Tissue-specific regulatory network extractor (TS-REX): a database and software resource for the tissue and cell type-specific investigation of transcription factor-gene networks

The prediction of transcription factor binding sites in genomic sequences is in principle very useful to identify upstream regulatory factors. However, when applying this concept to genomes of multicellular organisms such as mammals, one has to deal with a large number of false positive predictions since many transcription factor genes are only expressed in specific tissues or cell types. We developed TS-REX, a database/software system that supports the analysis of tissue and cell type-specific transcription factor-gene networks based on expressed sequence tag abundance of transcription factor-encoding genes in UniGene EST libraries. The use of expression levels of transcription factor-encoding genes according to hierarchical anatomical classifications covering different tissues and cell types makes it possible to filter out irrelevant binding site predictions and to identify candidates of potential functional importance for further experimental testing. TS-REX covers ESTs from H. sapiens and M. musculus, and allows the characterization of both presence and specificity of transcription factors in user-specified tissues or cell types. The software allows users to interactively visualize transcription factor-gene networks, as well as to export data for further processing. TS-REX was applied to predict regulators of Polycomb group genes in six human tumor tissues and in human embryonic stem cells

Charakterisierung von CD133-positiven Zellen in Stammzellregionen des embryonalen und adulten murinen Zentralen Nervensystems

Das Oberflächenprotein CD133 wurde auf frühen humanen und murinen neuralen Stammzellen und bestimmten humanen Tumorstammzellen des Zentralnervensystems (ZNS) detektiert. Ob CD133 ein genereller Marker für neurale Stammzellpopulationen des ZNS ist, blieb noch zu zeigen. Der erste Teil dieser Doktorarbeit befasst sich mit der Identifizierung von CD133-positiven Zellen in Stammzellregionen des embryonalen, postnatalen und adulten murinen ZNS. Mehrere CD133-positive Zelltypen wurden gefunden: Eine Subpopulation von Radialen Gliazellen (RGZ) und intermediäre Radiale Glia/Ependymale Zellen in der embryonalen und postnatalen Seitenventrikelregion, sowie Ependymzellen der Seitenventrikelwand (SVW) und des Zentralkanals im Rückenmark (RM) im adulten ZNS. Des Weiteren wurden CD133-positive Ventrikellumen-kontaktierende Typ B Zellen in der adulten SVW entdeckt. Die anschließende Evaluierung der in vitro Selbsterneuerung und Multipotenz von CD133-positiven und CD133-negativen Zellen der untersuchten Stammzellregionen zeigte, dass nicht alle CD133-positiven Zellen Stamm/Vorläuferzelleigenschaften aufwiesen, und darüber hinaus, dass nicht alle neuralen Stammzellen CD133-positiv waren. Im zweiten Teil dieser Arbeit wurden CD133-positive Ependymzellen zweier adulter muriner Stammzellnischen, nämlich der SVW und dem Zentralkanal des RM vergleichend analysiert. Der direkte Vergleich der isolierten Ependymzellen unter identischen Kulturbedingungen zeigte, dass sich RM-Ependymzellen langfristig selbst erneuern können und multipotent sind, während SVW-Ependymzellen keine Stamm/Vorläuferzelleigenschaften aufweisen. Mit Hilfe von Microarray Analysen wurde die molekulare Basis dieser funktionellen Unterschiede untersucht. Einige Gene, die in RM-Ependymzellen stärker exprimiert wurden, könnten von potentieller Relevanz für ihre Stammzelleigenschaften sein, da sie Proteine kodieren, die an der Regulierung des Zellzyklus, dem Erhalt von Telomeren sowie an der Apoptoseinduktion beteiligt sind. Eine potentielle Regulation von RM-Ependymzellen über Retinsäure wurde gefunden. Darüber hinaus wurde eine Gruppe von Genen mit höheren Expressionswerten in Zellen mit Stamm/Vorläufer-zelleigenschaften, nämlich RM-Ependymzellen, RGZ und/oder Neurospären, aber niedrigeren Transkriptionswerten in SVW-Ependymzellen identifiziert. Diesen Genen könnte eine funktionale Rolle in der Erhaltung des undifferenzierten Status von neuralen Stammzellen zukommen.The surface protein CD133 was found on early human and murine neural stem cells during development and certain human tumor stem cells in the central nervous system (CNS). Whether CD133 is a general marker for neural stem cell populations in the CNS remained to be determined. The first part of this thesis focussed on the identification of CD133-positive cells in stem cell regions of the developing and adult murine CNS. Several CD133-positive cell types were identified: A subpopulation of radial glial cells (RGC) and intermediate radial glial/ependymal cells in the embryonic and postnatal lateral ventricle region, as well as ependymal cells from the lateral ventricle wall (LVW) and the spinal cord central canal in the adult CNS. In addition, CD133-positive ventricle-contacting type B cells were found in the adult LVW. The evaluation of in vitro self-renewal and multipotency of CD133-positive and CD133-negative cells from the investigated stem cell regions revealed, that not all CD133-positive cells possessed stem/progenitor cell properties and furthermore, that not all neural stem/progenitor cells were CD133-positive. In the second part of this thesis, CD133-positive ependymal cells from two adult murine stem cell niches, the LVW and the central canal of the spinal cord, were compared. The investigation of their functional properties under identical culture conditions showed that spinal cord ependymal cells can self-renew long-term and are multipotent, whereas ependymal cells from the LVW lack those stem/progenitor cell features. Microarray experiments were performed to determine the underlying molecular basis of these functional differences. Several genes, which were higher expressed in spinal cord ependymal cells, possibly contribute to their stem cell properties, as they encode proteins associated with cell cycle regulation, telomere maintenance and induction of apoptosis. A potential regulation of adult spinal cord ependymal cells by retinoic acid was identified. Furthermore, a group of genes with higher expression in cells with stem/progenitor cell features, namely spinal cord ependymal cells, RGC and / or neurospheres, but lower transcript levels in LVW ependymal cells, was identified. These genes could be of functional importance for the immature state of neural stem cells

