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Sample records for cell lineage analysis

  1. Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissue-inducer cell lineage.

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    Ishizuka, Isabel E; Chea, Sylvestre; Gudjonson, Herman; Constantinides, Michael G; Dinner, Aaron R; Bendelac, Albert; Golub, Rachel

    2016-03-01

    The precise lineage relationship between innate lymphoid cells (ILCs) and lymphoid tissue-inducer (LTi) cells is poorly understood. Using single-cell multiplex transcriptional analysis of 100 lymphoid genes and single-cell cultures of fetal liver precursor cells, we identified the common proximal precursor to these lineages and found that its bifurcation was marked by differential induction of the transcription factors PLZF and TCF1. Acquisition of individual effector programs specific to the ILC subsets ILC1, ILC2 and ILC3 was initiated later, at the common ILC precursor stage, by transient expression of mixed ILC1, ILC2 and ILC3 transcriptional patterns, whereas, in contrast, the development of LTi cells did not go through multilineage priming. Our findings provide insight into the divergent mechanisms of the differentiation of the ILC lineage and LTi cell lineage and establish a high-resolution 'blueprint' of their development.

  2. Differential Protein Network Analysis of the Immune Cell Lineage

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    Trevor Clancy

    2014-01-01

    Full Text Available Recently, the Immunological Genome Project (ImmGen completed the first phase of the goal to understand the molecular circuitry underlying the immune cell lineage in mice. That milestone resulted in the creation of the most comprehensive collection of gene expression profiles in the immune cell lineage in any model organism of human disease. There is now a requisite to examine this resource using bioinformatics integration with other molecular information, with the aim of gaining deeper insights into the underlying processes that characterize this immune cell lineage. We present here a bioinformatics approach to study differential protein interaction mechanisms across the entire immune cell lineage, achieved using affinity propagation applied to a protein interaction network similarity matrix. We demonstrate that the integration of protein interaction networks with the most comprehensive database of gene expression profiles of the immune cells can be used to generate hypotheses into the underlying mechanisms governing the differentiation and the differential functional activity across the immune cell lineage. This approach may not only serve as a hypothesis engine to derive understanding of differentiation and mechanisms across the immune cell lineage, but also help identify possible immune lineage specific and common lineage mechanism in the cells protein networks.

  3. Cell lineage analysis of the mammalian female germline.

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    Yitzhak Reizel

    Full Text Available Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote. We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.

  4. Cell lineage analysis of the mammalian female germline.

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    Reizel, Yitzhak; Itzkovitz, Shalev; Adar, Rivka; Elbaz, Judith; Jinich, Adrian; Chapal-Ilani, Noa; Maruvka, Yosef E; Nevo, Nava; Marx, Zipora; Horovitz, Inna; Wasserstrom, Adam; Mayo, Avi; Shur, Irena; Benayahu, Dafna; Skorecki, Karl; Segal, Eran; Dekel, Nava; Shapiro, Ehud

    2012-01-01

    Fundamental aspects of embryonic and post-natal development, including maintenance of the mammalian female germline, are largely unknown. Here we employ a retrospective, phylogenetic-based method for reconstructing cell lineage trees utilizing somatic mutations accumulated in microsatellites, to study female germline dynamics in mice. Reconstructed cell lineage trees can be used to estimate lineage relationships between different cell types, as well as cell depth (number of cell divisions since the zygote). We show that, in the reconstructed mouse cell lineage trees, oocytes form clusters that are separate from hematopoietic and mesenchymal stem cells, both in young and old mice, indicating that these populations belong to distinct lineages. Furthermore, while cumulus cells sampled from different ovarian follicles are distinctly clustered on the reconstructed trees, oocytes from the left and right ovaries are not, suggesting a mixing of their progenitor pools. We also observed an increase in oocyte depth with mouse age, which can be explained either by depth-guided selection of oocytes for ovulation or by post-natal renewal. Overall, our study sheds light on substantial novel aspects of female germline preservation and development.

  5. Long-term live cell imaging and automated 4D analysis of drosophila neuroblast lineages.

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    Catarina C F Homem

    Full Text Available The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.

  6. Single-cell analysis of mixed-lineage states leading to a binary cell fate choice.

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    Olsson, Andre; Venkatasubramanian, Meenakshi; Chaudhri, Viren K; Aronow, Bruce J; Salomonis, Nathan; Singh, Harinder; Grimes, H Leighton

    2016-09-29

    Delineating hierarchical cellular states, including rare intermediates and the networks of regulatory genes that orchestrate cell-type specification, are continuing challenges for developmental biology. Single-cell RNA sequencing is greatly accelerating such research, given its power to provide comprehensive descriptions of genomic states and their presumptive regulators. Haematopoietic multipotential progenitor cells, as well as bipotential intermediates, manifest mixed-lineage patterns of gene expression at a single-cell level. Such mixed-lineage states may reflect the molecular priming of different developmental potentials by co-expressed alternative-lineage determinants, namely transcription factors. Although a bistable gene regulatory network has been proposed to regulate the specification of either neutrophils or macrophages, the nature of the transition states manifested in vivo, and the underlying dynamics of the cell-fate determinants, have remained elusive. Here we use single-cell RNA sequencing coupled with a new analytic tool, iterative clustering and guide-gene selection, and clonogenic assays to delineate hierarchical genomic and regulatory states that culminate in neutrophil or macrophage specification in mice. We show that this analysis captured prevalent mixed-lineage intermediates that manifested concurrent expression of haematopoietic stem cell/progenitor and myeloid progenitor cell genes. It also revealed rare metastable intermediates that had collapsed the haematopoietic stem cell/progenitor gene expression programme, instead expressing low levels of the myeloid determinants, Irf8 and Gfi1 (refs 9, 10, 11, 12, 13). Genetic perturbations and chromatin immunoprecipitation followed by sequencing revealed Irf8 and Gfi1 as key components of counteracting myeloid-gene-regulatory networks. Combined loss of these two determinants 'trapped' the metastable intermediate. We propose that mixed-lineage states are obligatory during cell-fate specification

  7. Single cell lineage analysis of mouse embryonic stem cells at the exit from pluripotency

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    Jamie Trott

    2013-08-01

    Understanding how interactions between extracellular signalling pathways and transcription factor networks influence cellular decision making will be crucial for understanding mammalian embryogenesis and for generating specialised cell types in vitro. To this end, pluripotent mouse Embryonic Stem (mES cells have proven to be a useful model system. However, understanding how transcription factors and signalling pathways affect decisions made by individual cells is confounded by the fact that measurements are generally made on groups of cells, whilst individual mES cells differentiate at different rates and towards different lineages, even in conditions that favour a particular lineage. Here we have used single-cell measurements of transcription factor expression and Wnt/β-catenin signalling activity to investigate their effects on lineage commitment decisions made by individual cells. We find that pluripotent mES cells exhibit differing degrees of heterogeneity in their expression of important regulators from pluripotency, depending on the signalling environment to which they are exposed. As mES cells differentiate, downregulation of Nanog and Oct4 primes cells for neural commitment, whilst loss of Sox2 expression primes cells for primitive streak commitment. Furthermore, we find that Wnt signalling acts through Nanog to direct cells towards a primitive streak fate, but that transcriptionally active β-catenin is associated with both neural and primitive streak commitment. These observations confirm and extend previous suggestions that pluripotency genes influence lineage commitment and demonstrate how their dynamic expression affects the direction of lineage commitment, whilst illustrating two ways in which the Wnt signalling pathway acts on this network during cell fate assignment.

  8. Transcriptional analysis of early lineage commitment in human embryonic stem cells

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    Wormald Sam

    2007-03-01

    Full Text Available Abstract Background The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells. Results We used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation. Conclusion These findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo.

  9. Transcriptional analysis of early lineage commitment in human embryonic stem cells

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    Laslett, Andrew L; Grimmond, Sean; Gardiner, Brooke; Stamp, Lincon; Lin, Adelia; Hawes, Susan M; Wormald, Sam; Nikolic-Paterson, David; Haylock, David; Pera, Martin F

    2007-01-01

    Background The mechanisms responsible for the maintenance of pluripotency in human embryonic stem cells, and those that drive their commitment into particular differentiation lineages, are poorly understood. In fact, even our knowledge of the phenotype of hESC is limited, because the immunological and molecular criteria presently used to define this phenotype describe the properties of a heterogeneous population of cells. Results We used a novel approach combining immunological and transcriptional analysis (immunotranscriptional profiling) to compare gene expression in hESC populations at very early stages of differentiation. Immunotranscriptional profiling enabled us to identify novel markers of stem cells and their differentiated progeny, as well as novel potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripotent state are downregulated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum during the early stages of stem cell differentiation. Conclusion These findings, that show that maintenance of pluripotency and lineage commitment are dynamic, interactive processes in hESC cultures, have important practical implications for propagation and directed differentiation of these cells, and for the interpretation of mechanistic studies of hESC renewal and commitment. Since embryonic stem cells at defined stages of commitment can be isolated in large numbers by immunological means, they provide a powerful model for studying molecular genetics of stem cell commitment in the embryo. PMID:17335568

  10. Single-Cell Transcriptomic Analysis Defines Heterogeneity and Transcriptional Dynamics in the Adult Neural Stem Cell Lineage

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    Ben W. Dulken

    2017-01-01

    Full Text Available Neural stem cells (NSCs in the adult mammalian brain serve as a reservoir for the generation of new neurons, oligodendrocytes, and astrocytes. Here, we use single-cell RNA sequencing to characterize adult NSC populations and examine the molecular identities and heterogeneity of in vivo NSC populations. We find that cells in the NSC lineage exist on a continuum through the processes of activation and differentiation. Interestingly, rare intermediate states with distinct molecular profiles can be identified and experimentally validated, and our analysis identifies putative surface markers and key intracellular regulators for these subpopulations of NSCs. Finally, using the power of single-cell profiling, we conduct a meta-analysis to compare in vivo NSCs and in vitro cultures, distinct fluorescence-activated cell sorting strategies, and different neurogenic niches. These data provide a resource for the field and contribute to an integrative understanding of the adult NSC lineage.

  11. SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells.

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    Stylianidou, Stella; Brennan, Connor; Nissen, Silas B; Kuwada, Nathan J; Wiggins, Paul A

    2016-11-01

    Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame-to-frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB-based image processing package well-suited to quantitative analysis of high-throughput live-cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine-learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame-to-frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell-cycle dynamics in bacteria as well as cell-contact mediated phenomena. This package has a range of built-in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution.

  12. Amniotic fluid stem cells morph into a cardiovascular lineage: analysis of a chemically induced cardiac and vascular commitment.

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    Maioli, Margherita; Contini, Giovanni; Santaniello, Sara; Bandiera, Pasquale; Pigliaru, Gianfranco; Sanna, Raimonda; Rinaldi, Salvatore; Delitala, Alessandro P; Montella, Andrea; Bagella, Luigi; Ventura, Carlo

    2013-01-01

    Mouse embryonic stem cells were previously observed along with mesenchymal stem cells from different sources, after being treated with a mixed ester of hyaluronan with butyric and retinoic acids, to show a significant increase in the yield of cardiogenic and vascular differentiated elements. The aim of the present study was to determine if stem cells derived from primitive fetal cells present in human amniotic fluid (hAFSCs) and cultured in the presence of a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids show a higher yield of differentiation toward the cardiovascular phenotype as compared with untreated cells. During the differentiation process elicited by exposure to HA + BU + RA, genes controlling pluripotency and plasticity of stem cells, such as Sox2, Nanog, and Oct4, were significantly downregulated at the transcriptional level. At this point, a significant increase in expression of genes controlling the appearance of cardiogenic and vascular lineages in HA + BU + RA-treated cells was observed. The protein expression levels typical of cardiac and vascular phenotypes, evaluated by Western blotting, immunofluorescence, and flow cytometry, were higher in hAFSCs cultured in the presence of HA + BU + RA, as compared with untreated control cells. Appearance of the cardiac phenotype was further inferred by ultrastructural analysis using transmission and scanning electron microscopy. These results demonstrate that a mixture of HA + BU + RA significantly increased the yield of elements committed toward cardiac and vascular phenotypes, confirming what we have previously observed in other cellular types.

  13. New Functional Signatures for Understanding Melanoma Biology from Tumor Cell Lineage-Specific Analysis

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    Florian Rambow

    2015-10-01

    Full Text Available Molecular signatures specific to particular tumor types are required to design treatments for resistant tumors. However, it remains unclear whether tumors and corresponding cell lines used for drug development share such signatures. We developed similarity core analysis (SCA, a universal and unsupervised computational framework for extracting core molecular features common to tumors and cell lines. We applied SCA to mRNA/miRNA expression data from various sources, comparing melanoma cell lines and metastases. The signature obtained was associated with phenotypic characteristics in vitro, and the core genes CAPN3 and TRIM63 were implicated in melanoma cell migration/invasion. About 90% of the melanoma signature genes belong to an intrinsic network of transcription factors governing neural development (TFAP2A, DLX2, ALX1, MITF, PAX3, SOX10, LEF1, and GAS7 and miRNAs (211-5p, 221-3p, and 10a-5p. The SCA signature effectively discriminated between two subpopulations of melanoma patients differing in overall survival, and classified MEKi/BRAFi-resistant and -sensitive melanoma cell lines.

  14. Single-Cell Network Analysis Identifies DDIT3 as a Nodal Lineage Regulator in Hematopoiesis

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    Cristina Pina

    2015-06-01

    Full Text Available We explore cell heterogeneity during spontaneous and transcription-factor-driven commitment for network inference in hematopoiesis. Since individual genes display discrete OFF states or a distribution of ON levels, we compute and combine pairwise gene associations from binary and continuous components of gene expression in single cells. Ddit3 emerges as a regulatory node with positive linkage to erythroid regulators and negative association with myeloid determinants. Ddit3 loss impairs erythroid colony output from multipotent cells, while forcing Ddit3 in granulo-monocytic progenitors (GMPs enhances self-renewal and impedes differentiation. Network analysis of Ddit3-transduced GMPs reveals uncoupling of myeloid networks and strengthening of erythroid linkages. RNA sequencing suggests that Ddit3 acts through development or stabilization of a precursor upstream of GMPs with inherent Meg-E potential. The enrichment of Gata2 target genes in Ddit3-dependent transcriptional responses suggests that Ddit3 functions in an erythroid transcriptional network nucleated by Gata2.

  15. Lineage Analysis in Pulmonary Arterial Hypertension

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    2012-06-01

    at Stanford) express membrane-targeted tandem dimer Tomato (mT) fluorescent protein in all cells prior to Cre-mediated excision, and membrane...Tie-2 Cre x mT/mG excises dTomato ( red ) and switches on GFP expression in endothelial cells. A. CD31 immunostaining (cyan). B. VE-Cadherin...the smooth muscle genetic lineage marking of some but not all, vascular lining cells (see red unrecombined cells adjacent to green cells in Figure 5A

  16. Cytomegalovirus immune evasion of myeloid lineage cells.

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    Brinkmann, Melanie M; Dağ, Franziska; Hengel, Hartmut; Messerle, Martin; Kalinke, Ulrich; Čičin-Šain, Luka

    2015-06-01

    Cytomegalovirus (CMV) evades the immune system in many different ways, allowing the virus to grow and its progeny to spread in the face of an adverse environment. Mounting evidence about the antiviral role of myeloid immune cells has prompted the research of CMV immune evasion mechanisms targeting these cells. Several cells of the myeloid lineage, such as monocytes, dendritic cells and macrophages, play a role in viral control, but are also permissive for CMV and are naturally infected by it. Therefore, CMV evasion of myeloid cells involves mechanisms that qualitatively differ from the evasion of non-CMV-permissive immune cells of the lymphoid lineage. The evasion of myeloid cells includes effects in cis, where the virus modulates the immune signaling pathways within the infected myeloid cell, and those in trans, where the virus affects somatic cells targeted by cytokines released from myeloid cells. This review presents an overview of CMV strategies to modulate and evade the antiviral activity of myeloid cells in cis and in trans.

  17. Mesenchymal progenitor cells for the osteogenic lineage.

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    Ono, Noriaki; Kronenberg, Henry M

    2015-09-01

    Mesenchymal progenitors of the osteogenic lineage provide the flexibility for bone to grow, maintain its function and homeostasis. Traditionally, colony-forming-unit fibroblasts (CFU-Fs) have been regarded as surrogates for mesenchymal progenitors; however, this definition cannot address the function of these progenitors in their native setting. Transgenic murine models including lineage-tracing technologies based on the cre-lox system have proven to be useful in delineating mesenchymal progenitors in their native environment. Although heterogeneity of cell populations of interest marked by a promoter-based approach complicates overall interpretation, an emerging complexity of mesenchymal progenitors has been revealed. Current literatures suggest two distinct types of bone progenitor cells; growth-associated mesenchymal progenitors contribute to explosive growth of bone in early life, whereas bone marrow mesenchymal progenitors contribute to the much slower remodeling process and response to injury that occurs mainly in adulthood. More detailed relationships of these progenitors need to be studied through further experimentation.

  18. Evolution of two prototypic T cell lineages.

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    Das, Sabyasachi; Li, Jianxu; Hirano, Masayuki; Sutoh, Yoichi; Herrin, Brantley R; Cooper, Max D

    2015-07-01

    Jawless vertebrates, which occupy a unique position in chordate phylogeny, employ leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLR) for antigen recognition. During the assembly of the VLR genes (VLRA, VLRB and VLRC), donor LRR-encoding sequences are copied in a step-wise manner into the incomplete germ-line genes. The assembled VLR genes are differentially expressed by discrete lymphocyte lineages: VLRA- and VLRC-producing cells are T-cell like, whereas VLRB-producing cells are B-cell like. VLRA(+) and VLRC(+) lymphocytes resemble the two principal T-cell lineages of jawed vertebrates that express the αβ or γδ T-cell receptors (TCR). Reminiscent of the interspersed nature of the TCRα/TCRδ locus in jawed vertebrates, the close proximity of the VLRA and VLRC loci facilitates sharing of donor LRR sequences during VLRA and VLRC assembly. Here we discuss the insight these findings provide into vertebrate T- and B-cell evolution, and the alternative types of anticipatory receptors they use for adaptive immunity.

  19. Trophoblast lineage cells derived from human induced pluripotent stem cells

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    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  20. Single-cell sequencing analysis characterizes common and cell-lineage-specific mutations in a muscle-invasive bladder cancer

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    Li, Yingrui; Xu, Xun; Song, Luting

    2012-01-01

    Background Cancers arise through an evolutionary process in which cell populations are subjected to selection; however, to date, the process of bladder cancer, which is one of the most common cancers in the world, remains unknown at a single-cell level. Results We carried out single-cell exome se...

  1. Embryonic origins of hull cells in the flatworm Macrostomum lignano through cell lineage analysis : developmental and phylogenetic implications

    NARCIS (Netherlands)

    Willems, Maxime; Egger, Bernhard; Wolff, Carsten; Mouton, Stijn; Houthoofd, Wouter; Fonderie, Pamela; Couvreur, Marjolein; Artois, Tom; Borgonie, Gaetan

    2009-01-01

    The development of macrostomid flatworms is of interest for evolutionary developmental biology research because these taxa combine characteristics of the canonical spiral cleavage pattern with significant deviations from this pattern. One such deviation is the formation of hull cells, which surround

  2. Intestinal lineage commitment of embryonic stem cells.

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    Cao, Li; Gibson, Jason D; Miyamoto, Shingo; Sail, Vibhavari; Verma, Rajeev; Rosenberg, Daniel W; Nelson, Craig E; Giardina, Charles

    2011-01-01

    Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.

  3. Lineage mapper: A versatile cell and particle tracker

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    Chalfoun, Joe; Majurski, Michael; Dima, Alden; Halter, Michael; Bhadriraju, Kiran; Brady, Mary

    2016-11-01

    The ability to accurately track cells and particles from images is critical to many biomedical problems. To address this, we developed Lineage Mapper, an open-source tracker for time-lapse images of biological cells, colonies, and particles. Lineage Mapper tracks objects independently of the segmentation method, detects mitosis in confluence, separates cell clumps mistakenly segmented as a single cell, provides accuracy and scalability even on terabyte-sized datasets, and creates division and/or fusion lineages. Lineage Mapper has been tested and validated on multiple biological and simulated problems. The software is available in ImageJ and Matlab at isg.nist.gov.

  4. Quantifying Selective Pressures Driving Bacterial Evolution Using Lineage Analysis

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    Lambert, Guillaume; Kussell, Edo

    2015-01-01

    Organisms use a variety of strategies to adapt to their environments and maximize long-term growth potential, but quantitative characterization of the benefits conferred by the use of such strategies, as well as their impact on the whole population's rate of growth, remains challenging. Here, we use a path-integral framework that describes how selection acts on lineages—i.e., the life histories of individuals and their ancestors—to demonstrate that lineage-based measurements can be used to quantify the selective pressures acting on a population. We apply this analysis to Escherichia coli bacteria exposed to cyclical treatments of carbenicillin, an antibiotic that interferes with cell-wall synthesis and affects cells in an age-dependent manner. While the extensive characterization of the life history of thousands of cells is necessary to accurately extract the age-dependent selective pressures caused by carbenicillin, the same measurement can be recapitulated using lineage-based statistics of a single surviving cell. Population-wide evolutionary pressures can be extracted from the properties of the surviving lineages within a population, providing an alternative and efficient procedure to quantify the evolutionary forces acting on a population. Importantly, this approach is not limited to age-dependent selection, and the framework can be generalized to detect signatures of other trait-specific selection using lineage-based measurements. Our results establish a powerful way to study the evolutionary dynamics of life under selection and may be broadly useful in elucidating selective pressures driving the emergence of antibiotic resistance and the evolution of survival strategies in biological systems.

  5. A continuum of cell states spans pluripotency and lineage commitment in human embryonic stem cells.

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    Shelley R Hough

    Full Text Available BACKGROUND: Commitment in embryonic stem cells is often depicted as a binary choice between alternate cell states, pluripotency and specification to a particular germ layer or extraembryonic lineage. However, close examination of human ES cell cultures has revealed significant heterogeneity in the stem cell compartment. METHODOLOGY/PRINCIPAL FINDINGS: We isolated subpopulations of embryonic stem cells using surface markers, then examined their expression of pluripotency genes and lineage specific transcription factors at the single cell level, and tested their ability to regenerate colonies of stem cells. Transcript analysis of single embryonic stem cells showed that there is a gradient and a hierarchy of expression of pluripotency genes in the population. Even cells at the top of the hierarchy generally express only a subset of the stem cell genes studied. Many cells co-express pluripotency and lineage specific genes. Cells along the continuum show a progressively decreasing likelihood of self renewal as their expression of stem cell surface markers and pluripotency genes wanes. Most cells that are positive for stem cell surface markers express Oct-4, but only those towards the top of the hierarchy express the nodal receptor TDGF-1 and the growth factor GDF3. SIGNIFICANCE: These findings on gene expression in single embryonic stem cells are in concert with recent studies of early mammalian development, which reveal molecular heterogeneity and a stochasticity of gene expression in blastomeres. Our work indicates that only a small fraction of the population resides at the top of the hierarchy, that lineage priming (co-expression of stem cell and lineage specific genes characterizes pluripotent stem cell populations, and that extrinsic signaling pathways are upstream of transcription factor networks that control pluripotency.

  6. Foetal stem cell derivation & characterization for osteogenic lineage

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    A Mangala Gowri

    2013-01-01

    Full Text Available Background & objectives: Mesencymal stem cells (MSCs derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications. Methods: Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR analysis and confirmed by sequencing using genetic analyzer. Results: Ovine foetal samples were processed to obtain mononuclear (MNC cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45 - /CD14 - was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. Interpretation & conclusions: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established

  7. Polycomb enables primitive endoderm lineage priming in embryonic stem cells

    DEFF Research Database (Denmark)

    Illingworth, Robert S; Hölzenspies, Jurriaan J; Roske, Fabian V

    2016-01-01

    Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver...... polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision....

  8. Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells

    Science.gov (United States)

    Aiba, Kazuhiro; Nedorezov, Timur; Piao, Yulan; Nishiyama, Akira; Matoba, Ryo; Sharova, Lioudmila V.; Sharov, Alexei A.; Yamanaka, Shinya; Niwa, Hitoshi; Ko, Minoru S. H.

    2009-01-01

    Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. PMID:19112179

  9. Analysis of Breast Cell-Lineage Response Differences to Taxol Using a Novel Co-Culture System

    Science.gov (United States)

    2005-06-01

    and personal observations). I HMS 50-d OH U Figure 2. Growth curves for parent, noS and WM elt 1IN4 IME nrmal HME adH S celsrains. 3 Homogeneous cell...Business Media, LLC 2006 56 Abstract Cancer is one of the most common diseases af- 1 Introduction 79 57 icting humans. The use of biomarkers speci c for

  10. A novel lineage transcription factor based analysis reveals differences in T helper cell subpopulation development in infected and intrauterine growth restricted (IUGR) piglets.

    Science.gov (United States)

    Ebner, F; Rausch, S; Scharek-Tedin, L; Pieper, R; Burwinkel, M; Zentek, J; Hartmann, S

    2014-10-01

    Research in mouse and human clearly identified subsets of T helper (Th) cells based on nuclear expression of specific lineage transcription factors. In swine, however, transcription factor based detection of functional subpopulations of porcine Th cells by flow cytometry is so far limited to regulatory T cells via Foxp3. T-bet and GATA-3 are the transcription factors that regulate commitment to Th1 or Th2 cells, respectively. In this study we prove GATA-3 and T-bet expression in porcine CD4(+) cells polarized in vitro. Importantly, GATA-3 and T-bet expressing cells were detectable in pigs infected with pathogens associated with Th2 and Th1 immune responses. Increased frequencies of GATA-3 positive CD4(+) cells are found in vivo in pigs experimentally infected with the nematode Trichuris suis, whereas porcine reproductive and respiratory syndrome virus (PRRSV) infection elicited T-bet positive CD4(+) T cells. Analysing the immune status of pre-weaning piglets with intrauterine growth restriction (IUGR) we found an increased expression of Foxp3, T-bet and GATA-3 in CD4(+) and CD4(+)CD8(+) double-positive T cells in systemic and intestinal compartments of IUGR piglets. Hence, we established the detection of porcine Th1 and Th2 cells via T-bet and GATA-3 and show that the porcine lineage transcription factors are differentially regulated very early in life depending on the developmental status.

  11. Lineage-specific reprogramming as a strategy for cell therapy.

    Science.gov (United States)

    Darabi, Radbod; Perlingeiro, Rita C R

    2008-06-15

    Embryonic stem (ES) cells are endowed with extensive ability for self renewal and differentiation. These features make them a promising candidate for cell therapy. However, despite the enthusiasm and hype surrounding the potential therapeutic use of human ES cells and more recently induced pluripotent stem (iPS) cells, to date few reports have documented successful therapeutic outcome with ES-derived cell populations. This is probably due to two main caveats associated with ES cells, their capacity to form teratomas and the challenge of isolating the appropriate therapeutic cell population from differentiating ES cells. We have focused our efforts on the derivation of skeletal muscle progenitors from ES cells and here we will discuss the strategy of reprogramming lineage choices by overexpression of a master regulator, which has proven successful for the generation of the skeletal myogenic lineage from mouse ES cells.

  12. Renin Lineage Cells Repopulate the Glomerular Mesangium after Injury

    OpenAIRE

    Starke, Charlotte; Betz, Hannah; Hickmann, Linda; Lachmann, Peter; Neubauer, Björn; Kopp, Jeffrey B.; Sequeira-Lopez, Maria Luisa S; Gomez, R. Ariel; Hohenstein, Bernd; Todorov, Vladimir T.; Hugo, Christian P. M.

    2014-01-01

    Mesangial cell injury has a major role in many CKDs. Because renin-positive precursor cells give rise to mesangial cells during nephrogenesis, this study tested the hypothesis that the same phenomenon contributes to glomerular regeneration after murine experimental mesangial injury. Mesangiolysis was induced by administration of an anti-mesangial cell serum in combination with LPS. In enhanced green fluorescent protein–reporter mice with constitutively labeled renin lineage cells, the size of...

  13. Differentiation into Endoderm Lineage: Pancreatic differentiation from Embryonic Stem Cells

    OpenAIRE

    2011-01-01

    The endoderm gives rise to digestive and respiratory tracts, thyroid, liver, and pancreas. Representative disease of endoderm lineages is type 1 diabetes resulting from destruction of the insulin-producing β cells. Generation of functional β cells from human embryonic stem (ES) cells in vitro can be practical, renewable cell source for replacement cell therapy for type 1 diabetes. It has been achieved by progressive instructive differentiation through each of the developmental stages. In this...

  14. Stem cell lineage specification: you become what you eat.

    Science.gov (United States)

    Folmes, Clifford D L; Terzic, Andre

    2014-09-02

    Nutrient availability and intermediate metabolism are increasingly recognized to govern stem cell behavior. Oburoglu et al. (2014) now demonstrate that glutamine- and glucose-dependent nucleotide synthesis segregate erythroid versus myeloid differentiation during hematopoietic stem cell specification, implicating a metabolism-centric regulation of lineage choices.

  15. Cell fate determination in the Caenorhabditis elegans epidermal lineages

    NARCIS (Netherlands)

    Soete, G.A.J.

    2007-01-01

    The starting point for this work was to use the hypodermal seam of C. elegans as a model system to study cell fate determination. Even though the seam is a relatively simple developmental system, the mechanisms that control cell fate determination in the seam lineages are connected in a highly compl

  16. Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq.

    Science.gov (United States)

    Treutlein, Barbara; Brownfield, Doug G; Wu, Angela R; Neff, Norma F; Mantalas, Gary L; Espinoza, F Hernan; Desai, Tushar J; Krasnow, Mark A; Quake, Stephen R

    2014-05-15

    The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

  17. Independent Stem Cell Lineages Regulate Adipose Organogenesis and Adipose Homeostasis

    Directory of Open Access Journals (Sweden)

    Yuwei Jiang

    2014-11-01

    Full Text Available Adipose tissues have striking plasticity, highlighted by childhood and adult obesity. Using adipose lineage analyses, smooth muscle actin (SMA-mural cell-fate mapping, and conditional PPARγ deletion to block adipocyte differentiation, we find two phases of adipocyte generation that emanate from two independent adipose progenitor compartments: developmental and adult. These two compartments are sequentially required for organ formation and maintenance. Although both developmental and adult progenitors are specified during the developmental period and express PPARγ, they have distinct microanatomical, functional, morphogenetic, and molecular profiles. Furthermore, the two compartments derive from different lineages; whereas adult adipose progenitors fate-map from an SMA+ mural lineage, developmental progenitors do not. Remarkably, the adult progenitor compartment appears to be specified earlier than the developmental cells and then enters the already developmentally formed adipose depots. Thus, two distinct cell compartments control adipose organ development and organ homeostasis, which may provide a discrete therapeutic target for childhood and adult obesity.

  18. Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila.

    Science.gov (United States)

    Biteau, Benoît; Jasper, Heinrich

    2014-06-26

    In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. Here, we identify a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. We find that EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, we show that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. Our work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. Our results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage.

  19. Slit/Robo Signaling Regulates Cell Fate Decisions in the Intestinal Stem Cell Lineage of Drosophila

    Directory of Open Access Journals (Sweden)

    Benoît Biteau

    2014-06-01

    Full Text Available In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. Here, we identify a signal from enteroendocrine cells (EEs that controls lineage specification in the Drosophila intestine. We find that EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, we show that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. Our work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. Our results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage.

  20. Developmental origin and lineage plasticity of endogenous cardiac stem cells.

    Science.gov (United States)

    Santini, Maria Paola; Forte, Elvira; Harvey, Richard P; Kovacic, Jason C

    2016-04-15

    Over the past two decades, several populations of cardiac stem cells have been described in the adult mammalian heart. For the most part, however, their lineage origins and in vivo functions remain largely unexplored. This Review summarizes what is known about different populations of embryonic and adult cardiac stem cells, including KIT(+), PDGFRα(+), ISL1(+)and SCA1(+)cells, side population cells, cardiospheres and epicardial cells. We discuss their developmental origins and defining characteristics, and consider their possible contribution to heart organogenesis and regeneration. We also summarize the origin and plasticity of cardiac fibroblasts and circulating endothelial progenitor cells, and consider what role these cells have in contributing to cardiac repair.

  1. Gene pair signatures in cell type transcriptomes reveal lineage control

    Science.gov (United States)

    Heinäniemi, Merja; Nykter, Matti; Kramer, Roger; Wienecke-Baldacchino, Anke; Sinkkonen, Lasse; Zhou, Joseph Xu; Kreisberg, Richard; Kauffman, Stuart A.; Huang, Sui; Shmulevich, Ilya

    2013-01-01

    The distinct cell types of multicellular organisms arise due to constraints imposed by gene regulatory networks on the collective change of gene expression across the genome, creating self-stabilizing expression states, or attractors. We compiled a resource of curated human expression data comprising 166 cell types and 2,602 transcription regulating genes and developed a data driven method built around the concept of expression reversal defined at the level of gene pairs, such as those participating in toggle switch circuits. This approach allows us to organize the cell types into their ontogenetic lineage-relationships and to reflect regulatory relationships among genes that explain their ability to function as determinants of cell fate. We show that this method identifies genes belonging to regulatory circuits that control neuronal fate, pluripotency and blood cell differentiation, thus offering a novel large-scale perspective on lineage specification. PMID:23603899

  2. Renin Lineage Cells Repopulate the Glomerular Mesangium after Injury

    Science.gov (United States)

    Starke, Charlotte; Betz, Hannah; Hickmann, Linda; Lachmann, Peter; Neubauer, Björn; Kopp, Jeffrey B.; Sequeira-Lopez, Maria Luisa S.; Gomez, R. Ariel; Hohenstein, Bernd; Hugo, Christian P.M.

    2015-01-01

    Mesangial cell injury has a major role in many CKDs. Because renin-positive precursor cells give rise to mesangial cells during nephrogenesis, this study tested the hypothesis that the same phenomenon contributes to glomerular regeneration after murine experimental mesangial injury. Mesangiolysis was induced by administration of an anti-mesangial cell serum in combination with LPS. In enhanced green fluorescent protein–reporter mice with constitutively labeled renin lineage cells, the size of the enhanced green fluorescent protein–positive area in the glomerular tufts increased after mesangial injury. Furthermore, we generated a novel Tet-on inducible triple-transgenic LacZ reporter line that allowed selective labeling of renin cells along renal afferent arterioles of adult mice. Although no intraglomerular LacZ expression was detected in healthy mice, about two-thirds of the glomerular tufts became LacZ positive during the regenerative phase after severe mesangial injury. Intraglomerular renin descendant LacZ-expressing cells colocalized with mesangial cell markers α8-integrin and PDGF receptor-β but not with endothelial, podocyte, or parietal epithelial cell markers. In contrast with LacZ-positive cells in the afferent arterioles, LacZ-positive cells in the glomerular tuft did not express renin. These data demonstrate that extraglomerular renin lineage cells represent a major source of repopulating cells for reconstitution of the intraglomerular mesangium after injury. PMID:24904091

  3. Bridging the gap between postembryonic cell lineages and identified embryonic neuroblasts in the ventral nerve cord of Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Oliver Birkholz

    2015-03-01

    Full Text Available The clarification of complete cell lineages, which are produced by specific stem cells, is fundamental for understanding mechanisms, controlling the generation of cell diversity and patterning in an emerging tissue. In the developing Central Nervous System (CNS of Drosophila, neural stem cells (neuroblasts exhibit two periods of proliferation: During embryogenesis they produce primary lineages, which form the larval CNS. After a phase of mitotic quiescence, a subpopulation of them resumes proliferation in the larva to give rise to secondary lineages that build up the CNS of the adult fly. Within the ventral nerve cord (VNC detailed descriptions exist for both primary and secondary lineages. However, while primary lineages have been linked to identified neuroblasts, the assignment of secondary lineages has so far been hampered by technical limitations. Therefore, primary and secondary neural lineages co-existed as isolated model systems. Here we provide the missing link between the two systems for all lineages in the thoracic and abdominal neuromeres. Using the Flybow technique, embryonic neuroblasts were identified by their characteristic and unique lineages in the living embryo and their further development was traced into the late larval stage. This comprehensive analysis provides the first complete view of which embryonic neuroblasts are postembryonically reactivated along the anterior/posterior-axis of the VNC, and reveals the relationship between projection patterns of primary and secondary sublineages.

  4. Cell lineage analysis of the mandibular segment of the amphipod Orchestia cavimana reveals that the crustacean paragnaths are sternal outgrowths and not limbs

    Directory of Open Access Journals (Sweden)

    Scholtz Gerhard

    2006-12-01

    Full Text Available Abstract The question of arthropod head segmentation has become one of the central issues in Evolutionary Developmental Biology. The number of theories pertaining to head segments progressively enlarges, old concepts have been revitalized, and nearly every conceivable composition of the arthropod head has at some point received discussion. One contentious issue involves a characteristic mouthpart in crustaceans – the lower lips or the so-called paragnaths. The paragnaths build the posterior border of the mouth region antagonistic to the upper lip – the labrum. We show here the development of the appendage-like structures in the mandibular region of the amphipod crustacean Orchestia cavimana at a high level of cellular resolution. The embryos are examined during development of the mouthparts using in vivo labeling. An invariant cell division pattern of the mandibular segment was detected by 4D-microscopy and a preliminary model for pattern of the first cleavages in the mandibular region created. With this indispensable precondition single ectodermal cells of the grid-like pattern were labeled with DiI – a lipophilic fluorescent dye – to trace cell lineages and determine the clonal composition of the developing mouthparts, especially the mandibular segment. From our data it is evident that the paragnaths are sternal outgrowths of the mandible segment. The assumption of the limb nature of paragnaths and the presence of an additional head segment between the mandibular and the second antennal segments are clearly refuted by our data. Our results show the power of cell lineage and clonal analyses for inferences on the nature, origin and thus homology of morphological structures. With this kind of investigation morphological and gene expression data can be complemented. We discuss notable similarities of paragnath anlagen to those of the hypopharynx complex in myriapods and hexapods. The fact that both structures grow out as two lateral buds

  5. Cell lineages, growth and repair of the mouse heart.

    Science.gov (United States)

    Lescroart, Fabienne; Meilhac, Sigolène M

    2012-01-01

    The formation of the heart involves diversification of lineages which differentiate into distinct cardiac cell types or contribute to different regions such as the four cardiac chambers. The heart is the first organ to form in the embryo. However, in parallel with the growth of the organism, before or after birth, the heart has to adapt its size to maintain pumping efficiency. The adult heart has only a mild regeneration potential; thus, strategies to repair the heart after injury are based on the mobilisation of resident cardiac stem cells or the transplantation of external sources of stem cells. We discuss current knowledge on these aspects and raise questions for future research.

  6. Optical imaging for stem cell differentiation to neuronal lineage.

    Science.gov (United States)

    Hwang, Do Won; Lee, Dong Soo

    2012-03-01

    In regenerative medicine, the prospect of stem cell therapy holds great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell- or tissue-specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating into a neuronal lineage. The detection limit of weak promoters or reporter genes can be greatly enhanced by adopting a yeast GAL4 amplification system or an engineering-enhanced luciferase reporter gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  7. Lineage Analysis in Pulmonary Arterial Hypertension

    Science.gov (United States)

    2013-06-01

    endothelial cells 12, 13. At the level of small pulmonary arteries, the occlusion by neointimal formation significantly exceeds muscularization of the...pyrrole. A, B) Normal muscular pulmonary artery (PA) adjacent to bronchiole (Br) A) hematoxylin and eosin stain (H&E), B) elastin-van Gieson stain (EVG...Iyer NV, Huso DL, Sun X, McWilliams R, Beaty T, Sham JS, Wiener CM, Sylvester JT, Semenza GL. Impaired physiological responses to chronic hypoxia in

  8. Polycomb enables primitive endoderm lineage priming in embryonic stem cells

    Science.gov (United States)

    Illingworth, Robert S; Hölzenspies, Jurriaan J; Roske, Fabian V; Bickmore, Wendy A; Brickman, Joshua M

    2016-01-01

    Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver of these coordinated changes, known as lineage priming, in a process that exploits novel polycomb activities. We find that intragenic levels of the polycomb mark H3K27me3 anti-correlate with changes in transcription, irrespective of the gene’s developmental trajectory or identity as a polycomb target. In contrast, promoter proximal H3K27me3 is markedly higher for PrEn priming genes. Consequently, depletion of this modification stimulates the degree to which ESCs are primed towards PrEn when challenged to differentiate, but has little effect on gene expression in self-renewing ESC culture. These observations link polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision. DOI: http://dx.doi.org/10.7554/eLife.14926.001 PMID:27723457

  9. Thymopentin enhances the generation of T-cell lineage derived from human embryonic stem cells in vitro.

    Science.gov (United States)

    Zhu, Ming-Xia; Wan, Wen-Li; Li, Hai-Shen; Wang, Jing; Chen, Gui-An; Ke, Xiao-Yan

    2015-02-15

    Thymopentin is a group of biologically active peptide secreted mainly by the epithelial cells of thymic cortex and medulla. Whether it promotes T cells production from human embryonic stem cells(hESCs) in vitro remains an elusive issue. In the present study, we develop a novel strategy that enhances T-cell lineage differentiation of hESCs in collagen matrix culture by sequential cytokine cocktails treatment combined with thymopentin stimulation. We observed that approximately 30.75% cells expressed CD34 on day 14 of the cultures and expressed the surface markers of erythroid, lymphoid and myeloid lineages. The results of colony assays and gene expressions by RT-PCR analysis also demonstrated that hematopoietic progenitor cells (HPCs) derived from hESCs were capable of multi-lineage differentiation. Further study revealed that culturing with thymopentin treatment, the CD34(+)CD45RA(+)CD7(+) cells sorted from HPCs expressed T-cell-related genes, IKAROS, DNTT, TCRγ and TCRβ, and T-cell surface markers, CD3, cytoplasmic CD3, CD5, CD27, TCRγδ, CD4 and CD8. The differentiated cells produced the cytokines including IFN-γ, IL-2 and TNF-α in response to stimulation, providing the evidence for T-cell function of these cells. In conclusion, thymopentin enhances T-cell lineage differentiation from hESCs in vitro by mimicking thymus peptide environment in vivo.

  10. Transcriptional repressor Tbx3 is required for the hormone-sensing cell lineage in mammary epithelium.

    Directory of Open Access Journals (Sweden)

    Kamini Kunasegaran

    Full Text Available The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production. Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells.

  11. Early cell lineage specification in a marsupial: a case for diverse mechanisms among mammals.

    Science.gov (United States)

    Frankenberg, Stephen; Shaw, Geoff; Freyer, Claudia; Pask, Andrew J; Renfree, Marilyn B

    2013-03-01

    Early cell lineage specification in eutherian mammals results in the formation of a pluripotent inner cell mass (ICM) and trophoblast. By contrast, marsupials have no ICM. Here, we present the first molecular analysis of mechanisms of early cell lineage specification in a marsupial, the tammar wallaby. There was no overt differential localisation of key lineage-specific transcription factors in cleavage and early unilaminar blastocyst stages. Pluriblast cells (equivalent to the ICM) became distinguishable from trophoblast cells by differential expression of POU5F1 and, to a greater extent, POU2, a paralogue of POU5F1. Unlike in the mouse, pluriblast-trophoblast differentiation coincided with a global nuclear-to-cytoplasmic transition of CDX2 localisation. Also unlike in the mouse, Hippo pathway factors YAP and WWTR1 showed mutually distinct localisation patterns that suggest non-redundant roles. NANOG and GATA6 were conserved as markers of epiblast and hypoblast, respectively, but some differences to the mouse were found in their mode of differentiation. Our results suggest that there is considerable evolutionary plasticity in the mechanisms regulating early lineage specification in mammals.

  12. Differentiation of monkey embryonic stem cells into neural lineages.

    Science.gov (United States)

    Kuo, Hung-Chih; Pau, K-Y Francis; Yeoman, Richard R; Mitalipov, Shoukhrat M; Okano, Hideyuki; Wolf, Don P

    2003-05-01

    Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.

  13. Optical Imaging for Stem Cell Differentiation to Neuronal Lineage

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Do Won; Lee, Dong Soo [Seoul National Univ., Seoul (Korea, Republic of)

    2012-03-15

    In regenerative medicine, the prospect of stem cell therapy hold great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell or tissue specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating a yeast GAL4 amplification system or an engineering-enhanced luciferase reported gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  14. Evolutionary modification of cell lineage in the direct-developing sea urchin Heliocidaris erythrogramma.

    Science.gov (United States)

    Wray, G A; Raff, R A

    1989-04-01

    The sea urchin Heliocidaris erythrogramma undergoes direct development, bypassing the usual echinoid pluteus larva. We present an analysis of cell lineage in H. erythrogramma as part of a definition of the mechanistic basis for this evolutionary change in developmental mode. Microinjection of fluoresceinated tracer dye and surface marking with vital dye are used to follow larval fates of 2-cell, 8-cell, and 16-cell blastomeres, and to examine axial specification. The animal-vegetal axis and adult dorsoventral axis are basically unmodified in H. erythrogramma. Animal cell fates are very similar to those of typically developing species; however, vegetal cell fates in H. erythrogramma are substantially altered. Radial differences exist among vegetal blastomere fates in the 8-cell embryo: dorsal vegetal blastomeres contribute proportionately more descendants to ectodermal and fewer to mesodermal fates, while ventral vegetal blastomeres have a complementary bias in fates. In addition, vegetal cell fates are more variable than in typical developers. There are no cells in H. erythrogramma with fates comparable to those of the micromeres and macromeres of typically developing echinoids. Instead, all vegetal cells in the 16-cell embryo can contribute progeny to ectoderm and gut. Alterations have thus arisen in cleavage patterns and timing of cell lineage partitioning during the evolution of direct development in H. erythrogramma.

  15. Histone deacetylase 1 and 3 regulate the mesodermal lineage commitment of mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Weiying Lv

    Full Text Available The important role of histone acetylation alteration has become increasingly recognized in mesodermal lineage differentiation and development. However, the contribution of individual histone deacetylases (HDACs to mesoderm specification remains poorly understood. In this report, we found that trichostatin A (TSA, an inhibitor of histone deacetylase (HDACi, could induce early differentiation of embryonic stem cells (ESCs and promote mesodermal lineage differentiation. Further analysis showed that the expression levels of HDAC1 and 3 are decreased gradually during ESCs differentiation. Ectopic expression of HDAC1 or 3 significantly inhibited differentiation into the mesodermal lineage. By contrast, loss of either HDAC1 or 3 enhanced the mesodermal differentiation of ESCs. Additionally, we demonstrated that the activity of HDAC1 and 3 is indeed required for the regulation of mesoderm gene expression. Furthermore, HDAC1 and 3 were found to interact physically with the T-box transcription factor T/Bry, which is critical for mesodermal lineage commitment. These findings indicate a key mechanism for the specific role of HDAC1 and 3 in mammalian mesoderm specification.

  16. Plectus - a stepping stone in embryonic cell lineage evolution of nematodes

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    Schulze Jens

    2012-07-01

    Full Text Available Abstract Background Recent studies have challenged the widespread view that the pattern of embryogenesis found in Caenorhabditis elegans (clade 9 is characteristic of nematodes in general. To understand this still largely unexplored landscape of developmental events, we set out to examine more distantly related nematodes in detail for temporospatial differences in pattern formation and cell specification. Members of the genus Plectus (clade 6 seem to be suitable candidates to show variety, with certain idiosyncratic features during early development and the convenient availability of cultivatable species. Methods The study was conducted using 4-D lineage analysis, 3-D modeling of developing embryos and laser-induced ablation of individual blastomeres. Results Detailed cell lineage studies of several Plectus species reveal that pattern formation and cell fate assignment differ markedly from C. elegans. Descendants of the first somatic founder cell S1 (AB - but not the progeny of other founder cells - demonstrate extremely variable spatial arrangements illustrating that here distinct early cell-cell interactions between invariant partners, as found in C. elegans, cannot take place. Different from C. elegans, in Plectus alternative positional variations among early S1 blastomeres resulting in a ‘situs inversus’ pattern, nevertheless give rise to adults with normal left-right asymmetries. In addition, laser ablations of early blastomeres uncover inductions between variable cell partners. Conclusions Our results suggest that embryonic cell specification in Plectus is not correlated with cell lineage but with position. With this peculiarity, Plectus appears to occupy an intermediate position between basal nematodes displaying a variable early development and the C. elegans-like invariant pattern. We suggest that indeterminate pattern formation associated with late, position-dependent fate assignment represents a plesiomorphic character among

  17. Micropatterned matrix directs differentiation of human mesenchymal stem cells towards myocardial lineage.

    Science.gov (United States)

    Tay, Chor Yong; Yu, Haiyang; Pal, Mintu; Leong, Wen Shing; Tan, Nguan Soon; Ng, Kee Woei; Leong, David Tai; Tan, Lay Poh

    2010-04-15

    Stem cell response can be influenced by a multitude of chemical, topological and mechanical physiochemical cues. While extensive studies have been focused on the use of soluble factors to direct stem cell differentiation, there are growing evidences illustrating the potential to modulate stem cell differentiation via precise engineering of cell shape. Fibronectin were printed on poly(lactic-co-glycolic acid) (PLGA) thin film forming spatially defined geometries as a means to control the morphology of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs that were cultured on unpatterned substrata adhered and flattened extensively (approximately 10,000 microm(2)) while cells grown on 20 microm micropatterend wide adhesive strips were highly elongated with much smaller area coverage of approximately 2000 microm(2). Gene expression analysis revealed up-regulation of several hallmark markers associated to neurogenesis and myogenesis for cells that were highly elongated while osteogenic markers were specifically down-regulated or remained at its nominal level. Even though there is clearly upregulated levels of both neuronal and myogenic lineages but at the functionally relevant level of protein expression, the myogenic lineage is dominant within the time scale studied as determined by the exclusive expression of cardiac myosin heavy chain for the micropatterned cells. Enforced cell shape distortion resulting in large scale rearrangement of cytoskeletal network and altered nucleus shape has been proposed as a physical impetus by which mechanical deformation is translated into biochemical response. These results demonstrated for the first time that cellular shape modulation in the absence of any induction factors may be a viable strategy to coax lineage-specific differentiation of stem cells.

  18. CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages

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    Joannah R. Fergusson

    2014-11-01

    Full Text Available The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR expression and cell lineage.

  19. Lineage Specification of Ovarian Theca Cells Requires Multi-Cellular Interactions via Oocyte and Granulosa Cells

    Science.gov (United States)

    Liu, Chang; Peng, Jia; Matzuk, Martin M.; Yao, Humphrey H-C

    2015-01-01

    Organogenesis of the ovary is a highly orchestrated process involving multiple lineage determinations of ovarian surface epithelium, granulosa cells, and theca cells. While the sources of ovarian surface epithelium and granulosa cells are known, the origin(s) of theca progenitor cells have not been definitively identified. Here we show that theca cells derive from two sources: Wt1+ cells indigenous to the ovary and Gli1+ mesenchymal cells migrated from the mesonephros. These progenitors acquire theca lineage marker Gli1 in response to paracrine signals Desert hedgehog (Dhh) and Indian hedgehog (Ihh) from granulosa cells. Ovaries lacking Dhh/Ihh exhibit theca layer loss, blunted steroid production, arrested folliculogenesis, and failure to form corpora lutea. Production of Dhh/Ihh in granulosa cells requires Growth differentiation factor 9 (GDF9) from the oocyte. Our studies provide the first genetic evidence for the origins of theca cells and reveal a multicellular interaction critical for the formation of a functional theca. PMID:25917826

  20. Active enhancers are delineated de novo during hematopoiesis, with limited lineage fidelity among specified primary blood cells.

    Science.gov (United States)

    Luyten, Annouck; Zang, Chongzhi; Liu, X Shirley; Shivdasani, Ramesh A

    2014-08-15

    Tissues may adopt diverse strategies to establish specific transcriptional programs in daughter lineages. In intestinal crypts, enhancers for genes expressed in both major cell types appear broadly permissive in stem and specified progenitor cells. In blood, another self-renewing tissue, it is unclear when chromatin becomes permissive for transcription of genes expressed in distinct terminal lineages. Using chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) to profile activating histone marks, we studied enhancer dynamics in primary mouse blood stem, progenitor, and specified cells. Stem and multipotent progenitor cells show scant H3K4me2 marking at enhancers bound by specific transcription factors in their committed progeny. Rather, enhancers are modulated dynamically and serially, with substantial loss and gain of H3K4me2, at each cellular transition. Quantitative analysis of these dynamics accurately modeled hematopoiesis according to Waddington's notion of epigenotypes. Delineation of enhancers in terminal blood lineages coincides with cell specification, and enhancers active in single lineages show well-positioned H3K4me2- and H3K27ac-marked nucleosomes and DNaseI hypersensitivity in other cell types, revealing limited lineage fidelity. These findings demonstrate that enhancer chronology in blood cells differs markedly from that in intestinal crypts. Chromatin dynamics in hematopoiesis provide a useful foundation to consider classical observations such as cellular reprogramming and multilineage locus priming.

  1. Expression and function of mixed lineage kinases in dendritic cells.

    Science.gov (United States)

    Handley, Matthew E; Rasaiyaah, Jane; Barnett, James; Thakker, Manish; Pollara, Gabriele; Katz, David R; Chain, Benjamin M

    2007-08-01

    Dendritic cells (DCs) sense the presence of conserved microbial structures in their local microenvironment via specific pattern recognition receptors (PRRs). This leads to a programme of changes, which include migration and activation, and enables them to induce adaptive T cell immunity. Mitogen-activated protein kinases (MAPKs) are implicated in this response, but the pathways leading from PRR ligation to MAPK activation, and hence DC activation, are not fully understood. Recent studies in the nervous system have suggested that the mixed lineage kinase (MLK) family of MAPK kinase kinase proteins may be involved as an intermediary step between PRRs and MAPKs. Therefore, in this study, we have used a well-established DC model to explore the role of MLKs in these cells. Messenger RNA for MLKs 2, 3, 4 and DLK and protein for MLKs 2, 3 and DLK are found in DC. DC activation in response to model PRR ligands, such as LPS or poly (I:C), is accompanied by phosphorylation of MLK3. In contrast, another known PRR ligand, zymosan, induces little MLK3 phosphorylation. Inhibition of MLK activity using a pharmacological inhibitor, CEP11004, blocks p38 and Jun N-terminal kinase (JNK) MAPK activation in response to LPS and poly (I:C), but not zymosan. The inhibition is associated with a block in DC activation as measured by cell-surface marker expression and cytokine secretion. Thus, MLKs are expressed in DC, and are implicated in DC activation, and the involvement of MLKs appears to be selective, depending on the nature of the DC stimulus.

  2. Pan-Genome Analysis of Brazilian Lineage A Amoebal Mimiviruses

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    Felipe L. Assis

    2015-06-01

    Full Text Available Since the recent discovery of Samba virus, the first representative of the family Mimiviridae from Brazil, prospecting for mimiviruses has been conducted in different environmental conditions in Brazil. Recently, we isolated using Acanthamoeba sp. three new mimiviruses, all of lineage A of amoebal mimiviruses: Kroon virus from urban lake water; Amazonia virus from the Brazilian Amazon river; and Oyster virus from farmed oysters. The aims of this work were to sequence and analyze the genome of these new Brazilian mimiviruses (mimi-BR and update the analysis of the Samba virus genome. The genomes of Samba virus, Amazonia virus and Oyster virus were 97%–99% similar, whereas Kroon virus had a low similarity (90%–91% with other mimi-BR. A total of 3877 proteins encoded by mimi-BR were grouped into 974 orthologous clusters. In addition, we identified three new ORFans in the Kroon virus genome. Additional work is needed to expand our knowledge of the diversity of mimiviruses from Brazil, including if and why among amoebal mimiviruses those of lineage A predominate in the Brazilian environment.

  3. Heterochronic misexpression of Ascl1 in the Atoh7 retinal cell lineage blocks cell cycle exit.

    Science.gov (United States)

    Hufnagel, Robert B; Riesenberg, Amy N; Quinn, Malgorzata; Brzezinski, Joseph A; Glaser, Tom; Brown, Nadean L

    2013-05-01

    Retinal neurons and glia arise from a common progenitor pool in a temporal order, with retinal ganglion cells (RGCs) appearing first, and Müller glia last. The transcription factors Atoh7/Math5 and Ascl1/Mash1 represent divergent bHLH clades, and exhibit distinct spatial and temporal retinal expression patterns, with little overlap during early development. Here, we tested the ability of Ascl1 to change the fate of cells in the Atoh7 lineage when misexpressed from the Atoh7 locus, using an Ascl1-IRES-DsRed2 knock-in allele. In Atoh7(Ascl1KI/+) and Atoh7(Ascl1KI/Ascl1KI) embryos, ectopic Ascl1 delayed cell cycle exit and differentiation, even in cells coexpressing Atoh7. The heterozygous retinas recovered, and eventually produced a normal complement of RGCs, while homozygous substitution of Ascl1 for Atoh7 did not promote postnatal retinal fates precociously, nor rescue Atoh7 mutant phenotypes. However, our analyses revealed two unexpected findings. First, ectopic Ascl1 disrupted cell cycle progression within the marked Atoh7 lineage, but also nonautonomously in other retinal cells. Second, the size of the Atoh7 retinal lineage was unaffected, supporting the idea of a compensatory shift of the non-proliferative cohort to maintain lineage size. Overall, we conclude that Ascl1 acts dominantly to block cell cycle exit, but is incapable of redirecting the fates of early RPCs.

  4. Proteome analysis of early lineage specification in bovine embryos.

    Science.gov (United States)

    Demant, Myriam; Deutsch, Daniela R; Fröhlich, Thomas; Wolf, Eckhard; Arnold, Georg J

    2015-02-01

    During mammalian embryo development, the zygote undergoes embryonic cleavage in the oviduct and reaches the uterus at the morula stage, when compaction and early lineage specification take place. To increase knowledge about the associated changes of the embryonic protein repertoire, we performed a comprehensive proteomic analysis of in vitro produced bovine morulae and blastocysts (six biological replicates), using an iTRAQ-based approach. A total of 560 proteins were identified of which 502 were quantified. The abundance of 140 proteins was significantly different between morulae and blastocysts, among them nucleophosmin (NPM1), eukaryotic translation initiation factor 5A-1 (EIF5A), receptor of activated protein kinase C 1 (GNB2L1/RACK1), and annexin A6 (ANXA6) with increased, and glutathione S-transferase mu 3 (GSTM3), peroxiredoxin 2 (PRDX2), and aldo-keto reductase family 1 member B1 (AKR1B1) with decreased abundance in blastocysts. Seventy-three percent of abundance altered proteins increased, reflecting an increase of translation activity in this period. This is further supported by an increase in the abundance of proteins involved in the translation machinery and the synthesis of ATP. Additionally, a complementary 2D saturation DIGE analysis led to the detection of protein isoforms, e.g. of GSTM3 and PRDX2, relevant for this period of mammalian development, and exemplarily verified the results of the iTRAQ approach. In summary, our systematic differential proteome analysis of bovine morulae and blastocysts revealed new molecular correlates of early lineage specification and differentiation events during bovine embryogenesis.

  5. Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages.

    Science.gov (United States)

    Hilty, Markus; Wüthrich, Daniel; Salter, Susannah J; Engel, Hansjürg; Campbell, Samuel; Sá-Leão, Raquel; de Lencastre, Hermínia; Hermans, Peter; Sadowy, Ewa; Turner, Paul; Chewapreecha, Claire; Diggle, Mathew; Pluschke, Gerd; McGee, Lesley; Köseoğlu Eser, Özgen; Low, Donald E; Smith-Vaughan, Heidi; Endimiani, Andrea; Küffer, Marianne; Dupasquier, Mélanie; Beaudoing, Emmanuel; Weber, Johann; Bruggmann, Rémy; Hanage, William P; Parkhill, Julian; Hathaway, Lucy J; Mühlemann, Kathrin; Bentley, Stephen D

    2014-12-04

    The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated S. pneumoniae (non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if non-Ec-Sp evolve sporadically, if they have high antibiotic nonsusceptiblity rates and a unique, specific gene content. Here, whole-genome sequencing of 131 non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multilocus sequences types (STs) ST344 (n = 39) and ST448 (n = 40). All ST344 and nine ST448 isolates had high nonsusceptiblity rates to β-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P = 0.005). In contrast, sporadic non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of PCV, non-Ec-Sp may become more prevalent.

