative structures were elucidated by spectral means, and the absolute configurations were confirmed by asymmetric syntheses of 1 and 2. These furanodictines potently induce neuronal differentiation of rat pheochromocytoma (PC-12) cells.

Cellular Factors Targeting APCs to Modulate Adaptive T Cell Immunity

PubMed Central

Do, Jeongsu; Min, Booki

2014-01-01

The fate of adaptive T cell immunity is determined by multiple cellular and molecular factors, among which the cytokine milieu plays the most important role in this process. Depending on the cytokines present during the initial T cell activation, T cells become effector cells that produce different effector molecules and execute adaptive immune functions. Studies thus far have primarily focused on defining how these factors control T cell differentiation by targeting T cells themselves. However, other non-T cells, particularly APCs, also express receptors for the factors and are capable of responding to them. In this review, we will discuss how APCs, by responding to those cytokines, influence T cell differentiation and adaptive immunity. PMID:25126585

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules.

PubMed

Kramer, Jan; Steinhoff, Jürgen; Klinger, Matthias; Fricke, Lutz; Rohwedel, Jürgen

2006-03-01

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.

In vitro hepatic differentiation of human endometrial stromal stem cells.

PubMed

Yang, Xin-yuan; Wang, Wei; Li, Xu

2014-02-01

Human endometrial stromal stem cells (hESSCs) can differentiate into mesodermal and ectodermal cellular lineages in the endometrium. However, whether hESSCs can differentiate into functional hepatic-like cells is unknown. In this study, we developed a multiple-step induction protocol to differentiate hESSCs into functional hepatic-like cells in vitro. Endometrial stromal cells were isolated by magnetic affinity sorting using anti-epithelial cell adhesion molecule-coated Dynabeads. The enriched hESSCs were analyzed by flow cytometry and were able to differentiate into osteoblasts or adipocytes under proper induction media. To differentiate into hepatic-like cells, hESSCs were cultured in a stepwise system containing hepatocyte growth factor, fibroblast growth factor-4, oncostatin M, and trichostatin A for a total of 24 d. The hepatic-like cell differentiation was analyzed by confocal microscopy and immunocytochemical staining. Glycogen storage, cellular urea synthesis, and ammonia concentrations were measured. Hepatic-like cells were successfully generated from hESSCs and were identified by their epithelial-like shape characteristics and expression of specific biomarkers albumin and cytokeratin 8 accompanied with a reduction of alpha-fetoprotein and alpha-smooth muscle actin expression. The hepatic-like cells generated were functional as evidenced by urea synthesis and glycogen storage. Our study demonstrated that hESSCs were able to differentiate into hepatic-like cells in vitro. Thus, endometrial stromal cells may be used as an easily accessible alternative source of stem cells for potential therapeutic applications in liver disease.

Effects of cellular origin on differentiation of human induced pluripotent stem cell–derived endothelial cells

PubMed Central

Zhao, Ming-Tao; Jahanbani, Fereshteh; Lee, Won Hee; Snyder, Michael P.

2016-01-01

Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. PMID:27398408

Dickkopf-3 maintains the PANC-1 human pancreatic tumor cells in a dedifferentiated state.

PubMed

Zenzmaier, Christoph; Hermann, Martin; Hengster, Paul; Berger, Peter

2012-01-01

Pancreatic cancer (PaCa) is the fourth leading cause of cancer deaths in Western societies, with pancreatic ductal adenocarcinomas (PDACs) accounting for >90% of such cases. PDAC is a heterogeneous disease that includes a subset showing overexpression of the secreted glycoprotein Dickkopf-related protein 3 (Dkk-3), a protein shown to be downregulated in various cancers of different tissues. The biological function of Dkk-3 in this subset was studied using the Dkk-3 expressing PANC-1 cell line as a model for PDACs. The influence of Dkk-3 overexpression and knockdown on cellular differentiation and proliferation of PANC-1 was investigated. Confocal microscopy showed that Dkk-3 was expressed in a fraction of PANC-1 cells. While lentiviral-mediated overexpression of DKK3 did not alter cellular proliferation, knockdown of DKK3 resulted in significant reduction of cellular proliferation and concomitant induction of cell cycle inhibitors CDKN2B (p15INK4b), CDKN1A (p21CIP1) and CDKN1B (p27KIP1). In parallel, pancreatic epithelial cell differentiation markers AMY2A, CELA1, CTRB1, GCG, GLB1 and INS were significantly upregulated. PANC-1 cells differentiated using exendin-4 showed analogous induction of cell cycle inhibitors and differentiation markers. Thus, we conclude that Dkk-3 is required to maintain a highly dedifferentiated and consequently proliferative state in PANC-1, indicating a similar function in the Dkk-3 overexpressing subset of PDACs. Therefore, Dkk-3 represents a potential target for the treatment of Dkk-3-positive subtypes of PaCa to drive cells into cell cycle arrest and differentiation.

Cellular Location and Expression of Na+, K+-ATPase α Subunits Affect the Anti-Proliferative Activity of Oleandrin

PubMed Central

Yang, Peiying; Cartwright, Carrie; Efuet, Ekem; Hamilton, Stanley R.; Wistuba, Ignacio Ivan; Menter, David; Addington, Crandell; Shureiqi, Imad; Newman, Robert A.

2015-01-01

The purpose of this study was to investigate whether intracellular distribution of Na+, K+-ATPase α3 subunit, a receptor for cardiac glycosides including oleandrin, is differentially altered in cancer versus normal cells and whether this altered distribution can be therapeutically targeted to inhibit cancer cell survival. The cellular distribution of Na+, K+-ATPase α3 isoform was investigated in paired normal and cancerous mucosa biopsy samples from patients with lung and colorectal cancers by immunohistochemical staining. The effects of oleandrin on α3 subunit intracellular distribution, cell death, proliferation, and EKR phosphorylation were examined in differentiated and undifferentiated human colon cancer CaCO-2 cells. While Na+, K+-ATPase α3 isoform was predominantly located near the cytoplasmic membrane in normal human colon and lung epithelia, the expression of this subunit in their paired cancer epithelia was shifted to a peri-nuclear position in both a qualitative and quantitative manner. Similarly, distribution of α3 isoform was also shifted from a cytoplasmic membrane location in differentiated human colon cancer CaCO-2 cells to a peri-nuclear position in undifferentiated CaCO-2 cells. Intriguingly, oleandrin exerted threefold stronger anti-proliferative activity in undifferentiated CaCO-2 cells (IC50, 8.25 nM) than in differentiated CaCO-2 cells (IC50, >25 nM). Oleandrin (10 to 20 nM) caused an autophagic cell death and altered ERK phosphorylation in undifferentiated but not in differentiated CaCO-2 cells. These data demonstrate that the intracellular location of Na+, K+-ATPase α3 isoform is altered in human cancer versus normal cells. These changes in α3 cellular location and abundance may indicate a potential target of opportunity for cancer therapy. PMID:23073998

The Role of Auxiliary Subunits for the Functional Diversity of Voltage-Gated Calcium Channels

PubMed Central

Campiglio, Marta; Flucher, Bernhard E

2015-01-01

Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc. PMID:25820299

Cell-Adhesive Matrices Composed of RGD Peptide-Displaying M13 Bacteriophage/Poly(lactic-co-glycolic acid) Nanofibers Beneficial to Myoblast Differentiation.

PubMed

Shin, Yong Cheol; Lee, Jong Ho; Jin, Linhua; Kim, Min Jeong; Kim, Chuntae; Hong, Suck Won; Oh, Jin Woo; Han, Dong-Wook

2015-10-01

Recently, there has been considerable effort to develop suitable scaffolds for tissue engineering applications. Cell adhesion is a prerequisite for cells to survive. In nature, the extracellular matrix (ECM) plays this role. Therefore, an ideal scaffold should be structurally similar to the natural ECM and have biocompatibility and biodegradability. In addition, the scaffold should have biofunctionality, which provides the potent ability to enhance the cellular behaviors, such as adhesion, proliferation and differentiation. This study concentrates on fabricating cell-adhesive matrices composed of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) nanofibers. Long rod-shaped M13 bacteriophages are non-toxic and can express many desired proteins on their surface. A genetically engineered M13 phage was constructed to display RGD peptides on its surface. PLGA is a biodegradable polymer with excellent biocompatibility and suitable physicochemical property for adhesive matrices. In this study, RGD-M13 phage/PLGA hybrid nanofiber matrices were fabricated by electrospinning. The physicochemical properties of these matrices were characterized by scanning electron microscopy, atomic force microscopy, Raman spectroscopy, and contact angle measurement. In addition, the cellular behaviors, such as the initial attachment, proliferation and differentiation, were analyzed by a CCK-8 assay and immunofluorescence staining to evaluate the potential application of these matrices to tissue engineering scaffolds. The RGD-M13 phage/PLGA nanofiber matrices could enhance the cellular behaviors and promote the differentiation of C2C12 myoblasts. These results suggest that the RGD-M13 phage/PLGA nanofiber matrices are beneficial to myoblast differentiation and can serve as effective tissue engineering scaffolds.

An introduction to the molecular basics of aryl hydrocarbon receptor biology.

PubMed

Abel, Josef; Haarmann-Stemmann, Thomas

2010-11-01

Depending on their chemical structure and properties, environmental chemicals and other xenobiotics that enter the cell can affect cellular function by either nonselective binding to cellular macromolecules or by interference with cellular receptors, which would initiate a more defined cell biological response. One of these intracellular chemosensor molecules is the aryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family that is known to mediate the biochemical and toxic effects of dioxins, polyaromatic hydrocarbons and related compounds. Numerous investigations have revealed that the AhR is not only a master regulator of drug metabolism activated by anthropogenic chemicals, but is also triggered by natural and endogenous ligands and can influence cell biological endpoints such as growth and differentiation. Cutting-edge research has identified new intriguing functions of the AhR, such as during proteasomal degradation of steroid hormone receptors, the cellular UVB stress response and the differentiation of certain T-cell subsets. In this review we provide both a survey of the fundamental basics of AhR biology and an insight into new functional aspects of AhR signaling to further stimulate research on this intriguing transcription factor at the interface between toxicology, cell biology and immunology.

Differanisole A, an inducer of the differentiation of Friend leukemic cells, induces stalk cell differentiation in Dictyostelium discoideum.

PubMed

Kubohara, Y; Okamoto, K; Tanaka, Y; Asahi, K; Sakurai, A; Takahashi, N

1993-05-03

Differanisole A isolated from the conditioned medium of a soil microorganism, Chaetomium strain RB-001, is an inducer of the differentiation of the Friend leukemic cells (mouse leukemia cells). The chemical structure of this substance is very similar to that of stalk cell differentiation-inducing factor (DIF) isolated from the cellular slime mould, Dictyostelium discoideum. We examined the effects of differanisole A on Dictyostelium HM44 cells, a mutant strain which is defective in DIF production, and found this substance to be an inducer of stalk cell differentiation in D. discoideum.

Differentiation of human SH-SY5Y neuroblastoma cells by all-trans retinoic acid activates the interleukin-18 system.

PubMed

Sallmon, Hannes; Hoene, Victoria; Weber, Sven C; Dame, Christof

2010-02-01

The clinical prognosis of children with high-stage neuroblastoma is still poor. Therapeutic approaches include surgery and cellular differentiation by retinoic acid, but also experimental interleukin-based immune modulation. However, the molecular mechanisms of all-trans retinoic acid (ATRA)-induced differentiation of neuroblastoma cells are incompletely understood. Herein, we examined the effect of ATRA on the activity of the interleukin-18 (IL-18) system in human SH-SY5Y neuroblastoma cells. It is shown that SH-SY5Y cells express IL-18 receptor (IL-18R) and the secreted antagonist IL-18-binding protein (IL-18BP), but no IL-18. SH-SY5Y cells are highly sensitive to ATRA treatment and react by cellular differentiation from a neuroblastic toward a more neuronal phenotype. This was associated with induction of IL-18 and reduction of IL-18BP expression, while IL-18R expression remained stable. Thereby, we identified the IL-18 system as a novel target of ATRA in neuroblastoma cells that might contribute to the therapeutic properties of retinoids in treatment of neuroblastoma.

Optical quantification of forces at play during stem cell differentiation

NASA Astrophysics Data System (ADS)

Ritter, Christine M.; Brickman, Joshua M.; Oddershede, Lene B.

2016-03-01

A cell is in constant interaction with its environment, it responds to external mechanical, chemical and biological signals. The response to these signals can be of various nature, for instance intra-cellular mechanical re-arrangements, cell-cell interactions, or cellular reinforcements. Optical methods are quite attractive for investigating the mechanics inside living cells as, e.g., optical traps are amongst the only nanotools that can reach and manipulate, measure forces, inside a living cell. In the recent years it has become increasingly evident that not only biochemical and biomolecular cues, but also that mechanical ones, play an important roles in stem cell differentiation. The first evidence for the importance of mechanical cues emerged from studies showing that substrate stiffness had an impact on stem cell differentiation. Recently, techniques such as optical tweezers and stretchers have been applied to stem cells, producing new insights into the role of mechanics in regulating renewal and differentiation. Here, we describe how optical tweezers and optical stretchers can be applied as a tool to investigate stem cell mechanics and some of the recent results to come out of this work.

Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

NASA Technical Reports Server (NTRS)

Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

2000-01-01

To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

Glucose metabolism regulates T cell activation, differentiation, and functions.

PubMed

Palmer, Clovis S; Ostrowski, Matias; Balderson, Brad; Christian, Nicole; Crowe, Suzanne M

2015-01-01

The adaptive immune system is equipped to eliminate both tumors and pathogenic microorganisms. It requires a series of complex and coordinated signals to drive the activation, proliferation, and differentiation of appropriate T cell subsets. It is now established that changes in cellular activation are coupled to profound changes in cellular metabolism. In addition, emerging evidence now suggest that specific metabolic alterations associated with distinct T cell subsets may be ancillary to their differentiation and influential in their immune functions. The "Warburg effect" originally used to describe a phenomenon in which most cancer cells relied on aerobic glycolysis for their growth is a key process that sustain T cell activation and differentiation. Here, we review how different aspects of metabolism in T cells influence their functions, focusing on the emerging role of key regulators of glucose metabolism such as HIF-1α. A thorough understanding of the role of metabolism in T cell function could provide insights into mechanisms involved in inflammatory-mediated conditions, with the potential for developing novel therapeutic approaches to treat these diseases.

Intestinal development and differentiation

PubMed Central

Noah, Taeko K.; Donahue, Bridgitte; Shroyer, Noah F.

2011-01-01

In this review, we present an overview of intestinal development and cellular differentiation of the intestinal epithelium. The review is separated into two sections: Section one summarizes organogenesis of the small and large intestines, including endoderm and gut tube formation in early embryogenesis, villus morphogenesis, and crypt formation. Section two reviews cell fate specification and differentiation of each cell type within the intestinal epithelium. Growth factor and transcriptional networks that regulate these developmental processes are summarized. PMID:21978911

Decreased expression of the stress protein HSP70 is an early event in murine erythroleukemic cell differentiation.

PubMed Central

Hensold, J O; Housman, D E

1988-01-01

Two-dimensional protein gels were used to systematically assess changes in gene expression in Friend erythroleukemia cells after exposure to inducers of differentiation. A rapid decrease in expression of the stress protein HSP70 was observed after exposure to inducers. The kinetics of this change suggest that it may be related to the cellular events that regulate the onset of differentiation. Images PMID:3164440

The TCP4 transcription factor regulates trichome cell differentiation by directly activating GLABROUS INFLORESCENCE STEMS in Arabidopsis thaliana.

PubMed

Vadde, Batthula Vijaya Lakshmi; Challa, Krishna Reddy; Nath, Utpal

2018-01-01

Trichomes are the first cell type to be differentiated during the morphogenesis of leaf epidermis and serve as an ideal model to study cellular differentiation. Many genes involved in the patterning and differentiation of trichome cells have been studied over the past decades, and the majority of these genes encode transcription factors that specifically regulate epidermal cell development. However, the upstream regulators of these genes that link early leaf morphogenesis with cell type differentiation are less studied. The TCP proteins are the plant-specific transcription factors involved in regulating diverse aspects of plant development including lateral organ morphogenesis by modulating cell proliferation and differentiation. Here, we show that the miR319-regulated class II TCP proteins, notably TCP4, suppress trichome branching in Arabidopsis leaves and inflorescence stem by direct transcriptional activation of GLABROUS INFLORESCENCE STEMS (GIS), a known negative regulator of trichome branching. The trichome branch number is increased in plants with reduced TCP activity and decreased in the gain-of-function lines of TCP4. Biochemical analyses show that TCP4 binds to the upstream regulatory region of GIS and activates its expression. Detailed genetic analyses show that GIS and TCP4 work in same pathway and GIS function is required for TCP4-mediated regulation of trichome differentiation. Taken together, these results identify a role for the class II TCP genes in trichome differentiation, thus providing a connection between organ morphogenesis and cellular differentiation. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Evolution of Microbial Quorum Sensing to Human Global Quorum Sensing: An Insight into How Gap Junctional Intercellular Communication Might Be Linked to the Global Metabolic Disease Crisis

PubMed Central

Trosko, James E.

2016-01-01

The first anaerobic organism extracted energy for survival and reproduction from its source of nutrients, with the genetic means to ensure protection of its individual genome but also its species survival. While it had a means to communicate with its community via simple secreted molecules (“quorum sensing”), the eventual shift to an aerobic environment led to multi-cellular metazoan organisms, with evolutionary-selected genes to form extracellular matrices, stem cells, stem cell niches, and a family of gap junction or “connexin” genes. These germinal and somatic stem cells responded to extracellular signals that triggered intra-cellular signaling to regulate specific genes out of the total genome. These extra-cellular induced intra-cellular signals also modulated gap junctional intercellular communication (GJIC) in order to regulate the new cellular functions of symmetrical and asymmetrical cell division, cell differentiation, modes of cell death, and senescence. Within the hierarchical and cybernetic concepts, differentiated by neurons organized in the brain of the Homo sapiens, the conscious mind led to language, abstract ideas, technology, myth-making, scientific reasoning, and moral decision–making, i.e., the creation of culture. Over thousands of years, this has created the current collision between biological and cultural evolution, leading to the global “metabolic disease” crisis. PMID:27314399

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

The Calcium-Sensing Receptor and Integrins in Cellular Differentiation and Migration

PubMed Central

Tharmalingam, Sujeenthar; Hampson, David R.

2016-01-01

The calcium-sensing receptor (CaSR) is a widely expressed homodimeric G-protein coupled receptor structurally related to the metabotropic glutamate receptors and GPRC6A. In addition to its well characterized role in maintaining calcium homeostasis and regulating parathyroid hormone release, evidence has accumulated linking the CaSR with cellular differentiation and migration, brain development, stem cell engraftment, wound healing, and tumor growth and metastasis. Elevated expression of the CaSR in aggressive metastatic tumors has been suggested as a potential novel prognostic marker for predicting metastasis, especially to bone tissue where extracellular calcium concentrations may be sufficiently high to activate the receptor. Recent evidence supports a model whereby CaSR-mediated activation of integrins promotes cellular migration. Integrins are single transmembrane spanning heterodimeric adhesion receptors that mediate cell migration by binding to extracellular matrix proteins. The CaSR has been shown to form signaling complexes with the integrins to facilitate both the movement and differentiation of cells, such as neurons during normal brain development and tumor cells under pathological circumstances. Thus, CaSR/integrin complexes may function as a universal cell migration or homing complex. Manipulation of this complex may be of potential interest for treating metastatic cancers, and for developmental disorders pertaining to aberrant neuronal migration. PMID:27303307

Mesenchymal cell differentiation and diseases: involvement of translin/TRAX complexes and associated proteins.

PubMed

Kasai, Masataka; Ishida, Reiko; Nakahara, Kazuhiko; Okumura, Ko; Aoki, Katsunori

2018-05-08

Translin and translin-associated factor X (translin/TRAX) proteins have been implicated in a variety of cellular activities central to nucleic acid metabolism. Accumulating evidence indicates that translin/TRAX complexes participate in processes ensuring the replication of DNA, as well as cell division. Significant progress has been made in understanding the roles of translin/TRAX complexes in RNA metabolism, such as through RNA-induced silencing complex activation or the microRNA depletion that occurs in Dicer deficiency. At the cellular level, translin-deficient (Tsn -/- ) mice display delayed endochondral ossification or progressive bone marrow failure with ectopic osteogenesis and adipogenesis, suggesting involvement in mesenchymal cell differentiation. In this review, we summarize the molecular and cellular functions of translin homo-octamer and translin/TRAX hetero-octamer. Finally, we discuss the multifaceted roles of translin, TRAX, and associated proteins in the healthy and disease states. © 2018 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals, Inc. on behalf of The New York Academy of Sciences.

Degradation-mediated cellular traction directs stem cell fate in covalently crosslinked three-dimensional hydrogels

NASA Astrophysics Data System (ADS)

Khetan, Sudhir; Guvendiren, Murat; Legant, Wesley R.; Cohen, Daniel M.; Chen, Christopher S.; Burdick, Jason A.

2013-05-01

Although cell-matrix adhesive interactions are known to regulate stem cell differentiation, the underlying mechanisms, in particular for direct three-dimensional encapsulation within hydrogels, are poorly understood. Here, we demonstrate that in covalently crosslinked hyaluronic acid (HA) hydrogels, the differentiation of human mesenchymal stem cells (hMSCs) is directed by the generation of degradation-mediated cellular traction, independently of cell morphology or matrix mechanics. hMSCs within HA hydrogels of equivalent elastic moduli that permit (restrict) cell-mediated degradation exhibited high (low) degrees of cell spreading and high (low) tractions, and favoured osteogenesis (adipogenesis). Moreover, switching the permissive hydrogel to a restrictive state through delayed secondary crosslinking reduced further hydrogel degradation, suppressed traction, and caused a switch from osteogenesis to adipogenesis in the absence of changes to the extended cellular morphology. Furthermore, inhibiting tension-mediated signalling in the permissive environment mirrored the effects of delayed secondary crosslinking, whereas upregulating tension induced osteogenesis even in the restrictive environment.

Metabolic regulation of inflammation.

PubMed

Gaber, Timo; Strehl, Cindy; Buttgereit, Frank

2017-05-01

Immune cells constantly patrol the body via the bloodstream and migrate into multiple tissues where they face variable and sometimes demanding environmental conditions. Nutrient and oxygen availability can vary during homeostasis, and especially during the course of an immune response, creating a demand for immune cells that are highly metabolically dynamic. As an evolutionary response, immune cells have developed different metabolic programmes to supply them with cellular energy and biomolecules, enabling them to cope with changing and challenging metabolic conditions. In the past 5 years, it has become clear that cellular metabolism affects immune cell function and differentiation, and that disease-specific metabolic configurations might provide an explanation for the dysfunctional immune responses seen in rheumatic diseases. This Review outlines the metabolic challenges faced by immune cells in states of homeostasis and inflammation, as well as the variety of metabolic configurations utilized by immune cells during differentiation and activation. Changes in cellular metabolism that contribute towards the dysfunctional immune responses seen in rheumatic diseases are also briefly discussed.

Operating principles of tristable circuits regulating cellular differentiation

NASA Astrophysics Data System (ADS)

Jia, Dongya; Jolly, Mohit Kumar; Harrison, William; Boareto, Marcelo; Ben-Jacob, Eshel; Levine, Herbert

2017-06-01

Many cell-fate decisions during embryonic development are governed by a motif comprised of two transcription factors (TFs) A and B that mutually inhibit each other and may self-activate. This motif, called as a self-activating toggle switch (SATS), can typically have three stable states (phenotypes)—two corresponding to differentiated cell fates, each of which has a much higher level of one TF than the other—≤ft(A,~B\\right)=≤ft(1,~0\\right) or ≤ft(0,~1\\right) —and the third state corresponding to an ‘undecided’ stem-like state with similar levels of both A and B—≤ft(A,~B\\right)=≤ft(1/2,1/2\\right) . Furthermore, two or more SATSes can be coupled together in various topologies in different contexts, thereby affecting the coordination between multiple cellular decisions. However, two questions remain largely unanswered: (a) what governs the co-existence and relative stability of these three stable states? (b) What orchestrates the decision-making of coupled SATSes? Here, we first demonstrate that the co-existence and relative stability of the three stable states in an individual SATS can be governed by the relative strength of self-activation, external signals activating and/or inhibiting A and B, and mutual degradation between A and B. Simultaneously, we investigate the effects of these factors on the decision-making of two coupled SATSes. Our results offer novel understanding into the operating principles of individual and coupled tristable self-activating toggle switches (SATSes) regulating cellular differentiation and can yield insights into synthesizing three-way genetic circuits and understanding of cellular reprogramming.

Discrimination of Dysplastic Nevi from Common Melanocytic Nevi by Cellular and Molecular Criteria.

PubMed

Mitsui, Hiroshi; Kiecker, Felix; Shemer, Avner; Cannizzaro, Maria Vittoria; Wang, Claire Q F; Gulati, Nicholas; Ohmatsu, Hanako; Shah, Kejal R; Gilleaudeau, Patricia; Sullivan-Whalen, Mary; Cueto, Inna; McNutt, Neil Scott; Suárez-Fariñas, Mayte; Krueger, James G

2016-10-01

Dysplastic nevi (DNs), also known as Clark's nevi or atypical moles, are distinguished from common melanocytic nevi by variegation in pigmentation and clinical appearance, as well as differences in tissue patterning. However, cellular and molecular differences between DNs and common melanocytic nevi are not completely understood. Using cDNA microarray, quantitative RT-PCR, and immunohistochemistry, we molecularly characterized DNs and analyzed the difference between DNs and common melanocytic nevi. A total of 111 probesets (91 annotated genes, fold change > 2.0 and false discovery rate < 0.25) were differentially expressed between the two lesions. An unexpected finding in DNs was altered differentiation and activation of epidermal keratinocytes with increased expression of hair follicle-related molecules (keratin 25, trichohyalin, ribonuclease, RNase A family, 7) and inflammation-related molecules (S100A7, S100A8) at both genomic and protein levels. The immune microenvironment of DNs was characterized by an increase of T helper type 1 (IFNγ) and T helper type 2 (IL13) cytokines as well as an upregulation of oncostatin M and CXCL1. DUSP3, which regulates cellular senescence, was identified as one of the disease discriminative genes between DNs and common melanocytic nevi by three independent statistical approaches and its altered expression was confirmed by immunohistochemistry. The molecular and cellular changes in which the epidermal-melanin unit undergoes follicular differentiation as well as upregulation of defined cytokines could drive complex immune, epidermal, and pigmentary alterations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Chitosan based hydrogels: characteristics and pharmaceutical applications

PubMed Central

Ahmadi, F.; Oveisi, Z.; Samani, S. Mohammadi; Amoozgar, Z.

2015-01-01

Hydrogel scaffolds serve as semi synthetic or synthetic extra cellular matrix to provide an amenable environment for cellular adherence and cellular remodeling in three dimensional structures mimicking that of natural cellular environment. Additionally, hydrogels have the capacity to carry small molecule drugs and/or proteins, growth factors and other necessary components for cell growth and differentiation. In the context of drug delivery, hydrogels can be utilized to localize drugs, increase drugs concentration at the site of action and consequently reduce off-targeted side effects. The current review aims to describe and classify hydrogels and their methods of production. The main highlight is chitosan-based hydrogels as biocompatible and medically relevant hydrogels for drug delivery. PMID:26430453

Novel chlorinated dibenzofurans isolated from the cellular slime mold, Polysphondylium filamentosum, and their biological activities.

PubMed

Kikuchi, Haruhisa; Kubohara, Yuzuru; Nguyen, Van Hai; Katou, Yasuhiro; Oshima, Yoshiteru

2013-08-01

Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of space relevant radiation exposure on differentiation and mineralization of murine osteoprogenitor cells

NASA Astrophysics Data System (ADS)

Lau, Patrick; Hu, Yueyuan; Hellweg, Christine; Baumstark-Khan, Christa; Reitz, Guenther

Extended exposure to altered gravity conditions like during long-term space flight results in significant bone loss. Exposure to ionizing radiation for cancer therapy causes bone damage and may increase the risk of fractures. Similarly, besides altered gravity conditions, astronauts on exploratory missions beyond low-Earth orbit will be exposed to high-energy heavy ions in addition to proton and photon radiation, although for prolonged periods and at lower doses and dose rates compared with therapy. Space conditions may place astronauts at a greater risk for mission-critical fractures. Until now, little is known about the effects of space radiation on the skeletal system especially on osteoprogenitor cells. Accelerator facilities are used to simulate parts of the radiation environment in space. Heavy ion accelerators therefore could be used to assess radiation risks for astronauts who will be exposed to higher radiation doses e.g. on a Mars mission. The aim of the present study was to determine the biological effects of spaceflight-relevant radiation exposure on the cellular level using murine osteoprogenitor cell lines compared to nonirradiated controls. To gain a deeper understanding of bone cell differenti-ation and mineralization after exposure to heavy ions, we examined gene expression modulation of bone specific transcription factors, osteoblast specific marker genes as well as genes function as coupling factors that link bone resorption to bone formation. We investigated the transcrip-tional modulation of type I collagen (Col I), osteocalcin (Ocn), Transforming growth factor-β1 (TGF-β1), interleukin-6 (IL-6) and the bone specific transcription factor Runx2 (Cbfa1). To gain deeper insight into potential cellular mechanisms involved in cellular response after ex-posure to heavy ions, we investigated gene expression modulations after exposure to energetic carbon ions (35 MeV/u, 73.2 keV/µm), iron ions (1000 MeV/u, 150 keV/µm) and lead ions (29 MeV/u, 9600 keV/µm) versus low LET X-rays. Exposure to X-irradiation dose-dependently increased the mRNA levels of Runx2 (cbfa1) whereas expression values of OCN and TGF-β1 were elevated at later time points. Exposure to heavy ions provoked a more marked effect on bone specific gene expression within the differentiation process. Collectively, our results indi-cate that heavy ions facilitate differentiation more effectively than X-rays as a major response in the progeny of irradiated osteoprogenitor cells. The data presented allow us to suggest that exposure to ionizing radiation interferes with bone formation at the level of cellular differenti-ation. In this regard, further experiments are needed to investigate gene expression patterns in mammalian cells that respond to differentiation after exposure to ionizing radiation.

Gene regulatory and signaling networks exhibit distinct topological distributions of motifs

NASA Astrophysics Data System (ADS)

Ferreira, Gustavo Rodrigues; Nakaya, Helder Imoto; Costa, Luciano da Fontoura

2018-04-01

The biological processes of cellular decision making and differentiation involve a plethora of signaling pathways and gene regulatory circuits. These networks in turn exhibit a multitude of motifs playing crucial parts in regulating network activity. Here we compare the topological placement of motifs in gene regulatory and signaling networks and observe that it suggests different evolutionary strategies in motif distribution for distinct cellular subnetworks.

An essential role for UTX in resolution and activation of bivalent promoters

PubMed Central

Dhar, Shilpa S.; Lee, Sung-Hun; Chen, Kaifu; Zhu, Guangjing; Oh, WonKyung; Allton, Kendra; Gafni, Ohad; Kim, Young Zoon; Tomoiga, Alin S.; Barton, Michelle Craig; Hanna, Jacob H.; Wang, Zhibin; Li, Wei; Lee, Min Gyu

2016-01-01

Trimethylated histone H3 lysine 27 (H3K27me3) is linked to gene silencing, whereas H3K4me3 is associated with gene activation. These two marks frequently co-occupy gene promoters, forming bivalent domains. Bivalency signifies repressed but activatable states of gene expression and can be resolved to active, H3K4me3-prevalent states during multiple cellular processes, including differentiation, development and epithelial mesenchymal transition. However, the molecular mechanism underlying bivalency resolution remains largely unknown. Here, we show that the H3K27 demethylase UTX (also called KDM6A) is required for the resolution and activation of numerous retinoic acid (RA)-inducible bivalent genes during the RA-driven differentiation of mouse embryonic stem cells (ESCs). Notably, UTX loss in mouse ESCs inhibited the RA-driven bivalency resolution and activation of most developmentally critical homeobox (Hox) a–d genes. The UTX-mediated resolution and activation of many bivalent Hox genes during mouse ESC differentiation were recapitulated during RA-driven differentiation of human NT2/D1 embryonal carcinoma cells. In support of the importance of UTX in bivalency resolution, Utx-null mouse ESCs and UTX-depleted NT2/D1 cells displayed defects in RA-driven cellular differentiation. Our results define UTX as a bivalency-resolving histone modifier necessary for stem cell differentiation. PMID:26762983

DNMT1 maintains progenitor function in self-renewing somatic tissue.

PubMed

Sen, George L; Reuter, Jason A; Webster, Daniel E; Zhu, Lilly; Khavari, Paul A

2010-01-28

Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

Sequential Delivery of BMP-2 and IGF-1 Using a Chitosan Gel with gelatin Microspheres Enhances Early osteoblastic Differentiation

DTIC Science & Technology

2012-01-18

Vunjak- Novakovic G, Freed LE. Growth factors for sequential cellular de- and re-differentiation in tissue engineering. Biochem Biophys Res Commun...2002;294(1):149–54. [35] Martin I, Suetterlin R, Baschong W, Heberer M, Vunjak- Novakovic G, Freed LE. Enhanced cartilage tissue engineering by sequential

De novo generation of HSCs from somatic and pluripotent stem cell sources

PubMed Central

Vo, Linda T.

2015-01-01

Generating human hematopoietic stem cells (HSCs) from autologous tissues, when coupled with genome editing technologies, is a promising approach for cellular transplantation therapy and for in vitro disease modeling, drug discovery, and toxicology studies. Human pluripotent stem cells (hPSCs) represent a potentially inexhaustible supply of autologous tissue; however, to date, directed differentiation from hPSCs has yielded hematopoietic cells that lack robust and sustained multilineage potential. Cellular reprogramming technologies represent an alternative platform for the de novo generation of HSCs via direct conversion from heterologous cell types. In this review, we discuss the latest advancements in HSC generation by directed differentiation from hPSCs or direct conversion from somatic cells, and highlight their applications in research and prospects for therapy. PMID:25762177

Efficient generation of hepatic cells from mesenchymal stromal cells by an innovative bio-microfluidic cell culture device.

PubMed

Yen, Meng-Hua; Wu, Yuan-Yi; Liu, Yi-Shiuan; Rimando, Marilyn; Ho, Jennifer Hui-Chun; Lee, Oscar Kuang-Sheng

2016-08-19

Mesenchymal stromal cells (MSCs) are multipotent and have great potential in cell therapy. Previously we reported the differentiation potential of human MSCs into hepatocytes in vitro and that these cells can rescue fulminant hepatic failure. However, the conventional static culture method neither maintains growth factors at an optimal level constantly nor removes cellular waste efficiently. In addition, not only is the duration of differentiating hepatocyte lineage cells from MSCs required to improve, but also the need for a large number of hepatocytes for cell therapy has not to date been addressed fully. The purpose of this study is to design and develop an innovative microfluidic device to overcome these shortcomings. We designed and fabricated a microfluidic device and a culture system for hepatic differentiation of MSCs using our protocol reported previously. The microfluidic device contains a large culture chamber with a stable uniform flow to allow homogeneous distribution and expansion as well as efficient induction of hepatic differentiation for MSCs. The device enables real-time observation under light microscopy and exhibits a better differentiation efficiency for MSCs compared with conventional static culture. MSCs grown in the microfluidic device showed a higher level of hepatocyte marker gene expression under hepatic induction. Functional analysis of hepatic differentiation demonstrated significantly higher urea production in the microfluidic device after 21 days of hepatic differentiation. The microfluidic device allows the generation of a large number of MSCs and induces hepatic differentiation of MSCs efficiently. The device can be adapted for scale-up production of hepatic cells from MSCs for cellular therapy.

ZNF281 inhibits neuronal differentiation and is a prognostic marker for neuroblastoma.

PubMed

Pieraccioli, Marco; Nicolai, Sara; Pitolli, Consuelo; Agostini, Massimiliano; Antonov, Alexey; Malewicz, Michal; Knight, Richard A; Raschellà, Giuseppe; Melino, Gerry

2018-06-25

Derangement of cellular differentiation because of mutation or inappropriate expression of specific genes is a common feature in tumors. Here, we show that the expression of ZNF281, a zinc finger factor involved in several cellular processes, decreases during terminal differentiation of murine cortical neurons and in retinoic acid-induced differentiation of neuroblastoma (NB) cells. The ectopic expression of ZNF281 inhibits the neuronal differentiation of murine cortical neurons and NB cells, whereas its silencing causes the opposite effect. Furthermore, TAp73 inhibits the expression of ZNF281 through miR34a. Conversely, MYCN promotes the expression of ZNF281 at least in part by inhibiting miR34a. These findings imply a functional network that includes p73, MYCN, and ZNF281 in NB cells, where ZNF281 acts by negatively affecting neuronal differentiation. Array analysis of NB cells silenced for ZNF281 expression identified GDNF and NRP2 as two transcriptional targets inhibited by ZNF281. Binding of ZNF281 to the promoters of these genes suggests a direct mechanism of repression. Bioinformatic analysis of NB datasets indicates that ZNF281 expression is higher in aggressive, undifferentiated stage 4 than in localized stage 1 tumors supporting a central role of ZNF281 in affecting the differentiation of NB. Furthermore, patients with NB with high expression of ZNF281 have a poor clinical outcome compared with low-expressors. These observations suggest that ZNF281 is a controller of neuronal differentiation that should be evaluated as a prognostic marker in NB. Copyright © 2018 the Author(s). Published by PNAS.

Nostopeptolide plays a governing role during cellular differentiation of the symbiotic cyanobacterium Nostoc punctiforme.

PubMed

Liaimer, Anton; Helfrich, Eric J N; Hinrichs, Katrin; Guljamow, Arthur; Ishida, Keishi; Hertweck, Christian; Dittmann, Elke

2015-02-10

Nostoc punctiforme is a versatile cyanobacterium that can live either independently or in symbiosis with plants from distinct taxa. Chemical cues from plants and N. punctiforme were shown to stimulate or repress, respectively, the differentiation of infectious motile filaments known as hormogonia. We have used a polyketide synthase mutant that accumulates an elevated amount of hormogonia as a tool to understand the effect of secondary metabolites on cellular differentiation of N. punctiforme. Applying MALDI imaging to illustrate the reprogramming of the secondary metabolome, nostopeptolides were identified as the predominant difference in the pks2(-) mutant secretome. Subsequent differentiation assays and visualization of cell-type-specific expression of nostopeptolides via a transcriptional reporter strain provided evidence for a multifaceted role of nostopeptolides, either as an autogenic hormogonium-repressing factor or as a chemoattractant, depending on its extracellular concentration. Although nostopeptolide is constitutively expressed in the free-living state, secreted levels dynamically change before, during, and after the hormogonium differentiation phase. The metabolite was found to be strictly down-regulated in symbiosis with Gunnera manicata and Blasia pusilla, whereas other metabolites are up-regulated, as demonstrated via MALDI imaging, suggesting plants modulate the fine-balanced cross-talk network of secondary metabolites within N. punctiforme.

Nostopeptolide plays a governing role during cellular differentiation of the symbiotic cyanobacterium Nostoc punctiforme

PubMed Central

Liaimer, Anton; Helfrich, Eric J. N.; Hinrichs, Katrin; Guljamow, Arthur; Ishida, Keishi; Hertweck, Christian; Dittmann, Elke

2015-01-01

Nostoc punctiforme is a versatile cyanobacterium that can live either independently or in symbiosis with plants from distinct taxa. Chemical cues from plants and N. punctiforme were shown to stimulate or repress, respectively, the differentiation of infectious motile filaments known as hormogonia. We have used a polyketide synthase mutant that accumulates an elevated amount of hormogonia as a tool to understand the effect of secondary metabolites on cellular differentiation of N. punctiforme. Applying MALDI imaging to illustrate the reprogramming of the secondary metabolome, nostopeptolides were identified as the predominant difference in the pks2− mutant secretome. Subsequent differentiation assays and visualization of cell-type-specific expression of nostopeptolides via a transcriptional reporter strain provided evidence for a multifaceted role of nostopeptolides, either as an autogenic hormogonium-repressing factor or as a chemoattractant, depending on its extracellular concentration. Although nostopeptolide is constitutively expressed in the free-living state, secreted levels dynamically change before, during, and after the hormogonium differentiation phase. The metabolite was found to be strictly down-regulated in symbiosis with Gunnera manicata and Blasia pusilla, whereas other metabolites are up-regulated, as demonstrated via MALDI imaging, suggesting plants modulate the fine-balanced cross-talk network of secondary metabolites within N. punctiforme. PMID:25624477

Effects of organophosphates on the regulation of mesenchymal stem cell proliferation and differentiation.

PubMed

Prugh, Amber M; Cole, Stephanie D; Glaros, Trevor; Angelini, Daniel J

2017-03-25

Mesenchymal stem cells (MSCs) are multipotent cells located within various adult tissues. Recent literature has reported that human bone marrow-derived MSCs express active acetylcholinesterase (AChE) and that disruption of AChE activity by organophosphate (OP) chemicals decreases the ability of MSCs to differentiate into osteoblasts. The potential role of AChE in regulating MSC proliferation and differentiation is currently unknown. In the present study, we demonstrate that MSCs exposed to OPs have both decreased AChE activity and abundance. In addition, exposure to these OPs induced cellular death while decreasing cellular proliferation. Exposures to these compounds also reduced the adipogenic/osteogenic differentiation potentials of the MSCs. To elucidate the possible role of AChE in MSCs signaling following OP exposure, we captured potential AChE binding partners by performing polyhistidine (His 8 )-tagged AChE pulldowns, followed by protein identification using liquid chromatography mass spectrometry (LC-MS). Using this method, we determined that the focal adhesion protein, vinculin, is a potential binding partner with AChE in MSCs and these initial findings were confirmed with follow-up co-immunoprecipitation experiments. Identifying AChE binding partners helps to determine potential pathways associated with MSC proliferation and differentiation, and this understanding could lead to the development of future MSC-based tissue repair therapies. Published by Elsevier B.V.

Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells.

PubMed

Chen, Jong-Hang; Chou, Chin-Cheng

2015-08-01

This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

2014-07-10

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

Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1

PubMed Central

Wagenknecht, Nadine; Reuter, Nina; Scherer, Myriam; Reichel, Anna; Müller, Regina; Stamminger, Thomas

2015-01-01

Promyelocytic leukemia nuclear bodies, also termed nuclear domain 10 (ND10), have emerged as nuclear protein accumulations mediating an intrinsic cellular defense against viral infections via chromatin-based mechanisms, however, their contribution to the control of herpesviral latency is still controversial. In this study, we utilized the monocytic cell line THP-1 as an in vitro latency model for human cytomegalovirus infection (HCMV). Characterization of THP-1 cells by immunofluorescence and Western blot analysis confirmed the expression of all major ND10 components. THP-1 cells with a stable, individual knockdown of PML, hDaxx or Sp100 were generated. Importantly, depletion of the major ND10 proteins did not prevent the terminal cellular differentiation of THP-1 monocytes. After construction of a recombinant, endotheliotropic human cytomegalovirus expressing IE2-EYFP, we investigated whether the depletion of ND10 proteins affects the onset of viral IE gene expression. While after infection of differentiated, THP-1-derived macrophages as well as during differentiation-induced reactivation from latency an increase in the number of IE-expressing cells was readily detectable in the absence of the major ND10 proteins, no effect was observed in non-differentiated monocytes. We conclude that PML, hDaxx and Sp100 primarily act as cellular restriction factors during lytic HCMV replication and during the dynamic process of reactivation but do not serve as key determinants for the establishment of HCMV latency. PMID:26057166

Cellular Microcultures: Programming Mechanical and Physicochemical Properties of 3D Hydrogel Cellular Microcultures via Direct Ink Writing (Adv. Healthcare Mater. 9/2016).

PubMed

McCracken, Joselle M; Badea, Adina; Kandel, Mikhail E; Gladman, A Sydney; Wetzel, David J; Popescu, Gabriel; Lewis, Jennifer A; Nuzzo, Ralph G

2016-05-01

R. Nuzzo and co-workers show on page 1025 how compositional differences in hydrogels are used to tune their cellular compliance by controlling their polymer mesh properties and subsequent uptake of the protein poly-l-lysine (green spheres in circled inset). The cover image shows pyramid micro-scaffolds prepared using direct ink writing (DIW) that differentially direct fibroblast and preosteoblast growth in 3D, depending on cell motility and surface treatment. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hantaviruses induce cell type- and viral species-specific host microRNA expression signatures

PubMed Central

Shin, Ok Sarah; Kumar, Mukesh; Yanagihara, Richard; Song, Jin-Won

2014-01-01

The mechanisms of hantavirus-induced modulation of host cellular immunity remain poorly understood. Recently, microRNAs (miRNAs) have emerged as a class of essential regulators of host immune response genes. To ascertain if differential host miRNA expression toward representative hantavirus species correlated with immune response genes, miRNA expression profiles were analyzed in human endothelial cells, macrophages and epithelial cells infected with pathogenic and nonpathogenic rodent- and shrew-borne hantaviruses. Distinct miRNA expression profiles were observed in a cell type- and viral species-specific pattern. A subset of miRNAs, including miR-151-5p and miR-1973, were differentially expressed between Hantaan virus and Prospect Hill virus. Pathway analyses confirmed that the targets of selected miRNAs were associated with inflammatory responses and innate immune receptor-mediated signaling pathways. Our data suggest that differential immune responses following hantavirus infection may be regulated in part by cellular miRNA through dysregulation of genes critical to the inflammatory process. PMID:24074584

Photospheric Magnetic Flux Transport - Supergranules Rule

NASA Technical Reports Server (NTRS)

Hathaway, David H.; Rightmire-Upton, Lisa

2012-01-01

Observations of the transport of magnetic flux in the Sun's photosphere show that active region magnetic flux is carried far from its origin by a combination of flows. These flows have previously been identified and modeled as separate axisymmetric processes: differential rotation, meridional flow, and supergranule diffusion. Experiments with a surface convective flow model reveal that the true nature of this transport is advection by the non-axisymmetric cellular flows themselves - supergranules. Magnetic elements are transported to the boundaries of the cells and then follow the evolving boundaries. The convective flows in supergranules have peak velocities near 500 m/s. These flows completely overpower the superimposed 20 m/s meridional flow and 100 m/s differential rotation. The magnetic elements remain pinned at the supergranule boundaries. Experiments with and without the superimposed axisymmetric photospheric flows show that the axisymmetric transport of magnetic flux is controlled by the advection of the cellular pattern by underlying flows representative of deeper layers. The magnetic elements follow the differential rotation and meridional flow associated with the convection cells themselves -- supergranules rule!

