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Characterization of CD133-Positive Cells in Stem Cell Regions of the Developing and Adult Murine Central Nervous System

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Characterization of CD133-Positive Cells in Stem Cell Regions of the Developing and Adult Murine Central Nervous System Characterization of CD133-positive cells in stem cell regions of the developing and adult murine central nervous system vorgelegt von Diplom-Ingenieurin Cosima Viola Pfenninger aus Erlangen Von der Fakultät III – Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktorin der Naturwissenschaften – Dr. rer. nat. – genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Peter Neubauer Berichter: Prof. Dr. Roland Lauster Berichter: Prof. Dr. Ulrike Nuber Tag der wissenschaftlichen Aussprache: 11.12. 2009 Berlin 2010 D 83 Table of Content................................................................................................................ I Abbreviations..................................................................................................................... IV 1. Introduction................................................................................................................... 1 1.1 Definition of stem and progenitor cells..................................................................... 1 1.2 In vitro neural stem/progenitor cell assay.................................................................. 1 1.3 Stem and progenitor cells in the murine central nervous system.............................. 2 1.3.1 Neural stem and progenitor cells in the developing forebrain.......................... 2 1.3.2 Origin of neurogenic astrocytes and ependymal cells...................................... 4 1.3.3 Neurogenesis in the adult forebrain.................................................................. 5 1.3.3.1 Composition of the neurogenic region in the adult lateral ventricle wall.......................... 5 1.3.3.2 Identity of neural stem cells in the adult lateral ventricle wall........................................ 7 1.3.4 Neural stem and progenitor cells in the adult spinal cord................................. 8 1.4 Ependymal cells of the adult LVW and spinal cord.................................................. 9 1.5 The transmembrane protein CD133.......................................................................... 11 1.5.1 Function of CD133............................................................................................ 12 1.5.2 CD133 distribution in the central nervous system............................................ 13 1.5.3 CD133 as a tumor stem cell marker in the CNS?............................................. 13 2. Aims of the Thesis.......................................................................................................... 15 3. Materials and Methods................................................................................................. 17 3.1 Animals...................................................................................................................... 17 3.2 Cell isolation and cultivation..................................................................................... 17 3.2.1 Cell culture media............................................................................................. 17 3.2.2 Isolation of embryonic and postnatal tissue...................................................... 19 3.2.3 Isolation of adult LVW tissue........................................................................... 20 3.2.4 Isolation of adult spinal cord tissue................................................................... 20 3.2.5 Cultivation of FACS- and MACS-isolated cells............................................... 21 3.2.6 Cultivation of adult LVW and spinal cord cells under different culture conditions.......................................................................................................... 22 3.2.7 Co-culture of CD133-positive and CD133-negative adult LVW cells............. 22 3.2.8 Cultivation of adult spinal cord ependymal cells with retinoic acid................. 23 3.3 Statistical analysis and illustration of data................................................................. 23 3.4 Fluorescence and magnetic activated cell sorting..................................................... 23 I 3.4.1 Fluorescence activated cell sorting................................................................... 23 3.4.2 Magnetic activated cell sorting......................................................................... 24 3.5 Immunostaining......................................................................................................... 25 3.6 RNA isolation and amplification............................................................................... 26 3.7 Gene expression microarray and data analysis.......................................................... 27 3.8 Multiplex reverse transcriptase PCR (rtPCR)............................................................ 27 3.9 Web resources............................................................................................................ 29 4. Results............................................................................................................................. 31 4.1 Identification and functional characterization of CD133-positive cells in stem cell regions of the murine central nervous system............................................................ 31 4.1.1 Localization of CD133 in stem cell regions of the developing forebrain and in the adult central nervous system................................................................... 31 4.1.1.1 Localization of CD133 in the LVW region during development...................................... 31 4.1.1.2 Localization of CD133 in the adult LVW and spinal cord.............................................. 32 4.1.2 Establishment of flow cytometry-based cell isolation from the adult LVW with CD133 antibodies..................................................................................... 35 4.1.3 Functional characterization of CD133-positive cells in stem cell regions of the developing and adult brain.......................................................................... 36 4.1.3.1 Functional properties of CD133-positive cells from the developing brain in vitro.............. 36 4.1.3.2 Functional properties of CD133-positive cells from the adult LVW in vitro...................... 37 4.1.3.3 Influence of extracellular signals on the NSP frequency of CD133-positive adult LVW cells.................................................................................................... 39 4.1.3.4 Influence of culture conditions on the NSP-forming potential of CD133-positive adult LVW cells.................................................................................................... 39 4.2 Comparison of CD133-positive ependymal cells from the adult murine LVW and spinal cord........................................................................................................... 41 4.2.1 Functional properties of adult LVW and spinal cord ependymal cells............. 41 4.2.1.1 Preparation of adult spinal cord cells for flow cytometry and optimization of culture conditions................................................................................................. 41 4.2.1.2 Ependymal cell isolation by a combination of surface markers....................................... 42 4.2.1.3 Functional properties of CD133+/CD24+/CD45-/CD34- LVW and spinal cord ependymal cells in vitro.......................................................................................... 42 4.2.1.4 Functional properties of CD133+/CD24- adult LVW cells in vitro................................... 46 4.2.2 Gene expression profile of adult LVW and spinal cord ependymal cells........ 47 4.2.2.1 Gene expression pattern specific for spinal cord ependymal cells................................... 51 II 4.2.2.2 Gene expression pattern specific for LVW ependymal cells............................................ 54 4.2.2.3 Comparison of gene expression data from LVW and spinal cord ependymal cells, RGC and spinal cord-derived NSPs.......................................................................... 55 5. Discussion....................................................................................................................... 58 5.1 Identification and functional characterization of CD133-positive cells in stem cell regions of the murine central nervous system............................................................ 58 5.1.1 CD133-positive cells in the developing forebrain and adult CNS.................... 58 5.1.2 Stem/progenitor cell properties of CD133-positive cells.................................. 60 5.1.3 Lineage relationship between CD133-positive tumor stem cells and CD133- positive CNS cells?........................................................................................... 62 5.2 Comparison of CD133-positive ependymal cells from the adult murine LVW and spinal cord........................................................................................................... 66 5.2.1 Stem/progenitor cell properties of ependymal cells in the adult LVW and spinal cord......................................................................................................... 67 5.2.2 Transcriptional profiling of adult LVW and spinal cord ependymal cells....... 69 5.2.3 Genes associated with stem cell properties of adult spinal cord ependymal cells................................................................................................................... 70 5.2.4 Genes associated with tumorigenesis in adult spinal cord ependymal
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