  6. Mesenchymal stem cells, neural lineage potential, heparan sulfate proteoglycans and the matrix.

    Science.gov (United States)

    Okolicsanyi, Rachel K; Griffiths, Lyn R; Haupt, Larisa M

    2014-04-01

    Along with the tri-lineage of bone, cartilage and fat, human mesenchymal stem cells (hMSCs) retain neural lineage potential. Multiple factors have been described that influence lineage fate of hMSCs including the extracellular microenvironment or niche. The niche includes the extracellular matrix (ECM) providing structural composition, as well as other associated proteins and growth factors, which collectively influence hMSC stemness and lineage specification. As such, lineage specific differentiation of MSCs is mediated through interactions including cell-cell and cell-matrix, as well as through specific signalling pathways triggering downstream events. Proteoglycans (PGs) are ubiquitous within this microenvironment and can be localised to the cell surface or embedded within the ECM. In addition, the heparan sulfate (HS) and chondroitin sulfate (CS) families of PGs interact directly with a number of growth factors, signalling pathways and ECM components including FGFs, Wnts and fibronectin. With evidence supporting a role for HSPGs and CSPGs in the specification of hMSCs down the osteogenic, chondrogenic and adipogenic lineages, along with the localisation of PGs in development and regeneration, it is conceivable that these important proteins may also play a role in the differentiation of hMSCs toward the neuronal lineage. Here we summarise the current literature and highlight the potential for HSPG directed neural lineage fate specification in hMSCs, which may provide a new model for brain damage repair.

  7. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing.

    Directory of Open Access Journals (Sweden)

    Liwen Chen

    Full Text Available Bone marrow derived mesenchymal stem cells (BM-MSCs have been shown to enhance wound healing; however, the mechanisms involved are barely understood. In this study, we examined paracrine factors released by BM-MSCs and their effects on the cells participating in wound healing compared to those released by dermal fibroblasts. Analyses of BM-MSCs with Real-Time PCR and of BM-MSC-conditioned medium by antibody-based protein array and ELISA indicated that BM-MSCs secreted distinctively different cytokines and chemokines, such as greater amounts of VEGF-alpha, IGF-1, EGF, keratinocyte growth factor, angiopoietin-1, stromal derived factor-1, macrophage inflammatory protein-1alpha and beta and erythropoietin, compared to dermal fibroblasts. These molecules are known to be important in normal wound healing. BM-MSC-conditioned medium significantly enhanced migration of macrophages, keratinocytes and endothelial cells and proliferation of keratinocytes and endothelial cells compared to fibroblast-conditioned medium. Moreover, in a mouse model of excisional wound healing, where concentrated BM-MSC-conditioned medium was applied, accelerated wound healing occurred compared to administration of pre-conditioned or fibroblast-conditioned medium. Analysis of cell suspensions derived from the wound by FACS showed that wounds treated with BM-MSC-conditioned medium had increased proportions of CD4/80-positive macrophages and Flk-1-, CD34- or c-kit-positive endothelial (progenitor cells compared to wounds treated with pre-conditioned medium or fibroblast-conditioned medium. Consistent with the above findings, immunohistochemical analysis of wound sections showed that wounds treated with BM-MSC-conditioned medium had increased abundance of macrophages. Our results suggest that factors released by BM-MSCs recruit macrophages and endothelial lineage cells into the wound thus enhancing wound healing.

  8. Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion

    DEFF Research Database (Denmark)

    Nygren, J.M.; Liuba, K.; Breitbach, M.;

    2008-01-01

    and Purkinje neurons. However, through lineage fate-mapping we demonstrate that such in vivo fusion of lymphoid and myeloid blood cells does not occur to an appreciable extent in steady-state adult tissues or during normal development. Rather, fusion of blood cells with different non-haematopoietic cell types...... is induced by organ-specific injuries or whole-body irradiation, which has been used in previous studies to condition recipients of bone marrow transplants. Our findings demonstrate that blood cells of the lymphoid and myeloid lineages contribute to various non-haematopoietic tissues by forming rare fusion......Recent studies have suggested that regeneration of non-haematopoietic cell lineages can occur through heterotypic cell fusion with haematopoietic cells of the myeloid lineage. Here we show that lymphocytes also form heterotypic-fusion hybrids with cardiomyocytes, skeletal muscle, hepatocytes...

  9. Origins of adult pigmentation: diversity in pigment stem cell lineages and implications for pattern evolution.

    Science.gov (United States)

    Parichy, David M; Spiewak, Jessica E

    2015-01-01

    Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage, and mate choice and have played important roles in speciation. Here, we review studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post-embryonic, peripheral nerve-associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow-orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns.

  10. Differentiation of Equine Mesenchymal Stromal Cells into Cells of Neural Lineage: Potential for Clinical Applications

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    Claudia Cruz Villagrán

    2014-01-01

    Full Text Available Mesenchymal stromal cells (MSCs are able to differentiate into extramesodermal lineages, including neurons. Positive outcomes were obtained after transplantation of neurally induced MSCs in laboratory animals after nerve injury, but this is unknown in horses. Our objectives were to test the ability of equine MSCs to differentiate into cells of neural lineage in vitro, to assess differences in morphology and lineage-specific protein expression, and to investigate if horse age and cell passage number affected the ability to achieve differentiation. Bone marrow-derived MSCs were obtained from young and adult horses. Following demonstration of stemness, MSCs were neurally induced and microscopically assessed at different time points. Results showed that commercially available nitrogen-coated tissue culture plates supported proliferation and differentiation. Morphological changes were immediate and all the cells displayed a neural crest-like cell phenotype. Expression of neural progenitor proteins, was assessed via western blot or immunofluorescence. In our study, MSCs generated from young and middle-aged horses did not show differences in their ability to undergo differentiation. The effect of cell passage number, however, is inconsistent and further experiments are needed. Ongoing work is aimed at transdifferentiating these cells into Schwann cells for transplantation into a peripheral nerve injury model in horses.

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

    Directory of Open Access Journals (Sweden)

    Hourinaz Behesti

    2013-01-01

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

  12. High glucose suppresses embryonic stem cell differentiation into neural lineage cells.

    Science.gov (United States)

    Yang, Penghua; Shen, Wei-bin; Reece, E Albert; Chen, Xi; Yang, Peixin

    2016-04-01

    Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25 mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5 mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14 cells. Under low glucose conditions, the GR-E14 cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14 cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14 cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14 cell line is a useful in vitro model for assessing the adverse effect of high glucose on the development of the central nervous system.

  13. High glucose suppresses embryonic stem cell differentiation into neural lineage cells

    Science.gov (United States)

    Yang, Penghua; Shen, Wei-bin; Reece, E. Albert; Chen, Xi; Yang, Peixin

    2017-01-01

    Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25 mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5 mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14 cells. Under low glucose conditions, the GR-E14 cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14 cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14 cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14 cell line is a useful in vitro model for assessing the adverse effect of high glucose on the development of the central nervous system. PMID:26940741

  14. Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU.1.

    Science.gov (United States)

    Champhekar, Ameya; Damle, Sagar S; Freedman, George; Carotta, Sebastian; Nutt, Stephen L; Rothenberg, Ellen V

    2015-04-15

    The ETS family transcription factor PU.1 is essential for the development of several blood lineages, including T cells, but its function in intrathymic T-cell precursors has been poorly defined. In the thymus, high PU.1 expression persists through multiple cell divisions in early stages but then falls sharply during T-cell lineage commitment. PU.1 silencing is critical for T-cell commitment, but it has remained unknown how PU.1 activities could contribute positively to T-cell development. Here we employed conditional knockout and modified antagonist PU.1 constructs to perturb PU.1 function stage-specifically in early T cells. We show that PU.1 is needed for full proliferation, restricting access to some non-T fates, and controlling the timing of T-cell developmental progression such that removal or antagonism of endogenous PU.1 allows precocious access to T-cell differentiation. Dominant-negative effects reveal that this repression by PU.1 is mediated indirectly. Genome-wide transcriptome analysis identifies novel targets of PU.1 positive and negative regulation affecting progenitor cell signaling and cell biology and indicating distinct regulatory effects on different subsets of progenitor cell transcription factors. Thus, in addition to supporting early T-cell proliferation, PU.1 regulates the timing of activation of the core T-lineage developmental program.

  15. Related pituitary cell lineages develop into interdigitated 3D cell networks.

    Science.gov (United States)

    Budry, Lionel; Lafont, Chrystel; El Yandouzi, Taoufik; Chauvet, Norbert; Conéjero, Geneviève; Drouin, Jacques; Mollard, Patrice

    2011-07-26

    The pituitary gland has long been considered to be a random patchwork of hormone-producing cells. By using pituitary-scale tridimensional imaging for two of the least abundant cell lineages, the corticotropes and gonadotropes, we have now uncovered highly organized and interdigitated cell networks that reflect homotypic and heterotypic interactions between cells. Although newly differentiated corticotrope cells appear on the ventral surface of the gland, they rapidly form homotypic strands of cells that extend from the lateral tips of the anterior pituitary along its ventral surface and into the medial gland. As the corticotrope network is established away from the microvasculature, cell morphology changes from rounded, to polygonal, and finally to cells with long cytoplasmic processes or cytonemes that connect corticotropes to the perivascular space. Gonadotropes differentiate later and are positioned in close proximity to corticotropes and capillaries. Blockade of corticotrope terminal differentiation produced by knockout of the gene encoding the transcription factor Tpit results in smaller gonadotropes within an expanded cell network, particularly in the lateral gland. Thus, pituitary-scale tridimensional imaging reveals highly structured cell networks of unique topology for each pituitary lineage. The sequential development of interdigitated cell networks during organogenesis indicate that extensive cell:cell interactions lead to a highly ordered cell positioning rather than random patchwork.

  16. Proliferative cell types in embryonic lineages of the central complex of the grasshopper Schistocerca gregaria.

    Science.gov (United States)

    Boyan, George; Williams, Leslie; Legl, Andrea; Herbert, Zsofia

    2010-08-01

    The central complex of the grasshopper Schistocerca gregaria develops to completion during embryogenesis. A major cellular contribution to the central complex is from the w, x, y, z lineages of the pars intercerebralis, each of which comprises over 100 cells, making them by far the largest in the embryonic protocerebrum. Our focus has been to find a cellular mechanism that allows such a large number of cell progeny to be generated within a restricted period of time. Immunohistochemical visualization of the chromosomes of mitotically active cells has revealed an almost identical linear array of proliferative cells present simultaneously in each w, x, y, z lineage at 50% of embryogenesis. This array is maintained relatively unchanged until almost 70% of embryogenesis, after which mitotic activity declines and then ceases. The array is absent from smaller lineages of the protocerebrum not associated with the central complex. The proliferative cells are located apically to the zone of ganglion mother cells and amongst the progeny of the neuroblast. Comparisons of cell morphology, immunoreactivity (horseradish peroxidase, repo, Prospero), location in lineages and spindle orientation have allowed us to distinguish the proliferative cells in an array from neuroblasts, ganglion mother cells, neuronal progeny and glia. Our data are consistent with the proliferative cells being secondary (amplifying) progenitors and originating from a specific subtype of ganglion mother cell. We propose a model of the way that neuroblasts, ganglion mother cells and secondary progenitors together produce the large cell numbers found in central complex lineages.

  17. Changes in glycosphingolipid composition during differentiation of human embryonic stem cells to ectodermal or endodermal lineages.

    Science.gov (United States)

    Liang, Yuh-Jin; Yang, Bei-Chia; Chen, Jin-Mei; Lin, Yu-Hsing; Huang, Chia-Lin; Cheng, Yuan-Yuan; Hsu, Chi-Yen; Khoo, Kay-Hooi; Shen, Chia-Ning; Yu, John

    2011-12-01

    Glycosphingolipids (GSLs) are ubiquitous components of cell membranes that can act as mediators of cell adhesion and signal transduction and can possibly be used as cell type-specific markers. Our previous study indicated that there was a striking switch in the core structures of GSLs during differentiation of human embryonic stem cells (hESCs) into embryoid body (EB), suggesting a close association of GSLs with cell differentiation. In this study, to further clarify if alterations in GSL patterns are correlated with lineage-specific differentiation of hESCs, we analyzed changes in GSLs as hESCs were differentiated into neural progenitors or endodermal cells by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and tandem mass spectrometry (MS/MS) analyses. During hESC differentiation into neural progenitor cells, we found that the core structures of GSLs switched from globo- and lacto- to mostly ganglio-series dominated by GD3. On the other hand, when hESCs were differentiated into endodermal cells, patterns of GSLs totally differed from those observed in EB outgrowth and neural progenitors. The most prominent GSL identified by the MALDI-MS and MS/MS analysis was Gb(4) Ceramide, with no appreciable amount of stage-specific embryonic antigens 3 or 4, or GD3, in endodermal cells. These changes in GSL profiling were accompanied by alterations in the biosynthetic pathways of expressions of key glycosyltransferases. Our findings suggest that changes in GSLs are closely associated with lineage specificity and differentiation of hESCs.

  18. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

    Institute of Scientific and Technical Information of China (English)

    Li-Wei Zheng; Logan Linthicum; Pamela K DenBesten; Yan Zhang

    2013-01-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCI) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which ,vas also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  19. Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans.

    Science.gov (United States)

    Mold, Jeff E; Venkatasubrahmanyam, Shivkumar; Burt, Trevor D; Michaëlsson, Jakob; Rivera, Jose M; Galkina, Sofiya A; Weinberg, Kenneth; Stoddart, Cheryl A; McCune, Joseph M

    2010-12-17

    Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or "layering") of distinct hematopoietic stem cells (HSCs) that give rise to distinct lymphocyte lineages at different stages of development. Here we provide evidence of an analogous layered immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSCs that are present at different stages of development. We also provide evidence that the fetal T cell lineage is biased toward immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.

  20. Lineage stability and phenotypic plasticity of Foxp3⁺ regulatory T cells.

    Science.gov (United States)

    Hori, Shohei

    2014-05-01

    Regulatory T (Treg) cells expressing the transcription factor forkhead box protein 3 (Foxp3) constitute a unique T-cell lineage committed to suppressive functions. While their differentiation state is remarkably stable in the face of various perturbations from the extracellular environment, they are able to adapt to diverse and fluctuating tissue environments by changing their phenotype. The lineage stability and phenotypic plasticity of Treg cells thus ensure the robustness of self-tolerance and tissue homeostasis. Recent studies have suggested, however, that Treg cells may retain lineage plasticity, the ability to switch their cell fate to various effector T-cell types under certain circumstances such as inflammation, a notion that remains highly contentious. While accumulating evidence indicates that some Treg cells can downregulate Foxp3 expression and/or acquire effector T-helper cell-like phenotypes, results from my laboratory have shown that Treg cells retain epigenetic memory of, and thus remain committed to, Foxp3 expression and suppressive functions despite such phenotypic plasticity. It has also become evident that Foxp3 can be promiscuously and transiently expressed in activated T cells. Here, I argue that the current controversy stems partly from the lack of the lineage specificity of Foxp3 expression and also from the confusion between phenotypic plasticity and lineage plasticity, and discuss implications of our findings in Treg cell fate determination and maintenance.

  1. In vivo clonal analysis reveals lineage-restricted progenitor characteristics in mammalian kidney development, maintenance, and regeneration

    NARCIS (Netherlands)

    Rinkevich, Y.; Montoro, D.T.; Contreras-Trujillo, H.; Harari-Steinberg, O.; Newman, A.M.; Tsai, J.M.; Lim, X.; van Amerongen, R.; Bowman, A.; Januszyk, M.; Pleniceanu, O.; Nusse, R.; Longaker, M.T.; Weissman, I.L.; Dekel, B.

    2014-01-01

    The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maint

  2. Lineage relationship of prostate cancer cell types based on gene expression

    Directory of Open Access Journals (Sweden)

    Ware Carol B

    2011-05-01

    Full Text Available Abstract Background Prostate tumor heterogeneity is a major factor in disease management. Heterogeneity could be due to multiple cancer cell types with distinct gene expression. Of clinical importance is the so-called cancer stem cell type. Cell type-specific transcriptomes are used to examine lineage relationship among cancer cell types and their expression similarity to normal cell types including stem/progenitor cells. Methods Transcriptomes were determined by Affymetrix DNA array analysis for the following cell types. Putative prostate progenitor cell populations were characterized and isolated by expression of the membrane transporter ABCG2. Stem cells were represented by embryonic stem and embryonal carcinoma cells. The cancer cell types were Gleason pattern 3 (glandular histomorphology and pattern 4 (aglandular sorted from primary tumors, cultured prostate cancer cell lines originally established from metastatic lesions, xenografts LuCaP 35 (adenocarcinoma phenotype and LuCaP 49 (neuroendocrine/small cell carcinoma grown in mice. No detectable gene expression differences were detected among serial passages of the LuCaP xenografts. Results Based on transcriptomes, the different cancer cell types could be clustered into a luminal-like grouping and a non-luminal-like (also not basal-like grouping. The non-luminal-like types showed expression more similar to that of stem/progenitor cells than the luminal-like types. However, none showed expression of stem cell genes known to maintain stemness. Conclusions Non-luminal-like types are all representatives of aggressive disease, and this could be attributed to the similarity in overall gene expression to stem and progenitor cell types.

  3. Novel origins of lineage founder cells in the direct-developing sea urchin Heliocidaris erythrogramma.

    Science.gov (United States)

    Wray, G A; Raff, R A

    1990-09-01

    The lineage and fate of each blastomere in the 32-cell embryo of the direct-developing sea urchin Heliocidaris erythrogramma have been traced by microinjection of tetramethylrhodamine-dextran. The results reveal substantive evolutionary modifications of the ancestral cell lineage pattern of indirect sea urchin development. Significant among these modifications are changes in the time and order of cell lineage segregation: vegetal ectodermal founder cells consistently arise earlier than during indirect development, while internal founder cells generally segregate later and in a different sequence. Modifications have also arisen in proportions of the embryo fated to become various cell types and larval structures. Ectodermal fates, particularly vestibular ectoderm, comprise a greater proportion of the total cellular volume in H. erythrogramma. Among internal cell types, coelom consumes more and endoderm less of the remaining cellular volume than during indirect sea urchin development. Evolutionary modifications are also apparent in the positional origin of larval cell types and structures in H. erythrogramma. These include an apparent tilt in the axis of prospective cell fate relative to the animal-vegetal axis as defined by cleavage planes. Together these evolutionary changes in the cell lineage of H. erythrogramma produce an accelerated loss of dorsoventral symmetry in cell fate relative to indirect development. The extent and diversity of rearrangements in its cell lineage indicate that the non-feeding larva of H. erythrogramma is a highly modified, novel form rather than a degenerate pluteus larva. These same modifications underscore the evolutionarily flexible relationship between cell lineage, gene expression, and larval morphology in sea urchin development.

  4. A review of adipocyte lineage cells and dermal papilla cells in hair follicle regeneration

    Directory of Open Access Journals (Sweden)

    Peipei Zhang

    2014-10-01

    Full Text Available Alopecia is an exceedingly prevalent problem effecting men and women of all ages. The standard of care for alopecia involves either transplanting existing hair follicles to bald areas or attempting to stimulate existing follicles with topical and/or oral medication. Yet, these treatment options are fraught with problems of cost, side effects, and, most importantly, inadequate long-term hair coverage. Innovative cell-based therapies have focused on the dermal papilla cell as a way to grow new hair in previously bald areas. However, despite this attention, many obstacles exist, including retention of dermal papilla inducing ability and maintenance of dermal papilla productivity after several passages of culture. The use of adipocyte lineage cells, including adipose-derived stem cells, has shown promise as a cell-based solution to regulate hair regeneration and may help in maintaining or increasing dermal papilla cells inducing hair ability. In this review, we highlight recent advances in the understanding of the cellular contribution and regulation of dermal papilla cells and summarize adipocyte lineage cells in hair regeneration.

  5. Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells.

    Directory of Open Access Journals (Sweden)

    Foad J Rouhani

    2016-04-01

    Full Text Available The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50-70 de novo single nucleotide variants (SNVs between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs, their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.

  6. Cell lineage, axis formation, and the origin of germ layers in the amphipod crustacean Orchestia cavimana.

    Science.gov (United States)

    Wolff, Carsten; Scholtz, Gerhard

    2002-10-01

    Embryos of the amphipod crustacean Orchestia cavimana are examined during cleavage, gastrulation, and segmentation by using in vivo labelling. Single blastomeres of the 8- and 16-cell stages were labelled with DiI to trace cell lineages. Early cleavage follows a distinct pattern and the a/p and d/v body axes are already determined at the 4- and 8-cell stages, respectively. In these stages, the germinal rudiment and the naupliar mesoderm can be traced back to a single blastomere each. In addition, the ectoderm and the postnaupliar mesoderm are separated into right and left components. At the16-cell stage, naupliar ectoderm is divided from the postnaupliar ectoderm, and extraembryonic lineages are separated from postnaupliar mesoderm and endoderm. From our investigation, it is evident that the cleavage pattern and cell lineage of Orchestia cavimana are not of the spiral type. Furthermore, the results of the labelling show many differences to cleavage patterns and cell lineages in other crustaceans, in particular, other Malacostraca. The cleavage and cell lineage patterns of the amphipod Orchestia are certainly derived within Malacostraca, whose ancestral cleavage mode was most likely of the superficial type. On the other hand, Orchestia exhibits a stereotyped cell division pattern during formation and differentiation of the germ band that is typical for malacostracans. Hence, a derived (apomorphic) early cleavage pattern is the ontogenetic basis for an evolutionarily older cell division pattern of advanced developmental stages. O. cavimana offers the possibility to trace the lineages and the fates of cells from early developmental stages up to the formation of segmental structures, including neurogenesis at a level of resolution that is not matched by any other arthropod system.

  7. Stochastic differentiation into an osteoclast lineage from cloned macrophage-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Shin-Ichi, E-mail: shayashi@med.tottori-u.ac.jp [Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503 (Japan); Murata, Akihiko; Okuyama, Kazuki; Shimoda, Yuhki; Hikosaka, Mari [Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503 (Japan); Yasuda, Hisataka [Planning and Development, Bioindustry Division, Oriental Yeast Co., Ltd, Itabashi-Ku, Tokyo 174-8505 (Japan); Yoshino, Miya [Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Cho, Yonago, Tottori 683-8503 (Japan)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer The frequency of C7 differentiation into osteoclast was low and constant. Black-Right-Pointing-Pointer Only extended C7 cell cultures exponentially increased osteoclast+ cultures. Black-Right-Pointing-Pointer C7 cell differentiation into committed osteoclast precursors is on 'autopilot'. Black-Right-Pointing-Pointer The system may maintain the stem cell self-renewal and differentiation. -- Abstract: Differentiation into osteoclasts is induced by a macrophage colony-stimulating factor and receptor activator of nuclear-factor {kappa}B ligand. The macrophage-like cell line, C7 has the potential to differentiate into osteoclasts when it is cultured with both factors for 6 days. Although C7 is an established cell line, the frequency of differentiation into this lineage was less than 10%, and the ratio was maintained at a constant level, even after repeated cloning. In this study, to increase the differentiation of C7 cells to osteoclasts, C7 derivative treatments with several activators and/or inhibitors were performed for 3 days prior to setting osteoclast induction analysis; however, a reagent to significantly up-regulate the frequency of differentiation was not found. Only extended cultures for osteoclastogenesis exponentially increased the frequency of osteoclast precursors. It is likely that C7 cell differentiation into committed osteoclast precursors is on 'autopilot' rather than requiring specific signals to drive this process.

  8. Isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages.

    Science.gov (United States)

    Crigler, Lauren; Kazhanie, Amita; Yoon, Tae-Jin; Zakhari, Julia; Anders, Joanna; Taylor, Barbara; Virador, Victoria M

    2007-07-01

    The skin contains two known subpopulations of stem cells/epidermal progenitors: a basal keratinocyte population found in the interfollicular epithelium and cells residing in the bulge region of the hair follicle. The major role of the interfollicular basal keratinocyte population may be epidermal renewal, whereas the bulge population may only be activated and recruited to form a cutaneous epithelium in case of trauma. Using 3-dimensional cultures of murine skin under stress conditions in which only reserve epithelial cells would be expected to survive and expand, we demonstrate that a mesenchymal population resident in neonatal murine dermis has the unique potential to develop an epidermis in vitro. In monolayer culture, this dermal subpopulation has long-term survival capabilities in restricted serum and an inducible capacity to evolve into multiple cell lineages, both epithelial and mesenchymal, depending on culture conditions. When grafted subcutaneously, this dermal subpopulation gave rise to fusiform structures, reminiscent of disorganized muscle, that stained positive for smooth muscle actin and desmin; on typical epidermal grafts, abundant melanocytes appeared throughout the dermis that were not associated with hair follicles. The multipotential cells can be repeatedly isolated from neonatal murine dermis by a sequence of differential centrifugation and selective culture conditions. These results suggest that progenitors capable of epidermal differentiation exist in the mesenchymal compartment of an abundant tissue source and may have a function in mesenchymal-epithelial transition upon insult. Moreover, these cells could be available in sufficient quantities for lineage determination or tissue engineering applications.

  9. Cooperation of B cell lineages in induction of HIV-1-broadly neutralizing antibodies.

    Science.gov (United States)

    Gao, Feng; Bonsignori, Mattia; Liao, Hua-Xin; Kumar, Amit; Xia, Shi-Mao; Lu, Xiaozhi; Cai, Fangping; Hwang, Kwan-Ki; Song, Hongshuo; Zhou, Tongqing; Lynch, Rebecca M; Alam, S Munir; Moody, M Anthony; Ferrari, Guido; Berrong, Mark; Kelsoe, Garnett; Shaw, George M; Hahn, Beatrice H; Montefiori, David C; Kamanga, Gift; Cohen, Myron S; Hraber, Peter; Kwong, Peter D; Korber, Bette T; Mascola, John R; Kepler, Thomas B; Haynes, Barton F

    2014-07-31

    Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make bnAbs is a key strategy for immunogen design. Here, we study the B cell response in a bnAb-producing individual and report cooperation between two B cell lineages to drive bnAb development. We isolated a virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization-traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both helper and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.

  10. MicroRNA expression profiles in umbilical cord blood cell lineages.

    Science.gov (United States)

    Merkerova, Michaela; Vasikova, Alzbeta; Belickova, Monika; Bruchova, Hana

    2010-01-01

    MicroRNAs (miRNAs), important regulators of cellular processes, show specific expression signatures in different blood cell lineages and stages of hematopoietic stem cell (HSC) differentiation, indicating their role in the control of hematopoiesis. Because neonatal blood displays various features of immaturity, we might expect differential miRNA regulation. Herein, we determined miRNA expression profiles of umbilical cord blood (UCB) cell lineages and compared them to those of bone marrow (BM) and peripheral blood (PB) cell counterparts. Further, we determined mRNA expression profiles using whole-genome microarrays. An approach combining bioinformatic prediction of miRNA targets with mRNA expression profiling was used to search for putative targets of miRNAs with potential functions in UCB. We pointed out several differentially expressed miRNAs and associated their expression with the target transcript levels. miR-148a expression was suppressed in HSCs and its level inversely correlated with the previously verified target, DNA methyltransferase 3B, suggesting dependence of de novo DNA methylation in HSCs on miR-148a. Prolonged cell survival of UCB HSCs may be associated with low expression of miR-143 and miR-145 and up-regulation of their downstream targets (high expression of c-MYC and miR-17-92 and following repression of TGFBR2). In HSCs, we monitored significant up-regulation of eight miRNAs, which were previously verified as regulators of HOX genes. Further, miR-146b may be associated with immaturity of neonatal immune system because it is strongly up-regulated in UCB granulocytes and T lymphocytes compared to PB cell counterparts. Comparative analysis revealed 13 miRNAs significantly altered between UCB and BM CD34(+) cells. In UCB CD34(+) cells, we monitored up-regulation of miR-520h, promoting differentiation of HSCs into progenitor cells, and reduction of miR-214, whose expression might support HSC survival. In conclusion, UCB cells show specific mi

  11. Ezh2 represses the basal cell lineage during lung endoderm development.

    Science.gov (United States)

    Snitow, Melinda E; Li, Shanru; Morley, Michael P; Rathi, Komal; Lu, Min Min; Kadzik, Rachel S; Stewart, Kathleen M; Morrisey, Edward E

    2015-01-01

    The development of the lung epithelium is regulated in a stepwise fashion to generate numerous differentiated and stem cell lineages in the adult lung. How these different lineages are generated in a spatially and temporally restricted fashion remains poorly understood, although epigenetic regulation probably plays an important role. We show that the Polycomb repressive complex 2 component Ezh2 is highly expressed in early lung development but is gradually downregulated by late gestation. Deletion of Ezh2 in early lung endoderm progenitors leads to the ectopic and premature appearance of Trp63+ basal cells that extend the entire length of the airway. Loss of Ezh2 also leads to reduced secretory cell differentiation. In their place, morphologically similar cells develop that express a subset of basal cell genes, including keratin 5, but no longer express high levels of either Trp63 or of standard secretory cell markers. This suggests that Ezh2 regulates the phenotypic switch between basal cells and secretory cells. Together, these findings show that Ezh2 restricts the basal cell lineage during normal lung endoderm development to allow the proper patterning of epithelial lineages during lung formation.

  12. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Morizane, Ryuji [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Monkawa, Toshiaki, E-mail: monkawa@sc.itc.keio.ac.jp [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Itoh, Hiroshi [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  13. Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior

    Science.gov (United States)

    Cai, Li; Hayes, Nancy L.; Takahashi, Takao; Caviness, Verne S Jr; Nowakowski, Richard S.

    2002-01-01

    Mechanisms that regulate neuron production in the developing mouse neocortex were examined by using a retroviral lineage marking method to determine the sizes of the lineages remaining in the proliferating population of the ventricular zone during the period of neuron production. The distribution of clade sizes obtained experimentally in four different injection-survival paradigms (E11-E13, E11-E14, E11-E15, and E12-E15) from a total of over 500 labeled lineages was compared with that obtained from three models in which the average behavior of the proliferating population [i.e., the proportion of cells remaining in the proliferative population (P) vs. that exiting the proliferative population (Q)] was quantitatively related to lineage size distribution. In model 1, different proportions of asymmetric, symmetric terminal, and symmetric nonterminal cell divisions coexisted during the entire developmental period. In model 2, the developmental period was divided into two epochs: During the first, asymmetric and symmetric nonterminal cell divisions occurred, but, during the second, asymmetric and symmetric terminal cell divisions occurred. In model 3, the shifts in P and Q are accounted for by changes in the proportions of the two types of symmetric cell divisions without the inclusion of any asymmetric cell divisions. The results obtained from the retroviral experiments were well accounted for by model 1 but not by model 2 or 3. These findings demonstrate that: 1) asymmetric and both types of symmetric cell divisions coexist during the entire period of neurogenesis in the mouse, 2) neuron production is regulated in the proliferative population by the independent decisions of the two daughter cells to reenter S phase, and 3) neurons are produced by both asymmetric and symmetric terminal cell divisions. In addition, the findings mean that cell death and/or tangential movements of cells in the proliferative population occur at only a low rate and that there are no

  14. Lineage specific expression of Polycomb Group Proteins in human embryonic stem cells in vitro.

    Science.gov (United States)

    Pethe, Prasad; Pursani, Varsha; Bhartiya, Deepa

    2015-05-01

    Human embryonic (hES) stem cells are an excellent model to study lineage specification and differentiation into various cell types. Differentiation necessitates repression of specific genes not required for a particular lineage. Polycomb Group (PcG) proteins are key histone modifiers, whose primary function is gene repression. PcG proteins form complexes called Polycomb Repressive Complexes (PRCs), which catalyze histone modifications such as H2AK119ub1, H3K27me3, and H3K9me3. PcG proteins play a crucial role during differentiation of stem cells. The expression of PcG transcripts during differentiation of hES cells into endoderm, mesoderm, and ectoderm lineage is yet to be shown. In-house derived hES cell line KIND1 was differentiated into endoderm, mesoderm, and ectoderm lineages; followed by characterization using RT-PCR for HNF4A, CDX2, MEF2C, TBX5, SOX1, and MAP2. qRT-PCR and western blotting was performed to compare expression of PcG transcripts and proteins across all the three lineages. We observed that cells differentiated into endoderm showed upregulation of RING1B, BMI1, EZH2, and EED transcripts. Mesoderm differentiation was characterized by significant downregulation of all PcG transcripts during later stages. BMI1 and RING1B were upregulated while EZH2, SUZ12, and EED remained low during ectoderm differentiation. Western blotting also showed distinct expression of BMI1 and EZH2 during differentiation into three germ layers. Our study shows that hES cells differentiating into endoderm, mesoderm, and ectoderm lineages show distinct PcG expression profile at transcript and protein level.

  15. Genome sequencing of normal cells reveals developmental lineages and mutational processes

    NARCIS (Netherlands)

    Behjati, Sam; Huch, Meritxell; van Boxtel, Ruben; Karthaus, Wouter; Wedge, David C; Tamuri, Asif U; Martincorena, Iñigo; Petljak, Mia; Alexandrov, Ludmil B; Gundem, Gunes; Tarpey, Patrick S; Roerink, Sophie; Blokker, Joyce; Maddison, Mark; Mudie, Laura; Robinson, Ben; Nik-Zainal, Serena; Campbell, Peter; Goldman, Nick; van de Wetering, Marc; Cuppen, Edwin; Clevers, Hans; Stratton, Michael R

    2014-01-01

    The somatic mutations present in the genome of a cell accumulate over the lifetime of a multicellular organism. These mutations can provide insights into the developmental lineage tree, the number of divisions that each cell has undergone and the mutational processes that have been operative. Here w

  16. Pox neuro control of cell lineages that give rise to larval poly-innervated external sensory organs in Drosophila.

    Science.gov (United States)

    Jiang, Yanrui; Boll, Werner; Noll, Markus

    2015-01-15

    The Pox neuro (Poxn) gene of Drosophila plays a crucial role in the development of poly-innervated external sensory (p-es) organs. However, how Poxn exerts this role has remained elusive. In this study, we have analyzed the cell lineages of all larval p-es organs, namely of the kölbchen, papilla 6, and hair 3. Surprisingly, these lineages are distinct from any previously reported cell lineages of sensory organs. Unlike the well-established lineage of mono-innervated external sensory (m-es) organs and a previously proposed model of the p-es lineage, we demonstrate that all wild-type p-es lineages exhibit the following features: the secondary precursor, pIIa, gives rise to all three support cells-socket, shaft, and sheath, whereas the other secondary precursor, pIIb, is neuronal and gives rise to all neurons. We further show that in one of the p-es lineages, that of papilla 6, one cell undergoes apoptosis. By contrast in Poxn null mutants, all p-es lineages have a reduced number of cells and their pattern of cell divisions is changed to that of an m-es organ, with the exception of a lineage in a minority of mutant kölbchen that retains a second bipolar neuron. Indeed, the role of Poxn in p-es lineages is consistent with the specification of the developmental potential of secondary precursors and the regulation of cell division but not apoptosis.

  17. Murine inner cell mass-derived lineages depend on Sall4 function

    Science.gov (United States)

    Elling, Ulrich; Klasen, Christian; Eisenberger, Tobias; Anlag, Katrin; Treier, Mathias

    2006-01-01

    Sall4 is a mammalian Spalt transcription factor expressed by cells of the early embryo and germ cells, an expression pattern similar to that of both Oct4 and Sox2, which play essential roles during early murine development. We show that the activity of Sall4 is cell-autonomously required for the development of the epiblast and primitive endoderm from the inner cell mass. Furthermore, no embryonic or extraembryonic endoderm stem cell lines could be established from Sall4-deficient blastocysts. In contrast, neither the development of the trophoblast lineage nor the ability to generate trophoblast cell lines from murine blastocysts was impaired in the absence of Sall4. These data establish Sall4 as an essential transcription factor required for the early development of inner cell mass-derived cell lineages. PMID:17060609

  18. Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction

    Directory of Open Access Journals (Sweden)

    Minsuk eKwak

    2013-02-01

    Full Text Available Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

  19. Lineage extrinsic and intrinsic control of immunoregulatory cell numbers by SHIP.

    Science.gov (United States)

    Collazo, Michelle M; Paraiso, Kim H T; Park, Mi-Young; Hazen, Amy L; Kerr, William G

    2012-07-01

    We previously showed that germline or induced SHIP deficiency expands immuno-regulatory cell numbers in T lymphoid and myeloid lineages. We postulated these increases could be interrelated. Here, we show that myeloid-specific ablation of SHIP leads to the expansion of both myeloid-derived suppressor cell (MDSC) and regulatory T (Treg) cell numbers, indicating SHIP-dependent control of Treg-cell numbers by a myeloid cell type. Conversely, T-lineage specific ablation of SHIP leads to expansion of Treg-cell numbers, but not expansion of the MDSC compartment, indicating SHIP also has a lineage intrinsic role in limiting Treg-cell numbers. However, the SHIP-deficient myeloid cell that promotes MDSC and Treg-cell expansion is not an MDSC as they lack SHIP protein expression. Thus, regulation of MDSC numbers in vivo must be controlled in a cell-extrinsic fashion by another myeloid cell type. We had previously shown that G-CSF levels are profoundly increased in SHIP(-/-) mice, suggesting this myelopoietic growth factor could promote MDSC expansion in a cell-extrinsic fashion. Consistent with this hypothesis, we find that G-CSF is required for expansion of the MDSC splenic compartment in mice rendered SHIP-deficient as adults. Thus, SHIP controls MDSC numbers, in part, by limiting production of the myelopoietic growth factor G-CSF.

  20. Regulatory effects on the population dynamics and wave propagation in a cell lineage model.

    Science.gov (United States)

    Wang, Mao-Xiang; Ma, Yu-Qiang; Lai, Pik-Yin

    2016-03-21

    We consider the interplay of cell proliferation, cell differentiation (and de-differentiation), cell movement, and the effect of feedback regulations on the population and propagation dynamics of different cell types in a cell lineage model. Cells are assumed to secrete and respond to negative feedback molecules which act as a control on the cell lineage. The cell densities are described by coupled reaction-diffusion partial differential equations, and the propagating wave front solutions in one dimension are investigated analytically and by numerical solutions. In particular, wavefront propagation speeds are obtained analytically and verified by numerical solutions of the equations. The emphasis is on the effects of the feedback regulations on different stages in the cell lineage. It is found that when the progenitor cell is negatively regulated, the populations of the cell lineage are strongly down-regulated with the steady growth rate of the progenitor cell being driven to zero beyond a critical regulatory strength. An analytic expression for the critical regulation strength in terms of the model parameters is derived and verified by numerical solutions. On the other hand, if the inhibition is acting on the differentiated cells, the change in the population dynamics and wave propagation speed is small. In addition, it is found that only the propagating speed of the progenitor cells is affected by the regulation when the diffusion of the differentiated cells is large. In the presence of de-differentiation, the effect on down-regulating the progenitor population is weakened and there is no effect on the propagation speed due to regulation, suggesting that the effect of regulatory control is diminished by de-differentiation pathways.

  1. Expression and regulation of versican in neural precursor cells and their lineages

    Institute of Scientific and Technical Information of China (English)

    Wen-li GU; Sai-li FU; Yan-xia WANG; Ying LI; Xiao-fei WANG; Xiao-ming XU; Pei-hua LU

    2007-01-01

    Aim: To have a better understanding of the expression and regulation of versican isoforms in neural precursor cells (NPC) and oligodendrogliogenesis. Methods:By immunocytochemistry, RT-PCR, and real-time PCR, we examined the temporal expression of versican in NPC isolated from embryonic d 16 rats as well as in oligodendrocyte (OL) lineage cells induced to differentiate from NPC,which mimicked the oligodendrogliogenesis in vivo. Results: We found that versican was constitutively expressed in NPC and their lineage cells, including neurons, astrocytes, and OL. In addition, 2 versican isoforms, V1/V0 and V2,were found to express at low levels in NPC, but at significantly higher levels in OL lineage cells. The peak expression of versican V2 was found at the oligodendrocyte precursor cell stage. Furthermore, the treatment of 2 pro-inflammatory cytokines, TNF-α and IFN-γ, enhanced the transcription of versican V2 in NPC in a dose-dependent manner, but showed no effect on V1/V0 expression.Conclusion: Taken together, our results demonstrate that versican, particularly the inhibitory V2 isoform, is increasingly expressed in OL lineage cells induced to differentiate from NPC. An increase in versican V2 expression after cytokine stimulation implies the interplay between the injury-induced upregulation of inflammatory cytokines and chondroitin sulfate proteoglycan-mediated inhibition of axonal regeneration after central nervous system injury.

  2. Lineage development of cell fusion hybrids upon somatic reprogramming

    OpenAIRE

    2011-01-01

    Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2011 Somatic cell reprogramming has been extensively studied over the last years and opened new perspectives in the use of pluripotent cells for regenerative biomedical purposes. Spontaneous cell fusion has been suggested to be involved in regenerative processes in vivo. Strong evidences support the hypothesis that the reprogrammed hybrids resulting from the fusion between a pluripote...

  3. Selective cell targeting and lineage tracing of human induced pluripotent stem cells using recombinant avian retroviruses.

    Science.gov (United States)

    Hildebrand, Laura; Seemann, Petra; Kurtz, Andreas; Hecht, Jochen; Contzen, Jörg; Gossen, Manfred; Stachelscheid, Harald

    2015-12-01

    Human induced pluripotent stem cells (hiPSC) differentiate into multiple cell types. Selective cell targeting is often needed for analyzing gene function by overexpressing proteins in a distinct population of hiPSC-derived cell types and for monitoring cell fate in response to stimuli. However, to date, this has not been possible, as commonly used viruses enter the hiPSC via ubiquitously expressed receptors. Here, we report for the first time the application of a heterologous avian receptor, the tumor virus receptor A (TVA), to selectively transduce TVA(+) cells in a mixed cell population. Expression of the TVA surface receptor via genetic engineering renders cells susceptible for infection by avian leucosis virus (ALV). We generated hiPSC lines with this stably integrated, ectopic TVA receptor gene that expressed the receptor while retaining pluripotency. The undifferentiated hiPSC(TVA+) as well as their differentiating progeny could be infected by recombinant ALV (so-called RCAS virus) with high efficiency. Due to incomplete receptor blocking, even sequential infection of differentiating or undifferentiated TVA(+) cells was possible. In conclusion, the TVA/RCAS system provides an efficient and gentle gene transfer system for hiPSC and extends our possibilities for selective cell targeting and lineage tracing studies.

  4. Cell lineage relationship in the stomach of normal and genetically manipulated mice

    Directory of Open Access Journals (Sweden)

    S.M. Karam

    1998-02-01

    Full Text Available The oxyntic mucosa of the mouse stomach is lined with a heterogeneous population of cells that form numerous short pits continuous with long tubular glands. Tritiated thymidine radioautography has made it possible to pinpoint the origin of all cell types and to follow the differentiation/migration of different cell lineages along the pit-gland unit. The proliferating multipotent stem cells functionally anchored in the upper glandular region, the isthmus, give rise to three main lineage precursors: 1 pre-pit cells, which migrate upward to the pit while differentiating into mucus-producing pit cells; 2 pre-neck cells, which migrate downward to the glandular neck while differentiating into mucus-producing neck cells that, by approaching the glandular base, gradually change their phenotype into pepsinogen- and intrinsic factor-producing zymogenic cells; 3 pre-parietal cells, which differentiate into acid-producing parietal cells in the isthmus and then undergo bipolar migration towards the pit and the glandular base. Thus, parietal cells are the only cells that complete their differentiation in the isthmus and then migrate to be scattered throughout the pit-gland unit. To determine whether parietal cells play a role in controlling decisions about cell fate within the pit-gland unit, the gastric epithelium has been examined in transgenic mice expressing the H,K-ATPase ß-subunit-1035 to +24/simian virus 40 large T antigen fusion gene. The blockade in parietal cell differentiation in these mice produces an amplification of lineage precursors, a marked depletion of zymogenic cells and an increase in pit cell census. Ablation of parietal cells in another transgenic mouse model expressing the H,K-ATPase ß-subunit-1035 to +24/diphtheria toxin fragment A fusion gene also produces amplification of lineage precursors, and similar effects on zymogenic and pit cell census. These findings strongly suggest that parietal cells produce regulatory signals that

  5. A reaction–diffusion mechanism influences cell lineage progression as a basis for formation, regeneration, and stability of intestinal crypts

    Directory of Open Access Journals (Sweden)

    Zhang Lei

    2012-07-01

    Full Text Available Abstract Background Colon crypts, a single sheet of epithelia cells, consist of a periodic pattern of stem cells, transit-amplifying cells, and terminally differentiated cells that constantly renew and turnover. Experimental evidence suggests that Wnt signaling promotes and regulates stem cell division, differentiation, and possible cell migrations while intestinal BMP signaling inhibits stem cell self-renewal and repression in crypt formation. As more molecular details on Wnt and BMP in crypts are being discovered, little is still known about how complex interactions among Wnt, BMP, and different types of cells, and surrounding environments may lead to de novo formation of multiple crypts or how such interactions affect regeneration and stability of crypts. Results We present a mathematical model that contains Wnt and BMP, a cell lineage, and their feedback regulations to study formation, regeneration, and stability of multiple crypts. The computational explorations and linear stability analysis of the model suggest a reaction–diffusion mechanism, which exhibits a short-range activation of Wnt plus a long-range inhibition with modulation of BMP signals in a growing tissue of cell lineage, can account for spontaneous formation of multiple crypts with the spatial and temporal pattern observed in experiments. Through this mechanism, the model can recapitulate some distinctive and important experimental findings such as crypt regeneration and crypt multiplication. BMP is important in maintaining stability of crypts and loss of BMP usually leads to crypt multiplication with a fingering pattern. Conclusions The study provides a mechanism for de novo formation of multiple intestinal crypts and demonstrates a synergetic role of Wnt and BMP in regeneration and stability of intestinal crypts. The proposed model presents a robust framework for studying spatial and temporal dynamics of cell lineages in growing tissues driven by multiple signaling

  6. C. elegans BED domain transcription factor BED-3 controls lineage-specific cell proliferation during organogenesis

    OpenAIRE

    Inoue, Takao; Sternberg, Paul W.

    2010-01-01

    The control of cell division is critical to organogenesis, but how this control is achieved is not fully understood. We found that mutations in bed-3, encoding a BED Zn-finger domain transcription factor, confer a phenotype where a specific set of cell divisions during vulval organogenesis is lost. Unlike general cell cycle regulators in Caenorhabditis elegans, the function of bed-3 is restricted to specific lineages. Transcriptional reporters suggest that bed-3 is expressed in a limited numb...

  7. Conversion of embryonic stem cells into extraembryonic lineages by CRISPR-mediated activators

    OpenAIRE

    2016-01-01

    The recently emerged CRISPR/Cas9 technique has opened a new perspective on readily editing specific genes. When combined with transcription activators, it can precisely manipulate endogenous gene expression. Here, we enhanced the expression of endogenous Cdx2 and Gata6 genes by CRISPR-mediated activators, thus mouse embryonic stem cells (ESCs) were directly converted into two extraembryonic lineages, i.e., typical trophoblast stem cells (TSCs) and extraembryonic endoderm cells (XENCs), which ...

  8. Epigenetic Control of Smooth Muscle Cell Identity and Lineage Memory.

    Science.gov (United States)

    Gomez, Delphine; Swiatlowska, Pamela; Owens, Gary K

    2015-12-01

    Vascular smooth muscle cells (SMCs), like all cells, acquire a cell-specific epigenetic signature during development that includes acquisition of a unique repertoire of histone and DNA modifications. These changes are postulated to induce an open chromatin state (referred to as euchromatin) on the repertoire of genes that are expressed in differentiated SMC, including SMC-selective marker genes like Acta2 and Myh11, as well as housekeeping genes expressed by most cell types. In contrast, genes that are silenced in differentiated SMC acquire modifications associated with a closed chromatin state (ie, heterochromatin) and transcriptional silencing. Herein, we review mechanisms that regulate epigenetic control of the differentiated state of SMC. In addition, we identify some of the major limitations in the field and future challenges, including development of innovative new tools and approaches, for performing single-cell epigenetic assays and locus-selective editing of the epigenome that will allow direct studies of the functional role of specific epigenetic controls during development, injury repair, and disease, including major cardiovascular diseases, such as atherosclerosis, hypertension, and microvascular disease, associated with diabetes mellitus.

  9. MYSM1-dependent checkpoints in B cell lineage differentiation and B cell-mediated immune response.

    Science.gov (United States)

    Förster, Michael; Farrington, Kyo; Petrov, Jessica C; Belle, Jad I; Mindt, Barbara C; Witalis, Mariko; Duerr, Claudia U; Fritz, Jörg H; Nijnik, Anastasia

    2017-03-01

    MYSM1 is a chromatin-binding histone deubiquitinase. MYSM1 mutations in humans result in lymphopenia whereas loss of Mysm1 in mice causes severe hematopoietic abnormalities, including an early arrest in B cell development. However, it remains unknown whether MYSM1 is required at later checkpoints in B cell development or for B cell-mediated immune responses. We analyzed conditional mouse models Mysm1(fl/fl)Tg.mb1-cre, Mysm1(fl/fl)Tg.CD19-cre, and Mysm1(fl/fl)Tg.CD21-cre with inactivation of Mysm1 at prepro-B, pre-B, and follicular B cell stages of development. We show that loss of Mysm1 at the prepro-B cell stage in Mysm1(fl/fl)Tg.mb1-cre mice results in impaired B cell differentiation, with an ∼2-fold reduction in B cell numbers in the lymphoid organs. Mysm1(fl/fl)Tg.mb1-cre B cells also showed increased expression of activation markers and impaired survival and proliferation. In contrast, Mysm1 was largely dispensable from the pre-B cell stage onward, with Mysm1(fl/fl)Tg.CD19-cre and Mysm1(fl/fl)Tg.CD21-cre mice showing no alterations in B cell numbers and largely normal responses to stimulation. MYSM1, therefore, has an essential role in B cell lineage specification but is dispensable at later stages of development. Importantly, MYSM1 activity at the prepro-B cell stage of development is important for the normal programming of B cell responses to stimulation once they complete their maturation process.

  10. B-cell Lineage Study in Patients with Juvenile Idiopathic Arthritis

    Directory of Open Access Journals (Sweden)

    Hossein Asgarian-Omran

    2008-12-01

    Full Text Available Objective: Juvenile idiopathic arthritis (JIA is the most common rheumatic disease in children. The exact causes of disease are still poorly understood. It seems that B cells have several functions in JIA, including production of autoantibodies, antigen presentation, production of cytokines, and activation of T cells. Here, we aimed to evaluate B-cell lineage and its precursors in the bone marrow of patients with JIA. Methods: Twenty consecutive patients with JIA were enrolled in this study. JIA is subdivided into three groups of Pauciarticular, Polyarticular, and Systemic JIA. Bone marrow mononuclear cells were separated. Then we analyzed the immunophenotype of the JIA patients by flow cytometry. After separation, the mononuclear cells were stained specific for B cell lineage [CD10, CD19 and CD20], T cell lineage [CD3] and non specific lineage [CD34, HLA-DR and TdT]. Findings: Flow cytometric study of bone marrow showed that JIA patients had low level of CD10, CD19, and CD20. Polyarticular patients had lower level of D10, CD19, and CD20 than pauciarticular JIA patients and systemic onset JIA patients had lower levels than both of them. Conclusion: Decreasing of B cell precursor in bone marrow is one of mechanisms for pathogenesis of JIA and the more decreased B cell precursors in bone marrow are, the worst severity of the disease is. Significant differences in CD10 content of bone marrow were detected between the polyarticular and pauciarticular groups.So, it seems that polyarticular JIA patients had lower percentage of pre B cell stage.

  11. Near Equilibrium Calculus of Stem Cells in Application to the Airway Epithelium Lineage

    Science.gov (United States)

    Sun, Zheng; Plikus, Maksim V.; Komarova, Natalia L.

    2016-01-01

    Homeostatic maintenance of tissues is orchestrated by well tuned networks of cellular signaling. Such networks regulate, in a stochastic manner, fates of all cells within the respective lineages. Processes such as symmetric and asymmetric divisions, differentiation, de-differentiation, and death have to be controlled in a dynamic fashion, such that the cell population is maintained at a stable equilibrium, has a sufficiently low level of stochastic variation, and is capable of responding efficiently to external damage. Cellular lineages in real tissues may consist of a number of different cell types, connected by hierarchical relationships, albeit not necessarily linear, and engaged in a number of different processes. Here we develop a general mathematical methodology for near equilibrium studies of arbitrarily complex hierarchical cell populations, under regulation by a control network. This methodology allows us to (1) determine stability properties of the network, (2) calculate the stochastic variance, and (3) predict how different control mechanisms affect stability and robustness of the system. We demonstrate the versatility of this tool by using the example of the airway epithelium lineage. Recent research shows that airway epithelium stem cells divide mostly asymmetrically, while the so-called secretory cells divide predominantly symmetrically. It further provides quantitative data on the recovery dynamics of the airway epithelium, which can include secretory cell de-differentiation. Using our new methodology, we demonstrate that while a number of regulatory networks can be compatible with the observed recovery behavior, the observed division patterns of cells are the most optimal from the viewpoint of homeostatic lineage stability and minimizing the variation of the cell population size. This not only explains the observed yet poorly understood features of airway tissue architecture, but also helps to deduce the information on the still largely hypothetical

  12. Downregulation of the transcription factor KLF4 is required for the lineage commitment of T cells

    Institute of Scientific and Technical Information of China (English)

    Xiaomin Wen; Haifeng Liu; Gang Xiao; Xiaolong Liu

    2011-01-01

    The roles of the reprogramming factors Oct4,Sox2,c-Myc and Klf4 in early T cell development are incompletely defined.Here,we show that Klf4 is the only reprogramming factor whose expression is downregulated when early thymic progenitors (ETPs) differentiate into T cells.Enforced expression of Klf4 in uncommitted progenitors severely impaired T cell development mainly at the DN2-to-DN3 transition when T cell lineage commitment occurs and affected the transcription of a variety of genes with crucial functions in early T cell development,including genes involved in microenvironmental signaling (IL-7Rα),Notch target genes (Deltexl),and essential T cell lineage regulatory or inhibitory genes (Bcllla,SpiB,and ldl).The survival of thymocytes and the rearrangement at the Tcrb locus were impaired in the presence of enforced Klf4 expression.The defects in the DN1-to-DN2 and DN2-to-DN3 transitions in Klf4 transgenic mice could not be rescued by the introduction of a TCR transgene,but was partially rescued by restoring the expression of IL-7Rα.Thus,our data indicate that the downregulation of Klf4 is a prerequisite for T cell lineage commitment.

  13. Thymic anlage is colonized by progenitors restricted to T, NK, and dendritic cell lineages.

    Science.gov (United States)

    Masuda, Kyoko; Itoi, Manami; Amagai, Takashi; Minato, Nagahiro; Katsura, Yoshimoto; Kawamoto, Hiroshi

    2005-03-01

    It remains controversial whether the thymus-colonizing progenitors are committed to the T cell lineage. A major problem that has impeded the characterization of thymic immigrants has been that the earliest intrathymic progenitors thus far identified do not necessarily represent the genuine thymic immigrants, because their developmental potential should have been influenced by contact with the thymic microenvironment. In the present study, we examined the developmental potential of the ontogenically earliest thymic progenitors of day 11 murine fetus. These cells reside in the surrounding mesenchymal region and have not encountered thymic epithelial components. Flow cytometric and immunohistochemical analyses demonstrated that these cells are exclusively Lin(-)c-kit(+)IL-7R(+). Limiting dilution analyses disclosed that the progenitors with T cell potential were abundant, while those with B cell potential were virtually absent in the region of day 11 thymic anlage. Clonal analyses reveled that they are restricted to T, NK, and dendritic cell lineages. Each progenitor was capable of forming a large number of precursors that may clonally accommodate highly diverse TCRbeta chains. These results provide direct evidence that the progenitors restricted to the T/NK/dendritic cell lineage selectively immigrate into the thymus.

  14. Hacking cell differentiation: transcriptional rerouting in reprogramming, lineage infidelity and metaplasia.

    Science.gov (United States)

    Regalo, Gonçalo; Leutz, Achim

    2013-08-01

    Initiating neoplastic cell transformation events are of paramount importance for the comprehension of regeneration and vanguard oncogenic processes but are difficult to characterize and frequently clinically overlooked. In epithelia, pre-neoplastic transformation stages are often distinguished by the appearance of phenotypic features of another differentiated tissue, termed metaplasia. In haemato/lymphopoietic malignancies, cell lineage ambiguity is increasingly recorded. Both, metaplasia and biphenotypic leukaemia/lymphoma represent examples of dysregulated cell differentiation that reflect a history of trans-differentiation and/or epigenetic reprogramming. Here we compare the similarity between molecular events of experimental cell trans-differentiation as an emerging therapeutic concept, with lineage confusion, as in metaplasia and dysplasia forecasting tumour development.

  15. The roles of titanium surface micro/nanotopography and wettability on the differential response of human osteoblast lineage cells.

    Science.gov (United States)

    Gittens, Rolando A; Olivares-Navarrete, Rene; Cheng, Alice; Anderson, David M; McLachlan, Taylor; Stephan, Ingrid; Geis-Gerstorfer, Jürgen; Sandhage, Kenneth H; Fedorov, Andrei G; Rupp, Frank; Boyan, Barbara D; Tannenbaum, Rina; Schwartz, Zvi

    2013-04-01

    Surface micro- and nanostructural modifications of dental and orthopedic implants have shown promising in vitro, in vivo and clinical results. Surface wettability has also been suggested to play an important role in osteoblast differentiation and osseointegration. However, the available techniques to measure surface wettability are not reliable on clinically relevant, rough surfaces. Furthermore, how the differentiation state of osteoblast lineage cells impacts their response to micro/nanostructured surfaces, and the role of wettability on this response, remain unclear. In the current study, surface wettability analyses (optical sessile drop analysis, environmental scanning electron microscopic analysis and the Wilhelmy technique) indicated hydrophobic static responses for deposited water droplets on microrough and micro/nanostructured specimens, while hydrophilic responses were observed with dynamic analyses of micro/nanostructured specimens. The maturation and local factor production of human immature osteoblast-like MG63 cells was synergistically influenced by nanostructures superimposed onto microrough titanium (Ti) surfaces. In contrast, human mesenchymal stem cells cultured on micro/nanostructured surfaces in the absence of exogenous soluble factors exhibited less robust osteoblastic differentiation and local factor production compared to cultures on unmodified microroughened Ti. Our results support previous observations using Ti6Al4V surfaces showing that recognition of surface nanostructures and subsequent cell response is dependent on the differentiation state of osteoblast lineage cells. The results also indicate that this effect may be partly modulated by surface wettability. These findings support the conclusion that the successful osseointegration of an implant depends on contributions from osteoblast lineage cells at different stages of osteoblast commitment.