Gravity-regulated differential auxin transport from columella to lateral root cap cells

NASA Technical Reports Server (NTRS)

Ottenschlager, Iris; Wolff, Patricia; Wolverton, Chris; Bhalerao, Rishikesh P.; Sandberg, Goran; Ishikawa, Hideo; Evans, Mike; Palme, Klaus

2003-01-01

Gravity-induced root curvature has long been considered to be regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients, and the transport mechanisms involved, remain to be identified. Here, we describe a GFP-based auxin biosensor to monitor auxin during Arabidopsis root gravitropism at cellular resolution. We identify elevated auxin levels at the root apex in columella cells, the site of gravity perception, and an asymmetric auxin flux from these cells to the lateral root cap (LRC) and toward the elongation zone after gravistimulation. We differentiate between an efflux-dependent lateral auxin transport from columella to LRC cells, and an efflux- and influx-dependent basipetal transport from the LRC to the elongation zone. We further demonstrate that endogenous gravitropic auxin gradients develop even in the presence of an exogenous source of auxin. Live-cell auxin imaging provides unprecedented insights into gravity-regulated auxin flux at cellular resolution, and strongly suggests that this flux is a prerequisite for root gravitropism.

Ubiquitination dynamics in the early-branching eukaryote Giardia intestinalis

PubMed Central

Niño, Carlos A; Chaparro, Jenny; Soffientini, Paolo; Polo, Simona; Wasserman, Moises

2013-01-01

Ubiquitination is a highly dynamic and versatile posttranslational modification that regulates protein function, stability, and interactions. To investigate the roles of ubiquitination in a primitive eukaryotic lineage, we utilized the early-branching eukaryote Giardia intestinalis. Using a combination of biochemical, immunofluorescence-based, and proteomics approaches, we assessed the ubiquitination status during the process of differentiation in Giardia. We observed that different types of ubiquitin modifications present specific cellular and temporal distribution throughout the Giardia life cycle from trophozoites to cyst maturation. Ubiquitin signal was detected in the wall of mature cysts, and enzymes implicated in cyst wall biogenesis were identified as substrates for ubiquitination. Interestingly, inhibition of proteasome activity did not affect trophozoite replication and differentiation, while it caused a decrease in cyst viability, arguing for proteasome involvement in cyst wall maturation. Using a proteomics approach, we identified around 200 high-confidence ubiquitinated candidates that vary their ubiquitination status during differentiation. Our results indicate that ubiquitination is critical for several cellular processes in this primitive eukaryote. PMID:23613346

Root graviresponsiveness and cellular differentiation in wild-type and a starchless mutant of Arabidopsis thaliana

NASA Technical Reports Server (NTRS)

Moore, R.

1989-01-01

Primary roots of a starchless mutant of Arabidopsis thaliana L. are strongly graviresponsive despite lacking amyloplasts in their columella cells. The ultrastructures of calyptrogen and peripheral cells in wild-type as compared to mutant seedlings are not significantly different. The largest difference in cellular differentiation in caps of mutant and wild-type roots is the relative volume of plastids in columella cells. Plastids occupy 12.3% of the volume of columella cells in wild-type seedlings, but only 3.69% of columella cells in mutant seedlings. These results indicate that: (1) amyloplasts and starch are not necessary for root graviresponsiveness; (2) the increase in relative volume of plastids that usually accompanies differentiation of columella cells is not necessary for root graviresponsiveness; and (3) the absence of starch and amyloplasts does not affect the structure of calyptrogen (i.e. meristematic) and secretory (i.e. peripheral) cells in root caps. These results are discussed relative to proposed models for root gravitropism.

PMA Induces SnoN Proteolysis and CD61 Expression through an Autocrine Mechanism

PubMed Central

Li, Chonghua; Peart, Natoya; Xuan, Zhenyu; Lewis, Dorothy E; Xia, Yang; Jin, Jianping

2014-01-01

Phorbol-12-myristate-13-acetate, also called PMA, is a small molecule that activates protein kinase C and functions to differentiate hematologic lineage cells. However, the mechanism of PMA-induced cellular differentiation is not fully understood. We found that PMA triggers global enhancement of protein ubiquitination in K562, a myelogenous leukemia cell line and one of the enhanced-ubiquitination targets is SnoN, an inhibitor of the Smad signaling pathway. Our data indicated that PMA stimulated the production of Activin A, a cytokine of the TGF-β family. Activin A then activated the phosphorylation of both Smad2 and Smad3. In consequence, SnoN is ubiquitinated by the APCCdh1 ubiquitin ligase with the help of phosphorylated Smad2. Furthermore, we found that SnoN proteolysis is important for the expression of CD61, a marker of megakaryocyte. These results indicate that protein ubiquitination promotes megakaryopoiesis via degrading SnoN, an inhibitor of CD61 expression, strengths the roles of ubiquitination in cellular differentiation. PMID:24637302

Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

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

Biemann, Ronald, E-mail: ronald.biemann@medizin.uni-halle.de; Navarrete Santos, Anne; Navarrete Santos, Alexander

Highlights: Black-Right-Pointing-Pointer Endocrine disrupting chemicals affect adipogenesis in mesenchymal stem cells (MSC). Black-Right-Pointing-Pointer The adipogenic impact depends strongly on the window of exposure. Black-Right-Pointing-Pointer Bisphenol A reduces the potential of MSC to differentiate into adipocytes. Black-Right-Pointing-Pointer DEHP and TBT trigger the adipogenic differentiation of mesenchymal stem cells. Black-Right-Pointing-Pointer BPA, DEHP and TBT did not affect adipogenesis in embryonic stem cells. -- Abstract: Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study,more » we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPAR{gamma}2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 {mu}M) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 {mu}M) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.« less

Evidence that differentiation-inducing factor-1 controls chemotaxis and cell differentiation, at least in part, via mitochondria in D. discoideum.

PubMed

Kubohara, Yuzuru; Kikuchi, Haruhisa; Nguyen, Van Hai; Kuwayama, Hidekazu; Oshima, Yoshiteru

2017-06-15

Differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one (DIF-1)] is an important regulator of cell differentiation and chemotaxis in the development of the cellular slime mold Dictyostelium discoideum However, the entire signaling pathways downstream of DIF-1 remain to be elucidated. To characterize DIF-1 and its potential receptor(s), we synthesized two fluorescent derivatives of DIF-1, boron-dipyrromethene (BODIPY)-conjugated DIF-1 (DIF-1-BODIPY) and nitrobenzoxadiazole (NBD)-conjugated DIF-1 (DIF-1-NBD), and investigated their biological activities and cellular localization. DIF-1-BODIPY (5 µM) and DIF-1 (2 nM) induced stalk cell differentiation in the DIF-deficient strain HM44 in the presence of cyclic adenosine monosphosphate (cAMP), whereas DIF-1-NBD (5 µM) hardly induced stalk cell differentiation under the same conditions. Microscopic analyses revealed that the biologically active derivative, DIF-1-BODIPY, was incorporated by stalk cells at late stages of differentiation and was localized to mitochondria. The mitochondrial uncouplers carbonyl cyanide m -chlorophenylhydrazone (CCCP), at 25-50 nM, and dinitrophenol (DNP), at 2.5-5 µM, induced partial stalk cell differentiation in HM44 in the presence of cAMP. DIF-1-BODIPY (1-2 µM) and DIF-1 (10 nM), as well as CCCP and DNP, suppressed chemotaxis in the wild-type strain Ax2 in shallow cAMP gradients. These results suggest that DIF-1-BODIPY and DIF-1 induce stalk cell differentiation and modulate chemotaxis, at least in part, by disturbing mitochondrial activity. © 2017. Published by The Company of Biologists Ltd.

Evidence that differentiation-inducing factor-1 controls chemotaxis and cell differentiation, at least in part, via mitochondria in D. discoideum

PubMed Central

Kikuchi, Haruhisa; Nguyen, Van Hai; Kuwayama, Hidekazu; Oshima, Yoshiteru

2017-01-01

ABSTRACT Differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one (DIF-1)] is an important regulator of cell differentiation and chemotaxis in the development of the cellular slime mold Dictyostelium discoideum. However, the entire signaling pathways downstream of DIF-1 remain to be elucidated. To characterize DIF-1 and its potential receptor(s), we synthesized two fluorescent derivatives of DIF-1, boron-dipyrromethene (BODIPY)-conjugated DIF-1 (DIF-1-BODIPY) and nitrobenzoxadiazole (NBD)-conjugated DIF-1 (DIF-1-NBD), and investigated their biological activities and cellular localization. DIF-1-BODIPY (5 µM) and DIF-1 (2 nM) induced stalk cell differentiation in the DIF-deficient strain HM44 in the presence of cyclic adenosine monosphosphate (cAMP), whereas DIF-1-NBD (5 µM) hardly induced stalk cell differentiation under the same conditions. Microscopic analyses revealed that the biologically active derivative, DIF-1-BODIPY, was incorporated by stalk cells at late stages of differentiation and was localized to mitochondria. The mitochondrial uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), at 25–50 nM, and dinitrophenol (DNP), at 2.5–5 µM, induced partial stalk cell differentiation in HM44 in the presence of cAMP. DIF-1-BODIPY (1–2 µM) and DIF-1 (10 nM), as well as CCCP and DNP, suppressed chemotaxis in the wild-type strain Ax2 in shallow cAMP gradients. These results suggest that DIF-1-BODIPY and DIF-1 induce stalk cell differentiation and modulate chemotaxis, at least in part, by disturbing mitochondrial activity. PMID:28619991

Tracking the Spatial and Functional Gradient of Monocyte-To-Macrophage Differentiation in Inflamed Lung.

PubMed

Sen, Debasish; Jones, Stephen M; Oswald, Erin M; Pinkard, Henry; Corbin, Kaitlin; Krummel, Matthew F

2016-01-01

Myeloid-derived cells such as monocytes, dendritic cells (DCs), and macrophages are at the heart of the immune effector function in an inflammatory response. But because of the lack of an efficient imaging system to trace these cells live during their migration and maturation in their native environment at sub-cellular resolution, our knowledge is limited to data available from specific time-points analyzed by flow cytometry, histology, genomics and other immunological methods. Here, we have developed a ratiometric imaging method for measuring monocyte maturation in inflamed mouse lungs in situ using real-time using 2-photon imaging and complementary methods. We visualized that while undifferentiated monocytes were predominantly found only in the vasculature, a semi-differentiated monocyte/macrophage population could enter the tissue and resembled more mature and differentiated populations by morphology and surface phenotype. As these cells entered and differentiated, they were already selectively localized near inflamed airways and their entry was associated with changes in motility and morphology. We were able to visualize these during the act of differentiation, a process that can be demonstrated in this way to be faster on a per-cell basis under inflammatory conditions. Finally, our in situ analyses demonstrated increases, in the differentiating cells, for both antigen uptake and the ability to mediate interactions with T cells. This work, while largely confirming proposed models for in situ differentiation, provides important in situ data on the coordinated site-specific recruitment and differentiation of these cells and helps elaborate the predominance of immune pathology at the airways. Our novel imaging technology to trace immunogenic cell maturation in situ will complement existing information available on in situ differentiation deduced from other immunological methods, and assist better understanding of the spatio-temporal cellular behavior during an inflammatory response.

Cellular signaling identifiability analysis: a case study.

PubMed

Roper, Ryan T; Pia Saccomani, Maria; Vicini, Paolo

2010-05-21

Two primary purposes for mathematical modeling in cell biology are (1) simulation for making predictions of experimental outcomes and (2) parameter estimation for drawing inferences from experimental data about unobserved aspects of biological systems. While the former purpose has become common in the biological sciences, the latter is less common, particularly when studying cellular and subcellular phenomena such as signaling-the focus of the current study. Data are difficult to obtain at this level. Therefore, even models of only modest complexity can contain parameters for which the available data are insufficient for estimation. In the present study, we use a set of published cellular signaling models to address issues related to global parameter identifiability. That is, we address the following question: assuming known time courses for some model variables, which parameters is it theoretically impossible to estimate, even with continuous, noise-free data? Following an introduction to this problem and its relevance, we perform a full identifiability analysis on a set of cellular signaling models using DAISY (Differential Algebra for the Identifiability of SYstems). We use our analysis to bring to light important issues related to parameter identifiability in ordinary differential equation (ODE) models. We contend that this is, as of yet, an under-appreciated issue in biological modeling and, more particularly, cell biology. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

A 3D Cellular Automaton for Cell Differentiation in a Solid Tumor with Plasticity

NASA Astrophysics Data System (ADS)

Margarit, David H.; Romanelli, Lilia; Fendrik, Alejandro J.

A model with spherical symmetry is proposed. We analyze the appropriate parameters of cell differentiation for different kinds of cells (Cancer Stem Cells (CSC) and Differentiated Cells (DC)). The plasticity (capacity to return from a DC to its previous state of CSC) is taken into account. Following this hypothesis, the dissemination of CSCs to another organ is analyzed. The location of the cells in the tumor and the plasticity range for possible metastasis is discussed.

Anodic Aluminum Oxide (AAO) Membranes for Cellular Devices

NASA Astrophysics Data System (ADS)

Ventura, Anthony P.

Anodic Aluminum Oxide (AAO) membranes can be fabricated with a highly tunable pore structure making them a suitable candidate for cellular hybrid devices with single-molecule selectivity. The objective of this study was to characterize the cellular response of AAO membranes with varying pore sizes to serve as a proof-of-concept for an artificial material/cell synapse system. AAO membranes with pore diameters ranging from 34-117 nm were achieved via anodization at a temperature of -1°C in a 2.7% oxalic acid electrolyte. An operating window was established for this setup to create membranes with through-pore and disordered pore morphologies. C17.2 neural stem cells were seeded onto the membranes and differentiated via serum withdrawal. The data suggests a highly tunable correlation between AAO pore diameter and differentiated cell populations. Analysis of membranes before and after cell culture indicated no breakdown of the through-pore structure. Immunocytochemistry (ICC) showed that AAO membranes had increased neurite outgrowth when compared to tissue culture treated (TCT) glass, and neurite outgrowth varied with pore diameter. Additionally, lower neuronal percentages were found on AAO as compared to TCT glass; however, neuronal population was also found to vary with pore diameter. Scanning electron microscopy (SEM) and ICC images suggested the presence of a tissue-like layer with a mixed-phenotype population. AAO membranes appear to be an excellent candidate for cellular devices, but more work must be completed to understand the surface chemistry of the AAO membranes as it relates to cellular response.

Slug is upregulated during wound healing and regulates cellular phenotypes in corneal epithelial cells.

PubMed

Aomatsu, Keiichi; Arao, Tokuzo; Abe, Kosuke; Kodama, Aya; Sugioka, Koji; Matsumoto, Kazuko; Kudo, Kanae; Kimura, Hideharu; Fujita, Yoshihiko; Hayashi, Hidetoshi; Nagai, Tomoyuki; Shimomura, Yoshikazu; Nishio, Kazuto

2012-02-16

The involvement of the epithelial mesenchymal transition (EMT) in the process of corneal wound healing remains largely unclear. The purpose of the present study was to gain insight into Slug expression and corneal wound healing. Slug expression during wound healing in the murine cornea was evaluated using fluorescence staining in vivo. Slug or Snail was stably introduced into human corneal epithelial cells (HCECs). These stable transfectants were evaluated for the induction of the EMT, cellular growth, migration activity, and expression changes in differentiation-related molecules. Slug, but not Snail, was clearly expressed in the nuclei of corneal epithelial cells in basal lesion of the corneal epithelium during wound healing in vivo. The overexpression of Slug or Snail induced an EMT-like cellular morphology and cadherin switching in HCECs, indicating that these transcription factors were able to mediate the typical EMT in HCECs. The overexpression of Slug or Snail suppressed cellular proliferation but enhanced the migration activity. Furthermore, ABCG2, TP63, and keratin 19, which are known as stemness-related molecules, were downregulated in these transfectants. It was found that Slug is upregulated during corneal wound healing in vivo. The overexpression of Slug mediated a change in the cellular phenotype affecting proliferation, migration, and expression levels of differentiation-related molecules. This is the first evidence that Slug is regulated during the process of corneal wound healing in the corneal epithelium in vivo, providing a novel insight into the EMT and Slug expression in corneal wound healing.

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.

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

PubMed

Soncin, Francesca; Mohamet, Lisa; Eckardt, Dominik; Ritson, Sarah; Eastham, Angela M; Bobola, Nicoletta; Russell, Angela; Davies, Steve; Kemler, Rolf; Merry, Catherine L R; Ward, Christopher M

2009-09-01

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell-cell contact in ES cells using gene knockout (Ecad(-/-)), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad(-/-) ES cells is dependent on the beta-catenin binding domain of E-cadherin, we show that beta-catenin null (betacat(-/-)) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad(-/-) ES cells is unlikely to be via beta-catenin signaling. Exposure of Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor beta family signaling in the self-renewal of Ecad(-/-) ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis.

PubMed

Hemphill, D D; McIlwraith, C W; Slayden, R A; Samulski, R J; Goodrich, L R

2016-05-01

IGF-I is one of several anabolic factors being investigated for the treatment of osteoarthritis (OA). Due to the short biological half-life, extended administration is required for more robust cartilage healing. Here we create a self-complimentary adeno-associated virus (AAV) gene therapy vector utilizing the transgene for IGF-I. Various biochemical assays were performed to investigate the cellular response to scAAVIGF-I treatment vs an scAAVGFP positive transduction control and a negative for transduction control culture. RNA-sequencing analysis was also performed to establish a differential regulation profile of scAAVIGF-I transduced chondrocytes. Biochemical analyses indicated an average media IGF-I concentration of 608 ng/ml in the scAAVIGF-I transduced chondrocytes. This increase in IGF-I led to increased expression of collagen type II and aggrecan and increased protein concentrations of cellular collagen type II and media glycosaminoglycan vs both controls. RNA-seq revealed a global regulatory pattern consisting of 113 differentially regulated GO categories including those for chondrocyte and cartilage development and regulation of apoptosis. This research substantiates that scAAVIGF-I gene therapy vector increased production of IGF-I to clinically relevant levels with a biological response by chondrocytes conducive to increased cartilage healing. The RNA-seq further established a set of differentially expressed genes and gene ontologies induced by the scAAVIGF-I vector while controlling for AAV infection. This dataset provides a static representation of the cellular transcriptome that, while only consisting of one time point, will allow for further gene expression analyses to compare additional cartilage healing therapeutics or a transient cellular response. Copyright © 2015. Published by Elsevier Ltd.

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

PubMed Central

Sohel, Md. Mahmodul Hasan; Hoelker, Michael; Noferesti, Sina Seifi; Salilew-Wondim, Dessie; Tholen, Ernst; Looft, Christian; Rings, Franca; Uddin, Muhammad Jasim; Spencer, Thomas E.; Schellander, Karl; Tesfaye, Dawit

2013-01-01

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment. PMID:24223816

RhoA-Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate Topography Dependent.

PubMed

Ogino, Yoichiro; Liang, Ruiwei; Mendonça, Daniela B S; Mendonça, Gustavo; Nagasawa, Masako; Koyano, Kiyoshi; Cooper, Lyndon F

2016-03-01

Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho-family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M), and nanotopography (N) to evaluate the effect of surface topography on RhoA-mediated functions (cell spreading, adhesion, migration, and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S < M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness dependent (S < M, N). RhoA activity and -mediated functions are influenced by surface topography. Smooth surface adherent cells appear highly sensitive to RhoA function, while nano-scale topography adherent cell may utilize alternative cellular signaling pathway(s) to influence adherent cellular functions regardless of RhoA activity. © 2015 Wiley Periodicals, Inc.

The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components.

PubMed

García-Cruz, Karla V; García-Ponce, Berenice; Garay-Arroyo, Adriana; Sanchez, María De La Paz; Ugartechea-Chirino, Yamel; Desvoyes, Bénédicte; Pacheco-Escobedo, Mario A; Tapia-López, Rosalinda; Ransom-Rodríguez, Ivan; Gutierrez, Crisanto; Alvarez-Buylla, Elena R

2016-07-29

Morphogenesis depends on the concerted modulation of cell proliferation and differentiation. Such modulation is dynamically adjusted in response to various external and internal signals via complex transcriptional regulatory networks that mediate between such signals and regulation of cell-cycle and cellular responses (proliferation, growth, differentiation). In plants, which are sessile, the proliferation/differentiation balance is plastically adjusted during their life cycle and transcriptional networks are important in this process. MADS-box genes are key developmental regulators in eukaryotes, but their role in cell proliferation and differentiation modulation in plants remains poorly studied. We characterize the XAL1 loss-of-function xal1-2 allele and overexpression lines using quantitative cellular and cytometry analyses to explore its role in cell cycle, proliferation, stem-cell patterning and transition to differentiation. We used quantitative PCR and cellular markers to explore if XAL1 regulates cell-cycle components and PLETHORA1 (PLT1) gene expression, as well as confocal microscopy to analyse stem-cell niche organization. We previously showed that XAANTAL1 (XAL1/AGL12) is necessary for Arabidopsis root development as a promoter of cell proliferation in the root apical meristem. Here, we demonstrate that XAL1 positively regulates the expression of PLT1 and important components of the cell cycle: CYCD3;1, CYCA2;3, CYCB1;1, CDKB1;1 and CDT1a In addition, we show that xal1-2 mutant plants have a premature transition to differentiation with root hairs appearing closer to the root tip, while endoreplication in these plants is partially compromised. Coincidently, the final size of cortex cells in the mutant is shorter than wild-type cells. Finally, XAL1 overexpression-lines corroborate that this transcription factor is able to promote cell proliferation at the stem-cell niche. XAL1 seems to be an important component of the networks that modulate cell proliferation/differentiation transition and stem-cell proliferation during Arabidopsis root development; it also regulates several cell-cycle components. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Mechanisms of crystalline silica-induced pulmonary toxicity revealed by global gene expression profiling

PubMed Central

Sellamuthu, Rajendran; Umbright, Christina; Li, Shengqiao; Kashon, Michael; Joseph, Pius

2015-01-01

A proper understanding of the mechanisms underlying crystalline silica-induced pulmonary toxicity has implications in the management and potential prevention of the adverse health effects associated with silica exposure including silicosis, cancer and several auto-immune diseases. Human lung type II epithelial cells and rat lungs exposed to crystalline silica were employed as experimental models to determine global gene expression changes in order to understand the molecular mechanisms underlying silica-induced pulmonary toxicity. The differential gene expression profile induced by silica correlated with its toxicity in the A549 cells. The biological processes perturbed by silica exposure in the A549 cells and rat lungs, as identified by the bioinformatics analysis of the differentially expressed genes, demonstrated significant similarity. Functional categorization of the differentially expressed genes identified cancer, cellular movement, cellular growth and proliferation, cell death, inflammatory response, cell cycle, cellular development, and genetic disorder as top ranking biological functions perturbed by silica exposure in A549 cells and rat lungs. Results of our study, in addition to confirming several previously identified molecular targets and mechanisms involved in silica toxicity, identified novel molecular targets and mechanisms potentially involved in silica-induced pulmonary toxicity. Further investigations, including those focused on the novel molecular targets and mechanisms identified in the current study may result in better management and, possibly, reduction and/or prevention of the potential adverse health effects associated with crystalline silica exposure. PMID:22087542

Galectin-3 in autoimmunity and autoimmune diseases

PubMed Central

de Oliveira, Felipe L; Gatto, Mariele; Bassi, Nicola; Luisetto, Roberto; Ghirardello, Anna; Punzi, Leonardo

2015-01-01

Galectin-3 (gal-3) is a β-galactoside-binding lectin, which regulates cell–cell and extracellular interactions during self/non-self-antigen recognition and cellular activation, proliferation, differentiation, migration and apoptosis. It plays a significant role in cellular and tissue pathophysiology by organizing niches that drive inflammation and immune responses. Gal-3 has some therapeutic potential in several diseases, including chronic inflammatory disorders, cancer and autoimmune diseases. Gal-3 exerts a broad spectrum of functions which differs according to its intra- or extracellular localization. Recombinant gal-3 strategy has been used to identify potential mode of action of gal-3; however, exogenous gal-3 may not reproduce the functions of the endogenous gal-3. Notably, gal-3 induces monocyte–macrophage differentiation, interferes with dendritic cell fate decision, regulates apoptosis on T lymphocytes and inhibits B-lymphocyte differentiation into immunoglobulin secreting plasma cells. Considering the influence of these cell populations in the pathogenesis of several autoimmune diseases, gal-3 seems to play a role in development of autoimmunity. Gal-3 has been suggested as a potential therapeutic agent in patients affected with some autoimmune disorders. However, the precise role of gal-3 in driving the inflammatory process in autoimmune or immune-mediated disorders remains elusive. Here, we reviewed the involvement of gal-3 in cellular and tissue events during autoimmune and immune-mediated inflammatory diseases. PMID:26142116

The Role of Cellular Proliferation in Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

PubMed

Marquez, Maribel P; Alencastro, Frances; Madrigal, Alma; Jimenez, Jossue Loya; Blanco, Giselle; Gureghian, Alex; Keagy, Laura; Lee, Cecilia; Liu, Robert; Tan, Lun; Deignan, Kristen; Armstrong, Brian; Zhao, Yuanxiang

2017-11-01

Mitotic clonal expansion has been suggested as a prerequisite for adipogenesis in murine preadipocytes, but the precise role of cell proliferation during human adipogenesis is unclear. Using adipose tissue-derived human mesenchymal stem cells as an in vitro cell model for adipogenic study, a group of cell cycle regulators, including Cdk1 and CCND1, were found to be downregulated as early as 24 h after adipogenic initiation and consistently, cell proliferation activity was restricted to the first 48 h of adipogenic induction. Cell proliferation was either further inhibited using siRNAs targeting cell cycle genes or enhanced by supplementing exogenous growth factor, basic fibroblast growth factor (bFGF), at specific time intervals during adipogenesis. Expression knockdown of Cdk1 at the initiation of adipogenic induction resulted in significantly increased adipocytes, even though total number of cells was significantly reduced compared to siControl-treated cells. bFGF stimulated proliferation throughout adipogenic differentiation, but exerted differential effect on adipogenic outcome at different phases, promoting adipogenesis during mitotic phase (first 48 h), but significantly inhibiting adipogenesis during adipogenic commitment phase (days 3-6). Our results demonstrate that cellular proliferation is counteractive to adipogenic commitment in human adipogenesis. However, cellular proliferation stimulation can be beneficial for adipogenesis during the mitotic phase by increasing the population of cells capable of committing to adipocytes before adipogenic commitment.

Cell differentiation and germ-soma separation in Ediacaran animal embryo-like fossils

NASA Astrophysics Data System (ADS)

Chen, Lei; Xiao, Shuhai; Pang, Ke; Zhou, Chuanming; Yuan, Xunlai

2014-12-01

Phosphorites of the Ediacaran Doushantuo Formation (~600 million years old) yield spheroidal microfossils with a palintomic cell cleavage pattern. These fossils have been variously interpreted as sulphur-oxidizing bacteria, unicellular protists, mesomycetozoean-like holozoans, green algae akin to Volvox, and blastula embryos of early metazoans or bilaterian animals. However, their complete life cycle is unknown and it is uncertain whether they had a cellularly differentiated ontogenetic stage, making it difficult to test their various phylogenetic interpretations. Here we describe new spheroidal fossils from black phosphorites of the Doushantuo Formation that have been overlooked in previous studies. These fossils represent later developmental stages of previously published blastula-like fossils, and they show evidence for cell differentiation, germ-soma separation, and programmed cell death. Their complex multicellularity is inconsistent with a phylogenetic affinity with bacteria, unicellular protists, or mesomycetozoean-like holozoans. Available evidence also indicates that the Doushantuo fossils are unlikely crown-group animals or volvocine green algae. We conclude that an affinity with cellularly differentiated multicellular eukaryotes, including stem-group animals or algae, is likely but more data are needed to constrain further the exact phylogenetic affinity of the Doushantuo fossils.

Cell differentiation and germ-soma separation in Ediacaran animal embryo-like fossils.

PubMed

Chen, Lei; Xiao, Shuhai; Pang, Ke; Zhou, Chuanming; Yuan, Xunlai

2014-12-11

Phosphorites of the Ediacaran Doushantuo Formation (∼600 million years old) yield spheroidal microfossils with a palintomic cell cleavage pattern. These fossils have been variously interpreted as sulphur-oxidizing bacteria, unicellular protists, mesomycetozoean-like holozoans, green algae akin to Volvox, and blastula embryos of early metazoans or bilaterian animals. However, their complete life cycle is unknown and it is uncertain whether they had a cellularly differentiated ontogenetic stage, making it difficult to test their various phylogenetic interpretations. Here we describe new spheroidal fossils from black phosphorites of the Doushantuo Formation that have been overlooked in previous studies. These fossils represent later developmental stages of previously published blastula-like fossils, and they show evidence for cell differentiation, germ-soma separation, and programmed cell death. Their complex multicellularity is inconsistent with a phylogenetic affinity with bacteria, unicellular protists, or mesomycetozoean-like holozoans. Available evidence also indicates that the Doushantuo fossils are unlikely crown-group animals or volvocine green algae. We conclude that an affinity with cellularly differentiated multicellular eukaryotes, including stem-group animals or algae, is likely but more data are needed to constrain further the exact phylogenetic affinity of the Doushantuo fossils.

Modulation of Dendritic Cell Activation and Subsequent Th1 Cell Polarization by Lidocaine

PubMed Central

Chung, Yeonseok

2015-01-01

Dendritic cells play an essential role in bridging innate and adaptive immunity by recognizing cellular stress including pathogen- and damage-associated molecular patterns and by shaping the types of antigen-specific T cell immunity. Although lidocaine is widely used in clinical settings that trigger cellular stress, it remains unclear whether such treatment impacts the activation of innate immune cells and subsequent differentiation of T cells. Here we showed that lidocaine inhibited the production of IL–6, TNFα and IL–12 from dendritic cells in response to toll-like receptor ligands including lipopolysaccharide, poly(I:C) and R837 in a dose-dependent manner. Notably, the differentiation of Th1 cells was significantly suppressed by the addition of lidocaine while the same treatment had little effect on the differentiation of Th17, Th2 and regulatory T cells in vitro. Moreover, lidocaine suppressed the ovalbumin-specific Th1 cell responses in vivo induced by the adoptive transfer of ovalbumin-pulsed dendritic cells. These results demonstrate that lidocaine inhibits the activation of dendritic cells in response to toll-like receptor signals and subsequently suppresses the differentiation of Th1 cell responses. PMID:26445366

Morphometric analysis of epidermal differentiation in primary roots of Zea mays

NASA Technical Reports Server (NTRS)

Moore, R.; Smith, H. S.

1990-01-01

Epidermal differentiation in primary roots of Zea mays was divided into six cell types based on cellular shape and cytoplasmic appearance. These six cell types are: 1) apical protoderm, located at the tip of the root pole and characterized by periclinally flattened cells; 2) cuboidal protoderm, located approximately 230 microns from the root pole and characterized by cuboidal cells; 3) tabular epidermis, located approximately 450 microns from the root pole and characterized by anticlinally flattened cells; 4) cuboidal epidermis, located approximately 900 microns from the root pole and characterized by cuboidal cells having numerous small vacuoles; 5) vacuolate cuboidal epidermis, located approximately 1,500 microns from the root pole and characterized by cuboidal cells containing several large vacuoles; and 6) columnar epidermis, located approximately 2,200 microns from the root pole (i.e., at the beginning of the zone of elongation) and characterized by elongated cells. We also used stereology to quantify the cellular changes associated with epidermal differentiation. The quiescent center and the apical protoderm have significantly different ultrastructures. The relative volume of dictyosomes increases dramatically during the early stages of epidermal differentiation. This increase correlates inversely with the amount of coverage provided by the root cap and mucilage.

Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.

PubMed

Glass, Donald A; Bialek, Peter; Ahn, Jong Deok; Starbuck, Michael; Patel, Millan S; Clevers, Hans; Taketo, Mark M; Long, Fanxin; McMahon, Andrew P; Lang, Richard A; Karsenty, Gerard

2005-05-01

Inactivation of beta-catenin in mesenchymal progenitors prevents osteoblast differentiation; inactivation of Lrp5, a gene encoding a likely Wnt coreceptor, results in low bone mass (osteopenia) by decreasing bone formation. These observations indicate that Wnt signaling controls osteoblast differentiation and suggest that it may regulate bone formation in differentiated osteoblasts. Here, we study later events and find that stabilization of beta-catenin in differentiated osteoblasts results in high bone mass, while its deletion from differentiated osteoblasts leads to osteopenia. Surprisingly, histological analysis showed that these mutations primarily affect bone resorption rather than bone formation. Cellular and molecular studies showed that beta-catenin together with TCF proteins regulates osteoblast expression of Osteoprotegerin, a major inhibitor of osteoclast differentiation. These findings demonstrate that beta-catenin, and presumably Wnt signaling, promote the ability of differentiated osteoblasts to inhibit osteoclast differentiation; thus, they broaden our knowledge of the functions Wnt proteins have at various stages of skeletogenesis.

Membrane order in the plasma membrane and endocytic recycling compartment.

PubMed

Iaea, David B; Maxfield, Frederick R

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

Mercury-induced biochemical and proteomic changes in rice roots.

PubMed

Chen, Yun-An; Chi, Wen-Chang; Huang, Tsai-Lien; Lin, Chung-Yi; Quynh Nguyeh, Thi Thuy; Hsiung, Yu-Chywan; Chia, Li-Chiao; Huang, Hao-Jen

2012-06-01

Mercury (Hg) is a serious environmental pollution threats to the planet. Accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. We investigated cellular, biochemical and proteomic changes in rice roots under Hg stress. Root growth rate was decreased and Hg, reactive oxygen species (ROS), and malondialdehyde (MDA) content and lipoxygenase activity were increased significantly with increasing Hg concentration in roots. We revealed a time-dependent alteration in total glutathione content and enzymatic activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) during Hg stress. 2-D electrophoresis revealed differential expression of 25 spots with Hg treatment of roots: 14 spots were upregulated and 11 spots downregulated. These differentially expressed proteins were identified by ESI-MS/MS to be involved in cellular functions including redox and hormone homeostasis, chaperone activity, metabolism, and transcription regulation. These results may provide new insights into the molecular basis of the Hg stress response in plants. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

The cellular slime mold: eukaryotic model microorganism.

PubMed

Urushihara, Hideko

2009-04-01

Cellular slime molds are eukaryotic microorganisms in the soil. They feed on bacteria as solitary amoebae but conditionally construct multicellular forms in which cell differentiation takes place. Therefore, they are attractive for the study of fundamental biological phenomena such as phagocytosis, cell division, chemotactic movements, intercellular communication, cell differentiation, and morphogenesis. The most widely used species, Dictyostelium discoideum, is highly amenable to experimental manipulation and can be used with most recent molecular biological techniques. Its genome and cDNA analyses have been completed and well-annotated data are publicly available. A larger number of orthologues of human disease-related genes were found in D. discoideum than in yeast. Moreover, some pathogenic bacteria infect Dictyostelium amoebae. Thus, this microorganism can also offer a good experimental system for biomedical research. The resources of cellular slime molds, standard strains, mutants, and genes are maintained and distributed upon request by the core center of the National BioResource Project (NBRP-nenkin) to support Dictyostelium community users as well as new users interested in new platforms for research and/or phylogenic consideration.

Cytoskeleton-interacting LIM-domain protein CRP1 suppresses cell proliferation and protects from stress-induced cell death

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

Latonen, Leena; Jaervinen, Paeivi M.; Haartman Institute, University of Helsinki, FIN-00014 Helsinki

2008-02-15

Members of the cysteine-rich protein (CRP) family are actin cytoskeleton-interacting LIM-domain proteins known to act in muscle cell differentiation. We have earlier found that CRP1, a founding member of this family, is transcriptionally induced by UV radiation in human diploid fibroblasts [M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res. 31 (2003) 4779-4790]. Here we show that CRP1 is induced by growth-inhibitory signals, such as increased cellular density, and cytotoxic stress induced by UV radiation or staurosporine. We found that high levels of CRP1more » correlate with differentiation-associated morphology towards the myofibroblast lineage and that expression of ectopic CRP1 suppresses cell proliferation. Following UV- and staurosporine-induced stresses, expression of CRP1 provides a survival advantage evidenced by decreased cellular death and increased cellular metabolic activity and attachment. Our studies identify that CRP1 is a novel stress response factor, and provide evidence for its growth-inhibitory and cytoprotective functions.« less

The making of a lymphocyte: the choice among disparate cell fates and the IKAROS enigma.

PubMed

Georgopoulos, Katia

2017-03-01

Lymphocyte differentiation is set to produce myriad immune effector cells with the ability to respond to multitudinous foreign substances. The uniqueness of this developmental system lies in not only the great diversity of cellular functions that it can generate but also the ability of its differentiation intermediates and mature effector cells to expand upon demand, thereby providing lifelong immunity. Surprisingly, the goals of this developmental system are met by a relatively small group of DNA-binding transcription factors that work in concert to control the timing and magnitude of gene expression and fulfill the demands for cellular specialization, expansion, and maintenance. The cellular and molecular mechanisms through which these lineage-promoting transcription factors operate have been a focus of basic research in immunology. The mechanisms of development discerned in this effort are guiding clinical research on disorders with an immune cell base. Here, I focus on IKAROS, one of the earliest regulators of lymphoid lineage identity and a guardian of lymphocyte homeostasis. © 2017 Georgopoulos; Published by Cold Spring Harbor Laboratory Press.

MYCN Promotes the Expansion of Phox2B-Positive Neuronal Progenitors to Drive Neuroblastoma Development

PubMed Central

Alam, Goleeta; Cui, Hongjuan; Shi, Huilin; Yang, Liqun; Ding, Jane; Mao, Ling; Maltese, William A.; Ding, Han-Fei

2009-01-01

Amplification of the oncogene MYCN is a tumorigenic event in the development of a subset of neuroblastomas that commonly consist of undifferentiated or poorly differentiated neuroblasts with unfavorable clinical outcome. The cellular origin of these neuroblasts is unknown. Additionally, the cellular functions and target cells of MYCN in neuroblastoma development remain undefined. Here we examine the cell types that drive neuroblastoma development in TH-MYCN transgenic mice, an animal model of the human disease. Neuroblastoma development in these mice begins with hyperplastic lesions in early postnatal sympathetic ganglia. We show that both hyperplasia and primary tumors are composed predominantly of highly proliferative Phox2B+ neuronal progenitors. MYCN induces the expansion of these progenitors by both promoting their proliferation and preventing their differentiation. We further identify a minor population of undifferentiated nestin+ cells in both hyperplastic lesions and primary tumors that may serve as precursors of Phox2B+ neuronal progenitors. These findings establish the identity of neuroblasts that characterize the tumor phenotype and suggest a cellular pathway by which MYCN can promote neuroblastoma development. PMID:19608868

Whole-Body Single-Cell Sequencing Reveals Transcriptional Domains in the Annelid Larval Body

PubMed Central

Achim, Kaia; Eling, Nils; Vergara, Hernando Martinez; Bertucci, Paola Yanina; Musser, Jacob; Vopalensky, Pavel; Brunet, Thibaut; Collier, Paul; Benes, Vladimir; Marioni, John C; Arendt, Detlev

2018-01-01

Abstract Animal bodies comprise diverse arrays of cells. To characterize cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii. We identify five transcriptionally distinct groups of differentiated cells, each expressing a unique set of transcription factors and effector genes that implement cellular phenotypes. Spatial mapping of cells into a cellular expression atlas, and wholemount in situ hybridization of group-specific genes reveals spatially coherent transcriptional domains in the larval body, comprising, for example, apical sensory-neurosecretory cells versus neural/epidermal surface cells. These domains represent new, basic subdivisions of the annelid body based entirely on differential gene expression, and are composed of multiple, transcriptionally similar cell types. They do not represent clonal domains, as revealed by developmental lineage analysis. We propose that the transcriptional domains that subdivide the annelid larval body represent families of related cell types that have arisen by evolutionary diversification. Their possible evolutionary conservation makes them a promising tool for evo–devo research. PMID:29373712

Membrane order in the plasma membrane and endocytic recycling compartment

PubMed Central

Iaea, David B.; Maxfield, Frederick R.

2017-01-01

The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles. PMID:29125865

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

PubMed

Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

2018-06-01

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

Wavelet-based multiscale analysis of bioimpedance data measured by electric cell-substrate impedance sensing for classification of cancerous and normal cells.

PubMed

Das, Debanjan; Shiladitya, Kumar; Biswas, Karabi; Dutta, Pranab Kumar; Parekh, Aditya; Mandal, Mahitosh; Das, Soumen

2015-12-01

The paper presents a study to differentiate normal and cancerous cells using label-free bioimpedance signal measured by electric cell-substrate impedance sensing. The real-time-measured bioimpedance data of human breast cancer cells and human epithelial normal cells employs fluctuations of impedance value due to cellular micromotions resulting from dynamic structural rearrangement of membrane protrusions under nonagitated condition. Here, a wavelet-based multiscale quantitative analysis technique has been applied to analyze the fluctuations in bioimpedance. The study demonstrates a method to classify cancerous and normal cells from the signature of their impedance fluctuations. The fluctuations associated with cellular micromotion are quantified in terms of cellular energy, cellular power dissipation, and cellular moments. The cellular energy and power dissipation are found higher for cancerous cells associated with higher micromotions in cancer cells. The initial study suggests that proposed wavelet-based quantitative technique promises to be an effective method to analyze real-time bioimpedance signal for distinguishing cancer and normal cells.

Immunophenotypic and Molecular Analysis of Human Dental Pulp Stem Cells Potential for Neurogenic Differentiation

PubMed Central

Fatima, Nikhat; Khan, Aleem A.; Vishwakarma, Sandeep K.

2017-01-01

Background: Growing evidence shows that dental pulp (DP) tissues could be a potential source of adult stem cells for the treatment of devastating neurological diseases and several other conditions. Aims: Exploration of the expression profile of several key molecular markers to evaluate the molecular dynamics in undifferentiated and differentiated DP-derived stem cells (DPSCs) in vitro. Settings and Design: The characteristics and multilineage differentiation ability of DPSCs were determined by cellular and molecular kinetics. DPSCs were further induced to form adherent (ADH) and non-ADH (NADH) neurospheres under serum-free condition which was further induced into neurogenic lineage cells and characterized for their molecular and cellular diversity at each stage. Statistical Analysis Used: Statistical analysis used one-way analysis of variance, Student's t-test, Livak method for relative quantification, and R programming. Results: Immunophenotypic analysis of DPSCs revealed >80% cells positive for mesenchymal markers CD90 and CD105, >70% positive for transferring receptor (CD71), and >30% for chemotactic factor (CXCR3). These cells showed mesodermal differentiation also and confirmed by specific staining and molecular analysis. Activation of neuronal lineage markers and neurogenic growth factors was observed during lineage differentiation of cells derived from NADH and ADH spheroids. Greater than 80% of cells were found to express β-tubulin III in both differentiation conditions. Conclusions: The present study reported a cascade of immunophenotypic and molecular markers to characterize neurogenic differentiation of DPSCs under serum-free condition. These findings trigger the future analyses for clinical applicability of DP-derived cells in regenerative applications. PMID:28566856

DNMT1 Maintains Progenitor Function in Self-Renewing Somatic Tissue

PubMed Central

Sen, George L.; Reuter, Jason A.; Webster, Daniel E.; Zhu, Lilly; Khavari, Paul A.

2010-01-01

Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation1,2. DNA methylation3,4,5 provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1)6,7 maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance,8 a clear role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unknown. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis revealed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, we show that UHRF1,9,10 a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A11,12 and B13, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue. PMID:20081831

The transcription factor Nerfin-1 prevents reversion of neurons into neural stem cells.

PubMed

Froldi, Francesca; Szuperak, Milan; Weng, Chen-Fang; Shi, Wei; Papenfuss, Anthony T; Cheng, Louise Y

2015-01-15

Cellular dedifferentiation is the regression of a cell from a specialized state to a more multipotent state and is implicated in cancer. However, the transcriptional network that prevents differentiated cells from reacquiring stem cell fate is so far unclear. Neuroblasts (NBs), the Drosophila neural stem cells, are a model for the regulation of stem cell self-renewal and differentiation. Here we show that the Drosophila zinc finger transcription factor Nervous fingers 1 (Nerfin-1) locks neurons into differentiation, preventing their reversion into NBs. Following Prospero-dependent neuronal specification in the ganglion mother cell (GMC), a Nerfin-1-specific transcriptional program maintains differentiation in the post-mitotic neurons. The loss of Nerfin-1 causes reversion to multipotency and results in tumors in several neural lineages. Both the onset and rate of neuronal dedifferentiation in nerfin-1 mutant lineages are dependent on Myc- and target of rapamycin (Tor)-mediated cellular growth. In addition, Nerfin-1 is required for NB differentiation at the end of neurogenesis. RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) analysis show that Nerfin-1 administers its function by repression of self-renewing-specific and activation of differentiation-specific genes. Our findings support the model of bidirectional interconvertibility between neural stem cells and their post-mitotic progeny and highlight the importance of the Nerfin-1-regulated transcriptional program in neuronal maintenance. © 2015 Froldi et al.; Published by Cold Spring Harbor Laboratory Press.

