Brain Power: Build It


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Francis G. Szele, Fellow and Tutor in Developmental Neurobiology, St. Anne’s College, Oxford.

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  • Mouse 3.5 day embryo. Shown in yellow is the inner cell mass of the blastocyst - stem cells.
  • Three major classes of cells in the nervous system
  • The Neural plate folds up to from the neural tube Three basic layers during development; ectoderm, mesoderm, and endoderm. The nervous sytem is derived from the ectoderm. Neural tube gives rise to CNS. Neural crest gives rise to PNS: Neurons and glia of dorsal root and cranial ganglia, autonomic ganglia both sympathetic and parasympathetic, Schwann cells of the peripheral nervous system, adrenal medulla, melanocytes and other structures in the periphery.
  • Much of what is known about cell migration in the brain has been through study of the cerebral cortex. The adult cerebral cortex is a 6 layered structure which forms through an unusual inside-out mode of migration. The bottom layers are formed first, and then the upper layers by cells moving through the bottom layers. Note that the E 8.5 ventricular zone is comprised of cells going through interkinetic nuclear migration (blue) and mitosis (green). Cells then migrate along radial glia to form the postmitotic layers. The first postmitotic layer is the preplate (PP). Then an intermediate zone (IZ) appears which is populated by incoming axons from more caudal structures. These axons may be a substrate for tangential migration. The Cajal-Retzius cells secrete reelin which inhibits neuronal migration of dorsally migrating cells and are responsible for cortical layerig. Later in development, a second layer of mitotically active cells, the subventricular zone (SVZ), appears (it should be called supra-ventricular zone because it is above the VZ). It is also termed the. Its cells are distinguishable from VZ because 1) they do not exhibit interkinetic movement of the nucleus and, 2) they go through the cell cycle at a slower rate.
  • Brain Power: Build It

    1. 1. Brain Power: Build It Francis Szele, PhD St. Annes College Physiology, Anatomy and Genetics University of Oxford [email_address] Nam et al., 2007 Magdalena Zernicka-Goetz Group
    3. 3. Stem cells give rise to the brain! ?
    4. 4. Three major classes of nerve cells Astrocytes Oligodendrocytes Neurons Glia
    5. 5. How Do We Know These Things?
    6. 6. Neurulation : The neural plate folds up to from the neural tube
    7. 7. Early ventricular zone contains many stem cells <ul><li>At this early stage neural tube is one cell layer thick. </li></ul><ul><li>Processes touch ventricular surface and pial surface </li></ul>
    8. 8. The development of stem cells in the CNS <ul><li>Stem cells divide symmetrically first to give rise to large numbers of cells. </li></ul><ul><li>The number of cell cycles influences growth of the brain. </li></ul><ul><li>Switch to an assymetric mode of division which generates different progeny. </li></ul>
    9. 9. Development of the Cerebral Cortex
    11. 11. Definition of Stem Cells <ul><li>A cell that is able to self-renew indefinitely </li></ul><ul><li>A cell that is able to proliferate </li></ul><ul><li>A cell that is multipotent (able to give rise to a variety of cells) </li></ul><ul><li>Forces analysis of behavior and retrospective assigment to “stem cell” category </li></ul>
    12. 12. Stem cells - defying gravity?
    13. 13. iPSC - induced pluripotential stem cells <ul><li>Oct3/4, Sox2, klf4, c-Myc sufficient to render adult fibroblasts (and other cells) “embryonic stem-like” </li></ul><ul><li>Obviates need and ethics of human embryos </li></ul><ul><li>Unlimited supply </li></ul><ul><li>Replicated in multiple labs and species. </li></ul><ul><li>Generate a wide variety of cells in vitro. </li></ul><ul><li>Generate all three germ layers in vivo. </li></ul><ul><li>Succesfully raised iPSC from ALS adults! </li></ul>
    14. 14. Problems with iPSC’s <ul><li>Genes introduced with retroviral vectors - potential for insertional mutagenesis. </li></ul><ul><li>c-Myc is an oncogene - 50% of chimeric mice generated with iPSC’s form tumours. </li></ul>
    15. 15. “ I NEED MORE NERVE CELLS, please”
    16. 16. The subventricular zone contains stem cell and migratory cells Granule neuron in olfactory bulb Nestin-eGFP Astrocytic stem cell Rapidly dividing progenitor Migrating neuroblasts Microglia Ependymal cells
    17. 17. Testing stems with neurospheres subdivide neurospheres: stem cell self-renewal assays of secondary floating neurospheres, stem cell fate potential assays of plated neurospheres, assays of migration from plated neurospheres, assays of cell surface molecule expression in plated neurospheres. day 8 in vitro Lesion Microdissect SVZ, culture floating neurospheres 5 days day 7 in vitro Determine number and size of floating neurospheres +EGF/FGF2 Floating neurospheres Plated neurospheres astrocytes neurons oligodendrocytes
    18. 18. 2 (multi)-photon time-lapse microscopy <ul><li>Features </li></ul><ul><ul><li>Excitation in the far red spectrum </li></ul></ul><ul><ul><li>Coincident excitation of fluorophore by 2-photons </li></ul></ul><ul><li>Advantages </li></ul><ul><ul><li>Less damaging - longer sampling </li></ul></ul><ul><ul><li>Deeper visualization </li></ul></ul><ul><li>Experimental Approach </li></ul><ul><ul><li>300  m sagittal slices </li></ul></ul><ul><ul><li>50-75 optical sections, every 3-7 min </li></ul></ul><ul><ul><li>4 hrs - 18 hrs </li></ul></ul><ul><ul><li>aCSF, 35 o C, 1 ml/min </li></ul></ul>Integrated Microscopy Resource, U. Wisconsin-Madison 2-photon confocal
    19. 19. Fluorescent mice to view stem cells and migration
    20. 20. 4-dimensional quantification
    21. 21. Do Stem Cells Move? Is Dcx necessary for SVZ motility? Nestin-eGFP
    22. 22. Do Human Brains Have Stem Cells? Pinel, Biopsychology, 4th Ed. <ul><li>The subventricular zone contains stem cells: </li></ul><ul><li>Self-renew </li></ul><ul><li>Multipotential </li></ul>Effects of Injury Increased Proliferation Increased SVZ neurogenesis Some migration towards injury Very little survival and integration
    23. 23. Stem Cells to Treat Multiple Sclerosis
    24. 24. Conclusions <ul><li>Neural stem cells give rise to brain during embryogenesis. </li></ul><ul><li>Neural stem cells self-renew and are multipotent. </li></ul><ul><li>Adult mammalian brain harbors stem cells. </li></ul><ul><li>Adult neural stem cells may become therapeutically relevant. </li></ul>