  16. Stepwise renal lineage differentiation of mouse embryonic stem cells tracing in vivo development

    Energy Technology Data Exchange (ETDEWEB)

    Nishikawa, Masaki, E-mail: masakiwestriver@gmail.com [Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA (United States); University of California at Los Angeles, David Geffen School of Medicine, Los Angeles, CA 91343 (United States); Yanagawa, Naomi [Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA (United States); University of California at Los Angeles, David Geffen School of Medicine, Los Angeles, CA 91343 (United States); Kojima, Nobuhiko [Institute of Industrial Science (IIS), University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505 (Japan); Yuri, Shunsuke; Hauser, Peter V.; Jo, Oak D.; Yanagawa, Norimoto [Medical and Research Services, Greater Los Angeles Veterans Affairs Healthcare System at Sepulveda, North Hills, CA (United States); University of California at Los Angeles, David Geffen School of Medicine, Los Angeles, CA 91343 (United States)

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer We induced renal lineages from mESCs by following the in vivo developmental cues. Black-Right-Pointing-Pointer We induced nephrogenic intermediate mesoderm by stepwise addition of factors. Black-Right-Pointing-Pointer We induced two types of renal progenitor cells by reciprocal conditioned media. Black-Right-Pointing-Pointer We propose the potential role of CD24 for the enrichment of renal lineage cells. -- Abstract: The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0-2, A plus BMP-4 (4) during days 2-4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4-6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6-8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was

  17. Lineage-specific expression of bestrophin-2 and bestrophin-4 in human intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Go Ito

    Full Text Available Intestinal epithelial cells (IECs regulate the absorption and secretion of anions, such as HCO3(- or Cl(-. Bestrophin genes represent a newly identified group of calcium-activated Cl(- channels (CaCCs. Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2 (BEST2 and bestrophin-4 (BEST4 might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes, remains largely unknown. Here, we show that BEST2 and BEST4 are expressed in vivo, each in a distinct, lineage-specific manner, in human IECs. While BEST2 was expressed exclusively in colonic goblet cells, BEST4 was expressed in the absorptive cells of both the small intestine and the colon. In addition, we found that BEST2 expression is significantly down-regulated in the active lesions of ulcerative colitis, where goblet cells were depleted, suggesting that BEST2 expression is restricted to goblet cells under both normal and pathologic conditions. Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells. Conversely, the induction of absorptive cell differentiation up-regulated the expression of BEST4 in villin-positive Caco-2 cells. In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells. These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.

  18. Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data.

    Science.gov (United States)

    Amat, Fernando; Lemon, William; Mossing, Daniel P; McDole, Katie; Wan, Yinan; Branson, Kristin; Myers, Eugene W; Keller, Philipp J

    2014-09-01

    The comprehensive reconstruction of cell lineages in complex multicellular organisms is a central goal of developmental biology. We present an open-source computational framework for the segmentation and tracking of cell nuclei with high accuracy and speed. We demonstrate its (i) generality by reconstructing cell lineages in four-dimensional, terabyte-sized image data sets of fruit fly, zebrafish and mouse embryos acquired with three types of fluorescence microscopes, (ii) scalability by analyzing advanced stages of development with up to 20,000 cells per time point at 26,000 cells min(-1) on a single computer workstation and (iii) ease of use by adjusting only two parameters across all data sets and providing visualization and editing tools for efficient data curation. Our approach achieves on average 97.0% linkage accuracy across all species and imaging modalities. Using our system, we performed the first cell lineage reconstruction of early Drosophila melanogaster nervous system development, revealing neuroblast dynamics throughout an entire embryo.

  19. The role of monocyte-lineage cells in human immuno-deficiency virus persistence: mechanisms and progress

    Institute of Scientific and Technical Information of China (English)

    WU Li

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1) persistence is a major barrier to the successful treatment and eradication of acquired immunodeficiency syndrome (AIDS). In addition to resting CD4+ T cells, a significant long-lived compartment of HIV-1 infection in vivo includes blood monocytes and tissue macrophages. Studying HIV-1 persistence in monocyte-lineage cells is critical because these cells are important HIV-1 target cells in vivo. Monocyte-lineage cells, including monocytes, dendritic cells (DCs) and macrophages, play a significant role in HIV-1 infection and transmission. These cells have been implicated as viral reservoirs that facilitate HIV-1 latency and persistence. A better understanding of HIV-1 interactions with monocyte-lineage cells can potentially aid in the development of new approaches for intervention. This minireview highlights the latest advances in understanding the role of monocyte-lineage cells in HIV-1 persistence and emphasizes new insights into the mechanisms underlying viral persistence.

  20. Directed differentiation into neural lineages and therapeutic potential of porcine embryonic stem cells in rat Parkinson's disease model.

    Science.gov (United States)

    Yang, Jenn-Rong; Liao, Chia-Hsin; Pang, Cheng-Yoong; Huang, Lynn Ling-Huei; Lin, Yu-Ting; Chen, Yi-Ling; Shiue, Yow-Ling; Chen, Lih-Ren

    2010-08-01

    This study was conducted to direct porcine embryonic stem (pES) cells differentiating into neural lineages and to investigate therapeutic potential of GFP-expressing pES (pES/GFP(+)) in the rat model of Parkinson's disease (PD). Directed differentiation of pES into neural lineages was induced by suspension culture in medium containing RA, SHH, and FGF combinations without going through embryoid body formation. A high yield of nestin-expressing neural precursors was found in all treatments on day 2 after the 12-day induction. On day 6 after replating, more than 86.2 and 83.4% of the differentiated cells stained positively for NFL and MAP2, respectively. The expression of TH, ChAT, and GABA specific markers were also observed in these NFL-positive neural cells. The undifferentiated pES/GFP(+) cells and their neuronal differentiation derivatives were transplanted into the Sprague-Dawley (SD) rat's brain, and their survival and development was determined by using live animal fluorescence optical imaging system every 15 days. The results showed that fluorescent signals from the injection site of SD rats' brain could be detected through the experimental period of 3 months. The level of fluorescent signal detected in the treatment group was twofold that of the control group. The results of behavior analysis showed that PD rats exhibited stably decreased asymmetric rotations after transplantation with pES/GFP(+)-derived D18 neuronal progenitors. The dopaminergic differentiation of grafted cells in the brain was further confirmed by immunohistochemical staining with anti-TH, anti-DA, and anti-DAT antibodies. These results suggested that the differentiation approach we developed would direct pES cells to differentiate into neural lineages and benefit the development of novel therapeutics involving stem cell transplantation.

  1. Integrin αv in the mechanical response of osteoblast lineage cells

    Energy Technology Data Exchange (ETDEWEB)

    Kaneko, Keiko [Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan); Ito, Masako [Medical Work-Life-Balance Center, Nagasaki University Hospital, Nagasaki 852-8501 (Japan); Naoe, Yoshinori [Department of Mechanism of Aging, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan); Lacy-Hulbert, Adam [Department of Pediatrics, Massachusetts General Hospital, Boston, MA 02114 (United States); Ikeda, Kyoji, E-mail: kikeda@ncgg.go.jp [Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511 (Japan)

    2014-05-02

    Highlights: • Deletion of integrin αv in osteoblast lineage results in an impaired SOST response to loading in vivo. • c-Src–p130Cas–JNK–YAP/TAZ is activated via integrin αv on osteoblasts in response to FSS. • Deletion of integrin αv in osteoblasts results in impaired responses to mechanical stimulation. • Integrin αv is a key component of the mechanosensing machinery in bone. - Abstract: Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src–JNK–YAP/TAZ pathway in response to mechanical stimulation.

  2. Ontogeny and distribution of cells in B lineage in the American leopard frog, Rana pipiens.

    Science.gov (United States)

    Zettergren, L D

    1982-01-01

    Two-color immunofluorescence techniques were used in order to trace the development and distribution of cells expressing immunoglobulin in Rana pipiens. Evidence is provided which suggests that (i) embryo-larval urogenital tissues are sites of generation of cells in B lineage, (ii) during ontogeny, there is a sequential expression of immunoglobulin isotypes on B cell surfaces, (iii) larvae are able to produce the full range of immunoglobulin clases found in adults, and (iv) at least two subpopulations of lymphocytes exist in Rana pipiens, sIg+ and sIg-; thymocytes and presumably peripheral T cells lack conventional surface immunoglobulin. Some ontogenetic and phylogenetic implications are discussed.

  3. Intermittent electrical stimuli for guidance of human mesenchymal stem cell lineage commitment towards neural-like cells on electroconductive substrates.

    Science.gov (United States)

    Thrivikraman, Greeshma; Madras, Giridhar; Basu, Bikramjit

    2014-08-01

    In the context of the role of multiple physical factors in dictating stem cell fate, the present paper demonstrates the effectiveness of the intermittently delivered external electric field stimulation towards switching the stem cell fate to specific lineage, when cultured in the absence of biochemical growth factors. In particular, our findings present the ability of human mesenchymal stem cells (hMSCs) to respond to the electric stimuli by adopting extended neural-like morphology on conducting polymeric substrates. Polyaniline (PANI) is selected as the model system to demonstrate this effect, as the electrical conductivity of the polymeric substrates can be systematically tailored over a broad range (10(-9) to 10 S/cm) from highly insulating to conducting by doping with varying concentrations (10(-5) to 1 m) of HCl. On the basis of the culture protocol involving the systematic delivery of intermittent electric field (dc) stimulation, the parametric window of substrate conductivity and electric field strength was established to promote significant morphological extensions, with minimal cellular damage. A time dependent morphological change in hMSCs with significant filopodial elongation was observed after 7 days of electrically stimulated culture. Concomitant with morphological changes, a commensurate increase in the expression of neural lineage commitment markers such as nestin and βIII tubulin was recorded from hMSCs grown on highly conducting substrates, as revealed from the mRNA expression analysis using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) as well as by immune-fluorescence imaging. Therefore, the present work establishes the key role of intermittent and systematic delivery of electric stimuli as guidance cues in promoting neural-like differentiation of hMSCs, when grown on electroconductive substrates.

  4. Differences in CART expression and cell cycle behavior discriminate sympathetic neuroblast from chromaffin cell lineages in mouse sympathoadrenal cells.

    Science.gov (United States)

    Chan, Wing Hei; Gonsalvez, David G; Young, Heather M; Southard-Smith, E Michelle; Cane, Kylie N; Anderson, Colin R

    2016-02-01

    Adrenal medullary chromaffin cells and peripheral sympathetic neurons originate from a common sympathoadrenal (SA) progenitor cell. The timing and phenotypic changes that mark this lineage diversification are not fully understood. The present study investigated the expression patterns of phenotypic markers, and cell cycle dynamics, in the adrenal medulla and the neighboring suprarenal ganglion of embryonic mice. The noradrenergic marker, tyrosine hydroxylase (TH), was detected in both presumptive adrenal medulla and sympathetic ganglion cells, but with significantly stronger immunostaining in the former. There was intense cocaine and amphetamine-regulated transcript (CART) peptide immunostaining in most neuroblasts, whereas very few adrenal chromaffin cells showed detectable CART immunostaining. This phenotypic segregation appeared as early as E12.5, before anatomical segregation of the two cell types. Cell cycle dynamics were also examined. Initially, 88% of Sox10 positive (+) neural crest progenitors were proliferating at E10.5. Many SA progenitor cells withdrew from the cell cycle at E11.5 as they started to express TH. Whereas 70% of neuroblasts (TH+/CART+ cells) were back in the cell cycle at E12.5, only around 20% of chromaffin (CART negative) cells were in the cell cycle at E12.5 and subsequent days. Thus, chromaffin cell and neuroblast lineages showed differences in proliferative behavior from their earliest appearance. We conclude that the intensity of TH immunostaining and the expression of CART permit early discrimination of chromaffin cells and sympathetic neuroblasts, and that developing chromaffin cells exhibit significantly lower proliferative activity relative to sympathetic neuroblasts.

  5. A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice.

    Science.gov (United States)

    Amitai-Lange, Aya; Berkowitz, Eran; Altshuler, Anna; Dbayat, Noora; Nasser, Waseem; Suss-Toby, Edith; Tiosano, Beatrice; Shalom-Feuerstein, Ruby

    2015-12-18

    Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

  6. Expression Patterns of Cancer-Testis Antigens in Human Embryonic Stem Cells and Their Cell Derivatives Indicate Lineage Tracks

    OpenAIRE

    Nadya Lifantseva; Anna Koltsova; Tatyana Krylova; Tatyana Yakovleva; Galina Poljanskaya; Olga Gordeeva

    2011-01-01

    Pluripotent stem cells can differentiate into various lineages but undergo genetic and epigenetic changes during long-term cultivation and, therefore, require regular monitoring. The expression patterns of cancer-testis antigens (CTAs) MAGE-A2, -A3, -A4, -A6, -A8, -B2, and GAGE were examined in undifferentiated human embryonic stem (hES) cells, their differentiated derivatives, teratocarcinoma (hEC) cells, and cancer cell lines of neuroectodermal and mesodermal origin. Undifferentiated hES ce...

  7. Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing Suppressor of Fused expression

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    Pozniak, Christine D.; Langseth, Abraham J.; Dijkgraaf, Gerrit J. P.; Choe, Youngshik; Werb, Zena; Pleasure, Samuel J.

    2010-01-01

    Oligodendrocyte precursor cells (OPCs) are lineage-restricted progenitors generally limited in vivo to producing oligodendrocytes. Mechanisms controlling genesis of OPCs are of interest because of their importance in myelin development and their potential for regenerative therapies in multiple sclerosis and dysmyelinating syndromes. We show here that the SoxE transcription factors (comprising Sox8, 9, and 10) induce multipotent neural precursor cells (NPCs) from the early postnatal subventricular zone (SVZ) to become OPCs in an autonomous manner. We performed a chromatin immunoprecipitation-based bioinformatic screen and identified Suppressor of Fused (Sufu) as a direct target of repression by Sox10. In vitro, overexpression of Sufu blocked OPC production, whereas RNAi-mediated inhibition augmented OPC production. Furthermore, mice heterozygous for Sufu have increased numbers of OPCs in the telencephalon during development. We conclude that Sox10 acts to restrict the potential of NPCs toward the oligodendrocyte lineage in part by regulating the expression of Sufu. PMID:21098272

  8. cKit Lineage Hemogenic Endothelium-Derived Cells Contribute to Mesenteric Lymphatic Vessels

    Directory of Open Access Journals (Sweden)

    Lukas Stanczuk

    2015-03-01

    Full Text Available Pathological lymphatic diseases mostly affect vessels in specific tissues, yet little is known about organ-specific regulation of the lymphatic vasculature. Here, we show that the vascular endothelial growth factor receptor 3 (VEGFR-3/p110α PI3-kinase signaling pathway is selectively required for the formation of mesenteric lymphatic vasculature. Using genetic lineage tracing, we demonstrate that part of the mesenteric lymphatic vasculature develops from cKit lineage cells of hemogenic endothelial origin through a process we define as lymphvasculogenesis. This is contrary to the current dogma that all mammalian lymphatic vessels form by sprouting from veins. Our results reveal vascular-bed-specific differences in the origin and mechanisms of vessel formation, which may critically underlie organ-specific manifestation of lymphatic dysfunction in disease. The progenitor cells identified in this study may be exploited to restore lymphatic function following cancer surgery, lymphedema, or tissue trauma.

  9. Histone Deacetylase (HDAC Inhibitors Down-Regulate Endothelial Lineage Commitment of Umbilical Cord Blood Derived Endothelial Progenitor Cells

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    Horia Maniu

    2012-11-01

    Full Text Available To test the involvement of histone deacetylases (HDACs activity in endothelial lineage progression, we investigated the effects of HDAC inhibitors on endothelial progenitors cells (EPCs derived from umbilical cord blood (UCB. Adherent EPCs, that expressed the endothelial marker proteins (PCAM-1, CD105, CD133, and VEGFR2 revealed by flow cytometry were treated with three HDAC inhibitors: Butyrate (BuA, Trichostatin A (TSA, and Valproic acid (VPA. RT-PCR assay showed that HDAC inhibitors down-regulated the expression of endothelial genes such as VE-cadherin, CD133, CXCR4 and Tie-2. Furthermore, flow cytometry analysis illustrated that HDAC inhibitors selectively reduce the expression of VEGFR2, CD117, VE-cadherin, and ICAM-1, whereas the expression of CD34 and CD45 remained unchanged, demonstrating that HDAC is involved in endothelial differentiation of progenitor cells. Real-Time PCR demonstrated that TSA down-regulated telomerase activity probably via suppression of hTERT expression, suggesting that HDAC inhibitor decreased cell proliferation. Cell motility was also decreased after treatment with HDAC inhibitors as shown by wound-healing assay. The balance of acethylation/deacethylation kept in control by the activity of HAT (histone acetyltransferases/HDAC enzymes play an important role in differentiation of stem cells by regulating proliferation and endothelial lineage commitment.

  10. Notch signalling inhibits CD4 expression during initiation and differentiation of human T cell lineage.

    Directory of Open Access Journals (Sweden)

    Stephen M Carlin

    Full Text Available The Delta/Notch signal transduction pathway is central to T cell differentiation from haemopoietic stem cells (HSCs. Although T cell development is well characterized using expression of cell surface markers, the detailed mechanisms driving differentiation have not been established. This issue becomes central with observations that adult HSCs exhibit poor differentiation towards the T cell lineage relative to neonatal or embryonic precursors. This study investigates the contribution of Notch signalling and stromal support cells to differentiation of adult and Cord Blood (CB human HSCs, using the Notch signalling OP9Delta co-culture system. Co-cultured cells were assayed at weekly intervals during development for phenotype markers using flow cytometry. Cells were also assayed for mRNA expression at critical developmental stages. Expression of the central thymocyte marker CD4 was initiated independently of Notch signalling, while cells grown with Notch signalling had reduced expression of CD4 mRNA and protein. Interruption of Notch signalling in partially differentiated cells increased CD4 mRNA and protein expression, and promoted differentiation to CD4(+ CD8(+ T cells. We identified a set of genes related to T cell development that were initiated by Notch signalling, and also a set of genes subsequently altered by Notch signal interruption. These results demonstrate that while Notch signalling is essential for establishment of the T cell lineage, at later stages of differentiation, its removal late in differentiation promotes more efficient DP cell generation. Notch signalling adds to signals provided by stromal cells to allow HSCs to differentiate to T cells via initiation of transcription factors such as HES1, GATA3 and TCF7. We also identify gene expression profile differences that may account for low generation of T cells from adult HSCs.

  11. E2F4 modulates differentiation and gene expression in hematopoietic progenitor cells during commitment to the lymphoid lineage.

    Science.gov (United States)

    Enos, Megan E; Bancos, Simona A; Bushnell, Timothy; Crispe, Ian N

    2008-03-15

    The E2F4 protein is involved in gene repression and cell cycle exit, and also has poorly understood effects in differentiation. We analyzed the impact of E2F4 deficiency on early steps in mouse hematopoietic development, and found defects in early hematopoietic progenitor cells that were propagated through common lymphoid precursors to the B and T lineages. In contrast, the defects in erythromyeloid precursor cells were self-correcting over time. This suggests that E2F4 is important in early stages of commitment to the lymphoid lineage. The E2F4-deficient progenitor cells showed reduced expression of several key lymphoid-lineage genes, and overexpression of two erythromyeloid lineage genes. However, we did not detect effects on cell proliferation. These findings emphasize the significance of E2F4 in controlling gene expression and cell fate.

  12. Lineage-specific expression of bestrophin-2 and bestrophin-4 in human intestinal epithelial cells

    DEFF Research Database (Denmark)

    Ito, Go; Okamoto, Ryuichi; Murano, Tatsuro

    2013-01-01

    Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3(-) or Cl(-). Bestrophin genes represent a newly identified group of calcium-activated Cl(-) channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2...... (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes...

  13. Testicular cell-conditioned medium supports embryonic stem cell differentiation toward germ lineage and to spermatocyte- and oocyte-like cells.

    Science.gov (United States)

    Shah, Syed M; Saini, Neha; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan S

    2016-08-01

    Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P embryonic development and progressed through two-cell, four-cell, eight-cell, morula, and blastocyst-like structures, indicative of their developmental competence

  14. C. elegans BED domain transcription factor BED-3 controls lineage-specific cell proliferation during organogenesis.

    Science.gov (United States)

    Inoue, Takao; Sternberg, Paul W

    2010-02-15

    The control of cell division is critical to organogenesis, but how this control is achieved is not fully understood. We found that mutations in bed-3, encoding a BED Zn-finger domain transcription factor, confer a phenotype where a specific set of cell divisions during vulval organogenesis is lost. Unlike general cell cycle regulators in Caenorhabditis elegans, the function of bed-3 is restricted to specific lineages. Transcriptional reporters suggest that bed-3 is expressed in a limited number of cell types including vulval cells whose divisions are affected in bed-3 mutants. A bed-3 mutation also affects the expression pattern of the cdh-3 cadherin gene in the vulva. The phenotype of bed-3 mutants is similar to the phenotype caused by mutations in cog-1 (Nkx6), a component of a gene regulatory network controlling cell type specific gene expression in the vulval lineage. These results suggest that bed-3 is a key component linking the gene regulatory network controlling cell-type specification to control of cell division during vulval organogenesis.

  15. Tracking the elusive fibrocyte: identification and characterization of collagen-producing hematopoietic lineage cells during murine wound healing.

    Science.gov (United States)

    Suga, Hirotaka; Rennert, Robert C; Rodrigues, Melanie; Sorkin, Michael; Glotzbach, Jason P; Januszyk, Michael; Fujiwara, Toshihiro; Longaker, Michael T; Gurtner, Geoffrey C

    2014-05-01

    Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However, putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and nonhematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein and nonhematopoietic cells with red fluorescent protein. Use of single-cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45- and Cd11b-, representing a previously unidentified population. Both cell types were found to initially peak, then reduce posthealing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together, we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time course of fibrocytes in cutaneous healing.

  16. Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Chanson, L. [Ecole Polytechnique Federale de Lausanne (Switzerland). Inst. of Bioengineering; Brownfield, D. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering; Garbe, J. C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; Kuhn, I. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; Stampfer, M. R. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; Bissell, M. J. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.; LaBarge, M. A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Div.

    2011-02-07

    Loss of organization is a principle feature of cancers; therefore it is important to understand how normal adult multilineage tissues, such as bilayered secretory epithelia, establish and maintain their architectures. The self-organization process that drives heterogeneous mixtures of cells to form organized tissues is well studied in embryology and with mammalian cell lines that were abnormal or engineered. Here we used a micropatterning approach that confined cells to a cylindrical geometry combined with an algorithm to quantify changes of cellular distribution over time to measure the ability of different cell types to self-organize relative to each other. Using normal human mammary epithelial cells enriched into pools of the two principal lineages, luminal and myoepithelial cells, we demonstrated that bilayered organization in mammary epithelium was driven mainly by lineage-specific differential E-cadherin expression, but that P-cadherin contributed specifically to organization of the myoepithelial layer. Disruption of the actomyosin network or of adherens junction proteins resulted in either prevention of bilayer formation or loss of preformed bilayers, consistent with continual sampling of the local microenvironment by cadherins. Together these data show that self-organization is an innate and reversible property of communities of normal adult human mammary epithelial cells.

  17. Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations.

    Science.gov (United States)

    Loh, Kyle M; Ang, Lay Teng; Zhang, Jingyao; Kumar, Vibhor; Ang, Jasmin; Auyeong, Jun Qiang; Lee, Kian Leong; Choo, Siew Hua; Lim, Christina Y Y; Nichane, Massimo; Tan, Junru; Noghabi, Monireh Soroush; Azzola, Lisa; Ng, Elizabeth S; Durruthy-Durruthy, Jens; Sebastiano, Vittorio; Poellinger, Lorenz; Elefanty, Andrew G; Stanley, Edouard G; Chen, Qingfeng; Prabhakar, Shyam; Weissman, Irving L; Lim, Bing

    2014-02-01

    Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of "pre-enhancer" states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.

  18. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells

    Directory of Open Access Journals (Sweden)

    Frederic B. Thalheimer

    2014-07-01

    Full Text Available The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however, the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely, the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that, once GADD45G is expressed, the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here, we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis.

  19. Lymphoid lineage differentiation potential of mouse nuclear transfer embryonic stem cells.

    Science.gov (United States)

    Eslami-Arshaghi, Tarlan; Salehi, Mohammad; Soleimani, Masoud; Gholipourmalekabadi, Mazaher; Mossahebi-Mohammadi, Majid; Ardeshirylajimi, Abdolreza; Rajabi, Hoda

    2015-09-01

    Stem cells therapy is considered as an efficient strategy for the treatment of some diseases. Nevertheless, some obstacles such as probability of rejection by the immune system limit applications of this strategy. Therefore, several efforts have been made to overcome this among which using the induced pluripotent stem cells (iPSCs) and nuclear transfer embryonic stem cell (nt-ESCs) are the most efficient strategies. The objective of this study was to evaluate the differentiation potential of the nt-ESCs to lymphoid lineage in the presence of IL-7, IL-3, FLT3-ligand and TPO growth factors in vitro. To this end, the nt-ESCs cells were prepared and treated with aforementioned growth factors for 7 and 14 days. Then, the cells were examined for expression of lymphoid markers (CD3, CD25, CD127 and CD19) by quantitative PCR (q-PCR) and flow cytometry. An increased expression of CD19 and CD25 markers was observed in the treated cells compared with the negative control samples by day 7. After 14 days, the expression level of all the tested CD markers significantly increased in the treated groups in comparison with the control. The current study reveals the potential of the nt-ESCs in differentiation to lymphoid lineage in the presence of defined growth factors.

  20. Does cell lineage in the developing cerebral cortex contribute to its columnar organization?

    Directory of Open Access Journals (Sweden)

    Marcos R Costa

    2010-06-01

    Full Text Available Since the pioneer work of Lorente de Nó, Ramón y Cajal, Brodmann, Mountcastle, Hubel and Wiesel and others, the cerebral cortex has been seen as a jigsaw of anatomic and functional modules involved in the processing of different sets of information. In fact, a columnar distribution of neurons displaying similar functional properties throughout the cerebral cortex has been observed by many researchers. Although it has been suggested that much of the anatomical substrate for such organization would be already specified at early developmental stages, before activity-dependent mechanisms could take place, it is still unclear whether gene expression in the ventricular zone could play a role in the development of discrete functional units, such as minicolumns or columns. Cell lineage experiments using replication-incompetent retroviral vectors have shown that the progeny of a single neuroepithelial/radial glial cell in the dorsal telencephalon is organized into discrete radial clusters of sibling excitatory neurons, which have a higher propensity for developing chemical synapses with each other rather than with neighbouring non-siblings. Here, we will discuss the possibility that the cell lineage of single neuroepithelial/radial glia cells could contribute for the columnar organization of the neocortex by generating radial columns of sibling, interconnected neurons. Borrowing some concepts from the studies on cell-cell recognition and transcription factor networks, we will also touch upon the potential molecular mechanisms involved in the establishment of sibling-neuron circuits.

  1. A mex3 homolog is required for differentiation during planarian stem cell lineage development.

    Science.gov (United States)

    Zhu, Shu Jun; Hallows, Stephanie E; Currie, Ko W; Xu, ChangJiang; Pearson, Bret J

    2015-06-26

    Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment.

  2. Mixed Lineage Kinase 3 negatively regulates IKK activity and enhances etoposide-induced cell death

    OpenAIRE

    Cole, Eric T.; Zhan, Yu; Abi Saab, Widian F.; Korchnak, Amanda C.; Ashburner, Brian P.; Chadee, Deborah N.

    2009-01-01

    Mixed Lineage Kinase 3 (MLK3) is a mitogen activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK signaling pathways. Nuclear factor kappa B (NF-κB) is a transcription factor that has important functions in inflammation, immunity and cell survival. We found that silencing mlk3 expression with RNA interference (RNAi) in SKOV3 human ovarian cancer epithelial cells and NIH-3T3 murine fibroblasts led to a reduction in the level of the inhibitor of kappa B alpha (IκBα) protein...

  3. Evolution in vitro: analysis of a lineage of ribozymes

    Science.gov (United States)

    Lehman, N.; Joyce, G. F.

    1993-01-01

    Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population. Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme. Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

  4. Distribution of neurosensory progenitor pools during inner ear morphogenesis unveiled by cell lineage reconstruction

    Science.gov (United States)

    Dyballa, Sylvia; Savy, Thierry; Germann, Philipp; Mikula, Karol; Remesikova, Mariana; Špir, Róbert; Zecca, Andrea; Peyriéras, Nadine; Pujades, Cristina

    2017-01-01

    Reconstructing the lineage of cells is central to understanding how the wide diversity of cell types develops. Here, we provide the neurosensory lineage reconstruction of a complex sensory organ, the inner ear, by imaging zebrafish embryos in vivo over an extended timespan, combining cell tracing and cell fate marker expression over time. We deliver the first dynamic map of early neuronal and sensory progenitor pools in the whole otic vesicle. It highlights the remodeling of the neuronal progenitor domain upon neuroblast delamination, and reveals that the order and place of neuroblasts’ delamination from the otic epithelium prefigure their position within the SAG. Sensory and non-sensory domains harbor different proliferative activity contributing distinctly to the overall growth of the structure. Therefore, the otic vesicle case exemplifies a generic morphogenetic process where spatial and temporal cues regulate cell fate and functional organization of the rudiment of the definitive organ. DOI: http://dx.doi.org/10.7554/eLife.22268.001 PMID:28051766

  5. Does Cell Lineage in the Developing Cerebral Cortex Contribute to its Columnar Organization?

    Science.gov (United States)

    Costa, Marcos R.; Hedin-Pereira, Cecilia

    2010-01-01

    Since the pioneer work of Lorente de Nó, Ramón y Cajal, Brodmann, Mountcastle, Hubel and Wiesel and others, the cerebral cortex has been seen as a jigsaw of anatomic and functional modules involved in the processing of different sets of information. In fact, a columnar distribution of neurons displaying similar functional properties throughout the cerebral cortex has been observed by many researchers. Although it has been suggested that much of the anatomical substrate for such organization would be already specified at early developmental stages, before activity-dependent mechanisms could take place, it is still unclear whether gene expression in the ventricular zone (VZ) could play a role in the development of discrete functional units, such as minicolumns or columns. Cell lineage experiments using replication-incompetent retroviral vectors have shown that the progeny of a single neuroepithelial/radial glial cell in the dorsal telencephalon is organized into discrete radial clusters of sibling excitatory neurons, which have a higher propensity for developing chemical synapses with each other rather than with neighboring non-siblings. Here, we will discuss the possibility that the cell lineage of single neuroepithelial/radial glia cells could contribute for the columnar organization of the neocortex by generating radial columns of sibling, interconnected neurons. Borrowing some concepts from the studies on cell–cell recognition and transcription factor networks, we will also touch upon the potential molecular mechanisms involved in the establishment of sibling-neuron circuits. PMID:20676384

  6. From Adult Bone Marrow Cells to Other Cell Lineages:Transdifferentiation or Cells Fusion

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Recent studies have demonstrated that intravenous transplantation or local injection of bone marrow cells can induce unexpected changes of their fate. The results of these experiments showed that after transplantation or injecton, some of tissue specific somatic cells such as hepatocytes, skeleton, cardiac muscle cells and brain cells expressed the donor cell-specific genes, such as Y chromosome. There are two hypotheses that can explain this phenomenon. One is bone marrow stem cell transdifferentiation and the other is spontaneous cell fusion.

  7. Ex vivo differentiation of human bone marrow-derived stem cells into neuronal cell-like lineages

    Directory of Open Access Journals (Sweden)

    Al-Zoubi A

    2016-06-01

    Full Text Available Adeeb Al-Zoubi,1,2 Feras Altwal,3 Farah Khalifeh,2 Jamil Hermas,4 Ziad Al-Zoubi,5 Emad Jafar,5 Mohammed El-Khateeb,6,7 1Department of Surgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 2Stem Cells of Arabia, Amman, Jordan; 3Department of Neuroscience, School of Graduate and Postdoctoral Studies, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA; 4Stem Cell Division, Al-Yamama Company, 5Jordan Orthopedic and Spinal Center, 6National Center for Diabetes, Endocrinology and Genetics, 7Department of Pathology, Faculty of Medicine, University of Jordan, Amman, Jordan Background: Methods to obtain safe and practical populations of stem cells (SCs at a clinical grade that are able to differentiate into neuronal cell lineages are yet to be developed. In a previous study, we showed that mouse bone marrow-derived SCs (BM-SCs differentiated into neuronal cell-like lineages when put in a neuronal-like environment, which is a special media supplemented with the necessary growth factors needed for the differentiation of SCs into neuronal cell-like lineages. Aim: In this study, we aim to assess the potentials of adult human CD34+ and CD133+ SCs to differentiate into neuronal cell-like lineages ex vivo when placed in a neuronal-like microenvironment. Methods: The neuronal-like microenvironment was created by culturing cells in nonhematopoietic expansion media (NHEM supplemented with growth factors that favor differentiation into neuronal cell lineages (low-affinity nerve growth factor [LNGF], mouse spinal cord extract [mSpE], or both. Cultured cells were assessed for neuronal differentiation by cell morphologies and by expression of GFAP. Results: Our results show that culturing unpurified human BM-derived mononuclear cells (hBM-MNCs in NHEM+LNGF+mSpE did not lead to neuronal differentiation. In contrast, culturing of purified CD34+ hBM-SCs in NHEM+LNGF+mSpE favored their differentiation into astrocyte

  8. Generation of priming mesenchymal stem cells with enhanced potential to differentiate into specific cell lineages using extracellular matrix proteins.

    Science.gov (United States)

    Han, Na Rae; Yun, Jung Im; Park, Young Hyun; Ahn, Ji Yeon; Kim, Choonghyo; Choi, Jung Hoon; Lee, Eunsong; Lim, Jeong Mook; Lee, Seung Tae

    2013-07-01

    Poor understanding of the differentiation of mesenchymal stem cells (MSCs) has resulted in a low differentiation yield, and has hindered their application in medicine. As a solution, priming MSCs sensitive to signaling, thus stimulating differentiation into a specific cell lineage, may improve the differentiation yield. To demonstrate this, priming MSCs were produced by using a gelatin matrix for the isolation of primary MSCs from bone-marrow-derived primary cells. Subsequently, cellular characteristics and sensitivity to specific differentiation signals were analyzed at passage five. Compared to non-priming MSCs, priming MSCs showed no significant differences in cellular characteristics, but demonstrated a significant increase in sensitivity to neurogenic differentiation signals. These results demonstrate that generation of priming MSCs by specific extracellular signaling increases the rate of differentiation into a cell-specific lineage.

  9. Neural-Competent Cells of Adult Human Dermis Belong to the Schwann Lineage

    Science.gov (United States)

    Etxaniz, Usue; Pérez-San Vicente, Adrián; Gago-López, Nuria; García-Dominguez, Mario; Iribar, Haizea; Aduriz, Ariane; Pérez-López, Virginia; Burgoa, Izaskun; Irizar, Haritz; Muñoz-Culla, Maider; Vallejo-Illarramendi, Ainara; Leis, Olatz; Matheu, Ander; Martín, Angel G.; Otaegui, David; López-Mato, María Paz; Gutiérrez-Rivera, Araika; MacLellan, Robb; Izeta, Ander

    2014-01-01

    Summary Resident neural precursor cells (NPCs) have been reported for a number of adult tissues. Understanding their physiological function or, alternatively, their activation after tissue damage or in vitro manipulation remains an unsolved issue. Here, we investigated the source of human dermal NPCs in adult tissue. By following an unbiased, comprehensive approach employing cell-surface marker screening, cell separation, transcriptomic characterization, and in vivo fate analyses, we found that p75NTR+ precursors of human foreskin can be ascribed to the Schwann (CD56+) and perivascular (CD56−) cell lineages. Moreover, neural differentiation potential was restricted to the p75NTR+CD56+ Schwann cells and mediated by SOX2 expression levels. Double-positive NPCs were similarly obtained from human cardiospheres, indicating that this phenomenon might be widespread. PMID:25418723

  10. Neural-Competent Cells of Adult Human Dermis Belong to the Schwann Lineage

    Directory of Open Access Journals (Sweden)

    Usue Etxaniz

    2014-11-01

    Full Text Available Resident neural precursor cells (NPCs have been reported for a number of adult tissues. Understanding their physiological function or, alternatively, their activation after tissue damage or in vitro manipulation remains an unsolved issue. Here, we investigated the source of human dermal NPCs in adult tissue. By following an unbiased, comprehensive approach employing cell-surface marker screening, cell separation, transcriptomic characterization, and in vivo fate analyses, we found that p75NTR+ precursors of human foreskin can be ascribed to the Schwann (CD56+ and perivascular (CD56− cell lineages. Moreover, neural differentiation potential was restricted to the p75NTR+CD56+ Schwann cells and mediated by SOX2 expression levels. Double-positive NPCs were similarly obtained from human cardiospheres, indicating that this phenomenon might be widespread.

  11. Neural-competent cells of adult human dermis belong to the Schwann lineage.

    Science.gov (United States)

    Etxaniz, Usue; Pérez-San Vicente, Adrián; Gago-López, Nuria; García-Dominguez, Mario; Iribar, Haizea; Aduriz, Ariane; Pérez-López, Virginia; Burgoa, Izaskun; Irizar, Haritz; Muñoz-Culla, Maider; Vallejo-Illarramendi, Ainara; Leis, Olatz; Matheu, Ander; Martín, Angel G; Otaegui, David; López-Mato, María Paz; Gutiérrez-Rivera, Araika; MacLellan, Robb; Izeta, Ander

    2014-11-11

    Resident neural precursor cells (NPCs) have been reported for a number of adult tissues. Understanding their physiological function or, alternatively, their activation after tissue damage or in vitro manipulation remains an unsolved issue. Here, we investigated the source of human dermal NPCs in adult tissue. By following an unbiased, comprehensive approach employing cell-surface marker screening, cell separation, transcriptomic characterization, and in vivo fate analyses, we found that p75NTR(+) precursors of human foreskin can be ascribed to the Schwann (CD56(+)) and perivascular (CD56(-)) cell lineages. Moreover, neural differentiation potential was restricted to the p75NTR(+)CD56(+) Schwann cells and mediated by SOX2 expression levels. Double-positive NPCs were similarly obtained from human cardiospheres, indicating that this phenomenon might be widespread.

  12. Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes.

    Science.gov (United States)

    Tan, Zhaoli; Chen, Keyan; Shao, Yong; Gao, Lihua; Wang, Yan; Xu, Jianming; Jin, Yang; Hu, Xianwen; Wang, Youliang

    2016-09-01

    Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2-Cre or VE-cadherin-Cre constructs to facilitate fate-mapping of LSECs in liver regeneration. Some YFP-positive LSECs were observed to convert into hepatocytes following a two-thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non-hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte-like (iHep) cells may provide a new approach to tissue engineering.

  13. Mast Cell-activated Bone Marrow Mesenchymal Stromal Cells Regulate Proliferation and Lineage Commitment of CD34+ Progenitor cells

    Directory of Open Access Journals (Sweden)

    Zoulfia eAllakhverdi

    2013-12-01

    Full Text Available Background: Shortly after allergen exposure, the number of bone marrow and circulating CD34+ progenitors increases. We aim to analyze the possible mechanism whereby the allergic reaction stimulates bone marrow to release these effector cells in increased numbers. We hypothesize that mast cells may play a predominant role in this process. Objective: To examine the effect of IgE-activated mast cells on bone marrow mesenchymal stromal cells which regulate proliferation and differentiation of CD34+ progenitors. Methods: Primary mast cells were derived from CD34+ precursors and activated with IgE/anti-IgE. Bone marrow mesenchymal stromal cells were co-cultured with CD34+ progenitor cells and stimulated with IL1/TNF or IgE/anti-IgE activated mast cells in Transwell system. Results: Bone marrow mesenchymal stromal cells produce low level of TSLP under steady state conditions, which is markedly increased by stimulation with proinflammatory cytokines IL-1 and TNF or IgE-activated mast cells. The latter also triggers BM-MSCs production of G-CSF, and GM-CSF while inhibiting SDF-1. Mast cell-activated mesenchymal stromal cells stimulate CD34+ cells to proliferate and to regulate their expression of early allergy-associated genes. Conclusion and Clinical Relevance: This in vitro study indicates that IgE-activated mast cells trigger bone marrow mesenchymal stromal cells to release TSLP and hematopoietic growth factors and to regulate the proliferation and lineage commitment of CD34+ precursor cells. The data predict that the effective inhibition of mast cells should impair mobilization and accumulation of allergic effector cells and thereby reduce the severity of allergic diseases.

  14. Epigenetic control of Ccr7 expression in distinct lineages of lung dendritic cells.

    Science.gov (United States)

    Moran, Timothy P; Nakano, Hideki; Kondilis-Mangum, Hrisavgi D; Wade, Paul A; Cook, Donald N

    2014-11-15

    Adaptive immune responses to inhaled allergens are induced following CCR7-dependent migration of precursor of dendritic cell (pre-DC)-derived conventional DCs (cDCs) from the lung to regional lymph nodes. However, monocyte-derived (moDCs) in the lung express very low levels of Ccr7 and consequently do not migrate efficiently to LN. To investigate the molecular mechanisms that underlie this dichotomy, we studied epigenetic modifications at the Ccr7 locus of murine cDCs and moDCs. When expanded from bone marrow precursors, moDCs were enriched at the Ccr7 locus for trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with transcriptional repression. Similarly, moDCs prepared from the lung also displayed increased levels of H3K27me3 at the Ccr7 promoter compared with migratory cDCs from that organ. Analysis of DC progenitors revealed that epigenetic modification of Ccr7 does not occur early during DC lineage commitment because monocytes and pre-DCs both had low levels of Ccr7-associated H3K27me3. Rather, Ccr7 is gradually silenced during the differentiation of monocytes to moDCs. Thus, epigenetic modifications of the Ccr7 locus control the migration and therefore the function of DCs in vivo. These findings suggest that manipulating epigenetic mechanisms might be a novel approach to control DC migration and thereby improve DC-based vaccines and treat inflammatory diseases of the lung.

  15. Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages.

    Science.gov (United States)

    Casero, David; Sandoval, Salemiz; Seet, Christopher S; Scholes, Jessica; Zhu, Yuhua; Ha, Vi Luan; Luong, Annie; Parekh, Chintan; Crooks, Gay M

    2015-12-01

    To elucidate the transcriptional 'landscape' that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNAs (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus.

  16. Lineage-specific diversification of killer cell Ig-like receptors in the owl monkey, a New World primate.

    Science.gov (United States)

    Cadavid, Luis F; Lun, Cheng-Man

    2009-01-01

    Killer cell Ig-like receptors (KIRs) modulate the cytotoxic effects of natural killer cells. In primates, the KIRs are highly diverse as a consequence of variation in gene content, alternative domain composition, and loci polymorphism. We analyzed a bacterial artificial chromosome (BAC) clone draft sequence spanning the owl monkey KIR cluster. The draft sequence had seven ordered yet unconnected contigs containing six full-length and two partial gene models, flanked by the LILRB and FcAR framework genes. Gene models were predicted to encode KIRs with inhibitory, activating, or dual functionality. Four gene models encoded three Ig domain receptors, while three others encoded molecules with four Ig domains. The additional domain resulted from an insertion in tandem of a 2,101 bp fragment containing the last 289 bp of intron 2, exon 3, and intron 3, resulting in molecules with two D0 domains. Re-screening of the owl monkey BAC library and sequencing of partial cDNAs from an owl monkey yielded five additional KIRs, four of which encoded receptors with short cytoplasmic domains with premature stop codons due to either a single nucleotide substitution or deletion or the absence of exon 8. Phylogenetic analysis by domains showed that owl monkey KIRs were monophyletic, clustering independently from other primate KIR lineages. Retroelements found in introns, however, were shared by KIRs from different primate lineages. This suggests that the owl monkey inherited a KIR cluster with a rich history of exon shuffling upon which positive selection for ligand binding operated to diversify the receptors in a lineage-specific fashion.

  17. Differentiation of human embryonic stem cells along a hepatocyte lineage and its application in liver regeneration

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Hepatocyte transplantation and bioartificial liver(BAL)as alternatives to liver transplantation offer the possibility of effective treatment for many inherited and acquired hepatic disorders.Unfortunately,the limited availability of donated livers and the variability of their derived hepatocytes make it difficult to obtain enough viable human hepatocytes for the hepatocyte-based therapies.Embryonic stem cells (ESCs),which could be isolated directly from the blastocyst inner cell mass,have permanent self-renewal capability and developmental pluripotency and therefore might be an ideal cell source in the treatment of hepatic discords.However,differentiation of hESCS into hepatocytes with significant numbers remains a challenge.This review updates our current understanding of differentiation of ESCs into hepatic lineage cells,their future therapeutic uses and problems in liver regeneration.

  18. Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation.

    Science.gov (United States)

    Yamamuro, Chizuko; Miki, Daisuke; Zheng, Zhimin; Ma, Jun; Wang, Jing; Yang, Zhenbiao; Dong, Juan; Zhu, Jian-Kang

    2014-06-05

    DNA methylation is a reversible epigenetic mark regulating genome stability and function in many eukaryotes. In Arabidopsis, active DNA demethylation depends on the function of the ROS1 subfamily of genes that encode 5-methylcytosine DNA glycosylases/lyases. ROS1-mediated DNA demethylation plays a critical role in the regulation of transgenes, transposable elements and some endogenous genes; however, there have been no reports of clear developmental phenotypes in ros1 mutant plants. Here we report that, in the ros1 mutant, the promoter region of the peptide ligand gene EPF2 is hypermethylated, which greatly reduces EPF2 expression and thereby leads to a phenotype of overproduction of stomatal lineage cells. EPF2 gene expression in ros1 is restored and the defective epidermal cell patterning is suppressed by mutations in genes in the RNA-directed DNA methylation pathway. Our results show that active DNA demethylation combats the activity of RNA-directed DNA methylation to influence the initiation of stomatal lineage cells.

  19. Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

    Science.gov (United States)

    Xie, Wenjie; Lorenz, Sebastian; Dolder, Silvia; Hofstetter, Willy

    2016-03-01

    Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

  20. Mechanical modulation of nascent stem cell lineage commitment in tissue engineering scaffolds.

    Science.gov (United States)

    Song, Min Jae; Dean, David; Knothe Tate, Melissa L

    2013-07-01

    Taking inspiration from tissue morphogenesis in utero, this study tests the concept of using tissue engineering scaffolds as delivery devices to modulate emergent structure-function relationships at early stages of tissue genesis. We report on the use of a combined computational fluid dynamics (CFD) modeling, advanced manufacturing methods, and experimental fluid mechanics (micro-piv and strain mapping) for the prospective design of tissue engineering scaffold geometries that deliver spatially resolved mechanical cues to stem cells seeded within. When subjected to a constant magnitude global flow regime, the local scaffold geometry dictates the magnitudes of mechanical stresses and strains experienced by a given cell, and in a spatially resolved fashion, similar to patterning during morphogenesis. In addition, early markers of mesenchymal stem cell lineage commitment relate significantly to the local mechanical environment of the cell. Finally, by plotting the range of stress-strain states for all data corresponding to nascent cell lineage commitment (95% CI), we begin to "map the mechanome", defining stress-strain states most conducive to targeted cell fates. In sum, we provide a library of reference mechanical cues that can be delivered to cells seeded on tissue engineering scaffolds to guide target tissue phenotypes in a temporally and spatially resolved manner. Knowledge of these effects allows for prospective scaffold design optimization using virtual models prior to prototyping and clinical implementation. Finally, this approach enables the development of next generation scaffolds cum delivery devices for genesis of complex tissues with heterogenous properties, e.g., organs, joints or interface tissues such as growth plates.

  1. Mechanical Modulation of Nascent Stem Cell Lineage Commitment in Tissue Engineering Scaffolds

    Science.gov (United States)

    Song, Min Jae; Dean, David; Tate, Melissa L. Knothe

    2013-01-01

    Taking inspiration from tissue morphogenesis in utero, this study tests the concept of using tissue engineering scaffolds as delivery devices to modulate emergent structure-function relationships at early stages of tissue genesis. We report on the use of a combined computational fluid dynamics (CFD) modeling, advanced manufacturing methods, and experimental fluid mechanics (micro-piv and strain mapping) for the prospective design of tissue engineering scaffold geometries that deliver spatially resolved mechanical cues to cells seeded within. When subjected to a constant magnitude global flow regime, the local scaffold geometry dictates the magnitudes of mechanical stresses and strains experienced by a given cell, and in a spatially resolved fashion, similar to patterning during morphogenesis. In addition, early markers of mesenchymal stem cell lineage commitment relate significantly to the local mechanical environment of the cell. Finally, by plotting the range of stress-strain states for all data corresponding to nascent cell lineage commitment (95% CI), we begin to “map the mechanome”, defining stress-strain states most conducive to targeted cell fates. In sum, we provide a library of reference mechanical cues that can be delivered to cells seeded on tissue engineering scaffolds to guide target tissue phenotypes in a temporally and spatially resolved manner. Knowledge of these effects allows for prospective scaffold design optimization using virtual models prior to prototyping and clinical implementation. Finally, this approach enables the development of next generation scaffolds cum delivery devices for genesis of complex tissues with heterogenous properties, e.g., organs, joints or interface tissues such as growth plates. PMID:23660249

  2. Traumatic brain injury reveals novel cell lineage relationships within the subventricular zone

    Directory of Open Access Journals (Sweden)

    Gretchen M. Thomsen

    2014-07-01

    Full Text Available The acute response of the rodent subventricular zone (SVZ to traumatic brain injury (TBI involves a physical expansion through increased cell proliferation. However, the cellular underpinnings of these changes are not well understood. Our analyses have revealed that there are two distinct transit-amplifying cell populations that respond in opposite ways to injury. Mash1+ transit-amplifying cells are the primary SVZ cell type that is stimulated to divide following TBI. In contrast, the EGFR+ population, which has been considered to be a functionally equivalent progenitor population to Mash1+ cells in the uninjured brain, becomes significantly less proliferative after injury. Although normally quiescent GFAP+ stem cells are stimulated to divide in SVZ ablation models, we found that the GFAP+ stem cells do not divide more after TBI. We found, instead, that TBI results in increased numbers of GFAP+/EGFR+ stem cells via non-proliferative means—potentially through the dedifferentiation of progenitor cells. EGFR+ progenitors from injured brains only were competent to revert to a stem cell state following brief exposure to growth factors. Thus, our results demonstrate previously unknown changes in lineage relationships that differ from conventional models and likely reflect an adaptive response of the SVZ to maintain endogenous brain repair after TBI.

  3. Implementation of erythroid lineage analysis by flow cytometry in diagnostic models for myelodysplastic syndromes

    Science.gov (United States)

    Cremers, Eline M.P.; Westers, Theresia M.; Alhan, Canan; Cali, Claudia; Visser-Wisselaar, Heleen A.; Chitu, Dana A.; van der Velden, Vincent H.J.; te Marvelde, Jeroen G.; Klein, Saskia K.; Muus, Petra; Vellenga, Edo; de Greef, Georgina E.; Legdeur, Marie-Cecile C.J.C.; Wijermans, Pierre W.; Stevens-Kroef, Marian J.P.L.; da Silva-Coelho, Pedro; Jansen, Joop H.; Ossenkoppele, Gert J.; van de Loosdrecht, Arjan A.

    2017-01-01

    Flow cytometric analysis is a recommended tool in the diagnosis of myelodysplastic syndromes. Current flow cytometric approaches evaluate the (im)mature myelo-/monocytic lineage with a median sensitivity and specificity of ~71% and ~93%, respectively. We hypothesized that the addition of erythroid lineage analysis could increase the sensitivity of flow cytometry. Hereto, we validated the analysis of erythroid lineage parameters recommended by the International/European LeukemiaNet Working Group for Flow Cytometry in Myelodysplastic Syndromes, and incorporated this evaluation in currently applied flow cytometric models. One hundred and sixty-seven bone marrow aspirates were analyzed; 106 patients with myelodysplastic syndromes, and 61 cytopenic controls. There was a strong correlation between presence of erythroid aberrancies assessed by flow cytometry and the diagnosis of myelodysplastic syndromes when validating the previously described erythroid evaluation. Furthermore, addition of erythroid aberrancies to two different flow cytometric models led to an increased sensitivity in detecting myelodysplastic syndromes: from 74% to 86% for the addition to the diagnostic score designed by Ogata and colleagues, and from 69% to 80% for the addition to the integrated flow cytometric score for myelodysplastic syndromes, designed by our group. In both models the specificity was unaffected. The high sensitivity and specificity of flow cytometry in the detection of myelodysplastic syndromes illustrates the important value of flow cytometry in a standardized diagnostic approach. The trial is registered at www.trialregister.nl as NTR1825; EudraCT n.: 2008-002195-10 PMID:27658438

  4. Morphometric analysis of Sphaeroidinellopsis: new biostratigraphic information from gradually evolving lineages

    Energy Technology Data Exchange (ETDEWEB)

    Gary, A.C.; Healy-Williams, N.; Williams, D.F.

    1986-05-01

    Gradually evolving lineages have not been routinely used biostratigraphically because morphological changes are difficult to define through time. However, using sophisticated shape-analysis systems, the authors developed a zonation based on gradual evolutionary changes in morphology. In this study, they analyzed morphologic changes in specimens of the sphaeroidinellopsis lineage in middle Miocene to Pleistocene sediments from the DeSoto Canyon area of the Gulf of Mexico. The authors quantified morphology with an automated video digitizer and Fourier analysis, which breaks the total shape into specific shape components. Fourier analysis accurately and objectively quantifies the morphology of various microfossils. They used EXTENDED CABFAC/EXTENDED Q-MODEL to define the morphologic substructure (i.e., existence of distinct morpho-types) of the Sphaeroidinellopsis population within time intervals. Comparing morphotypes between samples, they found the following. (1) Morphotypes are time continuous. When a morphotype disappears it does not reappear up section. (2) The progressive change in morphotypes through time follows a shape continuum. Morphotypes become increasingly more spherical with decreasing age. (3) The number of morphotypes present in any given sample is generally greater than the number of species possibly present. A zonation scheme based on gradual evolutionary changes in the morphology of foraminiferal lineages could complement traditional biostratigraphic approaches by significantly increasing the resolution of correlations in late Tertiary sediments.

  5. Fibroblast growth factor receptor-3 regulates Paneth cell lineage allocation and accrual of epithelial stem cells during murine intestinal development.

    Science.gov (United States)

    Vidrich, Alda; Buzan, Jenny M; Brodrick, Brooks; Ilo, Chibuzo; Bradley, Leigh; Fendig, Kirstin Skaar; Sturgill, Thomas; Cohn, Steven M

    2009-07-01

    Fibroblast growth factor receptor 3 (FGFR-3) is expressed in the lower crypt epithelium, where stem cells of the intestine reside. The role of FGFR-3 signaling in regulating features of intestinal morphogenesis was examined in FGFR-3-null (FGFR-3(-/-)) mice. FGFR-3(-/-) mice had only about half the number of intestinal crypts and a marked decrease in the number of functional clonogenic stem cells, as assessed by an in vivo microcolony-forming assay, compared with wild-type littermates. A marked deficit in allocation of progenitor cells to Paneth cell differentiation was noted, although all the principal epithelial lineages were represented in FGFR-3(-/-) mice. The total cellular content and nuclear localization of beta-catenin protein were reduced in FGFR-3(-/-) mice, as was expression of cyclin D1 and matrix metalloproteinase-7, major downstream targets of beta-catenin/T cell factor-4 (Tcf-4) signaling. Activation of FGFR-3 in Caco-2 cells, an intestinal epithelial cell line, abrogated the fall in beta-catenin/Tcf-4 signaling activity that is normally observed in these cells as cultures become progressively more confluent. These findings are consistent with the hypothesis that, during intestinal development, FGFR-3 signaling regulates crypt epithelial stem cell expansion and crypt morphogenesis, as well as Paneth cell lineage specification, through beta-catenin/Tcf-4-dependent and -independent pathways.

  6. Concise Review: Primary Cilia: Control Centers for Stem Cell Lineage Specification and Potential Targets for Cell-Based Therapies.

    Science.gov (United States)

    Bodle, Josephine C; Loboa, Elizabeth G

    2016-06-01

    Directing stem cell lineage commitment prevails as the holy grail of translational stem cell research, particularly to those interested in the application of mesenchymal stem cells and adipose-derived stem cells in tissue engineering. However, elucidating the mechanisms underlying their phenotypic specification persists as an active area of research. In recent studies, the primary cilium structure has been intimately associated with defining cell phenotype, maintaining stemness, as well as functioning in a chemo, electro, and mechanosensory capacity in progenitor and committed cell types. Many hypothesize that the primary cilium may indeed be another important player in defining and controlling cell phenotype, concomitant with lineage-dictated cytoskeletal dynamics. Many of the studies on the primary cilium have emerged from disparate areas of biological research, and crosstalk amongst these areas of research is just beginning. To date, there has not been a thorough review of how primary cilia fit into the current paradigm of stem cell differentiation and this review aims to summarize the current cilia work in this context. The goal of this review is to highlight the cilium's function and integrate this knowledge into the working knowledge of stem cell biologists and tissue engineers developing regenerative medicine technologies. Stem Cells 2016;34:1445-1454.

  7. FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans.

    Science.gov (United States)

    Minor, Paul J; He, Ting-Fang; Sohn, Chang Ho; Asthagiri, Anand R; Sternberg, Paul W

    2013-09-01

    The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Here, we show that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/β-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, we show that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans.

  8. In silico analysis of stomach lineage specific gene set expression pattern in gastric cancer.

    Science.gov (United States)

    Pandi, Narayanan Sathiya; Suganya, Sivagurunathan; Rajendran, Suriliyandi

    2013-10-04

    Stomach lineage specific gene products act as a protective barrier in the normal stomach and their expression maintains the normal physiological processes, cellular integrity and morphology of the gastric wall. However, the regulation of stomach lineage specific genes in gastric cancer (GC) is far less clear. In the present study, we sought to investigate the role and regulation of stomach lineage specific gene set (SLSGS) in GC. SLSGS was identified by comparing the mRNA expression profiles of normal stomach tissue with other organ tissue. The obtained SLSGS was found to be under expressed in gastric tumors. Functional annotation analysis revealed that the SLSGS was enriched for digestive function and gastric epithelial maintenance. Employing a single sample prediction method across GC mRNA expression profiles identified the under expression of SLSGS in proliferative type and invasive type gastric tumors compared to the metabolic type gastric tumors. Integrative pathway activation prediction analysis revealed a close association between estrogen-α signaling and SLSGS expression pattern in GC. Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. In conclusion, our results highlight that estrogen mediated regulation of SLSGS in gastric tumor is a molecular predictor of metabolic type GC and prognostic factor in GC.

  9. The potential of dental stem cells differentiating into neurogenic cell lineage after cultivation in different modes in vitro.

    Science.gov (United States)

    Yang, Chao; Sun, Liang; Li, Xinghan; Xie, Li; Yu, Mei; Feng, Lian; Jiang, Zongting; Guo, Weihua; Tian, Weidong

    2014-10-01

    Trauma or degenerative diseases of the central nervous system (CNS) cause the loss of neurons or glial cells. Stem cell transplantation has become a vital strategy for CNS regeneration. It is necessary to effectively induce nonneurogenic stem cells to differentiate into neurogenic cell lineages because of the limited source of neurogenic stem cells, relatively difficult cultivation, and ethical issues. Previous studies have found that dental stem cells can be used for transplantation therapy. The aim of this study was to explore a better inductive mode and time point for dental stem cells to differentiate into neural-like cells and evaluate a better candidate cell. In this study, dental follicle stem cells (DFSCs), dental papilla stem cells (DPSCs), and stem cells from apical papilla (SCAPs) were cultivated in five different modes. The proliferation ability, morphology, and expression of neural marker genes were analyzed. Results showed that DFSCs showed a higher proliferation potential. The proliferation was decreased after cultivation in chemical inductive medium as cultivation modes 3 and 5. The cells could present neural-like cell morphology after cultivation with human epidermal growth factor (EGF) and fibroblast growth factor-basic (bFGF) as cultivation modes 4 and 5. The vast majority of DFSCs gene expression levels in mode 4 on the third day was upregulated significantly. In conclusion, our data suggested that different dental stem cells exhibited different neural differentiation potentials. DFSCs might be the better candidate cell type. Furthermore, cultivation mode 4 and timing of the third day may promote differentiation into neurogenic cell lineages more effectively before transplantation to treat neurological diseases.

  10. Very Small Embryonic-Like Stem Cells: A Potential Developmental Link Between Germinal Lineage and Hematopoiesis in Humans.