BAG3 is involved in neuronal differentiation and migration.

PubMed

Santoro, Antonietta; Nicolin, Vanessa; Florenzano, Fulvio; Rosati, Alessandra; Capunzo, Mario; Nori, Stefania L

2017-05-01

Bcl2-associated athanogene 3 (BAG3) protein belongs to the family of co-chaperones interacting with several heat shock proteins. It plays a key role in protein quality control and mediates the clearance of misfolded proteins. Little is known about the expression and cellular localization of BAG3 during nervous system development and differentiation. Therefore, we analyze the subcellular distribution and expression of BAG3 in nerve-growth-factor-induced neurite outgrowth in PC12 cells and in developing and adult cortex of mouse brain. In differentiated PC12 cells, BAG3 was localized mainly in the neuritic domain rather than the cell body, whereas in control cells, it appeared to be confined to the cytoplasm near the nuclear membrane. Interestingly, the change of BAG3 localization during neuronal differentiation was associated only with a slight increase in total BAG3 expression. These data were coroborated by transmission electron microscopy showing that BAG3 was confined mainly within large dense-core vesicles of the axon in differentiated PC12 cells. In mouse developing cortex, BAG3 appeared to be intensely expressed in cellular processes of migrating cells, whereas in adult brain, a diffuse expression of low to medium intensity was detected in neuronal cell bodies. These findings suggest that BAG3 expression is required for neuronal differentiation and migration and that its role is linked to a change in its distribution pattern rather than to an increase in its protein expression levels.

A Method for the Direct Identification of Differentiating Muscle Cells by a Fluorescent Mitochondrial Dye

PubMed Central

Miyake, Tetsuaki; McDermott, John C.; Gramolini, Anthony O.

2011-01-01

Identification of differentiating muscle cells generally requires fixation, antibodies directed against muscle specific proteins, and lengthy staining processes or, alternatively, transfection of muscle specific reporter genes driving GFP expression. In this study, we examined the possibility of using the robust mitochondrial network seen in maturing muscle cells as a marker of cellular differentiation. The mitochondrial fluorescent tracking dye, MitoTracker, which is a cell-permeable, low toxicity, fluorescent dye, allowed us to distinguish and track living differentiating muscle cells visually by epi-fluorescence microscopy. MitoTracker staining provides a robust and simple detection strategy for living differentiating cells in culture without the need for fixation or biochemical processing. PMID:22174849

Microfluidic resonant waveguide grating biosensor system for whole cell sensing

NASA Astrophysics Data System (ADS)

Zaytseva, Natalya; Miller, William; Goral, Vasily; Hepburn, Jerry; Fang, Ye

2011-04-01

We report on a fluidic resonant waveguide grating (RWG) biosensor system that enables medium throughput measurements of cellular responses under microfluidics in a 32-well format. Dynamic mass redistribution assays under microfluidics differentiate the cross-desensitization process between the β2-adrenoceptor agonist epinephrine and the adenylate cyclase activator forskolin mediated signaling. This system opens new possibility to study cellular processes that are otherwise difficult to achieve using conventional RWG configurations.

Incorporating prior information into differential network analysis using non-paranormal graphical models.

PubMed

Zhang, Xiao-Fei; Ou-Yang, Le; Yan, Hong

2017-08-15

Understanding how gene regulatory networks change under different cellular states is important for revealing insights into network dynamics. Gaussian graphical models, which assume that the data follow a joint normal distribution, have been used recently to infer differential networks. However, the distributions of the omics data are non-normal in general. Furthermore, although much biological knowledge (or prior information) has been accumulated, most existing methods ignore the valuable prior information. Therefore, new statistical methods are needed to relax the normality assumption and make full use of prior information. We propose a new differential network analysis method to address the above challenges. Instead of using Gaussian graphical models, we employ a non-paranormal graphical model that can relax the normality assumption. We develop a principled model to take into account the following prior information: (i) a differential edge less likely exists between two genes that do not participate together in the same pathway; (ii) changes in the networks are driven by certain regulator genes that are perturbed across different cellular states and (iii) the differential networks estimated from multi-view gene expression data likely share common structures. Simulation studies demonstrate that our method outperforms other graphical model-based algorithms. We apply our method to identify the differential networks between platinum-sensitive and platinum-resistant ovarian tumors, and the differential networks between the proneural and mesenchymal subtypes of glioblastoma. Hub nodes in the estimated differential networks rediscover known cancer-related regulator genes and contain interesting predictions. The source code is at https://github.com/Zhangxf-ccnu/pDNA. szuouyl@gmail.com. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Adsorption of enamel matrix proteins to a bovine-derived bone grafting material and its regulation of cell adhesion, proliferation, and differentiation.

PubMed

Miron, Richard J; Bosshardt, Dieter D; Hedbom, Erik; Zhang, Yufeng; Haenni, Beat; Buser, Daniel; Sculean, Anton

2012-07-01

The use of various combinations of enamel matrix derivative (EMD) and grafting materials has been shown to promote periodontal wound healing/regeneration. However, the downstream cellular behavior of periodontal ligament (PDL) cells and osteoblasts has not yet been studied. Furthermore, it is unknown to what extent the bleeding during regenerative surgery may influence the adsorption of exogenous proteins to the surface of bone grafting materials and the subsequent cellular behavior. In the present study, the aim is to test EMD adsorption to the surface of natural bone mineral (NBM) particles in the presence of blood and determine the effect of EMD coating to NBM particles on downstream cellular pathways, such as adhesion, proliferation, and differentiation of primary human osteoblasts and PDL cells. NBM particles were precoated in various settings with EMD or human blood and analyzed for protein adsorption patterns via fluorescent imaging and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using fluorescent double-stranded DNA-binding dye. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding runt-related transcription factor 2, alkaline phosphatase (ALP), osteocalcin (OC), and collagen1α1 (COL1A1), and mineralization was assessed using red dye staining. Analysis of cell attachment and cell proliferation revealed significantly higher osteoblast and PDL cell attachment on EMD-coated surfaces when compared with control and blood-coated surfaces. EMD also stimulated release of growth factors and cytokines, including bone morphogenetic protein 2 and transforming growth factor β1. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers, including COL1A1, ALP, and OC, in osteoblasts and PDL cells cultured on EMD-coated NBM particles. The present results suggest that 1) EMD enhances osteoblast and PDL cell attachment, proliferation, and differentiation on NBM particles, and 2) blood contamination of the grafting material before mixing with EMD may inhibit EMD adsorption.

Towards decrypting cryptobiosis--analyzing anhydrobiosis in the tardigrade Milnesium tardigradum using transcriptome sequencing.

PubMed

Wang, Chong; Grohme, Markus A; Mali, Brahim; Schill, Ralph O; Frohme, Marcus

2014-01-01

Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair.

Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade Milnesium tardigradum Using Transcriptome Sequencing

PubMed Central

Wang, Chong; Grohme, Markus A.; Mali, Brahim; Schill, Ralph O.; Frohme, Marcus

2014-01-01

Background Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. Results A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Conclusions Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair. PMID:24651535

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

PubMed

Cerino, Giulia; Gaudiello, Emanuele; Grussenmeyer, Thomas; Melly, Ludovic; Massai, Diana; Banfi, Andrea; Martin, Ivan; Eckstein, Friedrich; Grapow, Martin; Marsano, Anna

2016-01-01

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment. © 2015 Wiley Periodicals, Inc.

Regional and temporal differences in gene expression of LH(BETA)T(AG) retinoblastoma tumors.

PubMed

Houston, Samuel K; Pina, Yolanda; Clarke, Jennifer; Koru-Sengul, Tulay; Scott, William K; Nathanson, Lubov; Schefler, Amy C; Murray, Timothy G

2011-07-23

The purpose of this study was to evaluate by microarray the hypothesis that LH(BETA)T(AG) retinoblastoma tumors exhibit regional and temporal variations in gene expression. LH(BETA)T(AG) mice aged 12, 16, and 20 weeks were euthanatized (n = 9). Specimens were taken from five tumor areas (apex, anterior lateral, center, base, and posterior lateral). Samples were hybridized to gene microarrays. The data were preprocessed and analyzed, and genes with a P < 0.01, according to the ANOVA models, and a log(2)-fold change >2.5 were considered to be differentially expressed. Differentially expressed genes were analyzed for overlap with known networks by using pathway analysis tools. There were significant temporal (P < 10(-8)) and regional differences in gene expression for LH(BETA)T(AG) retinoblastoma tumors. At P < 0.01 and log(2)-fold change >2.5, there were significant changes in gene expression of 190 genes apically, 84 genes anterolaterally, 126 genes posteriorly, 56 genes centrally, and 134 genes at the base. Differentially expressed genes overlapped with known networks, with significant involvement in regulation of cellular proliferation and growth, response to oxygen levels and hypoxia, regulation of cellular processes, cellular signaling cascades, and angiogenesis. There are significant temporal and regional variations in the LH(BETA)T(AG) retinoblastoma model. Differentially expressed genes overlap with key pathways that may play pivotal roles in murine retinoblastoma development. These findings suggest the mechanisms involved in tumor growth and progression in murine retinoblastoma tumors and identify pathways for analysis at a functional level, to determine significance in human retinoblastoma. Microarray analysis of LH(BETA)T(AG) retinal tumors showed significant regional and temporal variations in gene expression, including dysregulation of genes involved in hypoxic responses and angiogenesis.

Exposure of differentiated airway epithelial cells to volatile smoke in vitro.

PubMed

Beisswenger, Christoph; Platz, Juliane; Seifart, Carola; Vogelmeier, Claus; Bals, Robert

2004-01-01

Cigarette smoke (CS) is the predominant pathogenetic factor in the development of chronic bronchitis and chronic obstructive pulmonary disease. The knowledge about the cellular and molecular mechanisms underlying the smoke-induced inflammation in epithelial cells is limited. The aim of this study was to develop an in vitro model to monitor the effects of volatile CS on differentiated airway epithelial cells. The airway epithelial cell line MM-39 and primary human bronchial epithelial cells were cultivated as air-liquid interface cultures and exposed directly to volatile CS. We used two types of exposure models, one using ambient air, the other using humidified and warm air. Cytokine levels were measured by quantitative PCR and ELISA. Phosphorylation of p38 MAP kinase was assessed by Western blot analysis. To reduce the smoke-induced inflammation, antisense oligonucleotides directed against the p65 subunit of NF-kappaB were applied. Exposure of epithelia to cold and dry air resulted in a significant inflammatory response. In contrast, exposure to humidified warm air did not elicit a cellular response. Stimulation with CS resulted in upregulation of mRNA for IL-6 and IL-8 and protein release. Exposure to CS combined with heat-inactivated bacteria synergistically increased levels of the cytokines. Reactions of differentiated epithelial cells to smoke are mediated by the MAP kinase p38 and the transcription factor NF-kappaB. We developed an exposure model to examine the consequences of direct exposure of differentiated airway epithelial cells to volatile CS. The model enables to measure the cellular reactions to smoke exposure and to determine the outcome of therapeutic interventions. Copyright 2004 S. Karger AG, Basel

Cross-talk between T Cells and Hematopoietic Stem Cells during Adoptive Cellular Therapy for Malignant Glioma.

PubMed

Wildes, Tyler J; Grippin, Adam; Dyson, Kyle A; Wummer, Brandon M; Damiani, David J; Abraham, Rebecca S; Flores, Catherine T; Mitchell, Duane A

2018-04-30

Purpose: Adoptive T-cell immunotherapy (ACT) has emerged as a viable therapeutic for peripheral and central nervous system (CNS) tumors. In peripheral cancers, optimal efficacy of ACT is reliant on dendritic cells (DCs) in the tumor microenvironment. However, the CNS is largely devoid of resident migratory DCs to function as antigen-presenting cells during immunotherapy. Herein, we demonstrate that cellular interactions between adoptively transferred tumor-reactive T cells and bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) lead to the generation of potent intratumoral DCs within the CNS compartment. Experimental Design: We evaluated HSPC differentiation during ACT in vivo in glioma-bearing hosts and HSPC proliferation and differentiation in vitro using a T-cell coculture system. We utilized FACS, ELISAs, and gene expression profiling to study the phenotype and function of HSPC-derived cells ex vivo and in vivo. To demonstrate the impact of HSPC differentiation and function on antitumor efficacy, we performed survival experiments. Results: Transfer of HSPCs with concomitant ACT led to the production of activated CD86 + CD11c + MHCII + cells consistent with DC phenotype and function within the brain tumor microenvironment. These intratumoral DCs largely supplanted abundant host myeloid-derived suppressor cells. We determined that during ACT, HSPC-derived cells in gliomas rely on T-cell-released IFNγ to differentiate into DCs, activate T cells, and reject intracranial tumors. Conclusions: Our data support the use of HSPCs as a novel cellular therapy. Although DC vaccines induce robust immune responses in the periphery, our data demonstrate that HSPC transfer uniquely generates intratumoral DCs that potentiate T-cell responses and promote glioma rejection in situ Clin Cancer Res; 1-12. ©2018 AACR. ©2018 American Association for Cancer Research.

UVA and UVB Irradiation Differentially Regulate microRNA Expression in Human Primary Keratinocytes

PubMed Central

Kraemer, Anne; Chen, I-Peng; Henning, Stefan; Faust, Alexandra; Volkmer, Beate; Atkinson, Michael J.; Moertl, Simone; Greinert, Ruediger

2013-01-01

MicroRNA (miRNA)-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2), which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis. PMID:24391759

Exploring the mechanisms regulating regeneration of deer antlers.

PubMed Central

Price, J; Allen, S

2004-01-01

Deer antlers are the only mammalian appendages capable of repeated rounds of regeneration; every year they are shed and regrow from a blastema into large branched structures of cartilage and bone that are used for fighting and display. Longitudinal growth is by a process of modified endochondral ossification and in some species this can exceed 2 cm per day, representing the fastest rate of organ growth in the animal kingdom. However, despite their value as a unique model of mammalian regeneration the underlying mechanisms remain poorly understood. We review what is currently known about the local and systemic regulation of antler regeneration and some of the many unsolved questions of antler physiology are discussed. Molecules that we have identified as having potentially important local roles in antlers include parathyroid hormone-related peptide and retinoic acid (RA). Both are present in the blastema and in the rapidly growing antler where they regulate the differentiation of chondrocytes, osteoblasts and osteoclasts in vitro. Recent studies have shown that blockade of RA signalling can alter cellular differentiation in the blastema in vivo. The trigger that regulates the expression of these local signals is likely to be changing levels of sex steroids because the process of antler regeneration is linked to the reproductive cycle. The natural assumption has been that the most important hormone is testosterone, however, at a cellular level oestrogen may be a more significant regulator. Our data suggest that exogenous oestrogen acts as a 'brake', inhibiting the proliferation of progenitor cells in the antler tip while stimulating their differentiation, thus inhibiting continued growth. Deciphering the mechanism(s) by which sex steroids regulate cell-cycle progression and cellular differentiation in antlers may help to address why regeneration is limited in other mammalian tissues. PMID:15293809

Cellular fatty acids and aldehydes of oral Eubacterium.

PubMed

Itoh, U; Sato, M; Tsuchiya, H; Namikawa, I

1995-02-01

The cellular fatty acids and aldehydes of oral Eubacterium species were determined by gas chromatography-mass spectrometry. E. brachy and E. lentum contained mainly branched-chain fatty acids, whereas the others contained straight-chain acids. E. brachy, E. lentum, E. yurii ssp. yurii, E. yurii spp. margaretiae, E. limosum, E. plauti and E. aerofaciens also contained aldehydes with even carbon numbers. In addition to species-specific components, the compositional ratios of fatty acids and aldehydes characterized each individual species. The 10 species tested were divided into 5 groups by the principal component analysis. Cellular fatty acids and aldehydes would be chemical markers for interspecies differentiation of oral Eubacterium.

The effect of space and parabolic flight on macrophage hematopoiesis and function

NASA Technical Reports Server (NTRS)

Armstrong, J. W.; Gerren, R. A.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1995-01-01

We used weak electric fields to monitor macrophage spreading in microgravity. Using this technique, we demonstrated that bone marrow-derived macrophages responded to microgravity within 8 s. We also showed that microgravity differentially altered two processes associated with bone marrow-derived macrophage development. Spaceflight enhanced cellular proliferation and inhibited differentiation. These data indicate that the space/microgravity environment significantly affects macrophages.

Interleukin-6 inhibits early differentiation of ATDC5 chondrogenic progenitor cells.

PubMed

Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

2009-08-01

Interleukin (IL)-6 is a causative agent of systemic juvenile idiopathic arthritis (sJIA), a chronic inflammatory disease complicated with severe growth impairment. Recent trials of anti-IL-6 receptor monoclonal antibody, tocilizumab, indicated that tocilizumab blocks IL-6/IL-6 receptor-mediated inflammation, and induces catch-up growth in children with sJIA. This study evaluates the effects of IL-6 on chondrogenesis by ATDC5 cells, a clonal murine chondrogenic cell line that provides an excellent model for studying endochondral ossification at growth plate. ATDC5 cells were examined for the expression of IL-6 receptor and gp130 by fluorescence-activated cell sorting analysis. Recombinant murine IL-6 was added to ATDC5 cultures to observe cell differentiation, using a quantitative RT-PCR for the chondrogenic differentiation markers type II collagen, aggrecan, and type X collagen. To block IL-6, the anti-mouse IL-6 receptor monoclonal antibody MR16-1 was added. As a result, the cells expressed IL-6 receptor and gp130. The expression of chondrogenic differentiation marker gene was reduced by IL-6, but this was abrogated by MR16-1. We conclude that IL-6 inhibits early chondrogenesis of ATDC5 cells suggesting that IL-6 may affect committed stem cells at a cellular level during chondrogenic differentiation of growth plate chondrocytes, and that IL-6 may be a cellular-level factor in growth impairment in sJIA.

Glycosaminoglycan-Mimetic Signals Direct the Osteo/Chondrogenic Differentiation of Mesenchymal Stem Cells in a Three-Dimensional Peptide Nanofiber Extracellular Matrix Mimetic Environment.

PubMed

Arslan, Elif; Guler, Mustafa O; Tekinay, Ayse B

2016-04-11

Recent efforts in bioactive scaffold development focus strongly on the elucidation of complex cellular responses through the use of synthetic systems. Designing synthetic extracellular matrix (ECM) materials must be based on understanding of cellular behaviors upon interaction with natural and artificial scaffolds. Hence, due to their ability to mimic both the biochemical and mechanical properties of the native tissue environment, supramolecular assemblies of bioactive peptide nanostructures are especially promising for development of bioactive ECM-mimetic scaffolds. In this study, we used glycosaminoglycan (GAG) mimetic peptide nanofiber gel as a three-dimensional (3D) platform to investigate how cell lineage commitment is altered by external factors. We observed that amount of fetal bovine serum (FBS) presented in the cell media had synergistic effects on the ability of GAG-mimetic nanofiber gel to mediate the differentiation of mesenchymal stem cells into osteogenic and chondrogenic lineages. In particular, lower FBS concentration in the culture medium was observed to enhance osteogenic differentiation while higher amount FBS promotes chondrogenic differentiation in tandem with the effects of the GAG-mimetic 3D peptide nanofiber network, even in the absence of externally administered growth factors. We therefore demonstrate that mesenchymal stem cell differentiation can be specifically controlled by the combined influence of growth medium components and a 3D peptide nanofiber environment.

Melatonin antagonizes interleukin-18-mediated inhibition on neural stem cell proliferation and differentiation.

PubMed

Li, Zheng; Li, Xingye; Chan, Matthew T V; Wu, William Ka Kei; Tan, DunXian; Shen, Jianxiong

2017-09-01

Neural stem cells (NSCs) are self-renewing, pluripotent and undifferentiated cells which have the potential to differentiate into neurons, oligodendrocytes and astrocytes. NSC therapy for tissue regeneration, thus, gains popularity. However, the low survivals rate of the transplanted cell impedes its utilities. In this study, we tested whether melatonin, a potent antioxidant, could promote the NSC proliferation and neuronal differentiation, especially, in the presence of the pro-inflammatory cytokine interleukin-18 (IL-18). Our results showed that melatonin per se indeed exhibited beneficial effects on NSCs and IL-18 inhibited NSC proliferation, neurosphere formation and their differentiation into neurons. All inhibitory effects of IL-18 on NSCs were significantly reduced by melatonin treatment. Moreover, melatonin application increased the production of both brain-derived and glial cell-derived neurotrophic factors (BDNF, GDNF) in IL-18-stimulated NSCs. It was observed that inhibition of BDNF or GDNF hindered the protective effects of melatonin on NSCs. A potentially protective mechanism of melatonin on the inhibition of NSC's differentiation caused IL-18 may attribute to the up-regulation of these two major neurotrophic factors, BNDF and GNDF. The findings indicate that melatonin may play an important role promoting the survival of NSCs in neuroinflammatory diseases. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Phytocalpain controls the proliferation and differentiation fates of cells in plant organ development.

PubMed

Ahn, Joon-Woo; Kim, Moonil; Lim, Jeong Hwa; Kim, Gyung-Tae; Pai, Hyun-Sook

2004-06-01

Calpain, a calcium-dependent cysteine protease, plays an essential role in basic cellular processes in animal cells, including cell proliferation, apoptosis, and differentiation. NbDEK encodes the calpain homolog of N. benthamiana. In this study, virus-induced gene silencing (VIGS) of NbDEK resulted in arrested organ development and hyperplasia in all the major plant organs examined. The epidermal layers of the leaves and stems were covered with hyperproliferating cell masses, and stomata and trichome development was severely inhibited. During flower development, a single dome-like structure was grown from the flower meristem to generate a large cylinder-shaped flower lacking any floral organs. At the cellular level, cell division was sustained in tissues that were otherwise already differentiated, and cell differentiation was severely hampered. NbDEK is ubiquitously expressed in all the plant tissues examined. In the abnormal organs of the NbDEK VIGS lines, protein levels of D-type cyclins (CycD)2, CycD3, and proliferating cell nuclear antigen (PCNA) were greatly elevated, and transcription of E2F (E2 promoter binding factor), E2F-regulated genes, retinoblastoma (Rb), and KNOTTED1 (KN1)-type homeobox genes was also stimulated. These results suggest that phytocalpain is a key regulator of cell proliferation and differentiation during plant organogenesis, and that it acts partly by controlling the CycD/Rb pathway.

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed Central

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-01-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes. PMID:9121442

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-04-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Morin hydrate promotes inner ear neural stem cell survival and differentiation and protects cochlea against neuronal hearing loss.

PubMed

He, Qiang; Jia, Zhanwei; Zhang, Ying; Ren, Xiumin

2017-03-01

We aimed to investigate the effect of morin hydrate on neural stem cells (NSCs) isolated from mouse inner ear and its potential in protecting neuronal hearing loss. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and bromodeoxyuridine incorporation assays were employed to assess the effect of morin hydrate on the viability and proliferation of in vitro NSC culture. The NSCs were then differentiated into neurons, in which neurosphere formation and differentiation were evaluated, followed by neurite outgrowth and neural excitability measurements in the subsequent in vitro neuronal network. Mechanotransduction of cochlea ex vivo culture and auditory brainstem responses threshold and distortion product optoacoustic emissions amplitude in mouse ototoxicity model were also measured following gentamicin treatment to investigate the protective role of morin hydrate against neuronal hearing loss. Morin hydrate improved viability and proliferation, neurosphere formation and neuronal differentiation of inner ear NSCs, and promoted in vitro neuronal network functions. In both ex vivo and in vivo ototoxicity models, morin hydrate prevented gentamicin-induced neuronal hearing loss. Morin hydrate exhibited potent properties in promoting growth and differentiation of inner ear NSCs into functional neurons and protecting from gentamicin ototoxicity. Our study supports its clinical potential in treating neuronal hearing loss. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Notch3 is necessary for neuronal differentiation and maturation in the adult spinal cord.

PubMed

Rusanescu, Gabriel; Mao, Jianren

2014-10-01

Notch receptors are key regulators of nervous system development and promoters of neural stem cells renewal and proliferation. Defects in the expression of Notch genes result in severe, often lethal developmental abnormalities. Notch3 is generally thought to have a similar proliferative, anti-differentiation and gliogenic role to Notch1. However, in some cases, Notch3 has an opposite, pro-differentiation effect. Here, we show that Notch3 segregates from Notch1 and is transiently expressed in adult rat and mouse spinal cord neuron precursors and immature neurons. This suggests that during the differentiation of adult neural progenitor cells, Notch signalling may follow a modified version of the classical lateral inhibition model, involving the segregation of individual Notch receptors. Notch3 knockout mice, otherwise neurologically normal, are characterized by a reduced number of mature inhibitory interneurons and an increased number of highly excitable immature neurons in spinal cord laminae I-II. As a result, these mice have permanently lower nociceptive thresholds, similar to chronic pain. These results suggest that defective neuronal differentiation, for example as a result of reduced Notch3 expression or activation, may underlie human cases of intractable chronic pain, such as fibromyalgia and neuropathic pain. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Microarray Analysis of Gene Expression Alteration in Human Middle Ear Epithelial Cells Induced by Asian Sand Dust.

PubMed

Go, Yoon Young; Park, Moo Kyun; Kwon, Jee Young; Seo, Young Rok; Chae, Sung-Won; Song, Jae-Jun

2015-12-01

The primary aim of this study is to evaluate the gene expression profile of Asian sand dust (ASD)-treated human middle ear epithelial cell (HMEEC) using microarray analysis. The HMEEC was treated with ASD (400 µg/mL) and total RNA was extracted for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed. For selected genes, the changes in gene expression were confirmed by real-time polymerase chain reaction. A total of 1,274 genes were differentially expressed by ASD. Among them, 1,138 genes were 2 folds up-regulated, whereas 136 genes were 2 folds down-regulated. Up-regulated genes were mainly involved in cellular processes, including apoptosis, cell differentiation, and cell proliferation. Down-regulated genes affected cellular processes, including apoptosis, cell cycle, cell differentiation, and cell proliferation. The 10 genes including ADM, CCL5, EDN1, EGR1, FOS, GHRL, JUN, SOCS3, TNF, and TNFSF10 were identified as main modulators in up-regulated genes. A total of 11 genes including CSF3, DKK1, FOSL1, FST, TERT, MMP13, PTHLH, SPRY2, TGFBR2, THBS1, and TIMP1 acted as main components of pathway associated with 2-fold down regulated genes. We identified the differentially expressed genes in ASD-treated HMEEC. Our work indicates that air pollutant like ASD, may play an important role in the pathogenesis of otitis media.

Molecular mechanism of G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells.

PubMed

Tao, Yan-Fang; Wang, Na-Na; Xu, Li-Xiao; Li, Zhi-Heng; Li, Xiao-Lu; Xu, Yun-Yun; Fang, Fang; Li, Mei; Qian, Guang-Hui; Li, Yan-Hong; Li, Yi-Ping; Wu, Yi; Ren, Jun-Li; Du, Wei-Wei; Lu, Jun; Feng, Xing; Wang, Jian; He, Wei-Qi; Hu, Shao-Yan; Pan, Jian

2017-01-01

Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16 INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G 1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16 INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G 1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.

Hypoxia modulates the differentiation potential of stem cells of the apical papilla.

PubMed

Vanacker, Julie; Viswanath, Aiswarya; De Berdt, Pauline; Everard, Amandine; Cani, Patrice D; Bouzin, Caroline; Feron, Olivier; Diogenes, Anibal; Leprince, Julian G; des Rieux, Anne

2014-09-01

Stem cells from the apical papilla (SCAP) are a population of mesenchymal stem cells likely involved in regenerative endodontic procedures and have potential use as therapeutic agents in other tissues. In these situations, SCAP are exposed to hypoxic conditions either within a root canal devoid of an adequate blood supply or in a scaffold material immediately after implantation. However, the effect of hypoxia on SCAP proliferation and differentiation is largely unknown. Therefore, the objective of this study was to evaluate the effect of hypoxia on the fate of SCAP. SCAP were cultured under normoxia (21% O2) or hypoxia (1% O2) in basal or differentiation media. Cellular proliferation, gene expression, differentiation, and protein secretion were analyzed by live imaging, quantitative reverse-transcriptase polymerase chain reaction, cellular staining, and enzyme-linked immunosorbent assay, respectively. Hypoxia had no effect on SCAP proliferation, but it evoked the up-regulation of genes specific for osteogenic differentiation (runt-related transcription factor 2, alkaline phosphatase, and transforming growth factor-β1), neuronal differentiation ( 2'-3'-cyclic nucleotide 3' phosphodiesterase, SNAIL, neuronspecific enolase, glial cell-derived neurotrophic factor and neurotrophin 3), and angiogenesis (vascular endothelial growth factor A and B). Hypoxia also increased the sustained production of VEGFa by SCAP. Moreover, hypoxia augmented the neuronal differentiation of SCAP in the presence of differentiation exogenous factors as detected by the up-regulation of NSE, VEGFB, and GDNF and the expression of neuronal markers (PanF and NeuN). This study shows that hypoxia induces spontaneous differentiation of SCAP into osteogenic and neurogenic lineages while maintaining the release of the proangiogenic factor VEGFa. This highlights the potential of SCAP to promote pulp-dentin regeneration. Moreover, SCAP may represent potential therapeutic agents for neurodegenerative conditions because of their robust differentiation potential. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Canine mammary minute oncocytomas with neuroendocrine differentiation associated with multifocal acinar cell oncocytic metaplasia.

PubMed

Nagahara, Rei; Kimura, Masayuki; Itahashi, Megu; Sugahara, Go; Kawashima, Masashi; Murayama, Hirotada; Yoshida, Toshinori; Shibutani, Makoto

2016-11-01

Two solitary and minute tumors of 1 and 1.5 mm diameter were identified by microscopy in the left fourth mammary gland of a 13-year-old female Labrador Retriever dog, in addition to multiple mammary gland tumors. The former tumors were well circumscribed and were composed of small-to-large polyhedral neoplastic oncocytes with finely granular eosinophilic cytoplasm, and were arranged in solid nests separated by fine fibrovascular septa. Scattered lumina of variable sizes containing eosinophilic secretory material were evident. Cellular atypia was minimal, and no mitotic figures were visible. One tumor had several oncocytic cellular foci revealing cellular transition, with perivascular pseudorosettes consisting of columnar epithelial cells surrounding the fine vasculature. Scattered foci of mammary acinar cell hyperplasia showing oncocytic metaplasia were also observed. Immunohistochemically, the cytoplasm of neoplastic cells of the 2 microtumors showed diffuse immunoreactivity to anti-cytokeratin antibody AE1/AE3, and finely granular immunoreactivity for 60-kDa heat shock protein, mitochondrial membrane ATP synthase complex V beta subunit, and chromogranin A. One tumor also had oncocytic cellular foci forming perivascular pseudorosettes showing cellular membrane immunoreactivity for neural cell adhesion molecule. The tumors were negative for smooth muscle actin, neuron-specific enolase, vimentin, desmin, S100, and synaptophysin. Ultrastructural observation confirmed the abundant mitochondria in the cytoplasm of both neoplastic and hyperplastic cells, the former cells also having neuroendocrine granule-like electron-dense bodies. From these results, our case was diagnosed with mammary oncocytomas accompanied by neuroendocrine differentiation. Scattered foci of mammary oncocytosis might be related to the multicentric occurrence of these oncocytomas. © 2016 The Author(s).

A gradual update method for simulating the steady-state solution of stiff differential equations in metabolic circuits.

PubMed

Shiraishi, Emi; Maeda, Kazuhiro; Kurata, Hiroyuki

2009-02-01

Numerical simulation of differential equation systems plays a major role in the understanding of how metabolic network models generate particular cellular functions. On the other hand, the classical and technical problems for stiff differential equations still remain to be solved, while many elegant algorithms have been presented. To relax the stiffness problem, we propose new practical methods: the gradual update of differential-algebraic equations based on gradual application of the steady-state approximation to stiff differential equations, and the gradual update of the initial values in differential-algebraic equations. These empirical methods show a high efficiency for simulating the steady-state solutions for the stiff differential equations that existing solvers alone cannot solve. They are effective in extending the applicability of dynamic simulation to biochemical network models.

Evaluation of uridine 5'-eicosylphosphate as a stimulant of cyclic AMP-dependent cellular function.

PubMed

Yutani, Masahiro; Ogita, Akira; Fujita, Ken-Ichi; Usuki, Yoshinosuke; Tanaka, Toshio

2011-03-01

Sporulation of the yeast Saccharomyces cerevisiae is negatively regulated by cyclic AMP (cAMP). This microbial cell differentiation process was applied for the screening of a substance that can elevate the intracellular cAMP level. Among nucleoside 5'-alkylphosphates, uridine 5'-eicosylphosphate (UMPC20) selectively and predominantly inhibited ascospore formation of the yeast cells. We suppose the inhibitory effect of UMPC20 could indeed reflect the elevation of the cellular cAMP level.

Almost periodic cellular neural networks with neutral-type proportional delays

NASA Astrophysics Data System (ADS)

Xiao, Songlin

2018-03-01

This paper presents a new result on the existence, uniqueness and generalised exponential stability of almost periodic solutions for cellular neural networks with neutral-type proportional delays and D operator. Based on some novel differential inequality techniques, a testable condition is derived to ensure that all the state trajectories of the system converge to an almost periodic solution with a positive exponential convergence rate. The effectiveness of the obtained result is illustrated by a numerical example.

A Jagged 1-Notch 4 molecular switch mediates airway inflammation induced by ultrafine particles.

PubMed

Xia, Mingcan; Harb, Hani; Saffari, Arian; Sioutas, Constantinos; Chatila, Talal A

2018-04-05

Exposure to traffic-related particulate matter promotes asthma and allergic diseases. However, the precise cellular and molecular mechanisms by which particulate matter exposure acts to mediate these effects remain unclear. We sought to elucidate the cellular targets and signaling pathways critical for augmentation of allergic airway inflammation induced by ambient ultrafine particles (UFP). We used in vitro cell-culture assays with lung-derived antigen-presenting cells and allergen-specific T cells and in vivo mouse models of allergic airway inflammation with myeloid lineage-specific gene deletions, cellular reconstitution approaches, and antibody inhibition studies. We identified lung alveolar macrophages (AM) as the key cellular target of UFP in promoting airway inflammation. Aryl hydrocarbon receptor-dependent induction of Jagged 1 (Jag1) expression in AM was necessary and sufficient for augmentation of allergic airway inflammation by UFP. UFP promoted T H 2 and T H 17 cell differentiation of allergen-specific T cells in a Jag1- and Notch 4-dependent manner. Treatment of mice with an anti-Notch 4 antibody abrogated exacerbation of allergic airway inflammation induced by UFP. UFP exacerbate allergic airway inflammation by promoting a Jag1-Notch 4-dependent interaction between AM and allergen-specific T cells, leading to augmented T H cell differentiation. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Heparin concentration is critical for cell culture with human platelet lysate.

PubMed

Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang

2013-09-01

Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

A Unique Fungal Two-Component System Regulates Stress Responses, Drug Sensitivity, Sexual Development, and Virulence of Cryptococcus neoformans

PubMed Central

Bahn, Yong-Sun; Kojima, Kaihei; Cox, Gary M.

2006-01-01

The stress-activated mitogen-activated protein kinase (MAPK) pathway is widely used by eukaryotic organisms as a central conduit via which cellular responses to the environment effect growth and differentiation. The basidiomycetous human fungal pathogen Cryptococcus neoformans uniquely uses the stress-activated Pbs2-Hog1 MAPK system to govern a plethora of cellular events, including stress responses, drug sensitivity, sexual reproduction, and virulence. Here, we characterized a fungal “two-component” system that controls these fundamental cellular functions via the Pbs2-Hog1 MAPK cascade. A typical response regulator, Ssk1, modulated all Hog1-dependent phenotypes by controlling Hog1 phosphorylation, indicating that Ssk1 is the major upstream signaling component of the Pbs2-Hog1 pathway. A second response regulator, Skn7, governs sensitivity to Na+ ions and the antifungal agent fludioxonil, negatively controls melanin production, and functions independently of Hog1 regulation. To control these response regulators, C. neoformans uses multiple sensor kinases, including two-component–like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. Our findings highlight unique adaptations of this global two-component MAPK signaling cascade in a ubiquitous human fungal pathogen. PMID:16672377

Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin.

PubMed

Sada, Aiko; Jacob, Fadi; Leung, Eva; Wang, Sherry; White, Brian S; Shalloway, David; Tumbar, Tudorita

2016-06-01

The interfollicular epidermis regenerates from heterogeneous basal skin cell populations that divide at different rates. It has previously been presumed that infrequently dividing basal cells known as label-retaining cells (LRCs) are stem cells, whereas non-LRCs are short-lived progenitors. Here we employ the H2B-GFP pulse-chase system in adult mouse skin and find that epidermal LRCs and non-LRCs are molecularly distinct and can be differentiated by Dlx1(CreER) and Slc1a3(CreER) genetic marking, respectively. Long-term lineage tracing and mathematical modelling of H2B-GFP dilution data show that LRCs and non-LRCs constitute two distinct stem cell populations with different patterns of proliferation, differentiation and upward cellular transport. During homeostasis, these populations are enriched in spatially distinct skin territories and can preferentially produce unique differentiated lineages. On wounding or selective killing, they can temporarily replenish each other's territory. These two discrete interfollicular stem cell populations are functionally interchangeable and intrinsically well adapted to thrive in distinct skin environments.

The epithelial-mesenchymal interactions: insights into physiological and pathological aspects of oral tissues.

PubMed

Santosh, Arvind Babu Rajendra; Jones, Thaon Jon

2014-03-17

In the human biological system, the individual cells divide and form tissues and organs. These tissues are hetero-cellular. Basically any tissue consists of an epithelium and the connective tissue. The latter contains mainly mesenchymally-derived tissues with a diversified cell population. The cell continues to grow and differentiate in a pre-programmed manner using a messenger system. The epithelium and the mesenchymal portion of each tissue have two different origins and perform specific functions, but there is a well-defined interaction mechanism, which mediates between them. Epithelial mesenchymal interactions (EMIs) are part of this mechanism, which can be regarded as a biological conversation between epithelial and mesenchymal cell populations involved in the cellular differentiation of one or both cell populations. EMIs represent a process that is essential for cell growth, cell differentiation and cell multiplication. EMIs are associated with normal physiological processes in the oral cavity, such as odontogenesis, dentino-enamel junction formation, salivary gland development, palatogenesis, and also pathological processes, such as oral cancer. This paper focuses the role EMIs in odontogenesis, salivary gland development, palatogenesis and oral cancer.

Plasticity for axolotl lens regeneration is associated with age‐related changes in gene expression

PubMed Central

Sousounis, Konstantinos; Athippozhy, Antony T.; Voss, S. Randal

2014-01-01

Abstract Mexican axolotls lose potential for lens regeneration 2 weeks after hatching. We used microarrays to identify differently expressed genes before and after this critical time, using RNA isolated from iris. Over 3700 genes were identified as differentially expressed in response to lentectomy between young (7 days post‐hatching) and old (3 months post‐hatching) axolotl larvae. Strikingly, many of the genes were only expressed in the early or late iris. Genes that were highly expressed in young iris significantly enriched electron transport chain, transcription, metabolism, and cell cycle gene ontologies, all of which are associated with lens regeneration. In contrast, genes associated with cellular differentiation and tissue maturation were uniquely expressed in old iris. Many of these expression differences strongly suggest that young and old iris samples were collected before and after the spleen became developmentally competent to produce and secrete cells with humoral and innate immunity functions. Our study establishes the axolotl as a powerful model to investigate age‐related cellular differentiation and immune system ontogeny within the context of tissue regeneration. PMID:27499863

Functional diversification and specialization of cytosolic 70-kDa heat shock proteins.

PubMed

McCallister, Chelsea; Siracusa, Matthew C; Shirazi, Farzaneh; Chalkia, Dimitra; Nikolaidis, Nikolas

2015-03-20

A fundamental question in molecular evolution is how protein functional differentiation alters the ability of cells and organisms to cope with stress and survive. To answer this question we used two paralogous Hsp70s from mouse and explored whether these highly similar cytosolic molecular chaperones, which apart their temporal expression have been considered functionally interchangeable, are differentiated with respect to their lipid-binding function. We demonstrate that the two proteins bind to diverse lipids with different affinities and therefore are functionally specialized. The observed lipid-binding patterns may be related with the ability of both Hsp70s to induce cell death by binding to a particular plasma-membrane lipid, and the potential of only one of them to promote cell survival by binding to a specific lysosomal-membrane lipid. These observations reveal that two seemingly identical proteins differentially modulate cellular adaptation and survival by having acquired specialized functions via sequence divergence. Therefore, this study provides an evolutionary paradigm, where promiscuity, specificity, sub- and neo-functionalization orchestrate one of the most conserved systems in nature, the cellular stress-response.

Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells.

PubMed

Soto-Cerrato, Vanessa; Manuel-Manresa, Pilar; Hernando, Elsa; Calabuig-Fariñas, Silvia; Martínez-Romero, Alicia; Fernández-Dueñas, Víctor; Sahlholm, Kristoffer; Knöpfel, Thomas; García-Valverde, María; Rodilla, Ananda M; Jantus-Lewintre, Eloisa; Farràs, Rosa; Ciruela, Francisco; Pérez-Tomás, Ricardo; Quesada, Roberto

2015-12-23

Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.

Reactivity of commercially available monoclonal antibodies to human CD antigens with peripheral blood leucocytes of dromedary camels (Camelus dromedarius)

PubMed Central

Hussen, Jamal; Shawaf, Turke; Al-herz, Abdulkareem Imran; Alturaifi, Hussain R.; Alluwaimi, Ahmed M.

2017-01-01

Monoclonal antibodies (mAbs) to cell surface molecules have been proven as a key tool for phenotypic and functional characterization of the cellular immune response. One of the major difficulties in studying camel cellular immunity consists in the lack of mAbs that dtect their leukocyte differentiation antigens. In the present study two-parameter flow cytometry was used to screen existing commercially available mAbs to human leukocyte antigens and major histocompatibility molecules (MHC) for their reactivity with camel leukocytes. The comparison of patterns of reactivity obtained after labelling human and camel leukocytes have shown that mAbs specific to human cluster of differentiation (CD) 18, CD11a, CD11b and CD14 are predicted to be cross-reactive with homologous camel antigens. PMID:28652982

Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

NASA Astrophysics Data System (ADS)

Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

2005-05-01

Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( μg) may modulate the proliferation and differentiation. We investigated the application of μg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated μg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated μg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

Non-linear optical measurements using a scanned, Bessel beam

NASA Astrophysics Data System (ADS)

Collier, Bradley B.; Awasthi, Samir; Lieu, Deborah K.; Chan, James W.

2015-03-01

Oftentimes cells are removed from the body for disease diagnosis or cellular research. This typically requires fluorescent labeling followed by sorting with a flow cytometer; however, possible disruption of cellular function or even cell death due to the presence of the label can occur. This may be acceptable for ex vivo applications, but as cells are more frequently moving from the lab to the body, label-free methods of cell sorting are needed to eliminate these issues. This is especially true of the growing field of stem cell research where specialized cells are needed for treatments. Because differentiation processes are not completely efficient, cells must be sorted to eliminate any unwanted cells (i.e. un-differentiated or differentiated into an unwanted cell type). In order to perform label-free measurements, non-linear optics (NLO) have been increasingly utilized for single cell analysis because of their ability to not disrupt cellular function. An optical system was developed for the measurement of NLO in a microfluidic channel similar to a flow cytometer. In order to improve the excitation efficiency of NLO, a scanned Bessel beam was utilized to create a light-sheet across the channel. The system was tested by monitoring twophoton fluorescence from polystyrene microbeads of different sizes. Fluorescence intensity obtained from light-sheet measurements were significantly greater than measurements made using a static Gaussian beam. In addition, the increase in intensity from larger sized beads was more evident for the light-sheet system.

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

T Cell LEGO: Identifying the Master Builders and What They Do.

PubMed

Li, Jasmine; Turner, Stephen J

2018-02-20

Understanding how cell fate decisions are made during cellular differentiation and the mechanisms that drive them is a holy grail of cell biology. In this issue of Immunity, Hu et al. (2018) and Johnson et al. (2018) demonstrate that key transcriptional regulators and global changes in nuclear architecture underlie differentiation decisions during T cell development. Copyright © 2018 Elsevier Inc. All rights reserved.

CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells

PubMed Central

Valli, Emanuele; Trazzi, Stefania; Fuchs, Claudia; Erriquez, Daniela; Bartesaghi, Renata; Perini, Giovanni; Ciani, Elisabetta

2012-01-01

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G0/G1 phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5. PMID:22921766

Self-organization of human embryonic stem cells on micropatterns

PubMed Central

Deglincerti, Alessia; Etoc, Fred; Guerra, M. Cecilia; Martyn, Iain; Metzger, Jakob; Ruzo, Albert; Simunovic, Mijo; Yoney, Anna; Brivanlou, Ali H.; Siggia, Eric; Warmflash, Aryeh

2018-01-01

Fate allocation in the gastrulating embryo is spatially organized as cells differentiate to specialized cell types depending on their positions with respect to the body axes. There is a need for in vitro protocols that allow the study of spatial organization associated with this developmental transition. While embryoid bodies and organoids can exhibit some spatial organization of differentiated cells, these methods do not yield consistent and fully reproducible results. Here, we describe a micropatterning approach where human embryonic stem cells are confined to disk-shaped, sub-millimeter colonies. After 42 hours of BMP4 stimulation, cells form self-organized differentiation patterns in concentric radial domains, which express specific markers associated with the embryonic germ layers, reminiscent of gastrulating embryos. Our protocol takes 3 days; it uses commercial microfabricated slides (CYTOO), human laminin-521 (LN-521) as extra-cellular matrix coating, and either conditioned or chemically-defined medium (mTeSR). Differentiation patterns within individual colonies can be determined by immunofluorescence and analyzed with cellular resolution. Both the size of the micropattern and the type of medium affect the patterning outcome. The protocol is appropriate for personnel with basic stem cell culture training. This protocol describes a robust platform for quantitative analysis of the mechanisms associated with pattern formation at the onset of gastrulation. PMID:27735934

Oligodendroglial p130Cas Is a Target of Fyn Kinase Involved in Process Formation, Cell Migration and Survival

PubMed Central

Gonsior, Constantin; Binamé, Fabien; Frühbeis, Carsten; Bauer, Nina M.; Hoch-Kraft, Peter; Luhmann, Heiko J.; Trotter, Jacqueline; White, Robin

2014-01-01

Oligodendrocytes are the myelinating glial cells of the central nervous system. In the course of brain development, oligodendrocyte precursor cells migrate, scan the environment and differentiate into mature oligodendrocytes with multiple cellular processes which recognize and ensheath neuronal axons. During differentiation, oligodendrocytes undergo dramatic morphological changes requiring cytoskeletal rearrangements which need to be tightly regulated. The non-receptor tyrosine kinase Fyn plays a central role in oligodendrocyte differentiation and myelination. In order to improve our understanding of the role of oligodendroglial Fyn kinase, we have identified Fyn targets in these cells. Purification and mass-spectrometric analysis of tyrosine-phosphorylated proteins in response to overexpressed active Fyn in the oligodendrocyte precursor cell line Oli-neu, yielded the adaptor molecule p130Cas. We analyzed the function of this Fyn target in oligodendroglial cells and observed that reduction of p130Cas levels by siRNA affects process outgrowth, the thickness of cellular processes and migration behavior of Oli-neu cells. Furthermore, long term p130Cas reduction results in decreased cell numbers as a result of increased apoptosis in cultured primary oligodendrocytes. Our data contribute to understanding the molecular events taking place during oligodendrocyte migration and morphological differentiation and have implications for myelin formation. PMID:24586768

Establishment of immortal multipotent rat salivary progenitor cell line toward salivary gland regeneration.

PubMed

Yaniv, Adi; Neumann, Yoav; David, Ran; Stiubea-Cohen, Raluca; Orbach, Yoav; Lang, Stephan; Rotter, Nicole; Dvir-Ginzberg, Mona; Aframian, Doron J; Palmon, Aaron

2011-01-01

Adult salivary gland stem cells are promising candidates for cell therapy and tissue regeneration in cases of irreversible damage to salivary glands in head and neck cancer patients undergoing irradiation therapy. At present, the major restriction in handling such cells is their relatively limited life span during in vitro cultivation, resulting in an inadequate experimental platform to explore the salivary gland-originated stem cells as candidates for future clinical application in therapy. We established a spontaneous immortal integrin α6β1-expressing cell line of adult salivary progenitor cells from rats (rat salivary clone [RSC]) and investigated their ability to sustain cellular properties. This line was able to propagate for more than 400 doublings without loss of differentiation potential. RSC could differentiate in vitro to both acinar- and ductal-like structures and could be further manipulated upon culturing on a 3D scaffolds with different media supplements. Moreover, RSC expressed salivary-specific mRNAs and proteins as well as epithelial stem cell markers, and upon differentiation process their expression was changed. These results suggest RSC as a good model for further studies exploring cellular senescence, differentiation, and in vitro tissue engineering features as a crucial step toward reengineering irradiation-impaired salivary glands.

Photostimulation of osteogenic differentiation on silk scaffolds by plasma arc light source.

PubMed

Çakmak, Anıl Sera; Çakmak, Soner; Vatansever, H Seda; Gümüşderelioğlu, Menemşe

2018-05-01

Low-level laser therapy (LLLT) has been used for more than 30 years to heal wounds. In recent years, LLLT or photostimulation has been indicated as an effective tool for regenerative and dental medicine by using monochromatic light. The aim of this study is to indicate the usability of plasma arc light source for bone regeneration. This is why we used polychromatic light source providing effective wavelengths in the range of 590-1500 nm for cellular response and investigated photostimulation effects on osteogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D silk scaffolds. Cellular responses were examined by using cell culture methods in terms of proliferation, differentiation, and morphological analyses. The results showed that photostimulation with a polychromatic light source (applied for 5 min from the 3rd day after seeding up to the 28th day in 2-day intervals with 92-mW/cm 2 power from 10-cm distance to the cells) enhanced osteogenic differentiation of hMSCs according to higher alkaline phosphatase (ALP) activity, collagen and calcium content, osteogenic gene expressions, and matrix mineralization. In conclusion, we suggest that the plasma arc light source that was used here has a great potential for bone regeneration.

Disease relevant modifications of the methylome and transcriptome by particulate matter (PM2.5) from biomass combustion.

PubMed

Heßelbach, Katharina; Kim, Gwang-Jin; Flemming, Stephan; Häupl, Thomas; Bonin, Marc; Dornhof, Regina; Günther, Stefan; Merfort, Irmgard; Humar, Matjaz

2017-09-01

Exposure to particulate matter (PM) is recognized as a major health hazard, but molecular responses are still insufficiently described. We analyzed the epigenetic impact of ambient PM 2.5 from biomass combustion on the methylome of primary human bronchial epithelial BEAS-2B cells using the Illumina HumanMethylation450 BeadChip. The transcriptome was determined by the Affymetrix HG-U133 Plus 2.0 Array. PM 2.5 induced genome wide alterations of the DNA methylation pattern, including differentially methylated CpGs in the promoter region associated with CpG islands. Gene ontology analysis revealed that differentially methylated genes were significantly clustered in pathways associated with the extracellular matrix, cellular adhesion, function of GTPases, and responses to extracellular stimuli, or were involved in ion binding and shuttling. Differential methylations also affected tandem repeats. Additionally, 45 different miRNA CpG loci showed differential DNA methylation, most of them proximal to their promoter. These miRNAs are functionally relevant for lung cancer, inflammation, asthma, and other PM-associated diseases. Correlation of the methylome and transcriptome demonstrated a clear bias toward transcriptional activation by hypomethylation. Genes that exhibited both differential methylation and expression were functionally linked to cytokine and immune responses, cellular motility, angiogenesis, inflammation, wound healing, cell growth, differentiation and development, or responses to exogenous matter. Disease ontology of differentially methylated and expressed genes indicated their prominent role in lung cancer and their participation in dominant cancer related signaling pathways. Thus, in lung epithelial cells, PM 2.5 alters the methylome of genes and noncoding transcripts or elements that might be relevant for PM- and lung-associated diseases.

Disease relevant modifications of the methylome and transcriptome by particulate matter (PM2.5) from biomass combustion

PubMed Central

Heßelbach, Katharina; Kim, Gwang-Jin; Flemming, Stephan; Häupl, Thomas; Bonin, Marc; Dornhof, Regina; Günther, Stefan; Merfort, Irmgard; Humar, Matjaz

2017-01-01

ABSTRACT Exposure to particulate matter (PM) is recognized as a major health hazard, but molecular responses are still insufficiently described. We analyzed the epigenetic impact of ambient PM2.5 from biomass combustion on the methylome of primary human bronchial epithelial BEAS-2B cells using the Illumina HumanMethylation450 BeadChip. The transcriptome was determined by the Affymetrix HG-U133 Plus 2.0 Array. PM2.5 induced genome wide alterations of the DNA methylation pattern, including differentially methylated CpGs in the promoter region associated with CpG islands. Gene ontology analysis revealed that differentially methylated genes were significantly clustered in pathways associated with the extracellular matrix, cellular adhesion, function of GTPases, and responses to extracellular stimuli, or were involved in ion binding and shuttling. Differential methylations also affected tandem repeats. Additionally, 45 different miRNA CpG loci showed differential DNA methylation, most of them proximal to their promoter. These miRNAs are functionally relevant for lung cancer, inflammation, asthma, and other PM-associated diseases. Correlation of the methylome and transcriptome demonstrated a clear bias toward transcriptional activation by hypomethylation. Genes that exhibited both differential methylation and expression were functionally linked to cytokine and immune responses, cellular motility, angiogenesis, inflammation, wound healing, cell growth, differentiation and development, or responses to exogenous matter. Disease ontology of differentially methylated and expressed genes indicated their prominent role in lung cancer and their participation in dominant cancer related signaling pathways. Thus, in lung epithelial cells, PM2.5 alters the methylome of genes and noncoding transcripts or elements that might be relevant for PM- and lung-associated diseases. PMID:28742980

Characterization of tumor cells and stem cells by differential nuclear methylation imaging

NASA Astrophysics Data System (ADS)

Tajbakhsh, Jian; Wawrowsky, Kolja A.; Gertych, Arkadiusz; Bar-Nur, Ori; Vishnevsky, Eugene; Lindsley, Erik H.; Farkas, Daniel L.

2008-02-01

DNA methylation plays a key role in cellular differentiation. Aberrant global methylation patterns are associated with several cancer types, as a result of changes in long-term activation status of up to 50% of genes, including oncogenes and tumor-suppressor genes, which are regulated by methylation and demethylation of promoter region CpG dinucleotides (CpG islands). Furthermore, DNA methylation also occurs in nonisland CpG sites (> 95% of the genome), present once per 80 dinucleotides on average. Nuclear DNA methylation increases during the course of cellular differentiation while cancer cells usually show a net loss in methylation. Given the large dynamic range in DNA methylation load, the methylation pattern of a cell can provide a valuable distinction as to its status during differentiation versus the disease state. By applying immunofluorescence, confocal microscopy and 3D image analysis we assessed the potential of differential nuclear distribution of methylated DNA to be utilized as a biomarker to characterize cells during development and when diseased. There are two major fields that may immediately benefit from this development: (1) the search for factors that contribute to pluripotency and cell fate in human embryonic stem cell expansion and differentiation, and (2) the characterization of tumor cells with regard to their heterogeneity in molecular composition and behavior. We performed topological analysis of the distribution of methylated CpG-sites (MeC) versus heterochromatin. This innovative approach revealed significant differences in colocalization patterns of MeC and heterochromatin-derived signals between undifferentiated and differentiated human embryonic stem cells, as well as untreated AtT20 mouse pituitary tumor cells compared to a subpopulation of these cells treated with 5-azacytidine for 48 hours.

Phosphorylation of serine 779 in fibroblast growth factor receptor 1 and 2 by protein kinase C(epsilon) regulates Ras/mitogen-activated protein kinase signaling and neuronal differentiation.

PubMed

Lonic, Ana; Powell, Jason A; Kong, Yang; Thomas, Daniel; Holien, Jessica K; Truong, Nhan; Parker, Michael W; Guthridge, Mark A

2013-05-24

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser(779) in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser(779) in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser(779) was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCε can phosphorylate Ser(779) in vitro, whereas overexpression of PKCε results in constitutive Ser(779) phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCε reduces both growth factor-induced Ser(779) phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser(779), can quantitatively control Ras/MAPK signaling to promote specific cellular responses.

Radioembolization with 90Y glass microspheres for hepatocellular carcinoma: significance of pretreatment 11C-acetate and 18F-FDG PET/CT and posttreatment 90Y PET/CT in individualized dose prescription.

PubMed

Ho, Chi Lai; Chen, Sirong; Cheung, Shing Kee; Leung, Yim Lung; Cheng, Kam Chau; Wong, Ka Nin; Wong, Yuet Hung; Leung, Thomas Wai Tong

2018-06-11

The aim of this study was to establish an algorithm for the prescription of 90 Y glass microsphere radioembolization ( 90 Y-GMRE) of HCC in individual patients based on the relationship between tumour dose (TD) and response validated by 90 Y PET/CT dosimetry and dual-tracer PET/CT metabolic parameters. The study group comprised 62 HCC patients prospectively recruited for 90 Y-GMRE who underwent pretreatment dual-tracer ( 11 C-acetate and 18 F-FDG) PET/CT as surrogate markers of HCC cellular differentiation. Pretreatment tumour-to-nontumour ratio on 99m Tc-MAA SPECT/CT (T/NT MAA ) was correlated with posttreatment 90 Y PET/CT T/NT 90Y after quantification validation. The TD-response relationship for HCC of different tracer groups was assessed on follow-up PET/CT 2 months after treatment. 90 Y PET/CT was accurate in the measurement of recovery of injected 90 Y activity (81.9-99.9%, median 94.8%). Pretreatment SPECT/CT T/NT MAA was strongly correlated with posttreatment 90 Y PET/CT T/NT 90Y (5.6 ± 3.2 versus 5.9 ± 3.5, T/NT 90Y 1.01 × T/NT MAA  + 0.161, r = 0.918, P < 0.05). The response rates were 72.4% (21/29), 70.6% (12/17) and 25% (4/16) for well, moderately and poorly differentiated HCC, respectively. The cut-off TD for a good response was significantly different between poorly differentiated and well/moderately differentiated HCC (262 Gy versus 152/174 Gy) with 89.2% sensitivity and 88% specificity. At a limiting tolerated liver dose of 70 Gy, the T/NT MAA thresholds for predicting a good response in poorly differentiated and well/moderately differentiated HCC were 3.5 and 2.0/2.3. Disregarding HCC cellular differentiation, the cut-off TD became 170 Gy, with lower sensitivity (70.3%) and specificity (76%). 90 Y PET/CT can provide accurate dosimetry for 90 Y-GMRE. Pretreatment T/NT MAA predicts posttreatment T/NT 90Y . The TD thresholds for a good response are tracer-dependent, with a strong correlation between HCC radiosensitivity and cellular differentiation and other PET-based parameters. These cytokinetic factors improve treatment efficacy while minimizing organ damage for the prescription of personalized 90 Y-GMRE.

Dual functions of silver nanoparticles in F9 teratocarcinoma stem cells, a suitable model for evaluating cytotoxicity- and differentiation-mediated cancer therapy.

PubMed

Han, Jae Woong; Gurunathan, Sangiliyandi; Choi, Yun-Jung; Kim, Jin-Hoi

2017-01-01

Silver nanoparticles (AgNPs) exhibit strong antibacterial and anticancer activity owing to their large surface-to-volume ratios and crystallographic surface structure. Owing to their various applications, understanding the mechanisms of action, biological interactions, potential toxicity, and beneficial effects of AgNPs is important. Here, we investigated the toxicity and differentiation-inducing effects of AgNPs in teratocarcinoma stem cells. AgNPs were synthesized and characterized using various analytical techniques such as UV-visible spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. The cellular responses of AgNPs were analyzed by a series of cellular and biochemical assays. Gene and protein expressions were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The AgNPs showed typical crystalline structures and spherical shapes (average size =20 nm). High concentration of AgNPs induced cytotoxicity in a dose-dependent manner by increasing lactate dehydrogenase leakage and reactive oxygen species. Furthermore, AgNPs caused mitochondrial dysfunction, DNA fragmentation, increased expression of apoptotic genes, and decreased expression of antiapoptotic genes. Lower concentrations of AgNPs induced neuronal differentiation by increasing the expression of differentiation markers and decreasing the expression of stem cell markers. Cisplatin reduced the viability of F9 cells that underwent AgNPs-induced differentiation. The results showed that AgNPs caused differentially regulated cytotoxicity and induced neuronal differentiation of F9 cells in a concentration-dependent manner. Therefore, AgNPs can be used for differentiation therapy, along with chemotherapeutic agents, for improving cancer treatment by targeting specific chemotherapy-resistant cells within a tumor. Furthermore, understanding the molecular mechanisms of apoptosis and differentiation in stem cells could also help in developing new strategies for cancer stem cell (CSC) therapies. The findings of this study could significantly contribute to the nanomedicine because this study is the first of its kind, and our results will lead to new strategies for cancer and CSC therapies.

Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.

PubMed

Hsu, Yi-Chao; Wu, Yu-Ting; Yu, Ting-Hsien; Wei, Yau-Huei

2016-04-01

Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying mechanisms in the regulation of mitochondrial metabolism of MSCs may ultimately improve therapeutic outcomes of stem cell therapy in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

Neuron-like differentiation of mesenchymal stem cells on silicon nanowires

NASA Astrophysics Data System (ADS)

Kim, Hyunju; Kim, Ilsoo; Choi, Heon-Jin; Kim, So Yeon; Yang, Eun Gyeong

2015-10-01

The behavior of mammalian cells on vertical nanowire (NW) arrays, including cell spreading and the dynamic distribution of focal adhesions and cytoskeletal proteins, has been intensively studied to extend the implications for cellular manipulations in vitro. Prompted by the result that cells on silicon (Si) NWs showed morphological changes and reduced migration rates, we have explored the transition of mesenchymal stem cells into a neuronal lineage by using SiNWs with varying lengths. When human mesenchymal stem cells (hMSCs) were cultured on the longest SiNWs for 3 days, most of the cells exhibited elongated shapes with neurite-like extensions and dot-like focal adhesions that were prominently observed along with actin filaments. Under these circumstances, the cell motility analyzed by live cell imaging was found to decrease due to the presence of SiNWs. In addition, the slowed growth rate, as well as the reduced population of S phase cells, suggested that the cell cycle was likely arrested in response to the differentiation process. Furthermore, we measured the mRNA levels of several lineage-specific markers to confirm that the SiNWs actually induced neuron-like differentiation of the hMSCs while hampering their osteogenic differentiation. Taken together, our results implied that SiNWs were capable of inducing active reorganization of cellular behaviors, collectively guiding the fate of hMSCs into the neural lineage even in the absence of any inducing reagent.The behavior of mammalian cells on vertical nanowire (NW) arrays, including cell spreading and the dynamic distribution of focal adhesions and cytoskeletal proteins, has been intensively studied to extend the implications for cellular manipulations in vitro. Prompted by the result that cells on silicon (Si) NWs showed morphological changes and reduced migration rates, we have explored the transition of mesenchymal stem cells into a neuronal lineage by using SiNWs with varying lengths. When human mesenchymal stem cells (hMSCs) were cultured on the longest SiNWs for 3 days, most of the cells exhibited elongated shapes with neurite-like extensions and dot-like focal adhesions that were prominently observed along with actin filaments. Under these circumstances, the cell motility analyzed by live cell imaging was found to decrease due to the presence of SiNWs. In addition, the slowed growth rate, as well as the reduced population of S phase cells, suggested that the cell cycle was likely arrested in response to the differentiation process. Furthermore, we measured the mRNA levels of several lineage-specific markers to confirm that the SiNWs actually induced neuron-like differentiation of the hMSCs while hampering their osteogenic differentiation. Taken together, our results implied that SiNWs were capable of inducing active reorganization of cellular behaviors, collectively guiding the fate of hMSCs into the neural lineage even in the absence of any inducing reagent. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05787f

p21-activated kinase signaling in breast cancer.

PubMed

Gururaj, Anupama E; Rayala, Suresh K; Kumar, Rakesh

2005-01-01

The p21-activated kinases signal through a number of cellular pathways fundamental to growth, differentiation and apoptosis. A wealth of information has accumulated at an impressive pace in the recent past, both with regard to previously identified targets for p21-activated kinases that regulate the actin cytoskeleton and cellular stress pathways and with regard to newly identified targets and their role in cancer. Emerging data also provide new clues towards a previously unappreciated link between these various cellular processes. The present review attempts to provide a quick tutorial to the reader about the evolving significance of p21-activated kinases and small GTPases in breast cancer, using information from mouse models, tissue culture studies, and human materials.

Analysis of cellular signal transduction from an information theoretic approach.

PubMed

Uda, Shinsuke; Kuroda, Shinya

2016-03-01

Signal transduction processes the information of various cellular functions, including cell proliferation, differentiation, and death. The information for controlling cell fate is transmitted by concentrations of cellular signaling molecules. However, how much information is transmitted in signaling pathways has thus far not been investigated. Shannon's information theory paves the way to quantitatively analyze information transmission in signaling pathways. The theory has recently been applied to signal transduction, and mutual information of signal transduction has been determined to be a measure of information transmission. We review this work and provide an overview of how signal transduction transmits informational input and exerts biological output. Copyright © 2015 Elsevier Ltd. All rights reserved.

Holographic techniques for cellular fluorescence microscopy

NASA Astrophysics Data System (ADS)

Kim, Myung K.

2017-04-01

We have constructed a prototype instrument for holographic fluorescence microscopy (HFM) based on self-interference incoherent digital holography (SIDH) and demonstrate novel imaging capabilities such as differential 3D fluorescence microscopy and optical sectioning by compressive sensing.

Vitamin A

USDA-ARS?s Scientific Manuscript database

Vitamin A is essential during embryonic development and, in the adult, it is necessary for vision, immunity, metabolism, cellular proliferation, differentiation, and apoptosis. Recently, additional functions of vitamin A such as regulation of energy balance, insulin signaling and nervous system acti...

Therapeutic cloning and cellular reprogramming.

PubMed

Rodriguez, Ramon M; Ross, Pablo J; Cibelli, Jose B

2012-01-01

Embryonic stem cells are capable of differentiating into any cell-type present in an adult organism, and constitute a renewable source of tissue for regenerative therapies. The transplant of allogenic stem cells is challenging due to the risk of immune rejection. Nevertheless, somatic cell reprogramming techniques allow the generation of isogenic embryonic stem cells, genetically identical to the patient. In this chapter we will discuss the cellular reprogramming techniques in the context of regenerative therapy and the biological and technical barriers that they will need to overcome before clinical use.

Epigenetic Therapy in Lung Cancer - Role of microRNAs.

PubMed

Rothschild, Sacha I

2013-01-01

Lung cancer is the leading cause of cancer deaths worldwide. microRNAs (miRNAs) are a class of small non-coding RNA species that have been implicated in the control of many fundamental cellular and physiological processes such as cellular differentiation, proliferation, apoptosis, and stem cell maintenance. Some miRNAs have been categorized as "oncomiRs" as opposed to "tumor suppressor miRs." This review focuses on the role of miRNAs in the lung cancer carcinogenesis and their potential as diagnostic, prognostic, or predictive markers.

Mitochondrial oxidative stress caused by Sod2 deficiency promotes cellular senescence and aging phenotypes in the skin

PubMed Central

Velarde, Michael C.; Flynn, James M.; Day, Nicholas U.; Melov, Simon; Campisi, Judith

2012-01-01

Cellular senescence arrests the proliferation of mammalian cells at risk for neoplastic transformation, and is also associated with aging. However, the factors that cause cellular senescence during aging are unclear. Excessive reactive oxygen species (ROS) have been shown to cause cellular senescence in culture, and accumulated molecular damage due to mitochondrial ROS has long been thought to drive aging phenotypes in vivo. Here, we test the hypothesis that mitochondrial oxidative stress can promote cellular senescence in vivo and contribute to aging phenotypes in vivo, specifically in the skin. We show that the number of senescent cells, as well as impaired mitochondrial (complex II) activity increase in naturally aged mouse skin. Using a mouse model of genetic Sod2 deficiency, we show that failure to express this important mitochondrial anti-oxidant enzyme also impairs mitochondrial complex II activity, causes nuclear DNA damage, and induces cellular senescence but not apoptosis in the epidermis. Sod2 deficiency also reduced the number of cells and thickness of the epidermis, while increasing terminal differentiation. Our results support the idea that mitochondrial oxidative stress and cellular senescence contribute to aging skin phenotypes in vivo. PMID:22278880

Brca1 regulates in vitro differentiation of mammary epithelial cells.

PubMed

Kubista, Marion; Rosner, Margit; Kubista, Ernst; Bernaschek, Gerhard; Hengstschläger, Markus

2002-07-18

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing.

PubMed

Aberdam, Edith; Petit, Isabelle; Sangari, Linda; Aberdam, Daniel

2017-01-01

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing

PubMed Central

Aberdam, Edith; Petit, Isabelle; Sangari, Linda

2017-01-01

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests. PMID:28640863

Conversion of one cell type into another: implications for understanding organ development, pathogenesis of cancer and generating cells for therapy.

PubMed

Corbett, James L; Tosh, David

2014-06-01

Metaplasia is the irreversible conversion of one differentiated cell or tissue type into another. Metaplasia usually occurs in tissues that undergo regeneration, and may, in a pathological context, predispose to an increased risk of disease. Studying the conditions leading to the development of metaplasia is therefore of significant clinical interest. In contrast, transdifferentiation (or cellular reprogramming) is a subset of metaplasia that describes the permanent conversion of one differentiated cell type into another, and generally occurs between cells that arise from neighbouring regions of the same germ layer. Transdifferentiation, although rare, has been shown to occur in Nature. New insights into the signalling pathways involved in normal tissue development may be obtained by investigating the cellular and molecular mechanisms in metaplasia and transdifferentiation, and additional identification of key molecular regulators in transdifferentiation and metaplasia could provide new targets for therapeutic treatment of diseases such as cancer, as well as generating cells for transplantation into patients with degenerative disorders. In the present review, we focus on the transdifferentiation of pancreatic cells into hepatocyte-like cells, the development of Barrett's metaplasia in the oesophagus, and the cellular and molecular mechanisms underlying both processes.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries.

PubMed

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-09-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries

PubMed Central

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-01-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development. PMID:18544560

Heterogeneity of Metazoan Cells and Beyond: To Integrative Analysis of Cellular Populations at Single-Cell Level.

PubMed

Barteneva, Natasha S; Vorobjev, Ivan A

2018-01-01

In this paper, we review some of the recent advances in cellular heterogeneity and single-cell analysis methods. In modern research of cellular heterogeneity, there are four major approaches: analysis of pooled samples, single-cell analysis, high-throughput single-cell analysis, and lately integrated analysis of cellular population at a single-cell level. Recently developed high-throughput single-cell genetic analysis methods such as RNA-Seq require purification step and destruction of an analyzed cell often are providing a snapshot of the investigated cell without spatiotemporal context. Correlative analysis of multiparameter morphological, functional, and molecular information is important for differentiation of more uniform groups in the spectrum of different cell types. Simplified distributions (histograms and 2D plots) can underrepresent biologically significant subpopulations. Future directions may include the development of nondestructive methods for dissecting molecular events in intact cells, simultaneous correlative cellular analysis of phenotypic and molecular features by hybrid technologies such as imaging flow cytometry, and further progress in supervised and non-supervised statistical analysis algorithms.

Cellular dynamics of bovine aortic smooth muscle cells measured using MEMS force sensors

NASA Astrophysics Data System (ADS)

Tsukagoshi, Takuya; Nguyen, Thanh-Vinh; Hirayama Shoji, Kayoko; Takahashi, Hidetoshi; Matsumoto, Kiyoshi; Shimoyama, Isao

2018-04-01

Adhesive cells perceive the mechanical properties of the substrates to which they adhere, adjusting their cellular mechanical forces according to their biological characteristics. This mechanical interaction subsequently affects the growth, locomotion, and differentiation of the cell. However, little is known about the detailed mechanism that underlies this interaction between adherent cells and substrates because dynamically measuring mechanical phenomena is difficult. Here, we utilize microelectromechamical systems force sensors that can measure cellular traction forces with high temporal resolution (~2.5 µs) over long periods (~3 h). We found that the cellular dynamics reflected physical phenomena with time scales from milliseconds to hours, which contradicts the idea that cellular motion is slow. A single focal adhesion (FA) generates an average force of 7 nN, which disappears in ms via the action of trypsin-ethylenediaminetetraacetic acid. The force-changing rate obtained from our measurements suggests that the time required for an FA to decompose was nearly proportional to the force acting on the FA.

Phenotypic Characterization of Retinoic Acid Differentiated SH-SY5Y Cells by Transcriptional Profiling

PubMed Central

Korecka, Joanna A.; van Kesteren, Ronald E.; Blaas, Eva; Spitzer, Sonia O.; Kamstra, Jorke H.; Smit, August B.; Swaab, Dick F.; Verhaagen, Joost; Bossers, Koen

2013-01-01

Multiple genetic and environmental factors play a role in the development and progression of Parkinson’s disease (PD). The main neuropathological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta. To study genetic and molecular contributors to the disease process, there is a great need for readily accessible cells with prominent DAergic features that can be used for reproducible in vitro cellular screening. Here, we investigated the molecular phenotype of retinoic acid (RA) differentiated SH-SY5Y cells using genome wide transcriptional profiling combined with gene ontology, transcription factor and molecular pathway analysis. We demonstrated that RA induces a general neuronal differentiation program in SH-SY5Y cells and that these cells develop a predominantly mature DAergic-like neurotransmitter phenotype. This phenotype is characterized by increased dopamine levels together with a substantial suppression of other neurotransmitter phenotypes, such as those for noradrenaline, acetylcholine, glutamate, serotonin and histamine. In addition, we show that RA differentiated SH-SY5Y cells express the dopamine and noradrenalin neurotransmitter transporters that are responsible for uptake of MPP(+), a well known DAergic cell toxicant. MPP(+) treatment alters mitochondrial activity according to its proposed cytotoxic effect in DAergic neurons. Taken together, RA differentiated SH-SY5Y cells have a DAergic-like phenotype, and provide a good cellular screening tool to find novel genes or compounds that affect cytotoxic processes that are associated with PD. PMID:23724009

Differentiated NSC-34 cells as an in vitro cell model for VX.

PubMed

Kanjilal, Baishali; Keyser, Brian M; Andres, Devon K; Nealley, Eric; Benton, Betty; Melber, Ashley A; Andres, Jaclynn F; Letukas, Valerie A; Clark, Offie; Ray, Radharaman

2014-10-01

The US military has placed major emphasis on developing therapeutics against nerve agents (NA). Current efforts are hindered by the lack of effective in vitro cellular models to aid in the preliminary screening of potential candidate drugs/antidotes. The development of an in vitro cellular model to aid in discovering new NA therapeutics would be highly beneficial. In this regard, we have examined the response of a differentiated hybrid neuronal cell line, NSC-34, to the NA VX. VX-induced apoptosis of differentiated NSC-34 cells was measured by monitoring the changes in caspase-3 and caspase-9 activity post-exposure. Differentiated NSC-34 cells showed an increase in caspase-3 activity in a manner dependent on both time (17-23 h post-exposure) and dose (10-100 nM). The maximal increase in caspase-3 activity was found to be at 20-h post-exposure. Caspase-9 activity was also measured in response to VX and was found to be elevated at all concentrations (10-100 nM) tested. VX-induced cell death was also observed by utilizing annexin V/propidium iodide flow cytometry. Finally, VX-induced caspase-3 or -9 activities were reduced with the addition of pralidoxime (2-PAM), one of the current therapeutics used against NA toxicity, and dizocilpine (MK-801). Overall the data presented here show that differentiated NSC-34 cells are sensitive to VX-induced cell death and could be a viable in vitro cell model for screening NA candidate therapeutics.

microRNA expression profiling in fetal single ventricle malformation identified by deep sequencing.

PubMed

Yu, Zhang-Bin; Han, Shu-Ping; Bai, Yun-Fei; Zhu, Chun; Pan, Ya; Guo, Xi-Rong

2012-01-01

microRNAs (miRNAs) have emerged as key regulators in many biological processes, particularly cardiac growth and development, although the specific miRNA expression profile associated with this process remains to be elucidated. This study aimed to characterize the cellular microRNA profile involved in the development of congenital heart malformation, through the investigation of single ventricle (SV) defects. Comprehensive miRNA profiling in human fetal SV cardiac tissue was performed by deep sequencing. Differential expression of 48 miRNAs was revealed by sequencing by oligonucleotide ligation and detection (SOLiD) analysis. Of these, 38 were down-regulated and 10 were up-regulated in differentiated SV cardiac tissue, compared to control cardiac tissue. This was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Predicted target genes of the 48 differentially expressed miRNAs were analyzed by gene ontology and categorized according to cellular process, regulation of biological process and metabolic process. Pathway-Express analysis identified the WNT and mTOR signaling pathways as the most significant processes putatively affected by the differential expression of these miRNAs. The candidate genes involved in cardiac development were identified as potential targets for these differentially expressed microRNAs and the collaborative network of microRNAs and cardiac development related-mRNAs was constructed. These data provide the basis for future investigation of the mechanism of the occurrence and development of fetal SV malformations.

Diacylglycerol kinase ζ generates dipalmitoyl-phosphatidic acid species during neuroblastoma cell differentiation.

PubMed

Mizuno, Satoru; Kado, Sayaka; Goto, Kaoru; Takahashi, Daisuke; Sakane, Fumio

2016-12-01

Phosphatidic acid (PA) is one of the phospholipids composing the plasma membrane and acts as a second messenger to regulate a wide variety of important cellular events, including mitogenesis, migration and differentiation. PA consists of various molecular species with different acyl chains at the sn- 1 and sn -2 positions. However, it has been poorly understood what PA molecular species are produced during such cellular events. Here we identified the PA molecular species generated during retinoic acid (RA)-induced neuroblastoma cell differentiation using a newly established liquid chromatography/mass spectrometry (LC/MS) method. Intriguingly, the amount of 32:0-PA species was dramatically and transiently increased in Neuro-2a neuroblastoma cells 24-48 h after RA-treatment. In addition, 30:0- and 34:0-PA species were also moderately increased. Moreover, similar results were obtained when Neuro-2a cells were differentiated for 24 h by serum starvation. MS/MS analysis revealed that 32:0-PA species contains two palmitic acids (16:0 s). RT-PCR analysis showed that diacylglycerol kinase (DGK) δ and DGKζ were highly expressed in Neuro-2a cells. The silencing of DGKζ expression significantly decreased the production of 32:0-PA species, whereas DGKδ-siRNA did not. Moreover, neurite outgrowth was also markedly attenuated by the deficiency of DGKζ. Taken together, these results indicate that DGKζ exclusively generates very restricted PA species, 16:0/16:0-PA, and up-regulates neurite outgrowth during the initial/early stage of neuroblastoma cell differentiation.

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.

Proliferation and differentiation of brown adipocytes from interstitial cells during cold acclimation

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

Bukowiecki, L.J.; Geloeen, A.; Collet, A.J.

1986-06-01

The mechanisms of brown adipocyte proliferation and differentiation during cold acclimation (and/or adaptation to hyperphagia) have been studied by quantitative photonic radioautography. (/sup 3/H)thymidine was injected to warm-acclimated (25/sup 0/C) rats and to animals exposed to 5/sup 0/C for 2 days. Samples of interscapular brown adipose tissue were collected for quantitative analysis of mitotic frequencies at various periods of time (4 h-15 days) after the injection of (/sup 3/H)thymidine, the rats being maintained at the temperatures to which they were initially exposed. It was found that cold exposure for 2 days markedly enhanced mitotic activity in endothelial cells, interstitial cells,more » and brown preadipocytes rather than in fully differentiated brown adipocytes. The total tissue labeling index (percent of labeled nuclei) increased approx.70 times over control values. The authors now report that cellular labeling progressively increased in mature brown adipocytes during cold acclimation, whereas it correspondingly decreased in interstitial cells and brown preadipocytes. This indicates that the sequence of events for cellular differentiation is interstitial cells ..-->.. brown preadipocytes ..-->.. mature brown adipocytes. Remarkable, labeling frequency did not change in endothelial cells during cold acclimation demonstrating that these cells cannot be considered as progenitors of brown adipocytes. It is suggested that brown adipocyte proliferation and differentiation from interstitial cells represent the fundamental phenomena explaining the enhanced capacity of cold-acclimated and/or hyperphagic rats to respond calorigenically to catecholamines.« less

Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

PubMed Central

Mills, Jason C.; Sansom, Owen J.

2016-01-01

It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

Coordination of cellular differentiation, polarity, mitosis and meiosis - New findings from early vertebrate oogenesis.

PubMed

Elkouby, Yaniv M; Mullins, Mary C

2017-10-15

A mechanistic dissection of early oocyte differentiation in vertebrates is key to advancing our knowledge of germline development, reproductive biology, the regulation of meiosis, and all of their associated disorders. Recent advances in the field include breakthroughs in the identification of germline stem cells in Medaka, in the cellular architecture of the germline cyst in mice, in a mechanistic dissection of chromosomal pairing and bouquet formation in meiosis in mice, in tracing oocyte symmetry breaking to the chromosomal bouquet of meiosis in zebrafish, and in the biology of the Balbiani body, a universal oocyte granule. Many of the major events in early oogenesis are universally conserved, and some are co-opted for species-specific needs. The chromosomal events of meiosis are of tremendous consequence to gamete formation and have been extensively studied. New light is now being shed on other aspects of early oocyte differentiation, which were traditionally considered outside the scope of meiosis, and their coordination with meiotic events. The emerging theme is of meiosis as a common groundwork for coordinating multifaceted processes of oocyte differentiation. In an accompanying manuscript we describe methods that allowed for investigations in the zebrafish ovary to contribute to these breakthroughs. Here, we review these advances mostly from the zebrafish and mouse. We discuss oogenesis concepts across established model organisms, and construct an inclusive paradigm for early oocyte differentiation in vertebrates. Copyright © 2017 Elsevier Inc. All rights reserved.

Loss of lysophosphatidic acid receptor LPA1 alters oligodendrocyte differentiation and myelination in the mouse cerebral cortex.

PubMed

García-Díaz, Beatriz; Riquelme, Raquel; Varela-Nieto, Isabel; Jiménez, Antonio Jesús; de Diego, Isabel; Gómez-Conde, Ana Isabel; Matas-Rico, Elisa; Aguirre, José Ángel; Chun, Jerold; Pedraza, Carmen; Santín, Luis Javier; Fernández, Oscar; Rodríguez de Fonseca, Fernando; Estivill-Torrús, Guillermo

2015-11-01

Lysophosphatidic acid (LPA) is an intercellular signaling lipid that regulates multiple cellular functions, acting through specific G-protein coupled receptors (LPA(1-6)). Our previous studies using viable Malaga variant maLPA1-null mice demonstrated the requirement of the LPA1 receptor for normal proliferation, differentiation, and survival of the neuronal precursors. In the cerebral cortex LPA1 is expressed extensively in differentiating oligodendrocytes, in parallel with myelination. Although exogenous LPA-induced effects have been investigated in myelinating cells, the in vivo contribution of LPA1 to normal myelination remains to be demonstrated. This study identified a relevant in vivo role for LPA1 as a regulator of cortical myelination. Immunochemical analysis in adult maLPA1-null mice demonstrated a reduction in the steady-state levels of the myelin proteins MBP, PLP/DM20, and CNPase in the cerebral cortex. The myelin defects were confirmed using magnetic resonance spectroscopy and electron microscopy. Stereological analysis limited the defects to adult differentiating oligodendrocytes, without variation in the NG2+ precursor cells. Finally, a possible mechanism involving oligodendrocyte survival was demonstrated by the impaired intracellular transport of the PLP/DM20 myelin protein which was accompanied by cellular loss, suggesting stress-induced apoptosis. These findings describe a previously uncharacterized in vivo functional role for LPA1 in the regulation of oligodendrocyte differentiation and myelination in the CNS, underlining the importance of the maLPA1-null mouse as a model for the study of demyelinating diseases.

Regulation of mammalian cell differentiation by long non-coding RNAs

PubMed Central

Hu, Wenqian; Alvarez-Dominguez, Juan R; Lodish, Harvey F

2012-01-01

Differentiation of specialized cell types from stem and progenitor cells is tightly regulated at several levels, both during development and during somatic tissue homeostasis. Many long non-coding RNAs have been recognized as an additional layer of regulation in the specification of cellular identities; these non-coding species can modulate gene-expression programmes in various biological contexts through diverse mechanisms at the transcriptional, translational or messenger RNA stability levels. Here, we summarize findings that implicate long non-coding RNAs in the control of mammalian cell differentiation. We focus on several representative differentiation systems and discuss how specific long non-coding RNAs contribute to the regulation of mammalian development. PMID:23070366

Enhanced reactive oxygen species overexpression by CuO nanoparticles in poorly differentiated hepatocellular carcinoma cells

NASA Astrophysics Data System (ADS)

Kung, Mei-Lang; Hsieh, Shu-Ling; Wu, Chih-Chung; Chu, Tian-Huei; Lin, Yu-Chun; Yeh, Bi-Wen; Hsieh, Shuchen

2015-01-01

Copper oxide nanoparticles (CuO NPs) are known to exhibit toxic effects on a variety of cell types and organs. To determine the oxidative impact of CuO NPs on hepatocellular carcinoma (HCC) cells, well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) cells were exposed to CuO NPs. Cell viability assay showed that the median inhibition concentration (IC50) for SK-Hep-1 and HepG2 cells was 25 μg ml-1 and 85 μg ml-1, respectively. Cellular fluorescence intensity using DCFH-DA staining analysis revealed significant intracellular reactive oxygen species (ROS) generation of up to 242% in SK-Hep-1 cells, compared with 86% in HepG2 cells. HPLC analysis demonstrated that a CuO NP treatment caused cellular GSH depletion of 58% and a GSH/GSSG ratio decrease to ~0.1 in SK-Hep-1 cells. The oxidative stress caused by enhanced superoxide anion production was observed in both HepG2 (146%) and SK-Hep-1 (192%) cells. The Griess assay verified that CuO NPs induced NO production (170%) in SK-Hep-1 cells. Comet assay and western blot further demonstrated that CuO NPs induced severe DNA strand breakage (70%) in SK-Hep-1 cells and caused DNA damage via increased γ-H2AX levels. These results suggest that well-differentiated HepG2 cells possess a robust antioxidant defense system against CuO NP-induced ROS stress and exhibit more tolerance to oxidative stress. Conversely, poorly differentiated SK-Hep-1 cells exhibited a deregulated antioxidant defense system that allowed accumulation of CuO NP-induced ROS and resulted in severe cytotoxicity.Copper oxide nanoparticles (CuO NPs) are known to exhibit toxic effects on a variety of cell types and organs. To determine the oxidative impact of CuO NPs on hepatocellular carcinoma (HCC) cells, well-differentiated (HepG2) and poorly differentiated (SK-Hep-1) cells were exposed to CuO NPs. Cell viability assay showed that the median inhibition concentration (IC50) for SK-Hep-1 and HepG2 cells was 25 μg ml-1 and 85 μg ml-1, respectively. Cellular fluorescence intensity using DCFH-DA staining analysis revealed significant intracellular reactive oxygen species (ROS) generation of up to 242% in SK-Hep-1 cells, compared with 86% in HepG2 cells. HPLC analysis demonstrated that a CuO NP treatment caused cellular GSH depletion of 58% and a GSH/GSSG ratio decrease to ~0.1 in SK-Hep-1 cells. The oxidative stress caused by enhanced superoxide anion production was observed in both HepG2 (146%) and SK-Hep-1 (192%) cells. The Griess assay verified that CuO NPs induced NO production (170%) in SK-Hep-1 cells. Comet assay and western blot further demonstrated that CuO NPs induced severe DNA strand breakage (70%) in SK-Hep-1 cells and caused DNA damage via increased γ-H2AX levels. These results suggest that well-differentiated HepG2 cells possess a robust antioxidant defense system against CuO NP-induced ROS stress and exhibit more tolerance to oxidative stress. Conversely, poorly differentiated SK-Hep-1 cells exhibited a deregulated antioxidant defense system that allowed accumulation of CuO NP-induced ROS and resulted in severe cytotoxicity. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05843g

Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm.

PubMed

Lu, Junjie; Baccei, Anna; Lummertz da Rocha, Edroaldo; Guillermier, Christelle; McManus, Sean; Finney, Lydia A; Zhang, Cheng; Steinhauser, Matthew L; Li, Hu; Lerou, Paul H

2018-04-01

Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Cultural and chemical characterization of CDC groups EO-2, M-5, and M-6, Moraxella (Moraxella) species, Oligella urethralis, Acinetobacter species, and Psychrobacter immobilis.

PubMed

Moss, C W; Wallace, P L; Hollis, D G; Weaver, R E

1988-03-01

We determined phenotypic characteristics, cellular fatty acid composition, and isoprenoid quinone content of representative strains of CDC groups EO-2, M-5, and M-6, Moraxella (Moraxella) species, Oligella urethralis, Acinetobacter species, and Psychrobacter immobilis. All organisms contained ubiquinone with eight isoprene units as the major isoprenolog, but distinct differences were observed in fatty acid composition. Twenty-eight of the original collection of CDC group EO-2 strains were further identified as P. immobilis, EO-2, or EO-3 by distinctive cellular fatty acid profiles, cellular morphology, and pigment production. The cellular fatty acid compositions of M-5 and M-6 were similar but were clearly different from those of other organisms. The genus Acinetobacter was differentiated from other organisms in the study by small amounts of 2-hydroxydodecanoic acid (2-OH-12:0), and P. immobilis was differentiated by small amounts of decanoic acid (10:0) and a branched-chain 17-carbon acid (i-17:0). All Moraxella species were distinguished by small amounts of decanoic acid (10:0) and the absence of i-17:0. M. bovis, M. nonliquefaciens, and some strains of M. lacunata formed a single fatty acid group, while M. osloensis, M. phenylpyruvica, M. atlantae, and other strains of M. lacunata (M. lacunata II) had species-specific fatty acid profiles. O. urethralis differed from Moraxella species by the presence of large amounts (49%) of cis-vaccenic acid (18:1 omega 7c), small amounts (1%) of 3-hydroxyhexadecanoate (3-OH-16:0), and the absence of 10:0 and 3-hydroxydodecanoate (3-OH-12:0). The combined use of chemical data and a small number of conventional tests permitted rapid identification and differentiation of these organisms from each other and from related organisms.

Cultural and chemical characterization of CDC groups EO-2, M-5, and M-6, Moraxella (Moraxella) species, Oligella urethralis, Acinetobacter species, and Psychrobacter immobilis.