Prospectively isolated CD133/CD24-positive ependymal cells from the adult spinal cord and lateral ventricle wall differ in their long-term in vitro self-renewal and in vivo gene expression.

In contrast to ependymal cells located above the subventricular zone (SVZ) of the adult lateral ventricle wall (LVW), adult spinal cord (SC) ependymal cells possess certain neural stem cell characteristics. The molecular basis of this difference is unknown. In this study, antibodies against multiple cell surface markers were applied to isolate pure populations of SC and LVW ependymal cells, which allowed a direct comparison of their in vitro behavior and in vivo gene expression profile. Isolated CD133(+)/CD24(+)/CD45(-)/CD34(-) ependymal cells from the SC displayed in vitro self-renewal and differentiation capacity, whereas those from the LVW did not. SC ependymal cells showed a higher expression of several genes involved in cell division, cell cycle regulation, and chromosome stability, which is consistent with a long-term self-renewal capacity, and shared certain transcripts with neural stem cells of the embryonic forebrain. They also expressed several retinoic acid (RA)-regulated genes and responded to RA exposure. LVW ependymal cells showed higher transcript levels of many genes regulated by transforming growth factor-β family members. Among them were Dlx2, Id2, Hey1, which together with Foxg1 could explain their potential to turn into neuroblasts under certain environmental conditions. © 2010 Wiley-Liss, Inc

Tracheal remodeling: comparison of different composite cultures consisting of human respiratory epithelial cells and human chondrocytes