    Science.gov (United States)

    Virant-Klun, Irma

    2016-01-15

    It has been suggested that hematopoietic stem/progenitor cells (HSPCs) could become specified from a population of migrating primordial germ cells (PGCs), precursors of gametes, during embryogenesis. Some recent experimental data demonstrated that the cell population that is usually considered to be PGCs, moving toward the gonadal ridges of an embryo, contains a subset of cells coexpressing several germ cell and hematopoietic markers and possessing hematopoietic activity. Experimental data showed that bone morphogenetic protein 4 (BMP4) generates PGCs from mouse bone marrow-derived pluripotent stem cells. Interestingly, functional reproductive hormone receptors have been identified in HSPCs, thus indicating their potential role in reproductive function. Several reports have demonstrated fertility restoration and germ cell generation after bone marrow transplantation in both animal models and humans. A potential link between HSPCs and germinal lineage might be represented by very small embryonic-like stem cells (VSELs), which have been found in adult human bone marrow, peripheral blood, and umbilical cord blood, express a specific pattern of pluripotency, germinal lineage, and hematopoiesis, and are proposed to persist in adult tissues and organs from the embryonic period of life. Stem cell populations, similar to VSELs, expressing several genes related to pluripotency and germinal lineage, especially to PGCs, have been discovered in adult human reproductive organs, ovaries and testicles, and were related to primitive germ cell-like cell development in vitro, thus supporting the idea of VSELs as a potential link between germinal lineage and hematopoiesis.

  11. In silico analysis of stomach lineage specific gene set expression pattern in gastric cancer

    Energy Technology Data Exchange (ETDEWEB)

    Pandi, Narayanan Sathiya, E-mail: sathiyapandi@gmail.com; Suganya, Sivagurunathan; Rajendran, Suriliyandi

    2013-10-04

    Highlights: •Identified stomach lineage specific gene set (SLSGS) was found to be under expressed in gastric tumors. •Elevated expression of SLSGS in gastric tumor is a molecular predictor of metabolic type gastric cancer. •In silico pathway scanning identified estrogen-α signaling is a putative regulator of SLSGS in gastric cancer. •Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. -- Abstract: Stomach lineage specific gene products act as a protective barrier in the normal stomach and their expression maintains the normal physiological processes, cellular integrity and morphology of the gastric wall. However, the regulation of stomach lineage specific genes in gastric cancer (GC) is far less clear. In the present study, we sought to investigate the role and regulation of stomach lineage specific gene set (SLSGS) in GC. SLSGS was identified by comparing the mRNA expression profiles of normal stomach tissue with other organ tissue. The obtained SLSGS was found to be under expressed in gastric tumors. Functional annotation analysis revealed that the SLSGS was enriched for digestive function and gastric epithelial maintenance. Employing a single sample prediction method across GC mRNA expression profiles identified the under expression of SLSGS in proliferative type and invasive type gastric tumors compared to the metabolic type gastric tumors. Integrative pathway activation prediction analysis revealed a close association between estrogen-α signaling and SLSGS expression pattern in GC. Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. In conclusion, our results highlight that estrogen mediated regulation of SLSGS in gastric tumor is a molecular predictor of metabolic type GC and prognostic factor in GC.

  12. A planarian p53 homolog regulates proliferation and self-renewal in adult stem cell lineages.

    Science.gov (United States)

    Pearson, Bret J; Sánchez Alvarado, Alejandro

    2010-01-01

    The functions of adult stem cells and tumor suppressor genes are known to intersect. However, when and how tumor suppressors function in the lineages produced by adult stem cells is unknown. With a large population of stem cells that can be manipulated and studied in vivo, the freshwater planarian is an ideal system with which to investigate these questions. Here, we focus on the tumor suppressor p53, homologs of which have no known role in stem cell biology in any invertebrate examined thus far. Planaria have a single p53 family member, Smed-p53, which is predominantly expressed in newly made stem cell progeny. When Smed-p53 is targeted by RNAi, the stem cell population increases at the expense of progeny, resulting in hyper-proliferation. However, ultimately the stem cell population fails to self-renew. Our results suggest that prior to the vertebrates, an ancestral p53-like molecule already had functions in stem cell proliferation control and self-renewal.

  13. B cell lymphomas express CX3CR1 a non-B cell lineage adhesion molecule

    DEFF Research Database (Denmark)

    Andreasson, U.; Ek, S.; Merz, H.

    2008-01-01

    To study the differential expression of cell membrane-bound receptors and their potential role in growth and/or survival of the tumor cells, highly purified follicular lymphoma cells were analyzed, using gene expression analysis, and compared to non-malignant B cell populations. Filtering...... the genome for overexpressed genes coding for cell membrane-bound proteins/receptors resulted in a hit list of 27 identified genes. Among these, we have focused on the aberrant over expression of CX3CR1, in different types of B cell lymphoma, as compared to non-malignant B cells. We show that CX3CR1, which...... normally is not expressed on B cells, is expressed both at the mRNA and protein level in several subtypes of lymphoma. CX3CR1 has also shown to be involved in the homing to specific tissues that express the ligand, CX3CL1, in breast and prostate cancer and may thus be involved in dissemination of lymphoma...

  14. Transmissible [corrected] dog cancer genome reveals the origin and history of an ancient cell lineage.

    Science.gov (United States)

    Murchison, Elizabeth P; Wedge, David C; Alexandrov, Ludmil B; Fu, Beiyuan; Martincorena, Inigo; Ning, Zemin; Tubio, Jose M C; Werner, Emma I; Allen, Jan; De Nardi, Andrigo Barboza; Donelan, Edward M; Marino, Gabriele; Fassati, Ariberto; Campbell, Peter J; Yang, Fengtang; Burt, Austin; Weiss, Robin A; Stratton, Michael R

    2014-01-24

    Canine transmissible venereal tumor (CTVT) is the oldest known somatic cell lineage. It is a transmissible cancer that propagates naturally in dogs. We sequenced the genomes of two CTVT tumors and found that CTVT has acquired 1.9 million somatic substitution mutations and bears evidence of exposure to ultraviolet light. CTVT is remarkably stable and lacks subclonal heterogeneity despite thousands of rearrangements, copy-number changes, and retrotransposon insertions. More than 10,000 genes carry nonsynonymous variants, and 646 genes have been lost. CTVT first arose in a dog with low genomic heterozygosity that may have lived about 11,000 years ago. The cancer spawned by this individual dispersed across continents about 500 years ago. Our results provide a genetic identikit of an ancient dog and demonstrate the robustness of mammalian somatic cells to survive for millennia despite a massive mutation burden.

  15. Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines

    Directory of Open Access Journals (Sweden)

    Nidal Ghosheh

    2016-01-01

    Full Text Available Human pluripotent stem cells- (hPSCs- derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4 which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.

  16. [Distribution of abnormal cell clone with deletion of chromosome 20q in marrow cell lineages and apoptosis cells in myelodysplastic syndrome].

    Science.gov (United States)

    Qin, Ling; Wang, Chun; Qin, You-Wen; Xie, Kuang-Cheng; Yan, Shi-Ke; Gao, Yan-Rong; Wang, Xiao-Rui; Zhao, Chu-Xian

    2008-06-01

    This study was aimed to investigate the distribution of abnormal clone in marrow cell lineages and apoptosis cells in myelodysplastic syndrome (MDS) with deletion of chromosome 20q. Monoclonal antibodies recognizing myeloid precursors (CD15), erythroid precursors (GPA), T cells (CD3(+)CD56(-)CD16(-)), B cells (CD19), NK cells (CD3(-)CD56(+)CD16(+)) were used to sort bone marrow cells in a MDS patient with del (20q) by fluorescence activated cell sorting (FACS). Annexin V-FITC and PI were used to sort bone marrow Annexin V(+)PI(-) and Annexin V(-)PI(-) cells by FACS. The sorted positive cells were detected by interphase dual-color fluorescence in situ hybridization (D-FISH) using a LSI D20S108 probe (Spectrum Orange) and a Telvysion TM 20p probe (Spectrum Green). FACS and FISH analysis were also performed on the samples from 4 cases with normal karyotype. The results showed that the proportions of MDS clone in the myeloid and erythroid precursors were 70.50% and 93.33% respectively, in the RAEB-1 patient with del (20q) and were obviously higher than that in control group (5.39% and 6.17%). The proportions of abnormal clone in T, B and NK cells were 3.23%, 4.32% and 5.77% respectively and were less than that in control group (5.76%, 4.85%, 6.36%). The percentage of apoptotic cells in the bone marrow nucleated cells was 16.09%. The proportions of MDS clone in Annexin V(+)PI(-) and Annexin V(-)PI(-) cells were 32.48% and 70.11%, respectively. It is concluded that most myeloid and erythroid precursors are originated from the abnormal clone in MDS with del (20q). A little part of apoptotic cells are derived from the abnormal clone.

  17. Collagen scaffold microenvironments modulate cell lineage commitment for differentiation of bone marrow cells into regulatory dendritic cells

    Science.gov (United States)

    Fang, Yongxiang; Wang, Bin; Zhao, Yannan; Xiao, Zhifeng; Li, Jing; Cui, Yi; Han, Sufang; Wei, Jianshu; Chen, Bing; Han, Jin; Meng, Qingyuan; Hou, Xianglin; Luo, Jianxun; Dai, Jianwu; Jing, Zhizhong

    2017-01-01

    The microenvironment plays a pivotal role for cell survival and functional regulation, and directs the cell fate determination. The biological functions of DCs have been extensively investigated to date. However, the influences of the microenvironment on the differentiation of bone marrow cells (BMCs) into dendritic cells (DCs) are not well defined. Here, we established a 3D collagen scaffold microenvironment to investigate whether such 3D collagen scaffolds could provide a favourable niche for BMCs to differentiate into specialised DCs. We found that BMCs embedded in the 3D collagen scaffold differentiated into a distinct subset of DC, exhibiting high expression of CD11b and low expression of CD11c, co-stimulator (CD40, CD80, CD83, and CD86) and MHC-II molecules compared to those grown in 2D culture. DCs cultured in the 3D collagen scaffold possessed weak antigen uptake ability and inhibited T-cell proliferation in vitro; in addition, they exhibited potent immunoregulatory function to alleviate allo-delay type hypersensitivity when transferred in vivo. Thus, DCs differentiated in the 3D collagen scaffold were defined as regulatory DCs, indicating that collagen scaffold microenvironments probably play an important role in modulating the lineage commitment of DCs and therefore might be applied as a promising tool for generation of specialised DCs. PMID:28169322

  18. DNA methylation restricts lineage-specific functions of transcription factor Gata4 during embryonic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Masaaki Oda

    2013-06-01

    Full Text Available DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.

  19. Scleraxis is required for cell lineage differentiation and extracellular matrix remodeling during murine heart valve formation in vivo.

    Science.gov (United States)

    Levay, Agata K; Peacock, Jacqueline D; Lu, Yinhui; Koch, Manuel; Hinton, Robert B; Kadler, Karl E; Lincoln, Joy

    2008-10-24

    Heart valve structures, derived from mesenchyme precursor cells, are composed of differentiated cell types and extracellular matrix arranged to facilitate valve function. Scleraxis (scx) is a transcription factor required for tendon cell differentiation and matrix organization. This study identified high levels of scx expression in remodeling heart valve structures at embryonic day 15.5 through postnatal stages using scx-GFP reporter mice and determined the in vivo function using mice null for scx. Scx(-/-) mice display significantly thickened heart valve structures from embryonic day 17.5, and valves from mutant mice show alterations in valve precursor cell differentiation and matrix organization. This is indicated by decreased expression of the tendon-related collagen type XIV, increased expression of cartilage-associated genes including sox9, as well as persistent expression of mesenchyme cell markers including msx1 and snai1. In addition, ultrastructure analysis reveals disarray of extracellular matrix and collagen fiber organization within the valve leaflet. Thickened valve structures and increased expression of matrix remodeling genes characteristic of human heart valve disease are observed in juvenile scx(-/-) mice. In addition, excessive collagen deposition in annular structures within the atrioventricular junction is observed. Collectively, our studies have identified an in vivo requirement for scx during valvulogenesis and demonstrate its role in cell lineage differentiation and matrix distribution in remodeling valve structures.

  20. A role for mixed lineage kinases in granule cell apoptosis induced by cytoskeletal disruption.

    Science.gov (United States)

    Müller, Georg Johannes; Geist, Marie Aavang; Veng, Lone Merete; Willesen, Mette Georgi; Johansen, Flemming Fryd; Leist, Marcel; Vaudano, Elisabetta

    2006-03-01

    Microtubule disruption by colchicine induces apoptosis in selected neuronal populations. However, little is known about the upstream death signalling events mediating the neurotoxicity. We investigated first whether colchicine-induced granule cell apoptosis activates the c-Jun N-terminal kinase (JNK) pathway. Cultured murine cerebellar granule cells were exposed to 1 microm colchicine for 24 h. Activation of the JNK pathway was detected by western blotting as well as immunocytochemistry using antibodies against phospho-c-Jun (p-c-Jun). Next, adult male rats were injected intracerebroventricularly with colchicine (10 microg), and JNK pathway activation in dentate granule cells (DGCs) was detected by antibodies against p-c-Jun. The second part of the study tested the involvement of mixed lineage kinases (MLK) as upstream activators of the JNK pathway in colchicine toxicity, using CEP-1347, a potent MLK inhibitor. In vitro, significant inhibition of the JNK pathway, activated by colchicine, was achieved by 100-300 nm CEP-1347, which blocked both activation of cell death proteases and apoptosis. Moreover, CEP-1347 markedly delayed neurite fragmentation and cell degeneration. In vivo, CEP-1347 (1 mg/kg) significantly prevented p-c-jun increase following injection of colchicine, and enhanced survival of DGCs. We conclude that colchicine-induced neuronal apoptosis involves the JNK/MLK pathway, and that protection of granule cells can be achieved by MLK inhibition.

  1. Stem Cell Differentiation Toward the Myogenic Lineage for Muscle Tissue Regeneration: A Focus on Muscular Dystrophy.

    Science.gov (United States)

    Ostrovidov, Serge; Shi, Xuetao; Sadeghian, Ramin Banan; Salehi, Sahar; Fujie, Toshinori; Bae, Hojae; Ramalingam, Murugan; Khademhosseini, Ali

    2015-12-01

    Skeletal muscle tissue engineering is one of the important ways for regenerating functionally defective muscles. Among the myopathies, the Duchenne muscular dystrophy (DMD) is a progressive disease due to mutations of the dystrophin gene leading to progressive myofiber degeneration with severe symptoms. Although current therapies in muscular dystrophy are still very challenging, important progress has been made in materials science and in cellular technologies with the use of stem cells. It is therefore useful to review these advances and the results obtained in a clinical point of view. This article focuses on the differentiation of stem cells into myoblasts, and their application in muscular dystrophy. After an overview of the different stem cells that can be induced to differentiate into the myogenic lineage, we introduce scaffolding materials used for muscular tissue engineering. We then described some widely used methods to differentiate different types of stem cell into myoblasts. We highlight recent insights obtained in therapies for muscular dystrophy. Finally, we conclude with a discussion on stem cell technology. We discussed in parallel the benefits brought by the evolution of the materials and by the expansion of cell sources which can differentiate into myoblasts. We also discussed on future challenges for clinical applications and how to accelerate the translation from the research to the clinic in the frame of DMD.

  2. Brain vascular pericytes following ischemia have multipotential stem cell activity to differentiate into neural and vascular lineage cells.

    Science.gov (United States)

    Nakagomi, Takayuki; Kubo, Shuji; Nakano-Doi, Akiko; Sakuma, Rika; Lu, Shan; Narita, Aya; Kawahara, Maiko; Taguchi, Akihiko; Matsuyama, Tomohiro

    2015-06-01

    Brain vascular pericytes (PCs) are a key component of the blood-brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries.

  3. IL-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells.

    Science.gov (United States)

    Nodland, Sonja E; Berkowska, Magdalena A; Bajer, Anna A; Shah, Nisha; de Ridder, Dick; van Dongen, Jacques J M; LeBien, Tucker W; van Zelm, Menno C

    2011-08-25

    IL-7 is an important cytokine for lymphocyte differentiation. Similar to what occurs in vivo, human CD19⁺ cells developing in human/murine xenogeneic cultures show differential expression of the IL-7 receptor α (IL-7Rα) chain (CD127). We now describe the relationship between CD127 expression/signaling and Ig gene rearrangement. In the present study, < 10% of CD19⁺CD127⁺ and CD19⁺CD127⁻ populations had complete VDJ(H) rearrangements. IGH locus conformation measurements by 3D FISH revealed that CD127⁺ and CD127⁻ cells were less contracted than pediatric BM pro-B cells that actively rearrange the IGH locus. Complete IGH rearrangements in CD127⁺ and CD127⁻ cells had smaller CDR3 lengths and fewer N-nucleotide insertions than pediatric BM B-lineage cells. Despite the paucity of VDJ(H) rearrangements, microarray analysis indicated that CD127⁺ cells resembled large pre-B cells, which is consistent with their low level of Ig light-chain rearrangements. Unexpectedly, CD127⁻ cells showed extensive Ig light-chain rearrangements in the absence of IGH rearrangements and resembled small pre-B cells. Neutralization of IL-7 in xenogeneic cultures led to an increase in Ig light-chain rearrangements in CD127⁺ cells, but no change in complete IGH rearrangements. We conclude that IL-7-mediated suppression of premature Ig light-chain rearrangement is the most definitive function yet described for IL-7 in human B-cell development.

  4. Enobosarm (GTx-024) Modulates Adult Skeletal Muscle Mass Independently of the Androgen Receptor in the Satellite Cell Lineage.

    Science.gov (United States)

    Dubois, Vanessa; Simitsidellis, Ioannis; Laurent, Michaël R; Jardi, Ferran; Saunders, Philippa T K; Vanderschueren, Dirk; Claessens, Frank

    2015-12-01

    Androgens increase skeletal muscle mass, but their clinical use is hampered by a lack of tissue selectivity and subsequent side effects. Selective androgen receptor modulators elicit muscle-anabolic effects while only sparingly affecting reproductive tissues. The selective androgen receptor modulator, GTx-024 (enobosarm), is being investigated for cancer cachexia, sarcopenia, and muscle wasting diseases. Here we investigate the role of muscle androgen receptor (AR) in the anabolic effect of GTx-024. In mice lacking AR in the satellite cell lineage (satARKO), the weight of the androgen-sensitive levator ani muscle was lower but was decreased further upon orchidectomy. GTx-024 was as effective as DHT in restoring levator ani weights to sham levels. Expression of the muscle-specific, androgen-responsive genes S-adenosylmethionine decarboxylase and myostatin was decreased by orchidectomy and restored by GTx-024 and DHT in control mice, whereas the expression was low and unaffected by androgen status in satARKO. In contrast, insulin-like growth factor 1Ea expression was not different between satARKO and control muscle, decreased upon castration, and was restored by DHT and GTx-024 in both genotypes. These data indicate that GTx-024 does not selectively modulate AR in the satellite cell lineage and that cells outside this lineage remain androgen responsive in satARKO muscle. Indeed, residual AR-positive cells were present in satARKO muscle, coexpressing the fibroblast-lineage marker vimentin. AR positive, muscle-resident fibroblasts could therefore be involved in the indirect effects of androgens on muscle. In conclusion, both DHT and GTx-024 target AR pathways in the satellite cell lineage, but cells outside this lineage also contribute to the anabolic effects of androgens.

  5. Small Molecules Greatly Improve Conversion of Human-Induced Pluripotent Stem Cells to the Neuronal Lineage

    Directory of Open Access Journals (Sweden)

    Sally K. Mak

    2012-01-01

    Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC lines from patients with Parkinson’s disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.

  6. Notch-mediated suppression of TSC2 expression regulates cell differentiation in the Drosophila intestinal stem cell lineage.

    Directory of Open Access Journals (Sweden)

    Subir Kapuria

    Full Text Available Epithelial homeostasis in the posterior midgut of Drosophila is maintained by multipotent intestinal stem cells (ISCs. ISCs self-renew and produce enteroblasts (EBs that differentiate into either enterocytes (ECs or enteroendocrine cells (EEs in response to differential Notch (N activation. Various environmental and growth signals dynamically regulate ISC activity, but their integration with differentiation cues in the ISC lineage remains unclear. Here we identify Notch-mediated repression of Tuberous Sclerosis Complex 2 (TSC2 in EBs as a required step in the commitment of EBs into the EC fate. The TSC1/2 complex inhibits TOR signaling, acting as a tumor suppressor in vertebrates and regulating cell growth. We find that TSC2 is expressed highly in ISCs, where it maintains stem cell identity, and that N-mediated repression of TSC2 in EBs is required and sufficient to promote EC differentiation. Regulation of TSC/TOR activity by N signaling thus emerges as critical for maintenance and differentiation in somatic stem cell lineages.

  7. Multiploid CD61+ cells are the pre-dominant cell lineage infected during acute dengue virus infection in bone marrow.

    Directory of Open Access Journals (Sweden)

    Kristina B Clark

    Full Text Available Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM. Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM and humans were utilized to identify the cell lineage(s that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.

  8. Aryl hydrocarbon receptors in osteoclast lineage cells are a negative regulator of bone mass.

    Directory of Open Access Journals (Sweden)

    Tai-yong Yu

    Full Text Available Aryl hydrocarbon receptors (AhRs play a critical role in various pathological and physiological processes. Although recent research has identified AhRs as a key contributor to bone metabolism following studies in systemic AhR knockout (KO or transgenic mice, the cellular and molecular mechanism(s in this process remain unclear. In this study, we explored the function of AhR in bone metabolism using AhR(RANKΔOc/ΔOc (RANK(Cre/+;AhR(flox/flox mice. We observed enhanced bone mass together with decreased resorption in both male and female 12 and 24-week-old AhR(RANKΔOc/ΔOc mice. Control mice treated with 3-methylcholanthrene (3MC, an AhR agonist, exhibited decreased bone mass and increased bone resorption, whereas AhR(CtskΔOc/ΔOc (Ctsk(Cre/+;AhR(flox/flox mice injected with 3MC appeared to have a normal bone phenotype. In vitro, bone marrow-derived macrophages (BMDMs from AhR(RANKΔOc/ΔOc mice exhibited impaired osteoclastogenesis and repressed differentiation with downregulated expression of B lymphocyte-induced maturation protein 1 (Blimp1, and cytochrome P450 genes Cyp1b1 and Cyp1a2. Collectively, our results not only demonstrated that AhR in osteoclast lineage cells is a physiologically relevant regulator of bone resorption, but also highlighted the need for further studies on the skeletal actions of AhR inhibitors in osteoclast lineage cells commonly associated with bone diseases, especially diseases linked to environmental pollutants known to induce bone loss.

  9. Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhan, Yu; Abi Saab, Widian F.; Modi, Nidhi; Stewart, Amanda M. [Department of Biological Sciences, The University of Toledo, 2801 W. Bancroft, Toledo, OH 43606 (United States); Liu, Jinsong [Department of Pathology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030 (United States); Chadee, Deborah N., E-mail: deborah.chadee@utoledo.edu [Department of Biological Sciences, The University of Toledo, 2801 W. Bancroft, Toledo, OH 43606 (United States)

    2012-08-15

    Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells. -- Highlights: Black-Right-Pointing-Pointer Ovarian cancer cell lines have high levels of total and phosphorylated MLK3. Black-Right-Pointing-Pointer MLK3 is required for MMP expression and activity in ovarian cancer cells. Black-Right-Pointing-Pointer MLK3 is required for invasion of SKOV3 and HEY1B ovarian cancer cells. Black-Right-Pointing-Pointer MLK3-dependent regulation of MMP-2 and MMP-9 activities requires ERK and JNK.

  10. Activation of arylhydrocarbon receptor (AhR) in T lineage cells inhibits cellular growth

    Energy Technology Data Exchange (ETDEWEB)

    Nohara, K.; Tomohiro, I.; Chiharu, T. [National Institute for Environmental Studies, Tsukuba (Japan)

    2004-09-15

    Dioxins, including the most toxic congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), exert their toxic effects by binding and activating the arylhydrocarbon receptor (AhR), a liganddependent transcription factor. Upon binding dioxins, the AhR in the cytoplasm is activated and translocated to the nucleus, where it heterodimerizes with another transcription factor, ARNT. The AhR/ARNT heterodimer modulates expressions of various genes by binding xenobiotic responsive elements (XREs) in their enhancer regions or modifies cellular functions through protein-protein interactions. The AhR activation by TCDD exposure induces various immunotoxic reactions including thymus involution and suppression of T cell-dependent antibody production. We have investigated the roles of AhR activation in T lineage cells and their underlying mechanisms by generating transgenic (Tg) mice expressing a constitutively active AhR (CA-AhR) mutant specifically in T cells and by transiently expressing the CA-AhR mutant in Jurkat T cells.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-06-10

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

  12. Analysis of the full genome of human group C rotaviruses reveals lineage diversification and reassortment.

    Science.gov (United States)

    Medici, Maria Cristina; Tummolo, Fabio; Martella, Vito; Arcangeletti, Maria Cristina; De Conto, Flora; Chezzi, Carlo; Fehér, Enikő; Marton, Szilvia; Calderaro, Adriana; Bányai, Krisztián

    2016-08-01

    Group C rotaviruses (RVC) are enteric pathogens of humans and animals. Whole-genome sequences are available only for few RVCs, leaving gaps in our knowledge about their genetic diversity. We determined the full-length genome sequence of two human RVCs (PR2593/2004 and PR713/2012), detected in Italy from hospital-based surveillance for rotavirus infection in 2004 and 2012. In the 11 RNA genomic segments, the two Italian RVCs segregated within separate intra-genotypic lineages showed variation ranging from 1.9 % (VP6) to 15.9 % (VP3) at the nucleotide level. Comprehensive analysis of human RVC sequences available in the databases allowed us to reveal the existence of at least two major genome configurations, defined as type I and type II. Human RVCs of type I were all associated with the M3 VP3 genotype, including the Italian strain PR2593/2004. Conversely, human RVCs of type II were all associated with the M2 VP3 genotype, including the Italian strain PR713/2012. Reassortant RVC strains between these major genome configurations were identified. Although only a few full-genome sequences of human RVCs, mostly of Asian origin, are available, the analysis of human RVC sequences retrieved from the databases indicates that at least two intra-genotypic RVC lineages circulate in European countries. Gathering more sequence data is necessary to develop a standardized genotype and intra-genotypic lineage classification system useful for epidemiological investigations and avoiding confusion in the literature.

  13. SDF-1/CXCR4 axis in Tie2-lineage cells including endothelial progenitor cells contributes to bone fracture healing.

    Science.gov (United States)

    Kawakami, Yohei; Ii, Masaaki; Matsumoto, Tomoyuki; Kuroda, Ryosuke; Kuroda, Tomoya; Kwon, Sang-Mo; Kawamoto, Atsuhiko; Akimaru, Hiroshi; Mifune, Yutaka; Shoji, Taro; Fukui, Tomoaki; Kurosaka, Masahiro; Asahara, Takayuki

    2015-01-01

    CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal-derived-factor 1 (SDF-1). SDF-1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF-1/CXCR4 pathway in Tie2-lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2-Cre(ER) CXCR4 knockout mice (CXCR4(-/-) ) and wild-type mice (WT). We report here that in vitro, EPCs derived from of CXCR4(-/-) mouse bone marrow demonstrated severe reduction of migration activity and EPC colony-forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4(-/-) group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4(-/-) group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4(-/-) group. Especially, CXCR4(-/-) group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real-time RT-PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE-cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4(-/-) group. In the gain-of-function study, the fracture in the SDF-1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF-1 injected CXCR4(-/-) group. We demonstrated that an EPC SDF-1/CXCR4 axis plays an

  14. Enhanced generation of myeloid lineages in hematopoietic differentiation from embryonic stem cells by silencing transcriptional repressor Twist-2.

    Science.gov (United States)

    Sharabi, Andrew B; Lee, Sung-Hyung; Goodell, Margaret A; Huang, Xue F; Chen, Si-Yi

    2009-12-01

    The self-renewal and multilineage differentiation of embryonic stem cells (ESC) is largely governed by transcription factors or repressors. Extensive efforts have focused on elucidating critical factors that control the differentiation of specific cell lineages, for instance, myeloid lineages in hematopoietic development. In this study, we found that Twist-2, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in inhibiting the differentiation of ESC. Murine ES cells, in which Twist-2 expression is silenced by lentivirally delivered shRNA, exhibit an enhanced formation of primary embryoid bodies (EB) and enhanced differentiation into mesodermally derived hematopoietic colonies. Furthermore, Twist-2 silenced (LV-siTwist-2) ESC display significantly increased generation of myeloid lineages (Gr-1(+) and F4/80(+) cells) during in vitro hematopoietic differentiation. Treatment with the Toll-like receptor (TLR) 4 ligand synergistically stimulates the generation of primary EB formation as well as of hematopoietic progenitors differentiated from LV-siTwist-2 ES cells. Thus, this study reveals the critical role of the transcriptional repressor Twist-2 in regulating the development of myeloid lineage in hematopoietic differentiation from ESC. This study also suggests a potential strategy for directional differentiation of ESC by inhibiting a transcriptional repressor.

  15. Genome Analysis of a Transmissible Lineage of Pseudomonas aeruginosa Reveals Pathoadaptive Mutations and Distinct Evolutionary Paths of Hypermutators

    DEFF Research Database (Denmark)

    Marvig, Rasmus Lykke; Johansen, Helle Krogh; Molin, Søren

    2013-01-01

    infections. For example, it remains unclear what genes are mutated to facilitate the establishment of long-term existence in the human host environment, and in which way acquisition of a hypermutator phenotype with enhanced rates of spontaneous mutations influences the evolutionary trajectory of the pathogen....... Here we perform a retrospective study of the DK2 clone type of P. aeruginosa isolated from Danish patients suffering from cystic fibrosis (CF), and analyze the genomes of 55 bacterial isolates collected from 21 infected individuals over 38 years. Our phylogenetic analysis of 8,530 mutations in the DK2...... targeted by mutations to optimize pathogen fitness (pathoadaptive mutations). These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages. The long...

  16. Contribution of cells derived from the area pellucida to extraembryonic mesodermal cell lineages in heterospecific quail chick blastodermal chimeras.

    Science.gov (United States)

    Karagenç, Levent; Sandikci, Mustafa

    2013-01-01

    The current study has two main objectives: first, to determine if cells derived from the area pellucida are able to populate extraembryonic membranes, and second, to determine if donor cells have the potential to differentiate to endothelial (EC) and hematopoietic cells (HC) in the yolk sac and allantois, the two extraembryonic membranes functioning as hematopoietic organs in the avian embryo. To this end, quail chick chimeras were constructed by transferring dissociated cells from the areae pellucidae of the stage X-XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in the allantois, yolk sac, amnion, and chorion of resulting putative chimeras was examined using quail cell-specific antibody against a perinuclear antigen (QCPN) after 6 days of incubation. The presence of EC, HC, and smooth muscle cells among the QCPN(+) donor cells was examined using QH-1, a quail-specific marker identifying HC and EC and an anti-α-smooth muscle actin antibody. Evidence gathered in the present study demonstrates that quail cells derived from the areae pellucidae are able to populate all of the extraembryonic membranes of resulting heterospecific quail chick chimeras and, most importantly, give rise to HC, EC, and smooth muscle cells, all of the three main mesodermal lineages derived from the posterior mesoderm both in the yolk sac and allantois.

  17. Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Chow, Paik Wah [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Abdul Hamid, Zariyantey, E-mail: zyantey@ukm.edu.my [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Chan, Kok Meng [Environmental Health and Industrial Safety Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Inayat-Hussain, Salmaan Hussain [Environmental Health and Industrial Safety Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Rajab, Nor Fadilah [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia)

    2015-04-01

    Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-forming unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e{sup +} cells but reduced the total counts of Sca-1{sup +}, CD11b{sup +}, Gr-1{sup +}, and CD45{sup +} cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and

  18. Scaffolds for 3D in vitro culture of neural lineage cells.

    Science.gov (United States)

    Murphy, Ashley R; Laslett, Andrew; O'Brien, Carmel M; Cameron, Neil R

    2017-03-01

    Understanding how neurodegenerative disorders develop is not only a key challenge for researchers but also for the wider society, given the rapidly aging populations in developed countries. Advances in this field require new tools with which to recreate neural tissue in vitro and produce realistic disease models. This in turn requires robust and reliable systems for performing 3D in vitro culture of neural lineage cells. This review provides a state of the art update on three-dimensional culture systems for in vitro development of neural tissue, employing a wide range of scaffold types including hydrogels, solid porous polymers, fibrous materials and decellularised tissues as well as microfluidic devices and lab-on-a-chip systems. To provide some context with in vivo development of the central nervous system (CNS), we also provide a brief overview of the neural stem cell niche, neural development and neural differentiation in vitro. We conclude with a discussion of future directions for this exciting and important field of biomaterials research.

  19. Aging-like Phenotype and Defective Lineage Specification in SIRT1-Deleted Hematopoietic Stem and Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Pauline Rimmelé

    2014-07-01

    Full Text Available Aging hematopoietic stem cells (HSCs exhibit defective lineage specification that is thought to be central to increased incidence of myeloid malignancies and compromised immune competence in the elderly. Mechanisms underlying these age-related defects remain largely unknown. We show that the deacetylase Sirtuin (SIRT1 is required for homeostatic HSC maintenance. Differentiation of young SIRT1-deleted HSCs is skewed toward myeloid lineage associated with a significant decline in the lymphoid compartment, anemia, and altered expression of associated genes. Combined with HSC accumulation of damaged DNA and expression patterns of age-linked molecules, these have striking overlaps with aged HSCs. We further show that SIRT1 controls HSC homeostasis via the longevity transcription factor FOXO3. These findings suggest that SIRT1 is essential for HSC homeostasis and lineage specification. They also indicate that SIRT1 might contribute to delaying HSC aging.

  20. Loss of CD44dim Expression from Early Progenitor Cells Marks T-Cell Lineage Commitment in the Human Thymus

    Science.gov (United States)

    Canté-Barrett, Kirsten; Mendes, Rui D.; Li, Yunlei; Vroegindeweij, Eric; Pike-Overzet, Karin; Wabeke, Tamara; Langerak, Anton W.; Pieters, Rob; Staal, Frank J. T.; Meijerink, Jules P. P.

    2017-01-01

    Human T-cell development is less well studied than its murine counterpart due to the lack of genetic tools and the difficulty of obtaining cells and tissues. Here, we report the transcriptional landscape of 11 immature, consecutive human T-cell developmental stages. The changes in gene expression of cultured stem cells on OP9-DL1 match those of ex vivo isolated murine and human thymocytes. These analyses led us to define evolutionary conserved gene signatures that represent pre- and post-αβ T-cell commitment stages. We found that loss of dim expression of CD44 marks human T-cell commitment in early CD7+CD5+CD45dim cells, before the acquisition of CD1a surface expression. The CD44−CD1a− post-committed thymocytes have initiated in frame T-cell receptor rearrangements that are accompanied by loss of capacity to differentiate toward myeloid, B- and NK-lineages, unlike uncommitted CD44dimCD1a− thymocytes. Therefore, loss of CD44 represents a previously unrecognized human thymocyte stage that defines the earliest committed T-cell population in the thymus. PMID:28163708

  1. A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

    Directory of Open Access Journals (Sweden)

    Xiangshan Zhao

    2011-01-01

    Full Text Available Introduction: Emerging evidence suggests a direct role of cancer stem cells (CSCs in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs immortalized with human telomerase reverse transcriptase (hTERT possess properties of mammary stem / progenitor cells. Materials and Methods: In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with g-radiation, share with hTERT, the ability to maintain mammary stem / progenitor cells. Results: The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Conclusions: Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

  2. Evolutionary Convergence of Cell-Specific Gene Expression in Independent Lineages of C4 Grasses1[W][OPEN

    Science.gov (United States)

    John, Christopher R.; Smith-Unna, Richard D.; Woodfield, Helen; Covshoff, Sarah; Hibberd, Julian M.

    2014-01-01

    Leaves of almost all C4 lineages separate the reactions of photosynthesis into the mesophyll (M) and bundle sheath (BS). The extent to which messenger RNA profiles of M and BS cells from independent C4 lineages resemble each other is not known. To address this, we conducted deep sequencing of RNA isolated from the M and BS of Setaria viridis and compared these data with publicly available information from maize (Zea mays). This revealed a high correlation (r = 0.89) between the relative abundance of transcripts encoding proteins of the core C4 pathway in M and BS cells in these species, indicating significant convergence in transcript accumulation in these evolutionarily independent C4 lineages. We also found that the vast majority of genes encoding proteins of the C4 cycle in S. viridis are syntenic to homologs used by maize. In both lineages, 122 and 212 homologous transcription factors were preferentially expressed in the M and BS, respectively. Sixteen shared regulators of chloroplast biogenesis were identified, 14 of which were syntenic homologs in maize and S. viridis. In sorghum (Sorghum bicolor), a third C4 grass, we found that 82% of these trans-factors were also differentially expressed in either M or BS cells. Taken together, these data provide, to our knowledge, the first quantification of convergence in transcript abundance in the M and BS cells from independent lineages of C4 grasses. Furthermore, the repeated recruitment of syntenic homologs from large gene families strongly implies that parallel evolution of both structural genes and trans-factors underpins the polyphyletic evolution of this highly complex trait in the monocotyledons. PMID:24676859

  3. In Vivo Clonal Analysis Reveals Lineage-Restricted Progenitor Characteristics in Mammalian Kidney Development, Maintenance, and Regeneration

    Directory of Open Access Journals (Sweden)

    Yuval Rinkevich

    2014-05-01

    Full Text Available The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

  4. The human fetal lymphocyte lineage: identification by CD27 and LIN28B expression in B cell progenitors

    Science.gov (United States)

    McWilliams, Laurie; Su, Kuei-Ying; Liang, Xiaoe; Liao, Dongmei; Floyd, Serina; Amos, Joshua; Moody, M. Anthony; Kelsoe, Garnett; Kuraoka, Masayuki

    2013-01-01

    CD27, a member of the TNFR superfamily, is used to identify human memory B cells. Nonetheless, CD27+ B cells are present in patients with HIGM1 syndrome who are unable to generate GCs or memory B cells. CD27+IgD+ fetal B cells are present in umbilical cord blood, and CD27 may also be a marker of the human B1-like B cells. To define the origin of naïve CD27+IgD+ human B cells, we studied B cell development in both fetal and adult tissues. In human FL, most CD19+ cells coexpressed CD10, a marker of human developing B cells. Some CD19+CD10+ B cells expressed CD27, and these fetal CD27+ cells were present in the pro-B, pre-B, and immature/transitional B cell compartments. Lower frequencies of phenotypically identical cells were also identified in adult BM. CD27+ pro-B, pre-B, and immature/transitional B cells expressed recombination activating gene-1, terminal deoxynucleotidyl transferase and Vpre-B mRNA comparably to their CD27− counterparts. CD27+ and CD27− developing B cells showed similar Ig heavy chain gene usage with low levels of mutations, suggesting that CD27+ developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27+ developing B cells differed from CD27− developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27+ pro-B cells efficiently generated IgM+IgD+ immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B cell development identifies a physiologic state or lineage for human B cell development distinct from the memory B cell compartment. PMID:23901121

  5. Lamins regulate cell trafficking and lineage maturation of adult human hematopoietic cells

    OpenAIRE

    Shin, Jae-Won; Spinler, Kyle R.; Swift, Joe; Chasis, Joel A.; Mohandas, Narla; Discher, Dennis E.

    2013-01-01

    Comparing human blood cell types, nuclear diversity is visually striking but unexplained: quasi-spherical nuclei in stem/progenitor cells and T cells contrast with multilobed nuclei in neutrophils, giant nuclei in megakaryocytes, and anuclear erythrocytes. We hypothesized broad roles for the major nuclear structure proteins—lamins—and developed mass spectrometry-calibrated intracellular flow cytometry to quantify lamin-A:B ratios. This ratio controls both nuclear viscoelasticity and cell traf...

  6. Phylogenetic analysis of P5 P-type ATPases, a eukaryotic lineage of secretory pathway pumps

    DEFF Research Database (Denmark)

    Møller, Annette; Asp, Torben; Holm, Preben Bach

    2008-01-01

    Eukaryotes encompass a remarkable variety of organisms and unresolved lineages. Different phylogenetic analyses have lead to conflicting conclusions as to the origin and associations between lineages and species. In this work, we investigated evolutionary relationship of a family of cation pumps...... exclusive for the secretory pathway of eukaryotes by combining the identification of lineage-specific genes with phylogenetic evolution of common genes. Sequences of P5 ATPases, which are regarded to be cation pumps in the endoplasmic reticulum (ER), were identified in all eukaryotic lineages but not in any...

  7. Human embryonic and fetal mesenchymal stem cells differentiate toward three different cardiac lineages in contrast to their adult counterparts.

    Science.gov (United States)

    Ramkisoensing, Arti A; Pijnappels, Daniël A; Askar, Saïd F A; Passier, Robert; Swildens, Jim; Goumans, Marie José; Schutte, Cindy I; de Vries, Antoine A F; Scherjon, Sicco; Mummery, Christine L; Schalij, Martin J; Atsma, Douwe E

    2011-01-01

    Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.

  8. Genetic characterization of human T-cell lymphotropic virus type 1 in Mozambique: transcontinental lineages drive the HTLV-1 endemic.

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    Ana Carolina P Vicente

    Full Text Available BACKGROUND: Human T-Cell Lymphotropic Virus Type 1 (HTLV-1 is the etiological agent of adult T-cell leukemia (ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP. It has been estimated that 10-20 million people are infected worldwide, but no successful treatment is available. Recently, the epidemiology of this virus was addressed in blood donors from Maputo, showing rates from 0.9 to 1.2%. However, the origin and impact of HTLV endemic in this population is unknown. OBJECTIVE: To assess the HTLV-1 molecular epidemiology in Mozambique and to investigate their relationship with HTLV-1 lineages circulating worldwide. METHODS: Blood donors and HIV patients were screened for HTLV antibodies by using enzyme immunoassay, followed by Western Blot. PCR and sequencing of HTLV-1 LTR region were applied and genetic HTLV-1 subtypes were assigned by the neighbor-joining method. The mean genetic distance of Mozambican HTLV-1 lineages among the genetic clusters were determined. Human mitochondrial (mt DNA analysis was performed and individuals classified in mtDNA haplogroups. RESULTS: LTR HTLV-1 analysis demonstrated that all isolates belong to the Transcontinental subgroup of the Cosmopolitan subtype. Mozambican HTLV-1 sequences had a high inter-strain genetic distance, reflecting in three major clusters. One cluster is associated with the South Africa sequences, one is related with Middle East and India strains and the third is a specific Mozambican cluster. Interestingly, 83.3% of HIV/HTLV-1 co-infection was observed in the Mozambican cluster. The human mtDNA haplotypes revealed that all belong to the African macrohaplogroup L with frequencies representatives of the country. CONCLUSIONS: The Mozambican HTLV-1 genetic diversity detected in this study reveals that although the strains belong to the most prevalent and worldwide distributed Transcontinental subgroup of the Cosmopolitan subtype, there is a high HTLV diversity that could be

  9. [Progress of Experimental Research on Differentiation of Muscle-Derived Stem Cells into Haematopoietic Lineages in Vitro -Review].

    Science.gov (United States)

    Wang, Juan-Juan; Gao, Xiao-Ning; Chen, Shan-Shan; Zhang, Pan-Pan; Wang, Tao; Dou, Hao-Ying

    2016-12-01

    Muscle-derived stem cells (MDSC) are a population of multipotent stem cells in the muscular tissue. It provide an excellent prospect of hemopathy treatment due to their superiorities, such as rich sources, convenient material resource and a high survival rate after transplantation and so on. However, there are great differences in sampling, separation, purification, and proliferation when MDSC were cultured in vitro. In addition, the proliferation conditions of the MDSC in vitro are yet unclear. The related regulatory mechanisms, which MDSC transformed into haematopoietic cells, need to be investigated. In this article, the experimental researches on the differentiation of MDSC into haematopoietic lineages are reviewed, the concrete problems discussed in this review are culture of MDSC in vitro, identification of MDSC, proleferation of MDSC, differention of MDSC in to hematopoietic lineages and so on.

  10. Lineage analysis of early and advanced tubular adenocarcinomas of the stomach: continuous or discontinuous?

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    Mukaisho Ken-ichi

    2010-06-01

    Full Text Available Abstract Background Eradication of early gastric carcinoma (GC is thought to contribute to reduction in the mortality of GC, given that most of the early GCs progress to the advanced GCs. However, early GC is alternatively considered a dormant variant of GC, and it infrequently progresses to advanced GC. The aim of this study was to clarify the extent of overlap of genetic lineages between early and advanced tubular adenocarcinomas (TUBs of the stomach. Methods Immunohistochemical staining for p53 was performed using 28 surgically resected stomachs with 13 intramucosal and 15 invasive TUBs. By chromosome- and array-based comparative genomic hybridization (CGH, genomic copy number constitution was compared between the mucosal and invasive parts of the invasive TUBs and between the mucosal parts of the invasive and intramucosal TUBs, using 25 and 22 TUBs, respectively. TP53 mutation in exons 5-8 was examined in 20 TUBs. Results Chromosomal CGH revealed that 4q+ and 11q+ were more common in advanced and early TUBs, respectively, whereas copy number changes in 8q and 17p showed no significant differences between early and advanced TUBs. However, array CGH revealed that, of the 13 intramucosal TUBs examined, loss of MYC (MYC- and gain of TP53 (TP53+ was detected in 9 TUBs and MYC+ and/or TP53- was detected in 3 TUBs. Of the mucosal samples of 9 invasive TUBs, 7 showed MYC-/TP53+ and none showed MYC+ and/or TP53-. Of the 9 samples from the invasive parts, 1 (from submucosal cancers showed MYC-/TP53+ and 6 (1 from submucosal and 5 from advanced cancers showed MYC+ and/or TP53-. The latter 6 tumours commonly showed a mutant pattern (diffuse or null in p53 immunohistochemistry, and 4 of the 6 tumours assessable for TP53 sequence analysis revealed mutations. The overall array CGH pattern indicated that, between the mucosal and invasive parts, genetic lineage was found discontinuous in 5 advanced cancers and continuous in 3 submucosal cancers. Conclusions

  11. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

    DEFF Research Database (Denmark)

    Re, Angela; Workman, Christopher; Waldron, Levi;

    2014-01-01

    The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...... interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...

  12. Identification and Characterization of Mouse Otic Sensory Lineage Genes

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    Byron H. Hartman

    2015-03-01

    Full Text Available Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5 as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting

  13. Directing lineage specification of human mesenchymal stem cells by decoupling electrical stimulation and physical patterning on unmodified graphene

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    Balikov, Daniel A.; Fang, Brian; Chun, Young Wook; Crowder, Spencer W.; Prasai, Dhiraj; Lee, Jung Bok; Bolotin, Kiril I.; Sung, Hak-Joon

    2016-07-01

    The organization and composition of the extracellular matrix (ECM) have been shown to impact the propagation of electrical signals in multiple tissue types. To date, many studies with electroactive biomaterial substrates have relied upon passive electrical stimulation of the ionic media to affect cell behavior. However, development of cell culture systems in which stimulation can be directly applied to the material - thereby isolating the signal to the cell-material interface and cell-cell contracts - would provide a more physiologically-relevant paradigm for investigating how electrical cues modulate lineage-specific stem cell differentiation. In the present study, we have employed unmodified, directly-stimulated, (un)patterned graphene as a cell culture substrate to investigate how extrinsic electrical cycling influences the differentiation of naïve human mesenchymal stem cells (hMSCs) without the bias of exogenous biochemicals. We first demonstrated that cyclic stimulation does not deteriorate the cell culture media or result in cytotoxic pH, which are critical experiments for correct interpretation of changes in cell behavior. We then measured how the expression of osteogenic and neurogenic lineage-specific markers were altered simply by exposure to electrical stimulation and/or physical patterns. Expression of the early osteogenic transcription factor RUNX2 was increased by electrical stimulation on all graphene substrates, but the mature marker osteopontin was only modulated when stimulation was combined with physical patterns. In contrast, the expression of the neurogenic markers MAP2 and β3-tubulin were enhanced in all electrical stimulation conditions, and were less responsive to the presence of patterns. These data indicate that specific combinations of non-biological inputs - material type, electrical stimulation, physical patterns - can regulate hMSC lineage specification. This study represents a substantial step in understanding how the interplay of

  14. Systems and Trans-System Level Analysis Identifies Conserved Iron Deficiency Responses in the Plant Lineage[W][OA

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    Urzica, Eugen I.; Casero, David; Yamasaki, Hiroaki; Hsieh, Scott I.; Adler, Lital N.; Karpowicz, Steven J.; Blaby-Haas, Crysten E.; Clarke, Steven G.; Loo, Joseph A.; Pellegrini, Matteo; Merchant, Sabeeha S.

    2012-01-01

    We surveyed the iron nutrition-responsive transcriptome of Chlamydomonas reinhardtii using RNA-Seq methodology. Presumed primary targets were identified in comparisons between visually asymptomatic iron-deficient versus iron-replete cells. This includes the known components of high-affinity iron uptake as well as candidates for distributive iron transport in C. reinhardtii. Comparison of growth-inhibited iron-limited versus iron-replete cells revealed changes in the expression of genes in chloroplastic oxidative stress response pathways, among hundreds of other genes. The output from the transcriptome was validated at multiple levels: by quantitative RT-PCR for assessing the data analysis pipeline, by quantitative proteomics for assessing the impact of changes in RNA abundance on the proteome, and by cross-species comparison for identifying conserved or universal response pathways. In addition, we assessed the functional importance of three target genes, VITAMIN C 2 (VTC2), MONODEHYDROASCORBATE REDUCTASE 1 (MDAR1), and CONSERVED IN THE GREEN LINEAGE AND DIATOMS 27 (CGLD27), by biochemistry or reverse genetics. VTC2 and MDAR1, which are key enzymes in de novo ascorbate synthesis and ascorbate recycling, respectively, are likely responsible for the 10-fold increase in ascorbate content of iron-limited cells. CGLD27/At5g67370 is a highly conserved, presumed chloroplast-localized pioneer protein and is important for growth of Arabidopsis thaliana in low iron. PMID:23043051

  15. Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters.

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    Beamish, Christine A; Strutt, Brenda J; Arany, Edith J; Hill, David J

    2016-04-18

    Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive, glucose-transporter-2-low (Ins(+)Glut2(LO)) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future.

  16. Lineage tracing of neuromesodermal progenitors reveals novel Wnt-dependent roles in trunk progenitor cell maintenance and differentiation.

    Science.gov (United States)

    Garriock, Robert J; Chalamalasetty, Ravindra B; Kennedy, Mark W; Canizales, Lauren C; Lewandoski, Mark; Yamaguchi, Terry P

    2015-05-01

    In the development of the vertebrate body plan, Wnt3a is thought to promote the formation of paraxial mesodermal progenitors (PMPs) of the trunk region while suppressing neural specification. Recent lineage-tracing experiments have demonstrated that these trunk neural progenitors and PMPs derive from a common multipotent progenitor called the neuromesodermal progenitor (NMP). NMPs are known to reside in the anterior primitive streak (PS) region; however, the extent to which NMPs populate the PS and contribute to the vertebrate body plan, and the precise role that Wnt3a plays in regulating NMP self-renewal and differentiation are unclear. To address this, we used cell-specific markers (Sox2 and T) and tamoxifen-induced Cre recombinase-based lineage tracing to locate putative NMPs in vivo. We provide functional evidence for NMP location primarily in the epithelial PS, and to a lesser degree in the ingressed PS. Lineage-tracing studies in Wnt3a/β-catenin signaling pathway mutants provide genetic evidence that trunk progenitors normally fated to enter the mesodermal germ layer can be redirected towards the neural lineage. These data, combined with previous PS lineage-tracing studies, support a model that epithelial anterior PS cells are Sox2(+)T(+) multipotent NMPs and form the bulk of neural progenitors and PMPs of the posterior trunk region. Finally, we find that Wnt3a/β-catenin signaling directs trunk progenitors towards PMP fates; however, our data also suggest that Wnt3a positively supports a progenitor state for both mesodermal and neural progenitors.

  17. Trophic factors from adipose tissue-derived multi-lineage progenitor cells promote cytodifferentiation of periodontal ligament cells

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    Sawada, Keigo [Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka (Japan); Takedachi, Masahide, E-mail: takedati@dent.osaka-u.ac.jp [Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka (Japan); Yamamoto, Satomi; Morimoto, Chiaki; Ozasa, Masao; Iwayama, Tomoaki [Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka (Japan); Lee, Chun Man [Medical Center for Translational Research, Osaka University Hospital, Osaka (Japan); Okura, Hanayuki; Matsuyama, Akifumi [Research on Disease Bioresources, Platform of Therapeutics for Rare Disease, National Institute of Biomedical Innovation, Osaka (Japan); Kitamura, Masahiro; Murakami, Shinya [Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka (Japan)

    2015-08-14

    Stem and progenitor cells are currently being investigated for their applicability in cell-based therapy for periodontal tissue regeneration. We recently demonstrated that the transplantation of adipose tissue-derived multi-lineage progenitor cells (ADMPCs) enhances periodontal tissue regeneration in beagle dogs. However, the molecular mechanisms by which transplanted ADMPCs induce periodontal tissue regeneration remain to be elucidated. In this study, trophic factors released by ADMPCs were examined for their paracrine effects on human periodontal ligament cell (HPDL) function. ADMPC conditioned medium (ADMPC-CM) up-regulated osteoblastic gene expression, alkaline phosphatase activity and calcified nodule formation in HPDLs, but did not significantly affect their proliferative response. ADMPCs secreted a number of growth factors, including insulin-like growth factor binding protein 6 (IGFBP6), hepatocyte growth factor and vascular endothelial growth factor. Among these, IGFBP6 was most highly expressed. Interestingly, the positive effects of ADMPC-CM on HPDL differentiation were significantly suppressed by transfecting ADMPCs with IGFBP6 siRNA. Our results suggest that ADMPCs transplanted into a defect in periodontal tissue release trophic factors that can stimulate the differentiation of HPDLs to mineralized tissue-forming cells, such as osteoblasts and cementoblasts. IGFBP6 may play crucial roles in ADMPC-induced periodontal regeneration. - Highlights: • ADMPC-derived humoral factors stimulate cytodifferentiation of HPDLs. • ADMPCs secret growth factors including IGFBP6, VEGF and HGF. • IGFBP6 is involved in the promotion effect of ADMPC-CM on HPDL cytodifferentiation.

  18. Cell lineage specific distribution of H3K27 trimethylation accumulation in an in vitro model for human implantation.

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    Gijs Teklenburg

    Full Text Available Female mammals inactivate one of their two X-chromosomes to compensate for the difference in gene-dosage with males that have just one X-chromosome. X-chromosome inactivation is initiated by the expression of the non-coding RNA Xist, which coats the X-chromosome in cis and triggers gene silencing. In early mouse development the paternal X-chromosome is initially inactivated in all cells of cleavage stage embryos (imprinted X-inactivation followed by reactivation of the inactivated paternal X-chromosome exclusively in the epiblast precursors of blastocysts, resulting temporarily in the presence of two active X-chromosomes in this specific lineage. Shortly thereafter, epiblast cells randomly inactivate either the maternal or the paternal X-chromosome. XCI is accompanied by the accumulation of histone 3 lysine 27 trimethylation (H3K27me3 marks on the condensed X-chromosome. It is still poorly understood how XCI is regulated during early human development. Here we have investigated lineage development and the distribution of H3K27me3 foci in human embryos derived from an in-vitro model for human implantation. In this system, embryos are co-cultured on decidualized endometrial stromal cells up to day 8, which allows the culture period to be extended for an additional two days. We demonstrate that after the co-culture period, the inner cell masses have relatively high cell numbers and that the GATA4-positive hypoblast lineage and OCT4-positive epiblast cell lineage in these embryos have segregated. H3K27me3 foci were observed in ∼25% of the trophectoderm cells and in ∼7.5% of the hypoblast cells, but not in epiblast cells. In contrast with day 8 embryos derived from the co-cultures, foci of H3K27me3 were not observed in embryos at day 5 of development derived from regular IVF-cultures. These findings indicate that the dynamics of H3K27me3 accumulation on the X-chromosome in human development is regulated in a lineage specific fashion.

  19. Genome analysis of a transmissible lineage of pseudomonas aeruginosa reveals pathoadaptive mutations and distinct evolutionary paths of hypermutators.

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    Rasmus Lykke Marvig

    Full Text Available Genome sequencing of bacterial pathogens has advanced our understanding of their evolution, epidemiology, and response to antibiotic therapy. However, we still have only a limited knowledge of the molecular changes in in vivo evolving bacterial populations in relation to long-term, chronic infections. For example, it remains unclear what genes are mutated to facilitate the establishment of long-term existence in the human host environment, and in which way acquisition of a hypermutator phenotype with enhanced rates of spontaneous mutations influences the evolutionary trajectory of the pathogen. Here we perform a retrospective study of the DK2 clone type of P. aeruginosa isolated from Danish patients suffering from cystic fibrosis (CF, and analyze the genomes of 55 bacterial isolates collected from 21 infected individuals over 38 years. Our phylogenetic analysis of 8,530 mutations in the DK2 genomes shows that the ancestral DK2 clone type spread among CF patients through several independent transmission events. Subsequent to transmission, sub-lineages evolved independently for years in separate hosts, creating a unique possibility to study parallel evolution and identification of genes targeted by mutations to optimize pathogen fitness (pathoadaptive mutations. These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages. The long-term co-existence of both normal and hypermutator populations enabled comparative investigations of the mutation dynamics in homopolymeric sequences in which hypermutators are particularly prone to mutations. We find a positive exponential correlation between the length of the homopolymer and its likelihood to acquire mutations and identify two homopolymer-containing genes preferentially mutated in hypermutators. This homopolymer facilitated differential

  20. Effects of combination of melatonin and laser irradiation on ovarian cancer cells and endothelial lineage viability.

    Science.gov (United States)

    Akbarzadeh, Maryam; Nouri, Mohammad; Banekohal, Maryam Vahidi; Cheraghi, Omid; Tajalli, Habib; Movassaghpour, Aliakbar; Soltani, Sina; Cheraghi, Hadi; Feizy, Navid; Montazersaheb, Soheila; Rahbarghazi, Reza; Samadi, Nasser

    2016-11-01

    The main goal of anti-cancer therapeutic approaches is to induce apoptosis in tumor masses but not in the normal tissues. Nevertheless, the combination of photodynamic irradiation with complementary oncostatic agents contributes to better therapeutic performance. Here, we applied two different cell lines; SKOV3 ovarian carcinoma cells and HUVECs umbilical cord cells as in vitro models to pinpoint whether pharmacological concentration of melatonin in combination with photodynamic therapy induces cell cytotoxicity. The cells were separately treated with various concentrations of melatonin (0 to 10 mM) and photodynamic irradiation alone or in combination. Cells were preliminary exposed to increasing concentrations of melatonin for 24 h and subsequently underwent laser irradiation for 60 s with an output power of 80 mW in continuous mode at 675 nm wavelength and a total light dose of 13.22 J/cm(2). Cell viability, apoptosis/necrosis rates, and reactive oxygen species levels as well as heat shock protein 70 expression were monitored after single and combined treatments. A statistical analysis was performed by applying one-way analysis of variance (ANOVA) and post hoc Tukey's test. Combination treatment of both cell lines caused a marked increase in apoptosis/necrosis rate, reactive oxygen species generation, and heat shock protein 70 expression compared to incubation of the cells with each agent alone (p melatonin as a potent stimulus for enhancing the efficacy of laser on induction of apoptosis in tumor cells.

  1. Characterization of the platelet-derived growth factor receptor-α-positive cell lineage during murine late lung development.

    Science.gov (United States)

    Ntokou, Aglaia; Klein, Friederike; Dontireddy, Daria; Becker, Sven; Bellusci, Saverio; Richardson, William D; Szibor, Marten; Braun, Thomas; Morty, Rory E; Seeger, Werner; Voswinckel, Robert; Ahlbrecht, Katrin

    2015-11-01

    A reduced number of alveoli is the structural hallmark of diseases of the neonatal and adult lung, where alveoli either fail to develop (as in bronchopulmonary dysplasia), or are progressively destroyed (as in chronic obstructive pulmonary disease). To correct the loss of alveolar septa through therapeutic regeneration, the mechanisms of septa formation must first be understood. The present study characterized platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cell populations during late lung development in mice. PDGFRα(+) cells (detected using a PDGFRα(GFP) reporter line) were noted around the proximal airways during the pseudoglandular stage. In the canalicular stage, PDGFRα(+) cells appeared in the more distal mesenchyme, and labeled α-smooth muscle actin-positive tip cells in the secondary crests and lipofibroblasts in the primary septa during alveolarization. Some PDGFRα(+) cells appeared in the mesenchyme of the adult lung. Over the course of late lung development, PDGFRα(+) cells consistently expressed collagen I, and transiently expressed markers of mesenchymal stem cells. With the use of both, a constitutive and a conditional PDGFRα(Cre) line, it was observed that PDGFRα(+) cells generated alveolar myofibroblasts including tip cells of the secondary crests, and lipofibroblasts. These lineages were committed before secondary septation. The present study provides new insights into the time-dependent commitment of the PDGFRα(+) cell lineage to lipofibroblasts and myofibroblasts during late lung development that is needed to better understand the cellular contribution to the process of alveolarization.