PubMed Central

Moss, C W; Wallace, P L; Hollis, D G; Weaver, R E

1988-01-01

We determined phenotypic characteristics, cellular fatty acid composition, and isoprenoid quinone content of representative strains of CDC groups EO-2, M-5, and M-6, Moraxella (Moraxella) species, Oligella urethralis, Acinetobacter species, and Psychrobacter immobilis. All organisms contained ubiquinone with eight isoprene units as the major isoprenolog, but distinct differences were observed in fatty acid composition. Twenty-eight of the original collection of CDC group EO-2 strains were further identified as P. immobilis, EO-2, or EO-3 by distinctive cellular fatty acid profiles, cellular morphology, and pigment production. The cellular fatty acid compositions of M-5 and M-6 were similar but were clearly different from those of other organisms. The genus Acinetobacter was differentiated from other organisms in the study by small amounts of 2-hydroxydodecanoic acid (2-OH-12:0), and P. immobilis was differentiated by small amounts of decanoic acid (10:0) and a branched-chain 17-carbon acid (i-17:0). All Moraxella species were distinguished by small amounts of decanoic acid (10:0) and the absence of i-17:0. M. bovis, M. nonliquefaciens, and some strains of M. lacunata formed a single fatty acid group, while M. osloensis, M. phenylpyruvica, M. atlantae, and other strains of M. lacunata (M. lacunata II) had species-specific fatty acid profiles. O. urethralis differed from Moraxella species by the presence of large amounts (49%) of cis-vaccenic acid (18:1 omega 7c), small amounts (1%) of 3-hydroxyhexadecanoate (3-OH-16:0), and the absence of 10:0 and 3-hydroxydodecanoate (3-OH-12:0). The combined use of chemical data and a small number of conventional tests permitted rapid identification and differentiation of these organisms from each other and from related organisms. Images PMID:3356788

Repression of anti-proliferative factor Tob1 in osteoarthritic cartilage

PubMed Central

Gebauer, Mathias; Saas, Joachim; Haag, Jochen; Dietz, Uwe; Takigawa, Masaharu; Bartnik, Eckart; Aigner, Thomas

2005-01-01

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal (n = 9) and osteoarthritic (n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1β and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage (P < 0.01). One of the significantly repressed genes, Tob1, encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo. Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression. PMID:15743474

Primitive neuroectodermal tumors of the central nervous system.

PubMed

Becker, L E; Hinton, D

1983-06-01

Primitive neuroectodermal tumors are morphologically similar malignant tumors arising in intracranial and peripheral sites of the nervous system, showing varying degrees of cellular differentiation with a tendency to disseminate along cerebrospinal fluid pathways. They occur primarily in children and young adults. Under the designation primitive neuroectodermal tumors are included medulloblastomas and tumors that may differentiate in other directions, such as medulloepithelioma, neuroblastoma, polar spongioblastoma, pineoblastoma, ependymoblastoma, retinoblastoma, and olfactory neuroblastoma. From a practical, histologic point of view, these tumors are often indistinguishable from one another and are best thought of as primitive neuroectodermal tumors with or without differentiating features.

Diffusion-weighted imaging for the cellularity assessment and matrix characterization of soft tissue tumour.

PubMed

Robba, Tiziana; Chianca, Vito; Albano, Domenico; Clementi, Valeria; Piana, Raimondo; Linari, Alessandra; Comandone, Alessandro; Regis, Guido; Stratta, Maurizio; Faletti, Carlo; Borrè, Alda

2017-11-01

To evaluate whether apparent diffusion coefficient (ADC) of diffusion-weighted imaging (DWI) is able to investigate the histological features of soft tissue tumours. We reviewed MRIs of soft tissue tumours performed from 2012 to 2015 to calculate the average ADCs. We included 46 patients (27 male; mean age: 57 years, range 12-85 years) with histologically proven soft tissue tumours (10 benign, 2 intermediate 34 malignant) grouped into eight tumour type classes. An experienced pathologist assigned a semi-quantitative cellularity score (very high, high, medium and low) and tumour grading. The t test, ANOVA and linear regression were used to correlate ADC with clinicopathological data. Approximate receiver operating characteristic curves were created to predict possible uses of ADC to differentiate benign from malignant tumours. There was a significant difference (p < 0.01) in ADCs between these three groups excluding myxoid sarcomas. A significant difference was also evident between the tumour type classes (p < 0.001), grade II and III myxoid lesions (p < 0.05), tumour grading classes (p < 0.001) and cellularity scores classes (p < 0.001), with the lowest ADCs in the very high cellularity. While the linear regression analysis showed a significant relationship between ADC and tumour cellularity (r = 0.590, p ≤ 0.05) and grading (r = 0.437, p ≤ 0.05), no significant relationship was found with age, gender, tumour size and histological subtype. An optimal cut-off ADC value of 1.45 × 10 -3 mm 2 /s with 76.8% accuracy was found to differentiate benign from malignant tumours. DWI may offer adjunctive information about soft tissue tumours, but its clinical role is still to be defined.

Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration.

PubMed

Sliogeryte, Kristina; Thorpe, Stephen D; Wang, Zhao; Thompson, Clare L; Gavara, Nuria; Knight, Martin M

2016-01-25

The actin cytoskeleton forms a dynamic structure involved in many fundamental cellular processes including the control of cell morphology, migration and biomechanics. Recently LifeAct-GFP (green fluorescent protein) has been proposed for visualising actin structure and dynamics in live cells as an alternative to actin-GFP which has been shown to affect cell mechanics. Here we compare the two approaches in terms of their effect on cellular mechanical behaviour. Human mesenchymal stem cells (hMSCs) were analysed using micropipette aspiration and the effective cellular equilibrium and instantaneous moduli calculated using the standard linear solid model. We show that LifeAct-GFP provides clearer visualisation of F-actin organisation and dynamics. Furthermore, LifeAct-GFP does not alter effective cellular mechanical properties whereas actin-GFP expression causes an increase in the cell modulus. Interestingly, LifeAct-GFP expression did produce a small (~10%) increase in the percentage of cells exhibiting aspiration-induced membrane bleb formation, whilst actin-GFP expression reduced blebbing. Further studies examined the influence of LifeAct-GFP in other cell types, namely chondrogenically differentiated hMSCs and murine chondrocytes. LifeAct-GFP also had no effect on the moduli of these non-blebbing cells for which mechanical properties are largely dependent on the actin cortex. In conclusion we show that LifeAct-GFP enables clearer visualisation of actin organisation and dynamics without disruption of the biomechanical properties of either the whole cell or the actin cortex. Thus the study provides new evidence supporting the use of LifeAct-GFP rather than actin-GFP for live cell microscopy and the study of cellular mechanobiology. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Digital gene expression analysis in hemocytes of the white shrimp Litopenaeus vannamei in response to low salinity stress.

PubMed

Zhao, Qun; Pan, Luqing; Ren, Qin; Hu, Dongxu

2015-02-01

The white shrimp Litopenaeus vannamei has been greatly impacted by low salinity stress. To gain knowledge on the immune response in L. vannamei under such stress, we investigated digital gene expression (DEG) in L. vannamei hemocytes using the deep-sequencing platform Illumina HiSeq 2000. In total, 38,155 high quality unigenes with average length 770 bp were generated; 145 and 79 genes were identified up- or down-regulated, respectively. Functional categorization and pathways of the differentially expressed genes revealed that immune signaling pathways, cellular immunity, humoral immunity, apoptosis, cellular protein synthesis, lipid transport and energy metabolism were the differentially regulated processes occurring during low salinity stress. These results will provide a resource for subsequent gene expression studies regarding environmental stress and a valuable gene information for a better understanding of immune mechanisms of L. vannamei under low salinity stress. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cellular, ultrastructural and molecular analyses of epidermal cell development in the planarian Schmidtea mediterranea.

PubMed

Cheng, Li-Chun; Tu, Kimberly C; Seidel, Chris W; Robb, Sofia M C; Guo, Fengli; Sánchez Alvarado, Alejandro

2018-01-15

The epidermis is essential for animal survival, providing both a protective barrier and cellular sensor to external environments. The generally conserved embryonic origin of the epidermis, but the broad morphological and functional diversity of this organ across animals is puzzling. We define the transcriptional regulators underlying epidermal lineage differentiation in the planarian Schmidtea mediterranea, an invertebrate organism that, unlike fruitflies and nematodes, continuously replaces its epidermal cells. We find that Smed-p53, Sox and Pax transcription factors are essential regulators of epidermal homeostasis, and act cooperatively to regulate genes associated with early epidermal precursor cell differentiation, including a tandemly arrayed novel gene family (prog) of secreted proteins. Additionally, we report on the discovery of distinct and previously undescribed secreted organelles whose production is dependent on the transcriptional activity of soxP-3, and which we term Hyman vesicles. Copyright © 2017 Elsevier Inc. All rights reserved.

Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment.

PubMed

Collins, Carina A; Leslie, Michelle E; Peck, Scott C; Heese, Antje

2017-01-01

The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

Involvement of autophagy in T cell biology.

PubMed

Oral, Ozlem; Yedier, Ozlem; Kilic, Seval; Gozuacik, Devrim

2017-01-01

Autophagy is an essential cellular pathway that sequesters various cytoplasmic components, including accumulated proteins, damaged organelles or invading microorganisms and delivers them to lysosomes for degradation. The function of autophagy has been reported in various tissues and systems, including its role in the regulation of cellular immunity. Autophagy plays a fundamental role at various stages of T cell maturation. It regulates the thymocyte selection and the generation of T cell repertoire by presenting intracellular antigens to MHC class molecules. Autophagy is crucial for metabolic regulation of T cells, and therefore supports cell survival and homeostasis, particularly in activated mature T cells. Furthermore, deletion of specific autophagy-related genes induces several immunological alterations including differentiation of activated T cells into regulatory, memory or natural killer T cells. In this review, we emphasize the impact of autophagy on T cell development, activation and differentiation, which is pivotal for the adaptive immune system.

Plasticity within stem cell hierarchies in mammalian epithelia.

PubMed

Tetteh, Paul W; Farin, Henner F; Clevers, Hans

2015-02-01

Tissue homeostasis and regeneration are fueled by resident stem cells that have the capacity to self-renew, and to generate all the differentiated cell types that characterize a particular tissue. Classical models of such cellular hierarchies propose that commitment and differentiation occur unidirectionally, with the arrows 'pointing away' from the stem cell. Recent studies, all based on genetic lineage tracing, describe various strategies employed by epithelial stem cell hierarchies to replace damaged or lost cells. While transdifferentiation from one tissue type into another ('metaplasia') appears to be generally forbidden in nonpathological contexts, plasticity within an individual tissue stem cell hierarchy may be much more common than previously appreciated. In this review, we discuss recent examples of such plasticity in selected mammalian epithelia, highlighting the different modes of regeneration and their implications for our understanding of cellular hierarchy and tissue self-renewal. Copyright © 2014 Elsevier Ltd. All rights reserved.

Application of differential interference contrast with inverted microscopes to the in vitro perfused nephron.

PubMed

Horster, M; Gundlach, H

1979-12-01

The study of in vitro perfused individual nephron segments requires a microscope which provides: (1) easy access to the specimen for measurement of cellular solute flux and voltage; (2) an image with high resolution and contrast; (3) optical sectioning of the object at different levels; and (4) rapid recording of the morphological phenomena. This paper describes an example of commercially available apparatus meeting the above requirements, and illustrates its efficiency. The microscope is of the inverted type (Zeiss IM 35) equipped with differential-interference-contrast (DIC) with a long working distance, and an automatically controlled camera system. The microscopic image exhibits cellular and intercellular details in the unstained transporting mammalian nephron segments despite their tubular structure and great thickness and makes obvious function-structure correlations (e.g. cell volume changes); luminal and contraluminal cell borders are well resolved for controlled microelectrode impalement.

A cellular automaton model for neurogenesis in Drosophila

NASA Astrophysics Data System (ADS)

Luthi, Pascal O.; Chopard, Bastien; Preiss, Anette; Ramsden, Jeremy J.

1998-07-01

A cellular automaton (CA) is constructed for the formation of the central nervous system of the Drosophila embryo. This is an experimentally well-studied system in which complex interactions between neighbouring cells appear to drive their differentiation into different types. It appears that all the cells initially have the potential to become neuroblasts, and all strive to this end, but those which differentiate first block their as yet undifferentiated neighbours from doing so. The CA makes use of observational evidence for a lateral inhibition mechanism involving signalling products S of the ‘proneural’ or neuralizing genes. The key concept of the model is that cells are continuously producing S, but the production rate is lowered by inhibitory signals received from neighbouring cells which have advanced further along the developmental pathway. Comparison with experimental data shows that it well accounts for the observed proportion of neuroectodermal cells delaminating as neuroblasts.

Optimal matrix rigidity for stress fiber polarization in stem cells

PubMed Central

Rehfeldt, F.; Brown, A. E. X.; Discher, D. E.; Safran, S. A.

2010-01-01

The shape and differentiation of human mesenchymal stem cells is especially sensitive to the rigidity of their environment; the physical mechanisms involved are unknown. A theoretical model and experiments demonstrate here that the polarization/alignment of stress-fibers within stem cells is a non-monotonic function of matrix rigidity. We treat the cell as an active elastic inclusion in a surrounding matrix whose polarizability, unlike dead matter, depends on the feedback of cellular forces that develop in response to matrix stresses. The theory correctly predicts the monotonic increase of the cellular forces with the matrix rigidity and the alignment of stress-fibers parallel to the long axis of cells. We show that the anisotropy of this alignment depends non-monotonically on matrix rigidity and demonstrate it experimentally by quantifying the orientational distribution of stress-fibers in stem cells. These findings offer a first physical insight for the dependence of stem cell differentiation on tissue elasticity. PMID:20563235

Nanotechnology in the regulation of stem cell behavior

NASA Astrophysics Data System (ADS)

Wu, King-Chuen; Tseng, Ching-Li; Wu, Chi-Chang; Kao, Feng-Chen; Tu, Yuan-Kun; So, Edmund C.; Wang, Yang-Kao

2013-10-01

Stem cells are known for their potential to repair damaged tissues. The adhesion, growth and differentiation of stem cells are likely controlled by the surrounding microenvironment which contains both chemical and physical cues. Physical cues in the microenvironment, for example, nanotopography, were shown to play important roles in stem cell fate decisions. Thus, controlling stem cell behavior by nanoscale topography has become an important issue in stem cell biology. Nanotechnology has emerged as a new exciting field and research from this field has greatly advanced. Nanotechnology allows the manipulation of sophisticated surfaces/scaffolds which can mimic the cellular environment for regulating cellular behaviors. Thus, we summarize recent studies on nanotechnology with applications to stem cell biology, including the regulation of stem cell adhesion, growth, differentiation, tracking and imaging. Understanding the interactions of nanomaterials with stem cells may provide the knowledge to apply to cell-scaffold combinations in tissue engineering and regenerative medicine.

In vitro cytotoxicity and differential cellular sensitivity of derivatives of diamino acids. II. N1-methyl, N1-allyl, N1-(2-chloroethyl) and N1-propargyl nitrosoureas.

PubMed

Dulude, H; Salvador, R; Gallant, G

1995-01-01

The in vitro cytotoxicity and differential cellular sensitivity of a series of new N1-methyl, N1-allyl, N1-2-chloroethyl and N1-propargyl nitrosourea derivatives of diamino acids were determined in the National Cancer Institute's primary antitumor drug screen. The compounds tested showed an in vitro anticancer activity similar to commercialized nitrosoureas such as CCNU, BCNU, MeCCNU, chlorozotocin, streptozotocin and PCNU. The alkylating moiety of the nitrosoureas seems to play a role in the general selectivity of our compounds. The N1-methyl and N1-2-chloroethyl nitrosourea derivatives are more selective for central nervous system cell lines, the N1-allyl nitrosourea derivatives are more selective for lung cancer cell lines and the N1-propargyl nitrosoureas are more selective for leukemia cell lines.

Role of Integrin in Mechanical Loading of Osteoblasts

NASA Technical Reports Server (NTRS)

Globus, Ruth; Demsky, Caroline

2000-01-01

Mechanical forces generated by gravity, weightbearing, and muscle contraction play a key role in the genesis and maintenance of skeletal structure. The molecular mechanisms that mediate changes in osteoblast activity in response to altered patterns of skeletal loading are not known, and a better understanding of these processes may be essential for developing effective treatment strategies to prevent disuse osteoporosis. We have elucidated specific integrin/ECM (extracellular matrix) interactions that are required for osteoblast differentiation and survival and have developed a useful loading system to further explore the molecular basis of mechano-sensitivity of osteoblasts. The long term goal of our collaborative research is to understand how the ECM and cell adhesion proteins and integrins interaction to mediate the response of osteoblasts and their progenitors to mechanical loading. We suggest that integrin/ECM interactions are crucial for basic cellular processes, including differentiation and survival, as well as to participate in detecting and mediating cellular responses to mechanical stimuli.

Molecular, metabolic, and genetic control: An introduction

NASA Astrophysics Data System (ADS)

Tyson, John J.; Mackey, Michael C.

2001-03-01

The living cell is a miniature, self-reproducing, biochemical machine. Like all machines, it has a power supply, a set of working components that carry out its necessary tasks, and control systems that ensure the proper coordination of these tasks. In this Special Issue, we focus on the molecular regulatory systems that control cell metabolism, gene expression, environmental responses, development, and reproduction. As for the control systems in human-engineered machines, these regulatory networks can be described by nonlinear dynamical equations, for example, ordinary differential equations, reaction-diffusion equations, stochastic differential equations, or cellular automata. The articles collected here illustrate (i) a range of theoretical problems presented by modern concepts of cellular regulation, (ii) some strategies for converting molecular mechanisms into dynamical systems, (iii) some useful mathematical tools for analyzing and simulating these systems, and (iv) the sort of results that derive from serious interplay between theory and experiment.

Evidence for Functional Differentiation among Drosophila Septins in Cytokinesis and Cellularization

PubMed Central

Adam, Jennifer C.; Pringle, John R.; Peifer, Mark

2000-01-01

The septins are a conserved family of proteins that are involved in cytokinesis and other aspects of cell-surface organization. In Drosophila melanogaster, null mutations in the pnut septin gene are recessive lethal, but homozygous pnut mutants complete embryogenesis and survive until the pupal stage. Because the completion of cellularization and other aspects of early development seemed likely to be due to maternally contributed Pnut product, we attempted to generate embryos lacking the maternal contribution in order to explore the roles of Pnut in these processes. We used two methods, the production of germline clones homozygous for a pnut mutation and the rescue of pnut homozygous mutant flies by a pnut+ transgene under control of the hsp70 promoter. Remarkably, the pnut germline-clone females produced eggs, indicating that stem-cell and cystoblast divisions in the female germline do not require Pnut. Moreover, the Pnut-deficient embryos obtained by either method completed early syncytial development and began cellularization of the embryo normally. However, during the later stages of cellularization, the organization of the actin cytoskeleton at the leading edge of the invaginating furrows became progressively more abnormal, and the embryos displayed widespread defects in cell and embryo morphology beginning at gastrulation. Examination of two other septins showed that Sep1 was not detectable at the cellularization front in the Pnut-deficient embryos, whereas Sep2 was still present in normal levels. Thus, it is possible that Sep2 (perhaps in conjunction with other septins such as Sep4 and Sep5) fulfills an essential septin role during the organization and initial ingression of the cellularization furrow even in the absence of Pnut and Sep1. Together, the results suggest that some cell-division events in Drosophila do not require septin function, that there is functional differentiation among the Drosophila septins, or both. PMID:10982405

Silver Nanoparticle-Mediated Cellular Responses in Various Cell Lines: An in Vitro Model

PubMed Central

Zhang, Xi-Feng; Shen, Wei; Gurunathan, Sangiliyandi

2016-01-01

Silver nanoparticles (AgNPs) have attracted increased interest and are currently used in various industries including medicine, cosmetics, textiles, electronics, and pharmaceuticals, owing to their unique physical and chemical properties, particularly as antimicrobial and anticancer agents. Recently, several studies have reported both beneficial and toxic effects of AgNPs on various prokaryotic and eukaryotic systems. To develop nanoparticles for mediated therapy, several laboratories have used a variety of cell lines under in vitro conditions to evaluate the properties, mode of action, differential responses, and mechanisms of action of AgNPs. In vitro models are simple, cost-effective, rapid, and can be used to easily assess efficacy and performance. The cytotoxicity, genotoxicity, and biocompatibility of AgNPs depend on many factors such as size, shape, surface charge, surface coating, solubility, concentration, surface functionalization, distribution of particles, mode of entry, mode of action, growth media, exposure time, and cell type. Cellular responses to AgNPs are different in each cell type and depend on the physical and chemical nature of AgNPs. This review evaluates significant contributions to the literature on biological applications of AgNPs. It begins with an introduction to AgNPs, with particular attention to their overall impact on cellular effects. The main objective of this review is to elucidate the reasons for different cell types exhibiting differential responses to nanoparticles even when they possess similar size, shape, and other parameters. Firstly, we discuss the cellular effects of AgNPs on a variety of cell lines; Secondly, we discuss the mechanisms of action of AgNPs in various cellular systems, and try to elucidate how AgNPs interact with different mammalian cell lines and produce significant effects; Finally, we discuss the cellular activation of various signaling molecules in response to AgNPs, and conclude with future perspectives on research into AgNPs. PMID:27669221

Evaluation of Cellular Phenotypes Implicated in Immunopathogenesis and Monitoring Immune Reconstitution Inflammatory Syndrome in HIV/Leprosy Cases

PubMed Central

Giacoia-Gripp, Carmem Beatriz Wagner; Sales, Anna Maria; Nery, José Augusto da Costa; Santos-Oliveira, Joanna Reis; de Oliveira, Ariane Leite; Sarno, Euzenir Nunes; Morgado, Mariza Gonçalves

2011-01-01

Background It is now evident that HAART-associated immunological improvement often leads to a variety of new clinical manifestations, collectively termed immune reconstitution inflammatory syndrome, or IRIS. This phenomenon has already been described in cases of HIV coinfection with Mycobacterium leprae, most of them belonging to the tuberculoid spectrum of leprosy disease, as observed in leprosy reversal reaction (RR). However, the events related to the pathogenesis of this association need to be clarified. This study investigated the immunological profile of HIV/leprosy patients, with special attention to the cellular activation status, to better understand the mechanisms related to IRIS/RR immunopathogenesis, identifying any potential biomarkers for IRIS/RR intercurrence. Methods/Principal Findings Eighty-five individuals were assessed in this study: HIV/leprosy and HIV-monoinfected patients, grouped according to HIV-viral load levels, leprosy patients without HIV coinfection, and healthy controls. Phenotypes were evaluated by flow cytometry for T cell subsets and immune differentiation/activation markers. As expected, absolute counts of the CD4+ and CD8+ T cells from the HIV-infected individuals changed in relation to those of the leprosy patients and controls. However, there were no significant differences among the groups, whether in the expression of cellular differentiation phenotypes or cellular activation, as reflected by the expression of CD38 and HLA-DR. Six HIV/leprosy patients identified as IRIS/RR were analyzed during IRIS/RR episodes and after prednisone treatment. These patients presented high cellular activation levels regarding the expression of CD38 in CD8+ cells T during IRIS/RR (median: 77,15%), dropping significantly (p<0,05) during post-IRIS/RR moments (median: 29,7%). Furthermore, an increase of cellular activation seems to occur prior to IRIS/RR. Conclusion/Significance These data suggest CD38 expression in CD8+ T cells interesting tool identifying HIV/leprosy individuals at risk for IRIS/RR. So, a comparative investigation to leprosy patients at RR should be conducted. PMID:22205964

Evaluation of cellular phenotypes implicated in immunopathogenesis and monitoring immune reconstitution inflammatory syndrome in HIV/leprosy cases.

PubMed

Giacoia-Gripp, Carmem Beatriz Wagner; Sales, Anna Maria; Nery, José Augusto da Costa; Santos-Oliveira, Joanna Reis; de Oliveira, Ariane Leite; Sarno, Euzenir Nunes; Morgado, Mariza Gonçalves

2011-01-01

It is now evident that HAART-associated immunological improvement often leads to a variety of new clinical manifestations, collectively termed immune reconstitution inflammatory syndrome, or IRIS. This phenomenon has already been described in cases of HIV coinfection with Mycobacterium leprae, most of them belonging to the tuberculoid spectrum of leprosy disease, as observed in leprosy reversal reaction (RR). However, the events related to the pathogenesis of this association need to be clarified. This study investigated the immunological profile of HIV/leprosy patients, with special attention to the cellular activation status, to better understand the mechanisms related to IRIS/RR immunopathogenesis, identifying any potential biomarkers for IRIS/RR intercurrence. Eighty-five individuals were assessed in this study: HIV/leprosy and HIV-monoinfected patients, grouped according to HIV-viral load levels, leprosy patients without HIV coinfection, and healthy controls. Phenotypes were evaluated by flow cytometry for T cell subsets and immune differentiation/activation markers. As expected, absolute counts of the CD4+ and CD8+ T cells from the HIV-infected individuals changed in relation to those of the leprosy patients and controls. However, there were no significant differences among the groups, whether in the expression of cellular differentiation phenotypes or cellular activation, as reflected by the expression of CD38 and HLA-DR. Six HIV/leprosy patients identified as IRIS/RR were analyzed during IRIS/RR episodes and after prednisone treatment. These patients presented high cellular activation levels regarding the expression of CD38 in CD8+ cells T during IRIS/RR (median: 77,15%), dropping significantly (p<0,05) during post-IRIS/RR moments (median: 29,7%). Furthermore, an increase of cellular activation seems to occur prior to IRIS/RR. These data suggest CD38 expression in CD8+ T cells interesting tool identifying HIV/leprosy individuals at risk for IRIS/RR. So, a comparative investigation to leprosy patients at RR should be conducted.

Complete horizontal skin cell resurfacing and delayed vertical cell infiltration into porcine reconstructive tissue matrix compared to bovine collagen matrix and human dermis.

PubMed

Mirastschijski, Ursula; Kerzel, Corinna; Schnabel, Reinhild; Strauss, Sarah; Breuing, Karl-Heinz

2013-10-01

Xenogenous dermal matrices are used for hernia repair and breast reconstruction. Full-thickness skin replacement is needed after burn or degloving injuries with exposure of tendons or bones. The authors used a human skin organ culture model to study whether porcine reconstructive tissue matrix (Strattice) is effective as a dermal tissue replacement. Skin cells or split-thickness skin grafts were seeded onto human deepidermized dermis, Strattice, and Matriderm. Cellular resurfacing and matrix infiltration were monitored by live fluorescence imaging, histology, and electron microscopy. Proliferation, apoptosis, cell differentiation, and adhesion were analyzed by immunohistochemistry. Epithelial resurfacing and vertical proliferation were reduced and delayed with both bioartificial matrices compared with deepidermized dermis; however, no differences in apoptosis, cell differentiation, or basement membrane formation were found. Vertical penetration was greatest on Matriderm, whereas no matrix infiltration was found on Strattice in the first 12 days. Uncompromised horizontal resurfacing was greatest with Strattice but was absent with Matriderm. Strattice showed no stimulatory effect on cellular inflammation. Matrix texture and surface properties governed cellular performance on tissues. Although dense dermal compaction delayed vertical cellular ingrowth for Strattice, it allowed uncompromised horizontal resurfacing. Dense dermal compaction may slow matrix decomposition and result in prolonged biomechanical stability of the graft. Reconstructive surgeons should choose the adequate matrix substitute depending on biomechanical requirements at the recipient site. Strattice may be suitable as a dermal replacement at recipient sites with high mechanical load requirements.

Mesenchymal Stem Cell-Derived Factors Restore Function to Human Frataxin-Deficient Cells.

PubMed

Kemp, Kevin; Dey, Rimi; Cook, Amelia; Scolding, Neil; Wilkins, Alastair

2017-08-01

Friedreich's ataxia is an inherited neurological disorder characterised by mitochondrial dysfunction and increased susceptibility to oxidative stress. At present, no therapy has been shown to reduce disease progression. Strategies being trialled to treat Friedreich's ataxia include drugs that improve mitochondrial function and reduce oxidative injury. In addition, stem cells have been investigated as a potential therapeutic approach. We have used siRNA-induced knockdown of frataxin in SH-SY5Y cells as an in vitro cellular model for Friedreich's ataxia. Knockdown of frataxin protein expression to levels detected in patients with the disorder was achieved, leading to decreased cellular viability, increased susceptibility to hydrogen peroxide-induced oxidative stress, dysregulation of key anti-oxidant molecules and deficiencies in both cell proliferation and differentiation. Bone marrow stem cells are being investigated extensively as potential treatments for a wide range of neurological disorders, including Friedreich's ataxia. The potential neuroprotective effects of bone marrow-derived mesenchymal stem cells were therefore studied using our frataxin-deficient cell model. Soluble factors secreted by mesenchymal stem cells protected against cellular changes induced by frataxin deficiency, leading to restoration in frataxin levels and anti-oxidant defences, improved survival against oxidative stress and stimulated both cell proliferation and differentiation down the Schwann cell lineage. The demonstration that mesenchymal stem cell-derived factors can restore cellular homeostasis and function to frataxin-deficient cells further suggests that they may have potential therapeutic benefits for patients with Friedreich's ataxia.

A novel image encryption algorithm using chaos and reversible cellular automata

NASA Astrophysics Data System (ADS)

Wang, Xingyuan; Luan, Dapeng

2013-11-01

In this paper, a novel image encryption scheme is proposed based on reversible cellular automata (RCA) combining chaos. In this algorithm, an intertwining logistic map with complex behavior and periodic boundary reversible cellular automata are used. We split each pixel of image into units of 4 bits, then adopt pseudorandom key stream generated by the intertwining logistic map to permute these units in confusion stage. And in diffusion stage, two-dimensional reversible cellular automata which are discrete dynamical systems are applied to iterate many rounds to achieve diffusion on bit-level, in which we only consider the higher 4 bits in a pixel because the higher 4 bits carry almost the information of an image. Theoretical analysis and experimental results demonstrate the proposed algorithm achieves a high security level and processes good performance against common attacks like differential attack and statistical attack. This algorithm belongs to the class of symmetric systems.

Multi-scale Imaging of Cellular and Sub-cellular Structures using Scanning Probe Recognition Microscopy.

NASA Astrophysics Data System (ADS)

Chen, Q.; Rice, A. F.

2005-03-01

Scanning Probe Recognition Microscopy is a new scanning probe capability under development within our group to reliably return to and directly interact with a specific nanobiological feature of interest. In previous work, we have successfully recognized and classified tubular versus globular biological objects from experimental atomic force microscope images using a method based on normalized central moments [ref. 1]. In this paper we extend this work to include recognition schemes appropriate for cellular and sub-cellular structures. Globular cells containing tubular actin filaments are under investigation. Thus there are differences in external/internal shapes and scales. Continuous Wavelet Transform with a differential Gaussian mother wavelet is employed for multi- scale analysis. [ref. 1] Q. Chen, V. Ayres and L. Udpa, ``Biological Investigation Using Scanning Probe Recognition Microscopy,'' Proceedings 3rd IEEE Conference on Nanotechnology, vol. 2, p 863-865 (2003).

Cellular schwannoma arising from the gastric wall misdiagnosed as a gastric stromal tumor: A case report.

PubMed

Wang, Guangyao; Chen, Ping; Zong, Liang; Shi, Lei; Zhao, Wei

2014-02-01

Cellular schwannomas have been previously described at almost every anatomic location of the human body, but reports in the gastric wall are rare. The current study presents a rare case of cellular schwannoma originating from the gastric wall. Computed tomography revealed a 5.6×5.3×4.0-cm 3 solid mass located in the posterior wall of the stomach. Open laparotomy confirmed its mesenchymal origin. Microscopically, the tissue was composed of spindle-shaped and fascicularly-arranged cells, but mitotic figures were rare. Immunohistochemical staining showed that the tumor was negative for cluster of differentiation (CD)117, CD34, smooth muscle actin and desmin, but positive for S-100 and Ki67. The patient presented no evidence of recurrence and metastasis during follow-up. Gastric cellular schwannomas may be diagnosed by clinical characteristics, histological observations and immunohistochemical markers.

Intracellular Ca2+ concentration in the N1E-115 neuronal cell line and its use for peripheric nerve regeneration.

PubMed

Rodrigues, J M; Luís, A L; Lobato, J V; Pinto, M V; Faustino, A; Hussain, N Sooraj; Lopes, M A; Veloso, A P; Freitas, M; Geuna, S; Santos, J D; Maurício, A C

2005-01-01

Entubulation repair of peripheral nerve injuries has a lengthy history. Several experimental and clinical studies have explored the effectiveness of many biodegradable and non-degradable tubes with or without addition of molecules and cells. The main objective of the present study was to develop an economical and also an easy way for culturing a neural cell line which is capable of growing, differentiating and producing locally nerve growth factors, that are otherwise extremely expensive, inside 90 PLA/10 PLG nerve guides. For this purpose the authors have chosen the N1E-115 cell line, a clone of cells derived from mouse neuroblastoma C-1300 with the perspective of using this differentiated cellular system to cover the inside of 90 PLA/10 PLG nerve guides placed to bridge a nerve gap of 10 mm in the rat sciatic nerve experimental model. The N1E-115 cells proliferate in normal culture medium but undergo neuronal differentiation in response to DMSO. Upon induction of differentiation, proliferation of N1E-115 cells ceases, extensive neurite outgrowth is observed and the membranes become highly excitable. While it is known that Ca2+ serves as an important intracellular signal for cellular various processes, such as growth and differentiation, be toxic to cells and be involved in the triggering of events leading to excitotoxic cell death in neurons. The [Ca2+]i in non-differentiated N1E-115 cells and after distinct periods of differentiation, have been determined by the epifluorescence technique using the Fura-2-AM probe. The results of this quantitative assessment, revealed that N1E-115 cells which undergo neuronal differentiation for 48 hours in the presence of 1.5% DMSO are best qualified to be used to cover the interior of the nerve guides since the [Ca2+]i was not found to be elevated indicating thus that the onset the cell death processes was not occurred.

Identification and Characterization of FGF2-Dependent mRNA: microRNA Networks During Lens Fiber Cell Differentiation

PubMed Central

Wolf, Louise; Gao, Chun S.; Gueta, Karen; Xie, Qing; Chevallier, Tiphaine; Podduturi, Nikhil R.; Sun, Jian; Conte, Ivan; Zelenka, Peggy S.; Ashery-Padan, Ruth; Zavadil, Jiri; Cvekl, Ales

2013-01-01

MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3′-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation. PMID:24142921

Methylation alterations are not a major cause of PTTG1 misregulation.

PubMed

Hidalgo, Manuel; Galan, Jose Jorge; Sáez, Carmen; Ferrero, Eduardo; Castilla, Carolina; Ramirez-Lorca, Reposo; Pelaez, Pablo; Ruiz, Agustin; Japón, Miguel A; Royo, Jose Luis

2008-04-21

On its physiological cellular context, PTTG1 controls sister chromatid segregation during mitosis. Within its crosstalk to the cellular arrest machinery, relies a checkpoint of integrity for which gained the over name of securin. PTTG1 was found to promote malignant transformation in 3T3 fibroblasts, and further found to be overexpressed in different tumor types. More recently, PTTG1 has been also related to different processes such as DNA repair and found to trans-activate different cellular pathways involving c-myc, bax or p53, among others. PTTG1 over-expression has been correlated to a worse prognosis in thyroid, lung, colorectal cancer patients, and it can not be excluded that this effect may also occur in other tumor types. Despite the clinical relevance and the increasing molecular characterization of PTTG1, the reason for its up-regulation remains unclear. We analysed PTTG1 differential expression in PC-3, DU-145 and LNCaP tumor cell lines, cultured in the presence of the methyl-transferase inhibitor 5-Aza-2'-deoxycytidine. We also tested whether the CpG island mapping PTTG1 proximal promoter evidenced a differential methylation pattern in differentiated thyroid cancer biopsies concordant to their PTTG1 immunohistochemistry status. Finally, we performed whole-genome LOH studies using Affymetix 50 K microarray technology and FRET analysis to search for allelic imbalances comprising the PTTG1 locus. Our data suggest that neither methylation alterations nor LOH are involved in PTTG1 over-expression. These data, together with those previously reported, point towards a post-transcriptional level of misregulation associated to PTTG1 over-expression.

Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress.

PubMed

Jamsheer K, Muhammed; Laxmi, Ashverya

2015-01-01

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

Nanofiber Orientation and Surface Functionalization Modulate Human Mesenchymal Stem Cell Behavior In Vitro

PubMed Central

Kolambkar, Yash M.; Bajin, Mehmet; Wojtowicz, Abigail; Hutmacher, Dietmar W.; García, Andrés J.

2014-01-01

Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ɛ-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration. PMID:24020454

The Extracellular Environment's Effect on Cellular Processes: An In Vitro Study of Mechanical and Chemical Cues on Human Mesenchymal Stem Cells and C17.2 Neural Stem Cells

NASA Astrophysics Data System (ADS)

Casey, Meghan E.

Stem cells are widely used in the area of tissue engineering. The ability of cells to interact with materials on the nano- and micro- level is important in the success of the biomaterial. It is well-known that cells respond to their micro- and nano-environments through a process termed chemo-mechanotransduction. It is important to establish standard protocols for cellular experiments, as chemical modifications to maintenance environments can alter long-term research results. In this work, the effects of different media compositions on human mesenchymal stem cells (hMSCs) throughout normal in vitro maintenance are investigated. Changes in RNA regulation, protein expression and proliferation are studied via quantitative polymerase chain reaction (qPCR), immunocytochemistry (ICC) and cell counts, respectively. Morphological differences are also observed throughout the experiment. Results of this study illustrate the dynamic response of hMSC maintenance to differences in growth medium and passage number. These experiments highlight the effect growth medium has on in vitro experiments and the need of consistent protocols in hMSC research. A substantial opportunity exists in neuronal research to develop a material platform that allows for both the proliferation and differentiation of stem cells into neurons and the ability to quantify the secretome of neuronal cells. Anodic aluminum oxide (AAO) membranes are fabricated in a two-step anodization procedure where voltage is varied to control the pore size and morphology of the membranes. C17.2 neural stem cells are differentiated on the membranes via serum-withdrawal. Cellular growth is characterized by scanning electron microscopy (SEM), ICC and qPCR. ImageJ software is used to obtain phenotypic cell counts and neurite outgrowth lengths. Results indicate a highly tunable correlation between AAO nanopore sizes and differentiated cell populations. By selecting AAO membranes with specific pore size ranges, control of neuronal network density and neurite outgrowth length is achievable. To understand differentiation marker expressions in C17.2 NSCs and how material stiffness affects differentiation, cells are cultured on substrates of varying stiffness. qPCR is used to analyze neural stem cell, neural progenitor cell, neuron-restricted progenitor and differentiated post-mitotic neuronal cell RNA expression. Results suggest a relationship between material stiffness and neuronal development in C17.2 neural stem cells.

A genetic framework controlling the differentiation of intestinal stem cells during regeneration in Drosophila

PubMed Central

Boquete, Jean-Philippe

2017-01-01

The speed of stem cell differentiation has to be properly coupled with self-renewal, both under basal conditions for tissue maintenance and during regeneration for tissue repair. Using the Drosophila midgut model, we analyze at the cellular and molecular levels the differentiation program required for robust regeneration. We observe that the intestinal stem cell (ISC) and its differentiating daughter, the enteroblast (EB), form extended cell-cell contacts in regenerating intestines. The contact between progenitors is stabilized by cell adhesion molecules, and can be dynamically remodeled to elicit optimal juxtacrine Notch signaling to determine the speed of progenitor differentiation. Notably, increasing the adhesion property of progenitors by expressing Connectin is sufficient to induce rapid progenitor differentiation. We further demonstrate that JAK/STAT signaling, Sox21a and GATAe form a functional relay to orchestrate EB differentiation. Thus, our study provides new insights into the complex and sequential events that are required for rapid differentiation following stem cell division during tissue replenishment. PMID:28662029

Adhesion molecules and receptors

USDA-ARS?s Scientific Manuscript database

Adhesion molecules are necessary for leukocyte trafficking and differentiation. They serve to initiate cell-cell interactions under conditions of shear, and they sustain the cell-cell and cell-matrix interactions needed for cellular locomotion. They also can serve directly as signaling molecules act...

Redox regulation in cancer stem cells

USDA-ARS?s Scientific Manuscript database

Reactive oxygen species (ROS) and ROS-dependent (redox regulation) signaling pathways and transcriptional activities are thought to be critical in stem cell self-renewal and differentiation during growth and organogenesis. Aberrant ROS burst and dysregulation of those ROS-dependent cellular processe...

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus

PubMed Central

2014-01-01

Introduction Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. Methods Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. Results A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)–negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. Conclusions Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease. PMID:24972717

Novel immortal human cell lines reveal subpopulations in the nucleus pulposus.

PubMed

van den Akker, Guus G H; Surtel, Don A M; Cremers, Andy; Rodrigues-Pinto, Ricardo; Richardson, Stephen M; Hoyland, Judith A; van Rhijn, Lodewijk W; Welting, Tim J M; Voncken, Jan Willem

2014-06-27

Relatively little is known about cellular subpopulations in the mature nucleus pulposus (NP). Detailed understanding of the ontogenetic, cellular and molecular characteristics of functional intervertebral disc (IVD) cell populations is pivotal to the successful development of cell replacement therapies and IVD regeneration. In this study, we aimed to investigate whether phenotypically distinct clonal cell lines representing different subpopulations in the human NP could be generated using immortalization strategies. Nondegenerate healthy disc material (age range, 8 to 15 years) was obtained as surplus surgical material. Early passage NP monolayer cell cultures were initially characterized using a recently established NP marker set. NP cells were immortalized by simian virus 40 large T antigen (SV40LTag) and human telomerase reverse transcriptase expression. Immortalized cells were clonally expanded and characterized based on collagen type I, collagen type II, α1 (COL2A1), and SRY-box 9 (SOX9) protein expression profiles, as well as on expression of a subset of established in vivo NP cell lineage markers. A total of 54 immortal clones were generated. Profiling of a set of novel NP markers (CD24, CA12, PAX1, PTN, FOXF1 and KRT19 mRNA) in a representative set of subclones substantiated successful immortalization of multiple cellular subpopulations from primary isolates and confirmed their NP origin and/or phenotype. We were able to identify two predominant clonal NP subtypes based on their morphological characteristics and their ability to induce SOX9 and COL2A1 under conventional differentiation conditions. In addition, cluster of differentiation 24 (CD24)-negative NP responder clones formed spheroid structures in various culture systems, suggesting the preservation of a more immature phenotype compared to CD24-positive nonresponder clones. Here we report the generation of clonal NP cell lines from nondegenerate human IVD tissue and present a detailed characterization of NP cellular subpopulations. Differential cell surface marker expression and divergent responses to differentiation conditions suggest that the NP subtypes may correspond to distinct maturation stages and represent distinct NP cell subpopulations. Hence, we provide evidence that the immortalization strategy that we applied is capable of detecting cell heterogeneity in the NP. Our cell lines yield novel insights into NP biology and provide promising new tools for studies of IVD development, cell function and disease.

Evaluation of Late Effects of Heavy-Ion Radiation on Mesenchymal Stem Cells

NASA Technical Reports Server (NTRS)

Gonda, S.R.; Behravesh, E.; Huff, J.L.; Johnson, F.

2005-01-01

The overall objective of this recently funded study is to utilize well-characterized model test systems to assess the impact of pluripotent stem cell differentiation on biological effects associated with high-energy charged particle radiation. These stem cells, specifically mesenchymal stem cells (MSCs), have the potential for differentiation into bone, cartilage, fat, tendons, and other tissue types. The characterization of the regulation mechanisms of MSC differentiation to the osteoblastic lineage by transcription factors, such as Runx2/Cbfa1 and Osterix, and osteoinductive proteins such as members of the bone morphogenic protein family are well established. More importantly, for late biological effects, MSCs have been shown to contribute to tissue restructuring and repair after tissue injury. The complex regulation of and interactions between inflammation and repair determine the eventual outcome of the responses to tissue injury, for which MSCs play a crucial role. Additionally, MSCs have been shown to respond to reactive oxygen species, a secondary effector of radiation, by differentiating. With this, we hypothesized that differentiation of MSCs can alter or exacerbate the damage initiated by radiation, which can ultimately lead to late biological effects of misrepair/fibrosis which may ultimately lead to carcinogenesis. Currently, studies are underway to examine high-energy X-ray radiation at low and high doses, approximately 20 and 200 Rad, respectively, on cytogenetic damage and gene modulation of isolated MSCs. These cells, positive for MSC surface markers, were obtained from three persons. In vitro cell samples were harvested during cellular proliferation and after both cellular recovery and differentiation. Future work will use established in vitro models of increasing complexity to examine the value of traditional 2D tissue-culture techniques, and utilize 3D in vitro tissue culture techniques that can better assess late effects associated with radiation.

Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation.

PubMed

Galve-Roperh, Ismael; Chiurchiù, Valerio; Díaz-Alonso, Javier; Bari, Monica; Guzmán, Manuel; Maccarrone, Mauro

2013-10-01

Cannabinoids, the active components of cannabis (Cannabis sativa) extracts, have attracted the attention of human civilizations for centuries, much earlier than the discovery and characterization of their substrate of action, the endocannabinoid system (ECS). The latter is an ensemble of endogenous lipids, their receptors [in particular type-1 (CB1) and type-2 (CB2) cannabinoid receptors] and metabolic enzymes. Cannabinoid signaling regulates cell proliferation, differentiation and survival, with different outcomes depending on the molecular targets and cellular context involved. Cannabinoid receptors are expressed and functional from the very early developmental stages, when they regulate embryonic and trophoblast stem cell survival and differentiation, and thus may affect the formation of manifold adult specialized tissues derived from the three different germ layers (ectoderm, mesoderm and endoderm). In the ectoderm-derived nervous system, both CB1 and CB2 receptors are present in neural progenitor/stem cells and control their self-renewal, proliferation and differentiation. CB1 and CB2 show opposite patterns of expression, the former increasing and the latter decreasing along neuronal differentiation. Recently, endocannabinoid (eCB) signaling has also been shown to regulate proliferation and differentiation of mesoderm-derived hematopoietic and mesenchymal stem cells, with a key role in determining the formation of several cell types in peripheral tissues, including blood cells, adipocytes, osteoblasts/osteoclasts and epithelial cells. Here, we will review these new findings, which unveil the involvement of eCB signaling in the regulation of progenitor/stem cell fate in the nervous system and in the periphery. The developmental regulation of cannabinoid receptor expression and cellular/subcellular localization, together with their role in progenitor/stem cell biology, may have important implications in human health and disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mitochondrial Stress Tests Using Seahorse Respirometry on Intact Dictyostelium discoideum Cells.