The reconstruction of extensive tracheal defects is still an unsolved challenge for thoracic surgery. Tissue engineering is a promising possibility to solve this problem through the generation of an autologous tracheal replacement from patients' own tissue. Therefore, this study investigated the potential of three different coculture systems, combining human respiratory epithelial cells and human chondrocytes. The coculture systems were analyzed by histological staining with alcian blue, immunohistochemical staining with the antibodies, 34betaE12 and CD44v6, and scanning electron microscopy. The first composite culture consisted of human respiratory epithelial cells seeded on human high-density chondrocyte pellets. For the second system, we used native articular cartilage chips as base for the respiratory epithelial cells. The third system consisted of a collagen membrane, seeded with respiratory epithelial cells and human chondrocytes onto different sides of the membrane, which achieved the most promising results. In combination with an air-liquid interface system and fibroblast-conditioned medium, an extended epithelial multilayer with differentiated epithelial cells could be generated. Our results suggest that at least three factors are necessary for the development towards a tracheal replacement: (1) a basal lamina equivalent, consisting of collagen fibers for cell-cell interaction and cell polarization, (2) extracellular factors of mesenchymal fibroblasts, and (3) the presence of an air-liquid interface system for proliferation and differentiation of the epithelial cells

CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells

Human brain tumor stem cells have been enriched using antibodies against the surface protein CD133. An antibody recognizing CD133 also served to isolate normal neural stem cells from fetal human brain, suggesting a possible lineage relationship between normal neural and brain tumor stem cells. Whether CD133-positive brain tumor stem cells can be derived from CD133-positive neural stem or progenitor cells still requires direct experimental evidence, and an important step toward such investigations is the identification and characterization of normal CD133-presenting cells in neurogenic regions of the embryonic and adult brain. Here, we present evidence that CD133 is a marker for embryonic neural stem cells, an intermediate radial glial/ependymal cell type in the early postnatal stage, and for ependymal cells in the adult brain, but not for neurogenic astrocytes in the adult subventricular zone. Our findings suggest two principal possibilities for the origin of brain tumor stem cells: a derivation from CD133-expressing cells, which are normally not present in the adult brain (embryonic neural stem cells and an early postnatal intermediate radial glial/ependymal cell type), or from CD133-positive ependymal cells in the adult brain, which are, however, generally regarded as postmitotic. Alternatively, brain tumor stem cells could be derived from proliferative but CD133-negative neurogenic astrocytes in the adult brain. In the latter case, brain tumor development would involve the production of CD133

Genomic basis for drought resistance in European beech forests threatened by climate change

In the course of global climate change, Central Europe is experiencing more frequent and prolonged periods of drought. The drought years 2018 and 2019 affected European beeches (Fagus sylvatica L.) differently: even in the same stand, drought-damaged trees neighboured healthy trees, suggesting that the genotype rather than the environment was responsible for this conspicuous pattern. We used this natural experiment to study the genomic basis of drought resistance with Pool-GWAS. Contrasting the extreme phenotypes identified 106 significantly associated single-nucleotide polymorphisms (SNPs) throughout the genome. Most annotated genes with associated SNPs (>70%) were previously implicated in the drought reaction of plants. Non-synonymous substitutions led either to a functional amino acid exchange or premature termination. An SNP assay with 70 loci allowed predicting drought phenotype in 98.6% of a validation sample of 92 trees. Drought resistance in European beech is a moderately polygenic trait that should respond well to natural selection, selective management, and breeding

Genomic basis for drought resistance in European beech forests threatened by climate change

In the course of global climate change, Central Europe is experiencing more frequent and prolonged periods of drought. The drought years 2018 and 2019 affected European beeches (Fagus sylvatica L.) differently: even in the same stand, drought-damaged trees neighboured healthy trees, suggesting that the genotype rather than the environment was responsible for this conspicuous pattern. We used this natural experiment to study the genomic basis of drought resistance with Pool-GWAS. Contrasting the extreme phenotypes identified 106 significantly associated single-nucleotide polymorphisms (SNPs) throughout the genome. Most annotated genes with associated SNPs (>70%) were previously implicated in the drought reaction of plants. Non-synonymous substitutions led either to a functional amino acid exchange or premature termination. An SNP assay with 70 loci allowed predicting drought phenotype in 98.6% of a validation sample of 92 trees. Drought resistance in European beech is a moderately polygenic trait that should respond well to natural selection, selective management, and breeding