  2. Efficient production of trophoblast lineage cells from human induced pluripotent stem cells.

    Science.gov (United States)

    Kojima, Junya; Fukuda, Atsushi; Taira, Hayato; Kawasaki, Tomoyuki; Ito, Hiroe; Kuji, Naoaki; Isaka, Keiichi; Umezawa, Akihiro; Akutsu, Hidenori

    2017-03-13

    Human induced pluripotent stem cells (hiPSCs) are potentially useful in both clinical applications and basic biological research. hiPSCs can differentiate into extra-embryonic cells in the presence of BMP4. However, the differentiation potential of hiPSCs can be affected by culture conditions or genetic variation. In this study, we investigated the effect of various BMP4 concentrations on the expression states of trophoblast markers and the optimal conditions for trophoblast induction. A high-fidelity gene expression assay using hiPSC lines showed that the expression levels of various trophoblast marker genes, such as KRT7, GCM1, CGB, and HLA-G, were upregulated by BMP4 in a dose-dependent manner in all types of hiPSCs used in this study. Treatment with high doses of BMP4 for prolonged periods increased the ratio of cells with trophoblast markers irrespective of the presence of bFGF. We found that the expression states of major pluripotency- and differentiation-related protein-coding genes in BMP4-treated cells depended on culture conditions rather than donor cell types. However, miRNA expression states were affected by donor cell types rather than BMP4 dose. Furthermore, the effect of the presence of bFGF on differentiation potential of KRT7-positive cells differed among iPSC types. Mechanistically, chromatin states around KRT7 promoter regions were comparable among the iPSC types used in this study, indicating that hiPSC chromatin state at these regions is not a parameter for cytotrophoblast differentiation potential. In conclusion, the optimal conditions for trophoblast differentiation from hiPSCs differ according to parental cell line.Laboratory Investigation advance online publication, 13 March 2017; doi:10.1038/labinvest.2016.159.

  3. Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions.

    Science.gov (United States)

    Gez, Swetlana; Crossett, Ben; Christopherson, Richard I

    2007-09-01

    Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.

  4. Phylogeny and FISH probe analysis of the “Candidatus Competibacter”-lineage in wastewater treatment systems

    DEFF Research Database (Denmark)

    Nittami, Tadashi; McIlroy, Simon Jon; Kanai, Eri

    . This study aimed at resolving the phylogeny and reassessing FISH probes for coverage of the diversity of the lineage in EBPR systems. Analysis of the level of 16S rRNA gene sequence diversity of lineage members (> 89%) suggests that it is more appropriately viewed as a family rather than a single genus......Our understanding of the microbial ecology of enhanced biological phosphorus removal (EBPR) wastewater treatment systems has been greatly advanced through the application of molecular methods such as fluorescence in situ hybridization (FISH). Considerable attention has been directed...... the gammaproteobacterial “Candidatus Competibacter”-lineage. The group is currently delineated by 8 FISH probe defined phylotypes, although further undescribed phylogenetic diversity beyond what is covered by these probes is evident. Where studied, marked differences in physiology between members are observed, including...

  5. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology.

    Science.gov (United States)

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-03-17

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.

  6. Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology

    Science.gov (United States)

    Hu, C; Cao, H; Pan, X; Li, J; He, J; Pan, Q; Xin, J; Yu, X; Li, J; Wang, Y; Zhu, D; Li, L

    2016-01-01

    Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated. PMID:26986509

  7. Establishment of a GM-CSF-dependent megakaryoblastic cell line with the potential to differentiate into an eosinophilic lineage in response to retinoic acids.

    Science.gov (United States)

    Ma, F; Koike, K; Higuchi, T; Kinoshita, T; Takeuchi, K; Mwamtemi, H H; Sawai, N; Kamijo, T; Shiohara, M; Horie, S; Kawa, S; Sasaki, Y; Hidaka, E; Yamagami, O; Yamashita, T; Koike, T; Ishii, E; Komiyama, A

    1998-02-01

    We recently established a human granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line (HML) from colony-constituent cells grown by peripheral blood cells of a patient with acute megakaryoblastic leukaemia. The HML cells possessed megakaryocytic features, as determined by cytochemical, electron microscopic and flow cytometric analysis. In the present study we examined the effects of retinoic acid (RA) on the development of HML cells. All-trans-RA, 13-cis-RA and 9-cis-RA at 10(-8) mol/l to 10(-5) mol/l inhibited the GM-CSF-dependent cell growth. Some of the RA-treated cells contained prominent azurophilic granules and were positive for peroxidase. They also reacted with Biebrich scarlet, Luxol fast blue and a monoclonal antibody against eosinophil peroxidase. In addition, exposure to RA increased the frequency and the intensity of major basic protein-positive cells. However, eosinophil-derived neurotoxin and eosinophil cationic protein were not detected or were only detected at a low level in the lysates of the HML cells treated with RA. Although IL-5 alone could not stimulate cell growth, the addition of IL-5 to the cultures containing stem cell factor + all-trans-RA was required for the expression of the eosinophilic phenotype. These results suggest that the HML cell line is a megakaryoblastic cell line with the potential to differentiate into the eosinophilic lineage. HML cells may be a useful model for elucidating the eosinophilic differentiation programme.

  8. Mixed lineage kinase 3 inhibits phorbol myristoyl acetate-induced DNA synthesis but not osteopontin expression in rat mesangial cells.

    Science.gov (United States)

    Parameswaran, Narayanan; Hall, Carolyn S; Bock, Barbara C; Sparks, Harvey V; Gallo, Kathleen A; Spielman, William S

    2002-12-01

    Mixed lineage kinase 3 (MLK 3) (also called SPRK or PTK-1) is a recently described member of the family of the mixed lineage kinase subfamily of Ser/Thr protein kinases that interacts with mitogen-activated protein kinase pathways. In order to test the biological relevance and potential interaction of MLK 3 with protein kinase C-mediated signaling pathways, human MLK 3 was stably expressed in rat glomerular mesangial cells using a retroviral vector (LXSN) and the effects of phorbol myristoyl acetate (PMA) on DNA synthesis and osteopontin mRNA expression were examined. In control (vector-transfected) mesangial cells PMA increased [3H]-thymidine incorporation in a concentration-dependent manner. In mesangial cells stably expressing MLK 3, the PMA-induced increase in [3H]-thymidine incorporation was significantly reduced (> 50%). However, the PMA-induced increase in osteopontin mRNA was not affected by MLK 3 expression. To determine the mechanisms of these effects, activation of ERK2, JNK1 and p38 in response to PMA was examined in both vector and MLK 3 transfected cells. ERK2 activation was increased several fold by PMA in control cells but was attenuated significantly in MLK 3 expressing cells, suggesting that MLK 3 expression in mesangial cells can negatively regulate the ERK pathway. PMA had no significant effect on JNK and P38 activation, in either vector- or MLK 3-expressing cells. PD98059, a MEK inhibitor blocked PMA-induced DNA synthesis without affecting osteopontin expression. These results suggest that while protein kinase C activation increases cellular proliferation and osteopontin mRNA expression, over-expression of MLK 3 affects only the PKC-induced DNA synthesis, probably through inhibition of ERK. These results also indicate a novel mechanism of growth regulation by a member of the mixed-lineage kinase family that might have significant therapeutic implications in proliferative glomerulonephritis.

  9. Comparative cell-specific transcriptomics reveals differentiation of C4 photosynthesis pathways in switchgrass and other C4 lineages.

    Science.gov (United States)

    Rao, Xiaolan; Lu, Nan; Li, Guifen; Nakashima, Jin; Tang, Yuhong; Dixon, Richard A

    2016-03-01

    Almost all C4 plants require the co-ordination of the adjacent and fully differentiated cell types, mesophyll (M) and bundle sheath (BS). The C4 photosynthetic pathway operates through two distinct subtypes based on how malate is decarboxylated in BS cells; through NAD-malic enzyme (NAD-ME) or NADP-malic enzyme (NADP-ME). The diverse or unique cell-specific molecular features of M and BS cells from separate C4 subtypes of independent lineages remain to be determined. We here provide an M/BS cell type-specific transcriptome data set from the monocot NAD-ME subtype switchgrass (Panicum virgatum). A comparative transcriptomics approach was then applied to compare the M/BS mRNA profiles of switchgrass, monocot NADP-ME subtype C4 plants maize and Setaria viridis, and dicot NAD-ME subtype Cleome gynandra. We evaluated the convergence in the transcript abundance of core components in C4 photosynthesis and transcription factors to establish Kranz anatomy, as well as gene distribution of biological functions, in these four independent C4 lineages. We also estimated the divergence between NAD-ME and NADP-ME subtypes of C4 photosynthesis in the two cell types within C4 species, including differences in genes encoding decarboxylating enzymes, aminotransferases, and metabolite transporters, and differences in the cell-specific functional enrichment of RNA regulation and protein biogenesis/homeostasis. We suggest that C4 plants of independent lineages in both monocots and dicots underwent convergent evolution to establish C4 photosynthesis, while distinct C4 subtypes also underwent divergent processes for the optimization of M and BS cell co-ordination. The comprehensive data sets in our study provide a basis for further research on evolution of C4 species.

  10. Small Molecule Cardiogenol C Upregulates Cardiac Markers and Induces Cardiac Functional Properties in Lineage-Committed Progenitor Cells

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    Agnes K. Mike

    2014-01-01

    Full Text Available Background/Aims: Cell transplantation into the heart is a new therapy after myocardial infarction. Its success, however, is impeded by poor donor cell survival and by limited transdifferentiation of the transplanted cells into functional cardiomyocytes. A promising strategy to overcome these problems is the induction of cardiomyogenic properties in donor cells by small molecules. Methods: Here we studied cardiomyogenic effects of the small molecule compound cardiogenol C (CgC, and structural derivatives thereof, on lineage-committed progenitor cells by various molecular biological, biochemical, and functional assays. Results: Treatment with CgC up-regulated cardiac marker expression in skeletal myoblasts. Importantly, the compound also induced cardiac functional properties: first, cardiac-like sodium currents in skeletal myoblasts, and secondly, spontaneous contractions in cardiovascular progenitor cell-derived cardiac bodies. Conclusion: CgC induces cardiomyogenic function in lineage-committed progenitor cells, and can thus be considered a promising tool to improve cardiac repair by cell therapy.

  11. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    Science.gov (United States)

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

  12. In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts

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    F Di Meglio

    2009-08-01

    Full Text Available The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR. The amount of proteins characteristic for cardiac cells (a-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively inclines toward an increase in both a-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart.

  13. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes.

    Science.gov (United States)

    Xiao, Xiangwei; Guo, Ping; Prasadan, Krishna; Shiota, Chiyo; Peirish, Lauren; Fischbach, Shane; Song, Zewen; Gaffar, Iljana; Wiersch, John; El-Gohary, Yousef; Husain, Sohail Z; Gittes, George K

    2014-12-01

    Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type- and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

  14. Single-cell analysis: Advances and future perspectives

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    Emir Hodzic

    2016-11-01

    Full Text Available The last several years have seen rapid development of technologies and methods that permit a detailed analysis of the genome and transcriptome of a single cell. Recent evidence from studies of single cells reveals that each cell type has a distinct lineage and function. The lineage and stage of development of each cell determine how they respond to each other and the environment. Experimental approaches that utilize single-cell analysis are effective means to understand how cell networks work in concert to coordinate a response at the population level; recent progress in single-cell analysis is offering a glimpse at the future.

  15. Fetal male lineage determination by analysis of Y-chromosome STR haplotype in maternal plasma.

    Science.gov (United States)

    Barra, Gustavo Barcelos; Santa Rita, Ticiane Henriques; Chianca, Camilla Figueiredo; Velasco, Lara Francielle Ribeiro; de Sousa, Claudia Ferreira; Nery, Lídia Freire Abdalla; Costa, Sandra Santana Soares

    2015-03-01

    The aim of this study is to determine the fetus Y-STR haplotype in maternal plasma during pregnancy and estimate, non-invasively, if the alleged father and fetus belong to the same male lineage. The study enrolled couples with singleton pregnancies and known paternity. All participants signed informed consent and the local ethics committee approved the study. Peripheral blood was collected in EDTA tubes (mother) and in FTA paper (father). Maternal plasma DNA was extracted by using NucliSens EasyMAG. Fetal gender was determined by qPCR targeting DYS-14 in maternal plasma and it was also confirmed after the delivery. From all included volunteers, the first consecutive 20 mothers bearing male fetuses and 10 mothers bearing female fetuses were selected for the Y-STR analysis. The median gestational age was 12 weeks (range 12-36). All DNA samples were subjected to PCR amplification by PowerPlex Y23, ampFLSTR Yfiler, and two in-house multiplexes, which together accounts for 27 different Y-STR. The PCR products were detected with 3500 Genetic Analyzer and they were analyzed using GeneMapper-IDX. Fetuses' haplotypes (Yfiler format) were compared to other 5328 Brazilian haplotypes available on Y-chromosome haplotypes reference database (YHRD). As a result, between 22 and 27 loci were successfully amplified from maternal plasma in all 20 cases of male fetuses. None of the women bearing female fetuses had a falsely amplified Y-STR haplotype. The haplotype detected in maternal plasma completely matched the alleged father haplotype in 16 out of the 20 cases. Four cases showed single mismatches and they did not configure exclusions; 1 case showed a mutation in the DYS 458 locus due to the loss of one repeat unit and 3 cases showed one DYS 385I/II locus dropout. All mismatches were confirmed after the delivery. Seventeen fetuses' haplotypes were not found in YHRD and one of them had a mutation, which corresponded to the paternity probability of 99.9812% and 95.7028%, respectively

  16. Ancestral trees for modeling stem cell lineages genetically rather than functionally: understanding mutation accumulation and distinguishing the restrictive cancer stem cell propagation theory and the unrestricted cell propagation theory of human tumorigenesis.

    Science.gov (United States)

    Shibata, Darryl K; Kern, Scott E

    2008-01-01

    Cancer stem cells either could be rare or common in tumors, constituting the major distinction between the two fundamentally opposed theoretical models of tumor progression: A newer and restrictive stem cell propagation model, in which the stem cells are a small and special minority of the tumor cells, and a standard older model, an unrestricted cell proliferation theory, in which many or most tumor cells are capable of indefinite generations of cell division. Stem cells of tumors are difficult to quantitate using functional assays, and the validity of the most common assays is seriously questioned. Nonetheless, stem cells are an essential component of any tumorigenesis model. Alternative approaches to studying tumor stem cells should be explored. Cell populations can be conceived of as having a genealogy, a relationship of cells to their ancestral lineage, from the zygote to the adult cells or neoplasms. Models using ancestral trees thus offer an anatomic and genetic means to "observe" stem cells independent of artificial conditions. Ancestral trees broaden our attention backward along a lineage, to the zygote stage, and thereby add insight into how the mutations of tumors accumulate. It is possible that a large fraction of mutations in a tumor originate from normal, endogenous, replication errors (nearly all being passenger mutations) occurring prior to the emergence of the first transformed cell. Trees can be constructed from experimental measurements - molecular clocks - of real human tissues and tumors. Detailed analysis of single-cell methylation patterns, heritable yet slightly plastic, now can provide this information in the necessary depth. Trees based on observations of molecular clocks may help us to distinguish between competing theories regarding the proliferative properties among cells of actual human tumors, to observe subtle and difficult phenomena such as the extinction of stem lineages, and to address the origins and rates of mutations in various

  17. Fibroblasts isolated from human middle turbinate mucosa cause neural progenitor cells to differentiate into glial lineage cells.

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    Xingjia Wu

    Full Text Available Transplantation of olfactory ensheathing cells (OECs is a potential therapy for repair of spinal cord injury (SCI. Autologous transplantation of OECs has been reported in clinical trials. However, it is still controversial whether purified OECs or olfactory mucosa containing OECs, fibroblasts and other cells should be used for transplantation. OECs and fibroblasts were isolated from olfactory mucosa of the middle turbinate from seven patients. The percentage of OECs with p75(NTR+ and GFAP(+ ranged from 9.2% to 73.2%. Fibroblasts were purified and co-cultured with normal human neural progenitors (NHNPs. Based on immunocytochemical labeling, NHNPs were induced into glial lineage cells when they were co-cultured with the mucosal fibroblasts. These results demonstrate that OECs can be isolated from the mucosa of the middle turbinate bone as well as from the dorsal nasal septum and superior turbinates, which are the typical sites for harvesting OECs. Transplantation of olfactory mucosa containing fibroblasts into the central nervous system (CNS needs to be further investigated before translation to clinical application.

  18. BRILIA: Integrated Tool for High-Throughput Annotation and Lineage Tree Assembly of B-Cell Repertoires

    Science.gov (United States)

    Lee, Donald W.; Khavrutskii, Ilja V.; Wallqvist, Anders; Bavari, Sina; Cooper, Christopher L.; Chaudhury, Sidhartha

    2017-01-01

    The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from Variable (V), Diversity (D), and Joining (J) (VDJ) recombination and somatic hypermutation (SHM) during B-cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable complementarity determining region 3 (CDR3), which plays a critical role in antigen specificity and binding affinity. Tracking the selection and mutation of the CDR3 can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present B-cell repertoire inductive lineage and immunosequence annotator (BRILIA), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94 and 70% accuracy, respectively, and it has a 90% SHM-positive prediction rate in the CDR3 of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B-cell clusters and yielded inconsistent SHM frequencies. We demonstrate

  19. Genomic Analysis of the Evolution and Global Spread of Hyper-invasive Meningococcal Lineage 5

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    Odile B. Harrison

    2015-03-01

    Interpretation: These data were inconsistent with a single point of origin followed by pandemic spread, rather suggesting that the sub-lineages had diversified and spread by asymptomatic transmission, with multiple distinct strains causing localised hyperendemic outbreaks.

  20. MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny

    NARCIS (Netherlands)

    Horton, S J; Jaques, J; Woolthuis, C; van Dijk, J; Mesuraca, M; Huls, G; Morrone, G; Vellenga, E; Schuringa, J J

    2013-01-01

    The MLL-AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of t

  1. High cell density and latent membrane protein 1 expression induce cleavage of the mixed lineage leukemia gene at 11q23 in nasopharyngeal carcinoma cell line

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    Sim Sai-Peng

    2010-09-01

    Full Text Available Abstract Background Nasopharyngeal carcinoma (NPC is commonly found in Southern China and South East Asia. Epstein-Barr virus (EBV infection is well associated with NPC and has been implicated in its pathogenesis. Moreover, various chromosome rearrangements were reported in NPC. However, the underlying mechanism of chromosome rearrangement remains unclear. Furthermore, the relationship between EBV and chromosome rearrangement with respect to the pathogenesis of NPC has not been established. We hypothesize that during virus- or stress-induced apoptosis, chromosomes are initially cleaved at the base of the chromatin loop domain structure. Upon DNA repair, cell may survive with rearranged chromosomes. Methods In this study, cells were seeded at various densities to induce apoptosis. Genomic DNA extracted was processed for Southern hybridization. In order to investigate the role of EBV, especially the latent membrane protein 1 (LMP1, LMP1 gene was overexpressed in NPC cells and chromosome breaks were analyzed by inverse polymerase chain (IPCR reaction. Results Southern analysis revealed that high cell density resulted in cleavage of the mixed lineage leukemia (MLL gene within the breakpoint cluster region (bcr. This high cell density-induced cleavage was significantly reduced by caspase inhibitor, Z-DEVD-FMK. Similarly, IPCR analysis showed that LMP1 expression enhanced cleavage of the MLL bcr. Breakpoint analysis revealed that these breaks occurred within the matrix attachment region/scaffold attachment region (MAR/SAR. Conclusions Since MLL locates at 11q23, a common deletion site in NPC, our results suggest a possibility of stress- or virus-induced apoptosis in the initiation of chromosome rearrangements at 11q23. The breakpoint analysis results also support the role of chromatin structure in defining the site of chromosome rearrangement.

  2. Aging alters bone-fat reciprocity by shifting in vivo mesenchymal precursor cell fate towards an adipogenic lineage.

    Science.gov (United States)

    Singh, Lakshman; Brennan, Tracy A; Russell, Elizabeth; Kim, Jung-Hoon; Chen, Qijun; Brad Johnson, F; Pignolo, Robert J

    2016-04-01

    Bone marrow derived mesenchymal progenitor cells (MPCs) play an important role in bone homeostasis. Age-related changes occur in bone resulting in a decrease in bone density and a relative increase in adipocity. Although in vitro studies suggest the existence of an age-related lineage switch between osteogenic and adipogenic fates, stem cell and microenvironmental contributions to this process have not been elucidated in vivo. In order to study the effects of MPC and microenvironmental aging on functional engraftment and lineage switching, transplantation studies were performed under non-myeloablative conditions in old recipients, with donor MPCs derived from young and old green fluorescent protein (GFP) transgenic mice. Robust engraftment by young MPCs or their progeny was observed in the marrow, bone-lining region and in the matrix of young recipients; however, significantly lower engraftment was seen at the same sites in old recipients transplanted with old MPCs. Differentiation of transplanted MPCs strongly favored adipogenesis over osteogenesis in old recipients irrespective of MPC donor age, suggesting that microenvironmental alterations that occur with in vivo aging are predominately responsible for MPC lineage switching. These data indicate that aging alters bone-fat reciprocity and differentiation of mesenchymal progenitors towards an adipogenic fate.

  3. Phylogeographic analysis reveals a deep lineage split within North Atlantic Littorina saxatilis.

    Science.gov (United States)

    Doellman, Meredith M; Trussell, Geoffrey C; Grahame, John W; Vollmer, Steve V

    2011-11-07

    Phylogeographic studies provide critical insight into the evolutionary histories of model organisms; yet, to date, range-wide data are lacking for the rough periwinkle Littorina saxatilis, a classic example of marine sympatric speciation. Here, we use mitochondrial DNA (mtDNA) sequence data to demonstrate that L. saxatilis is not monophyletic for this marker, but is composed of two distinct mtDNA lineages (I and II) that are shared with sister species Littorina arcana and Littorina compressa. Bayesian coalescent dating and phylogeographic patterns indicate that both L. saxatilis lineages originated in the eastern North Atlantic, around the British Isles, at approximately 0.64 Ma. Both lineages are now distributed broadly across the eastern, central and western North Atlantic, and show strong phylogeographic structure among regions. The Iberian Peninsula is genetically distinct, suggesting prolonged isolation from northeastern North Atlantic populations. Western North Atlantic populations of L. saxatilis lineages I and II predate the last glacial maximum and have been isolated from eastern North Atlantic populations since that time. This identification of two distinct, broadly distributed mtDNA lineages further complicates observed patterns of repeated incipient ecological speciation in L. saxatilis, because the sympatric origins of distinct ecotype pairs on eastern North Atlantic shores may be confounded by admixture of divergent lineages.

  4. Determining Lineage Pathways from Cellular Barcoding Experiments

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    Leïla Perié

    2014-02-01

    Full Text Available Cellular barcoding and other single-cell lineage-tracing strategies form experimental methodologies for analysis of in vivo cell fate that have been instrumental in several significant recent discoveries. Due to the highly nonlinear nature of proliferation and differentiation, interrogation of the resulting data for evaluation of potential lineage pathways requires a new quantitative framework complete with appropriate statistical tests. Here, we develop such a framework, illustrating its utility by analyzing data from barcoded multipotent cells of the blood system. This application demonstrates that the data require additional paths beyond those found in the classical model, which leads us to propose that hematopoietic differentiation follows a loss of potential mechanism and to suggest further experiments to test this deduction. Our quantitative framework can evaluate the compatibility of lineage trees with barcoded data from any proliferating and differentiating cell system.

  5. FGFR-1 is required by epicardium-derived cells for myocardial invasion and correct coronary vascular lineage differentiation.

    Science.gov (United States)

    Pennisi, David J; Mikawa, Takashi

    2009-04-01

    Critical steps in coronary vascular formation include the epithelial-mesenchyme transition (EMT) that epicardial cells undergo to become sub-epicardial; the invasion of the myocardium; and the differentiation of coronary lineages. However, the factors controlling these processes are not completely understood. Epicardial and coronary vascular precursors migrate to the avascular heart tube during embryogenesis via the proepicardium (PE). Here, we show that in the quail embryo fibroblast growth factor receptor (FGFR)-1 is expressed in a spatially and temporally restricted manner in the PE and epicardium-derived cells, including vascular endothelial precursors, and is up-regulated in epicardial cells after EMT. We used replication-defective retroviral vectors to over-express or knock-down FGFR-1 in the PE. FGFR-1 over-expression resulted in increased epicardial EMT. Knock-down of FGFR-1, however, did not inhibit epicardial EMT but greatly compromised the ability of PE progeny to invade the myocardium. The latter could, however, contribute to endothelia and smooth muscle of sub-epicardial vessels. Correct FGFR-1 levels were also important for correct coronary lineage differentiation with, at E12, an increase in the proportion of endothelial cells amongst FGFR-1 over-expressing PE progeny and a decrease in the proportion of smooth muscle cells in antisense FGFR-1 virus-infected PE progeny. Finally, in a heart explant system, constitutive activation of FGFR-1 signaling in epicardial cells resulted in increased delamination from the epicardium, invasion of the sub-epicardium, and invasion of the myocardium. These data reveal novel roles for FGFR-1 signaling in epicardial biology and coronary vascular lineage differentiation, and point to potential new therapeutic avenues.

  6. Lineage Tracing and Cell Ablation Identify a Post-Aire-Expressing Thymic Epithelial Cell Population

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    Todd C. Metzger

    2013-10-01

    Full Text Available Thymic epithelial cells in the medulla (mTECs play a critical role in enforcing central tolerance through expression and presentation of tissue-specific antigens (TSAs and deletion of autoreactive thymocytes. TSA expression requires autoimmune regulator (Aire, a transcriptional activator present in a subset of mTECs characterized by high CD80 and major histocompatibility complex II expression and a lack of potential for differentiation or proliferation. Here, using an Aire-DTR transgenic line, we show that short-term ablation specifically targets Aire+ mTECs, which quickly undergo RANK-dependent recovery. Repeated ablation also affects Aire− mTECs, and using an inducible Aire-Cre fate-mapping system, we find that this results from the loss of a subset of mTECs that showed prior expression of Aire, maintains intermediate TSA expression, and preferentially migrates toward the center of the medulla. These results clearly identify a distinct stage of mTEC development and underscore the diversity of mTECs that play a key role in maintaining tolerance.

  7. Revised lineage of larval photoreceptor cells in Ciona reveals archetypal collaboration between neural tube and neural crest in sensory organ formation.

    Science.gov (United States)

    Oonuma, Kouhei; Tanaka, Moeko; Nishitsuji, Koki; Kato, Yumiko; Shimai, Kotaro; Kusakabe, Takehiro G

    2016-12-01

    The Ciona intestinalis larva has two distinct photoreceptor organs, a conventional pigmented ocellus and a nonpigmented ocellus, that are asymmetrically situated in the brain. The ciliary photoreceptor cells of these ocelli resemble visual cells of the vertebrate retina. Precise elucidation of the lineage of the photoreceptor cells will be key to understanding the developmental mechanisms of these cells as well as the evolutionary relationships between the photoreceptor organs of ascidians and vertebrates. Photoreceptor cells of the pigmented ocellus have been thought to develop from anterior animal (a-lineage) blastomeres, whereas the developmental origin of the nonpigmented ocellus has not been determined. Here, we show that the photoreceptor cells of both ocelli develop from the right anterior vegetal hemisphere: those of the pigmented ocellus from the right A9.14 cell and those of the nonpigmented ocellus from the right A9.16 cell. The pigmented ocellus is formed by a combination of two lineages of cells with distinct embryonic origins: the photoreceptor cells originate from a medial portion of the A-lineage neural plate, while the pigment cell originates from the lateral edge of the a-lineage neural plate. In light of the recently proposed close evolutionary relationship between the ocellus pigment cell of ascidians and the cephalic neural crest of vertebrates, the ascidian ocellus may represent a prototypic contribution of the neural crest to a cranial sensory organ.

  8. ERK2 suppresses self-renewal capacity of embryonic stem cells, but is not required for multi-lineage commitment.

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    William B Hamilton

    Full Text Available Activation of the FGF-ERK pathway is necessary for naïve mouse embryonic stem (ES cells to exit self-renewal and commit to early differentiated lineages. Here we show that genetic ablation of Erk2, the predominant ERK isozyme expressed in ES cells, results in hyper-phosphorylation of ERK1, but an overall decrease in total ERK activity as judged by substrate phosphorylation and immediate-early gene (IEG induction. Normal induction of this subset of canonical ERK targets, as well as p90RSK phosphorylation, was rescued by transgenic expression of either ERK1 or ERK2 indicating a degree of functional redundancy. In contrast to previously published work, Erk2-null ES cells exhibited no detectable defect in lineage specification to any of the three germ layers when induced to differentiate in either embryoid bodies or in defined neural induction conditions. However, under self-renewing conditions Erk2-null ES cells express increased levels of the pluripotency-associated transcripts, Nanog and Tbx3, a decrease in Nanog-GFP heterogeneity, and exhibit enhanced self-renewal in colony forming assays. Transgenic add-back of ERK2 is capable of restoring normal pluripotent gene expression and self-renewal capacity. We show that ERK2 contributes to the destabilization of ES cell self-renewal by reducing expression of pluripotency genes, such as Nanog, but is not specifically required for the early stages of germ layer specification.

  9. Delta-like1-induced Notch1 signaling regulates the human plasmacytoid dendritic cell versus T-cell lineage decision through control of GATA-3 and Spi-B

    NARCIS (Netherlands)

    W. Dontje; R. Schotte; T. Cupedo; M. Nagasawa; F. Scheeren; R. Gimeno; H. Spits; B. Blom

    2006-01-01

    Human early thymic precursors have the potential to differentiate into multiple cell lineages, including T cells and plasmacytoid dendritic cells (pDCs). This decision is guided by the induction or silencing of lineage-specific transcription factors. The ETS family member Spi-B is a key regulator of

  10. Foxn1 regulates lineage progression in cortical and medullary thymic epithelial cells but is dispensable for medullary sublineage divergence.

    Directory of Open Access Journals (Sweden)

    Craig S Nowell

    2011-11-01

    Full Text Available The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development.

  11. Micropatterning Extracellular Matrix Proteins on Electrospun Fibrous Substrate Promote Human Mesenchymal Stem Cell Differentiation Toward Neurogenic Lineage.

    Science.gov (United States)

    Li, Huaqiong; Wen, Feng; Chen, Huizhi; Pal, Mintu; Lai, Yuekun; Zhao, Allan Zijian; Tan, Lay Poh

    2016-01-13

    In this study, hybrid micropatterned grafts constructed via a combination of microcontact printing and electrospinning techniques process were utilized to investigate the influencing of patterning directions on human mesenchymal stem cells (hMSCs) differentiation to desired phenotypes. We found that the stem cells could align and elongate along the direction of the micropattern, where they randomly distributed on nonmicropatterned surfaces. Concomitant with patterning effect of component on stem cell alignment, a commensurate increase on the expression of neural lineage commitment markers, such as microtubule associated protein 2 (MAP2), Nestin, NeuroD1, and Class III β-Tubulin, were revealed from mRNA expression by quantitative Real Time PCR (qRT-PCR) and MAP2 expression by immunostaining. In addition, the effect of electrospun fiber orientation on cell behaviors was further examined. An angle of 45° between the direction of micropatterning and orientation of aligned fibers was verified to greatly prompt the outgrowth of filopodia and neurogenesis of hMSCs. This study demonstrates that the significance of hybrid components and electrospun fiber alignment in modulating cellular behavior and neurogenic lineage commitment of hMSCs, suggesting promising application of porous scaffolds with smart component and topography engineering in clinical regenerative medicine.

  12. Phylogenetic analysis of Sicilian goats reveals a new mtDNA lineage.

    Science.gov (United States)

    Sardina, M T; Ballester, M; Marmi, J; Finocchiaro, R; van Kaam, J B C H M; Portolano, B; Folch, J M

    2006-08-01

    The mitochondrial hypervariable region 1 (HVR1) sequence of 67 goats belonging to the Girgentana, Maltese and Derivata di Siria breeds was partially sequenced in order to present the first phylogenetic characterization of Sicilian goat breeds. These sequences were compared with published sequences of Indian and Pakistani domestic goats and wild goats. Mitochondrial lineage A was observed in most of the Sicilian goats. However, three Girgentana haplotypes were highly divergent from the Capra hircus clade, indicating that a new mtDNA lineage in domestic goats was found.

  13. Canonical FGFs Prevent Osteogenic Lineage Commitment and Differentiation of Human Bone Marrow Stromal Cells Via ERK1/2 Signaling.

    Science.gov (United States)

    Simann, Meike; Le Blanc, Solange; Schneider, Verena; Zehe, Viola; Lüdemann, Martin; Schütze, Norbert; Jakob, Franz; Schilling, Tatjana

    2017-02-01

    Controlling the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) in favor of osteogenesis represents a promising approach for osteoporosis therapy and prevention. Previously, Fibroblast Growth Factor 1 (FGF1) and its subfamily member FGF2 were scored as leading candidates to exercise control over skeletal precursor commitment and lineage decision albeit literature results are highly inconsistent. We show here that FGF1 and 2 strongly prevent the osteogenic commitment and differentiation of hBMSCs. Mineralization of extracellular matrix (ECM) and mRNA expression of osteogenic marker genes Alkaline Phosphatase (ALP), Collagen 1A1 (COL1A1), and Integrin-Binding Sialoprotein (IBSP) were significantly reduced. Furthermore, master regulators of osteogenic commitment like Runt-Related Transcription Factor 2 (RUNX2) and Bone Morphogenetic Protein 4 (BMP4) were downregulated. When administered under adipogenic culture conditions, canonical FGFs did not support osteogenic marker expression. Moreover despite the presence of osteogenic differentiation factors, FGFs even disabled the pro-osteogenic lineage decision of pre-differentiated adipocytic cells. In contrast to FGF Receptor 2 (FGFR2), FGFR1 was stably expressed throughout osteogenic and adipogenic differentiation and FGF addition. Moreover, FGFR1 and Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) were found to be responsible for underlying signal transduction using respective inhibitors. Taken together, we present new findings indicating that canonical FGFR-ERK1/2 signaling entrapped hBMSCs in a pre-committed state and arrested further maturation of committed precursors. Our results might aid in unraveling and controlling check points relevant for ageing-associated aberrant adipogenesis with consequences for the treatment of degenerative diseases such as osteoporosis and for skeletal tissue engineering strategies. J. Cell. Biochem. 118: 263-275, 2017. © 2016 Wiley

  14. Loss of C/EBP alpha cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage

    DEFF Research Database (Denmark)

    Porse, Bo T; Bryder, David; Theilgaard-Mönch, Kim;

    2005-01-01

    CCAAT/enhancer binding protein (C/EBP)alpha is a myeloid-specific transcription factor that couples lineage commitment to terminal differentiation and cell cycle arrest, and is found mutated in 9% of patients who have acute myeloid leukemia (AML). We previously showed that mutations which......, accumulation of myeloblasts and promyelocytes, and expansion of myeloid progenitor populations--all characteristics of AML. Circulating myeloblasts and hepatic leukocyte infiltration were observed, but thrombocytopenia, anemia, and elevated leukocyte count--normally associated with AML-were absent...

  15. The ubiquitous transcription factor CTCF promotes lineage-specific epigenomic remodeling and establishment of transcriptional networks driving cell differentiation.

    Science.gov (United States)

    Dubois-Chevalier, Julie; Staels, Bart; Lefebvre, Philippe; Eeckhoute, Jérôme

    2015-01-01

    Cell differentiation relies on tissue-specific transcription factors (TFs) that cooperate to establish unique transcriptomes and phenotypes. However, the role of ubiquitous TFs in these processes remains poorly defined. Recently, we have shown that the CCCTC-binding factor (CTCF) is required for adipocyte differentiation through epigenomic remodelling of adipose tissue-specific enhancers and transcriptional activation of Peroxisome proliferator-activated receptor gamma (PPARG), the main driver of the adipogenic program (PPARG), and its target genes. Here, we discuss how these findings, together with the recent literature, illuminate a functional role for ubiquitous TFs in lineage-determining transcriptional networks.

  16. Cranial Radiation for Pediatric T-Lineage Acute Lymphoblastic Leukemia: A Systematic Review and Meta-analysis

    OpenAIRE

    Kelly, Michael J.; Thomas A. Trikalinos; Dahabreh, Issa J.; Gianferante, Matthew; Parsons, Susan K.

    2014-01-01

    There are heterogeneous approaches to cranial irradiation therapy (CRT) for T-lineage acute lymphoblastic leukemia (T-ALL). We performed a systematic review of studies that specified a radiation strategy and reported survival for pediatric T-ALL. Our analysis included 62 publications reporting 78 treatment groups (patient n=5844). The average event-free survival (EFS) was higher by 6% per 5 years (p

  17. SDF-1 and CXCR4 play an important role in adult SVZ lineage cell proliferation and differentiation.

    Science.gov (United States)

    Zhu, Chang; Yao, Wen-Long; Tan, Wei; Zhang, Chuan-Han

    2017-02-15

    Evidence has shown that stromal cell-derived factor (SDF-1/CXCL12) plays an important role in maintaining adult neural progenitor cells (NPCs). SDF-1 is also known to enhance recovery by recruiting NPCs to damaged regions and recent studies have revealed that SDF-1α exhibits pleiotropism, thereby differentially affecting NPC subpopulations. In this study, we investigated the role of SDF-1 in in vitro NPC self-renewal, proliferation and differentiation, following treatment with different concentrations of SDF-1 or a CXCR4 antagonist, AMD3100. We observed that AMD3100 inhibited the formation of primary neurospheres. However, SDF-1 and AMD3100 exhibited no effect on proliferation upon inclusion of growth factors in the media. Following growth factor withdrawal, AMD3100 and SDF-1 treatment resulted in differential effects on NPC proliferation. SDF-1, at a concentration of 500ng/ml, resulted in an increase in the relative proportion of oligodendrocytes following growth factor withdrawal-induced differentiation. Using CXCR4 knockout mice, we observed that SDF-1 affected NPC proliferation in the sub-ventricular zone (SVZ). We also investigated the occurrence of differential CXCR4 expression at different stages during lineage progression. These results clearly indicate that signaling interactions between SDF-1 and CXCR4 play an important role in adult SVZ lineage cell proliferation and differentiation.

  18. The roles of FGF and MAP kinase signaling in the segregation of the epiblast and hypoblast cell lineages in bovine and human embryos

    NARCIS (Netherlands)

    Kuijk, E.W.; van Tol, L.T.; te Velde, H.; Wubbolts, R.; Welling, M.; Geijsen, N.; Roelen, B.A.

    2012-01-01

    At the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell sign

  19. The Wnt receptor, Lrp5, is expressed by mouse mammary stem cells and is required to maintain the basal lineage.

    Directory of Open Access Journals (Sweden)

    Nisha M Badders

    Full Text Available BACKGROUND: Ectopic Wnt signaling induces increased stem/progenitor cell activity in the mouse mammary gland, followed by tumor development. The Wnt signaling receptors, Lrp5/6, are uniquely required for canonical Wnt activity. Previous data has shown that the absence of Lrp5 confers resistance to Wnt1-induced tumor development. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that all basal mammary cells express Lrp5, and co-express Lrp6 in a similar fashion. Though Wnt dependent transcription of key target genes is relatively unchanged in mammary epithelial cell cultures, the absence of Lrp5 specifically depletes adult regenerative stem cell activity (to less than 1%. Stem cell activity can be enriched by >200 fold (over 80% of activity, based on high Lrp5 expression alone. Though Lrp5 null glands have apparent normal function, the basal lineage is relatively reduced (from 42% basal/total epithelial cells to 22% and Lrp5-/- mammary epithelial cells show enhanced expression of senescence-associated markers in vitro, as measured by expression of p16(Ink4a and TA-p63. CONCLUSIONS/SIGNIFICANCE: This is the first single biomarker that has been demonstrated to be functionally involved in stem cell maintenance. Together, these results demonstrate that Wnt signaling through Lrp5 is an important component of normal mammary stem cell function.

  20. Identification and functional characterization of the miRNA-gene regulatory network in chronic myeloid leukemia lineage negative cells

    Science.gov (United States)

    Agatheeswaran, S.; Pattnayak, N. C.; Chakraborty, S.

    2016-09-01

    Chronic myeloid leukemia (CML) is maintained by leukemic stem cells (LSCs) which are resistant to the existing TKI therapy. Hence a better understanding of the CML LSCs is necessary to eradicate these cells and achieve complete cure. Using the miRNA-gene interaction networks from the CML lin(-) cells we identified a set of up/down-regulated miRNAs and corresponding target genes. Association studies (Pearson correlation) from the miRNA and gene expression data showed that miR-1469 and miR-1972 have significantly higher number of target genes, 75 and 50 respectively. We observed that miR-1972 induces G2-M cell cycle arrest and miR-1469 moderately arrested G1 cell cycle when overexpressed in KCL22 cells. We have earlier shown that a combination of imatinib and JAK inhibitor I can significantly bring down the proliferation of CML lineage negative cells. Here we observed that imatinib and JAK inhibitor I combination restored the expression pattern of the down-regulated miRNAs in primary CML lin(-) cells. Thus effective manipulation of the deregulated miRNAs can restore the miRNA-mRNA networks that can efficiently inhibit CML stem and progenitor cells and alleviate the disease.

  1. Deletion of FGFR3 in Osteoclast Lineage Cells Results in Increased Bone Mass in Mice by Inhibiting Osteoclastic Bone Resorption.

    Science.gov (United States)

    Su, Nan; Li, Xiaogang; Tang, Yubin; Yang, Jing; Wen, Xuan; Guo, Jingyuan; Tang, Junzhou; Du, Xiaolan; Chen, Lin

    2016-09-01

    Fibroblast growth factor receptor 3 (FGFR3) participates in bone remodeling. Both Fgfr3 global knockout and activated mice showed decreased bone mass with increased osteoclast formation or bone resorption activity. To clarify the direct effect of FGFR3 on osteoclasts, we specifically deleted Fgfr3 in osteoclast lineage cells. Adult mice with Fgfr3 deficiency in osteoclast lineage cells (mutant [MUT]) showed increased bone mass. In a drilled-hole defect model, the bone remodeling of the holed area in cortical bone was also impaired with delayed resorption of residual woven bone in MUT mice. In vitro assay demonstrated that there was no significant difference between the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts derived from wild-type and Fgfr3-deficient bone marrow monocytes, suggesting that FGFR3 had no remarkable effect on osteoclast formation. The bone resorption activity of Fgfr3-deficient osteoclasts was markedly decreased accompanying with downregulated expressions of Trap, Ctsk, and Mmp 9. The upregulated activity of osteoclastic bone resorption by FGF2 in vitro was also impaired in Fgfr3-deficient osteoclasts, indicating that FGFR3 may participate in the regulation of bone resorption activity of osteoclasts by FGF2. Reduced adhesion but not migration in osteoclasts with Fgfr3 deficiency may be responsible for the impaired bone resorption activity. Our study for the first time genetically shows the direct positive regulation of FGFR3 on osteoclastic bone resorption. © 2016 American Society for Bone and Mineral Research.

  2. All-trans retinoic acid promotes neural lineage entry by pluripotent embryonic stem cells via multiple pathways

    Directory of Open Access Journals (Sweden)

    Fang Bo

    2009-07-01

    Full Text Available Abstract Background All-trans retinoic acid (RA is one of the most important morphogens with pleiotropic actions. Its embryonic distribution correlates with neural differentiation in the developing central nervous system. To explore the precise effects of RA on neural differentiation of mouse embryonic stem cells (ESCs, we detected expression of RA nuclear receptors and RA-metabolizing enzymes in mouse ESCs and investigated the roles of RA in adherent monolayer culture. Results Upon addition of RA, cell differentiation was directed rapidly and exclusively into the neural lineage. Conversely, pharmacological interference with RA signaling suppressed this neural differentiation. Inhibition of fibroblast growth factor (FGF signaling did not suppress significantly neural differentiation in RA-treated cultures. Pharmacological interference with extracellular signal-regulated kinase (ERK pathway or activation of Wnt pathway effectively blocked the RA-promoted neural specification. ERK phosphorylation was enhanced in RA-treated cultures at the early stage of differentiation. Conclusion RA can promote neural lineage entry by ESCs in adherent monolayer culture systems. This effect depends on RA signaling and its crosstalk with the ERK and Wnt pathways.

  3. Lineage-specific detection of influenza B virus using real-time polymerase chain reaction with melting curve analysis.

    Science.gov (United States)

    Tewawong, Nipaporn; Chansaenroj, Jira; Klinfueng, Sirapa; Vichiwattana, Preeyaporn; Korkong, Sumeth; Thongmee, Thanunrat; Theamboonlers, Apiradee; Payungporn, Sunchai; Vongpunsawad, Sompong; Poovorawan, Yong

    2016-06-01

    Influenza B viruses comprise two lineages, Victoria (B/Vic) and Yamagata (B/Yam), which co-circulate globally. The surveillance data on influenza B virus lineages in many countries often underestimate the true prevalence due to the lack of a rapid, accurate, and cost-effective method for virus detection. We have developed a real-time PCR with melting curve analysis for lineage-specific differential detection of influenza B virus. By amplifying a region of the hemagglutinin gene using real-time PCR with SYBR Green I dye, B/Vic and B/Yam could be differentiated based on their melting temperature peaks. This method was efficient (B/Vic = 93.2 %; B/Yam 97.7 %), sensitive (B/Vic, 94.6 %; B/Yam, 96.3 %), and specific (B/Vic, 97.7 %; B/Yam, 97.1 %). The lower detection limit was 10(2) copies per microliter. The assay was evaluated using 756 respiratory specimens that were positive for influenza B virus, obtained between 2010 and 2015. The incidence of influenza B virus was approximately 18.9 % of all influenza cases, and the percentage was highest among children aged 6-17 years (7.57 %). The overall percentage of mismatched influenza B vaccine was 21.1 %. Our findings suggest that real-time PCR with melting curve analysis can provide a rapid, simple, and sensitive lineage-specific influenza B virus screening method to facilitate influenza surveillance.

  4. Tools and Techniques for Wt1-Based Lineage Tracing.

    Science.gov (United States)

    Wilm, Bettina; Muñoz-Chapuli, Ramon

    2016-01-01

    The spatiotemporal expression pattern of Wt1 has been extensively studied in a number of animal models to establish its function and the developmental fate of the cells expressing this gene. In this chapter, we review the available animal models for Wt1-expressing cell lineage analysis, including direct Wt1 expression reporters and systems for permanent Wt1 lineage tracing. We describe the presently used constitutive or inducible genetic lineage tracing approaches based on the Cre/loxP system utilizing Cre recombinase expression under control of a Wt1 promoter.To make these systems accessible, we provide laboratory protocols that include dissection and processing of the tissues for immunofluorescence and histopathological analysis of the lineage-labeled Wt1-derived cells within the embryo/tissue context.

  5. Differentiation of retinal ganglion cells and photoreceptor precursors from mouse induced pluripotent stem cells carrying an Atoh7/Math5 lineage reporter.

    Science.gov (United States)

    Xie, Bin-Bin; Zhang, Xiang-Mei; Hashimoto, Takao; Tien, Amy H; Chen, Andrew; Ge, Jian; Yang, Xian-Jie

    2014-01-01

    The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs). The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.

  6. Differentiation of retinal ganglion cells and photoreceptor precursors from mouse induced pluripotent stem cells carrying an Atoh7/Math5 lineage reporter.

    Directory of Open Access Journals (Sweden)

    Bin-Bin Xie

    Full Text Available The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs. The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.

  7. Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage: genome analysis and virulence mechanisms.

    Directory of Open Access Journals (Sweden)

    Makrina Totsika

    Full Text Available Escherichia coli strains causing urinary tract infection (UTI are increasingly recognized as belonging to specific clones. E. coli clone O25b:H4-ST131 has recently emerged globally as a leading multi-drug resistant pathogen causing urinary tract and bloodstream infections in hospitals and the community. While most molecular studies to date examine the mechanisms conferring multi-drug resistance in E. coli ST131, relatively little is known about their virulence potential. Here we examined E. coli ST131 clinical isolates from two geographically diverse collections, one representing the major pathogenic lineages causing UTI across the United Kingdom and a second representing UTI isolates from patients presenting at two large hospitals in Australia. We determined a draft genome sequence for one representative isolate, E. coli EC958, which produced CTX-M-15 extended-spectrum β-lactamase, CMY-23 type AmpC cephalosporinase and was resistant to ciprofloxacin. Comparative genome analysis indicated that EC958 encodes virulence genes commonly associated with uropathogenic E. coli (UPEC. The genome sequence of EC958 revealed a transposon insertion in the fimB gene encoding the activator of type 1 fimbriae, an important UPEC bladder colonization factor. We identified the same fimB transposon insertion in 59% of the ST131 UK isolates, as well as 71% of ST131 isolates from Australia, suggesting this mutation is common among E. coli ST131 strains. Insertional inactivation of fimB resulted in a phenotype resembling a slower off-to-on switching for type 1 fimbriae. Type 1 fimbriae expression could still be induced in fimB-null isolates; this correlated strongly with adherence to and invasion of human bladder cells and bladder colonisation in a mouse UTI model. We conclude that E. coli ST131 is a geographically widespread, antibiotic resistant clone that has the capacity to produce numerous virulence factors associated with UTI.

  8. A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production

    Directory of Open Access Journals (Sweden)

    Emanuel Joseph Paul Nazareth

    2016-05-01

    Full Text Available Human pluripotent stem cells (hPSCs exist in heterogeneous micro-environments with multiple subpopulations, convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors, measuring yield and purity outputs of undifferentiated, neuroectoderm, mesendoderm, and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4 and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically, small interfering RNA knockdown of RAPTOR, a component of mTOR complex 1, phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold, with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives.

  9. Dual small-molecule targeting of SMAD signaling stimulates human induced pluripotent stem cells toward neural lineages.

    Directory of Open Access Journals (Sweden)

    Methichit Wattanapanitch

    Full Text Available Incurable neurological disorders such as Parkinson's disease (PD, Huntington's disease (HD, and Alzheimer's disease (AD are very common and can be life-threatening because of their progressive disease symptoms with limited treatment options. To provide an alternative renewable cell source for cell-based transplantation and as study models for neurological diseases, we generated induced pluripotent stem cells (iPSCs from human dermal fibroblasts (HDFs and then differentiated them into neural progenitor cells (NPCs and mature neurons by dual SMAD signaling inhibitors. Reprogramming efficiency was improved by supplementing the histone deacethylase inhibitor, valproic acid (VPA, and inhibitor of p160-Rho associated coiled-coil kinase (ROCK, Y-27632, after retroviral transduction. We obtained a number of iPS colonies that shared similar characteristics with human embryonic stem cells in terms of their morphology, cell surface antigens, pluripotency-associated gene and protein expressions as well as their in vitro and in vivo differentiation potentials. After treatment with Noggin and SB431542, inhibitors of the SMAD signaling pathway, HDF-iPSCs demonstrated rapid and efficient differentiation into neural lineages. Six days after neural induction, neuroepithelial cells (NEPCs were observed in the adherent monolayer culture, which had the ability to differentiate further into NPCs and neurons, as characterized by their morphology and the expression of neuron-specific transcripts and proteins. We propose that our study may be applied to generate neurological disease patient-specific iPSCs allowing better understanding of disease pathogenesis and drug sensitivity assays.

  10. Disheveled mediated planar cell polarity signaling is required in the second heart field lineage for outflow tract morphogenesis.

    Science.gov (United States)

    Sinha, Tanvi; Wang, Bing; Evans, Sylvia; Wynshaw-Boris, Anthony; Wang, Jianbo

    2012-10-01

    Disheveled (Dvl) is a key regulator of both the canonical Wnt and the planar cell polarity (PCP) pathway. Previous genetic studies in mice indicated that outflow tract (OFT) formation requires Dvl1 and 2, but it was unclear which pathway was involved and whether Dvl1/2-mediated signaling was required in the second heart field (SHF) or the cardiac neural crest (CNC) lineage, both of which are critical for OFT development. In this study, we used Dvl1/2 null mice and a set of Dvl2 BAC transgenes that function in a pathway-specific fashion to demonstrate that Dvl1/2-mediated PCP signaling is essential for OFT formation. Lineage-specific gene-ablation further indicated that Dvl1/2 function is dispensable in the CNC, but required in the SHF for OFT lengthening to promote cardiac looping. Mutating the core PCP gene Vangl2 and non-canonical Wnt gene Wnt5a recapitulated the OFT morphogenesis defects observed in Dvl1/2 mutants. Consistent with genetic interaction studies suggesting that Wnt5a signals through the PCP pathway, Dvl1/2 and Wnt5a mutants display aberrant cell packing and defective actin polymerization and filopodia formation specifically in SHF cells in the caudal splanchnic mesoderm (SpM), where Wnt5a and Dvl2 are co-expressed specifically. Our results reveal a critical role of PCP signaling in the SHF during early OFT lengthening and cardiac looping and suggest that a Wnt5a→ Dvl PCP signaling cascade may regulate actin polymerization and protrusive cell behavior in the caudal SpM to promote SHF deployment, OFT lengthening and cardiac looping.

  11. HIV Skews the Lineage-Defining Transcriptional Profile of Mycobacterium tuberculosis-Specific CD4+ T Cells.

    Science.gov (United States)

    Riou, Catherine; Strickland, Natalie; Soares, Andreia P; Corleis, Björn; Kwon, Douglas S; Wherry, E John; Wilkinson, Robert J; Burgers, Wendy A

    2016-04-01

    HIV-infected persons are at greater risk of developing tuberculosis (TB) even before profound CD4 loss occurs, suggesting that HIV alters CD4(+) T cell functions capable of containing bacterial replication. An effective immune response to Mycobacterium tuberculosis most likely relies on the development of a balanced CD4 response, in which distinct CD4(+) Th subsets act in synergy to control the infection. To define the diversity of M. tuberculosis-specific CD4(+) Th subsets and determine whether HIV infection impacts such responses, the expression of lineage-defining transcription factors T-bet, Gata3, RORγt, and Foxp3 was measured in M. tuberculosis-specific CD4(+) T cells in HIV-uninfected (n = 20) and HIV-infected individuals (n = 20) with latent TB infection. Our results show that, upon 5-d restimulation in vitro, M. tuberculosis-specific CD4(+) T cells from healthy individuals have the ability to exhibit a broad spectrum of Th subsets, defined by specific patterns of transcription factor coexpression. These transcription factor profiles were skewed in HIV-infected individuals where the proportion of T-bet(high)Foxp3(+) M. tuberculosis-specific CD4(+) T cells was significantly decreased (p = 0.002) compared with HIV-uninfected individuals, a change that correlated inversely with HIV viral load (p = 0.0007) and plasma TNF-α (p = 0.027). Our data demonstrate an important balance in Th subset diversity defined by lineage-defining transcription factor coexpression profiles that is disrupted by HIV infection and suggest a role for HIV in impairing TB immunity by altering the equilibrium of M. tuberculosis-specific CD4(+) Th subsets.

  12. Mapping the dynamic expression of Wnt11 and the lineage contribution of Wnt11-expressing cells during early mouse development.

    Science.gov (United States)

    Sinha, Tanvi; Lin, Lizhu; Li, Ding; Davis, Jennifer; Evans, Sylvia; Wynshaw-Boris, Anthony; Wang, Jianbo

    2015-02-15

    Planar cell polarity (PCP) signaling is an evolutionarily conserved mechanism that coordinates polarized cell behavior to regulate tissue morphogenesis during vertebrate gastrulation, neurulation and organogenesis. In Xenopus and zebrafish, PCP signaling is activated by non-canonical Wnts such as Wnt11, and detailed understanding of Wnt11 expression has provided important clues on when, where and how PCP may be activated to regulate tissue morphogenesis. To explore the role of Wnt11 in mammalian development, we established a Wnt11 expression and lineage map with high spatial and temporal resolution by creating and analyzing a tamoxifen-inducible Wnt11-CreER BAC (bacterial artificial chromosome) transgenic mouse line. Our short- and long-term lineage tracing experiments indicated that Wnt11-CreER could faithfully recapitulate endogenous Wnt11 expression, and revealed for the first time that cells transiently expressing Wnt11 at early gastrulation were fated to become specifically the progenitors of the entire endoderm. During mid-gastrulation, Wnt11-CreER expressing cells also contribute extensively to the endothelium in both embryonic and extraembryonic compartments, and the endocardium in all chambers of the developing heart. In contrast, Wnt11-CreER expression in the myocardium starts from late-gastrulation, and occurs in three transient, sequential waves: first in the precursors of the left ventricular (LV) myocardium from E7.0 to 8.0; subsequently in the right ventricular (RV) myocardium from E8.0 to 9.0; and finally in the superior wall of the outflow tract (OFT) myocardium from E8.5 to 10.5. These results provide formal genetic proof that the majority of the endocardium and myocardium diverge by mid-gastrulation in the mouse, and suggest a tight spatial and temporal control of Wnt11 expression in the myocardial lineage to coordinate with myocardial differentiation in the first and second heart field progenitors to form the LV, RV and OFT. The insights gained

  13. Gene Regulation in M Cell Lineages: In Vitro and In Vivo

    OpenAIRE

    Wang, Jing

    2011-01-01

    M cells are specialized epithelial cells, which assist immune surveillance by transcytosis of particles and antigens to underlying lymphoid tissues. So far, three M cell phenotypes have been identified in airways and intestines. However, the mechanism of M cell differentiation is poorly understood, as well as the relationships between different M cell subtypes. To better understand these questions, we treated human (Caco-2BBe) and rodent (IEC-6) intestinal epithelial cell lines with lymphotox...

  14. Identification and Characterization of Lineage(-)CD45(-)Sca-1(+) VSEL Phenotypic Cells Residing in Adult Mouse Bone Tissue.

    Science.gov (United States)

    Nakatsuka, Ryusuke; Iwaki, Ryuji; Matsuoka, Yoshikazu; Sumide, Keisuke; Kawamura, Hiroshi; Fujioka, Tatsuya; Sasaki, Yutaka; Uemura, Yasushi; Asano, Hiroaki; Kwon, A-Hon; Sonoda, Yoshiaki

    2016-01-01

    Murine bone marrow (BM)-derived very small embryonic-like stem cells (BM VSELs), defined by a lineage-negative (Lin(-)), CD45-negative (CD45(-)), Sca-1-positive (Sca-1(+)) immunophenotype, were previously reported as postnatal pluripotent stem cells (SCs). We developed a highly efficient method for isolating Lin(-)CD45(-)Sca-1(+) small cells using enzymatic treatment of murine bone. We designated these cells as bone-derived VSELs (BD VSELs). The incidences of BM VSELs in the BM-derived nucleated cells and that of BD VSELs in bone-derived nucleated cells were 0.002% and 0.15%, respectively. These BD VSELs expressed a variety of hematopoietic stem cell (HSC), mesenchymal stem cell (MSC), and endothelial cell markers. The gene expression profile of the BD VSELs was clearly distinct from those of HSCs, MSCs, and ES cells. In the steady state, the BD VSELs proliferated slowly, however, the number of BD VSELs significantly increased in the bone after acute liver injury. Moreover, green fluorescent protein-mouse derived BD VSELs transplanted via tail vein injection after acute liver injury were detected in the liver parenchyma of recipient mice. Immunohistological analyses suggested that these BD VSELs might transdifferentiate into hepatocytes. This study demonstrated that the majority of the Lin(-)CD45(-)Sca-1(+) VSEL phenotypic cells reside in the bone rather than the BM. However, the immunophenotype and the gene expression profile of BD VSELs were clearly different from those of other types of SCs, including BM VSELs, MSCs, HSCs, and ES cells. Further studies will therefore be required to elucidate their cellular and/or SC characteristics and the potential relationship between BD VSELs and BM VSELs.