PubMed

Lay, Sui; Sanislav, Oana; Annesley, Sarah J; Fisher, Paul R

2016-01-01

Mitochondria not only play a critical and central role in providing metabolic energy to the cell but are also integral to the other cellular processes such as modulation of various signaling pathways. These pathways affect many aspects of cell physiology, including cell movement, growth, division, differentiation, and death. Mitochondrial dysfunction which affects mitochondrial bioenergetics and causes oxidative phosphorylation defects can thus lead to altered cellular physiology and manifest in disease. The assessment of the mitochondrial bioenergetics can thus provide valuable insights into the physiological state, and the alterations to the state of the cells. Here, we describe a method to successfully use the Seahorse XF(e)24 Extracellular Flux Analyzer to assess the mitochondrial respirometry of the cellular slime mold Dictyostelium discoideum.

Massive Broad Ligament Cellular Leiomyoma with Cystic Change: A Diagnostic Dilemma.

PubMed

Sharma, Preeti; Zaheer, Sufian; Yadav, Amit Kumar; Mandal, Ashish Kumar

2016-04-01

Leiomyomas are known to arise from uterus, but rarely from broad ligament. Further, cellular leiomyoma of broad ligament is the least common variant reported in literature. The diagnostic dilemma arises when leiomyomas undergo degenerative changes. This poses both clinical and radiological difficulty in differentiating with an ovarian tumour. We present an unusual case of a huge broad ligament mass measuring 29x19x09cm, mimicking an ovarian tumour both clinically and radiologically. Histopathology revealed cellular leiomyoma of broad ligament with cystic and myxoid degeneration hereby being the second case reported in literature. This case is being presented not only because of the rare incidence but also due to its diagnostic confusion with ovarian malignancy on clinical evaluation and radiological findings.

Involvement of CRF2 signaling in enterocyte differentiation

PubMed Central

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-01-01

AIM To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. METHODS For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. RESULTS CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. CONCLUSION Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases. PMID:28811708

Involvement of CRF2 signaling in enterocyte differentiation.

PubMed

Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-07-28

To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor krüppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells.

PubMed

Valli, Emanuele; Trazzi, Stefania; Fuchs, Claudia; Erriquez, Daniela; Bartesaghi, Renata; Perini, Giovanni; Ciani, Elisabetta

2012-01-01

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G(0)/G(1) phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5. Copyright © 2012 Elsevier B.V. All rights reserved.

Endothelial-monocyte activating polypeptide II disrupts alveolar epithelial type II to type I cell transdifferentiation

PubMed Central

2012-01-01

Background Distal alveolar morphogenesis is marked by differentiation of alveolar type (AT)-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP) II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII→ATI trans-differentiation has not been explored. Method In a controlled nonvascular environment, an in vitro model of ATII→ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation. Results Here, we show that EMAP II significantly blocked ATII→ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA. Conclusion Our findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia. PMID:22214516

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

Orfali, Nina; Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA.; McKenna, Sharon L.

Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL.more » Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects.« less

Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

PubMed Central

2010-01-01

Background Infection by infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 × 44 K Agilent chicken custom oligo microarrays. Results Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi). Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB), cell cycle regulation (cyclin B2, CDK1, and CKI3), matrix metalloproteinases (MMPs) and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death. Conclusion The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections. PMID:20663125

MicroRNA-432 contributes to dopamine cocktail and retinoic acid induced differentiation of human neuroblastoma cells by targeting NESTIN and RCOR1 genes.

PubMed

Das, Eashita; Bhattacharyya, Nitai Pada

2014-05-02

MicroRNA (miRNA) regulates expression of protein coding genes and has been implicated in diverse cellular processes including neuronal differentiation, cell growth and death. To identify the role of miRNA in neuronal differentiation, SH-SY5Y and IMR-32 cells were treated with dopamine cocktail and retinoic acid to induce differentiation. Detection of miRNAs in differentiated cells revealed that expression of many miRNAs was altered significantly. Among the altered miRNAs, human brain expressed miR-432 induced neurite projections, arrested cells in G0-G1, reduced cell proliferation and could significantly repress NESTIN/NES, RCOR1/COREST and MECP2. Our results reveal that miR-432 regulate neuronal differentiation of human neuroblastoma cells. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Autophagy dictates metabolism and differentiation of inflammatory immune cells

PubMed Central

Riffelmacher, Thomas; Richter, Felix Clemens; Simon, Anna Katharina

2018-01-01

ABSTRACT The role of macroautophagy/autophagy, a conserved lysosomal degradation pathway, during cellular differentiation has been well studied over the last decade. In particular, evidence for its role during immune cell differentiation is growing. Despite the description of a variety of dramatic immune phenotypes in tissue-specific autophagy knockout models, the underlying mechanisms are still under debate. One of the proposed mechanisms is the impact of autophagy on the altered metabolic states during immune cell differentiation. This concept is strengthened through novel molecular insights into how AMPK and MTOR signaling cascades affect both autophagy and metabolism. In this review, we discuss direct and indirect evidence linking autophagy, metabolic pathways and immune cell differentiation including T, B, and innate lymphocytes as well as in myeloid cells that are direct mediators of inflammation. Herein, we propose a model for autophagy-driven immunometabolism controlling immune cell differentiation. PMID:28806133

Nuclear Mechanics and Stem Cell Differentiation.

PubMed

Mao, Xinjian; Gavara, Nuria; Song, Guanbin

2015-12-01

Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

ent-Jungermannenone C Triggers Reactive Oxygen Species-Dependent Cell Differentiation in Leukemia Cells.

PubMed

Yue, Zongwei; Xiao, Xinhua; Wu, Jinbao; Zhou, Xiaozhou; Liu, Weilong; Liu, Yaxi; Li, Houhua; Chen, Guoqiang; Wu, Yingli; Lei, Xiaoguang

2018-02-23

Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by clonal proliferation of myeloid blasts. Despite the progress that has been made in the treatment of various malignant hematopoietic diseases, the effective treatment of AML remains very challenging. Differentiation therapy has emerged as a promising approach for leukemia treatment, and new and effective chemical agents to trigger the differentiation of AML cells, especially drug-resistant cells, are urgently required. Herein, the natural product jungermannenone C, a tetracyclic diterpenoid isolated from liverworts, is reported to induce cell differentiation in AML cells. Interestingly, the unnatural enantiomer of jungermannenone C (1) was found to be more potent than jungermannenone C in inducing cell differentiation. Furthermore, compound 1 targets peroxiredoxins I and II by selectively binding to the conserved cysteine residues and leads to cellular reactive oxygen species accumulation. Accordingly, ent-jungermannenone C (1) shows potential for further investigation as an effective differentiation therapy against AML.

[Effects of triterpenoid from Psidium guajava leaves ursolic acid on proliferation, differentiation of 3T3-L1 preadipocyte and insulin resistance].

PubMed

Lin, Juan-Na; Kuang, Qiao-Ting; Ye, Kai-He; Ye, Chun-Ling; Huang, Yi; Zhang, Xiao-Qi; Ye, Wen-Cai

2013-08-01

To investigate the influences of triterpenoid from Psidium guajava Leaves (ursolic acid) on the proliferation, differentiation of 3T3-L1 preadipocyte, and its possible mechanism treat for insulin resistance. 3T3-L1 preadipocyte was cultured in vitro. After adding ursolic acid to the culture medium for 48h, the cell viability was tested by MTT assay. Induced for 6 days, the lipid accumulation of adipocyte was measured by Oil Red O staining. The insulin resistant cell model was established with Dexamethasone. Cellular glucose uptake was determined with GOD-POD assays and FFA concentration was determined at the time of 48h. Secreted adiponectin were measured by ELISA. The protein levels of PPARgamma and PTP1B in insulin resistant adipocyte were measured by Western Blotting. Compared with medium control group, 30, 100 micromol/L ursolic acid could increase its proliferation and differentiation significantly (P < 0.05 or P < 0.01). Compared with the model group, ursolic acid at 100 micromol/L could enhance cellular glucose uptake of insulin resistant adipocyte significantly both in basic and insulin stimulation state (P < 0.01), while ursolic acid at 30 micromol/L could already enhance its glucose uptake significantly (P < 0.05), and could already decrease its FFA production significantly (P < 0.05). Ursolic acid at 30 micromol/L could increase the secretion of adiponectin on insulin resistant adipocyte significantly (P < 0.05), up-regulate the expression of PPARgamma protein (P < 0.05), but showed no effect on the PTP1B protein expression (P > 0.05). Ursolic acid can improve the proliferation and differentiation of 3T3-L1 preadipocyte, enhance cellular glucose uptake, inhibit the production of FFA, promote the secretion of adiponectin insulin resistant adipocyte, its mechanism may be related to upregulating the expression of PPARgamma protein.

Insulin-Like Growth Factor 1 Receptor and p38 Mitogen-Activated Protein Kinase Signals Inversely Regulate Signal Transducer and Activator of Transcription 3 Activity to Control Human Dental Pulp Stem Cell Quiescence, Propagation, and Differentiation

PubMed Central

Vandomme, Jerome; Touil, Yasmine; Ostyn, Pauline; Olejnik, Cecile; Flamenco, Pilar; El Machhour, Raja; Segard, Pascaline; Masselot, Bernadette; Bailliez, Yves; Formstecher, Pierre

2014-01-01

Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Ylow stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration. PMID:24266654

Cellular internalization of LiNbO3 nanocrystals for second harmonic imaging and the effects on stem cell differentiation

NASA Astrophysics Data System (ADS)

Li, Jianhua; Qiu, Jichuan; Guo, Weibo; Wang, Shu; Ma, Baojin; Mou, Xiaoning; Tanes, Michael; Jiang, Huaidong; Liu, Hong

2016-03-01

Second harmonic generation (SHG) nanocrystals have recently been reported to label cancer cells and other functional cell lines due to their unique double-frequency property. In this paper, we report for the first time the use of lithium niobate (LiNbO3, LN) nanocrystals as SHG labels for imaging stem cells. Rat mesenchymal stem cells (rMSCs) were labeled with LN nanocrystals in order to study the cellular internalization of the nanocrystals and the influence on stem cell differentiation. The results showed that LN nanocrystals were endocytosed by the rMSCs and the distribution of the internalized nanoparticles demonstrated a high consistency with the orientation of the actin filaments. Besides, LN-labeled rMSCs showed a concentration-dependent viability. Most importantly, rMSCs labeled with 50 μg per mL of LN nanocrystals retained their ability to differentiate into both osteogenic and adipogenic lineages. The results prove that LN nanocrystals can be used as a cytocompatible, near-infrared (NIR) light driven cell label for long-term imaging, without hindering stem cell differentiation. This work will promote the use of LN nanocrystals to broader applications like deep-tissue tracking, remote drug delivery and stem cell therapy.Second harmonic generation (SHG) nanocrystals have recently been reported to label cancer cells and other functional cell lines due to their unique double-frequency property. In this paper, we report for the first time the use of lithium niobate (LiNbO3, LN) nanocrystals as SHG labels for imaging stem cells. Rat mesenchymal stem cells (rMSCs) were labeled with LN nanocrystals in order to study the cellular internalization of the nanocrystals and the influence on stem cell differentiation. The results showed that LN nanocrystals were endocytosed by the rMSCs and the distribution of the internalized nanoparticles demonstrated a high consistency with the orientation of the actin filaments. Besides, LN-labeled rMSCs showed a concentration-dependent viability. Most importantly, rMSCs labeled with 50 μg per mL of LN nanocrystals retained their ability to differentiate into both osteogenic and adipogenic lineages. The results prove that LN nanocrystals can be used as a cytocompatible, near-infrared (NIR) light driven cell label for long-term imaging, without hindering stem cell differentiation. This work will promote the use of LN nanocrystals to broader applications like deep-tissue tracking, remote drug delivery and stem cell therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00785f

Quantifying the driving factors for language shift in a bilingual region.

PubMed

Prochazka, Katharina; Vogl, Gero

2017-04-25

Many of the world's around 6,000 languages are in danger of disappearing as people give up use of a minority language in favor of the majority language in a process called language shift. Language shift can be monitored on a large scale through the use of mathematical models by way of differential equations, for example, reaction-diffusion equations. Here, we use a different approach: we propose a model for language dynamics based on the principles of cellular automata/agent-based modeling and combine it with very detailed empirical data. Our model makes it possible to follow language dynamics over space and time, whereas existing models based on differential equations average over space and consequently provide no information on local changes in language use. Additionally, cellular automata models can be used even in cases where models based on differential equations are not applicable, for example, in situations where one language has become dispersed and retreated to language islands. Using data from a bilingual region in Austria, we show that the most important factor in determining the spread and retreat of a language is the interaction with speakers of the same language. External factors like bilingual schools or parish language have only a minor influence.

Periodontal Biology: Stem Cells, Bmp2 Gene, Transcriptional Enhancers, and Use of Sclerostin Antibody and Pth for Treatment of Periodontal Disease and Bone Loss

PubMed Central

Harris, Stephen E; Rediske, Michael; Neitzke, Rebecca; Rakian, Audrey

2017-01-01

The periodontium is a complex tissue with epithelial components and a complex set of mesodermal derived alveolar bone, cellular and a cellular cementum, and tendon like ligaments (PDL). The current evidence demonstrates that the major pool of periodontal stem cells is derived from a population of micro vascular associated aSMA-positive stem/progenitor (PSC) cells that by lineage tracing form all three major mesodermal derived components of the periodontium. With in vitro aSMA+ stem cells, transcriptome and chip- seq experiments, the gene network and enhancer maps were determined at several differentiation states of the PSC. Current work on the role of the Bmp2 gene in the periodontal stem cell differentiation demonstrated that this Wnt regulated gene, Bmp2, is necessary for differentiation to all three major mesodermal derived component of the periodontium. The mechanism and use of Sclerostin antibody as an activator of Wnt signaling and Bmp2 gene as a potential route to treat craniofacial bone loss is discussed. As well, the mechanism and use of Pth in the treatment of periodontal bone loss or other craniofacial bone loss is presented in this review. PMID:29457146

Manganese-Induced Neurotoxicity and Alterations in Gene Expression in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Gandhi, Deepa; Sivanesan, Saravanadevi; Kannan, Krishnamurthi

2018-06-01

Manganese (Mn) is an essential trace element required for many physiological functions including proper biochemical and cellular functioning of the central nervous system (CNS). However, exposure to excess level of Mn through occupational settings or from environmental sources has been associated with neurotoxicity. The cellular and molecular mechanism of Mn-induced neurotoxicity remains unclear. In the current study, we investigated the effects of 30-day exposure to a sub-lethal concentration of Mn (100 μM) in human neuroblastoma cells (SH-SY5Y) using transcriptomic approach. Microarray analysis revealed differential expression of 1057 transcripts in Mn-exposed SH-SY5Y cells as compared to control cells. Gene functional annotation cluster analysis exhibited that the differentially expressed genes were associated with several biological pathways. Specifically, genes involved in neuronal pathways including neuron differentiation and development, regulation of neurogenesis, synaptic transmission, and neuronal cell death (apoptosis) were found to be significantly altered. KEGG pathway analysis showed upregulation of p53 signaling pathways and neuroactive ligand-receptor interaction pathways, and downregulation of neurotrophin signaling pathway. On the basis of the gene expression profile, possible molecular mechanisms underlying Mn-induced neuronal toxicity were predicted.

Transcriptomic analysis of the dimorphic transition of Ustilago maydis induced in vitro by a change in pH.

PubMed

Martínez-Soto, Domingo; Ruiz-Herrera, José

2013-01-01

Dimorphism is the property of fungi to grow as budding yeasts or mycelium, depending on the environmental conditions. This phenomenon is important as a model of differentiation in eukaryotic organisms, and since a large number of fungal diseases are caused by dimorphic fungi, its study is important for practical reasons. In this work, we examined the transcriptome during the dimorphic transition of the basidiomycota phytopathogenic fungus Ustilago maydis using microarrays, utilizing yeast and mycelium monomorphic mutants as controls. This way, we thereby identified 154 genes of the fungus that are specifically involved in the dimorphic transition induced by a pH change. Of these, 82 genes were up-regulated, and 72 were down-regulated. Differential categorization of these genes revealed that they mostly belonged to the classes of metabolism, cell cycle and DNA processing, transcription and protein fate, transport and cellular communication, stress, cell differentiation and biogenesis of cellular components, while a significant number of them corresponded to unclassified proteins. The data reported in this work are important for our understanding of the molecular bases of dimorphism in U. maydis, and possibly of other fungi. Copyright © 2013. Published by Elsevier Inc.

High-content screening of small compounds on human embryonic stem cells.

PubMed

Barbaric, Ivana; Gokhale, Paul J; Andrews, Peter W

2010-08-01

Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

MOF maintains transcriptional programs regulating cellular stress response

PubMed Central

Sheikh, B N; Bechtel-Walz, W; Lucci, J; Karpiuk, O; Hild, I; Hartleben, B; Vornweg, J; Helmstädter, M; Sahyoun, A H; Bhardwaj, V; Stehle, T; Diehl, S; Kretz, O; Voss, A K; Thomas, T; Manke, T; Huber, T B; Akhtar, A

2016-01-01

MOF (MYST1, KAT8) is the major H4K16 lysine acetyltransferase (KAT) in Drosophila and mammals and is essential for embryonic development. However, little is known regarding the role of MOF in specific cell lineages. Here we analyze the differential role of MOF in proliferating and terminally differentiated tissues at steady state and under stress conditions. In proliferating cells, MOF directly binds and maintains the expression of genes required for cell cycle progression. In contrast, MOF is dispensable for terminally differentiated, postmitotic glomerular podocytes under physiological conditions. However, in response to injury, MOF is absolutely critical for podocyte maintenance in vivo. Consistently, we detect defective nuclear, endoplasmic reticulum and Golgi structures, as well as presence of multivesicular bodies in vivo in podocytes lacking Mof following injury. Undertaking genome-wide expression analysis of podocytes, we uncover several MOF-regulated pathways required for stress response. We find that MOF, along with the members of the non-specific lethal but not the male-specific lethal complex, directly binds to genes encoding the lysosome, endocytosis and vacuole pathways, which are known regulators of podocyte maintenance. Thus, our work identifies MOF as a key regulator of cellular stress response in glomerular podocytes. PMID:26387537

Phenotypic stability and plasticity in GMP-derived cells as determined by their underlying regulatory network.

PubMed

Ramírez, Carlos; Mendoza, Luis

2018-04-01

Blood cell formation has been recognized as a suitable system to study celular differentiation mainly because of its experimental accessibility, and because it shows characteristics such as hierarchical and gradual bifurcated patterns of commitment, which are present in several developmental processes. Although hematopoiesis has been extensively studied and there is a wealth of molecular and cellular data about it, it is not clear how the underlying molecular regulatory networks define or restrict cellular differentiation processes. Here, we infer the molecular regulatory network that controls the differentiation of a blood cell subpopulation derived from the granulocyte-monocyte precursor (GMP), comprising monocytes, neutrophils, eosinophils, basophils and mast cells. We integrate published qualitative experimental data into a model to describe temporal expression patterns observed in GMP-derived cells. The model is implemented as a Boolean network, and its dynamical behavior is studied. Steady states of the network can be clearly identified with the expression profiles of monocytes, mast cells, neutrophils, basophils, and eosinophils, under wild-type and mutant backgrounds. All scripts are publicly available at https://github.com/caramirezal/RegulatoryNetworkGMPModel. lmendoza@biomedicas.unam.mx. Supplementary data are available at Bioinformatics online.

Dynamical and Mechanistic Reconstructive Approaches of T Lymphocyte Dynamics: Using Visual Modeling Languages to Bridge the Gap between Immunologists, Theoreticians, and Programmers

PubMed Central

Thomas-Vaslin, Véronique; Six, Adrien; Ganascia, Jean-Gabriel; Bersini, Hugues

2013-01-01

Dynamic modeling of lymphocyte behavior has primarily been based on populations based differential equations or on cellular agents moving in space and interacting each other. The final steps of this modeling effort are expressed in a code written in a programing language. On account of the complete lack of standardization of the different steps to proceed, we have to deplore poor communication and sharing between experimentalists, theoreticians and programmers. The adoption of diagrammatic visual computer language should however greatly help the immunologists to better communicate, to more easily identify the models similarities and facilitate the reuse and extension of existing software models. Since immunologists often conceptualize the dynamical evolution of immune systems in terms of “state-transitions” of biological objects, we promote the use of unified modeling language (UML) state-transition diagram. To demonstrate the feasibility of this approach, we present a UML refactoring of two published models on thymocyte differentiation. Originally built with different modeling strategies, a mathematical ordinary differential equation-based model and a cellular automata model, the two models are now in the same visual formalism and can be compared. PMID:24101919

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

MOF maintains transcriptional programs regulating cellular stress response.

PubMed

Sheikh, B N; Bechtel-Walz, W; Lucci, J; Karpiuk, O; Hild, I; Hartleben, B; Vornweg, J; Helmstädter, M; Sahyoun, A H; Bhardwaj, V; Stehle, T; Diehl, S; Kretz, O; Voss, A K; Thomas, T; Manke, T; Huber, T B; Akhtar, A

2016-05-01

MOF (MYST1, KAT8) is the major H4K16 lysine acetyltransferase (KAT) in Drosophila and mammals and is essential for embryonic development. However, little is known regarding the role of MOF in specific cell lineages. Here we analyze the differential role of MOF in proliferating and terminally differentiated tissues at steady state and under stress conditions. In proliferating cells, MOF directly binds and maintains the expression of genes required for cell cycle progression. In contrast, MOF is dispensable for terminally differentiated, postmitotic glomerular podocytes under physiological conditions. However, in response to injury, MOF is absolutely critical for podocyte maintenance in vivo. Consistently, we detect defective nuclear, endoplasmic reticulum and Golgi structures, as well as presence of multivesicular bodies in vivo in podocytes lacking Mof following injury. Undertaking genome-wide expression analysis of podocytes, we uncover several MOF-regulated pathways required for stress response. We find that MOF, along with the members of the non-specific lethal but not the male-specific lethal complex, directly binds to genes encoding the lysosome, endocytosis and vacuole pathways, which are known regulators of podocyte maintenance. Thus, our work identifies MOF as a key regulator of cellular stress response in glomerular podocytes.

Epigenetic regulatory mechanisms in vertebrate eye development and disease

PubMed Central

Cvekl, A; Mitton, KP

2014-01-01

Eukaryotic DNA is organized as a nucleoprotein polymer termed chromatin with nucleosomes serving as its repetitive architectural units. Cellular differentiation is a dynamic process driven by activation and repression of specific sets of genes, partitioning the genome into transcriptionally active and inactive chromatin domains. Chromatin architecture at individual genes/loci may remain stable through cell divisions, from a single mother cell to its progeny during mitosis, and represents an example of epigenetic phenomena. Epigenetics refers to heritable changes caused by mechanisms distinct from the primary DNA sequence. Recent studies have shown a number of links between chromatin structure, gene expression, extracellular signaling, and cellular differentiation during eye development. This review summarizes recent advances in this field, and the relationship between sequence-specific DNA-binding transcription factors and their roles in recruitment of chromatin remodeling enzymes. In addition, lens and retinal differentiation is accompanied by specific changes in the nucleolar organization, expression of non-coding RNAs, and DNA methylation. Epigenetic regulatory mechanisms in ocular tissues represent exciting areas of research that have opened new avenues for understanding normal eye development, inherited eye diseases and eye diseases related to aging and the environment. PMID:20179734

Role of Passive Diffusion, Transporters, and Membrane Trafficking-Mediated Processes in Cellular Drug Transport.

PubMed

Cocucci, E; Kim, J Y; Bai, Y; Pabla, N

2017-01-01

Intracellular drug accumulation is thought to be dictated by two major processes, passive diffusion through the lipid membrane or membrane transporters. The relative role played by these distinct processes remains actively debated. Moreover, the role of membrane-trafficking in drug transport remains underappreciated and unexplored. Here we discuss the distinct processes involved in cellular drug distribution and propose that better experimental models are required to elucidate the differential contributions of various processes in intracellular drug accumulation. © 2016 American Society for Clinical Pharmacology and Therapeutics.

Hematopoiesis: an evolving paradigm for stem cell biology.

PubMed

Orkin, Stuart H; Zon, Leonard I

2008-02-22

Establishment and maintenance of the blood system relies on self-renewing hematopoietic stem cells (HSCs) that normally reside in small numbers in the bone marrow niche of adult mammals. This Review describes the developmental origins of HSCs and the molecular mechanisms that regulate lineage-specific differentiation. Studies of hematopoiesis provide critical insights of general relevance to other areas of stem cell biology including the role of cellular interactions in development and tissue homeostasis, lineage programming and reprogramming by transcription factors, and stage- and age-specific differences in cellular phenotypes.

BAP1 and Cancer

PubMed Central

Carbone, Michele; Yang, Haining; Pass, Harvey I.; Krausz, Thomas; Testa, Joseph R.; Gaudino, Giovanni

2013-01-01

Preface BAP1 is a deubiquitylase that is found associated with multi-protein complexes that regulate key cellular pathways, including the cell cycle, cellular differentiation, cell death, gluconeogenesis and the DNA damage response (DDR). Recent findings indicate that germline BAP1 mutations cause a novel cancer syndrome, characterized, at least in the affected families studied so far, by the onset at an early age of benign melanocytic skin tumours with mutated BAP1, and later in life by a high incidence of mesothelioma, uveal melanoma, cutaneous melanoma and possibly additional cancers. PMID:23550303

2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1

PubMed Central

Dhar, Jayeeta; Cuevas, Rolando A.; Goswami, Ramansu; Zhu, Jianzhong

2015-01-01

2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. PMID:26178980

Hardwiring stem cell communication through tissue structure

PubMed Central

Xin, Tianchi; Greco, Valentina; Myung, Peggy

2016-01-01

Adult stem cells across diverse organs self-renew and differentiate to maintain tissue homeostasis. How stem cells receive input to preserve tissue structure and function largely relies on their communication with surrounding cellular and non-cellular elements. As such, how tissues are organized and patterned not only reflects organ function but also inherently hardwires networks of communication between stem cells and their environment to direct tissue homeostasis and injury repair. This review highlights how different methods of stem cell communication reflect the unique organization and function of diverse tissues. PMID:26967287

Epigenetic Therapy in Lung Cancer – Role of microRNAs

PubMed Central

Rothschild, Sacha I.

2013-01-01

Lung cancer is the leading cause of cancer deaths worldwide. microRNAs (miRNAs) are a class of small non-coding RNA species that have been implicated in the control of many fundamental cellular and physiological processes such as cellular differentiation, proliferation, apoptosis, and stem cell maintenance. Some miRNAs have been categorized as “oncomiRs” as opposed to “tumor suppressor miRs.” This review focuses on the role of miRNAs in the lung cancer carcinogenesis and their potential as diagnostic, prognostic, or predictive markers. PMID:23802096

Celllular Uptake and Clearance of TIO2 Nanoparticles

EPA Science Inventory

Differential rates of cellular uptake and clearance of engineered nanomaterials may influence the propensity for tissue accumulation under chronic exposure conditions. A retinal pigment epithelial cell line (ARPE-19) was used to investigate 1) if Ti02 (Degussa, P25) nanoparticles...

Temporal competition between differentiation programs determines cell fate choice

NASA Astrophysics Data System (ADS)

Kuchina, Anna; Espinar, Lorena; Cagatay, Tolga; Balbin, Alejandro; Alvarado, Alma; Garcia-Ojalvo, Jordi; Suel, Gurol

2011-03-01

During pluripotent differentiation, cells adopt one of several distinct fates. The dynamics of this decision-making process are poorly understood, since cell fate choice may be governed by interactions between differentiation programs that are active at the same time. We studied the dynamics of decision-making in the model organism Bacillus subtilis by simultaneously measuring the activities of competing differentiation programs (sporulation and competence) in single cells. We discovered a precise switch-like point of cell fate choice previously hidden by cell-cell variability. Engineered artificial crosslinks between competence and sporulation circuits revealed that the precision of this choice is generated by temporal competition between the key players of two differentiation programs. Modeling suggests that variable progression towards a switch-like decision might represent a general strategy to maximize adaptability and robustness of cellular decision-making.

Diversity in TAF proteomics: consequences for cellular differentiation and migration.

PubMed

Kazantseva, Jekaterina; Palm, Kaia

2014-09-19

Development is a highly controlled process of cell proliferation and differentiation driven by mechanisms of dynamic gene regulation. Specific DNA binding factors for establishing cell- and tissue-specific transcriptional programs have been characterised in different cell and animal models. However, much less is known about the role of "core transcription machinery" during cell differentiation, given that general transcription factors and their spatiotemporally patterned activity govern different aspects of cell function. In this review, we focus on the role of TATA-box associated factor 4 (TAF4) and its functional isoforms generated by alternative splicing in controlling lineage-specific differentiation of normal mesenchymal stem cells and cancer stem cells. In the light of our recent findings, induction, control and maintenance of cell differentiation status implies diversification of the transcription initiation apparatus orchestrated by alternative splicing.

Effects of genistein, a soybean-derived isoflavone, on proliferation and differentiation of B16-BL6 mouse melanoma cells.

PubMed

Yan, C H; Chen, X G; Li, Y; Han, R

1999-01-01

Genistein, a soybean-derived isoflavone, may contribute to the lower cancer incidence in South Asian countries. In this study, the effects and molecular mechanisms of genistein on growth and differentiation of B16-BL6 mouse melanoma cells were investigated. Genistein suppressed the growth of these melanoma cells. The IC50 value is 15.5 microM. On the other hand, genistein induced the changes of cell shape and cytoskeletal network. The cytoskeletal filaments were induced to form a bundle along the direction of elongation of the cells. Moreover, tyrosine phosphorylation levels of cytoskeleton-associated proteins decreased after the cells were exposed to 20 or 30 microM of genistein for 3 days. All these morphological and molecular changes were accompanied by appearance of the differentiated phenotypes. Genistein induced the increase of cellular melanin content, enhancement of tyrosinase activity, and decrease of colonization potentials in soft agar in a time-dependent and dose-dependent manner. The effective concentration was no more than 10 microM after 3 days' exposure. The tumorigenic potentials of B16-BL6 cells in C57BL/6 mouse also decreased after exposure to 20 or 30 microM of genistein for 3 days. When expressions of tumor-related genes were investigated in the differentiation-induced cells, the content of P53 dramatically increased while that of c-Myc protein decreased. Therefore, due to its ability to induce cellular and molecular changes, genistein suppressed the growth and induced differentiated phenotypes in B16-BL6 melanoma cells.

Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation

PubMed Central

Santos, Jerran; Milthorpe, Bruce K; Herbert, Benjamin R; Padula, Matthew P

2017-01-01

Background Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results. Methods Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line. Results The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress. Conclusion The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs. PMID:28844130

Vascular Differentiation from Pluripotent Stem Cells in 3-D Auxetic Scaffolds.

PubMed

Song, Liqing; Ahmed, Mohammad Faisal; Li, Yan; Zeng, Changchun; Li, Yan

2018-05-10

Auxetic scaffolds, i.e. scaffolds that can display negative Poisson's ratio, have unique physical properties and can expand transversally when axially strained or contract under compression. Auxetic materials have been used for bioprostheses and artery stents due to the enhanced compressive strength and shear stiffness. In vascular tissue engineering, auxetic scaffolds allow the widening of blood vessels when blood flows through (creating compressive stress) to prevent the blockage. However, the influence of auxetic materials on the cellular fate decision in local environment is unclear. In this study, auxetic polyurethane foams were used to support vascular differentiation from pluripotent stem cells (PSCs). The expression of alkaline phosphatase, Oct-4 and Nanog was lower after four days of differentiation for the cells grown in auxetic scaffolds. Higher expression of vascular markers CD31 and VE-cadherin was observed for the cells from auxetic scaffolds compared to those from the scaffolds before auxetic conversion. Little influence on the expression of cardiac marker α-actinin was observed. The vascular cells secreted extracellular matrix proteins vitronectin and laminin and expressed membrane-bound matrix metalloproteinase 9. The examination of Yes-associated protein expression indicated more cytoplasmic retention in the cells from auxetic scaffolds compared to those from regular scaffolds, suggesting that the auxetic scaffolds may affect cellular contraction. This study demonstrates a novel 3-D culture based on auxetic scaffolds for vascular differentiation and provides a platform to study the influence of biophysical microenvironments on differentiation of PSCs. The outcome of this study has implications for regenerative medicine and drug discovery. This article is protected by copyright. All rights reserved.

Proteomic Analysis of Human Adipose Derived Stem Cells during Small Molecule Chemical Stimulated Pre-neuronal Differentiation.

PubMed

Santos, Jerran; Milthorpe, Bruce K; Herbert, Benjamin R; Padula, Matthew P

2017-11-30

Adipose derived stem cells (ADSCs) are acquired from abdominal liposuction yielding a thousand fold more stem cells per millilitre than those from bone marrow. A large research void exists as to whether ADSCs are capable of transdermal differentiation toward neuronal phenotypes. Previous studies have investigated the use of chemical cocktails with varying inconclusive results. Human ADSCs were treated with a chemical stimulant, beta-mercaptoethanol, to direct them toward a neuronal-like lineage within 24 hours. Quantitative proteomics using iTRAQ was then performed to ascertain protein abundance differences between ADSCs, beta-mercaptoethanol treated ADSCs and a glioblastoma cell line. The soluble proteome of ADSCs differentiated for 12 hours and 24 hours was significantly different from basal ADSCs and control cells, expressing a number of remodeling, neuroprotective and neuroproliferative proteins. However toward the later time point presented stress and shock related proteins were observed to be up regulated with a large down regulation of structural proteins. Cytokine profiles support a large cellular remodeling shift as well indicating cellular distress. The earlier time point indicates an initiation of differentiation. At the latter time point there is a vast loss of cell population during treatment. At 24 hours drastically decreased cytokine profiles and overexpression of stress proteins reveal that exposure to beta-mercaptoethanol beyond 24 hours may not be suitable for clinical application as our results indicate that the cells are in trauma whilst producing neuronal-like morphologies. The shorter treatment time is promising, indicating a reducing agent has fast acting potential to initiate neuronal differentiation of ADSCs.

Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells.

PubMed

Buravkova, L B; Rylova, Y V; Andreeva, E R; Kulikov, A V; Pogodina, M V; Zhivotovsky, B; Gogvadze, V

2013-10-01

Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs. Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide. We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs. Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state. These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

In serum veritas—in serum sanitas? Cell non-autonomous aging compromises differentiation and survival of mesenchymal stromal cells via the oxidative stress pathway

PubMed Central

Geißler, S; Textor, M; Schmidt-Bleek, K; Klein, O; Thiele, M; Ellinghaus, A; Jacobi, D; Ode, A; Perka, C; Dienelt, A; Klose, J; Kasper, G; Duda, G N; Strube, P

2013-01-01

Even tissues capable of complete regeneration, such as bone, show an age-related reduction in their healing capacity. Here, we hypothesized that this decline is primarily due to cell non-autonomous (extrinsic) aging mediated by the systemic environment. We demonstrate that culture of mesenchymal stromal cells (MSCs) in serum from aged Sprague–Dawley rats negatively affects their survival and differentiation ability. Proteome analysis and further cellular investigations strongly suggest that serum from aged animals not only changes expression of proteins related to mitochondria, unfolded protein binding or involved in stress responses, it also significantly enhances intracellular reactive oxygen species production and leads to the accumulation of oxidatively damaged proteins. Conversely, reduction of oxidative stress levels in vitro markedly improved MSC function. These results were validated in an in vivo model of compromised bone healing, which demonstrated significant increase regeneration in aged animals following oral antioxidant administration. These observations indicate the high impact of extrinsic aging on cellular functions and the process of endogenous (bone) regeneration. Thus, addressing the cell environment by, for example, systemic antioxidant treatment is a promising approach to enhance tissue regeneration and to regain cellular function especially in elderly patients. PMID:24357801

Systemic evaluation of cellular reprogramming processes exploiting a novel R-tool: eegc.

PubMed

Zhou, Xiaoyuan; Meng, Guofeng; Nardini, Christine; Mei, Hongkang

2017-08-15

Cells derived by cellular engineering, i.e. differentiation of induced pluripotent stem cells and direct lineage reprogramming, carry a tremendous potential for medical applications and in particular for regenerative therapies. These approaches consist in the definition of lineage-specific experimental protocols that, by manipulation of a limited number of biological cues-niche mimicking factors, (in)activation of transcription factors, to name a few-enforce the final expression of cell-specific (marker) molecules. To date, given the intricate complexity of biological pathways, these approaches still present imperfect reprogramming fidelity, with uncertain consequences on the functional properties of the resulting cells. We propose a novel tool eegc to evaluate cellular engineering processes, in a systemic rather than marker-based fashion, by integrating transcriptome profiling and functional analysis. Our method clusters genes into categories representing different states of (trans)differentiation and further performs functional and gene regulatory network analyses for each of the categories of the engineered cells, thus offering practical indications on the potential lack of the reprogramming protocol. eegc R package is released under the GNU General Public License within the Bioconductor project, freely available at https://bioconductor.org/packages/eegc/. christine.nardini.rsrc@gmail.com or hongkang.k.mei@gsk.com. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Systemic evaluation of cellular reprogramming processes exploiting a novel R-tool: eegc

PubMed Central

Zhou, Xiaoyuan; Meng, Guofeng; Nardini, Christine; Mei, Hongkang

2017-01-01

Abstract Motivation Cells derived by cellular engineering, i.e. differentiation of induced pluripotent stem cells and direct lineage reprogramming, carry a tremendous potential for medical applications and in particular for regenerative therapies. These approaches consist in the definition of lineage-specific experimental protocols that, by manipulation of a limited number of biological cues—niche mimicking factors, (in)activation of transcription factors, to name a few—enforce the final expression of cell-specific (marker) molecules. To date, given the intricate complexity of biological pathways, these approaches still present imperfect reprogramming fidelity, with uncertain consequences on the functional properties of the resulting cells. Results We propose a novel tool eegc to evaluate cellular engineering processes, in a systemic rather than marker-based fashion, by integrating transcriptome profiling and functional analysis. Our method clusters genes into categories representing different states of (trans)differentiation and further performs functional and gene regulatory network analyses for each of the categories of the engineered cells, thus offering practical indications on the potential lack of the reprogramming protocol. Availability and Implementation eegc R package is released under the GNU General Public License within the Bioconductor project, freely available at https://bioconductor.org/packages/eegc/. Contact christine.nardini.rsrc@gmail.com or hongkang.k.mei@gsk.com Supplementary information Supplementary data are available at Bioinformatics online. PMID:28398503

Regulatory RNA binding proteins contribute to the transcriptome-wide splicing alterations in human cellular senescence.

PubMed

Dong, Qiongye; Wei, Lei; Zhang, Michael Q; Wang, Xiaowo

2018-06-24

Dysregulation of mRNA splicing has been observed in certain cellular senescence process. However, the common splicing alterations on the whole transcriptome shared by various types of senescence are poorly understood. In order to systematically identify senescence-associated transcriptomic changes in genome-wide scale, we collected RNA sequencing datasets of different human cell types with a variety of senescence-inducing methods from public databases and performed meta-analysis. First, we discovered that a group of RNA binding proteins were consistently down-regulated in diverse senescent samples and identified 406 senescence-associated common differential splicing events. Then, eight differentially expressed RNA binding proteins were predicted to regulate these senescence-associated splicing alterations through an enrichment analysis of their RNA binding information, including motif scanning and enhanced cross-linking immunoprecipitation data. In addition, we constructed the splicing regulatory modules that might contribute to senescence-associated biological processes. Finally, it was confirmed that knockdown of the predicted senescence-associated potential splicing regulators through shRNAs in HepG2 cell line could result in senescence-like splicing changes. Taken together, our work demonstrated a broad range of common changes in mRNA splicing switches and detected their central regulatory RNA binding proteins during senescence. These findings would help to better understand the coordinating splicing alterations in cellular senescence.

Comparison of ventricular and lumbar cerebrospinal fluid T cells in non-inflammatory neurological disorder (NIND) patients.

PubMed

Provencio, J Javier; Kivisäkk, Pia; Tucky, Barbara H; Luciano, Mark G; Ransohoff, Richard M

2005-06-01

The aim of the present study was to define the cellular composition of ventricular, as compared with lumbar, cerebrospinal fluid (CSF) in patients with non-inflammatory neurological disorders (NIND). We addressed this issue by determining the cellular composition of lumbar CSF from patients with normal pressure hydrocephalus (NPH) who were undergoing lumbar CSF drainage during evaluation for shunting procedures, and evaluating ventricular CSF from a subset of these who underwent subsequent placement of ventriculoperitoneal shunts. We determined the cellular composition of lumbar CSF from 18 patients with NPH, and found that the leukocyte differentials, and relative proportions of CD4+ and CD8+ central memory (TCM), effector memory (TEM) and naive cell (TNaive) populations, were equivalent to those found previously in studies of CSF from patients with NIND. We further evaluated cells in the ventricular CSF of five patients who had previously undergone lumbar drainage. Leukocyte differential counts, as well as CD4+ and CD8+ TCM, TEM, and TNaive proportions, were equivalent in matched ventricular and lumbar CSF samples. These observations support the hypothesis that leukocytes enter the CSF in a selective fashion, at its site of formation in the choroid plexus. The results implicate CSF T cells in the immune surveillance of the central nervous system.

Requirement for the Murine Zinc Finger Protein ZFR in Perigastrulation Growth and Survival

PubMed Central

Meagher, Madeleine J.; Braun, Robert E.

2001-01-01

The transition from preimplantation to postimplantation development leads to the initiation of complex cellular differentiation and morphogenetic movements, a dramatic decrease in cell cycle length, and a commensurate increase in the size of the embryo. Accompanying these changes is the need for the transfer of nutrients from the mother to the embryo and the elaboration of sophisticated genetic networks that monitor genomic integrity and the homeostatic control of cellular growth, differentiation, and programmed cell death. To determine the function of the murine zinc finger protein ZFR in these events, we generated mice carrying a null mutation in the gene encoding it. Homozygous mutant embryos form normal-appearing blastocysts that implant and initiate the process of gastrulation. Mutant embryos form mesoderm but they are delayed in their development and fail to form normal anterior embryonic structures. Loss of ZFR function leads to both an increase in programmed cell death and a decrease in mitotic index, especially in the region of the distal tip of the embryonic ectoderm. Mutant embryos also have an apparent reduction in apical vacuoles in the columnar visceral endoderm cells in the extraembryonic region. Together, these cellular phenotypes lead to a dramatic development delay and embryonic death by 8 to 9 days of gestation, which are independent of p53 function. PMID:11283266

Voltage-Gated K+ Channel, Kv3.3 Is Involved in Hemin-Induced K562 Differentiation

PubMed Central

Song, Min Seok; Choi, Seon Young; Ryu, Pan Dong; Lee, So Yeong

2016-01-01

Voltage-gated K+ (Kv) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between Kv channels and cell differentiation remains poorly investigated. This study demonstrates that Kv3.3 is involved in K562 cell erythroid differentiation. Down-regulation of Kv3.3 using siRNA-Kv3.3 increased hemin-induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element-binding protein, and c-fos. Down-regulation of Kv3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The Kv channels, or at least Kv3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of Kv channel regulation of cell differentiation would provide important information regarding vital cellular processes. PMID:26849432

Cellular solitary fibrous tumor (hemangiopericytoma) with anaplasia at cerebellopontine angle--a case report.

PubMed

Zeng, Jianying; Ogera, Patricia; Benardete, Ethan A; Nicastri, Anthony D; Rao, Chandrakant

2012-08-15

Cellular solitary fibrous tumor is currently considered a synonym for hemangiopericytoma, as it became increasingly clear that the morphological and immunohistochemical features that separate these two entities have become tenuous, and evidence for a unifying concept has emerged. Furthermore, as no evidence of pericytic differentiation is given in most cases of hemangiopericytoma, this diagnostic term is waning in popularity. We present here a case of cellular solitary fibrous tumor in a 22-year-old man. Neuroimaging revealed a right cerebellopontine angle tumor. Most of the tumor was cellular although some less cellular areas were seen. Sinusoidally dilated large vessels, including staghorn type, were seen. Nuclear pleomorphism and increased mitotic activity (5 mitosis/10 high power field) were regarded as evidence of anaplasia. Diffuse CD34 immunoreactivity and focal positivity for Factor XIIIa were seen in the tumor, which was negative for EMA and S100. The tumor also displayed rich reticulin network. Solitary fibrous tumor at cerebellopontine angle is rare, and 20 such cases (five reported as hemangiopericytoma) have been reported in the English literature. Copyright © 2012 Elsevier GmbH. All rights reserved.

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

PubMed Central

Rhein, Cosima; Tripal, Philipp; Seebahn, Angela; Konrad, Alice; Kramer, Marcel; Nagel, Christine; Kemper, Jonas; Bode, Jens; Mühle, Christiane; Gulbins, Erich; Reichel, Martin; Becker, Cord-Michael; Kornhuber, Johannes

2012-01-01

Background Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. Methodology/Principal Findings We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. Conclusions/Significance These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity. PMID:22558155

Comparison of the Immune Response To Coxiella burnetii and Burkholderia mallei in Balb/C Mice: A Differential Response by Two Diverse Biological Agents

DTIC Science & Technology

2003-07-01

COMPARISON OF THE IMMUNE RESPONSE TO COXIELLA BURNETII AND BURKHOLDERIA MALLEI IN BALB/C MICE: A DIFFERENTIAL RESPONSE BY TWO DIVERSE...response of BALB/c mice to two biological agents, Coxiella burnetii and Burkholderia mallei . Both C. burnetii and B. mallei cell preparations induced a...response in mice to Burkholderia mallei and Coxiella burnetii to determine the differences in their response that might reveal why one one cellular

Dual functions of silver nanoparticles in F9 teratocarcinoma stem cells, a suitable model for evaluating cytotoxicity- and differentiation-mediated cancer therapy

PubMed Central

Han, Jae Woong; Gurunathan, Sangiliyandi; Choi, Yun-Jung; Kim, Jin-Hoi

2017-01-01

Background Silver nanoparticles (AgNPs) exhibit strong antibacterial and anticancer activity owing to their large surface-to-volume ratios and crystallographic surface structure. Owing to their various applications, understanding the mechanisms of action, biological interactions, potential toxicity, and beneficial effects of AgNPs is important. Here, we investigated the toxicity and differentiation-inducing effects of AgNPs in teratocarcinoma stem cells. Materials and methods AgNPs were synthesized and characterized using various analytical techniques such as UV–visible spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. The cellular responses of AgNPs were analyzed by a series of cellular and biochemical assays. Gene and protein expressions were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Results The AgNPs showed typical crystalline structures and spherical shapes (average size =20 nm). High concentration of AgNPs induced cytotoxicity in a dose-dependent manner by increasing lactate dehydrogenase leakage and reactive oxygen species. Furthermore, AgNPs caused mitochondrial dysfunction, DNA fragmentation, increased expression of apoptotic genes, and decreased expression of antiapoptotic genes. Lower concentrations of AgNPs induced neuronal differentiation by increasing the expression of differentiation markers and decreasing the expression of stem cell markers. Cisplatin reduced the viability of F9 cells that underwent AgNPs-induced differentiation. Conclusion The results showed that AgNPs caused differentially regulated cytotoxicity and induced neuronal differentiation of F9 cells in a concentration-dependent manner. Therefore, AgNPs can be used for differentiation therapy, along with chemotherapeutic agents, for improving cancer treatment by targeting specific chemotherapy-resistant cells within a tumor. Furthermore, understanding the molecular mechanisms of apoptosis and differentiation in stem cells could also help in developing new strategies for cancer stem cell (CSC) therapies. The findings of this study could significantly contribute to the nanomedicine because this study is the first of its kind, and our results will lead to new strategies for cancer and CSC therapies. PMID:29066898

Neural Differentiation of Embryonic Stem Cells In Vitro: A Road Map to Neurogenesis in the Embryo

PubMed Central

Abranches, Elsa; Silva, Margarida; Pradier, Laurent; Schulz, Herbert; Hummel, Oliver; Henrique, Domingos; Bekman, Evguenia

2009-01-01

Background The in vitro generation of neurons from embryonic stem (ES) cells is a promising approach to produce cells suitable for neural tissue repair and cell-based replacement therapies of the nervous system. Available methods to promote ES cell differentiation towards neural lineages attempt to replicate, in different ways, the multistep process of embryonic neural development. However, to achieve this aim in an efficient and reproducible way, a better knowledge of the cellular and molecular events that are involved in the process, from the initial specification of neuroepithelial progenitors to their terminal differentiation into neurons and glial cells, is required. Methodology/Principal Findings In this work, we characterize the main stages and transitions that occur when ES cells are driven into a neural fate, using an adherent monolayer culture system. We established improved conditions to routinely produce highly homogeneous cultures of neuroepithelial progenitors, which organize into neural tube-like rosettes when they acquire competence for neuronal production. Within rosettes, neuroepithelial progenitors display morphological and functional characteristics of their embryonic counterparts, namely, apico-basal polarity, active Notch signalling, and proper timing of production of neurons and glia. In order to characterize the global gene activity correlated with each particular stage of neural development, the full transcriptome of different cell populations that arise during the in vitro differentiation protocol was determined by microarray analysis. By using embryo-oriented criteria to cluster the differentially expressed genes, we define five gene expression signatures that correlate with successive stages in the path from ES cells to neurons. These include a gene signature for a primitive ectoderm-like stage that appears after ES cells enter differentiation, and three gene signatures for subsequent stages of neural progenitor development, from an early stage that follows neural induction to a final stage preceding terminal differentiation. Conclusions/Significance Overall, our work confirms and extends the cellular and molecular parallels between monolayer ES cell neural differentiation and embryonic neural development, revealing in addition novel aspects of the genetic network underlying the multistep process that leads from uncommitted cells to differentiated neurons. PMID:19621087

Assessment of the potential activity of major dietary compounds as selective estrogen receptor modulators in two distinct cell models for proliferation and differentiation

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

Lecomte, Sylvain; Lelong, Marie; Bourgine, Gaëlle

Estrogen receptors (ERs) α and β are distributed in most tissues of women and men. ERs are bound by estradiol (E2), a natural hormone, and mediate the pleiotropic and tissue-specific effects of E2, such as proliferation of breast epithelial cells or protection and differentiation of neuronal cells. Numerous environmental molecules, called endocrine disrupting compounds, also interact with ERs. Phytoestrogens belong to this large family and are considered potent therapeutic molecules that act through their selective estrogen receptor modulator (SERM) activity. Using breast cancer cell lines as a model of estrogen-dependent proliferation and a stably ER-expressing PC12 cell line as amore » model of neuronal differentiating cells, we studied the SERM activity of major dietary compounds, such as apigenin, liquiritigenin, daidzein, genistein, coumestrol, resveratrol and zearalenone. The ability of these compounds to induce ER-transactivation and breast cancer cell proliferation and enhance Nerve Growth Factor (NGF) -induced neuritogenesis was assessed. Surprisingly, although all compounds were able to activate the ER through an estrogen responsive element reporter gene, they showed differential activity toward proliferation or differentiation. Apigenin and resveratrol showed a partial or no proliferative effect on breast cancer cells but fully contributed to the neuritogenesis effect of NGF. However, daidzein and zearalenone showed full effects on cellular proliferation but did not induce cellular differentiation. In summary, our results suggest that the therapeutic potential of phytoestrogens can diverge depending on the molecule and the phenotype considered. Hence, apigenin and resveratrol might be used in the development of therapeutics for breast cancer and brain diseases. - Highlights: • SERM activity of dietary compounds on proliferation and differentiation is studied. • All the dietary compounds tested transactivate estrogen receptors. • Apigenin and resveratrol could be good candidates for future therapeutics. • Daidzein and zearalenone are to be avoided to maintain human health.« less

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development.

PubMed

Dupé, Aurélien; Dumas, Carole; Papadopoulou, Barbara

2015-01-01

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania.

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development

PubMed Central

Dupé, Aurélien; Dumas, Carole; Papadopoulou, Barbara

2015-01-01

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania. PMID:26334886

Reversible effect of all-trans-retinoic acid on AML12 hepatocyte proliferation and cell cycle progression

EPA Science Inventory

The role of all-trans-retinoic acid (atRA) in the regulation of cellular proliferation and differentiation is well documented. Numerous studies have established the cancer preventive propertiesofatRAwhichfunctionstoregulate levels ofcellcycleproteinsessentialfortheGliS transition...

MYC activation is a hallmark of cancer initiation and maintenance.

PubMed

Gabay, Meital; Li, Yulin; Felsher, Dean W

2014-06-02

The MYC proto-oncogene has been implicated in the pathogenesis of most types of human tumors. MYC activation alone in many normal cells is restrained from causing tumorigenesis through multiple genetic and epigenetically controlled checkpoint mechanisms, including proliferative arrest, apoptosis, and cellular senescence. When pathologically activated in a permissive epigenetic and/or genetic context, MYC bypasses these mechanisms, enforcing many of the "hallmark" features of cancer, including relentless tumor growth associated with DNA replication and transcription, cellular proliferation and growth, protein synthesis, and altered cellular metabolism. MYC mandates tumor cell fate, by inducing stemness and blocking cellular senescence and differentiation. Additionally, MYC orchestrates changes in the tumor microenvironment, including the activation of angiogenesis and suppression of the host immune response. Provocatively, brief or even partial suppression of MYC back to its physiological levels of activation can result in the restoration of intrinsic checkpoint mechanisms, resulting in acute and sustained tumor regression, associated with tumor cells undergoing proliferative arrest, differentiation, senescence, and apoptosis, as well as remodeling of the tumor microenvironment, recruitment of an immune response, and shutdown of angiogenesis. Hence, tumors appear to be "addicted" to MYC because of both tumor cell-intrinsic, cell-autonomous and host-dependent, immune cell-dependent mechanisms. Both the trajectory and persistence of many human cancers require sustained MYC activation. Multiscale mathematical modeling may be useful to predict when tumors will be addicted to MYC. MYC is a hallmark molecular feature of both the initiation and maintenance of tumorigenesis. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

PubMed

Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

Extreme cellular adaptations and cell differentiation required by a cyanobacterium for carbonate excavation

PubMed Central

Guida, Brandon Scott; Garcia-Pichel, Ferran

2016-01-01

Some cyanobacteria, known as euendoliths, excavate and grow into calcium carbonates, with their activity leading to significant marine and terrestrial carbonate erosion and to deleterious effects on coral reef and bivalve ecology. Despite their environmental relevance, the mechanisms by which they can bore have remained elusive and paradoxical, in that, as oxygenic phototrophs, cyanobacteria tend to alkalinize their surroundings, which will encourage carbonate precipitation, not dissolution. Therefore, cyanobacteria must rely on unique adaptations to bore. Studies with the filamentous euendolith, Mastigocoleus testarum, indicated that excavation requires both cellular energy and transcellular calcium transport, mediated by P-type ATPases, but the cellular basis for this phenomenon remains obscure. We present evidence that excavation in M. testarum involves two unique cellular adaptations. Long-range calcium transport is based on active pumping at multiple cells along boring filaments, orchestrated by the preferential localization of calcium ATPases at one cell pole, in a ring pattern, facing the cross-walls, and by repeating this placement and polarity, a pattern that breaks at branching and apical cells. In addition, M. testarum differentiates specialized cells we call calcicytes, that which accumulate calcium at concentrations more than 500-fold those found in other cyanobacteria, concomitantly and drastically lowering photosynthetic pigments and enduring severe cytoplasmatic alkalinization. Calcicytes occur commonly, but not exclusively, in apical parts of the filaments distal to the excavation front. We suggest that calcicytes allow for fast calcium flow at low, nontoxic concentrations through undifferentiated cells by providing buffering storage for excess calcium before final excretion to the outside medium. PMID:27140633

Cellular dynamics in the muscle satellite cell niche

PubMed Central

Bentzinger, C Florian; Wang, Yu Xin; Dumont, Nicolas A; Rudnicki, Michael A

2013-01-01

Satellite cells, the quintessential skeletal muscle stem cells, reside in a specialized local environment whose anatomy changes dynamically during tissue regeneration. The plasticity of this niche is attributable to regulation by the stem cells themselves and to a multitude of functionally diverse cell types. In particular, immune cells, fibrogenic cells, vessel-associated cells and committed and differentiated cells of the myogenic lineage have emerged as important constituents of the satellite cell niche. Here, we discuss the cellular dynamics during muscle regeneration and how disease can lead to perturbation of these mechanisms. To define the role of cellular components in the muscle stem cell niche is imperative for the development of cell-based therapies, as well as to better understand the pathobiology of degenerative conditions of the skeletal musculature. PMID:24232182

Papillomavirus E6 oncoproteins

PubMed Central

Vande Pol, Scott B.; Klingelhutz, Aloysius J.

2013-01-01

Papillomaviruses induce benign and malignant epithelial tumors, and the viral E6 oncoprotein is essential for full transformation. E6 contributes to transformation by associating with cellular proteins, docking on specific acidic LXXLL peptide motifs found on the associated cellular proteins. This review examines insights from recent studies of human and animal E6 proteins that determine the three-dimensional structure of E6 when bound to acidic LXXLL peptides. The structure of E6 is related to recent advances in the purification and identification of E6 associated protein complexes. These E6 protein-complexes, together with other proteins that bind to E6, alter a broad array of biological outcomes including modulation of cell survival, cellular transcription, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression. PMID:23711382

Cellular mechanisms of estradiol-mediated sexual differentiation of the brain.

PubMed

Wright, Christopher L; Schwarz, Jaclyn S; Dean, Shannon L; McCarthy, Margaret M

2010-09-01

Gonadal steroids organize the developing brain during a perinatal sensitive period and have enduring consequences for adult behavior. In male rodents testicular androgens are aromatized in neurons to estrogens and initiate multiple distinct cellular processes that ultimately determine the masculine phenotype. Within specific brain regions, overall cell number and dendritic morphology are the principal targets for hormonal organization. Recent advances have been made in elucidating the cellular mechanisms by which the neurological underpinnings of sexually dimorphic physiology and behavior are determined. These include estradiol-mediated prostaglandin synthesis, presynaptic release of glutamate, postsynaptic changes in glutamate receptors and changes in cell adhesion molecules. Sex differences in cell death are mediated by hormonal modulation of survival and death factors such as TNFalpha and Bcl-2/BAX. Copyright 2010 Elsevier Ltd. All rights reserved.

The extracellular matrix: Structure, composition, age-related differences, tools for analysis and applications for tissue engineering.

PubMed

Kular, Jaspreet K; Basu, Shouvik; Sharma, Ram I

2014-01-01

The extracellular matrix is a structural support network made up of diverse proteins, sugars and other components. It influences a wide number of cellular processes including migration, wound healing and differentiation, all of which is of particular interest to researchers in the field of tissue engineering. Understanding the composition and structure of the extracellular matrix will aid in exploring the ways the extracellular matrix can be utilised in tissue engineering applications especially as a scaffold. This review summarises the current knowledge of the composition, structure and functions of the extracellular matrix and introduces the effect of ageing on extracellular matrix remodelling and its contribution to cellular functions. Additionally, the current analytical technologies to study the extracellular matrix and extracellular matrix-related cellular processes are also reviewed.

RNASeq analysis of differentiated keratinocytes reveals a massive response to late events during human papillomavirus type 16 infection, including loss of epithelial barrier function.

PubMed

Klymenko, T; Gu, Q; Herbert, I; Stevenson, A; Iliev, V; Watkins, G; Pollock, C; Bhatia, R; Cuschieri, K; Herzyk, P; Gatherer, D; Graham, S V

2017-10-11

The human papillomavirus (HPV) replication cycle is tightly linked to epithelial cell differentiation. To examine HPV-associated changes in the keratinocyte transcriptome, RNAs isolated from undifferentiated and differentiated cell populations of normal, spontaneously immortalised, keratinocytes (NIKS), and NIKS stably transfected with HPV16 episomal genomes (NIKS16), were compared using RNASeq. HPV16 infection altered expression of 2862 cellular genes. Next, to elucidate the role of keratinocyte gene expression in late events during the viral life cycle, RNASeq was carried out on triplicate differentiated populations of NIKS (uninfected) and NIKS16 (infected). Of the top 966 genes altered (>log 2 = 1.8, 3.5-fold change) 670 genes were downregulated and 296 genes were up-regulated. HPV down-regulated many genes involved in epithelial barrier function that involves structural resistance to the environment and immunity to infectious agents. For example, HPV infection repressed expression of the differentiated keratinocyte-specific pattern recognition receptor TLR7, the Langerhans cell chemoattractant, CCL20, and proinflammatory cytokines, IL1A and IL1B. However, IRF1, IFNκ and viral restriction factors (IFIT1, 2, 3, 5, OASL, CD74, RTP4) were up-regulated. HPV infection abrogated gene expression associated with the physical epithelial barrier, including keratinocyte cytoskeleton, intercellular junctions and cell adhesion. qPCR and western blotting confirmed changes in expression of seven of the most significantly altered mRNAs. Expression of three genes showed statistically significant changes during cervical disease progression in clinical samples. Taken together, the data indicate that HPV infection manipulates the differentiating keratinocyte transcriptome to create an environment conducive to productive viral replication and egress. IMPORTANCE Human papillomavirus (HPV) genome amplification and capsid formation takes place in differentiated keratinocytes. The viral life cycle is intimately associated with host cell differentiation. Deep sequencing (RNASeq) of RNA from undifferentiated and differentiated uninfected and HPV16-positive keratinocytes showed that almost 3000 genes were differentially expressed in keratinocyte due to HPV16 infection. Strikingly, the epithelial barrier function of differentiated keratinocytes, comprising keratinocyte immune function and cellular structure, was found to be disrupted. These data provide new insights into virus-host interaction crucial for production of infectious virus and reveal that HPV infection remodels keratinocytes for completion of the virus replication cycle. Copyright © 2017 American Society for Microbiology.

Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress

PubMed Central

Jamsheer K, Muhammed; Laxmi, Ashverya

2015-01-01

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response. PMID:26442059

A selenium-deficient Caco-2 cell model for assessing differential incorporation of chemical or food selenium into glutathione peroxidase.

PubMed

Zeng, Huawei; Botnen, James H; Johnson, Luann K

2008-01-01

Assessing the ability of a selenium (Se) sample to induce cellular glutathione peroxidase (GPx) activity in Se-deficient animals is the most commonly used method to determine Se bioavailability. Our goal is to establish a Se-deficient cell culture model with differential incorporation of Se chemical forms into GPx, which may complement the in vivo studies. In the present study, we developed a Se-deficient Caco-2 cell model with a serum gradual reduction method. It is well recognized that selenomethionine (SeMet) is the major nutritional source of Se; therefore, SeMet, selenite, or methylselenocysteine (SeMSC) was added to cell culture media with different concentrations and treatment time points. We found that selenite and SeMSC induced GPx more rapidly than SeMet. However, SeMet was better retained as it is incorporated into proteins in place of methionine; compared with 8-, 24-, or 48-h treatment, 72-h Se treatment was a more sensitive time point to measure the potential of GPx induction in all tested concentrations. Based on induction of GPx activity, the cellular bioavailability of Se from an extract of selenobroccoli after a simulated gastrointestinal digestion was comparable with that of SeMSC and SeMet. These in vitro data are, for the first time, consistent with previous published data regarding selenite and SeMet bioavailability in animal models and Se chemical speciation studies with broccoli. Thus, Se-deficient Caco-2 cell model with differential incorporation of chemical or food forms of Se into GPx provides a new tool to study the cellular mechanisms of Se bioavailability.

Extracellular matrix of adipogenically differentiated mesenchymal stem cells reveals a network of collagen filaments, mostly interwoven by hexagonal structural units.

PubMed

Ullah, Mujib; Sittinger, Michael; Ringe, Jochen

2013-01-01

Extracellular matrix (ECM) is the non-cellular component of tissues, which not only provides biological shelter but also takes part in the cellular decisions for diverse functions. Every tissue has an ECM with unique composition and topology that governs the process of determination, differentiation, proliferation, migration and regeneration of cells. Little is known about the structural organization of matrix especially of MSC-derived adipogenic ECM. Here, we particularly focus on the composition and architecture of the fat ECM to understand the cellular behavior on functional bases. Thus, mesenchymal stem cells (MSC) were adipogenically differentiated, then, were transferred to adipogenic propagation medium, whereas they started the release of lipid droplets leaving bare network of ECM. Microarray analysis was performed, to indentify the molecular machinery of matrix. Adipogenesis was verified by Oil Red O staining of lipid droplets and by qPCR of adipogenic marker genes PPARG and FABP4. Antibody staining demonstrated the presence of collagen type I, II and IV filaments, while alkaline phosphatase activity verified the ossified nature of these filaments. In the adipogenic matrix, the hexagonal structures were abundant followed by octagonal structures, whereas they interwoven in a crisscross manner. Regarding molecular machinery of adipogenic ECM, the bioinformatics analysis revealed the upregulated expression of COL4A1, ITGA7, ITGA7, SDC2, ICAM3, ADAMTS9, TIMP4, GPC1, GPC4 and downregulated expression of COL14A1, ADAMTS5, TIMP2, TIMP3, BGN, LAMA3, ITGA2, ITGA4, ITGB1, ITGB8, CLDN11. Moreover, genes associated with integrins, glycoproteins, laminins, fibronectins, cadherins, selectins and linked signaling pathways were found. Knowledge of the interactive-language between cells and matrix could be beneficial for the artificial designing of biomaterials and bioscaffolds. © 2013.

Methylation alterations are not a major cause of PTTG1 missregulation

PubMed Central

Hidalgo, Manuel; Galan, Jose Jorge; Sáez, Carmen; Ferrero, Eduardo; Castilla, Carolina; Ramirez-Lorca, Reposo; Pelaez, Pablo; Ruiz, Agustin; Japón, Miguel A; Royo, Jose Luis

2008-01-01

Background On its physiological cellular context, PTTG1 controls sister chromatid segregation during mitosis. Within its crosstalk to the cellular arrest machinery, relies a checkpoint of integrity for which gained the over name of securin. PTTG1 was found to promote malignant transformation in 3T3 fibroblasts, and further found to be overexpressed in different tumor types. More recently, PTTG1 has been also related to different processes such as DNA repair and found to trans-activate different cellular pathways involving c-myc, bax or p53, among others. PTTG1 over-expression has been correlated to a worse prognosis in thyroid, lung, colorectal cancer patients, and it can not be excluded that this effect may also occur in other tumor types. Despite the clinical relevance and the increasing molecular characterization of PTTG1, the reason for its up-regulation remains unclear. Method We analysed PTTG1 differential expression in PC-3, DU-145 and LNCaP tumor cell lines, cultured in the presence of the methyl-transferase inhibitor 5-Aza-2'-deoxycytidine. We also tested whether the CpG island mapping PTTG1 proximal promoter evidenced a differential methylation pattern in differentiated thyroid cancer biopsies concordant to their PTTG1 immunohistochemistry status. Finally, we performed whole-genome LOH studies using Affymetix 50 K microarray technology and FRET analysis to search for allelic imbalances comprising the PTTG1 locus. Conclusion Our data suggest that neither methylation alterations nor LOH are involved in PTTG1 over-expression. These data, together with those previously reported, point towards a post-transcriptional level of missregulation associated to PTTG1 over-expression. PMID:18426563

Secretome of Differentiated PC12 Cells Restores the Monocrotophos-Induced Damages in Human Mesenchymal Stem Cells and SHSY-5Y Cells: Role of Autophagy and Mitochondrial Dynamics.

PubMed

Srivastava, A; Singh, S; Rajpurohit, C S; Srivastava, P; Pandey, A; Kumar, D; Khanna, V K; Pant, A B

2018-06-01

A perturbed cellular homeostasis is a key factor associated with xenobiotic exposure resulting in various ailments. The local cellular microenvironment enriched with secretory components aids in cell-cell communication that restores this homeostasis. Deciphering the underlying mechanism behind this restorative potential of secretome could serve as a possible solution to many health hazards. We, therefore, explored the protective efficacy of the secretome of differentiated PC12 cells with emphasis on induction of autophagy and mitochondrial biogenesis. Monocrotophos (MCP), a widely used neurotoxic organophosphate, was used as the test compound at sublethal concentration. The conditioned medium (CM) of differentiated PC12 cells comprising of their secretome restored the cell viability, oxidative stress and apoptotic cell death in MCP-challenged human mesenchymal stem cells and SHSY-5Y, a human neuroblastoma cell line. Delving further to identify the underlying mechanism of this restorative effect we observed a marked increase in the expression of autophagy markers LC3, Beclin-1, Atg5 and Atg7. Exposure to autophagy inhibitor, 3-methyladenine, led to a reduced expression of these markers with a concomitant increase in the expression of pro-apoptotic caspase-3. Besides that, the increased mitochondrial fission in MCP-exposed cells was balanced with increased fusion in the presence of CM facilitated by AMPK/SIRT1/PGC-1α signaling cascade. Mitochondrial dysfunctions are strongly associated with autophagy activation and as per our findings, cellular secretome too induces autophagy. Therefore, connecting these three potential apices can be a major breakthrough in repair and rescue of xenobiotic-damaged tissues and cells.

In vitro proliferation and osteogenic differentiation of mesenchymal stem cells on nanoporous alumina

PubMed Central

Song, Yuanhui; Ju, Yang; Song, Guanbin; Morita, Yasuyuki

2013-01-01

Cell adhesion, migration, and proliferation are significantly affected by the surface topography of the substrates on which the cells are cultured. Alumina is one of the most popular implant materials used in orthopedics, but few data are available concerning the cellular responses of mesenchymal stem cells (MSCs) grown on nanoporous structures. MSCs were cultured on smooth alumina substrates and nanoporous alumina substrates to investigate the interaction between surface topographies of nanoporous alumina and cellular behavior. Nanoporous alumina substrates with pore sizes of 20 nm and 100 nm were used to evaluate the effect of pore size on MSCs as measured by proliferation, morphology, expression of integrin β1, and osteogenic differentiation. An MTT assay was used to measure cell viability of MSCs on different substrates, and determined that cell viability decreased with increasing pore size. Scanning electron microscopy was used to investigate the effect of pore size on cell morphology. Extremely elongated cells and prominent cell membrane protrusions were observed in cells cultured on alumina with the larger pore size. The expression of integrin β1 was enhanced in MSCs cultured on porous alumina, revealing that porous alumina substrates were more favorable for cell growth than smooth alumina substrates. Higher levels of osteoblastic differentiation markers such as alkaline phosphatase, osteocalcin, and mineralization were detected in cells cultured on alumina with 100 nm pores compared with cells cultured on alumina with either 20 nm pores or smooth alumina. This work demonstrates that cellular behavior is affected by variation in pore size, providing new insight into the potential application of this novel biocompatible material for the developing field of tissue engineering. PMID:23935364

Effects of salinity on the cellular physiological responses of Natrinema sp. J7-2

PubMed Central

Mei, Yunjun; Liu, Huan; Zhang, Shunxi; Yang, Ming; Hu, Chun; Zhang, Jian; Shen, Ping; Chen, Xiangdong

2017-01-01

The halophilic archaea (haloarchaea) live in hyersaline environments such as salt lakes, salt ponds and marine salterns. To cope with the salt stress conditions, haloarchaea have developed two fundamentally different strategies: the "salt-in" strategy and the "compatible-solute" strategy. Although investigation of the molecular mechanisms underlying the tolerance to high salt concentrations has made outstanding achievements, experimental study from the aspect of transcription is rare. In the present study, we monitored cellular physiology of Natrinema sp. J7-2 cells incubated in different salinity media (15%, 25% and 30% NaCl) from several aspects, such as cellular morphology, growth, global transcriptome and the content of intracellular free amino acids. The results showed that the cells were polymorphic and fragile at a low salt concentration (15% NaCl) but had a long, slender rod shape at high salt concentrations (25% and 30% NaCl). The cells grew best in 25% NaCl, mediocre in 30% NaCl and struggled in 15% NaCl. An RNA-seq analysis revealed differentially expressed genes (DEGs) in various salinity media. A total of 1,148 genes were differentially expressed, consisting of 719 DEGs (348 up-regulated and 371 down-regulated genes) between cells in 15% vs 25% NaCl, and 733 DEGs (521 up-regulated and 212 down-regulated genes) between cells in 25% vs 30% NaCl. Moreover, 304 genes were commonly differentially expressed in both 15% vs 25% and 25% vs30% NaCl. The DEGs were enriched in different KEGG metabolic pathways, such as amino acids, glycerolipid, ribosome, nitrogen, protoporphyrin, porphyrin and porhiniods. The intracellular predominant free amino acids consisted of the glutamate family (Glu, Arg and Pro), aspartate family (Asp) and aromatic amino acids (Phe and Trp), especially Glu and Asp. PMID:28926633

Rewiring of the inferred protein interactome during blood development studied with the tool PPICompare.

PubMed

Will, Thorsten; Helms, Volkhard

2017-04-04

Differential analysis of cellular conditions is a key approach towards understanding the consequences and driving causes behind biological processes such as developmental transitions or diseases. The progress of whole-genome expression profiling enabled to conveniently capture the state of a cell's transcriptome and to detect the characteristic features that distinguish cells in specific conditions. In contrast, mapping the physical protein interactome for many samples is experimentally infeasible at the moment. For the understanding of the whole system, however, it is equally important how the interactions of proteins are rewired between cellular states. To overcome this deficiency, we recently showed how condition-specific protein interaction networks that even consider alternative splicing can be inferred from transcript expression data. Here, we present the differential network analysis tool PPICompare that was specifically designed for isoform-sensitive protein interaction networks. Besides detecting significant rewiring events between the interactomes of grouped samples, PPICompare infers which alterations to the transcriptome caused each rewiring event and what is the minimal set of alterations necessary to explain all between-group changes. When applied to the development of blood cells, we verified that a reasonable amount of rewiring events were reported by the tool and found that differential gene expression was the major determinant of cellular adjustments to the interactome. Alternative splicing events were consistently necessary in each developmental step to explain all significant alterations and were especially important for rewiring in the context of transcriptional control. Applying PPICompare enabled us to investigate the dynamics of the human protein interactome during developmental transitions. A platform-independent implementation of the tool PPICompare is available at https://sourceforge.net/projects/ppicompare/ .

Differential regulation of placental amino acid transport by saturated and unsaturated fatty acids.

PubMed

Lager, Susanne; Jansson, Thomas; Powell, Theresa L

2014-10-15

Fatty acids are critical for normal fetal development but may also influence placental function. We have previously reported that oleic acid (OA) stimulates amino acid transport in primary human trophoblasts (PHTs). In other tissues, saturated and unsaturated fatty acids have distinct effects on cellular signaling, for instance, palmitic acid (PA) but not OA reduces IκBα expression. We hypothesized that saturated and unsaturated fatty acids differentially affect trophoblast amino acid transport and cellular signaling. To test this hypothesis, PHTs were cultured in docosahexaenoic acid (DHA; 50 μM), OA (100 μM), or PA (100 μM). DHA and OA were also combined to test whether DHA could counteract the OA stimulatory effect on amino acid transport. The effects of fatty acids were compared against a vehicle control. Amino acid transport was measured by isotope-labeled tracers. Activation of inflammatory-related signaling pathways and the mechanistic target of rapamycin (mTOR) pathway were determined by Western blot analysis. Exposure of PHTs to DHA for 24 h reduced amino acid transport and phosphorylation of p38 MAPK, STAT3, mTOR, eukaryotic initiation factor 4E-binding protein 1, and ribosomal protein (rp)S6. In contrast, OA increased amino acid transport and phosphorylation of ERK, mTOR, S6 kinase 1, and rpS6. The combination of DHA with OA increased amino acid transport and rpS6 phosphorylation. PA did not affect amino acid transport but reduced IκBα expression. In conclusion, these fatty acids differentially regulated placental amino acid transport and cellular signaling. Taken together, these findings suggest that dietary fatty acids could alter the intrauterine environment by modifying placental function, thereby having long-lasting effects on the developing fetus. Copyright © 2014 the American Physiological Society.

Gene expression profiling of Japanese psoriatic skin reveals an increased activity in molecular stress and immune response signals.

PubMed

Kulski, Jerzy K; Kenworthy, William; Bellgard, Matthew; Taplin, Ross; Okamoto, Koichi; Oka, Akira; Mabuchi, Tomotaka; Ozawa, Akira; Tamiya, Gen; Inoko, Hidetoshi

2005-12-01

Gene expression profiling was performed on biopsies of affected and unaffected psoriatic skin and normal skin from seven Japanese patients to obtain insights into the pathways that control this disease. HUG95A Affymetrix DNA chips that contained oligonucleotide arrays of approximately 12,000 well-characterized human genes were used in the study. The statistical analysis of the Affymetrix data, based on the ranking of the Student t-test statistic, revealed a complex regulation of molecular stress and immune gene responses. The majority of the 266 induced genes in affected and unaffected psoriatic skin were involved with interferon mediation, immunity, cell adhesion, cytoskeleton restructuring, protein trafficking and degradation, RNA regulation and degradation, signalling transduction, apoptosis and atypical epidermal cellular proliferation and differentiation. The disturbances in the normal protein degradation equilibrium of skin were reflected by the significant increase in the gene expression of various protease inhibitors and proteinases, including the induced components of the ATP/ubiquitin-dependent non-lysosomal proteolytic pathway that is involved with peptide processing and presentation to T cells. Some of the up-regulated genes, such as TGM1, IVL, FABP5, CSTA and SPRR, are well-known psoriatic markers involved in atypical epidermal cellular organization and differentiation. In the comparison between the affected and unaffected psoriatic skin, the transcription factor JUNB was found at the top of the statistical rankings for the up-regulated genes in affected skin, suggesting that it has an important but as yet undefined role in psoriasis. Our gene expression data and analysis suggest that psoriasis is a chronic interferon- and T-cell-mediated immune disease of the skin where the imbalance in epidermal cellular structure, growth and differentiation arises from the molecular antiviral stress signals initiating inappropriate immune responses.

The Screening of Genes Sensitive to Long-Term, Low-Level Microwave Exposure and Bioinformatic Analysis of Potential Correlations to Learning and Memory.

PubMed

Zhao, Ya Li; Li, Ying Xian; Ma, Hong Bo; Li, Dong; Li, Hai Liang; Jiang, Rui; Kan, Guang Han; Yang, Zhen Zhong; Huang, Zeng Xin

2015-08-01

To gain a better understanding of gene expression changes in the brain following microwave exposure in mice. This study hopes to reveal mechanisms contributing to microwave-induced learning and memory dysfunction. Mice were exposed to whole body 2100 MHz microwaves with specific absorption rates (SARs) of 0.45 W/kg, 1.8 W/kg, and 3.6 W/kg for 1 hour daily for 8 weeks. Differentially expressing genes in the brains were screened using high-density oligonucleotide arrays, with genes showing more significant differences further confirmed by RT-PCR. The gene chip results demonstrated that 41 genes (0.45 W/kg group), 29 genes (1.8 W/kg group), and 219 genes (3.6 W/kg group) were differentially expressed. GO analysis revealed that these differentially expressed genes were primarily involved in metabolic processes, cellular metabolic processes, regulation of biological processes, macromolecular metabolic processes, biosynthetic processes, cellular protein metabolic processes, transport, developmental processes, cellular component organization, etc. KEGG pathway analysis showed that these genes are mainly involved in pathways related to ribosome, Alzheimer's disease, Parkinson's disease, long-term potentiation, Huntington's disease, and Neurotrophin signaling. Construction of a protein interaction network identified several important regulatory genes including synbindin (sbdn), Crystallin (CryaB), PPP1CA, Ywhaq, Psap, Psmb1, Pcbp2, etc., which play important roles in the processes of learning and memorye. Long-term, low-level microwave exposure may inhibit learning and memory by affecting protein and energy metabolic processes and signaling pathways relating to neurological functions or diseases. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

[The concept of cellular immortality, a myth or a reality. Example of "immortalized" articular chondrocytes].

PubMed

Adolphe, M; Thenet, S

1990-01-01

The concept of cellular immortality, which arose from the historical studies of A. Carrel, is getting a new start with the progress of virology. However, the definition of cell immortalization is still ambiguous. Although scientists agree that cells regarded as immortal have acquired an infinite growth capacity, the relationship of this change with the first stages of transformation is difficult to clearly define. Immortalized cell lines have already been obtained from numerous cell types by using viral infection or transfection with viral and cellular genes. Immortalization of cells is interesting for three main reasons: it permits study of the steps in progression to transformation, allows establishment of cell lines for producing biological products, and permits various cell types to maintain a part of their differentiated functions. For example, hypothalamic neurosecretory cells, macrophages, astrocytes and intestinal epithelial cells have been immortalized and these lines can be used for understanding the balance between division and differentiation, and also for pharmacotoxicological studies. In our laboratory, we immortalized rabbit articular chondrocytes by transfection with SV40 large T and little t encoding genes. At the 9th subculture, when the control culture was senescent, clones of polygonal cells appeared in the transfected cell cultures. Three clones have been selected and have been maintained in culture for two years. Growth curves of normal and SV40-transfected chondrocytes were compared and displayed similar doubling times (approximately 20 hours). The exponential phase of growth was longer for immortalized cells resulting in a 2-fold higher saturation density. These cells appear to be not fully transformed and maintain some properties of differentiated chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellularizing hydrogel-based scaffolds to repair bone tissue: How to create a physiologically relevant micro-environment?

PubMed Central

Maisani, Mathieu; Pezzoli, Daniele; Chassande, Olivier; Mantovani, Diego

2017-01-01

Tissue engineering is a promising alternative to autografts or allografts for the regeneration of large bone defects. Cell-free biomaterials with different degrees of sophistication can be used for several therapeutic indications, to stimulate bone repair by the host tissue. However, when osteoprogenitors are not available in the damaged tissue, exogenous cells with an osteoblast differentiation potential must be provided. These cells should have the capacity to colonize the defect and to participate in the building of new bone tissue. To achieve this goal, cells must survive, remain in the defect site, eventually proliferate, and differentiate into mature osteoblasts. A critical issue for these engrafted cells is to be fed by oxygen and nutrients: the transient absence of a vascular network upon implantation is a major challenge for cells to survive in the site of implantation, and different strategies can be followed to promote cell survival under poor oxygen and nutrient supply and to promote rapid vascularization of the defect area. These strategies involve the use of scaffolds designed to create the appropriate micro-environment for cells to survive, proliferate, and differentiate in vitro and in vivo. Hydrogels are an eclectic class of materials that can be easily cellularized and provide effective, minimally invasive approaches to fill bone defects and favor bone tissue regeneration. Furthermore, by playing on their composition and processing, it is possible to obtain biocompatible systems with adequate chemical, biological, and mechanical properties. However, only a good combination of scaffold and cells, possibly with the aid of incorporated growth factors, can lead to successful results in bone regeneration. This review presents the strategies used to design cellularized hydrogel-based systems for bone regeneration, identifying the key parameters of the many different micro-environments created within hydrogels. PMID:28634532

A secretome analysis reveals that PPARα is upregulated by fractionated-dose γ-irradiation in three-dimensional keratinocyte cultures

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

Lee, Jeeyong; Kim, Hyun-Ji; Yi, Jae Youn, E-mail: yjy_71@kcch.re.kr

Studies have shown that γ-irradiation induces various biological responses, including oxidative stress and apoptosis, as well as cellular repair and immune system responses. However, most such studies have been performed using traditional two-dimensional cell culture systems, which are limited in their ability to faithfully represent in vivo conditions. A three-dimensional (3D) environment composed of properly interconnected and differentiated cells that allow communication and cooperation among cells via secreted molecules would be expected to more accurately reflect cellular responses. Here, we investigated γ-irradiation–induced changes in the secretome of 3D-cultured keratinocytes. An analysis of keratinocyte secretome profiles following fractionated-dose γ-irradiation revealed changes inmore » genes involved in cell adhesion, angiogenesis, and the immune system. Notably, peroxisome proliferator-activated receptor-α (PPARα) was upregulated in response to fractionated-dose γ-irradiation. This upregulation was associated with an increase in the transcription of known PPARα target genes in secretome, including angiopoietin-like protein 4, dermokine and kallikrein-related peptide 12, which were differentially regulated by fractionated-dose γ-irradiation. Collectively, our data imply a mechanism linking γ-irradiation and secretome changes, and suggest that these changes could play a significant role in the coordinated cellular responses to harmful ionizing radiation, such as those associated with radiation therapy. This extension of our understanding of γ-irradiation-induced secretome changes has the potential to improve radiation therapy strategies. - Highlights: • γ-irradiation induced changes of cell adhesion, angiogenesis, and immune system in secretome of 3D-cultured keratinocytes. • Peroxisome proliferator-activated receptor-α (PPARα) was upregulated in response to fractionated-dose γ-irradiation. • The known PPARα target genes were differentially regulated by fractionated-dose γ-irradiation.« less

ACSL1 Is Associated With Fetal Programming of Insulin Sensitivity and Cellular Lipid Content

PubMed Central

Joseph, Roy; Poschmann, Jeremie; Sukarieh, Rami; Too, Peh Gek; Julien, Sofi G.; Xu, Feng; Teh, Ai Ling; Holbrook, Joanna D.; Ng, Kai Lyn; Chong, Yap Seng; Gluckman, Peter D.; Prabhakar, Shyam

2015-01-01

Individuals who are born small for gestational age (SGA) have a risk to develop various metabolic diseases during their life course. The biological memory of the prenatal state of growth restricted individuals may be reflected in epigenetic alterations in stem cell populations. Mesenchymal stem cells (MSCs) from the Wharton's jelly of umbilical cord tissue are multipotent, and we generated primary umbilical cord MSC isolates from SGA and normal neonates, which were subsequently differentiated into adipocytes. We established chromatin state maps for histone marks H3K27 acetylation and H3K27 trimethylation and tested whether enrichment of these marks was associated with gene expression changes. After validating gene expression levels for 10 significant chromatin immunoprecipitation sequencing candidate genes, we selected acyl-coenzyme A synthetase 1 (ACSL1) for further investigations due to its key roles in lipid metabolism. The ACSL1 gene was found to be highly associated with histone acetylation in adipocytes differentiated from MSCs with SGA background. In SGA-derived adipocytes, the ACSL1 expression level was also found to be associated with increased lipid loading as well as higher insulin sensitivity. ACSL1 depletion led to changes in expression of candidate genes such as proinflammatory chemokines and down-regulated both, the amount of cellular lipids and glucose uptake. Increased ACSL1, as well as modulated downstream candidate gene expression, may reflect the obese state, as detected in mice fed a high-fat diet. In summary, we believe that ACSL1 is a programmable mediator of insulin sensitivity and cellular lipid content and adipocytes differentiated from Wharton's jelly MSCs recapitulate important physiological characteristics of SGA individuals. PMID:25915184

ACSL1 Is Associated With Fetal Programming of Insulin Sensitivity and Cellular Lipid Content.

PubMed

Joseph, Roy; Poschmann, Jeremie; Sukarieh, Rami; Too, Peh Gek; Julien, Sofi G; Xu, Feng; Teh, Ai Ling; Holbrook, Joanna D; Ng, Kai Lyn; Chong, Yap Seng; Gluckman, Peter D; Prabhakar, Shyam; Stünkel, Walter

2015-06-01

Individuals who are born small for gestational age (SGA) have a risk to develop various metabolic diseases during their life course. The biological memory of the prenatal state of growth restricted individuals may be reflected in epigenetic alterations in stem cell populations. Mesenchymal stem cells (MSCs) from the Wharton's jelly of umbilical cord tissue are multipotent, and we generated primary umbilical cord MSC isolates from SGA and normal neonates, which were subsequently differentiated into adipocytes. We established chromatin state maps for histone marks H3K27 acetylation and H3K27 trimethylation and tested whether enrichment of these marks was associated with gene expression changes. After validating gene expression levels for 10 significant chromatin immunoprecipitation sequencing candidate genes, we selected acyl-coenzyme A synthetase 1 (ACSL1) for further investigations due to its key roles in lipid metabolism. The ACSL1 gene was found to be highly associated with histone acetylation in adipocytes differentiated from MSCs with SGA background. In SGA-derived adipocytes, the ACSL1 expression level was also found to be associated with increased lipid loading as well as higher insulin sensitivity. ACSL1 depletion led to changes in expression of candidate genes such as proinflammatory chemokines and down-regulated both, the amount of cellular lipids and glucose uptake. Increased ACSL1, as well as modulated downstream candidate gene expression, may reflect the obese state, as detected in mice fed a high-fat diet. In summary, we believe that ACSL1 is a programmable mediator of insulin sensitivity and cellular lipid content and adipocytes differentiated from Wharton's jelly MSCs recapitulate important physiological characteristics of SGA individuals.

AgNORs in hyperplasia, papilloma and oral squamous cell carcinoma.

PubMed

Fonseca, L M; do Carmo, M A

2000-01-01

Ten inflammatory fibrous hyperplasias, ten papillomas, and nineteen oral squamous cell carcinomas were analyzed by the AgNOR technique to determine if different disturbances of oral epithelia presented different AgNOR counts. The papilloma group showed higher mean AgNOR counts (3.15 +/- 0.58) than the hyperplasia group (1.98 +/- 0.24) and smaller than the well-differentiated oral squamous cell carcinoma group (6.56 +/- 1.25) and poorly differentiated oral squamous cell carcinoma group (7.07 +/- 1.60). The differences among the groups of lesions were statistically significant (P < 0.05) except between the well differentiated oral squamous cell carcinoma group and the poorly differentiated oral squamous cell carcinoma group. Our findings suggest that the cellular proliferation ratio in papillomas is greater than hyperplasias and smaller than carcinomas.

A metabolic switch controls intestinal differentiation downstream of Adenomatous polyposis coli (APC).

PubMed

Sandoval, Imelda T; Delacruz, Richard Glenn C; Miller, Braden N; Hill, Shauna; Olson, Kristofor A; Gabriel, Ana E; Boyd, Kevin; Satterfield, Christeena; Remmen, Holly Van; Rutter, Jared; Jones, David A

2017-04-11

Elucidating signaling pathways that regulate cellular metabolism is essential for a better understanding of normal development and tumorigenesis. Recent studies have shown that mitochondrial pyruvate carrier 1 (MPC1) , a crucial player in pyruvate metabolism, is downregulated in colon adenocarcinomas. Utilizing zebrafish to examine the genetic relationship between MPC1 and Adenomatous polyposis coli (APC), a key tumor suppressor in colorectal cancer, we found that apc controls the levels of mpc1 and that knock down of mpc1 recapitulates phenotypes of impaired apc function including failed intestinal differentiation. Exogenous human MPC1 RNA rescued failed intestinal differentiation in zebrafish models of apc deficiency. Our data demonstrate a novel role for apc in pyruvate metabolism and that pyruvate metabolism dictates intestinal cell fate and differentiation decisions downstream of apc .