  15. Human induced hepatic lineage-oriented stem cells: autonomous specification of human iPS cells toward hepatocyte-like cells without any exogenous differentiation factors.

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    Tetsuya Ishikawa

    Full Text Available Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG, conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5, transporters (SULT2A1, SLC13A5, and SLCO2B1, and urea cycle-related enzymes (ARG1 and CPS1. In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the

  16. Chemically defined serum-free and xeno-free media for multiple cell lineages

    OpenAIRE

    Usta, Sümeyra Naz; Scharer, Christopher D.; Xu, Jie; Frey, Teryl K.; Nash, Rodney J

    2014-01-01

    Cell culture is one of the most common methods used to recapitulate a human disease environment in a laboratory setting. Cell culture techniques are used to grow and maintain cells of various types including those derived from primary tissues, such as stem cells and cancer tumors. However, a major confounding factor with cell culture is the use of serum and animal (xeno) products in the media. The addition of animal products introduces batch and lot variations that lead to experimental variab...

  17. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

    Directory of Open Access Journals (Sweden)

    Bedayat Babak

    2010-02-01

    Full Text Available Abstract Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts. With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival.

  18. Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells.

    Science.gov (United States)

    Mathiasen, Joanne R; McKenna, Beth Ann W; Saporito, Michael S; Ghadge, Ghanashyam D; Roos, Raymond P; Holskin, Beverly P; Wu, Zhi-Liang; Trusko, Stephen P; Connors, Thomas C; Maroney, Anna C; Thomas, Beth Ann; Thomas, Jeffrey C; Bozyczko-Coyne, Donna

    2004-04-02

    The neuropathology of Parkinson's Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic cells in culture treated with the MPTP neurotoxic metabolite, MPP(+). MPTP through MPP(+) activates the stress-activated c-Jun N-terminal kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347 acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK 1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was examined in MPP(+)-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative adenoviral constructs to MLKs. CEP-1347 inhibited MPP(+)-induced cell death and the morphological features of apoptosis. CEP-1347 also prevented MPP(+)-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP(+)-mediated increase in activated JNK levels and inhibited neuronal death following MPP(+) addition compared to cultures infected with a control construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP(+)-induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP(+)-mediated SH-SY5Y cell death in culture and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.

  19. Calcium dependent CAMTA1 in adult stem cell commitment to a myocardial lineage.

    Directory of Open Access Journals (Sweden)

    Barbara Muller-Borer

    Full Text Available The phenotype of somatic cells has recently been found to be reversible. Direct reprogramming of one cell type into another has been achieved with transduction and over expression of exogenous defined transcription factors emphasizing their role in specifying cell fate. To discover early and novel endogenous transcription factors that may have a role in adult-derived stem cell acquisition of a cardiomyocyte phenotype, mesenchymal stem cells from human and mouse bone marrow and rat liver were co-cultured with neonatal cardiomyocytes as an in vitro cardiogenic microenvironment. Cell-cell communications develop between the two cell types as early as 24 hrs in co-culture and are required for elaboration of a myocardial phenotype in the stem cells 8-16 days later. These intercellular communications are associated with novel Ca(2+ oscillations in the stem cells that are synchronous with the Ca(2+ transients in adjacent cardiomyocytes and are detected in the stem cells as early as 24-48 hrs in co-culture. Early and significant up-regulation of Ca(2+-dependent effectors, CAMTA1 and RCAN1 ensues before a myocardial program is activated. CAMTA1 loss-of-function minimizes the activation of the cardiac gene program in the stem cells. While the expression of RCAN1 suggests involvement of the well-characterized calcineurin-NFAT pathway as a response to a Ca(2+ signal, the CAMTA1 up-regulated expression as a response to such a signal in the stem cells was unknown. Cell-cell communications between the stem cells and adjacent cardiomyocytes induce Ca(2+ signals that activate a myocardial gene program in the stem cells via a novel and early Ca(2+-dependent intermediate, up-regulation of CAMTA1.

  20. The Evaluation of Nerve Growth Factor Over Expression on Neural Lineage Specific Genes in Human Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Mortazavi Yousef

    2016-07-01

    Full Text Available Objective Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer’s disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells (MSCs as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor (NGF and assessed their neural lineage genes. Materials and Methods In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein (GFP gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 (HEK-293 packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction (PCR. Results We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor (GDNF, glial fibrillary acidic protein (GFAP and Microtubule-associated protein 2 (MAP2 genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor (BDNF genes reduced. Conclusion Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this

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

    Directory of Open Access Journals (Sweden)

    Kentaro Kato

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

  2. Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors

    Science.gov (United States)

    Roberts, Casey L.; Chen, Silvia S.; Murchison, Angela C.; Ogle, Rebecca A.; Francis, Michael P.; Ogle, Roy C.

    2017-01-01

    While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells' origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2–6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPARγ but similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells. PMID:28250775

  3. Blood and immune cell engineering: Cytoskeletal contractility and nuclear rheology impact cell lineage and localization: Biophysical regulation of hematopoietic differentiation and trafficking.

    Science.gov (United States)

    Shin, Jae-Won; Discher, Dennis E

    2015-06-01

    Clinical success with human hematopoietic stem cell (HSC) transplantation establishes a paradigm for regenerative therapies with other types of stem cells. However, it remains generally challenging to therapeutically treat tissues after engineering of stem cells in vitro. Recent studies suggest that stem and progenitor cells sense physical features of their niches. Here, we review biophysical contributions to lineage decisions, maturation, and trafficking of blood and immune cells. Polarized cellular contractility and nuclear rheology are separately shown to be functional markers of a hematopoietic hierarchy that predict the ability of a lineage to traffic in and out of the bone marrow niche. These biophysical determinants are regulated by a set of structural molecules, including cytoplasmic myosin-II and nuclear lamins, which themselves are modulated by a diverse range of transcriptional and post-translational mechanisms. Small molecules that target these mechanobiological circuits, along with novel bioengineering methods, could prove broadly useful in programming blood and immune cells for therapies ranging from blood transfusions to immune attack of tumors.

  4. EBIO Does Not Induce Cardiomyogenesis in Human Pluripotent Stem Cells but Modulates Cardiac Subtype Enrichment by Lineage-Selective Survival

    Directory of Open Access Journals (Sweden)

    Monica Jara-Avaca

    2017-02-01

    Full Text Available Subtype-specific human cardiomyocytes (CMs are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs in response to 1-ethyl-2-benzimidazolinone (EBIO, a chemical modulator of small-/intermediate-conductance Ca2+-activated potassium channels (SKs 1–4. Investigating EBIO in human pluripotent stem cells (PSCs, we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs, timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs, including an increase in nodal- and atrial-like phenotypes. However, our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and, subsequently, CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO, presumably via an SK-independent mechanism. Together, EBIO did not promote cardiogenic differentiation of PSCs, opposing previous findings, but triggered lineage-selective survival at a cardiac progenitor stage, which we propose as a pharmacological strategy to modulate CM subtype composition.

  5. Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging.

    Science.gov (United States)

    Kaverina, Natalya V; Kadoya, Hiroyuki; Eng, Diana G; Rusiniak, Michael E; Sequeira-Lopez, Maria Luisa S; Gomez, R Ariel; Pippin, Jeffrey W; Gross, Kenneth W; Peti-Peterdi, Janos; Shankland, Stuart J

    2017-01-01

    Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman's capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.

  6. Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells

    DEFF Research Database (Denmark)

    Oberwallner, Barbara; Brodarac, Andreja; Anić, Petra;

    2015-01-01

    lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell......OBJECTIVES: Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation...... that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure. METHODS: Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic...

  7. A new loss-of-function allele 28y reveals a role of ARGONAUTE1 in limiting asymmetric division of stomatal lineage ground cell

    Institute of Scientific and Technical Information of China (English)

    Kezhen Yangy; Min Jiangy; Jie Le

    2014-01-01

    In Arabidopsis thaliana L., stomata are produced through a series of divisions including asymmetric and symmetric divisions. Asymmetric entry division of meristemoid mother cellproduces two daughter cells, the smal er meristemoid and the larger sister cell, a stomatal lineage ground cell(SLGC). Stomatal lineage ground cells can differentiate into epidermal pavement cells but have the potential to divide asymmetrical y, spacing divisions, to create satel ite meristemoids. Peptide ligands and TOO MANY MOUTHS (TMM) and ERECTA family receptors regulate the initiation of stomatal lineages, activity, and orientation of spacing divisions. Here, we reported that a natural mutant 28y displayed an increased stomatal density and index. Using map-based cloning, we identified mutation in ARGONAUTE1 (AGO1) as the cause of 28y phenotypes. Time-lapse tracing of stomatal lineage cells reveals that stomatal overproduction in 28y is caused by the excessive asymmetric spacing division of SLGCs.Further genetic results demonstrated that AGO1 acts down-stream of TMM and negatively regulates the SPCH transcripts, but in a brassinosteroid-independent manner. Upregulation of AGAMOUS-LIKE16 (AGL16) in 28y mutants suggests that AGO1 is required to restrict AGL16-mediated stomatal spacing divisions, an miRNA pathway in addition to ligand-receptor signaling modules.

  8. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    Energy Technology Data Exchange (ETDEWEB)

    Takegahara, Yuki; Yamanouchi, Keitaro, E-mail: akeita@mail.ecc.u-tokyo.ac.jp; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.

  9. 5-Hydroxymethylcytosine Remodeling Precedes Lineage Specification during Differentiation of Human CD4(+) T Cells

    OpenAIRE

    Colm E. Nestor; Antonio Lentini; Cathrine Hägg Nilsson; Danuta R. Gawel; Mika Gustafsson; Lina Mattson; Hui Wang; Olof Rundquist; Richard R. Meehan; Bernward Klocke; Martin Seifert; Stefanie M. Hauck; Helmut Laumen; Huan Zhang; Mikael Benson

    2016-01-01

    5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4(+) T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe s...

  10. Origin and dynamic lineage characteristics of the developing Drosophila midgut stem cells.

    Science.gov (United States)

    Takashima, Shigeo; Aghajanian, Patrick; Younossi-Hartenstein, Amelia; Hartenstein, Volker

    2016-08-15

    Proliferating intestinal stem cells (ISCs) generate all cell types of the Drosophila midgut, including enterocytes, endocrine cells, and gland cells (e.g., copper cells), throughout the lifetime of the animal. Among the signaling mechanisms controlling the balance between ISC self-renewal and the production of different cell types, Notch (N) plays a pivotal role. In this paper we investigated the emergence of ISCs during metamorphosis and the role of N in this process. Precursors of the Drosophila adult intestinal stem cells (pISCs) can be first detected within the pupal midgut during the first hours after onset of metamorphosis as motile mesenchymal cells. pISCs perform 2-3 rounds of parasynchronous divisions. The first mitosis yields only an increase in pISC number. During the following rounds of mitosis, dividing pISCs give rise to more pISCs, as well as the endocrine cells that populate the midgut of the eclosing fly. Enterocytes do not appear among the pISC progeny until around the time of eclosion. The "proendocrine" gene prospero (pros), expressed from mid-pupal stages onward in pISCs, is responsible to advance the endocrine fate in these cells; following removal of pros, pISCs continue to proliferate, but endocrine cells do not form. Conversely, the onset of N activity that occurs around the stage when pros comes on restricts pros expression among pISCs. Loss of N abrogates proliferation and switches on an endocrine fate among all pISCs. Our results suggest that a switch depending on the activity of N and pros acts at the level of the pISC to decide between continued proliferation and endocrine differentiation.

  11. Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells.

    Science.gov (United States)

    Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan; Bronner, Marianne E

    2017-03-29

    The 'neural plate border' of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure.

  12. Identification of a negative regulatory role for spi-C in the murine B cell lineage.

    Science.gov (United States)

    Li, Stephen K H; Solomon, Lauren A; Fulkerson, Patricia C; DeKoter, Rodney P

    2015-04-15

    Spi-C is an E26 transformation-specific family transcription factor that is highly related to PU.1 and Spi-B. Spi-C is expressed in developing B cells, but its function in B cell development and function is not well characterized. To determine whether Spi-C functions as a negative regulator of Spi-B (encoded by Spib), mice were generated that were germline knockout for Spib and heterozygous for Spic (Spib(-/-)Spic(+/-)). Interestingly, loss of one Spic allele substantially rescued B cell frequencies and absolute numbers in Spib(-/-) mouse spleens. Spib(-/-)Spic(+/-) B cells had restored proliferation compared with Spib(-/-) B cells in response to anti-IgM or LPS stimulation. Investigation of a potential mechanism for the Spib(-/-)Spic(+/-) phenotype revealed that steady-state levels of Nfkb1, encoding p50, were elevated in Spib(-/-)Spic(+/-) B cells compared with Spib(-/-) B cells. Spi-B was shown to directly activate the Nfkb1 gene, whereas Spi-C was shown to repress this gene. These results indicate a novel role for Spi-C as a negative regulator of B cell development and function.

  13. In vitro immunogenicity of undifferentiated pluripotent stem cells (PSC) and derived lineages.

    Science.gov (United States)

    Kadereit, Suzanne; Trounson, Alan

    2011-11-01

    The observation that embryonic stem cells (ESCs) expressed reduced levels of major histocompatibility (MHC) class I genes, no MHC class II or costimulatory molecules suggested early on that pluripotent stem cells (PSCs) could be "immune-privileged" and were unable to induce immune reactions. However, soon it became apparent that in some instances, ESCs were recognized by immune cells but still could reduce an active and strong immune response. Similar results were obtained with other PSCs. Almost 10 years later, the exact mechanisms are still not well understood and seem to differ between the different human and rodent PSC lines (even between different murine cell lines). These differences could be due to differing experimental approaches, different derivation protocols (to obtain the PSC lines), species specificity, or genetic background of the cells lines. A better understanding of the immune regulatory mechanisms deployed by PSCs and early derivates may inform us on immune regulation and could be exploitable for regenerative medicine using allogeneic cells. As PSCs grow robustly in culture and can easily be gene-modified, one could envision the generation of cell lines that maintain these immune suppressive properties through terminal differentiation, thus generating universal donor cells.

  14. Pre-transplantation specification of stem cells to cardiac lineage for regeneration of cardiac tissue.

    Science.gov (United States)

    Mayorga, Maritza; Finan, Amanda; Penn, Marc

    2009-03-01

    Myocardial infarction (MI) is a lead cause of mortality in the Western world. Treatment of acute MI is focused on restoration of antegrade flow which inhibits further tissue loss, but does not restore function to damaged tissue. Chronic therapy for injured myocardial tissue involves medical therapy that attempts to minimize pathologic remodeling of the heart. End stage therapy for chronic heart failure (CHF) involves inotropic therapy to increase surviving cardiac myocyte function or mechanical augmentation of cardiac performance. Not until the point of heart transplantation, a limited resource at best, does therapy focus on the fundamental problem of needing to replace injured tissue with new contractile tissue. In this setting, the potential for stem cell therapy has garnered significant interest for its potential to regenerate or create new contractile cardiac tissue. While to date adult stem cell therapy in clinical trials has suggested potential benefit, there is waning belief that the approaches used to date lead to regeneration of cardiac tissue. As the literature has better defined the pathways involved in cardiac differentiation, preclinical studies have suggested that stem cell pretreatment to direct stem cell differentiation prior to stem cell transplantation may be a more efficacious strategy for inducing cardiac regeneration. Here we review the available literature on pre-transplantation conditioning of stem cells in an attempt to better understand stem cell behavior and their readiness in cell-based therapy for myocardial regeneration.

  15. Effects of dose rates on radiation-induced replenishment of intestinal stem cells determined by Lgr5 lineage tracing.

    Science.gov (United States)

    Otsuka, Kensuke; Iwasaki, Toshiyasu

    2015-07-01

    An understanding of the dynamics of intestinal Lgr5(+) stem cells is important for elucidating the mechanism of colonic cancer development. We previously established a method for evaluating Lgr5(+) stem cells by tamoxifen-dependent Lgr5-lineage tracing and showed that high-dose-rate radiation stimulated replenishment of colonic stem cells. In this study, we evaluated the effects of low-dose-rate radiation on stem cell maintenance. Tamoxifen (4OHT)-injected Lgr5-EGFP-IRES-Cre(ERT2) × ROSA-LSL-LacZ mice were used, LacZ-labeled colonic crypts were enumerated, and the loss of LacZ(+) crypts under low-dose-rate radiation was estimated. After 4OHT treatment, the number of LacZ-labeled Lgr5(+) stem cells was higher in the colon of infant mice than in adult mice. The percentage of LacZ-labeled crypts in infant mice rapidly decreased after 4OHT treatment. However, the percentage of labeled crypts plateaued at ∼2% at 4 weeks post-treatment and remained unchanged for up to 7 months. Thus, it will be advantageous to evaluate the long-term effects of low-dose-rate radiation. Next, we determined the percentages of LacZ-labeled crypts irradiated with 1 Gy administered at different dose rates. As reported in our previous study, mice exposed to high-dose-rate radiation (30 Gy/h) showed a marked replenishment (P = 0.04). However, mice exposed to low-dose-rate radiation (0.003 Gy/h) did not exhibit accelerated stem-cell replenishment (P = 0.47). These findings suggest the percentage of labeled crypts can serve as a useful indicator of the effects of dose rate on the stem cell pool.

  16. Retroposon analysis of major cetacean lineages: the monophyly of toothed whales and the paraphyly of river dolphins.

    Science.gov (United States)

    Nikaido, M; Matsuno, F; Hamilton, H; Brownell, R L; Cao, Y; Ding, W; Zuoyan, Z; Shedlock, A M; Fordyce, R E; Hasegawa, M; Okada, N

    2001-06-19

    SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram, and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: (i) Odontocetes are monophyletic; (ii) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; (iii) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and (iv) the rapid radiation of extant cetacean lineages occurred some 28-33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.

  17. Metagenome Sequence Analysis of Filamentous Microbial Communities Obtained from Geochemically Distinct Geothermal Channels Reveals Specialization of Three Aquificales Lineages

    Directory of Open Access Journals (Sweden)

    Cristina eTakacs-vesbach

    2013-05-01

    Full Text Available The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems worldwide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal ‘filamentous streamer’ communities (~40 Mbp per site, which targeted three different groups of Aquificales found in Yellowstone National Park (YNP. Unassembled metagenome sequence and PCR-amplified 16S rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobaculum (Aquificaceae populations, whereas the circumneutral pH (6.5 - 7.8 sites containing dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae. Thermocrinis (Aquificaceae populations were found primarily in the circumneutral sites with undetectable sulfide, and to a lesser extent in one sulfidic system at pH 8. Phylogenetic analysis of assembled sequence containing 16S rRNA genes as well as conserved protein-encoding genes revealed that the composition and function of these communities varied across geochemical conditions. Each Aquificales lineage contained genes for CO2 fixation by the reverse TCA cycle, but only the Sulfurihydrogenibium populations perform citrate cleavage using ATP citrate lyase (Acl. The Aquificaceae populations use an alternative pathway catalyzed by two separate enzymes, citryl CoA synthetase (Ccs and citryl CoA lyase (Ccl. All three Aquificales lineages contained evidence of aerobic respiration, albeit due to completely different types of heme Cu oxidases (subunit I involved in oxygen reduction. The distribution of Aquificales populations and differences among functional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2, O2 have resulted in niche specialization among members of the Aquificales.

  18. Zebrafish kruppel-like factor 4a represses intestinal cell proliferation and promotes differentiation of intestinal cell lineages.

    Directory of Open Access Journals (Sweden)

    I-Chen Li

    Full Text Available BACKGROUND: Mouse krüppel-like factor 4 (Klf4 is a zinc finger-containing transcription factor required for terminal differentiation of goblet cells in the colon. However, studies using either Klf4(-/- mice or mice with conditionally deleted Klf4 in their gastric epithelia showed different results in the role of Klf4 in epithelial cell proliferation. We used zebrafish as a model organism to gain further understanding of the role of Klf4 in the intestinal cell proliferation and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: We characterized the function of klf4a, a mammalian klf4 homologue by antisense morpholino oligomer knockdown. Zebrafish Klf4a shared high amino acid similarities with human and mouse Klf4. Phylogenetic analysis grouped zebrafish Klf4a together with both human and mouse Klf4 in a branch with high bootstrap value. In zebrafish, we demonstrate that Klf4a represses intestinal cell proliferation based on results of BrdU incorporation, p-Histone 3 immunostaining, and transmission electron microscopy analyses. Decreased PepT1 expression was detected in intestinal bulbs of 80- and 102-hours post fertilization (hpf klf4a morphants. Significant reduction of alcian blue-stained goblet cell number was identified in intestines of 102- and 120-hpf klf4a morphants. Embryos treated with γ-secretase inhibitor showed increased klf4a expression in the intestine, while decreased klf4a expression and reduction in goblet cell number were observed in embryos injected with Notch intracellular domain (NICD mRNA. We were able to detect recovery of goblet cell number in 102-hpf embryos that had been co-injected with both klf4a and Notch 1a NICD mRNA. CONCLUSIONS/SIGNIFICANCE: This study provides in vivo evidence showing that zebrafih Klf4a is essential for the repression of intestinal cell proliferation. Zebrafish Klf4a is required for the differentiation of goblet cells and the terminal differentiation of enterocytes. Moreover, the regulation of

  19. Yes-Associated Protein Inhibits Transcription of Myocardin and Attenuates Differentiation of Vascular Smooth Muscle Cell from Cardiovascular Progenitor Cell Lineage.

    Science.gov (United States)

    Wang, Lunchang; Qiu, Ping; Jiao, Jiao; Hirai, Hiroyuki; Xiong, Wei; Zhang, Jifeng; Zhu, Tianqing; Ma, Peter X; Chen, Y Eugene; Yang, Bo

    2017-02-01

    Vascular smooth muscle cells (VSMCs) derived from cardiovascular progenitor cell (CVPC) lineage populate the tunica media of the aortic root. Understanding differentiation of VSMCs from CVPC will further our understanding of the molecular mechanisms contributing to aortic root aneurysms, and thus, facilitate the development of novel therapeutic agents to prevent this devastating complication. It is established that the yes-associated protein (YAP) and Hippo pathway is important for VSMC proliferation and phenotype switch. To determine the role of YAP in differentiation of VSMCs from CVPCs, we utilized the in vitro monolayer lineage specific differentiation method by differentiating human embryonic stem cells into CVPCs, and then, into VSMCs. We found that expression of YAP decreased during differentiation of VSMC from CVPCs. Overexpression of YAP attenuated expression of VSMC contractile markers and impaired VSMC function. Knockdown of YAP increased expression of contractile proteins during CVPC-VSMCs differentiation. Importantly, expression of YAP decreased transcription of myocardin during this process. Overexpression of YAP in PAC1 SMC cell line inhibited luciferase activity of myocardin proximal promoter in a dose dependent and NKX2.5 dependent manners. YAP protein interacted with NKX2.5 protein and inhibited binding of NKX2.5 to the 5'-proximal promoter region of myocardin in CVPC-derived VSMCs. In conclusion, YAP negatively regulates differentiation of VSMCs from CVPCs by decreasing transcription of myocardin in a NKX2.5-dependent manner. Stem Cells 2017;35:351-361.

  20. Deleterious effects on MDAMB-231 breast adenocarcinoma cell lineage submitted to Ho-166 radioactive seeds at very low activity

    Energy Technology Data Exchange (ETDEWEB)

    Falcao, Patricia L.; Campos, Tarcisio P.R., E-mail: campos@nuclear.ufmg.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Engenharia Nuclear; Sarmento, Eduardo V. [Centro de Desenvolvimento de Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Cuperschmid, Ethel M. [Universidade Federal de Minas Gerais (CEMEMOR/UFMG), Belo Horizonte, BR (Brazil). Fac. de Medicina. Centro de Memoria da Medicina

    2011-07-01

    Herein, the deleterious effect of ionizing radiation provided by Ho-166 radioactive seeds at low activity were addressed, based on experimental in vitro assays at the MDA MB231 cell lineage, a breast adenocarcinoma, compared to PBMC - peripheral blood cells. The methodology involves of the MDBMB-231 and PBMC expansion in culture in suitable environment in 30mm well plates and T-25 flasks. Seeds were synthesized with Ho-165 incorporated and characterized previously. Activation was processed at IPR1 reactor at the peripheral table, at 8h exposition. Three groups of seeds were tested: 0,34 mCi, 0,12 mCi activity, and control group. Such seeds were placed on culture and held to a period of 05 half-lives of the radionuclide. The biological responses at these exposure were documented by inverse microscopic photographic in time. Also, MTT essay were performed. A fast response in producing deleterious effects at cancer cell was observed even if for the low activity seeds. Also, a biological response dependent to a radial distance of the seed was observed. At conclusion, viability clonogenic control of MDAMB231 is identified at the exposition to Ho-166 ceramic seeds, even if at low activity of 0,1 to 0,3mCi. (author)

  1. Transgenic mice overexpressing arginase 1 in monocytic cell lineage are affected by lympho-myeloproliferative disorders and disseminated intravascular coagulation.

    Science.gov (United States)

    Astigiano, Simonetta; Morini, Monica; Damonte, Patrizia; Fraternali Orcioni, Giulio; Cassanello, Michela; Puglisi, Andrea; Noonan, Douglas M; Bronte, Vincenzo; Barbieri, Ottavia

    2015-11-01

    Arginase (ARG) is a metabolic enzyme present in two isoforms that hydrolyze l-arginine to urea and ornithine. In humans, ARG isoform 1 is also expressed in cells of the myeloid lineage. ARG activity promotes tumour growth and inhibits T lymphocyte activation. However, the two ARG transgenic mouse lines produced so far failed to show such effects. We have generated, in two different genetic backgrounds, transgenic mice constitutively expressing ARG1 under the control of the CD68 promoter in macrophages and monocytes. Both heterozygous and homozygous transgenic mice showed a relevant increase in mortality at early age, compared with wild-type siblings (67/267 and 48/181 versus 8/149, respectively, both P < 0.005). This increase was due to high incidence of haematologic malignancies, in particular myeloid leukaemia, myeloid dysplasia, lymphomas and disseminated intravascular coagulation (DIC), diseases that were absent in wild-type mice. Atrophy of lymphoid organs due to reduction in T-cell compartment was also detected. Our results indicate that ARG activity may participate in the pathogenesis of lymphoproliferative and myeloproliferative disorders, suggest the involvement of alterations of L-arginine metabolism in the onset of DIC and confirm a role for the enzyme in regulating T-cell homeostasis.

  2. Stem-cell dynamics and lineage topology from in vivo fate mapping in the hematopoietic system.

    Science.gov (United States)

    Höfer, Thomas; Barile, Melania; Flossdorf, Michael

    2016-06-01

    In recent years, sophisticated fate-mapping tools have been developed to study the behavior of stem cells in the intact organism. These experimental approaches are beginning to yield a quantitative picture of how cell numbers are regulated during steady state and in response to challenges. Focusing on hematopoiesis and immune responses, we discuss how novel mathematical approaches driven by these fate-mapping data have provided insights into the dynamics and topology of cellular differentiation pathways in vivo. The combination of experiment and theory has allowed to quantify the degree of self-renewal in stem and progenitor cells, shown how native hematopoiesis differs fundamentally from post-transplantation hematopoiesis, and uncovered that the diversification of T lymphocytes during immune responses resembles tissue renewal driven by stem cells.

  3. Bone Marrow-Derived Mesenchymal Cell Differentiation toward Myogenic Lineages: Facts and Perspectives

    Directory of Open Access Journals (Sweden)

    Daniela Galli

    2014-01-01

    Full Text Available Bone marrow-derived mesenchymal stem cells (BM-MSCs are valuable platforms for new therapies based on regenerative medicine. BM-MSCs era is coming of age since the potential of these cells is increasingly demonstrated. In fact, these cells give origin to osteoblasts, chondroblasts, and adipocyte precursors in vitro, and they can also differentiate versus other mesodermal cell types like skeletal muscle precursors and cardiomyocytes. In our short review, we focus on the more recent manipulations of BM-MSCs toward skeletal and heart muscle differentiation, a growing field of obvious relevance considering the toll of muscle disease (i.e., muscular dystrophies, the heavier toll of heart disease in developed countries, and the still not completely understood mechanisms of muscle differentiation and repair.

  4. Thymopoiesis in mice depends on a Foxn1-positive thymic epithelial cell lineage

    OpenAIRE

    Corbeaux, Tatiana; Hess, Isabell; Swann, Jeremy B.; Kanzler, Benoît; Haas-Assenbaum, Annette; Boehm, Thomas

    2010-01-01

    The thymus is essential for T-cell development. Here, we focus on the role of the transcription factor Foxn1 in the development and function of thymic epithelial cells (TECs) of the mouse. TECs are of endodermal origin; they initially express Foxn1 and give rise to orthotopic (thoracic) and additional (cervical) thymi. Using Foxn1-directed cytoablation, we show that during embryogenesis, cervical thymi develop a few days after the thoracic lobes, and that bipotent epithelial progenitors of co...

  5. Transcription factor KLF7 regulates differentiation of neuroectodermal and mesodermal cell lineages

    Energy Technology Data Exchange (ETDEWEB)

    Caiazzo, Massimiliano, E-mail: caiazzo@igb.cnr.it [Institute of Genetics and Biophysics ' A. Buzzati-Traverso,' CNR, 80131 Naples (Italy); Istituto di diagnosi e cura ' Hermitage Capodimonte,' 80131 Naples (Italy); Colucci-D' Amato, Luca, E-mail: luca.colucci@unina2.it [Institute of Genetics and Biophysics ' A. Buzzati-Traverso,' CNR, 80131 Naples (Italy); Dipartimento di Scienze della Vita, Seconda Universita di Napoli, 81100 Caserta (Italy); Esposito, Maria T., E-mail: maria_teresa.esposito@kcl.ac.uk [CEINGE Biotecnologie Avanzate, 80145 Naples (Italy); Parisi, Silvia, E-mail: parisi@ceinge.unina.it [CEINGE Biotecnologie Avanzate, 80145 Naples (Italy); Stifani, Stefano, E-mail: stefano.stifani@mcgill.ca [Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4 (Canada); Ramirez, Francesco, E-mail: francesco.ramirez@mssm.edu [Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029 (United States); Porzio, Umberto di, E-mail: diporzio@igb.cnr.it [Institute of Genetics and Biophysics ' A. Buzzati-Traverso,' CNR, 80131 Naples (Italy)

    2010-08-15

    Previous gene targeting studies in mice have implicated the nuclear protein Krueppel-like factor 7 (KLF7) in nervous system development while cell culture assays have documented its involvement in cell cycle regulation. By employing short hairpin RNA (shRNA)-mediated gene silencing, here we demonstrate that murine Klf7 gene expression is required for in vitro differentiation of neuroectodermal and mesodermal cells. Specifically, we show a correlation of Klf7 silencing with down-regulation of the neuronal marker microtubule-associated protein 2 (Map2) and the nerve growth factor (NGF) tyrosine kinase receptor A (TrkA) using the PC12 neuronal cell line. Similarly, KLF7 inactivation in Klf7-null mice decreases the expression of the neurogenic marker brain lipid-binding protein/fatty acid-binding protein 7 (BLBP/FABP7) in neural stem cells (NSCs). We also report that Klf7 silencing is detrimental to neuronal and cardiomyocytic differentiation of embryonic stem cells (ESCs), in addition to altering the adipogenic and osteogenic potential of mouse embryonic fibroblasts (MEFs). Finally, our results suggest that genes that are key for self-renewal of undifferentiated ESCs repress Klf7 expression in ESCs. Together with previous findings, these results provide evidence that KLF7 has a broad spectrum of regulatory functions, which reflect the discrete cellular and molecular contexts in which this transcription factor operates.

  6. A conserved chromatin architecture marks and maintains the restricted germ cell lineage in worms and flies.

    Science.gov (United States)

    Schaner, Christine E; Deshpande, Girish; Schedl, Paul D; Kelly, William G

    2003-11-01

    In C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. We have identified a chromatin-based mechanism that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis.

  7. Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage.

    Science.gov (United States)

    Bischoff, D S; Zhu, J H; Makhijani, N S; Kumar, A; Yamaguchi, D T

    2008-02-15

    The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay.

  8. [Master Transcription Regulators Specifying Cell-Lineage Fates in Development As Possible Therapeutic Targets in Oncology].

    Science.gov (United States)

    Kondratyeva, L G; Vinogradova, T V; Chernov, I P; Sverdlov, E D

    2015-11-01

    The transformation of normal precursors into cancer cells is an intricately regulated, multistep process. The master regulatory genes that play a crucial role in the process of organism development may also play a key role in carcinogenesis. From such a point of view, cancer is not simply a genetic disease that is due to a progressive accumulation of mutation--it is also a disorder of the developmental system of the tissue in which cancer emerges. Master regulators and their genes disturb stem cell differentiation upon mutation and thus may serve as targets for cancer therapy, in addition to the classic oncogenes and suppressors of tumor formation. This review is an attempt to give a modern concept of master genes and their functions in adult stem cells of the organism and in carcinogenesis, with pancreatic cancer as an example.

  9. A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells.

    Science.gov (United States)

    Rieske, Piotr; Augelli, Brian J; Stawski, Robert; Gaughan, John; Azizi, S Ausim; Krynska, Barbara

    2009-02-01

    Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of betaIII-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors.

  10. A Dorsal SHH-Dependent Domain in the V-SVZ Produces Large Numbers of Oligodendroglial Lineage Cells in the Postnatal Brain

    Directory of Open Access Journals (Sweden)

    Cheuk Ka Tong

    2015-10-01

    Full Text Available Neural stem cells in different locations of the postnatal mouse ventricular-subventricular zone (V-SVZ generate different subtypes of olfactory bulb (OB interneurons. High Sonic hedgehog (SHH signaling in the ventral V-SVZ regulates the production of specific subtypes of neurons destined for the OB. Here we found a transient territory of high SHH signaling in the dorsal V-SVZ beneath the corpus callosum (CC. Using intersectional lineage tracing in neonates to label dorsal radial glial cells (RGCs expressing the SHH target gene Gli1, we demonstrate that this region produces many CC cells in the oligodendroglial lineage and specific subtypes of neurons in the OB. The number of oligodendroglial cells generated correlated with the levels of SHH signaling. This work identifies a dorsal domain of SHH signaling, which is an important source of oligodendroglial cells for the postnatal mammalian forebrain.

  11. A Dorsal SHH-Dependent Domain in the V-SVZ Produces Large Numbers of Oligodendroglial Lineage Cells in the Postnatal Brain.

    Science.gov (United States)

    Tong, Cheuk Ka; Fuentealba, Luis C; Shah, Jugal K; Lindquist, Robert A; Ihrie, Rebecca A; Guinto, Cristina D; Rodas-Rodriguez, Jose L; Alvarez-Buylla, Arturo

    2015-10-13

    Neural stem cells in different locations of the postnatal mouse ventricular-subventricular zone (V-SVZ) generate different subtypes of olfactory bulb (OB) interneurons. High Sonic hedgehog (SHH) signaling in the ventral V-SVZ regulates the production of specific subtypes of neurons destined for the OB. Here we found a transient territory of high SHH signaling in the dorsal V-SVZ beneath the corpus callosum (CC). Using intersectional lineage tracing in neonates to label dorsal radial glial cells (RGCs) expressing the SHH target gene Gli1, we demonstrate that this region produces many CC cells in the oligodendroglial lineage and specific subtypes of neurons in the OB. The number of oligodendroglial cells generated correlated with the levels of SHH signaling. This work identifies a dorsal domain of SHH signaling, which is an important source of oligodendroglial cells for the postnatal mammalian forebrain.

  12. A role for mixed lineage kinases in granule cell apoptosis induced by cytoskeletal disruption

    DEFF Research Database (Denmark)

    Müller, Georg Johannes; Geist, Marie Aavang; Veng, Lone Merete

    2006-01-01

    Microtubule disruption by colchicine induces apoptosis in selected neuronal populations. However, little is known about the upstream death signalling events mediating the neurotoxicity. We investigated first whether colchicine-induced granule cell apoptosis activates the c-Jun N-terminal kinase...

  13. Endogenous GABA controls oligodendrocyte lineage cell number, myelination, and CNS internode length

    DEFF Research Database (Denmark)

    Hamilton, Nicola B; Clarke, Laura E; Arancibia-Carcamo, I Lorena;

    2016-01-01

    Adjusting the thickness and internodal length of the myelin sheath is a mechanism for tuning the conduction velocity of axons to match computational needs. Interactions between oligodendrocyte precursor cells (OPCs) and developing axons regulate the formation of myelin around axons. We now show, ...

  14. The "Yin" and "Yang" of Cell Cycle Progression and Differentiation in the Oligodendroglial Lineage

    Science.gov (United States)

    Nguyen, Laurent; Borgs, Laurence; Vandenbosch, Renaud; Mangin, Jean-Marie; Beukelaers, Pierre; Moonen, Gustave; Gallo, Vittorio; Malgrange, Brigitte; Belachew, Shibeshih

    2006-01-01

    In white matter disorders such as leukodystrophies (LD), periventricular leucomalacia (PVL), or multiple sclerosis (MS), the hypomyelination or the remyelination failure by oligodendrocyte progenitor cells involves errors in the sequence of events that normally occur during development when progenitors proliferate, migrate through the white…

  15. A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification

    Directory of Open Access Journals (Sweden)

    Syandan Chakraborty

    2014-12-01

    Full Text Available Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior. We show that the fusion of two transactivation domains to Cas9 dramatically enhances gene activation to a level that is necessary to reprogram cell phenotype. Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes. The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.

  16. The synergistic effect of nanotopography and sustained dual release of hydrophobic and hydrophilic neurotrophic factors on human mesenchymal stem cell neuronal lineage commitment.

    Science.gov (United States)

    Teo, Benjamin Kim Kiat; Tan, Guo-Dong Sean; Yim, Evelyn K F

    2014-08-01

    A combination of nanotopography and controlled release is a potential platform for neuronal tissue engineering applications. Previous studies showed that combining both physical and chemical guidance was more effective than individual cues in the directional promotion of neurite outgrowth. Nanotopography can direct human mesenchymal stem cells (hMSCs) into neuronal lineage, while controlled release of neurotrophic factors can deliver temporally controlled biochemical signals. Hypothesizing that the synergistic effect will enhance neuronal lineage commitment of hMSCs, a fabrication method for multiple neurotrophic factors delivery from a single nanopatterned (350 nm gratings), poly-ɛ-caprolactone (PCL) film was developed and evaluated. Our results showed a synergistic effect on hMSC differentiation cultured on substrates with both nanotopographical and biochemical cues. The protein/drug encapsulation into PCL nanopatterned films was first optimized using a hydrophilic model protein, bovine serum albumin. The hydrophobic retinoic acid (RA) molecule was directly incorporated into PCL films. To achieve sustained release, hydrophilic nerve growth factor (NGF) was first encapsulated within polyelectrolyte complexation fibers before they were embedded within the nanopatterned PCL film. Our results showed that nanotopography on the fabricated polymer films remained intact, while release of bioactive RA and NGF was sustained over a period of 3 weeks. Under the combinatorial effect of physical and biochemical cues, we observed an enhanced upregulation of neuronal genes such as microtubule-associated protein 2 (MAP2) and neurofilament light (NFL) as compared with sustained delivery of individual cues and bolus delivery. Quantitative polymerase chain reaction analysis showed that MAP2 and NFL gene upregulation in hMSCs was most pronounced on the nanogratings with sustained release of both RA and NGF. The fabricated platforms supported the sustained delivery of multiple

  17. Extracellular Vesicles Derived from Osteogenically Induced Human Bone Marrow Mesenchymal Stem Cells Can Modulate Lineage Commitment

    Directory of Open Access Journals (Sweden)

    Margarida Martins

    2016-03-01

    Full Text Available The effective osteogenic commitment of human bone marrow mesenchymal stem cells (hBMSCs is critical for bone regenerative therapies. Extracellular vesicles (EVs derived from hBMSCs have a regenerative potential that has been increasingly recognized. Herein, the osteoinductive potential of osteogenically induced hBMSC-EVs was examined. hBMSCs secreted negatively charged nanosized vesicles (∼35 nm with EV-related surface markers. The yield of EVs over 7 days was dependent on an osteogenic stimulus (standard chemical cocktail or RUNX2 cationic-lipid transfection. These EVs were used to sequentially stimulate homotypic uncommitted cells during 7 days, matching the seeding density of EV parent cells, culture time, and stimuli. Osteogenically committed hBMSC-EVs induced an osteogenic phenotype characterized by marked early induction of BMP2, SP7, SPP1, BGLAP/IBSP, and alkaline phosphatase. Both EV groups outperformed the currently used osteoinductive strategies. These data show that naturally secreted EVs can guide the osteogenic commitment of hBMSCs in the absence of other chemical or genetic osteoinductors.

  18. Motor neurons and oligodendrocytes arise from distinct cell lineages by progenitor recruitment.

    Science.gov (United States)

    Ravanelli, Andrew M; Appel, Bruce

    2015-12-01

    During spinal cord development, ventral neural progenitor cells that express the transcription factors Olig1 and Olig2, called pMN progenitors, produce motor neurons and then oligodendrocytes. Whether motor neurons and oligodendrocytes arise from common or distinct progenitors in vivo is not known. Using zebrafish, we found that motor neurons and oligodendrocytes are produced sequentially by distinct progenitors that have distinct origins. When olig2(+) cells were tracked during the peak period of motor neuron formation, most differentiated as motor neurons without further cell division. Using time-lapse imaging, we found that, as motor neurons differentiated, more dorsally positioned neuroepithelial progenitors descended to the pMN domain and initiated olig2 expression. Inhibition of Hedgehog signaling during motor neuron differentiation blocked the ventral movement of progenitors, the progressive initiation of olig2 expression, and oligodendrocyte formation. We therefore propose that the motor neuron-to-oligodendrocyte switch results from Hedgehog-mediated recruitment of glial-fated progenitors to the pMN domain subsequent to neurogenesis.

  19. A major lineage of enteroendocrine cells coexpress CCK, secretin, GIP, GLP-1, PYY, and neurotensin but not somatostatin

    DEFF Research Database (Denmark)

    Egerod, Kristoffer Lihme; Engelstoft, Maja Storm; Grunddal, Kaare Villum

    2012-01-01

    in crypts and villi as demonstrated by immunohistochemistry and FACS analysis of separated cell populations. Single-cell quantitative PCR indicated that approximately half of the duodenal CCK-eGFP cells express one peptide precursor in addition to CCK, whereas an additional smaller fraction expresses two...

  20. Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

    Science.gov (United States)

    Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

    2016-01-01

    We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001 PMID:27644592

  1. Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin

    NARCIS (Netherlands)

    C.E. Wong (Christine); S. Paratore (Sabrina); M.T. Dours-Zimmermann (María); T. Rochat (Thierry); T. Pietri (Thomas); U. Suter (Ueli); D. Zimmermann (Dieter); S. Dufour (Sylvie); J.P. Thiery (Joachim); D.N. Meijer (Dies); C. Beermann (Christopher); Y. Barrandon (Yann); L. Sommer (Lukas)

    2006-01-01

    textabstractGiven their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell-like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and di

  2. Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage.

    Science.gov (United States)

    Quiroz, Felipe Garcia; Posada, Olga M; Gallego-Perez, Daniel; Higuita-Castro, Natalia; Sarassa, Carlos; Hansford, Derek J; Agudelo-Florez, Piedad; López, Luis E

    2010-04-01

    Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation. This work aimed to study the stability of the housekeeping genes beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A) during the osteogenic differentiation of hBMSCs. Their stability was evaluated via RT-qPCR in 14 and 20 day differentiation assays to the osteogenic lineage. Different normalization strategies were evaluated to quantify the osteogenic markers collagen type I, bone sialoprotein and osteonectin. Cell differentiation was confirmed via alizarin red staining. The results demonstrated up-regulation of beta-actin with maximum fold changes (MFC) of 4.38. GAPDH and RPL13A were not regulated by osteogenic media after 14 days and presented average fold changes lower than 2 in 20 day cultures. RPL13A (MFC < 2) had a greater stability when normalizing as a function of culture time compared with GAPDH (MFC cells characterization via RT-qPCR.

  3. Hematopoietic lineage cell specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation.

    Science.gov (United States)

    Kahner, Bryan N; Dorsam, Robert T; Mada, Sripal R; Kim, Soochong; Stalker, Timothy J; Brass, Lawrence F; Daniel, James L; Kitamura, Daisuke; Kunapuli, Satya P

    2007-10-01

    Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). We have investigated the contribution of hematopoietic lineage cell-specific protein 1 (HS1) downstream of GPVI in platelet activation. Stimulation of GPVI leads to tyrosine phosphorylation of HS1, which is blocked by Src-family kinase inhibitors. Coimmunoprecipitation experiments revealed that HS1 associates with Syk and phosphatidylinositol 3-kinases. HS1-null mice displayed increased bleeding times and increased time to occlusion in the FeCl(3) in vivo thrombosis model compared with their wild-type littermates. In addition, aggregation and secretion responses were diminished in HS1-null mouse platelets after stimulation of GPVI and protease-activated receptor 4 (PAR-4) agonists compared with wild-type littermate mouse platelets. Finally, Akt phosphorylation was diminished after GPVI or PAR-4 stimulation in platelets from HS1-null mice compared with their wild-type littermates. These results demonstrate that phosphorylation of the HS1 protein occurs downstream of GPVI stimulation and that HS1 plays a significant functional role in platelet activation downstream of GPVI and PARs.

  4. Identification of a cell lineage-specific gene coding for a sea urchin alpha 2(IV)-like collagen chain.

    Science.gov (United States)

    Exposito, J Y; Suzuki, H; Geourjon, C; Garrone, R; Solursh, M; Ramirez, F

    1994-05-06

    We report the isolation of several overlapping cDNAs from an embryonic library of Strongylocentrotus purpuratus coding for a novel sea urchin collagen chain. The conceptual amino acid translation of the cDNAs indicated that the protein displays the structural features of a vertebrate type IV-like collagen alpha chain. In addition to a putative 31-residue signal peptide, the sea urchin molecule contains a 14-residue amino-terminal non-collagenous segment, a discontinuous 1,477-amino acid triple helical domain, and a 225-residue carboxyl-terminal domain rich in cysteines. The amino- and carboxyl-terminal non-collagenous regions of the echinoid molecule are remarkably similar to the 7 S and carboxyl-terminal non-collagenous (NC1) domains of the alpha 1 and alpha 2 chains of vertebrate type IV collagen. The sequence similarity and distinct structural features of the 7 S and NC1 domains strongly suggest that the sea urchin polypeptide is evolutionarily related to the alpha 2(IV) class of collagen chains. Finally, in situ hybridizations revealed that expression of this collagen gene is restricted to the mesenchyme cell lineage of the developing sea urchin embryo.

  5. CD3 Ligation on Immature Thymocytes Generates Antagonist-like Signals Appropriate for CD8 Lineage Commitment, Independently of  T Cell Receptor Specificity

    Science.gov (United States)

    Albert Basson, M.; Bommhardt, Ursula; Cole, Michael S.; Tso, J. Yun; Zamoyska, Rose

    1998-01-01

    The signals that direct differentiation of T cells to the CD4 or CD8 lineages in the thymus remain poorly understood. Although it has been relatively easy to direct differentiation of CD4 single positive (CD4+) cells using combinations of antibodies and pharmacological agents that mimic receptor engagements, equivalent stimuli do not induce efficient maturation of CD8+ cells. Here we report that, irrespective of the MHC-restriction specificity of the TCR, differentiation of mature CD8+ thymocytes can be induced by ligation of CD3 polypeptides on immature thymocytes with a F(ab′)2 reagent (CD3fos-F(ab′)2). The tyrosine phosphorylation patterns stimulated by CD3fos-F(ab′)2 have been shown to resemble those delivered to mature T cells by antagonist peptides, which are known to direct positive selection of CD8+ cells, and we can show that this reagent exhibits potent antagonistic-like activity for primary T cell responses. Our results suggest a distinction in the signals that specify lineage commitment in the thymus. We present a model of thymocyte differentiation that proposes that the relative balance of signals delivered by TCR engagement and by p56lck activation is responsible for directing commitment to the CD8 or CD4 lineages. PMID:9547336

  6. Rapid detection of dendritic cell and monocyte disorders using CD4 as a lineage marker of the human peripheral blood antigen presenting cell compartment

    Directory of Open Access Journals (Sweden)

    Laura eJardine

    2013-12-01

    Full Text Available Dendritic cells (DCs and monocytes are critical regulators and effectors of innate and adaptive immune responses. Monocyte expansion has been described in many pathological states while monocyte and DC deficiency syndromes are relatively recent additions to the catalogue of human primary immunodeficiency disorders. Clinically applicable screening tests to diagnose and monitor these conditions are lacking. Conventional strategies for identifying human DCs and monocytes have been based on the use of a lineage gate to exclude lymphocytes, thus preventing simultaneous detection of DCs, monocytes and lymphocyte subsets. Here we demonstrate that CD4 is a reliable lineage marker for the human peripheral blood antigen presenting cell compartment that can be used to identify DCs and monocytes in parallel with lymphocytes. Based on this principle, simple modification of a standard lymphocyte phenotyping assay permits simultaneous enumeration of four lymphocyte and five DC/monocyte populations from a single sample. This approach is applicable to clinical samples and facilitates the diagnosis of DC and monocyte disorders in a wide range of clinical settings, including genetic deficiency, neoplasia and inflammation.

  7. The Peutz-Jeghers kinase LKB1 suppresses polyp growth from intestinal cells of a proglucagon-expressing lineage in mice

    Directory of Open Access Journals (Sweden)

    Sagen Zac-Varghese

    2014-11-01

    Full Text Available Liver kinase B1 (LKB1; also known as STK11 is a serine/threonine kinase and tumour suppressor that is mutated in Peutz-Jeghers syndrome (PJS, a premalignant syndrome associated with the development of gastrointestinal polyps. Proglucagon-expressing enteroendocrine cells are involved in the control of glucose homeostasis and the regulation of appetite through the secretion of gut hormones such as glucagon-like peptide-1 (GLP-1 and peptide tyrosine tyrosine (PYY. To determine the role of LKB1 in these cells, we bred mice bearing floxed alleles of Lkb1 against animals carrying Cre recombinase under proglucagon promoter control. These mice (GluLKB1KO were viable and displayed near-normal growth rates and glucose homeostasis. However, they developed large polyps at the gastro-duodenal junction, and displayed premature mortality (death from 120 days of age. Histological analysis of the polyps demonstrated that they had a PJS-like appearance with an arborising smooth-muscle core. Circulating GLP-1 levels were normal in GluLKB1KO mice and the polyps expressed low levels of the peptide, similar to levels in the neighbouring duodenum. Lineage tracing using a Rosa26tdRFP transgene revealed, unexpectedly, that enterocytes within the polyps were derived from non-proglucagon-expressing precursors, whereas connective tissue was largely derived from proglucagon-expressing precursors. Developmental studies in wild-type mice suggested that a subpopulation of proglucagon-expressing cells undergo epithelial-mesenchymal transition (EMT to become smooth-muscle-like cells. Thus, it is likely that polyps in the GluLKB1KO mice developed from a unique population of smooth-muscle-like cells derived from a proglucagon-expressing precursor. The loss of LKB1 within this subpopulation seems to be sufficient to drive tumorigenesis.

  8. A Gene Regulatory Network Cooperatively Controlled by Pdx1 and Sox9 Governs Lineage Allocation of Foregut Progenitor Cells

    DEFF Research Database (Denmark)

    Shih, Hung Ping; Seymour, Philip A; Patel, Nisha A;

    2015-01-01

    The generation of pancreas, liver, and intestine from a common pool of progenitors in the foregut endoderm requires the establishment of organ boundaries. How dorsal foregut progenitors activate pancreatic genes and evade the intestinal lineage choice remains unclear. Here, we identify Pdx1 and Sox...... pancreatic fate and sheds light on the gene regulatory circuitry that governs the development of distinct organs from multi-lineage-competent foregut progenitors....

  9. Chromatin dynamics in Pollen Mother Cells underpin a common scenario at the somatic-to-reproductive fate transition of both the male and female lineages in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Wenjing eShe

    2015-04-01

    Full Text Available Unlike animals, where the germline is established early during embryogenesis, plants set aside their reproductive lineage late in development in dedicated floral organs. The specification of pollen mother cells (PMCs committed to meiosis takes place in the sporogenous tissue in anther locules and marks the somatic-to-reproductive cell fate transition towards the male reproductive lineage. Here we show that Arabidopsis PMCs differentiation is accompanied by large-scale changes in chromatin organization. This is characterized by significant increase in nuclear volume, chromatin decondensation, reduction in heterochromatin, eviction of linker histones and the H2AZ histone variant. These structural alterations are accompanied by dramatic, quantitative changes in histone modifications levels compared to that of surrounding somatic cells that do not share a sporogenic fate. All these changes are highly reminiscent of those we have formerly described in female megaspore mother cells (MMCs. This indicates that chromatin reprogramming is a common underlying scenario in the somatic-to-reproductive cell fate transition in both male and female lineages.

  10. Chromatin dynamics in pollen mother cells underpin a common scenario at the somatic-to-reproductive fate transition of both the male and female lineages in Arabidopsis.

    Science.gov (United States)

    She, Wenjing; Baroux, Célia

    2015-01-01

    Unlike animals, where the germline is established early during embryogenesis, plants set aside their reproductive lineage late in development in dedicated floral organs. The specification of pollen mother cells (PMC) committed to meiosis takes place in the sporogenous tissue in anther locules and marks the somatic-to-reproductive cell fate transition toward the male reproductive lineage. Here we show that Arabidopsis PMC differentiation is accompanied by large-scale changes in chromatin organization. This is characterized by significant increase in nuclear volume, chromatin decondensation, reduction in heterochromatin, eviction of linker histones and the H2AZ histone variant. These structural alterations are accompanied by dramatic, quantitative changes in histone modifications levels compared to that of surrounding somatic cells that do not share a sporogenic fate. All these changes are highly reminiscent of those we have formerly described in female megaspore mother cells (MMC). This indicates that chromatin reprogramming is a common underlying scenario in the somatic-to-reproductive cell fate transition in both male and female lineages.

  11. Direct crosstalk between cancer and osteoblast lineage cells fuels metastatic growth in bone via auto-amplification of IL-6 and RANKL signaling pathways.

    Science.gov (United States)

    Zheng, Yu; Chow, Shu-Oi; Boernert, Katja; Basel, Dennis; Mikuscheva, Anastasia; Kim, Sarah; Fong-Yee, Colette; Trivedi, Trupti; Buttgereit, Frank; Sutherland, Robert L; Dunstan, Colin R; Zhou, Hong; Seibel, Markus J

    2014-09-01

    The bone microenvironment and its modification by cancer and host cell interactions is a key driver of skeletal metastatic growth. Interleukin-6 (IL-6) stimulates receptor activator of NF-κB ligand (RANKL) expression in bone cells, and serum IL-6 levels are associated with poor clinical outcomes in cancer patients. We investigated the effects of RANKL on cancer cells and the role of tumor-derived IL-6 within the bone microenvironment. Using human breast cancer cell lines to induce tumors in the bone of immune-deficient mice, we first determined whether RANKL released by cells of the osteoblast lineage directly promotes IL-6 expression by cancer cells in vitro and in vivo. We then disrupted of IL-6 signaling in vivo either via knockdown of IL-6 in tumor cells or through treatment with specific anti-human or anti-mouse IL-6 receptor antibodies to investigate the tumor effect. Finally, we tested the effect of RANK knockdown in cancer cells on cancer growth. We demonstrate that osteoblast lineage-derived RANKL upregulates secretion of IL-6 by breast cancers in vivo and in vitro. IL-6, in turn, induces expression of RANK by cancer cells, which sensitizes the tumor to RANKL and significantly enhances cancer IL-6 release. Disruption in vivo of this auto-amplifying crosstalk by knockdown of IL-6 or RANK in cancer cells, or via treatment with anti-IL-6 receptor antibodies, significantly reduces tumor growth in bone but not in soft tissues. RANKL and IL-6 mediate direct paracrine-autocrine signaling between cells of the osteoblast lineage and cancer cells, significantly enhancing the growth of metastatic breast cancers within bone.

  12. Lineage analysis of circulating Trypanosoma cruzi parasites and their association with clinical forms of Chagas disease in Bolivia.

    Directory of Open Access Journals (Sweden)

    Ramona del Puerto

    Full Text Available BACKGROUND: The causative agent of Chagas disease, Trypanosoma cruzi, is divided into 6 Discrete Typing Units (DTU: Tc I, IIa, IIb, IIc, IId and IIe. In order to assess the relative pathogenicities of different DTUs, blood samples from three different clinical groups of chronic Chagas disease patients (indeterminate, cardiac, megacolon from Bolivia were analyzed for their circulating parasites lineages using minicircle kinetoplast DNA polymorphism. METHODS AND FINDINGS: Between 2000 and 2007, patients sent to the Centro Nacional de Enfermedades Tropicales for diagnosis of Chagas from clinics and hospitals in Santa Cruz, Bolivia, were assessed by serology, cardiology and gastro-intestinal examinations. Additionally, patients who underwent colonectomies due to Chagasic magacolon at the Hospital Universitario Japonés were also included. A total of 306 chronic Chagas patients were defined by their clinical types (81 with cardiopathy, 150 without cardiopathy, 100 with megacolon, 144 without megacolon, 164 with cardiopathy or megacolon, 73 indeterminate and 17 cases with both cardiopathy and megacolon. DNA was extracted from 10 ml of peripheral venous blood for PCR analysis. The kinetoplast minicircle DNA (kDNA was amplified from 196 out of 306 samples (64.1%, of which 104 (53.3% were Tc IId, 4 (2.0% Tc I, 7 (3.6% Tc IIb, 1 (0.5% Tc IIe, 26 (13.3% Tc I/IId, 1 (0.5% Tc I/IIb/IId, 2 (1.0% Tc IIb/d and 51 (25.9% were unidentified. Of the 133 Tc IId samples, three different kDNA hypervariable region patterns were detected; Mn (49.6%, TPK like (48.9% and Bug-like (1.5%. There was no significant association between Tc types and clinical manifestations of disease. CONCLUSIONS: None of the identified lineages or sublineages was significantly associated with any particular clinical manifestations in the chronic Chagas patients in Bolivia.

  13. Dynamic formation of asexual diploid and polyploid lineages: multilocus analysis of Cobitis reveals the mechanisms maintaining the diversity of clones.

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    Karel Janko

    Full Text Available Given the hybrid genomic constitutions and increased ploidy of many asexual animals, the identification of processes governing the origin and maintenance of clonal diversity provides useful information about the evolutionary consequences of interspecific hybridization, asexuality and polyploidy. In order to understand the processes driving observed diversity of biotypes and clones in the Cobitis taenia hybrid complex, we performed fine-scale genetic analysis of Central European hybrid zone between two sexual species using microsatellite genotyping and mtDNA sequencing. We found that the hybrid zone is populated by an assemblage of clonally (gynogenetically reproducing di-, tri- and tetraploid hybrid lineages and that successful clones, which are able of spatial expansion, recruit from two ploidy levels, i.e. diploid and triploid. We further compared the distribution of observed estimates of clonal ages to theoretical distributions simulated under various assumptions and showed that new clones are most likely continuously recruited from ancestral populations. This suggests that the clonal diversity is maintained by dynamic equilibrium between origination and extinction of clonal lineages. On the other hand, an interclonal selection is implied by nonrandom spatial distribution of individual clones with respect to the coexisting sexual species. Importantly, there was no evidence for sexually reproducing hybrids or clonally reproducing non-hybrid forms. Together with previous successful laboratory synthesis of clonal Cobitis hybrids, our data thus provide the most compelling evidence that 1 the origin of asexuality is causally linked to interspecific hybridization; 2 successful establishment of clones is not restricted to one specific ploidy level and 3 the initiation of clonality and polyploidy may be dynamic and continuous in asexual complexes.