Undifferentiated Neuroblastoma Cells Are More Sensitive to Photogenerated Oxidative Stress Than Differentiated Cells.

PubMed

Lee, Chu-I; Perng, Jing-Huei; Chen, Huang-Yo; Hong, Yi-Ren; Wang, Jyh-Jye

2015-09-01

Neuroblastoma is one of the most aggressive cancers and has a complex form of differentiation. We hypothesized that advanced cellular differentiation may alter the susceptibility of neuroblastoma to photodynamic treatment (PDT) and confer selective survival advantage. We demonstrated that hematoporphyrin uptake by undifferentiated SH-SY5Y cells was lower than that of differentiated counterparts, yet the former were more susceptible to PDT-induced oxidative stress killing. Photogenerated reactive oxygen species (ROS) in undifferentiated cells efficiently stimulated cell cycle arrest at G2/M phase, mitochondrial apoptotic pathway activation, the sustained phosphorylation of Akt/GSK-3β and ERK. Differentiated cells with more resistance to PDT exhibited a ROS-independent and a prolonged activation of ERK. Both SH-SY5Y cells exposed to PDT exhibited ROS-independent p38 and JNK activation. These results may have important implications for neuroblastoma patients undergoing photodynamic therapy. © 2015 Wiley Periodicals, Inc.

Quantitative evaluation method for differentiation of C2C12 myoblasts by ultrasonic microscopy

NASA Astrophysics Data System (ADS)

Takanashi, Kyoichi; Washiya, Mamoru; Ota, Kazuki; Yoshida, Sachiko; Hozumi, Naohiro; Kobayashi, Kazuto

2017-07-01

Cell differentiation was evaluated by ultrasonic microscopy. However, there were some regions that showed a lower acoustic impedance than the culture liquid. It was considered that, in such regions, the cells were not perfectly in contact with the film substrate. Hence, a waveform analysis was performed, and compensated acoustic impedances in such regions were in a reasonable range of values. By the same analysis, the displacements of partially floated cells were also successfully calculated. The elapsed day transitions of the compensated acoustic impedances and displacements were successfully evaluated. In the process of differentiation, actin fibers comprising the cytoskeleton are supposed to loosen in order to induce cellular fusion. In addition, the progress in cell differentiation accompanied by a change into a three-dimensional structure can partially be assessed by the displacement between a cell and a cultured film. Hence, we believe that cell differentiation can be evaluated using an ultrasonic microscope.

TOPICAL REVIEW: Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

NASA Astrophysics Data System (ADS)

Amranul Haque, Md; Nagaoka, Masato; Hexig, Bayar; Akaike, Toshihiro

2010-02-01

Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

DNA methylation dynamics during in vivo differentiation of blood and skin stem cells

PubMed Central

Bock, Christoph; Beerman, Isabel; Lien, Wen-Hui; Smith, Zachary D.; Gu, Hongcang; Boyle, Patrick; Gnirke, Andreas; Fuchs, Elaine; Rossi, Derrick J.; Meissner, Alexander

2012-01-01

DNA methylation is a mechanism of epigenetic regulation that is common to all vertebrates. Functional studies underscore its relevance for tissue homeostasis, but the global dynamics of DNA methylation during in vivo differentiation remain underexplored. Here we report high-resolution DNA methylation maps of adult stem cell differentiation in mouse, focusing on 19 purified cell populations of the blood and skin lineages. DNA methylation changes were locus-specific and relatively modest in magnitude. They frequently overlapped with lineage-associated transcription factors and their binding sites, suggesting that DNA methylation may protect cells from aberrant transcription factor activation. DNA methylation and gene expression provided complementary information, and combining the two enabled us to infer the cellular differentiation hierarchy of the blood lineage directly from genomic data. In summary, these results demonstrate that in vivo differentiation of adult stem cells is associated with small but informative changes in the genomic distribution of DNA methylation. PMID:22841485

Sex determination: ciliates' self-censorship.

PubMed

Bloomfield, Gareth

2014-07-07

Differentiation involves the expression of certain latent cellular characteristics and the repression of others. A new study has revealed how Paramecium uses short RNAs to delete information from the somatic genome of one of its two sexes. Copyright © 2014 Elsevier Ltd. All rights reserved.

A rapid HPLC-APCI-MS method to detect fluoroacetate in plants

USDA-ARS?s Scientific Manuscript database

Many plant species worldwide can cause sudden death of grazing livestock. One diagnostic differential is the presence of monofluoroacetate (MFA) that is metabolised to fluorocitrate that subsequently inhibits the Kreb’s Cycle (the tricarboxylic acid cycle) leading to cellular respiration dysfunction...

Thermal proximity coaggregation for system-wide profiling of protein complex dynamics in cells.

PubMed

Tan, Chris Soon Heng; Go, Ka Diam; Bisteau, Xavier; Dai, Lingyun; Yong, Chern Han; Prabhu, Nayana; Ozturk, Mert Burak; Lim, Yan Ting; Sreekumar, Lekshmy; Lengqvist, Johan; Tergaonkar, Vinay; Kaldis, Philipp; Sobota, Radoslaw M; Nordlund, Pär

2018-03-09

Proteins differentially interact with each other across cellular states and conditions, but an efficient proteome-wide strategy to monitor them is lacking. We report the application of thermal proximity coaggregation (TPCA) for high-throughput intracellular monitoring of protein complex dynamics. Significant TPCA signatures observed among well-validated protein-protein interactions correlate positively with interaction stoichiometry and are statistically observable in more than 350 annotated human protein complexes. Using TPCA, we identified many complexes without detectable differential protein expression, including chromatin-associated complexes, modulated in S phase of the cell cycle. Comparison of six cell lines by TPCA revealed cell-specific interactions even in fundamental cellular processes. TPCA constitutes an approach for system-wide studies of protein complexes in nonengineered cells and tissues and might be used to identify protein complexes that are modulated in diseases. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Differential protein acetylation induced by novel histone deacetylase inhibitors.

PubMed

Glaser, K B; Li, J; Pease, L J; Staver, M J; Marcotte, P A; Guo, J; Frey, R R; Garland, R B; Heyman, H R; Wada, C K; Vasudevan, A; Michaelides, M R; Davidsen, S K; Curtin, M L

2004-12-17

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.

The Effect of Hypoxia on Mesenchymal Stem Cell Biology

PubMed Central

Ejtehadifar, Mostafa; Shamsasenjan, Karim; Movassaghpour, Aliakbar; Akbarzadehlaleh, Parvin; Dehdilani, Nima; Abbasi, Parvaneh; Molaeipour, Zahra; Saleh, Mahshid

2015-01-01

Although physiological and pathological role of hypoxia have been appreciated in mammalians for decades however the cellular biology of hypoxia more clarified in the past 20 years. Discovery of the transcription factor hypoxia-inducible factor (HIF)-1, in the 1990s opened a new window to investigate the mechanisms behind hypoxia. In different cellular contexts HIF-1 activation show variable results by impacting various aspects of cell biology such as cell cycle, apoptosis, differentiation and etc. Mesenchymal stem cells (MSC) are unique cells which take important role in tissue regeneration. They are characterized by self-renewal capacity, multilineage potential, and immunosuppressive property. Like so many kind of cells, hypoxia induces different responses in MSCs by HIF- 1 activation. The activation of this molecule changes the growth, multiplication, differentiation and gene expression profile of MSCs in their niche by a complex of signals. This article briefly discusses the most important effects of hypoxia in growth kinetics, signalling pathways, cytokine secretion profile and expression of chemokine receptors in different conditions. PMID:26236651

A bioactive triphasic ceramic-coated hydroxyapatite promotes proliferation and osteogenic differentiation of human bone marrow stromal cells.

PubMed

Nair, Manitha B; Bernhardt, Anne; Lode, Anja; Heinemann, Christiane; Thieme, Sebastian; Hanke, Thomas; Varma, Harikrishna; Gelinsky, Michael; John, Annie

2009-08-01

Hydroxyapatite (HA) ceramics are widely used as bone graft substitutes because of their biocompatibility and osteoconductivity. However, to enhance the success of therapeutic application, many efforts are undertaken to improve the bioactivity of HA. We have developed a triphasic, silica-containing ceramic-coated hydroxyapatite (HASi) and evaluated its performance as a scaffold for cell-based tissue engineering applications. Human bone marrow stromal cells (hBMSCs) were seeded on both HASi and HA scaffolds and cultured with and without osteogenic supplements for a period of 4 weeks. Cellular responses were determined in vitro in terms of cell adhesion, viability, proliferation, and osteogenic differentiation, where both materials exhibited excellent cytocompatibility. Nevertheless, an enhanced rate of cell proliferation and higher levels of both alkaline phosphatase expression and activity were observed for cells cultured on HASi with osteogenic supplements. These findings indicate that the bioactivity of HA endowed with a silica-containing coating has definitely influenced the cellular activity, projecting HASi as a suitable candidate material for bone regenerative therapy.

Cell fate determination dynamics in bacteria

NASA Astrophysics Data System (ADS)

Kuchina, Anna; Espinar, Lorena; Cagatay, Tolga; Garcia-Ojalvo, Jordi; Suel, Gurol

2010-03-01

The fitness of an organism depends on many processes that serve the purpose to adapt to changing environment in a robust and coordinated fashion. One example of such process is cellular fate determination. In the presence of a variety of alternative responses each cell adopting a particular fate represents a ``choice'' that must be tightly regulated to ensure the best survival strategy for the population taking into account the broad range of possible environmental challenges. We investigated this problem in the model organism B.Subtilis which under stress conditions differentiates terminally into highly resistant spores or initiates an alternative transient state of competence. The dynamics underlying cell fate choice remains largely unknown. We utilize quantitative fluorescent microscopy to track the activities of genes involved in these responses on a single-cell level. We explored the importance of temporal interactions between competing cell fates by re- engineering the differentiation programs. I will discuss how the precise dynamics of cellular ``decision-making'' governed by the corresponding biological circuits may enable cells to adjust to diverse environments and determine survival.

Ultrasensitive dual phosphorylation dephosphorylation cycle kinetics exhibits canonical competition behavior

NASA Astrophysics Data System (ADS)

Huang, Qingdao; Qian, Hong

2009-09-01

We establish a mathematical model for a cellular biochemical signaling module in terms of a planar differential equation system. The signaling process is carried out by two phosphorylation-dephosphorylation reaction steps that share common kinase and phosphatase with saturated enzyme kinetics. The pair of equations is particularly simple in the present mathematical formulation, but they are singular. A complete mathematical analysis is developed based on an elementary perturbation theory. The dynamics exhibits the canonical competition behavior in addition to bistability. Although widely understood in ecological context, we are not aware of a full range of biochemical competition in a simple signaling network. The competition dynamics has broad implications to cellular processes such as cell differentiation and cancer immunoediting. The concepts of homogeneous and heterogeneous multisite phosphorylation are introduced and their corresponding dynamics are compared: there is no bistability in a heterogeneous dual phosphorylation system. A stochastic interpretation is also provided that further gives intuitive understanding of the bistable behavior inside the cells.

KEEPING AN EYE ON RETINOBLASTOMA CONTROL OF HUMAN EMBRYONIC STEM CELLS

PubMed Central

Conklin, Jamie F.; Sage, Julien

2010-01-01

Human embryonic stem cells (hESCs) hold great promise in regenerative medicine. However, before the full potential of these cells is achieved, major basic biological questions need to be addressed. In particular, there are still gaps in our knowledge of the molecular mechanisms underlying the derivation of hESCs from blastocysts, the regulation of the undifferentiated, pluripotent state, and the control of differentiation into specific lineages. Furthermore, we still do not fully understand the tumorigenic potential of hESCs, limiting their use in regenerative medicine. The RB pathway is a key signaling module that controls cellular proliferation, cell survival, chromatin structure, and cellular differentiation in mammalian cells. Members of the RB pathway are important regulators of hESC biology and manipulation of the activity of this pathway may provide novel means to control the fate of hESCs. Here we review what is known about the expression and function of members of the RB pathway in hESCs and discuss areas of interest in this field. PMID:19760644

Modulation of occluding junctions alters the hematopoietic niche to trigger immune activation

PubMed Central

Khadilkar, Rohan J; Vogl, Wayne; Goodwin, Katharine

2017-01-01

Stem cells are regulated by signals from their microenvironment, or niche. During Drosophila hematopoiesis, a niche regulates prohemocytes to control hemocyte production. Immune challenges activate cell-signalling to initiate the cellular and innate immune response. Specifically, certain immune challenges stimulate the niche to produce signals that induce prohemocyte differentiation. However, the mechanisms that promote prohemocyte differentiation subsequent to immune challenges are poorly understood. Here we show that bacterial infection induces the cellular immune response by modulating occluding-junctions at the hematopoietic niche. Occluding-junctions form a permeability barrier that regulates the accessibility of prohemocytes to niche derived signals. The immune response triggered by infection causes barrier breakdown, altering the prohemocyte microenvironment to induce immune cell production. Moreover, genetically induced barrier ablation provides protection against infection by activating the immune response. Our results reveal a novel role for occluding-junctions in regulating niche-hematopoietic progenitor signalling and link this mechanism to immune cell production following infection. PMID:28841136

Cell-Cell Contact Area Affects Notch Signaling and Notch-Dependent Patterning.

PubMed

Shaya, Oren; Binshtok, Udi; Hersch, Micha; Rivkin, Dmitri; Weinreb, Sheila; Amir-Zilberstein, Liat; Khamaisi, Bassma; Oppenheim, Olya; Desai, Ravi A; Goodyear, Richard J; Richardson, Guy P; Chen, Christopher S; Sprinzak, David

2017-03-13

During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from micrometers to tens of micrometers. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes. Copyright © 2017 Elsevier Inc. All rights reserved.

The importance of controlling in vitro oxygen tension to accurately model in vivo neurophysiology.

PubMed

Bordt, Evan A

2018-05-01

The majority of in vitro studies modeling in vivo conditions are performed on the lab bench in atmospheric air. However, the oxygen tension (pO 2 ) present in atmospheric air (160mm Hg, ∼21% O 2 ) is in great excess to the pO 2 that permeates tissues within the brain (5-45mm Hg, ∼1-6% O 2 ). This review will discuss the differentiation between pO 2 in the in vivo environment and the pO 2 commonly used during in vitro experiments, and how this could affect assay outcomes. Also highlighted are studies linking changes in pO 2 to changes in cellular function, particularly the role of pO 2 in mitochondrial function, reactive oxygen species production, and cellular growth and differentiation. The role of hypoxia inducible factor 1 and oxygen sensing is also presented. Finally, emerging literature exploring sex differences in tissue oxygenation is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Cell-cell contact area affects Notch signaling and Notch-dependent patterning

PubMed Central

Shaya, Oren; Binshtok, Udi; Hersch, Micha; Rivkin, Dmitri; Weinreb, Sheila; Amir-Zilberstein, Liat; Khamaisi, Bassma; Oppenheim, Olya; Desai, Ravi A.; Goodyear, Richard J.; Richardson, Guy P.; Chen, Christopher S.; Sprinzak, David

2017-01-01

Summary During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from microns to tens of microns. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes. PMID:28292428

Nuclear matrix - structure, function and pathogenesis.

PubMed

Wasąg, Piotr; Lenartowski, Robert

2016-12-20

The nuclear matrix (NM), or nuclear skeleton, is the non-chromatin, ribonucleoproteinaceous framework that is resistant to high ionic strength buffers, nonionic detergents, and nucleolytic enzymes. The NM fulfills a structural role in eukaryotic cells and is responsible for maintaining the shape of the nucleus and the spatial organization of chromatin. Moreover, the NM participates in several cellular processes, such as DNA replication/repair, gene expression, RNA transport, cell signaling and differentiation, cell cycle regulation, apoptosis and carcinogenesis. Short nucleotide sequences called scaffold/matrix attachment regions (S/MAR) anchor the chromatin loops to the NM proteins (NMP). The NMP composition is dynamic and depends on the cell type and differentiation stage or metabolic activity. Alterations in the NMP composition affect anchoring of the S/MARs and thus alter gene expression. This review aims to systematize information about the skeletal structure of the nucleus, with particular emphasis on the organization of the NM and its role in selected cellular processes. We also discuss several diseases that are caused by aberrant NM structure or dysfunction of individual NM elements.

Therapeutic concentration of lithium stimulates complement C3 production in dendritic cells and microglia via GSK-3 inhibition.

PubMed

Yu, Zhiqian; Ono, Chiaki; Aiba, Setsuya; Kikuchi, Yoshie; Sora, Ichiro; Matsuoka, Hiroo; Tomita, Hiroaki

2015-02-01

Evidence indicates that widely prescribed mood stabilizer, lithium (Li), mediates cellular functions of differentiated monocytic cells, including microglial migration, monocyte-derived dendritic cell (MoDC) differentiation, and amelioration of monocytic malfunctions observed in neuropsychiatric diseases. Here, we surveyed molecules which take major roles in regulating these monocytic cellular functions. MoDCs treated with 1 and 5 mM Li, and microglia separated from Li-treated mice were subjected to microarray-based comprehensive gene expression analyses. Findings were validated using multiple experiments, including quantitative PCR, ELISA and immunostaining studies. Differing effects of Li on the two cell types were observed. Inflammation- and chemotaxis-relevant genes were significantly over-represented among Li-induced genes in MoDCs, whereas no specific category of genes was over-represented in microglia. The third component of complement (C3) was the only gene which was significantly induced by a therapeutic concentration of Li in both MoDCs and microglia. C3 production was increased by Li via GSK-3 inhibition. Li-induced C3 production was seen only in differentiated monocytic cells, but not in circulating monocytes. Our findings highlight a link between Li treatment and C3 production in differentiated monocytic cells, and reveal a regulatory role of GSK-3 in C3 production. Induction of microglial C3 production might be a novel neuroprotective mechanism of Li via regulating interactions between microglia and neurons. GLIA 2015;63:257-270. © 2014 Wiley Periodicals, Inc.

Bioinspired Nanocomplex for Spatiotemporal Imaging of Sequential mRNA Expression in Differentiating Neural Stem Cells

PubMed Central

2015-01-01

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions. PMID:25494492

Bioactive silica nanoparticles promote osteoblast differentiation through stimulation of autophagy and direct association with LC3 and p62.

PubMed

Ha, Shin-Woo; Weitzmann, M Neale; Beck, George R

2014-06-24

We recently identified an engineered bioactive silica-based nanoparticle formulation (designated herein as NP1) that stimulates in vitro differentiation and mineralization of osteoblasts, the cells responsible for bone formation, and increases bone mineral density in young mice in vivo. The results demonstrate that these nanoparticles have intrinsic biological activity; however, the intracellular fate and a complete understanding of the mechanism(s) involved remains to be elucidated. Here we investigated the cellular mechanism(s) by which NP1 stimulates differentiation and mineralization of osteoblasts. We show that NP1 enters the cells through a caveolae-mediated endocytosis followed by stimulation of the mitogen activated protein kinase ERK1/2 (p44/p42). Our findings further revealed that NP1 stimulates autophagy including the processing of LC3β-I to LC3β-II, a key protein involved in autophagosome formation, which is dependent on ERK1/2 signaling. Using a variant of NP1 with cobalt ferrite magnetic metal core (NP1-MNP) to pull down associated proteins, we found direct binding of LC3β and p62, two key proteins involved in autophagosome formation, with silica nanoparticles. Interestingly, NP1 specifically interacts with the active and autophagosome associated form of LC3β (LC3β-II). Taken together, the stimulation of autophagy and associated signaling suggests a cellular mechanism for the stimulatory effects of silica nanoparticles on osteoblast differentiation and mineralization.

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

PubMed

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

PubMed

Marín-Llera, Jessica Cristina; Chimal-Monroy, Jesús

2018-05-01

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues. © 2018 International Federation for Cell Biology.

Evaluation of human embryonic stem cells and their differentiated fibroblastic progenies as cellular models for in vitro genotoxicity screening.

PubMed

Vinoth, Kumar Jayaseelan; Manikandan, Jayapal; Sethu, Swaminathan; Balakrishnan, Lakshmidevi; Heng, Alexis; Lu, Kai; Hande, Manoor Prakash; Cao, Tong

2014-08-20

This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures. Copyright © 2014. Published by Elsevier B.V.

Bioactive Silica Nanoparticles Promote Osteoblast Differentiation through Stimulation of Autophagy and Direct Association with LC3 and p62

PubMed Central

2015-01-01

We recently identified an engineered bioactive silica-based nanoparticle formulation (designated herein as NP1) that stimulates in vitro differentiation and mineralization of osteoblasts, the cells responsible for bone formation, and increases bone mineral density in young mice in vivo. The results demonstrate that these nanoparticles have intrinsic biological activity; however, the intracellular fate and a complete understanding of the mechanism(s) involved remains to be elucidated. Here we investigated the cellular mechanism(s) by which NP1 stimulates differentiation and mineralization of osteoblasts. We show that NP1 enters the cells through a caveolae-mediated endocytosis followed by stimulation of the mitogen activated protein kinase ERK1/2 (p44/p42). Our findings further revealed that NP1 stimulates autophagy including the processing of LC3β-I to LC3β-II, a key protein involved in autophagosome formation, which is dependent on ERK1/2 signaling. Using a variant of NP1 with cobalt ferrite magnetic metal core (NP1-MNP) to pull down associated proteins, we found direct binding of LC3β and p62, two key proteins involved in autophagosome formation, with silica nanoparticles. Interestingly, NP1 specifically interacts with the active and autophagosome associated form of LC3β (LC3β-II). Taken together, the stimulation of autophagy and associated signaling suggests a cellular mechanism for the stimulatory effects of silica nanoparticles on osteoblast differentiation and mineralization. PMID:24806912

RNA Sequencing Reveals the Alteration of the Expression of Novel Genes in Ethanol-Treated Embryoid Bodies.

PubMed

Mandal, Chanchal; Kim, Sun Hwa; Chai, Jin Choul; Oh, Seon Mi; Lee, Young Seek; Jung, Kyoung Hwa; Chai, Young Gyu

2016-01-01

Fetal alcohol spectrum disorder is a collective term representing fetal abnormalities associated with maternal alcohol consumption. Prenatal alcohol exposure and related anomalies are well characterized, but the molecular mechanism behind this phenomenon is not well characterized. In this present study, our aim is to profile important genes that regulate cellular development during fetal development. Human embryonic carcinoma cells (NCCIT) are cultured to form embryoid bodies and then treated in the presence and absence of ethanol (50 mM). We employed RNA sequencing to profile differentially expressed genes in the ethanol-treated embryoid bodies from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH data sets. A total of 632, 205 and 517 differentially expressed genes were identified from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, respectively. Functional annotation using bioinformatics tools reveal significant enrichment of differential cellular development and developmental disorders. Furthermore, a group of 42, 15 and 35 transcription factor-encoding genes are screened from all of the differentially expressed genes obtained from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, respectively. We validated relative gene expression levels of several transcription factors from these lists by quantitative real-time PCR. We hope that our study substantially contributes to the understanding of the molecular mechanism underlying the pathology of alcohol-mediated anomalies and ease further research.

Integration of Proteomic, Transcriptional, and Interactome Data Reveals Hidden Signaling Components

PubMed Central

Huang, Shao-shan Carol; Fraenkel, Ernest

2009-01-01

Cellular signaling and regulatory networks underlie fundamental biological processes such as growth, differentiation, and response to the environment. Although there are now various high-throughput methods for studying these processes, knowledge of them remains fragmentary. Typically, the vast majority of hits identified by transcriptional, proteomic, and genetic assays lie outside of the expected pathways. These unexpected components of the cellular response are often the most interesting, because they can provide new insights into biological processes and potentially reveal new therapeutic approaches. However, they are also the most difficult to interpret. We present a technique, based on the Steiner tree problem, that uses previously reported protein-protein and protein-DNA interactions to determine how these hits are organized into functionally coherent pathways, revealing many components of the cellular response that are not readily apparent in the original data. Applied simultaneously to phosphoproteomic and transcriptional data for the yeast pheromone response, it identifies changes in diverse cellular processes that extend far beyond the expected pathways. PMID:19638617

Cellular reprogramming dynamics follow a simple 1D reaction coordinate

NASA Astrophysics Data System (ADS)

Teja Pusuluri, Sai; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2018-01-01

Cellular reprogramming, the conversion of one cell type to another, induces global changes in gene expression involving thousands of genes, and understanding how cells globally alter their gene expression profile during reprogramming is an ongoing problem. Here we reanalyze time-course data on cellular reprogramming from differentiated cell types to induced pluripotent stem cells (iPSCs) and show that gene expression dynamics during reprogramming follow a simple 1D reaction coordinate. This reaction coordinate is independent of both the time it takes to reach the iPSC state as well as the details of the experimental protocol used. Using Monte-Carlo simulations, we show that such a reaction coordinate emerges from epigenetic landscape models where cellular reprogramming is viewed as a ‘barrier-crossing’ process between cell fates. Overall, our analysis and model suggest that gene expression dynamics during reprogramming follow a canonical trajectory consistent with the idea of an ‘optimal path’ in gene expression space for reprogramming.

Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish.

PubMed

Kim, Dal Hyung; Kim, Jungsoo; Marques, João C; Grama, Abhinav; Hildebrand, David G C; Gu, Wenchao; Li, Jennifer M; Robson, Drew N

2017-11-01

Calcium imaging with cellular resolution typically requires an animal to be tethered under a microscope, which substantially restricts the range of behaviors that can be studied. To expand the behavioral repertoire amenable to imaging, we have developed a tracking microscope that enables whole-brain calcium imaging with cellular resolution in freely swimming larval zebrafish. This microscope uses infrared imaging to track a target animal in a behavior arena. On the basis of the predicted trajectory of the animal, we applied optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We combined this motion-cancellation system with differential illumination focal filtering, a variant of HiLo microscopy, which enabled us to image the brain of a freely swimming larval zebrafish for more than an hour. This work expands the repertoire of natural behaviors that can be studied with cellular-resolution calcium imaging to potentially include spatial navigation, social behavior, feeding and reward.

Wavefront cellular learning automata.

PubMed

Moradabadi, Behnaz; Meybodi, Mohammad Reza

2018-02-01

This paper proposes a new cellular learning automaton, called a wavefront cellular learning automaton (WCLA). The proposed WCLA has a set of learning automata mapped to a connected structure and uses this structure to propagate the state changes of the learning automata over the structure using waves. In the WCLA, after one learning automaton chooses its action, if this chosen action is different from the previous action, it can send a wave to its neighbors and activate them. Each neighbor receiving the wave is activated and must choose a new action. This structure for the WCLA is necessary in many dynamic areas such as social networks, computer networks, grid computing, and web mining. In this paper, we introduce the WCLA framework as an optimization tool with diffusion capability, study its behavior over time using ordinary differential equation solutions, and present its accuracy using expediency analysis. To show the superiority of the proposed WCLA, we compare the proposed method with some other types of cellular learning automata using two benchmark problems.

Wavefront cellular learning automata

NASA Astrophysics Data System (ADS)

Moradabadi, Behnaz; Meybodi, Mohammad Reza

2018-02-01

This paper proposes a new cellular learning automaton, called a wavefront cellular learning automaton (WCLA). The proposed WCLA has a set of learning automata mapped to a connected structure and uses this structure to propagate the state changes of the learning automata over the structure using waves. In the WCLA, after one learning automaton chooses its action, if this chosen action is different from the previous action, it can send a wave to its neighbors and activate them. Each neighbor receiving the wave is activated and must choose a new action. This structure for the WCLA is necessary in many dynamic areas such as social networks, computer networks, grid computing, and web mining. In this paper, we introduce the WCLA framework as an optimization tool with diffusion capability, study its behavior over time using ordinary differential equation solutions, and present its accuracy using expediency analysis. To show the superiority of the proposed WCLA, we compare the proposed method with some other types of cellular learning automata using two benchmark problems.

Differences in primary cellular factors influencing the metabolism and distribution of 3,5,3′-L-triiodothyronine and L-thyroxine

PubMed Central

Oppenheimer, Jack H.; Schwartz, Harold L.; Shapiro, Harvey C.; Bernstein, Gerald; Surks, Martin I.

1970-01-01

Administration of phenobarbital, which acts exclusively on cellular sites, results in an augmentation of the liver/plasma concentration ratio of L-thyroxine (T4) in rats but no change in the liver/plasma concentration ratio of L-triiodothyronine (T3). Whereas phenobarbital stimulates the fecal clearance rate both of T3 and T4, it increases the deiodinative clearance rate of T4 only. These findings suggest basic differences in the cellular metabolism of T3 and T4. Further evidence pointing to cellular differences was obtained from a comparison of the distribution and metabolism of these hormones with appropriate corrections for the effect of differential plasma binding. The percentage of total exchangeable cellular T4 within the liver (28.5) is significantly greater than the corresponding percentage of exchangeable cellular T3 within this organ (12.3). Extrahepatic tissues bind T3 twice as firmly as T4. The cellular metabolic clearance rate (= free hormone clearance rate) of T3 exceeds that of T4 by a factor 1.8 in the rat. The corresponding ratio in man, 2.4, was determined by noncompartmental analysis of turnover studies in four individuals after the simultaneous injection of T4-125I and T3-131I. The greater cellular metabolic clearance rate of T3 both in rat and man may be related to the higher specific hormonal potency of this iodothyronine. PMID:5441537

Transcriptional analysis of Volvox photoreceptors suggests the existence of different cell-type specific light-signaling pathways.

PubMed

Kianianmomeni, Arash; Hallmann, Armin

2015-02-01

Photosynthetic organisms, e.g., plants including green algae, use a sophisticated light-sensing system, composed of primary photoreceptors and additional downstream signaling components, to monitor changes in the ambient light environment towards adjust their growth and development. Although a variety of cellular processes, e.g., initiation of cleavage division and final cellular differentiation, have been shown to be light-regulated in the green alga Volvox carteri, little is known about the underlying light perception and signaling pathways. This multicellular alga possesses at least 12 photoreceptors, i.e., one phototropin (VcPhot), four cryptochromes (VcCRYa, VcCRYp, VcCRYd1, and VcCRYd2), and seven members of rhodopsin-like photoreceptors (VR1, VChR1, VChR2, VcHKR1, VcHKR2, VcHKR3, and VcHKR4), which display distinct light-dependent chemical processes based on their protein architectures and associated chromophores. Gene expression analyses could show that the transcript levels of some of the photoreceptor genes (e.g., VChR1 and VcHKR1) accumulate during division cleavages, while others (e.g., VcCRYa, VcCRYp, and VcPhot) accumulate during final cellular differentiation. However, the pattern of transcript accumulation changes when the alga switches to the sexual development. Eight photoreceptor genes, e.g., VcPhot, VcCRYp, and VcHKR1, are highly expressed in the somatic cells, while only the animal-type rhodopsin VR1 was found to be highly expressed in the reproductive cells/embryos during both asexual and sexual life cycles. Moreover, accumulation of VChR1 and VcCRYa transcripts is more sensitive to light and changes in response to more than one light quality. Obviously, different regulatory mechanisms underlying gene expression control transcript accumulation of photoreceptors not only during development, but also in a cell-type specific way and in response to various external signals such as light quality. The transcriptional patterns described in this study show that Volvox photoreceptors are mostly expressed in a cell-type specific manner. This gives reason to believe that cell-type specific light-signaling pathways allow differential regulation of cellular and developmental processes in response to the environmental light cues.

Cellular Responses to Mechanical Stress Selected Contribution: A Three-Dimensional Model for Assessment of in Vitro Toxicity in Balaena Mysticetus Renal Tissue

NASA Technical Reports Server (NTRS)

Goodwin, T. J.; Coate-Li, L.; Linnehan, R. M.; Hammond, T. G.

2000-01-01

This study established two- and three-dimensional renal proximal tubular cell cultures of the endangered species bowhead whale (Balaena mysticetus), developed SV40-transfected cultures, and cloned the 61-amino acid open reading frame for the metallothionein protein, the primary binding site for heavy metal contamination in mammals. Microgravity research, modulations in mechanical culture conditions (modeled microgravity), and shear stress have spawned innovative approaches to understanding the dynamics of cellular interactions, gene expression, and differentiation in several cellular systems. These investigations have led to the creation of ex vivo tissue models capable of serving as physiological research analogs for three-dimensional cellular interactions. These models are enabling studies in immune function, tissue modeling for basic research, and neoplasia. Three-dimensional cellular models emulate aspects of in vivo cellular architecture and physiology and may facilitate environmental toxicological studies aimed at elucidating biological functions and responses at the cellular level. Marine mammals occupy a significant ecological niche (72% of the Earth's surface is water) in terms of the potential for information on bioaccumulation and transport of terrestrial and marine environmental toxins in high-order vertebrates. Few ex vivo models of marine mammal physiology exist in vitro to accomplish the aforementioned studies. Techniques developed in this investigation, based on previous tissue modeling successes, may serve to facilitate similar research in other marine mammals.

Growth and cellular ion content of a salt-sensitive symbiotic system Azolla pinnata-Anabaena azollae under NaCl stress.

PubMed

Rai, Vandna; Sharma, Naveen Kumar; Rai, Ashwani K

2006-09-01

Salinity, at a concentration of 10 mM NaCl affected the growth of Azolla pinnata-Anabaena azollae association and became lethal at 40 mM. Plants exposed up to 30 mM NaCl exhibited longer roots than the control, especially during the beginning of incubation. Average root number in plants exposed to 10 and 20 mM NaCl remained almost the same as in control. A further rise in NaCl concentration to 30 mM reduced the root number, and roots shed off at 40 mM NaCl. Presence of NaCl in the nutrient solution increased the cellular Na+ of the intact association exhibiting differential accumulation by individual partners, while it reduced the cellular Ca2+ level. However, cellular K+ content did not show significant change. Cellular Na+ based on fresh weight of respective individual partners (host tissues and cyanobiont) remained higher in the host tissues than the cyanobiont, while reverse was true for K+ and Ca2+ contents. The contribution of A. azollae in the total cellular ion content of the association was a little because of meagre contribution of the cyanobiont mass (19-21%). High salt sensitivity of Azolla-Anabaena complex is due to an inability of the association to maintain low Na+ and high Ca2+ cellular level.

Tissue engineering bioreactor systems for applying physical and electrical stimulations to cells.

PubMed

Jin, GyuHyun; Yang, Gi-Hoon; Kim, GeunHyung

2015-05-01

Bioreactor systems in tissue engineering applications provide various types of stimulation to mimic the tissues in vitro and in vivo. Various bioreactors have been designed to induce high cellular activities, including initial cell attachment, cell growth, and differentiation. Although cell-stimulation processes exert mostly positive effects on cellular responses, in some cases such stimulation can also have a negative effect on cultured cells. In this review, we discuss various types of bioreactor and the positive and negative effects of stimulation (physical, chemical, and electrical) on various cultured cell types. © 2014 Wiley Periodicals, Inc.

2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

PubMed

Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

2015-10-01

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Deriving excitatory neurons of the neocortex from pluripotent stem cells

PubMed Central

Hansen, David V.; Rubenstein, John L.R.; Kriegstein, Arnold R.

2011-01-01

The human cerebral cortex is an immensely complex structure that subserves critical functions that can be disrupted in developmental and degenerative disorders. Recent innovations in cellular reprogramming and differentiation techniques have provided new ways to study the cellular components of the cerebral cortex. Here we discuss approaches to generate specific subtypes of excitatory cortical neurons from pluripotent stem cells. We review spatial and temporal aspects of cortical neuron specification that can guide efforts to produce excitatory neuron subtypes with increased resolution. Finally, we discuss distinguishing features of human cortical development and their translational ramifications for cortical stem cell technologies. PMID:21609822

Immediate Administration of Intraarticular Triamcinolone Acetonide after Joint Injury Modulates Molecular Outcomes Associated with Early Synovitis

PubMed Central

Sieker, Jakob T.; Ayturk, Ugur M.; Proffen, Benedikt L.; Weissenberger, Manuela H.; Kiapour, Ata M.; Murray, Martha M.

2016-01-01

Objective To test if intraarticular corticosteroid injection mitigates injury-induced synovitis and collagen degradation after anterior cruciate ligament (ACL) transection and characterize the synovial response using a functional genomics approach in a preclinical model of post- traumatic osteoarthritis. Methods Yorkshire pigs received untreated unilateral ACL transection (ACLT, n=6) or transection with immediate injection of 20mg triamcinolone acetonide (STEROID, n=6). Total synovial membrane cellularity and synovial fluid concentration of COL-2 3/4C short neoepitope bearing collagen fragments at 14 days post-injury were primary endpoints and compared between ACLT, STEROID and INTACT (n=6 uninjured knees). Cells were differentiated by histological phenotype and counted, while RNA-seq was used to quantify transcriptome-wide gene expression, monocyte, macrophage and lymphocyte markers. Results Total cellularity of 13% (95% confidence interval of 9–16) and COL-2 3/4C short levels of 0.24 Kg/ml (0.08–0.39) were determined in INTACT. Significant increases in total cellularity to 21% (16–27) and COL-2 3/4C short to 0.49 Kg/ml (0.39–0.59) were observed in ACLT. Compared to ACLT, total cellularity was non-significantly and COL-2 3/4C short was significantly decreased in STEROID to 17% (15–18, p=0.26) and 0.29 Kg/ml (0.23–0.35). Between ACLT and INTACT, 255 genes were differentially expressed and enriched pathways related to cellular immune response and proteolysis. Mononuclear leukocytes were the dominant cell type in cell dense areas. MARCO, SOCS3, CCR1, IL4R and MMP2 expression was significantly associated with COL-2 3/4C short levels. Conclusions Early intraarticular immunosuppression mitigated the injury-induced increase of collagen fragments, an outcome better predicted by specific marker expression than histological measures of synovitis. PMID:26866935

Biomimetics in thin film design: Niche-like wrinkles designed for i-cell progenitor cell differentiation.

PubMed

Major, Roman; Lackner, Juergen M; Sanak, Marek; Major, Boguslaw

2017-11-01

The future and development of science are in interdisciplinary areas, such as biomedical engineering. Self-assembled structures, similar to stem cell niches, inhibit rapid cellular division processes and enable the capture of stem cells from blood flow. By modifying the surface topography and stiffness properties, progenitor cells were differentiated towards the formation of endothelial cell monolayers to effectively inhibit the coagulation cascade. Wrinkled material layers in the form of thin polymeric coatings were prepared. An optimized surface topography led to proper cell differentiation and influenced the appropriate formation of endothelial cell monolayers. Blood activation was decelerated by the formed endothelium. Copyright © 2017 Elsevier B.V. All rights reserved.

Dysfunctions at human intestinal barrier by water-borne protozoan parasites: lessons from cultured human fully differentiated colon cancer cell lines.

PubMed

Liévin-Le Moal, Vanessa

2013-06-01

Some water-borne protozoan parasites induce diseases through their membrane-associated functional structures and virulence factors that hijack the host cellular molecules and signalling pathways leading to structural and functional lesions in the intestinal barrier. In this Microreview we analyse the insights on the mechanisms of pathogenesis of Entamoeba intestinalis, Giardia and Cryptosporidium observed in the human colon carcinoma fully differentiated colon cancer cell lines, cell subpopulations and clones expressing the structural and functional characteristics of highly specialized fully differentiated epithelial cells lining the intestinal epithelium and mimicking structurally and functionally an intestinal barrier. © 2013 John Wiley & Sons Ltd.

Male sex determination: insights into molecular mechanisms

PubMed Central

McClelland, Kathryn; Bowles, Josephine; Koopman, Peter

2012-01-01

Disorders of sex development often arise from anomalies in the molecular or cellular networks that guide the differentiation of the embryonic gonad into either a testis or an ovary, two functionally distinct organs. The activation of the Y-linked gene Sry (sex-determining region Y) and its downstream target Sox9 (Sry box-containing gene 9) triggers testis differentiation by stimulating the differentiation of Sertoli cells, which then direct testis morphogenesis. Once engaged, a genetic pathway promotes the testis development while actively suppressing genes involved in ovarian development. This review focuses on the events of testis determination and the struggle to maintain male fate in the face of antagonistic pressure from the underlying female programme. PMID:22179516

Quantifying the driving factors for language shift in a bilingual region

PubMed Central

Prochazka, Katharina; Vogl, Gero

2017-01-01

Many of the world’s around 6,000 languages are in danger of disappearing as people give up use of a minority language in favor of the majority language in a process called language shift. Language shift can be monitored on a large scale through the use of mathematical models by way of differential equations, for example, reaction–diffusion equations. Here, we use a different approach: we propose a model for language dynamics based on the principles of cellular automata/agent-based modeling and combine it with very detailed empirical data. Our model makes it possible to follow language dynamics over space and time, whereas existing models based on differential equations average over space and consequently provide no information on local changes in language use. Additionally, cellular automata models can be used even in cases where models based on differential equations are not applicable, for example, in situations where one language has become dispersed and retreated to language islands. Using data from a bilingual region in Austria, we show that the most important factor in determining the spread and retreat of a language is the interaction with speakers of the same language. External factors like bilingual schools or parish language have only a minor influence. PMID:28298530

The extracellular microenvironment explains variations in passive drug transport across different airway epithelial cell types.

PubMed

Min, Kyoung Ah; Talattof, Arjang; Tsume, Yasuhiro; Stringer, Kathleen A; Yu, Jing-Yu; Lim, Dong Hyun; Rosania, Gus R

2013-08-01

We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types. Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types. Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in the extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells. Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types.

Spontaneous Up states in vitro: a single-metric index of the functional maturation and regional differentiation of the cerebral cortex.

PubMed

Rigas, Pavlos; Adamos, Dimitrios A; Sigalas, Charalambos; Tsakanikas, Panagiotis; Laskaris, Nikolaos A; Skaliora, Irini

2015-01-01

Understanding the development and differentiation of the neocortex remains a central focus of neuroscience. While previous studies have examined isolated aspects of cellular and synaptic organization, an integrated functional index of the cortical microcircuit is still lacking. Here we aimed to provide such an index, in the form of spontaneously recurring periods of persistent network activity -or Up states- recorded in mouse cortical slices. These coordinated network dynamics emerge through the orchestrated regulation of multiple cellular and synaptic elements and represent the default activity of the cortical microcircuit. To explore whether spontaneous Up states can capture developmental changes in intracortical networks we obtained local field potential recordings throughout the mouse lifespan. Two independent and complementary methodologies revealed that Up state activity is systematically modified by age, with the largest changes occurring during early development and adolescence. To explore possible regional heterogeneities we also compared the development of Up states in two distinct cortical areas and show that primary somatosensory cortex develops at a faster pace than primary motor cortex. Our findings suggest that in vitro Up states can serve as a functional index of cortical development and differentiation and can provide a baseline for comparing experimental and/or genetic mouse models.

Methods and insights from the characterization of osteoprogenitor cells of bats (Mammalia: Chiroptera).

PubMed

Ball, H C; Moussa, F M; Mbimba, T; Orman, R; Safadi, F F; Cooper, L N

2016-07-01

Osteoprogenitor cells contribute to the development and maintenance of skeletal tissues. Bats are unique model taxa whose cellular processes are poorly understood, especially in regards to skeletal biology. Forelimb bones of bats, unlike those of terrestrial mammals, bend during flight and function in controlled deformation. As a first step towards understanding the molecular processes governing deposition of this flexible bone matrix, we provide the first method for isolation and differentiation of cell populations derived from the bone marrow and cortical bone of bats, and compare results with those harvested from C57BL/6J mice. Osteogenic capacity of these cells was assessed via absolute quantitative real-time PCR (qPCR) and through quantification of in vitro mineral deposition. Results indicate the differentiated bone cells of bats display significantly lower gene expression of known osteogenic markers (Runt-related transcription factor (RUNX2), osteocalcin (BGLAP) and osterix (SP7)), and deposit a less-mineralized matrix compared with murine controls. By characterizing the in vitro performance of osteoprogenitor cells throughout differentiation and matrix production, this study lays the ground work for in vitro manipulations of bat stem and osteoprogenitor cells and extends our understanding of the cellular diversity across mammals that occupy different habitats. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Myostatin Suppression of Akirin1 Mediates Glucocorticoid-Induced Satellite Cell Dysfunction

PubMed Central

Dong, Yanjun; Pan, Jenny S.; Zhang, Liping

2013-01-01

Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production. PMID:23516508

Cellular manganese content is developmentally regulated in human dopaminergic neurons

NASA Astrophysics Data System (ADS)

Kumar, Kevin K.; Lowe, Edward W., Jr.; Aboud, Asad A.; Neely, M. Diana; Redha, Rey; Bauer, Joshua A.; Odak, Mihir; Weaver, C. David; Meiler, Jens; Aschner, Michael; Bowman, Aaron B.

2014-10-01

Manganese (Mn) is both an essential biological cofactor and neurotoxicant. Disruption of Mn biology in the basal ganglia has been implicated in the pathogenesis of neurodegenerative disorders, such as parkinsonism and Huntington's disease. Handling of other essential metals (e.g. iron and zinc) occurs via complex intracellular signaling networks that link metal detection and transport systems. However, beyond several non-selective transporters, little is known about the intracellular processes regulating neuronal Mn homeostasis. We hypothesized that small molecules that modulate intracellular Mn could provide insight into cell-level Mn regulatory mechanisms. We performed a high throughput screen of 40,167 small molecules for modifiers of cellular Mn content in a mouse striatal neuron cell line. Following stringent validation assays and chemical informatics, we obtained a chemical `toolbox' of 41 small molecules with diverse structure-activity relationships that can alter intracellular Mn levels under biologically relevant Mn exposures. We utilized this toolbox to test for differential regulation of Mn handling in human floor-plate lineage dopaminergic neurons, a lineage especially vulnerable to environmental Mn exposure. We report differential Mn accumulation between developmental stages and stage-specific differences in the Mn-altering activity of individual small molecules. This work demonstrates cell-level regulation of Mn content across neuronal differentiation.