  14. Lineage Analysis of Circulating Trypanosoma cruzi Parasites and Their Association with Clinical Forms of Chagas Disease in Bolivia

    Science.gov (United States)

    del Puerto, Ramona; Nishizawa, Juan Eiki; Kikuchi, Mihoko; Iihoshi, Naomi; Roca, Yelin; Avilas, Cinthia; Gianella, Alberto; Lora, Javier; Gutierrez Velarde, Freddy Udalrico; Renjel, Luis Alberto; Miura, Sachio; Higo, Hiroo; Komiya, Norihiro; Maemura, Koji; Hirayama, Kenji

    2010-01-01

    Background The causative agent of Chagas disease, Trypanosoma cruzi, is divided into 6 Discrete Typing Units (DTU): Tc I, IIa, IIb, IIc, IId and IIe. In order to assess the relative pathogenicities of different DTUs, blood samples from three different clinical groups of chronic Chagas disease patients (indeterminate, cardiac, megacolon) from Bolivia were analyzed for their circulating parasites lineages using minicircle kinetoplast DNA polymorphism. Methods and Findings Between 2000 and 2007, patients sent to the Centro Nacional de Enfermedades Tropicales for diagnosis of Chagas from clinics and hospitals in Santa Cruz, Bolivia, were assessed by serology, cardiology and gastro-intestinal examinations. Additionally, patients who underwent colonectomies due to Chagasic magacolon at the Hospital Universitario Japonés were also included. A total of 306 chronic Chagas patients were defined by their clinical types (81 with cardiopathy, 150 without cardiopathy, 100 with megacolon, 144 without megacolon, 164 with cardiopathy or megacolon, 73 indeterminate and 17 cases with both cardiopathy and megacolon). DNA was extracted from 10 ml of peripheral venous blood for PCR analysis. The kinetoplast minicircle DNA (kDNA) was amplified from 196 out of 306 samples (64.1%), of which 104 (53.3%) were Tc IId, 4 (2.0%) Tc I, 7 (3.6%) Tc IIb, 1 (0.5%) Tc IIe, 26 (13.3%) Tc I/IId, 1 (0.5%) Tc I/IIb/IId, 2 (1.0%) Tc IIb/d and 51 (25.9%) were unidentified. Of the 133 Tc IId samples, three different kDNA hypervariable region patterns were detected; Mn (49.6%), TPK like (48.9%) and Bug-like (1.5%). There was no significant association between Tc types and clinical manifestations of disease. Conclusions None of the identified lineages or sublineages was significantly associated with any particular clinical manifestations in the chronic Chagas patients in Bolivia. PMID:20502516

  15. Analysis of complete nucleotide sequences of Angolan hepatitis B virus isolates reveals the existence of a separate lineage within genotype E.

    Directory of Open Access Journals (Sweden)

    Barbara V Lago

    Full Text Available Hepatitis B virus genotype E (HBV/E is highly prevalent in Western Africa. In this work, 30 HBV/E isolates from HBsAg positive Angolans (staff and visitors of a private hospital in Luanda were genetically characterized: 16 of them were completely sequenced and the pre-S/S sequences of the remaining 14 were determined. A high proportion (12/30, 40% of subjects tested positive for both HBsAg and anti-HBs markers. Deduced amino acid sequences revealed the existence of specific substitutions and deletions in the B- and T-cell epitopes of the surface antigen (pre-S1- and pre-S2 regions of the virus isolates derived from 8/12 individuals with concurrent HBsAg/anti-HBs. Phylogenetic analysis performed with 231 HBV/E full-length sequences, including 16 from this study, showed that all isolates from Angola, Namibia and the Democratic Republic of Congo (n = 28 clustered in a separate lineage, divergent from the HBV/E isolates from nine other African countries, namely Cameroon, Central African Republic, Côte d'Ivoire, Ghana, Guinea, Madagascar, Niger, Nigeria and Sudan, with a Bayesian posterior probability of 1. Five specific mutations, namely small S protein T57I, polymerase Q177H, G245W and M612L, and X protein V30L, were observed in 79-96% of the isolates of the separate lineage, compared to a frequency of 0-12% among the other HBV/E African isolates.

  16. Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers.

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    Wendy W Hwang-Verslues

    Full Text Available Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44(+/CD24(-/low and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR(+/ESA(+ cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44(+/CD24(-/low, ESA(+, CD133(+, CXCR4(+ and PROCR(+ in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

  17. Genetic Mosaics and the Germ Line Lineage

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    Mark E. Samuels

    2015-04-01

    Full Text Available Genetic mosaics provide information about cellular lineages that is otherwise difficult to obtain, especially in humans. De novo mutations act as cell markers, allowing the tracing of developmental trajectories of all descendants of the cell in which the new mutation arises. De novo mutations may arise at any time during development but are relatively rare. They have usually been observed through medical ascertainment, when the mutation causes unusual clinical signs or symptoms. Mutational events can include aneuploidies, large chromosomal rearrangements, copy number variants, or point mutations. In this review we focus primarily on the analysis of point mutations and their utility in addressing questions of germ line versus somatic lineages. Genetic mosaics demonstrate that the germ line and soma diverge early in development, since there are many examples of combined somatic and germ line mosaicism for de novo mutations. The occurrence of simultaneous mosaicism in both the germ line and soma also shows that the germ line is not strictly clonal but arises from at least two, and possibly multiple, cells in the embryo with different ancestries. Whole genome or exome DNA sequencing technologies promise to expand the range of studies of genetic mosaics, as de novo mutations can now be identified through sequencing alone in the absence of a medical ascertainment. These technologies have been used to study mutation patterns in nuclear families and in monozygotic twins, and in animal model developmental studies, but not yet for extensive cell lineage studies in humans.

  18. Green tea epigallocatechin-3-gallate modulates differentiation of naive CD4+ T cells into specific lineage effector cells

    Science.gov (United States)

    CD4+ T helper (Th) subsets Th1, Th9, and Th17 cells are implicated in inducing autoimmunity whereas regulatory T cells (Treg) have a protective effect. We previously showed that epigallocatechin-3-gallate (EGCG) attenuated experimental autoimmune encephalomyelitis (EAE) and altered CD4+ T cell subpo...

  19. Discriminating micropathogen lineages and their reticulate evolution through graph theory-based network analysis: the case of Trypanosoma cruzi, the agent of Chagas disease.

    Science.gov (United States)

    Arnaud-Haond, Sophie; Moalic, Yann; Barnabé, Christian; Ayala, Francisco José; Tibayrenc, Michel

    2014-01-01

    Micropathogens (viruses, bacteria, fungi, parasitic protozoa) share a common trait, which is partial clonality, with wide variance in the respective influence of clonality and sexual recombination on the dynamics and evolution of taxa. The discrimination of distinct lineages and the reconstruction of their phylogenetic history are key information to infer their biomedical properties. However, the phylogenetic picture is often clouded by occasional events of recombination across divergent lineages, limiting the relevance of classical phylogenetic analysis and dichotomic trees. We have applied a network analysis based on graph theory to illustrate the relationships among genotypes of Trypanosoma cruzi, the parasitic protozoan responsible for Chagas disease, to identify major lineages and to unravel their past history of divergence and possible recombination events. At the scale of T. cruzi subspecific diversity, graph theory-based networks applied to 22 isoenzyme loci (262 distinct Multi-Locus-Enzyme-Electrophoresis -MLEE) and 19 microsatellite loci (66 Multi-Locus-Genotypes -MLG) fully confirms the high clustering of genotypes into major lineages or "near-clades". The release of the dichotomic constraint associated with phylogenetic reconstruction usually applied to Multilocus data allows identifying putative hybrids and their parental lineages. Reticulate topology suggests a slightly different history for some of the main "near-clades", and a possibly more complex origin for the putative hybrids than hitherto proposed. Finally the sub-network of the near-clade T. cruzi I (28 MLG) shows a clustering subdivision into three differentiated lesser near-clades ("Russian doll pattern"), which confirms the hypothesis recently proposed by other investigators. The present study broadens and clarifies the hypotheses previously obtained from classical markers on the same sets of data, which demonstrates the added value of this approach. This underlines the potential of graph

  20. Metagenomic and whole-genome analysis reveals new lineages of gokushoviruses and biogeographic separation in the sea

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    Jessica Myriam Labonté

    2013-12-01

    Full Text Available Much remains to be learned about single-stranded (ss DNA viruses in natural systems, and the evolutionary relationships among them. One of the eight recognized families of ssDNA viruses is the Microviridae, a group of viruses infecting bacteria. In this study we used metagenomic analysis, genome assembly and amplicon sequencing of purified ssDNA to show that bacteriophages belonging to the subfamily Gokushovirinae within the Microviridae are genetically diverse and widespread members of marine microbial communities. Metagenomic analysis of coastal samples from the Gulf of Mexico and British Columbia, Canada, revealed numerous sequences belonging to gokushoviruses and allowed the assembly of five putative genomes with an organization similar to chlamydiamicroviruses. Fragment recruitment to these genomes from different metagenomic data sets is consistent with gokushovirus genotypes being restricted to specific oceanic regions. Conservation among the assembled genomes allowed the design of degenerate primers that target an 800 bp fragment from the gene encoding the major capsid protein. Sequences could be amplified from coastal temperate and subtropical waters, but not from samples collected from the Arctic Ocean, or freshwater lakes. Phylogenetic analysis revealed that most sequences were distantly related to those from cultured representatives. Moreover, the sequences fell into at least seven distinct evolutionary groups, most of which were represented by one of the assembled metagenomes. Our results greatly expand the known sequence space for gokushoviruses, and reveal biogeographic separation and new evolutionary lineages of gokushoviruses in the oceans.

  1. CKbeta8-1 alters expression of cyclin E in colony forming units-granulocyte macrophage (CFU-GM) lineage from human cord blood CD34+ cells.

    Science.gov (United States)

    Noh, Eui Kyu; Ra, Jae Sun; Lee, Seong Ae; Kwon, Byoung S; Han, In Seob

    2005-12-31

    A C6 beta-chemokine, CKbeta8-1, suppressed the colony formation of CD34+ cells of human cord blood (CB). Molecular mechanisms involved in CKbeta8-1-medicated suppression of colony formation of CD34+ cells are not known. To address this issue, the level of various G1/S cell cycle regulating proteins in CKbeta8-1-treated CD34+ cells were compared with those in untreated CD34+ cells. CKbeta8-1 did not significantly alter the expression of the G1/S cycle regulation proteins (cyclin D1, D3, and E), CDK inhibitor (p27and Rb), and other cell proliferation regulation protein (p53) in CB CD34+ cells. Here we describe an in vitro system in which CB CD34+ cells were committed to a multipotent progenitor lineage of colony forming units-granulocyte/macrophage (CFU-GM) by a simple combination of recombinant human (rh) GM-CSF and rhIL-3. In this culture system, we found that cyclin E protein appeared later and disappeared faster in the CKbeta8-1-treated cells than in the control cells during CFU-GM lineage development. These findings suggested that cyclin E may play a role in suppressing the colony formation of CFU-GM by CKbeta8-1.

  2. Targeting mixed lineage kinases in ER-positive breast cancer cells leads to G2/M cell cycle arrest and apoptosis.

    Science.gov (United States)

    Wang, Limin; Gallo, Kathleen A; Conrad, Susan E

    2013-08-01

    Estrogen receptor (ER)-positive tumors represent the most common type of breast cancer, and ER-targeted therapies such as antiestrogens and aromatase inhibitors have therefore been widely used in breast cancer treatment. While many patients have benefited from these therapies, both innate and acquired resistance continue to be causes of treatment failure. Novel targeted therapeutics that could be used alone or in combination with endocrine agents to treat resistant tumors or to prevent their development are therefore needed. In this report, we examined the effects of inhibiting mixed-lineage kinase (MLK) activity on ER-positive breast cancer cells and non-tumorigenic mammary epithelial cells. Inhibition of MLK activity with the pan-MLK inhibitor CEP-1347 blocked cell cycle progression in G2 and early M phase, and induced apoptosis in three ER-positive breast cancer cell lines, including one with acquired antiestrogen resistance. In contrast, it had no effect on the cell cycle or apoptosis in two non-tumorigenic mammary epithelial cell lines. CEP-1347 treatment did not decrease the level of active ERK or p38 in any of the cell lines tested. However, it resulted in decreased JNK and NF-κB activity in the breast cancer cell lines. A JNK inhibitor mimicked the effects of CEP-1347 in breast cancer cells, and overexpression of c-Jun rescued CEP-1347-induced Bax expression. These results indicate that proliferation and survival of ER-positive breast cancer cells are highly dependent on MLK activity, and suggest that MLK inhibitors may have therapeutic efficacy for ER-positive breast tumors, including ones that are resistant to current endocrine therapies.

  3. Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication.

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    Li-Jun Ma

    2009-07-01

    Full Text Available Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called "zygomycetes," R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs, comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11, could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.

  4. Genome characterization and phylogenetic analysis of a lineage IV peste des petits ruminants virus in southern China.

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    Li, Xiao-Peng; Zhai, Shao-Lun; He, Dong-Sheng; Guo, Peng-Ju; Lv, Dian-Hong; Wen, Xiao-Hui; Luo, Man-Lin; Chen, Rui-Ai; Wei, Wen-Kang

    2015-12-01

    Since 2013, the second outbreak of peste des petits ruminants (PPR) caused by Peste des petits ruminants virus (PPRV) has spread over more than 20 provinces, municipalities, and autonomous regions in China, resulting in major economic losses for livestock industry. In 2014, we encountered a clinical PPR case on a goat farm in Guangdong province, southern China. The complete genome of this PPRV strain, named CH/GDDG/2014, was sequenced to determine its similarities and differences with other strains. The CH/GDDG/2014 genome comprised 15,954 nucleotides (six nucleotides more than classical PPRVs identified before 2013, but complying with the rule of six) with six open reading frames encoding nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin, and large polymerase protein, respectively. The whole-genome-based alignment analysis indicated that CH/GDDG/2014 had the most proximate consensus (99.8 %) to China/XJYL/2013 and the least consensus (87.2 %) to KN5/2011. The phylogenetic analysis showed that CH/GDDG/2014 was clustered in one branch (lineage IV) with other emerging strains during the second outbreak. This study is the first report describing the whole-genome sequence of PPRV in Guangdong province, southern China and also suggests the PPR outbreak may be closely related to illegal cross-regional importation of goats.

  5. Analysis of the nucleoprotein gene identifies three distinct lineages of viral haemorrhagic septicemia virus (VHSV) within the European marine environment

    Science.gov (United States)

    Snow, M.; Cunningham, C.O.; Melvin, W.T.; Kurath, G.

    1999-01-01

    A ribonuclease (RNase) protection assay (RPA) has been used to detect nucleotide sequence variation within the nucleoprotein gene of 39 viral haemorrhagic septicaemia virus (VHSV) isolates of European marine origin. The classification of VHSV isolates based on RPA cleavage patterns permitted the identification of ten distinct groups of viruses based on differences at the molecular level. The nucleotide sequence of representatives of each of these groupings was determined and subjected to phylogenetic analysis. This revealed grouping of the European marine isolates of VHSV into three genotypes circulating within distinct geographic areas. A fourth genotype was identified comprising isolates originating from North America. Phylogenetic analyses indicated that VHSV isolates recovered from wild caught fish around the British Isles were genetically related to isolates responsible for losses in farmed turbot. Furthermore, a relationship between naturally occurring marine isolates and VHSV isolates causing mortality among rainbow trout in continental Europe was demonstrated. Analysis of the nucleoprotein gene identifies distinct lineages of viral haemorrhagic septicaemia virus within the European marine environment. Virus Res. 63, 35-44. Available from: 

  6. Whole genome sequence analysis of the arctic-lineage strain responsible for distemper in Italian wolves and dogs through a fast and robust next generation sequencing protocol.

    Science.gov (United States)

    Marcacci, Maurilia; Ancora, Massimo; Mangone, Iolanda; Teodori, Liana; Di Sabatino, Daria; De Massis, Fabrizio; Camma', Cesare; Savini, Giovanni; Lorusso, Alessio

    2014-06-01

    Dynamic surveillance and characterization of canine distemper virus (CDV) circulating strains are essential against possible vaccine breakthroughs events. This study describes the setup of a fast and robust next-generation sequencing (NGS) Ion PGM™ protocol that was used to obtain the complete genome sequence of a CDV isolate (CDV2784/2013). CDV2784/2013 is the prototype of CDV strains responsible for severe clinical distemper in dogs and wolves in Italy during 2013. CDV2784/2013 was isolated on cell culture and total RNA was used for NGS sample preparation. A total of 112.3 Mb of reads were assembled de novo using MIRA version 4.0rc4, which yielded a total number of 403 contigs with 12.1% coverage. The whole genome (15,690 bp) was recovered successfully and compared to those of existing CDV whole genomes. CDV2784/2013 was shown to have 92% nt identity with the Onderstepoort vaccine strain. This study describes for the first time a fast and robust Ion PGM™ platform-based whole genome amplification protocol for non-segmented negative stranded RNA viruses starting from total cell-purified RNA. Additionally, this is the first study reporting the whole genome analysis of an Arctic lineage strain that is known to circulate widely in Europe, Asia and USA.

  7. Clonal analysis of kit ligand a functional expression reveals lineage-specific competence to promote melanocyte rescue in the mutant regenerating caudal fin.

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    Robert C Tryon

    Full Text Available The study of regeneration in an in vivo vertebrate system has the potential to reveal targetable genes and pathways that could improve our ability to heal and repair damaged tissue. We have developed a system for clonal labeling of discrete cell lineages and independently inducing gene expression under control of the heat shock promoter in the zebrafish caudal fin. Consequently we are able to test the affects of overexpressing a single gene in the context of regeneration within each of the nine different cell lineage classes that comprise the caudal fin. This can test which lineage is necessary or sufficient to provide gene function. As a first example to demonstrate this approach, we explored which lineages were competent to functionally express the kit ligand a protein as assessed by the local complementation of the mutation in the sparse-like (kitlgatc244b background. We show that dermal fibroblast expression of kit ligand a robustly supports the rescue of melanocytes in the regenerating caudal fin. kit ligand a expression from skin and osteoblasts results in more modest and variable rescue of melanocytes, while lateral line expression was unable to complement the mutation.

  8. Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence

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    Natalie J. Dorà

    2015-11-01

    Full Text Available The limbal epithelial stem cell (LESC hypothesis proposes that LESCs in the corneal limbus maintain the corneal epithelium both during normal homeostasis and wound repair. The alternative corneal epithelial stem cell (CESC hypothesis proposes that LESCs are only involved in wound repair and CESCs in the corneal epithelium itself maintain the corneal epithelium during normal homeostasis. We used tamoxifen-inducible, CreER-loxP lineage tracing to distinguish between these hypotheses. Clones of labelled cells were induced in adult CAGG-CreER;R26R-LacZ reporter mice and their distributions analysed after different chase periods. Short-lived clones, derived from labelled transient amplifying cells, were shed during the chase period and long-lived clones, derived from stem cells, expanded. At 6 weeks, labelled clones appeared at the periphery, extended centripetally as radial stripes and a few reached the centre by 14 weeks. Stripe numbers depended on the age of tamoxifen treatment. Stripes varied in length, some were discontinuous, few reached the centre and almost half had one end at the limbus. Similar stripes extended across the cornea in CAGG-CreER;R26R-mT/mG reporter mice. The distributions of labelled clones are inconsistent with the CESC hypothesis and support the LESC hypothesis if LESCs cycle between phases of activity and quiescence, each lasting several weeks.

  9. Bioluminescence-based visualization of CD4 T cell dynamics using a T lineage-specific luciferase transgenic model1

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    Zinn Kurt R

    2009-08-01

    Full Text Available Abstract Background Rapid clonal expansion of T cells occurs in response to antigenic challenges. The kinetics of the T cell response has previously been described using tissue-based studies performed at defined time points. Luciferase bioluminescence has recently been utilized for non-invasive analysis of in vivo biologic processes in real-time. Results We have created a novel transgenic mouse model (T-Lux using a human CD2 mini-gene to direct luciferase expression specifically to the T cell compartment. T-Lux T cells demonstrated normal homing patterns within the intact mouse and following adoptive transfer. Bioluminescent signal correlated with T cell numbers in the whole body images as well as within specific organ regions of interest. Following transfer into lymphopenic (RAG2-/- recipients, homeostatic proliferation of T-Lux T cells was visualized using bioluminescent imaging. Real-time bioluminescent analysis of CD4+ T cell antigen-specific responses enabled real-time comparison of the kinetics and magnitude of clonal expansion and contraction in the inductive lymph node and tissue site of antigen injection. T cell expansion was dose-dependent despite the presence of supraphysiologic numbers of OVA-specific OT-II transgenic TCR T-Lux T cells. CD4+ T cells subsequently underwent a rapid (3–4 day contraction phase in the draining lymph node, with a delayed contraction in the antigen delivery site, with bioluminescent signal diminished below initial levels, representing TCR clonal frequency control. Conclusion The T-Lux mouse provides a novel, efficient model for tracking in vivo aspects of the CD4+ T cell response to antigen, providing an attractive approach for studies directed at immunotherapy or vaccine design.

  10. SNP-based differentiation of Phytophthora infestans clonal lineages using locked nucleic acid probes and high resolution melt analysis

    Science.gov (United States)

    Phytophthora infestans, the cause of the devastating late blight disease of potato and tomato, exhibits a clonal reproductive lifestyle in North America. Phenotypes such as fungicide sensitivity and host preference are conserved among individuals within clonal lineages, while substantial phenotypic ...

  11. miR-18b and miR-518b Target FOXN1 during epithelial lineage differentiation in pluripotent cells.

    Science.gov (United States)

    Kushwaha, Ritu; Thodima, Venkata; Tomishima, Mark J; Bosl, George J; Chaganti, R S K

    2014-05-15

    MicroRNAs (miRNAs) regulate myriad biological processes; however, their role in cell fate choice is relatively unexplored. Pluripotent NT2/D1 embryonal carcinoma cells differentiate into an epithelial/smooth muscle phenotype when treated with bone morphogenetic protein-2 (BMP-2). To identify miRNAs involved in epithelial cell development, we performed miRNA profiling of NT2/D1 cells treated with BMP-2 at 6, 12, and 24 h, and on days 6 and 10. Integration of the miRNA profiling data with previously obtained gene expression profiling (GEP) data of NT2/D1 cells treated with BMP-2 at the same time points identified miR-18b and miR-518b as the top two miRNAs with the highest number of up-regulated predicted targets with known functions in epithelial lineage development. Silencing of miR-18b and miR-518b in NT2/D1 cells revealed several up-regulated TFs with functions in epithelial lineage development; among these, target prediction programs identified FOXN1 as the only direct target of both miRNAs. FOXN1 has previously been shown to play an important role in keratinocyte differentiation and epithelial cell proliferation. NT2/D1 and H9 human embryonic stem cells with silenced miR-18b and miR-518b showed up-regulation of FOXN1 and the epithelial markers CDH1, EPCAM, KRT19, and KRT7. A 3'UTR luciferase assay confirmed FOXN1 to be a target of the two miRNAs, and up-regulation of FOXN1 in NT2/D1 cells led to the expression of epithelial markers. Overexpression of the two miRNAs in BMP-2-treated NT2/D1 cells led to down-regulation of FOXN1 and epithelial lineage markers. These results show that miR-18b and miR-518b are upstream controllers of FOXN1-directed epithelial lineage development.

  12. Functional clustering and lineage markers: insights into cellular differentiation and gene function from large-scale microarray studies of purified primary cell populations.

    Science.gov (United States)

    Hume, David A; Summers, Kim M; Raza, Sobia; Baillie, J Kenneth; Freeman, Thomas C

    2010-06-01

    Very large microarray datasets showing gene expression across multiple tissues and cell populations provide a window on the transcriptional networks that underpin the differences in functional activity between biological systems. Clusters of co-expressed genes provide lineage markers, candidate regulators of cell function and, by applying the principle of guilt by association, candidate functions for genes of currently unknown function. We have analysed a dataset comprising pure cell populations from hemopoietic and non-hemopoietic cell types (http://biogps.gnf.org). Using a novel network visualisation and clustering approach, we demonstrate that it is possible to identify very tight expression signatures associated specifically with embryonic stem cells, mesenchymal cells and hematopoietic lineages. Selected examples validate the prediction that gene function can be inferred by co-expression. One expression cluster was enriched in phagocytes, which, alongside endosome-lysosome constituents, contains genes that may make up a 'pathway' for phagocyte differentiation. Promoters of these genes are enriched for binding sites for the ETS/PU.1 and MITF families. Another cluster was associated with the production of a specific extracellular matrix, with high levels of gene expression shared by cells of mesenchymal origin (fibroblasts, adipocytes, osteoblasts and myoblasts). We discuss the limitations placed upon such data by the presence of alternative promoters with distinct tissue specificity within many protein-coding genes.

  13. Cranial radiation for pediatric T-lineage acute lymphoblastic leukemia: a systematic review and meta-analysis.

    Science.gov (United States)

    Kelly, Michael J; Trikalinos, Thomas A; Dahabreh, Issa J; Gianferante, Matthew; Parsons, Susan K

    2014-10-01

    There are heterogeneous approaches to cranial radiation therapy (CRT) for T-lineage acute lymphoblastic leukemia (T-ALL). We performed a systematic review of studies that specified a radiation strategy and reported survival for pediatric T-ALL. Our analysis included 62 publications reporting 78 treatment groups (patient n = 5844). The average event-free survival (EFS) was higher by 6% per 5 years (P reference group (CRT for all) which had a year-adjusted EFS of 65% (95% confidence interval, CI: 61-69%) the adjusted EFS was significantly worse (rate difference (RD) = -9%, 95% CI: -15 to -2%) among studies that used a risk-directed approach to CRT (P = 0.004). The adjusted EFS for the other strategies were not significantly different compared to the reference group: CRT for central nervous system positive patients only (RD = -3%, 95% CI: -14 to 7%, P = 0.49); CRT omitted for all patients (RD = 5%, 95% CI: -4 to 15%, P = 0.33). CRT may not be necessary with current chemotherapy for T-ALL. These findings, however, are susceptible to bias and caution should be applied in drawing conclusions on the comparative effectiveness of alternative CRT strategies.

  14. First multigene analysis of Archamoebae (Amoebozoa: Conosa) robustly reveals its phylogeny and shows that Entamoebidae represents a deep lineage of the group.

    Science.gov (United States)

    Pánek, Tomáš; Zadrobílková, Eliška; Walker, Giselle; Brown, Matthew W; Gentekaki, Eleni; Hroudová, Miluše; Kang, Seungho; Roger, Andrew J; Tice, Alexander K; Vlček, Čestmír; Čepička, Ivan

    2016-05-01

    Archamoebae is an understudied group of anaerobic free-living or endobiotic protists that constitutes the major anaerobic lineage of the supergroup Amoebozoa. Hitherto, the phylogeny of Archamoebae was based solely on SSU rRNA and actin genes, which did not resolve relationships among the main lineages of the group. Because of this uncertainty, several different scenarios had been proposed for the phylogeny of the Archamoebae. In this study, we present the first multigene phylogenetic analysis that includes members of Pelomyxidae, and Rhizomastixidae. The analysis clearly shows that Mastigamoebidae, Pelomyxidae and Rhizomastixidae form a clade of mostly free-living, amoeboid flagellates, here called Pelobiontida. The predominantly endobiotic and aflagellated Entamoebidae represents a separate, deep-branching lineage, Entamoebida. Therefore, two unique evolutionary events, horizontal transfer of the nitrogen fixation system from bacteria and transfer of the sulfate activation pathway to mitochondrial derivatives, predate the radiation of recent lineages of Archamoebae. The endobiotic lifestyle has arisen at least three times independently during the evolution of the group. We also present new ultrastructural data that clarifies the primary divergence among the family Mastigamoebidae which had previously been inferred from phylogenetic analyses based on SSU rDNA.

  15. Current lineages of the epithelioma papulosum cyprini (EPC) cell line are contaminated with fathead minnow, Pimephales promelas, cells

    Science.gov (United States)

    Winton, J.; Batts, W.; deKinkelin, P.; LeBerre, M.; Bremont, M.; Fijan, N.

    2010-01-01

    Initially established from proliferative skin lesions of the common carp, Cyprinus carpio L., the epithelioma papulosum cyprini (EPC) cell line (Fijan, Sulimanovic, Bearzotti, Muzinic, Zwillenberg, Chilmonczyk, Vautherot & de Kinkelin 1983) has become one of the most widely used tools for research on fish viruses and the diagnosis of fish viral diseases.

  16. The Molecular Signature of HIV-1-Associated Lipomatosis Reveals Differential Involvement of Brown and Beige/Brite Adipocyte Cell Lineages.

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    Rubén Cereijo

    Full Text Available Highly active antiretroviral therapy has remarkably improved quality of life of HIV-1-infected patients. However, this treatment has been associated with the so-called lipodystrophic syndrome, which conveys a number of adverse metabolic effects and morphological alterations. Among them, lipoatrophy of subcutaneous fat in certain anatomical areas and hypertrophy of visceral depots are the most common. Less frequently, lipomatous enlargements of subcutaneous fat at distinct anatomic areas occur. Lipomatous adipose tissue in the dorso-cervical area ("buffalo hump" has been associated with a partial white-to-brown phenotype transition and with increased cell proliferation, but, to date, lipomatous enlargements arising in other parts of the body have not been characterized. In order to establish the main molecular events associated with the appearance of lipomatosis in HIV-1 patients, we analyzed biopsies of lipomatous tissue from "buffalo hump" and from other anatomical areas in patients, in comparison with healthy subcutaneous adipose tissue, using a marker gene expression approach. Both buffalo-hump and non-buffalo-hump lipomatous adipose tissues exhibited similar patterns of non-compromised adipogenesis, unaltered inflammation, non-fibrotic phenotype and proliferative activity. Shorter telomere length, prelamin A accumulation and SA-β-Gal induction, reminiscent of adipocyte senescence, were also common to both types of lipomatous tissues. Buffalo hump biopsies showed expression of marker genes of brown adipose tissue (e.g. UCP1 and, specifically, of "classical" brown adipocytes (e.g. ZIC1 but not of beige/brite adipocytes. No such brown fat-related gene expression occurred in lipomatous tissues at other anatomical sites. In conclusion, buffalo hump and other subcutaneous adipose tissue enlargements from HIV-1-infected patients share a similar lipomatous character. However, a distorted induction of white-to-"classical brown adipocyte" phenotype

  17. The Zebrafish Anillin-eGFP Reporter Marks Late Dividing Retinal Precursors and Stem Cells Entering Neuronal Lineages

    Science.gov (United States)

    Guglielmi, Luca; Patzel, Eva; Sel, Saadettin; Auffarth, Gerd U.; Carl, Matthias; Poggi, Lucia

    2017-01-01

    Monitoring cycling behaviours of stem and somatic cells in the living animal is a powerful tool to better understand tissue development and homeostasis. The tg(anillin:anillin-eGFP) transgenic line carries the full-length zebrafish F-actin binding protein Anillin fused to eGFP from a bacterial artificial chromosome (BAC) containing Anillin cis-regulatory sequences. Here we report the suitability of the Anillin-eGFP reporter as a direct indicator of cycling cells in the late embryonic and post-embryonic retina. We show that combining the anillin:anillin-eGFP with other transgenes such as ptf1a:dsRed and atoh7:gap-RFP allows obtaining spatial and temporal resolution of the mitotic potentials of specific retinal cell populations. This is exemplified by the analysis of the origin of the previously reported apically and non-apically dividing late committed precursors of the photoreceptor and horizontal cell layers. PMID:28107513

  18. SOX2 expression is upregulated in adult spinal cord after contusion injury in both oligodendrocyte lineage and ependymal cells.

    Science.gov (United States)

    Lee, Hyun Joon; Wu, Junfang; Chung, Jumi; Wrathall, Jean R

    2013-02-01

    The upregulation of genes normally associated with development may occur in the adult after spinal cord injury (SCI). To test this, we performed real-time RT-PCR array analysis of mouse spinal cord mRNAs comparing embryonic day (E)14.5 spinal cord with intact adult and adult cord 1 week after a clinically relevant standardized contusion SCI. We found significantly increased expression of a large number of neural development- and stem cell-associated genes after SCI. These included Sox2 (sex determining region Y-box 2), a transcription factor that regulates self-renewal and potency of embryonic neural stem cells and is one of only a few key factors needed to induce pluripotency. In adult spinal cord of Sox2-EGFP mice, Sox2-EGFP was found mainly in the ependymal cells of the central canal. After SCI, both mRNA and protein levels of Sox2 were significantly increased at and near the injury site. By 1 day, Sox2 was upregulated in NG2(+) oligodendrocyte progenitor cells (OPC) in the spared white matter. By 3 days, Sox2-EGFP ependymal cells had increased proliferation and begun to form multiple layers and clusters of cells in the central lesion zone of the cord. Expression of Sox2 by NG2(+) cells had declined by 1 week, but increased numbers of other Sox2-expressing cells persisted for at least 4 weeks after SCI in both mouse and rat models. Thus, SCI upregulates many genes associated with development and neural stem cells, including the key transcription factor Sox2, which is expressed in a pool of cells that persists for weeks after SCI.

  19. Genetic ablation of androgen receptor signaling in fetal Leydig cell lineage affects Leydig cell functions in adult testis.

    Science.gov (United States)

    Kaftanovskaya, Elena M; Lopez, Carolina; Ferguson, Lydia; Myhr, Courtney; Agoulnik, Alexander I

    2015-06-01

    It is commonly accepted that androgen-producing fetal Leydig cells (FLC) are substituted by adult Leydig cells (ALC) during perinatal testis development. The mechanisms influencing this process are unclear. We used mice with a retinoid acid receptor 2 promoter-Cre recombinase transgene (Rarb-cre) expressed in embryonic FLC precursors, but not in postnatal testis, and a dual fluorescent Cre recombinase reporter to label FLC and ALC in vivo. All FLC in newborn testis had the recombinant, whereas the majority of LC in adult testis had the nonrecombinant reporter. Primary LC cultures from adult testis had either recombinant (20%) or nonrecombinant (80%) cells, demonstrating that the FLC survive in adult testis and their ontogeny is distinct from ALC. Conditional inactivation of androgen receptor (AR) allele using the Rarb-cre transgene resulted in a 50% increase of AR-negative LC in adult testis. The mutant males became infertile with age, with all LC in older testis showing signs of incomplete differentiation, such as a large number of big lipid droplets, an increase of finger-like protrusions, and a misexpression of steroidogenic or FLC- and ALC-specific genes. We propose that the antiandrogenic exposure during early development may similarly result in an increase of FLC in adult testis, leading to abnormal LC differentiation.

  20. Surface molecules that drive T cell development in vitro in the absence of thymic epithelium and in the absence of lineage-specific signals.

    Science.gov (United States)

    Cibotti, R; Punt, J A; Dash, K S; Sharrow, S O; Singer, A

    1997-03-01

    Differentiation of immature double positive (DP) CD4+ CD8+ thymocytes into single positive (SP) CD4+ and CD8+ T cells is referred to as positive selection and requires physical contact with thymic cortical epithelium. We now have identified "coinducer" molecules on DP thymocytes that, together with TCR, signal DP thymocytes to differentiate into SP T cells in vitro in the absence of thymic epithelium. A remarkable number of different molecules on DP thymocytes possessed "coinducing" activity, including CD2, CD5, CD24, CD28, CD49d, CD81, and TSA-1. Interestingly, in vitro differentiation occurred in the absence of lineage-specific signals, yet resulted in the selective generation of CD4+CD8- T cells. Thus, the present study has identified surface molecules that can signal DP thymocytes to differentiate into SP T cells in the absence of thymic epithelium and has characterized a default pathway for CD4+ T cell differentiation.

  1. Maternal exposure to 3,3'-iminodipropionitrile targets late-stage differentiation of hippocampal granule cell lineages to affect brain-derived neurotrophic factor signaling and interneuron subpopulations in rat offspring.

    Science.gov (United States)

    Itahashi, Megu; Abe, Hajime; Tanaka, Takeshi; Mizukami, Sayaka; Kikuchihara, Yoh; Yoshida, Toshinori; Shibutani, Makoto

    2015-08-01

    3,3'-Iminodipropionitrile (IDPN) causes neurofilament (NF)-filled swellings in the proximal segments of many large-caliber myelinated axons. This study investigated the effect of maternal exposure to IDPN on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 67 or 200 ppm IDPN in drinking water from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, female offspring subjected to analysis had decreased parvalbumin(+), reelin(+) and phospho-TrkB(+) interneurons in the dentate hilus at 200 ppm and increased granule cell populations expressing immediate-early gene products, Arc or c-Fos, at ≥  67 ppm. mRNA expression in the dentate gyrus examined at 200 ppm decreased with brain-derived neurotrophic factor (Bdnf) and very low density lipoprotein receptor. Immunoreactivity for phosphorylated NF heavy polypeptide decreased in the molecular layer of the dentate gyrus and the stratum radiatum of the cornu ammonis (CA) 3, portions showing axonal projections from mossy cells and pyramidal neurons, at 200 ppm on PND 21, whereas immunoreactivity for synaptophysin was unchanged in the dentate gyrus. Observed changes all disappeared on PND 77. There were no fluctuations in the numbers of apoptotic cells, proliferating cells and subpopulations of granule cell lineage in the subgranular zone on PND 21 and PND 77. Thus, maternal IDPN exposure may reversibly affect late-stage differentiation of granule cell lineages involving neuronal plasticity as evident by immediate-early gene responses to cause BDNF downregulation resulting in a reduction in parvalbumin(+) or reelin(+) interneurons and suppression of axonal plasticity in the mossy cells and CA3 pyramidal neurons.

  2. Up- or downregulation of tescalcin in HL-60 cells is associated with their differentiation to either granulocytic or macrophage-like lineage.

    Science.gov (United States)

    Levay, Konstantin; Slepak, Vladlen Z

    2010-04-15

    Tescalcin is a 25-kDa EF-hand Ca(2+)-binding protein that is differentially expressed in several mammalian tissues. Previous studies demonstrated that expression of this protein is essential for differentiation of hematopoietic precursor cell lines and primary stem cells into megakaryocytes. Here we show that tescalcin is expressed in primary human granulocytes and is upregulated in human promyelocytic leukemia HL-60 cells that have been induced to differentiate along the granulocytic lineage. However, during induced macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated. The decrease in expression is associated with a rapid drop in tescalcin mRNA level, whereas upregulation occurs via a post-transcriptional mechanism. Tescalcin is necessary for HL-60 differentiation into granulocytes as its knockdown by shRNA impairs the ability of HL-60 cells to acquire the characteristic phenotypes such as phagocytic activity and generation of reactive oxygen species measured by respiratory burst assay. Both up- and downregulation of tescalcin require activation of the MEK/ERK cascade. It appears that commitment of HL-60 cells toward granulocytic versus macrophage-like lineage correlates with expression of tescalcin and kinetics of ERK activation. In retinoic acid-induced granulocytic differentiation, the activation of ERK and upregulation of tescalcin occurs slowly (16-48 h). In contrast, in PMA-induced macrophage-like differentiation the activation of ERK is rapid (15-30 min) and tescalcin is downregulated. These studies indicate that tescalcin is one of the key gene products that is involved in switching differentiation program in some cell types.

  3. Inhibition of mixed-lineage kinase (MLK) activity during G2-phase disrupts microtubule formation and mitotic progression in HeLa cells.

    Science.gov (United States)

    Cha, Hyukjin; Dangi, Surabhi; Machamer, Carolyn E; Shapiro, Paul

    2006-01-01

    The mixed-lineage kinases (MLK) are serine/threonine protein kinases that regulate mitogen-activated protein (MAP) kinase signaling pathways in response to extracellular signals. Recent studies indicate that MLK activity may promote neuronal cell death through activation of the c-Jun NH2-terminal kinase (JNK) family of MAP kinases. Thus, inhibitors of MLK activity may be clinically useful for delaying the progression of neurodegenerative diseases, such as Parkinson's. In proliferating non-neuronal cells, MLK may have the opposite effect of promoting cell proliferation. In the current studies we examined the requirement for MLK proteins in regulating cell proliferation by examining MLK function during G2 and M-phase of the cell cycle. The MLK inhibitor CEP-11004 prevented HeLa cell proliferation by delaying mitotic progression. Closer examination revealed that HeLa cells treated with CEP-11004 during G2-phase entered mitosis similar to untreated G2-phase cells. However, CEP-11004 treated cells failed to properly exit mitosis and arrested in a pro-metaphase state. Partial reversal of the CEP-11004 induced mitotic arrest could be achieved by overexpression of exogenous MLK3. The effects of CEP-11004 treatment on mitotic events included the inhibition of histone H3 phosphorylation during prophase and prior to nuclear envelope breakdown and the formation of aberrant mitotic spindles. These data indicate that MLK3 might be a unique target to selectively inhibit transformed cell proliferation by disrupting mitotic spindle formation resulting in mitotic arrest.

  4. Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis.

    Science.gov (United States)

    Sun, Hongliang; Tsai, Ying; Nowak, Irena; Liesveld, Jane; Chen, Yuhchyau

    2012-09-01

    Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.

  5. Metagenome sequence analysis of filamentous microbial communities obtained from geochemically distinct geothermal channels reveals specialization of three aquificales lineages

    DEFF Research Database (Denmark)

    Takacs-Vesbach, Cristina; Inskeep, William P; Jay, Zackary J;

    2013-01-01

    containing 16S rRNA genes as well as conserved protein-encoding genes revealed that the composition and function of these communities varied across geochemical conditions. Each Aquificales lineage contained genes for CO2 fixation by the reverse-TCA cycle, but only the Sulfurihydrogenibium populations perform......The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems worldwide and are considered one of the earliest lineages of the domain Bacteria. We analyzed metagenome sequence obtained from six thermal "filamentous streamer" communities (∼40 Mbp per site), which targeted three...... types of heme Cu oxidases (subunit I) involved in oxygen reduction. The distribution of Aquificales populations and differences among functional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2, O2) have...

  6. Dasatinib targets B-lineage cells but does not provide an effective therapy for myeloproliferative disease in c-Cbl RING finger mutant mice.

    Directory of Open Access Journals (Sweden)

    Johanna M Duyvestyn

    Full Text Available This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL.

  7. Dasatinib Targets B-Lineage Cells but Does Not Provide an Effective Therapy for Myeloproliferative Disease in c-Cbl RING Finger Mutant Mice

    Science.gov (United States)

    Duyvestyn, Johanna M.; Taylor, Samuel J.; Dagger, Samantha A.; Orandle, Marlene; Morse, Herbert C.; Thien, Christine B. F.; Langdon, Wallace Y.

    2014-01-01

    This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL. PMID:24718698

  8. A novel role for mixed-lineage kinase-like mitogen-activated protein triple kinase alpha in neoplastic cell transformation and tumor development.

    Science.gov (United States)

    Cho, Yong-Yeon; Bode, Ann M; Mizuno, Hideya; Choi, Bu Young; Choi, Hong Seok; Dong, Zigang

    2004-06-01

    Previously, no member of the mixed-lineage kinase (MLK) protein family was known to function as an oncogene. Here, we demonstrate that MLK-like mitogen-activated protein triple kinase (MLTK)-alpha, a member of the MLK family, induced neoplastic cell transformation and tumorigenesis in athymic nude mice. Introduction of small interference RNA (siRNA)-MLTK-alpha into MLTK-alpha-overexpressing cells dramatically suppressed cell transformation. Nuclear accumulation of the pHisG-MLTK-alpha fusion protein was observed after epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate treatment. Phosphorylation of downstream mitogen-activated protein kinase-targeted transcription factors including c-Myc, Elk-1, c-Jun, and activating transcription factor (ATF) 2 was also differentially enhanced in MLTK-alpha-overexpressing cells exposed to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate stimulation compared with cells expressing mock vector or siRNA-MLTK-alpha. Very importantly, MLTK-alpha-overexpressing cells formed fibrosarcomas when injected s.c. into athymic nude mice, whereas almost no tumor formation was observed in mice that received injections of mock or siRNA-MLTK-alpha stably transfected cells. These results are the first to indicate that MLTK-alpha plays a key role in neoplastic cell transformation and cancer development.

  9. Mixed lineage kinase 3 inhibits platelet-derived growth factor-stimulated DNA synthesis and matrix mRNA expression in mesangial cells.

    Science.gov (United States)

    Parameswaran, Narayanan; Hall, Carolyn S; Böck, Barbara C; Sparks, Harvey V; Gallo, Kathleen A; Spielman, William S

    2002-01-01

    Mixed lineage kinase 3 (MLK 3) is a recently described member of the MLK subfamily of Ser/Thr protein kinases that interacts with MAPK pathways. The aim of this study was to test the potential interaction of MLK 3 with signaling pathways stimulated by PDGF in rat mesangial cells. We have established a stable cell line expressing human MLK 3 in rat glomerular mesangial cells. The effects of PDGF on proliferation and matrix mRNA expression were examined. In control (vector-transfected) mesangial cells PDGF increased [(3)H]-thymidine incorporation significantly in a concentration-dependent manner. In mesangial cells expressing MLK 3, PDGF-induced increase in DNA synthesis was significantly reduced. PDGF also induced fibronectin and collagen I mRNA expression in control cells, the effects of which were also significantly blocked in MLK 3-transfected cells. To understand the potential interaction of MLK 3 over expression with the MAPK pathways and to examine the potential mechanism of the effects of MLK 3 over expression on proliferation and matrix expression, activation of ERK2, JNK1 and p38 were examined. ERK2 activation was increased several fold by PDGF in control cells but was attenuated significantly in MLK 3 expressing cells. PDGF did not have any effect on JNK and p38 activation, in either cell types. Using the same stable-transfected cell line, identical results were obtained on proliferation and matrix expression with sarafotoxin-s6b (endothelin receptor agonist) another potent mitogenic and sclerotic agent for mesangial cells. These results indicate an important role for MLK 3 in the regulation of growth and matrix expression in mesangial cells.

  10. The neuro-glial properties of adipose-derived adult stromal (ADAS cells are not regulated by Notch 1 and are not derived from neural crest lineage.

    Directory of Open Access Journals (Sweden)

    Philip C Wrage

    Full Text Available We investigated whether adipose-derived adult stromal (ADAS are of neural crest origin and the extent to which Notch 1 regulates their growth and differentiation. Mouse ADAS cells cultured in media formulated for neural stem cells (NSC displayed limited capacity for self-renewal, clonogenicity, and neurosphere formation compared to NSC from the subventricular zone in the hippocampus. Although ADAS cells expressed Nestin, GFAP, NSE and Tuj1 in vitro, exposure to NSC differentiation supplements did not induce mature neuronal marker expression. In contrast, in mesenchymal stem cell (MSC media, ADAS cells retained their ability to proliferate and differentiate beyond 20 passages and expressed high levels of Nestin. In neuritizing cocktails, ADAS cells extended processes, downregulated Nestin expression, and displayed depolarization-induced Ca(2+ transients but no spontaneous or evoked neural network activity on Multi-Electrode Arrays. Deletion of Notch 1 in ADAS cell cultures grown in NSC proliferation medium did not significantly alter their proliferative potential in vitro or the differentiation-induced downregulation of Nestin. Co-culture of ADAS cells with fibroblasts that stably expressed the Notch ligand Jagged 1 or overexpression of the Notch intracellular domain (NICD did not alter ADAS cell growth, morphology, or cellular marker expression. ADAS cells did not display robust expression of neural crest transcription factors or genes (Sox, CRABP2, and TH; and lineage tracing analyses using Wnt1-Cre;Rosa26R-lacZ or -EYFP reporter mice confirmed that fewer than 2% of the ADAS cell population derived from a Wnt1-positive population during development. In summary, although media formulations optimized for MSCs or NSCs enable expansion of mouse ADAS cells in vitro, we find no evidence that these cells are of neural crest origin, that they can undergo robust terminal differentiation into functionally mature neurons, and that Notch 1 is likely to be

  11. Estimating ancestral distributions of lineages with uncertain sister groups: a statistical approach to Dispersal-Vicariance Analysis and a case using Aesculus L. (Sapindaceae) including fossils

    Institute of Scientific and Technical Information of China (English)

    A.J. HARRIS; Qiu-Yun (Jenny) XIANG

    2009-01-01

    We propose a simple statistical approach for using Dispersal-Vicariance Analysis (DIVA) software to infer biogeographic histories without fully bifurcating trees. In this approach, ancestral ranges are first optimized for a sample of Bayesian trees. The probability P of an ancestral range r at a node is then calculated as P(rY)= Σnt=1 F(rY)tPt where Y is a node, and F(rY) is the frequency of range r among all the optimal solutions resulting from DIVA optimization at node Y, t is one of n topologies optimized, and Pt is the probability of topology t. Node Y is a hypothesized ancestor shared by a specific crown lineage and the sister of that lineage "x", where x may vary due to phylogenetic uncertainty (polytomies and nodes with posterior probability <100%). Using this method, the ancestral distribution at Y can be estimated to provide inference of the geographic origins of the specific crown group of interest. This approach takes into account phylogenetic uncertainty as well as uncertainty from DIVA optimization. It is an extension of the previously described method called Bayes-DIVA, which pairs Bayesian phylogenetic analysis with biogeographic analysis using DIVA. Further, we show that the probability P of an ancestral range at Y calculated using this method does not equate to pp* F(rY) on the Bayesian consensus tree when both variables are < 100%, where pp is the posterior probability and F(rY) is the frequency of range r for the node containing the specific crown group. We tested our DIVA-Bayes approach using Aesculus L., which has major lineages unresolved as a polytomy. We inferred the most probable geographic origins of the five traditional sections of Aesculus and ofAesculus californica Nutt. and examined range subdivisions at parental nodes of these lineages.Additionally, we used the DIVA-Bayes data from Aesculus to quantify the effects on biogeographic inference of including two wildcard fossil taxa in phylogenetic analysis. Our analysis resolved the

  12. p38α MAPK Regulates Lineage Commitment and OPG Synthesis of Bone Marrow Stromal Cells to Prevent Bone Loss under Physiological and Pathological Conditions

    Directory of Open Access Journals (Sweden)

    Qian Cong

    2016-04-01

    Full Text Available Bone marrow-derived mesenchymal stromal cells (BM-MSCs are capable of differentiating into osteoblasts, chondrocytes, and adipocytes. Skewed differentiation of BM-MSCs contributes to the pathogenesis of osteoporosis. Yet how BM-MSC lineage commitment is regulated remains unclear. We show that ablation of p38α in Prx1+ BM-MSCs produced osteoporotic phenotypes, growth plate defects, and increased bone marrow fat, secondary to biased BM-MSC differentiation from osteoblast/chondrocyte to adipocyte and increased osteoclastogenesis and bone resorption. p38α regulates BM-MSC osteogenic commitment through TAK1-NF-κB signaling and osteoclastogenesis through osteoprotegerin (OPG production by BM-MSCs. Estrogen activates p38α to maintain OPG expression in BM-MSCs to preserve the bone. Ablation of p38α in BM-MSCs positive for Dermo1, a later BM-MSC marker, only affected osteogenic differentiation. Thus, p38α mitogen-activated protein kinase (MAPK in Prx1+ BM-MSCs acts to preserve the bone by promoting osteogenic lineage commitment and sustaining OPG production. This study thus unravels previously unidentified roles for p38α MAPK in skeletal development and bone remodeling.

  13. Glial cell lineage expression of mutant ataxin-1 and huntingtin induces developmental and late-onset neuronal pathologies in Drosophila models.

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    Takuya Tamura

    Full Text Available BACKGROUND: In several neurodegenerative disorders, toxic effects of glial cells on neurons are implicated. However the generality of the non-cell autonomous pathologies derived from glial cells has not been established, and the specificity among different neurodegenerative disorders remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We newly generated Drosophila models expressing human mutant huntingtin (hHtt103Q or ataxin-1 (hAtx1-82Q in the glial cell lineage at different stages of differentiation, and analyzed their morphological and behavioral phenotypes. To express hHtt103Q and hAtx1-82Q, we used 2 different Gal4 drivers, gcm-Gal4 and repo-Gal4. Gcm-Gal4 is known to be a neuroglioblast/glioblast-specific driver whose effect is limited to development. Repo-Gal4 is known to be a pan-glial driver and the expression starts at glioblasts and continues after terminal differentiation. Gcm-Gal4-induced hHtt103Q was more toxic than repo-Gal4-induced hHtt103Q from the aspects of development, locomotive activity and survival of flies. When hAtx1-82Q was expressed by gcm- or repo-Gal4 driver, no fly became adult. Interestingly, the head and brain sizes were markedly reduced in a part of pupae expressing hAtx1-82Q under the control of gcm-Gal4, and these pupae showed extreme destruction of the brain structure. The other pupae expressing hAtx1-82Q also showed brain shrinkage and abnormal connections of neurons. These results suggested that expression of polyQ proteins in neuroglioblasts provided a remarkable effect on the developmental and adult brains, and that glial cell lineage expression of hAtx1-82Q was more toxic than that of hHtt103Q in our assays. CONCLUSION/SIGNIFICANCE: All these studies suggested that the non-cell autonomous effect of glial cells might be a common pathology shared by multiple neurodegenerative disorders. In addition, the fly models would be available for analyzing molecular pathologies and developing novel therapeutics against

  14. Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP)

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yoon Jung [Dental Regenerative Biotechnology Major, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Lee, Jue Yeon [Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Lee, Seung Jin [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul (Korea, Republic of); Chung, Chong-Pyoung, E-mail: ccpperio@snu.ac.kr [Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Department of Periodontology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul (Korea, Republic of); Park, Yoon Jeong, E-mail: parkyj@snu.ac.kr [Dental Regenerative Biotechnology Major, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer CBP sequence is identified from BSP and has collagen binding activity. Black-Right-Pointing-Pointer CBP directly activates the MAPK signaling, especially ERK1/2. Black-Right-Pointing-Pointer CBP increase osteoblastic differentiation by the activation of Runx2. Black-Right-Pointing-Pointer CBP decrease adipogenic differentiation by the inhibition of PPAR{gamma}. -- Abstract: Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Several recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor {gamma}. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a

  15. Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells

    Science.gov (United States)

    Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan; Bronner, Marianne E

    2017-01-01

    The ‘neural plate border’ of vertebrate embryos contains precursors of neural crest and placode cells, both defining vertebrate characteristics. How these lineages segregate from neural and epidermal fates has been a matter of debate. We address this by performing a fine-scale quantitative temporal analysis of transcription factor expression in the neural plate border of chick embryos. The results reveal significant overlap of transcription factors characteristic of multiple lineages in individual border cells from gastrula through neurula stages. Cell fate analysis using a Sox2 (neural) enhancer reveals that cells that are initially Sox2+ cells can contribute not only to neural tube but also to neural crest and epidermis. Moreover, modulating levels of Sox2 or Pax7 alters the apportionment of neural tube versus neural crest fates. Our results resolve a long-standing question and suggest that many individual border cells maintain ability to contribute to multiple ectodermal lineages until or beyond neural tube closure. DOI: http://dx.doi.org/10.7554/eLife.21620.001 PMID:28355135

  16. Multi-lineage differentiation of hMSCs encapsulated in thermo-reversible hydrogel using a co-culture system with differentiated cells.

    Science.gov (United States)

    Park, Ji Sun; Yang, Han Na; Woo, Dae Gyun; Kim, Hyemin; Na, Kun; Park, Keun-Hong

    2010-10-01

    The micro-environment is an important factor in the differentiation of cultured stem cells for the purpose of site specific transplantation. In an attempt to optimize differentiation conditions, co-culture systems composed of both stem cells and primary cells or cell lines were used in hydrogel with in vitro and in vivo systems. Stem cells encapsulated in hydrogel, under certain conditions, can undergo increased differentiation both in vitro and in vivo; therefore, reconstruction of transplanted stem cells in a hydrogel co-culture system is important for tissue regeneration. In order to construct such a co-culture system, we attempted to create a hydrogel scaffold which could induce neo-tissue growth from the recipient bed into the material. This material would enable encapsulation of stem cells in vitro after which they could be transferred to an in vivo system utilizing nude mice. In this case, the hydrogel was implanted in the subfascial space of nude mice and excised 4 weeks later. Cross-sections of the excised samples were stained with von Kossa or safranin-O and tubular formations into the gel were observed with and tested by doppler imaging. The data showed that the hydrogel markedly induced growth of osteogenic, chondrogenic, and vascular-rich tissue into the hydrogel by 4 weeks, which surpassed that after transplantation in a co-culture system. Further, a co-culture system with differentiated cells and stem cells potentially enhanced chondrogenesis, osteogenesis, and vascularization. These findings suggest that a co-culture system with hydrogel as scaffold material for neo-tissue formation is a useful tools for multi-lineage stem cell differentiation.

  17. Commitment of Satellite Cells Expressing the Calcium Channel α2δ1 Subunit to the Muscle Lineage

    Directory of Open Access Journals (Sweden)

    Tammy Tamayo

    2012-01-01

    Full Text Available Satellite cells can maintain or repair muscle because they possess stem cell properties, making them a valuable option for cell therapy. However, cell transplants into skeletal muscle of patients with muscular dystrophy are limited by donor cell attachment, migration, and survival in the host tissue. Cells used for therapy are selected based on specific markers present in the plasma membrane. Although many markers have been identified, there is a need to find a marker that is expressed at different states in satellite cells, activated, quiescent, or differentiated cell. Furthermore, the marker has to be present in human tissue. Recently we reported that the plasma membrane α2δ1 protein is involved in cell attachment and migration in myoblasts. The α2δ1 subunit forms a part of the L-type voltage-dependent calcium channel in adult skeletal muscle. We found that the α2δ1 subunit is expressed in the majority of newly isolated satellite cells and that it appears earlier than the α1 subunits and at higher levels than the β or γ subunits. We also found that those cells that expressed α2δ1 would differentiate into muscle cells. This evidence indicates that the α2δ1 may be used as a marker of satellite cells that will differentiate into muscle.

  18. Shear Stress Induces Differentiation of Endothelial Lineage Cells to Protect Neonatal Brain from Hypoxic-Ischemic Injury through NRP1 and VEGFR2 Signaling

    Directory of Open Access Journals (Sweden)

    Chia-Wei Huang

    2015-01-01

    Full Text Available Neonatal hypoxic-ischemic (HI brain injuries disrupt the integrity of neurovascular structure and lead to lifelong neurological deficit. The devastating damage can be ameliorated by preserving the endothelial network, but the source for therapeutic cells is limited. We aim to evaluate the beneficial effect of mechanical shear stress in the differentiation of endothelial lineage cells (ELCs from adipose-derived stem cells (ASCs and the possible intracellular signals to protect HI injury using cell-based therapy in the neonatal rats. The ASCs expressed early endothelial markers after biochemical stimulation of endothelial growth medium. The ELCs with full endothelial characteristics were accomplished after a subsequential shear stress application for 24 hours. When comparing the therapeutic potential of ASCs and ELCs, the ELCs treatment significantly reduced the infarction area and preserved neurovascular architecture in HI injured brain. The transplanted ELCs can migrate and engraft into the brain tissue, especially in vessels, where they promoted the angiogenesis. The activation of Akt by neuropilin 1 (NRP1 and vascular endothelial growth factor receptor 2 (VEGFR2 was important for ELC migration and following in vivo therapeutic outcomes. Therefore, the current study demonstrated importance of mechanical factor in stem cell differentiation and showed promising protection of brain from HI injury using ELCs treatment.

  19. Phylogenetic lineages in Entomophthoromycota

    NARCIS (Netherlands)

    Gryganskyi, A.P.; Humber, R.A.; Smith, M.E.; Hodge, K.; Huang, B.; Voigt, K.; Vilgalys, R.

    2013-01-01

    Entomophthoromycota is one of six major phylogenetic lineages among the former phylum Zygomycota. These early terrestrial fungi share evolutionarily ancestral characters such as coenocytic mycelium and gametangiogamy as a sexual process resulting in zygospore formation. Previous molecular studies ha

  20. Biosensors for Cell Analysis.

    Science.gov (United States)

    Zhou, Qing; Son, Kyungjin; Liu, Ying; Revzin, Alexander

    2015-01-01

    Biosensors first appeared several decades ago to address the need for monitoring physiological parameters such as oxygen or glucose in biological fluids such as blood. More recently, a new wave of biosensors has emerged in order to provide more nuanced and granular information about the composition and function of living cells. Such biosensors exist at the confluence of technology and medicine and often strive to connect cell phenotype or function to physiological or pathophysiological processes. Our review aims to describe some of the key technological aspects of biosensors being developed for cell analysis. The technological aspects covered in our review include biorecognition elements used for biosensor construction, methods for integrating cells with biosensors, approaches to single-cell analysis, and the use of nanostructured biosensors for cell analysis. Our hope is that the spectrum of possibilities for cell analysis described in this review may pique the interest of biomedical scientists and engineers and may spur new collaborations in the area of using biosensors for cell analysis.

  1. Diversification of two lineages of symbiotic Photobacterium.

    Directory of Open Access Journals (Sweden)

    Henryk Urbanczyk

    Full Text Available Understanding of processes driving bacterial speciation requires examination of closely related, recently diversified lineages. To gain an insight into diversification of bacteria, we conducted comparative genomic analysis of two lineages of bioluminescent symbionts, Photobacterium leiognathi and 'P. mandapamensis'. The two lineages are evolutionary and ecologically closely related. Based on the methods used in bacterial taxonomy for classification of new species (DNA-DNA hybridization and ANI, genetic relatedness of the two lineages is at a cut-off point for species delineation. In this study, we obtained the whole genome sequence of a representative P. leiognathi strain lrivu.4.1, and compared it to the whole genome sequence of 'P. mandapamensis' svers.1.1. Results of the comparative genomic analysis suggest that P. leiognathi has a more plastic genome and acquired genes horizontally more frequently than 'P. mandapamensis'. We predict that different rates of recombination and gene acquisition contributed to diversification of the two lineages. Analysis of lineage-specific sequences in 25 strains of P. leiognathi and 'P. mandapamensis' found no evidence that bioluminescent symbioses with specific host animals have played a role in diversification of the two lineages.

  2. Diversification of two lineages of symbiotic Photobacterium.

    Science.gov (United States)

    Urbanczyk, Henryk; Urbanczyk, Yoshiko; Hayashi, Tetsuya; Ogura, Yoshitoshi

    2013-01-01

    Understanding of processes driving bacterial speciation requires examination of closely related, recently diversified lineages. To gain an insight into diversification of bacteria, we conducted comparative genomic analysis of two lineages of bioluminescent symbionts, Photobacterium leiognathi and 'P. mandapamensis'. The two lineages are evolutionary and ecologically closely related. Based on the methods used in bacterial taxonomy for classification of new species (DNA-DNA hybridization and ANI), genetic relatedness of the two lineages is at a cut-off point for species delineation. In this study, we obtained the whole genome sequence of a representative P. leiognathi strain lrivu.4.1, and compared it to the whole genome sequence of 'P. mandapamensis' svers.1.1. Results of the comparative genomic analysis suggest that P. leiognathi has a more plastic genome and acquired genes horizontally more frequently than 'P. mandapamensis'. We predict that different rates of recombination and gene acquisition contributed to diversification of the two lineages. Analysis of lineage-specific sequences in 25 strains of P. leiognathi and 'P. mandapamensis' found no evidence that bioluminescent symbioses with specific host animals have played a role in diversification of the two lineages.

  3. Genome Analysis of Clostridium difficile PCR Ribotype 014 Lineage in Australian Pigs and Humans Reveals a Diverse Genetic Repertoire and Signatures of Long-Range Interspecies Transmission

    Science.gov (United States)

    Knight, Daniel R.; Squire, Michele M.; Collins, Deirdre A.; Riley, Thomas V.

    2017-01-01

    Clostridium difficile PCR ribotype (RT) 014 is well-established in both human and porcine populations in Australia, raising the possibility that C. difficile infection (CDI) may have a zoonotic or foodborne etiology. Here, whole genome sequencing and high-resolution core genome phylogenetics were performed on a contemporaneous collection of 40 Australian RT014 isolates of human and porcine origin. Phylogenies based on MLST (7 loci, STs 2, 13, and 49) and core orthologous genes (1260 loci) showed clustering of human and porcine strains indicative of very recent shared ancestry. Core genome single nucleotide variant (SNV) analysis found 42% of human strains showed a clonal relationship (separated by ≤2 SNVs in their core genome) with one or more porcine strains, consistent with recent inter-host transmission. Clones were spread over a vast geographic area with 50% of the human cases occurring without recent healthcare exposure. These findings suggest a persistent community reservoir with long-range dissemination, potentially due to agricultural recycling of piggery effluent. We also provide the first pan-genome analysis for this lineage, characterizing its resistome, prophage content, and in silico virulence potential. The RT014 is defined by a large “open” pan-genome (7587 genes) comprising a core genome of 2296 genes (30.3% of the total gene repertoire) and an accessory genome of 5291 genes. Antimicrobial resistance genotypes and phenotypes varied across host populations and ST lineages and were characterized by resistance to tetracycline [tetM, tetA(P), tetB(P) and tetW], clindamycin/erythromycin (ermB), and aminoglycosides (aph3-III-Sat4A-ant6-Ia). Resistance was mediated by clinically important mobile genetic elements, most notably Tn6194 (harboring ermB) and a novel variant of Tn5397 (harboring tetM). Numerous clinically important prophages (Siphoviridae and Myoviridae) were identified as well as an uncommon accessory gene regulator locus (agr3

  4. mSEL-1L (Suppressor/enhancer Lin12-like) protein levels influence murine neural stem cell self-renewal and lineage commitment.

    Science.gov (United States)

    Cardano, Marina; Diaferia, Giuseppe R; Cattaneo, Monica; Dessì, Sara S; Long, Qiaoming; Conti, Luciano; Deblasio, Pasquale; Cattaneo, Elena; Biunno, Ida

    2011-05-27

    Murine SEL-1L (mSEL-1L) is a key component of the endoplasmic reticulum-associated degradation pathway. It is essential during development as revealed by the multi-organ dysfunction and in uterus lethality occurring in homozygous mSEL-1L-deficient mice. Here we show that mSEL-1L is highly expressed in pluripotent embryonic stem cells and multipotent neural stem cells (NSCs) but silenced in all mature neural derivatives (i.e. astrocytes, oligodendrocytes, and neurons) by mmu-miR-183. NSCs derived from homozygous mSEL-1L-deficient embryos (mSEL-1L(-/-) NSCs) fail to proliferate in vitro, show a drastic reduction of the Notch effector HES-5, and reveal a significant down-modulation of the early neural progenitor markers PAX-6 and OLIG-2, when compared with the wild type (mSEL-1L(+/+) NSCs) counterpart. Furthermore, these cells are almost completely deprived of the neural marker Nestin, display a significant decrease of SOX-2 expression, and rapidly undergo premature astrocytic commitment and apoptosis. The data suggest severe self-renewal defects occurring in these cells probably mediated by misregulation of the Notch signaling. The results reported here denote mSEL-1L as a primitive marker with a possible involvement in the regulation of neural progenitor stemness maintenance and lineage determination.

  5. mSEL-1L (Suppressor/Enhancer Lin12-like) Protein Levels Influence Murine Neural Stem Cell Self-renewal and Lineage Commitment*

    Science.gov (United States)

    Cardano, Marina; Diaferia, Giuseppe R.; Cattaneo, Monica; Dessì, Sara S.; Long, Qiaoming; Conti, Luciano; DeBlasio, Pasquale; Cattaneo, Elena; Biunno, Ida

    2011-01-01

    Murine SEL-1L (mSEL-1L) is a key component of the endoplasmic reticulum-associated degradation pathway. It is essential during development as revealed by the multi-organ dysfunction and in uterus lethality occurring in homozygous mSEL-1L-deficient mice. Here we show that mSEL-1L is highly expressed in pluripotent embryonic stem cells and multipotent neural stem cells (NSCs) but silenced in all mature neural derivatives (i.e. astrocytes, oligodendrocytes, and neurons) by mmu-miR-183. NSCs derived from homozygous mSEL-1L-deficient embryos (mSEL-1L−/− NSCs) fail to proliferate in vitro, show a drastic reduction of the Notch effector HES-5, and reveal a significant down-modulation of the early neural progenitor markers PAX-6 and OLIG-2, when compared with the wild type (mSEL-1L+/+ NSCs) counterpart. Furthermore, these cells are almost completely deprived of the neural marker Nestin, display a significant decrease of SOX-2 expression, and rapidly undergo premature astrocytic commitment and apoptosis. The data suggest severe self-renewal defects occurring in these cells probably mediated by misregulation of the Notch signaling. The results reported here denote mSEL-1L as a primitive marker with a possible involvement in the regulation of neural progenitor stemness maintenance and lineage determination. PMID:21454627

  6. The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours.

    Science.gov (United States)

    Kamata, Tamihiro; Jin, Hong; Giblett, Susan; Patel, Bipin; Patel, Falguni; Foster, Charles; Pritchard, Catrin

    2015-07-16

    The tumour microenvironment is known to play an integral role in facilitating cancer progression at advanced stages, but its function in some pre-cancerous lesions remains elusive. We have used the (V600) (E)BRAF-driven mouse lung model that develop premalignant lesions to understand stroma-tumour interactions during pre-cancerous development. In this model, we have found that immature macrophage-lineage cells (IMCs) producing PDGFA, TGFβ and CC chemokines are recruited to the stroma of premalignant lung adenomas through CC chemokine receptor 1 (CCR1)-dependent mechanisms. Stromal IMCs promote proliferation and transcriptional alterations suggestive of epithelial-mesenchymal transition in isolated premalignant lung tumour cells ex vivo, and are required for the maintenance of early-stage lung tumours in vivo. Furthermore, we have found that IMC recruitment to the microenvironment is restrained by the cholesterol-binding protein, Niemann-Pick type C2 (NPC2). Studies on isolated cells ex vivo confirm that NPC2 is secreted from tumour cells and is taken up by IMCs wherein it suppresses secretion of the CCR1 ligand CC chemokine 6 (CCL6), at least in part by facilitating its lysosomal degradation. Together, these findings show that NPC2 secreted by premalignant lung tumours suppresses IMC recruitment to the microenvironment in a paracrine manner, thus identifying a novel target for the development of chemopreventive strategies in lung cancer.

  7. Neuroprotective and antiapoptotic activity of lineage-negative bone marrow cells after intravitreal injection in a mouse model of acute retinal injury.

    Science.gov (United States)

    Machalińska, Anna; Rogińska, Dorota; Pius-Sadowska, Ewa; Kawa, Miłosz P; Paczkowska, Edyta; Rudnicki, Michał; Lejkowska, Renata; Baumert, Bartłomiej; Wiszniewska, Barbara; Machaliński, Bogusław

    2015-01-01

    We investigated effects of bone marrow-derived, lineage-negative cell (Lin(-)BMC) transplantation in acute retinal injury. Lin(-)BMCs were intravitreally injected into murine eyes at 24 h after NaIO3-induced injury. Morphology, function, and expression of apoptosis-related genes, including brain-derived neurotrophic factor (BDNF) and its receptor, were assessed in retinas at 7 days, 28 days, and 3 months after transplantation. Moreover, global gene expression at day 7 was analyzed by RNA arrays. We observed that Lin(-)BMCs integrated into outer retinal layers improving morphological retinal structure and induced molecular changes such as downregulation of proapoptotic caspase-3 gene, a decrease in BAX/BCL-2 gene ratio, and significant elevation of BDNF expression. Furthermore, transplanted Lin(-)BMCs differentiated locally into cells with a macrophage-like phenotype. Finally, Lin(-)BMCs treatment was associated with generation of two distinct transcriptomic patterns. The first relates to downregulated genes associated with regulation of neuron cell death and apoptosis, response to oxidative stress/hypoxia and external stimuli, and negative regulation of cell proliferation. The second relates to upregulated genes associated with neurological system processes and sensory perception. Collectively, our data demonstrate that transplanted Lin(-)BMCs exert neuroprotective function against acute retinal injury and this effect may be associated with their antiapoptotic properties and ability to express neurotrophic factors.

  8. Neuroprotective and Antiapoptotic Activity of Lineage-Negative Bone Marrow Cells after Intravitreal Injection in a Mouse Model of Acute Retinal Injury

    Directory of Open Access Journals (Sweden)

    Anna Machalińska

    2015-01-01

    Full Text Available We investigated effects of bone marrow-derived, lineage-negative cell (Lin−BMC transplantation in acute retinal injury. Lin−BMCs were intravitreally injected into murine eyes at 24 h after NaIO3-induced injury. Morphology, function, and expression of apoptosis-related genes, including brain-derived neurotrophic factor (BDNF and its receptor, were assessed in retinas at 7 days, 28 days, and 3 months after transplantation. Moreover, global gene expression at day 7 was analyzed by RNA arrays. We observed that Lin−BMCs integrated into outer retinal layers improving morphological retinal structure and induced molecular changes such as downregulation of proapoptotic caspase-3 gene, a decrease in BAX/BCL-2 gene ratio, and significant elevation of BDNF expression. Furthermore, transplanted Lin−BMCs differentiated locally into cells with a macrophage-like phenotype. Finally, Lin−BMCs treatment was associated with generation of two distinct transcriptomic patterns. The first relates to downregulated genes associated with regulation of neuron cell death and apoptosis, response to oxidative stress/hypoxia and external stimuli, and negative regulation of cell proliferation. The second relates to upregulated genes associated with neurological system processes and sensory perception. Collectively, our data demonstrate that transplanted Lin−BMCs exert neuroprotective function against acute retinal injury and this effect may be associated with their antiapoptotic properties and ability to express neurotrophic factors.

  9. Collagen-Coated Polytetrafluoroethane Membrane Inserts Enhances Chondrogenic Differentiation of Human Cord Blood Multi-Lineage Progenitor Cells

    DEFF Research Database (Denmark)

    Munir, Samir; Søballe, Kjeld; Ulrich-Vinther, Michael;

    Background: Articular chondrocytes and bone marrow-derived multipotent mesenchymal stromal cells (MSCs) are the favoured cells for cartilage tissue engineering. Umbilical cord blood has proven an alternative source of MSCs and moreover they may be more potent chondroprogenitor cells than bonemarrow...... MSCs. Purpose / Aim of Study: Multilineage progenitor cells (MLPCs) are clonal cord blood-derived MSCs and may therefore provide a cell source with more reproducible outcomes compared to heterogeneous primary MSC cultures. Materials and Methods: We evaluated the chondrogenic potency of MLPCs...... in standard micromass pellet system, layered on calcium polyphosphate (CPP), and on semi-permeable polytetrafluoroethane membranes with and without collagen type I, II or IV pre-coating. Findings / Results: The MPLC cell line used in this study possessed poor chondrogenic potency overall, but membrane...

  10. Expression of mixed lineage kinase-1 in pancreatic beta-cell lines at different stages of maturation and during embryonic pancreas development.

    Science.gov (United States)

    DeAizpurua, H J; Cram, D S; Naselli, G; Devereux, L; Dorow, D S

    1997-06-27

    Events controlling differentiation to insulin-secreting beta-cells in the pancreas are not well understood, although beta-cells are thought to arise from pluripotent ductal precursor cells. To search for signaling proteins that might be involved in beta-cell maturation, we analyzed protein kinase expression in two developmentally and functionally distinct pancreatic beta-cell lines, RIN-5AH and RIN-A12, by reverse transcriptase polymerase chain reaction. A number of tyrosine and serine/threonine kinases were identified in both lines. One protein kinase, mixed lineage kinase-1 (MLK-1), was expressed at both the RNA and protein levels in RIN-5AH cells, which display an immature beta-cell phenotype, but was not detected in the more mature RIN-A12 cells. Furthermore, levels of MLK-1 mRNA and protein were increased after brief stimulation of RIN-5AH cells with either the differentiation inducer, sodium butyrate, or with serum after serum starvation. These increases in expression were independent of phenotypic markers such as insulin secretion or surface expression of major histocompatibility class I- and A2B5-reactive ganglioside. In addition, increases in MLK-1 expression in the stimulated RIN-5AH cells were accompanied by phosphorylation of MLK-1 on serine but not tyrosine. Antisense oligonucleotides to two distinct regions of MLK-1 caused RIN-5AH cells, but not RIN-A12 cells, to adopt a highly undifferentiated morphology, with a reduction in DNA synthesis and MLK-1 protein levels and elevated glucagon mRNA levels, but with no effect on insulin mRNA. In an immunohistochemical survey of embryonic mouse tissues, we found that temporal expression of MLK-1 was regulated in a tissue-specific manner. In the embryonic pancreas, MLK-1 expression was evident in ductal cells from day 13 to 16 but was not detected in late stage gestation or neonatal pancreas. These data suggest that MLK-1 is regulated in immature pancreatic beta-cells and their ductal precursors at the level of

  11. Tracking human migrations by the analysis of the distribution of HLA alleles, lineages and haplotypes in closed and open populations.

    Science.gov (United States)

    Fernandez Vina, Marcelo A; Hollenbach, Jill A; Lyke, Kirsten E; Sztein, Marcelo B; Maiers, Martin; Klitz, William; Cano, Pedro; Mack, Steven; Single, Richard; Brautbar, Chaim; Israel, Shosahna; Raimondi, Eduardo; Khoriaty, Evelyne; Inati, Adlette; Andreani, Marco; Testi, Manuela; Moraes, Maria Elisa; Thomson, Glenys; Stastny, Peter; Cao, Kai

    2012-03-19

    The human leucocyte antigen (HLA) system shows extensive variation in the number and function of loci and the number of alleles present at any one locus. Allele distribution has been analysed in many populations through the course of several decades, and the implementation of molecular typing has significantly increased the level of diversity revealing that many serotypes have multiple functional variants. While the degree of diversity in many populations is equivalent and may result from functional polymorphism(s) in peptide presentation, homogeneous and heterogeneous populations present contrasting numbers of alleles and lineages at the loci with high-density expression products. In spite of these differences, the homozygosity levels are comparable in almost all of them. The balanced distribution of HLA alleles is consistent with overdominant selection. The genetic distances between outbred populations correlate with their geographical locations; the formal genetic distance measurements are larger than expected between inbred populations in the same region. The latter present many unique alleles grouped in a few lineages consistent with limited founder polymorphism in which any novel allele may have been positively selected to enlarge the communal peptide-binding repertoire of a given population. On the other hand, it has been observed that some alleles are found in multiple populations with distinctive haplotypic associations suggesting that convergent evolution events may have taken place as well. It appears that the HLA system has been under strong selection, probably owing to its fundamental role in varying immune responses. Therefore, allelic diversity in HLA should be analysed in conjunction with other genetic markers to accurately track the migrations of modern humans.

  12. Evidence of progenitor cells of glandular and myoepithelial cell lineages in the human adult female breast epithelium: a new progenitor (adult stem) cell concept.

    Science.gov (United States)

    Boecker, Werner; Buerger, Horst

    2003-10-01

    Although experimental data clearly confirm the existence of self-renewing mammary stem cells, the characteristics of such progenitor cells have never been satisfactorily defined. Using a double immunofluorescence technique for simultaneous detection of the basal cytokeratin 5, the glandular cytokeratins 8/18 and the myoepithelial differentiation marker smooth muscle actin (SMA), we were able to demonstrate the presence of CK5+ cells in human adult breast epithelium. These cells have the potential to differentiate to either glandular (CK8/18+) or myoepithelial cells (SMA+) through intermediary cells (CK5+ and CK8/18+ or SMA+). We therefore proceeded on the assumption that the CK5+ cells are phenotypically and behaviourally progenitor (committed adult stem) cells of human breast epithelium. Furthermore, we furnish evidence that most of these progenitor cells are located in the luminal epithelium of the ductal lobular tree. Based on data obtained in extensive analyses of proliferative breast disease lesions, we have come to regard usual ductal hyperplasia as a progenitor cell-derived lesion, whereas most breast cancers seem to evolve from differentiated glandular cells. Double immunofluorescence experiments provide a new tool to characterize phenotypically progenitor (adult stem) cells and their progenies. This model has been shown to be of great value for a better understanding not only of normal tissue regeneration but also of proliferative breast disease. Furthermore, this model provides a new tool for unravelling further the regulatory mechanisms that govern normal and pathological cell growth.

  13. Whole-genome analysis of human influenza A virus reveals multiple persistent lineages and reassortment among recent H3N2 viruses.

    Directory of Open Access Journals (Sweden)

    2005-09-01

    Full Text Available Understanding the evolution of influenza A viruses in humans is important for surveillance and vaccine strain selection. We performed a phylogenetic analysis of 156 complete genomes of human H3N2 influenza A viruses collected between 1999 and 2004 from New York State, United States, and observed multiple co-circulating clades with different population frequencies. Strikingly, phylogenies inferred for individual gene segments revealed that multiple reassortment events had occurred among these clades, such that one clade of H3N2 viruses present at least since 2000 had provided the hemagglutinin gene for all those H3N2 viruses sampled after the 2002-2003 influenza season. This reassortment event was the likely progenitor of the antigenically variant influenza strains that caused the A/Fujian/411/2002-like epidemic of the 2003-2004 influenza season. However, despite sharing the same hemagglutinin, these phylogenetically distinct lineages of viruses continue to co-circulate in the same population. These data, derived from the first large-scale analysis of H3N2 viruses, convincingly demonstrate that multiple lineages can co-circulate, persist, and reassort in epidemiologically significant ways, and underscore the importance of genomic analyses for future influenza surveillance.

  14. Analysis of methicillin-resistant Staphylococcus aureus major clonal lineages by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Zhang, Tingting; Ding, Jinya; Rao, Xiancai; Yu, Jingbo; Chu, Meiling; Ren, Wei; Wang, Lu; Xue, Wencheng

    2015-10-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen associated with nosocomial infections in many countries. Multilocus sequence typing (MLST) is one of the genetic typing methods used to type MRSA with a high discriminatory power, however, it is labor-intensive, timely, and costly. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled with ClinProTools is a potential tool to discover biomarker peaks and to generate a classification model based on highly sophisticated mathematical algorithms to discriminate clonal lineages. We investigated the performance of MALDI-TOF MS for discriminating 154 MRSA-ST239, 72 MRSA-ST5, 30 MRSA-ST59, 14 MRSA-ST45, and 20 MRSA-OST (other clonal lineages). Our results indicate that the model construction and validation have good potency to discriminate ST45 from other lineages with a sensitivity and a specificity of both 100%, and a sensitivity of 95.80% and a specificity of 94.62% to identify ST239. For Biotyper classification, the sensitivity and specificity were more than of 90% for ST239, ST59 and ST45, whereas only 81.94% sensitivity for ST5. By single-peak analysis, the peaks m/z 4808 and 9614 can correctly discriminate ST45 a sensitivity and a specificity of both 100%; the peak m/z 6554 can also discriminate ST239 with a sensitivity of 91.9% and a specificity of 85.4%. In conclusion, MALDI-TOF MS coupled with ClinProTools has a high detection performance for MRSA typing with obvious advantages of being rapid, highly accurate, and being a low cost in comparison with MLST.

  15. A boost of BMP4 accelerates the commitment of human embryonic stem cells to the endothelial lineage.

    Science.gov (United States)

    Goldman, Orit; Feraud, Olivier; Boyer-Di Ponio, Julie; Driancourt, Catherine; Clay, Denis; Le Bousse-Kerdiles, Marie-Caroline; Bennaceur-Griscelli, Annelise; Uzan, Georges

    2009-08-01

    Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12.

  16. Monocyte/macrophage lineage commitment and distribution are affected by the lack of regulatory T cells in scurfy mice.

    Science.gov (United States)

    Skuljec, Jelena; Cabanski, Maciej; Surdziel, Ewa; Lachmann, Nico; Brennig, Sebastian; Pul, Refik; Jirmo, Adan C; Habener, Anika; Visic, Julia; Dalüge, Kathleen; Hennig, Christian; Moritz, Thomas; Happle, Christine; Hansen, Gesine

    2016-07-01

    Foxp3(+) regulatory T (Treg) cells play a pivotal role in maintaining immunological tolerance. Loss-of-function mutations in the Foxp3 gene result in multiorgan inflammation known as immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in humans and scurfy (Sf) disease in mice. While the impact of missing Treg cells on adaptive immune cells is well documented, their role in regulation of myeloid cells remains unclear. Here we report that Sf mice exhibit an altered composition of stem and progenitor cells, characterized by increased numbers of myeloid precursors and higher efficiency of macrophage generation ex vivo. The proportion of monocytes/macrophages in the bone marrow, blood, and spleen was significantly elevated in Sf mice, which was accompanied with tissue-specific monocyte expression of homing receptor and phagocytic activity. Sf mice displayed high levels of M-CSF and other inflammatory cytokines, including monocyte-recruiting chemokines. Adoptive transfer of WT CD4(+) cells and in vivo neutralization of M-CSF normalized frequencies of monocyte subsets and their progenitors and reduced high levels of monocyte-related cytokines in Sf mice, while Treg cell transfer to RAG2(-/-) mice had no effect on myelopoiesis and monocyte/macrophage counts. Our findings illustrate that deregulated myelopoiesis in Sf mice is mainly caused by the inflammatory reaction resulting from the lack of Treg cells.

  17. The early human germ cell lineage does not express SOX2 during in vivo development or upon in vitro culture

    DEFF Research Database (Denmark)

    Perrett, Rebecca M; Turnpenny, Lee; Eckert, Judith J

    2008-01-01

    of redundancy within the group B family of human SOX genes. Although lacking SOX2, proliferative human germ cells can still be identified in situ during early development and are capable of culture in vitro. Surprisingly, with the exception of FGF4, many stem cell-restricted SOX2 target genes remained detected...

  18. Signaling networks regulating tooth organogenesis and regeneration, and the specification of dental mesenchymal and epithelial cell lineages.

    Science.gov (United States)

    Jussila, Maria; Thesleff, Irma

    2012-04-01

    Teeth develop as ectodermal appendages from epithelial and mesenchymal tissues. Tooth organogenesis is regulated by an intricate network of cell-cell signaling during all steps of development. The dental hard tissues, dentin, enamel, and cementum, are formed by unique cell types whose differentiation is intimately linked with morphogenesis. During evolution the capacity for tooth replacement has been reduced in mammals, whereas teeth have acquired more complex shapes. Mammalian teeth contain stem cells but they may not provide a source for bioengineering of human teeth. Therefore it is likely that nondental cells will have to be reprogrammed for the purpose of clinical tooth regeneration. Obviously this will require understanding of the mechanisms of normal development. The signaling networks mediating the epithelial-mesenchymal interactions during morphogenesis are well characterized but the molecular signatures of the odontogenic tissues remain to be uncovered.

  19. Lineage plasticity and cell biology of fibrocartilage and hyaline cartilage: Its significance in cartilage repair and replacement

    Energy Technology Data Exchange (ETDEWEB)

    Freemont, Anthony J. [Regenerative Medicine Research Group, University of Manchester, England (United Kingdom)]. E-mail: Tony.freemont@man.ac.uk; Hoyland, Judith [Regenerative Medicine Research Group, University of Manchester, England (United Kingdom)

    2006-01-15

    Cartilage repair is a major goal of modern tissue engineering. To produce novel engineered implants requires a knowledge of the basic biology of the tissues that are to be replaced or reproduced. Hyaline articular cartilage and meniscal fibrocartilage are two tissues that have excited attention because of the frequency with which they are damaged. A basic strategy is to re-engineer these tissues ex vivo by stimulating stem cells to differentiate into the cells of the mature tissue capable of producing an intact functional matrix. In this brief review, the sources of cells for tissue engineering cartilage and the culture conditions that have promoted differentiation are discussed within the context of natural cartilage repair. In particular, the role of cell density, cytokines, load, matrices and oxygen tension are discussed.

  20. A role for interleukin-1β in determining the lineage fate of embryonic rat hippocampal neural precursor cells.

    Science.gov (United States)

    Green, Holly F; Treacy, Eimear; Keohane, Aoife K; Sullivan, Aideen M; O'Keeffe, Gerard W; Nolan, Yvonne M

    2012-03-01

    Neurogenesis occurs in the hippocampus of the developing and adult brain due to the presence of multipotent stem cells and restricted precursor cells at different stages of differentiation. It has been proposed that they may be of potential benefit for use in cell transplantation approaches for neurodegenerative disorders and trauma. Prolonged release of interleukin-1β (IL-1β) from activated microglia has a deleterious effect on hippocampal neurons and is implicated in the impaired neurogenesis and cognitive dysfunction associated with aging, Alzheimer's disease and depression. This study assessed the effect of IL-1β on the proliferation and differentiation of embryonic rat hippocampal NPCs in vitro. We show that IL-1R1 is expressed on proliferating NPCs and that IL-1β treatment decreases cell proliferation and neurosphere growth. When NPCs were differentiated in the presence of IL-1β, a significant reduction in the percentages of newly-born neurons and post-mitotic neurons and a significant increase in the percentage of astrocytes was observed in these cultures. These effects were attenuated by IL-1 receptor antagonist. These data reveal that IL-1β exerts an anti-proliferative, anti-neurogenic and pro-gliogenic effect on embryonic hippocampal NPCs, which is mediated by IL-1R1. The present results emphasise the consequences of an inflammatory environment during NPC development, and indicate that strategies to inhibit IL-1β signalling may be necessary to facilitate effective cell transplantation approaches or in conditions where endogenous hippocampal neurogenesis is impaired.

  1. Differential expression of surface markers in mouse bone marrow mesenchymal stromal cell subpopulations with distinct lineage commitment.

    Directory of Open Access Journals (Sweden)

    Maria Rostovskaya

    Full Text Available Bone marrow mesenchymal stromal cells (BM MSCs represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.

  2. Improved outcome with hematopoietic stem cell transplantation in a poor prognostic subgroup of infants with mixed-lineage-leukemia (MLL)-rearranged acute lymphoblastic leukemia: results from the Interfant-99 Study

    DEFF Research Database (Denmark)

    Mann, Georg; Attarbaschi, Andishe; Schrappe, Martin;

    2010-01-01

    To define a role for hematopoietic stem cell transplantation (HSCT) in infants with acute lymphoblastic leukemia and rearrangements of the mixed-lineage-leukemia gene (MLL(+)), we compared the outcome of MLL(+) patients from trial Interfant-99 who either received chemotherapy only or HSCT. Of 376...

  3. C/EBPα Is Required for Long-Term Self-Renewal and Lineage Priming of Hematopoietic Stem Cells and for the Maintenance of Epigenetic Configurations in Multipotent Progenitors

    DEFF Research Database (Denmark)

    Hasemann, Marie S; Lauridsen, Felicia K B; Waage, Johannes;

    2014-01-01

    as a priming factor at the HSC level where it actively promotes myeloid differentiation and counteracts lymphoid lineage choice. Taken together, our results show that C/EBPα is a key regulator of HSC biology, which influences the epigenetic landscape of HSCs in order to balance different cell fate options....

  4. Analysis of 16 Y STR loci in the Finnish population reveals a local reduction in the diversity of male lineages.

    Science.gov (United States)

    Hedman, M; Pimenoff, V; Lukka, M; Sistonen, P; Sajantila, A

    2004-05-28

    We analysed samples of 400 Finnish males using nine Y-chromosomal short tandem repeat (STR) loci (minimal haplotype); for 200 of these subjects an additional seven Y-chromosomal STR loci were used. The geographical distribution of the observed haplotypes was determined from 200 individuals of known paternal origin within Finland. The observed number of alleles varied from 2 to 13 alleles per locus. A total of 146 minimal haplotypes were identified in our population sample. Interestingly, 90 (22.5%) individuals shared an identical haplotype. This haplotype was extremely frequent in the northern and eastern subpopulations of Savo, Pohjanmaa and Karjala (53, 42 and 37%, respectively). With the seven additional loci analysed in the sample of 200 individuals, 120 haplotypes were identified, and individuals sharing the most common haplotype decreased to 13.0%. However, in comparison to other European populations, the Finnish population showed decreased genetic diversity (GD) when the number of different minimal haplotypes in the population was divided by the sample size (36.5% in Finns versus 83.7% on average). Our results strongly support the earlier hypothesis of individual isolated Y-chromosomal lineages and population substructuring in Finland. For paternity testing, power of exclusion was 92% using minimal haplotype data, but including the seven additional loci this value increased to 97%.

  5. Genotypic lineages and restriction fragment length polymorphism of canine distemper virus isolates in Thailand.

    Science.gov (United States)

    Radtanakatikanon, Araya; Keawcharoen, Juthatip; Charoenvisal, Na Taya; Poovorawan, Yong; Prompetchara, Eakachai; Yamaguchi, Ryoji; Techangamsuwan, Somporn

    2013-09-27

    Canine distemper virus (CDV) is known to cause multisystemic disease in all families of terrestrial carnivores. Attenuated live vaccines have been used to control CDV in a variety of species for many decades, yet a number of CDV infections in vaccinated dogs are still observed. The aims of this study were to investigate the genetic diversity of CDV lineages based on phosphoprotein (P), hemagglutinin (H) and fusion protein (F) genes and to develop the restriction fragment length polymorphism (RFLP) technique for effective differentiation among individual wild-type and vaccine lineages in Thailand. Four commercial vaccine products, thirteen conjunctival swabs and various tissues from 9 necropsied dogs suspected of having CDV infections were included. Virus isolation was performed using Vero cell expressing canine signaling lymphocyte activation molecules (Vero-DST cells). Reverse-transcription polymerase chain reaction (RT-PCR) on 3 gene regions from the dog derived specimens and the vaccines were carried out, then RFLP analysis upon F-gene amplified fragments was developed. Nucleotide sequence and phylogenetic analysis were compared with other CDV lineages in Genbank. Phylogenetic relationships revealed that CDV field isolates were separated from the vaccine lineage and could be divided into two clusters; one of which belonged to the Asia-1 lineage and another, not related to any previous recognized lineages was proposed as 'Asia-4'. RFLP patterns demonstrating concordance with phylogenetic trees of the distemper virus allowed for differentiation between the Asia-1, Asia-4 and vaccine lineages. Thus, RFLP technique is able to effectively distinguish individual wild-type canine distemper virus from vaccine lineages in Thailand.

  6. Applications of myeloid-specific promoters in transgenic mice support in vivo imaging and functional genomics but do not support the concept of distinct macrophage and dendritic cell lineages or roles in immunity.

    Science.gov (United States)

    Hume, David A

    2011-04-01

    Myeloid lineage cells contribute to innate and acquired immunity, homeostasis, wound repair, and inflammation. There is considerable interest in manipulation of their function in transgenic mice using myeloid-specific promoters. This review considers the applications and specificity of some of the most widely studied transgenes, driven by promoter elements of the lysM, csf1r, CD11c, CD68, macrophage SRA, and CD11b genes, as well as several others. Transgenes have been used in mice to generate myeloid lineage-specific cell ablation, expression of genes of interest, including fluorescent reporters, or deletion via recombination. In general, the specificity of such transgenes has been overinterpreted, and none of them provide well-documented, reliable, differential expression in any specific myeloid cell subset, macrophages, granulocytes, or myeloid DCs. Nevertheless, they have proved valuable in cell isolation, functional genomics, and live imaging of myeloid cell behavior in many different pathologies.

  7. Targeting oncogene expression to endothelial cells induces proliferation of the myelo-erythroid lineage by repressing the Notch pathway.

    Science.gov (United States)

    Alghisi, E; Distel, M; Malagola, M; Anelli, V; Santoriello, C; Herwig, L; Krudewig, A; Henkel, C V; Russo, D; Mione, M C

    2013-11-01

    Human oncogenes involved in the development of hematological malignancies have been widely used to model experimental leukemia. However, models of myeloid leukemia rarely reproduce the human disease in full, due to genetic complexity or to difficulties in targeting leukemia initiating cells. Here, we used a zebrafish genetic model to induce the expression of oncogenic RAS in endothelial cells, including the hemogenic endothelium of the dorsal aorta that generates hematopoietic cells, and observed the development of a myelo-erythroid proliferative disorder. In larvae, the phenotype is characterized by disruption of the vascular system and prominent expansion of the caudal hematopoietic tissue. In few surviving juveniles, increased number of immature hematopoietic cells and arrest of myeloid maturation was found in kidney marrow. Peripheral blood showed increased erythroblasts and myeloid progenitors. We found that the abnormal phenotype is associated with a downregulation of the Notch pathway, whereas overexpressing an activated form of Notch together with the oncogene prevents the expansion of the myelo-erythroid compartment. This study identifies the downregulation of the Notch pathway following an oncogenic event in the hemogenic endothelium as an important step in the pathogenesis of myelo-erythroid disorders and describes a number of potential effectors of this transformation.

  8. IL-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells

    NARCIS (Netherlands)

    S.E. Nodland (Sonja E.); M.A. Berkowska (Magdalena); A.A. Bajer (Anna A.); N. Shah (Nisha); D. de Ridder (Dick); J.J.M. van Dongen (Jacques); T.W. LeBien (Tucker W.); M.C. van Zelm (Menno)

    2011-01-01

    textabstractIL-7 is an important cytokine for lymphocyte differentiation. Similar to what occurs in vivo, human CD19+cells developing in human/murine xenogeneic cultures show differential expression of the IL-7 receptor α (IL-7Rα) chain (CD127). We now describe the relationship between CD127 express

  9. IL-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells

    NARCIS (Netherlands)

    Nodland, Sonja E.; Berkowska, Magdalena A.; Bajer, Anna A.; Shah, Nisha; de Ridder, Dick; van Dongen, Jacques J. M.; LeBien, Tucker W.; van Zelm, Menno C.

    2011-01-01

    IL-7 is an important cytokine for lymphocyte differentiation. Similar to what occurs in vivo, human CD19(+) cells developing in human/murine xenogeneic cultures show differential expression of the IL-7 receptor alpha (IL-7R alpha) chain (CD127). We now describe the relationship between CD127 express

  10. Developmental stage-specific contribution of LGR5(+) cells to basal and luminal epithelial lineages in the postnatal mammary gland

    NARCIS (Netherlands)

    de Visser, K.E.; Ciampricotti, M.; Michalak, E.M.; Tan, D.W.; Speksnijder, E.N.; Hau, C.S.; Clevers, H.; Barker, N.; Jonkers, J.

    2012-01-01

    The leucine-rich repeat-containing heterotrimeric guanine nucleotide-binding protein-coupled receptor 5 (LGR5) has been identified as a marker of cycling stem cells in several epithelial tissues, including small intestine, colon, stomach and hair follicle. To investigate whether LGR5 also marks mamm

  11. Luminal progenitors restrict their lineage potential during mammary gland development.

    Directory of Open Access Journals (Sweden)

    Veronica Rodilla

    2015-02-01

    Full Text Available The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.

  12. Luminal progenitors restrict their lineage potential during mammary gland development.

    Science.gov (United States)

    Rodilla, Veronica; Dasti, Alessandro; Huyghe, Mathilde; Lafkas, Daniel; Laurent, Cécile; Reyal, Fabien; Fre, Silvia

    2015-02-01

    The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.

  13. High level PHGDH expression in breast is predominantly associated with keratin 5-positive cell lineage independently of malignancy

    DEFF Research Database (Denmark)

    Gromova, Irina; Gromov, Pavel; Honma, Naoko

    2015-01-01

    We have previously reported the 2D PAGE-based proteomic profiling of a prospective cohort of 78 triple negative breast cancer (TNBC) patients, and the establishment of a cumulative TNBC protein database. Analysis of this database identified a number of proteins as being specifically overexpressed...

  14. Soluble factors from bone marrow endothelial cells regulate differentiation and proliferation of hematopoietic and endothelial lineages and embryonic stem cells%骨髓内皮细胞来源的可溶性因子调节造血系、内皮系及胚胎干细胞的分化和增殖

    Institute of Scientific and Technical Information of China (English)

    王绮如; 阎琦

    2013-01-01

    We have established a bone marrow endothelial cell line.This review focuses on the elucidation and analysis of the effects of this bone marrow endothelial cell-conditioned medium (BMEC-CM) on the differentiation and proliferation of hematopoietic and endothelial progenitors as well as embryonic stem cells (ESCs).We will review that (1) BMEC-CM promotes proliferation and differentiation of hematopoietic lineage; (2) BMEC-CM promotes proliferation and differentiation of endothelial lineage; (3) BMECCM induces differentiation of hematopoietic stem cells/progenitors into endothelial progenitors; and (4) BMEC-CM induces differentiation of ESCs into hematopoietic cells and endothelial cells.We conclude that the soluble factors secreted by BMECs are able to support the proliferation and differentiation of hematopoietic and endothelium lineages.Moreover,these soluble factors induce hematopoietic cells to differentiate to endothelial cells,and induce ESCs to differentiate towards both endothelial cells and hematopoietic cells.Therefore,this work provides evidence that a close relationship involved in the development of hematopoietic and endothelial lineage.This disclosure will be beneficial for therapy strategy in the treatment of ischemic and tumor diseases,and improve our understanding of the relationship between hematopoietic and endothelial lineages.%本研究室建立了一支骨髓内皮细胞(bone marrow endothelial cell,BMEC)株.本文综述了这一内皮细胞株细胞的条件培养液(bone marrow endothelial cell-conditioned medium,BMEC-CM)对造血系、内皮系及胚胎干细胞的分化增殖的作用.(1)BMEC-CM促进造血系细胞的分化和增殖;(2) BMEC-CM促进内皮系细胞的分化和增殖;(3) BMEC-CM诱导造血干/祖细胞向内皮系细胞分化;(4) BMEC-CM诱导胚胎干细胞分化为造血细胞和内皮细胞.以上研究成果提示,骨髓内皮细胞分泌可溶性因子支持造血系及内皮系细胞的分化与增殖,促进造

  15. CRX is a diagnostic marker of retinal and pineal lineage tumors.

    Directory of Open Access Journals (Sweden)

    Sandro Santagata

    Full Text Available BACKGROUND: CRX is a homeobox transcription factor whose expression and function is critical to maintain retinal and pineal lineage cells and their progenitors. To determine the biologic and diagnostic potential of CRX in human tumors of the retina and pineal, we examined its expression in multiple settings. METHODOLOGY/PRINCIPAL FINDINGS: Using situ hybridization and immunohistochemistry we show that Crx RNA and protein expression are exquisitely lineage restricted to retinal and pineal cells during normal mouse and human development. Gene expression profiling analysis of a wide range of human cancers and cancer cell lines also supports that CRX RNA is highly lineage restricted in cancer. Immunohistochemical analysis of 22 retinoblastomas and 13 pineal parenchymal tumors demonstrated strong expression of CRX in over 95% of these tumors. Importantly, CRX was not detected in the majority of tumors considered in the differential diagnosis of pineal region tumors (n = 78. The notable exception was medulloblastoma, 40% of which exhibited CRX expression in a heterogeneous pattern readily distinguished from that seen in retino-pineal tumors. CONCLUSIONS/SIGNIFICANCE: These findings describe new potential roles for CRX in human cancers and highlight the general utility of lineage restricted transcription factors in cancer biology. They also identify CRX as a sensitive and specific clinical marker and a potential lineage dependent therapeutic target in retinoblastoma and pineoblastoma.

  16. 16S rDNA-based analysis reveals cosmopolitan occurrence but limited diversity of two cyanobacterial lineages with contrasted patterns of intracellular carbonate mineralization

    Directory of Open Access Journals (Sweden)

    Marie eRagon

    2014-07-01

    Full Text Available Cyanobacteria are mainly thought to induce carbonate precipitation extracellularly via their photosynthetic activity combined with the nucleation potential of exopolymeric substances. The discovery in microbialites of the alkaline lake Alchichica (Mexico of Candidatus Gloeomargarita lithophora, a cyanobacterium forming large amounts of intracellular Mg-Ca-Sr-Ba carbonate spherules, showed that intracellular biomineralization in cyanobacteria is also possible. A second cyanobacterium isolated from the same environment, Candidatus Synechococcus calcipolaris G9, has been recently shown to also form intracellular calcium carbonates at the cell poles, a capability shared by all cultured species of the Thermosynechococcus clade, to which it belongs. To explore the diversity of these two distant cyanobacterial lineages representing two different patterns of intracellular calcification, we designed specific primers against their 16S rRNA genes and looked for their occurrence in a wide variety of samples. We identified the presence of members of the Gloeomargarita and Thermosynechococcus/S. calcipolaris lineages in microbialites collected from Lake Alchichica and three other neighboring Mexican lakes. The two clades also occurred in karstic areas and in some thermophilic or hypersaline microbial mats collected in South America and/or Southern Europe. Surprisingly, the within-group diversity in the two clades was low, especially within the S. calcipolaris clade, with all 16S rRNA gene sequences retrieved sharing more than 97% identity. This suggests that these clades are composed of a limited number of species with cosmopolitan distribution. Moreover, scanning electron microscopy coupled with energy dispersive x-ray spectrometry showed the presence of intracellularly calcifying Gloeomargarita-like cyanobacteria in fresh samples where this clade was relatively abundant, suggesting that these cyanobacteria do precipitate carbonates intracellularly under

  17. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

    Science.gov (United States)

    Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

    2013-04-01

    Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

  18. Reconstructing a B-cell clonal lineage. I. Statistical inference of unobserved ancestors [v1; ref status: indexed, http://f1000r.es/z6

    Directory of Open Access Journals (Sweden)

    Thomas B Kepler

    2013-04-01

    Full Text Available One of the key phenomena in the adaptive immune response to infection and immunization is affinity maturation, during which antibody genes are mutated and selected, typically resulting in a substantial increase in binding affinity to the eliciting antigen. Advances in technology on several fronts have made it possible to clone large numbers of heavy-chain light-chain pairs from individual B cells and thereby identify whole sets of clonally related antibodies. These collections could provide the information necessary to reconstruct their own history - the sequence of changes introduced into the lineage during the development of the clone - and to study affinity maturation in detail. But the success of such a program depends entirely on accurately inferring the founding ancestor and the other unobserved intermediates. Given a set of clonally related immunoglobulin V-region genes, the method described here allows one to compute the posterior distribution over their possible ancestors, thereby giving a thorough accounting of the uncertainty inherent in the reconstruction. I demonstrate the application of this method on heavy-chain and light-chain clones, assess the reliability of the inference, and discuss the sources of uncertainty.

  19. Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Laura Tarnawski

    Full Text Available In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

  20. Phylogenetic approaches for the analysis of mitochondrial genome sequence data in the Hymenoptera--a lineage with both rapidly and slowly evolving mitochondrial genomes.

    Science.gov (United States)

    Dowton, Mark; Cameron, Stephen L; Austin, Andy D; Whiting, Michael F

    2009-08-01

    We entirely sequenced two new hymenopteran mitochondrial genomes (Cephus cinctus and Orussus occidentalis), and a substantial portion of another three hymenopterans (Schlettererius cinctipes, Venturia canescens, and Enicospilus). We analyze them together with nine others reported in the literature. We establish that the rate of genetic divergence is two to three times higher among some Hymenoptera when compared with others, making this a group with both long and short phylogenetic branches. We then assessed the ability of a range of phylogenetic approaches to recover seven uncontroversial relationships, when lineages show markedly different rates of molecular evolution. This range encompassed maximum parsimony and Bayesian analysis of (i) amino acid data, (ii) nucleotide data, and (iii) nucleotide data excluding third codon positions. Unpartitioned analyses were compared with partitioned analyses, with the data partitioned by codon position (ribosomal genes were placed in a separate partition). These analyses indicated that partitioned, Bayesian analysis of nucleotide data, excluding 3rd codon positions, recovered more of the uncontroversial relationships than any other approach. These results suggest that the analysis of complete mitochondrial genome sequences holds promise for the resolution of hymenopteran superfamily relationships.

  1. High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation.

    Science.gov (United States)

    Vega, Sebastián L; Liu, Er; Arvind, Varun; Bushman, Jared; Sung, Hak-Joon; Becker, Matthew L; Lelièvre, Sophie; Kohn, Joachim; Vidi, Pierre-Alexandre; Moghe, Prabhas V

    2017-02-01

    Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions.

  2. Microfluidics for single cell analysis

    DEFF Research Database (Denmark)

    Jensen, Marie Pødenphant

    Isolation and manipulation of single cells have gained an increasing interest from researchers because of the heterogeneity of cells from the same cell culture. Single cell analysis can ensure a better understanding of differences between individual cells and potentially solve a variety of clinic...

  3. Parallel emergence of negative epistasis across experimental lineages.

    Science.gov (United States)

    Zee, Peter C; Velicer, Gregory J

    2017-01-27

    Epistatic interactions can greatly impact evolutionary phenomena, particularly the process of adaptation. Here, we leverage four parallel experimentally evolved lineages to study the emergence and trajectories of epistatic interactions in the social bacterium Myxococcus xanthus. A social gene (pilA) necessary for effective group swarming on soft agar had been deleted from the common ancestor of these lineages. During selection for competitiveness at the leading edge of growing colonies, two lineages evolved qualitatively novel mechanisms for greatly increased swarming on soft agar, whereas the other two lineages evolved relatively small increases in swarming. By reintroducing pilA into different genetic backgrounds along the four lineages, we tested whether parallel lineages showed similar patterns of epistasis. In particular, we tested whether a pattern of negative epistasis between accumulating mutations and pilA previously found in the fastest lineage would be found only in the two evolved lineages with the fastest and most striking swarming phenotypes, or rather was due to common epistatic structure across all lineages arising from the generic fixation of adaptive mutations. Our analysis reveals the emergence of negative epistasis across all four independent lineages. Further, we present results showing that the observed negative epistasis is not due exclusively to evolving populations approaching a maximum phenotypic value that inherently limits positive effects of pilA reintroduction, but rather involves direct antagonistic interactions between accumulating mutations and the reintroduced social gene.

  4. Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti–factor VIII immune responses

    OpenAIRE

    Ettinger, Ruth A.; James, Eddie A.; Kwok, William W.; Arthur R Thompson; Pratt, Kathleen P.

    2009-01-01

    The development of neutralizing antibodies (inhibitors) after factor VIII (FVIII) infusions is a serious complication that affects approximately one-quarter of hemophilia A patients who have access to replacement therapy. To investigate the differentiation of naive T cells into FVIII-specific helper T cells that promote B-cell activation and antibody secretion, HLA-DRA-DRB1*0101-restricted T-cell clones that respond to a specific epitope in FVIII were isolated from a mild hemophilia A subject...

  5. Analysis of Y-chromosome STRs in Chile confirms an extensive introgression of European male lineages in urban populations.

    Science.gov (United States)

    Toscanini, Ulises; Brisighelli, Francesca; Moreno, Fabián; Pantoja-Astudillo, Jaime A; Morales, Eugenia Aguirre; Bustos, Patricio; Pardo-Seco, Jacobo; Salas, Antonio

    2016-03-01

    We analyzed the Y chromosome haplotypes (Yfiler) of 978 non-related Chilean males grouped in five sampling regions (Iquique, Santiago de Chile, Concepción, Temuco and Punta Arenas) covering main geo-political regions. Overall, 803 different haplotypes and 688 singletons were observed. Molecular diversity was moderately lower than in other neighboring countries (e.g. Argentina); and AMOVA analysis on Y-STR haplotypes showed that among variation within Chile accounted for only 0.25% of the total variation. Punta Arenas, in the southern cone, showed the lowest haplotype diversity, and discrimination capacity, and also the highest matching probability of the five Chilean samples, probably reflecting its more marked geographic isolation compared to the other regions. Multidimensional scaling (MDS) analysis based on RST genetic distances suggested a close proximity of Chilean Y-chromosome profiles to European ones. Consistently, haplogroups inferred from Y-STR profiles revealed that the Native American component constituted only 8% of all the haplotypes, and this component ranged from 5% in the Centre of the country to 9-10% in the South and 13% in the North, which is in good agreement with the distribution of Native American communities in these regions. AMOVA computed on inferred haplogroups confirmed the very low among variation observed in Chilean populations. The present project provides the first Chilean dataset to the international Y-chromosome STR Haplotype Reference Database (YHRD) and it is also the first reference database for Y-chromosome forensic casework of the country.

  6. PEG10 Activation by Co-Stimulation of CXCR5 and CCR7 Essentially Contributes to Resistance to Apoptosis in CD19+CD34+ B Cells from Patients with B Cell Lineage Acute and Chronic Lymphocytic Leukemia

    Institute of Scientific and Technical Information of China (English)

    ChunsongHu; JeiXiong; LinjeiZhang; BaojunHuang; QiupingZhang; QunLi; MingzhenYang; YaouWu; QunWu; QianShen; QingpingGao; KejianZhang; ZhiminSun; JunyanLin; YouxinJin

    2004-01-01

    We investigated CD19+CD34+ and CD19+CD34 B cells from cord blood (CB) and typical patients with B cell lineage acute and chronic lymphocytic leukemia (B-ALL and B-CLL) in terms of expression and functions of CXCR5/CXCL13 and CCR7/CCL19. CXCR5 and CCR7 were selectively frequent expressed on B-ALL, B-CLL and CB CD19+CD34+ B cells, but not on CD19+CD34- B cells. Instead of induction of impressive chemotactic responsiveness, CXCL13 and CCL19 together induced significant resistance to TNF-α-mediated apoptosis in B-ALL and B-CLL but not CB CD19+CD34+ B cells. B-ALL and B-CLL CD19+CD34+ B cells expressed elevatedlevel of Paternally Expressed Gene 10 (PEG10), and CXCL13 and CCL19 together significantly up-regulated PEG10 expression in the cells. We found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulated PEG10 expression and function, subsequent stabilized caspase-3 and caspase-8 in B-ALL and B-CLL CD19+CD34+ B cells, and rescued the cells from TNF-α-mediated apoptosis. We suggested that normal lymphocytes, especially naive B and T cells, utilized CXCR5/CXCL13 and CCR7/CCL19 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoid tissues organogenesis. Meanwhile certain malignant cells took advantages of CXCR5/CXCL13 and CCR7/CCL19 for infiltration, resistance to apoptosis, and inappropriate proliferation. Cellular & Molecular Immunology.

  7. PEG10 Activation by Co-Stimulation of CXCR5 and CCR7 Essentially Contributes to Resistance to Apoptosis in CD19+CD34+ B Cells from Patients with B Cell Lineage Acute and Chronic Lymphocytic Leukemia

    Institute of Scientific and Technical Information of China (English)

    Chunsong Hu; Qian Shen; Qingping Gao; Kejian Zhang; Zhimin Sun; Junyan Liu; Youxin Jin; Jinquan Tan; Jei Xiong; Linjei zhang; Baojun Huang; Qiuping Zhang; Qun Li; Mingzhen Yang; Yaou Wu; Qun Wu

    2004-01-01

    We investigated CD19+CD34+ and CD19+CD34- B cells from cord blood (CB) and typical patients with B cell lineage acute and chronic lymphocytic leukemia (B-ALL and B-CLL) in terms of expression and functions of CXCR5/CXCL13 and CCR7/CCL19. CXCR5 and CCR7 were selectively frequent expressed on B-ALL, B-CLL and CB CD19+CD34+ B cells, but not on CD19+CD34- B cells. Instead of induction of impressive chemotactic responsiveness, CXCL13 and CCL19 together induced significant resistance to TNF-α-mediated apoptosis in B-ALL and B-CLL but not CB CD19+CD34+ B cells. B-ALL and B-CLL CD19+CD34+ B cells expressed elevated level of Paternally Expressed Gene 10 (PEG10), and CXCL13 and CCL19 together significantly up-regulated PEG10 expression in the cells. We found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulated PEG10 expression and function, subsequent stabilized caspase-3 and caspase-8 in B-ALL and B-CLL CD19+CD34+ B cells, and rescued the cells from TNF-α-mediated apoptosis. We suggested that normal lymphocytes, especially na(I)ve B and T cells, utilized CXCR5/CXCL13 and CCR7/CCL19 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoid tissues organogenesis.Meanwhile certain malignant cells took advantages of CXCR5/CXCL13 and CCR7/CCL19 for infiltration,resistance to apoptosis, and inappropriate proliferation.

  8. CellTracks cell analysis system for rare cell detection

    NARCIS (Netherlands)

    Kagan, Michael T.; Trainer, Michael N.; Bendele, Teresa; Rao, Chandra; Horton, Allen; Tibbe, Arjan G.; Greve, Jan; Terstappen, Leon W.M.M.

    2002-01-01

    The CellTracks system is a Compact Disk-based cell analyzer that, similar to flow cytometry, differentiates cells that are aligned while passing through focused laser beams. In CellTracks, only immuno-magnetically labeled cells are aligned and remain in position for further analysis. This feature is

  9. Ultrastructural characterization and multilocus sequence analysis (MLSA) of 'Candidatus Rickettsiella isopodorum', a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda).

    Science.gov (United States)

    Kleespies, Regina G; Federici, Brian A; Leclerque, Andreas

    2014-07-01

    The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation 'Candidatus Rickettsiella isopodorum' is introduced for this new lineage of isopod-associated Rickettsiella bacteria.

  10. Spatial and temporal lineage analysis of a Pitx3-driven Cre-recombinase knock-in mouse model.

    Directory of Open Access Journals (Sweden)

    Marten P Smidt

    Full Text Available Development and function of mesodiencephalic dopaminergic (mdDA neurons has received a lot of scientific interest since these neurons are critically involved in neurological diseases as Parkinson and psychiatric diseases as schizophrenia, depression and attention deficit hyperactivity disorder (ADHD. The understanding of the molecular processes that lead to normal development and function of mdDA neurons has provided insight in the pathology and provided critical information on new treatment paradigms. In order to be able to study specific genetic ablation in mdDA neurons a new tools was developed that drives Cre-recombinase under the control of the Pitx3 locus. The Pitx3 gene is well known for its specific expression in mdDA neurons and is present at the onset of terminal differentiation. Analysis of newly generated Pitx3-Cre knock-in mice shows that Cre expression, measured through the activation of eYfp by removal of a "Stop" signal (LoxP-Stop-LoxP-eYfp reporter mouse, is present at the onset of terminal differentiation and mimics closely the native Pitx3 expression domain. In conclusion, we present here a new Cre-driver mouse model to be used in the restricted ablation of interesting genes in mdDA neurons in order to improve our understanding of the underlying molecular programming.

  11. Spatial and Temporal Lineage Analysis of a Pitx3-Driven Cre-Recombinase Knock-In Mouse Model

    Science.gov (United States)

    Smidt, Marten P.; von Oerthel, Lars; Hoekstra, Elisa J.; Schellevis, Raymond D.; Hoekman, Marco F. M.

    2012-01-01

    Development and function of mesodiencephalic dopaminergic (mdDA) neurons has received a lot of scientific interest since these neurons are critically involved in neurological diseases as Parkinson and psychiatric diseases as schizophrenia, depression and attention deficit hyperactivity disorder (ADHD). The understanding of the molecular processes that lead to normal development and function of mdDA neurons has provided insight in the pathology and provided critical information on new treatment paradigms. In order to be able to study specific genetic ablation in mdDA neurons a new tools was developed that drives Cre-recombinase under the control of the Pitx3 locus. The Pitx3 gene is well known for its specific expression in mdDA neurons and is present at the onset of terminal differentiation. Analysis of newly generated Pitx3-Cre knock-in mice shows that Cre expression, measured through the activation of eYfp by removal of a “Stop” signal (LoxP-Stop-LoxP-eYfp reporter mouse), is present at the onset of terminal differentiation and mimics closely the native Pitx3 expression domain. In conclusion, we present here a new Cre-driver mouse model to be used in the restricted ablation of interesting genes in mdDA neurons in order to improve our understanding of the underlying molecular programming. PMID:22870339

  12. Y-chromosome analysis reveals genetic divergence and new founding native lineages in Athapaskan- and Eskimoan-speaking populations

    Science.gov (United States)

    Dulik, Matthew C.; Owings, Amanda C.; Gaieski, Jill B.; Vilar, Miguel G.; Andre, Alestine; Lennie, Crystal; Mackenzie, Mary Adele; Kritsch, Ingrid; Snowshoe, Sharon; Wright, Ruth; Martin, James; Gibson, Nancy; Andrews, Thomas D.; Schurr, Theodore G.; Adhikarla, Syama; Adler, Christina J.; Balanovska, Elena; Balanovsky, Oleg; Bertranpetit, Jaume; Clarke, Andrew C.; Comas, David; Cooper, Alan; Der Sarkissian, Clio S. I.; GaneshPrasad, ArunKumar; Haak, Wolfgang; H