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Neural stem cells: Biological characterization and potential applications 2017 Emília Madarász Inst. of Experimental Medicine of Hungarian Academy of Sciences Terminally differentiated cells Axon Symmetric mitosis Asymmetric mitosis Sejttest + + Dendrit Agykérgi piramis-sejt Funder cells: new phenotype Amplifying cells Inner cell mass trophoblasts Fertilized oocyte Day 3 Early blastocyte (day 5) Embryonic stem cells, tissue stem cells, induced pluripotent stem cells In vivo Inner cell mass (ICM) oviduct Embedding trophoectoderm blastocyte trophoblasts In vitro Early blastocyte Embryonic stem cells (ESCs) ES cell lines Tissue stem cells ICM Uterus wall Day 32; 6 mm Day 13 0.4 mm Day 18 1.25 mm Induced pluripotent stem cells (IPSCs): introduction of „stemness genes” to non-differentiated tissue cells Heterogenous tissue stem cell populations from the beginning of embryo development ESTABLISHMENT OF BODY AXES 14 day human embryo ICM epiblast hypoblast Neural plate nodus Primitive streak Body axis Primitive streak Body axis Neural tube Roof plate Alar plate Basal plate ~16 day after fertilization Differential gene expression determines: Dopaminerg idegsejtek HC: hippocampus ST: striatum SN: substantia nigra (A8, A9) VTA: ventral tegmental area (A10) B.olf.: Bulbus olfactorius (A16) • the boundaries of brain regions • the neurotransmitter-phenotype of neurons • the position of fibre tracks Noradrenerg idegsejtek LC: locus coeruleus T: thalamus Hy: hyppocampus LTA: lateral tegmental area Szerotonerg idegsejtek SCN: suprachiasmatic nucleus T: thalamus HC: hyppocampus LTA: lateral tegmental area R: raphe magok Kolinerg idegsejtek Septum medialia: Ch1 nucl.fasc. diagonalis: Ch2, Ch3 nucl. basalis Meynerti : Ch4 nucl. pedunculopontis: Ch5 nucl. tegment. Laterodorsalis:Ch6 medialis habenula : Ch7 Nucl. parabigeminal : Ch8. Ventral layers of the spinal cord: Motoneurons, g-efferents, vegetative „motoneurons” Symmetric mitosis Asymmetric mitosis + + Pia - ECM Radial neuroepithel cell Lateral induction / inhibition ventricle Radial glia ECM Symmetric asymmetric mitoses G.C. Schoenwolf , 2001 Neuronal precursor Primary germinative zone Notch Hes ErbB2 BLBP Delta-1 Mash/Ngn1,2 NRG-1 1. Notch/Delta system 1. 3. 2. Differentiating cells Non-differentiating cell …differences between neighbouring cells caused by stochastic events and intrinsic or extrinsic factors are stabilized or amplified through Notch and Delta signals… Notch/Delta system Lateral inhibition (Drosophila proneural cluster) Embryonic cell migration from the primary germinative zone 17 day human Migrating neuronal precursors radial glia Rakic P. J. Comp. Neurol. 1972, 145: 61-84 Halfter, W. et al. J. Neurosci. 2002; Neuronal precursors derived from the primary germinative zone migrate along the radial glia cells The secondary germinative zone derives from the primary zone: Astroglia, Oligodendroglia Local interneuron Small projecting neuron Projecting neuron Radial glia SVZ VZ ependyma Primary germinative layer prenatal Secondary germinative layer postnatal E10.5 E1 0, 5 E1 0,5 Alvarez-Buylla 1998 E10,5 E10, 5 Adult Shuurmans 2004 The forebrain cortex composed by neurons derived from both, the primary and secondary germinative zones Adult-hood neurogenic zones ependyma agyszövet kamra Forebrain subventricular zone (SVZ) nmfhé Hippocampus subgranular zone (SGZ) Embryonic neuroectodermal stem cells Neural groove Neural plate Neural crest = Neuroepithelial stem cells 17 day human Embryonic/fetal neural tissue stem cells Neural tube Primary neural stem cells = radial glia cells in the ventricular zone Secondary neural stem cells = neural stem cells in the subventricular zone E 14.5 mouse forebran Adult-hood neural stem cells subventricular zone subgranular zone In case of injury, the subependymal zone can produce cells along the entire neuroaxis Large number of different neural stem cell populations exist synchroneously Characterization of the different neural stem cells is poor Terminally differentiated neurons X ? Radial glia-like neural stem cells Embryonic neuroectodermal stem cells Amplifying progenitor → Stem cell 4 ? Lateral Induction/inhibition Amplifying progenitor → Stem cell 3 ? ? Stem cell 1’ Amplifying progenitor → Stem cell 2 Radial glia 1 self-renewing + 1 more differentiated progeny Symmetric mitosis Asymmetric mitosis + + In response to tissue damages, large-scale cell production occurs in the CNS Jorfi et al., 2015. J.Neur.Engin Resident progenitors are scattered in the fibre tracks Zadori et al., 2011 Their fate???? mainly scar formation Baumann 2007. Phys.Rev. Neuron characteristics Arborizing processes extremely large cell surface in comparison to volume • special intracellular transport machinery • local substance production and protein synthesis • large number of adhesion points (pre- and postsynapses, astroglia-neuron connections, oligodendroglia tight junctions) Rapid information forwarding to distant cell parts • axon – dendrit polarity • voltage-dependent ion channels and special ion pumps: characteristic types and arrangements Specialised secretory cell • more than one secreted material (neurotransmitter, neuromodulator, neuropeptide) • different release mechanisms (normal exocytosis, synaptic release) Specialised chamical receptor cell • characteristic receptor areas in the cell membrane (postsynapses) The neuronal surface is a mozaic of specialised membrane areas The terminally differentiated neuron is incapable • • to divide to migrate Cell motility composed by elongation and generation of processes The process-endings and synapses possess self-standing regulatory mechanisms: plasticity of process elongation and synaptic reorganization Stereotype anatomical structure Bascet cell; rat hippocampus CA1 region axon s.r. s.p. dendrite 100 mm s.o. Zemankovics Rita rekonstrukciója; MTA KOKI 1013 neurons x 104 synapses = 1017 neuron-neuron connections J. Spacek; http://synapses.mcg.edu/atlas/ The axon growth cone follows extracellular instructions Phase-contrast video-microscopy Jürgen Löschinger felvétele Electronmicroscopy on growing neurites *: growth cone; elongating process Growth cones attach preferably to neighboring processes: The neurites compose fascicles The processes of cortical neurons guide the thalamic processes toward the future cortical targets Zoltán Molnár „Timed” receptor-ligandum interactions forbid defined process-elongation routes Slit ↔ Robo P2 P17 Stanfield; O’Leary; 1985-95 Retrográd labelling from the medulla : P2: the entire layer V P17: the sensomotory cortex only Double labelling on P2 and on P17 : Not the cells, only the non-appropriate processes do disappear during development: Developmental elimination of excess (non-used) processes Kémiai szinapszis 1949! target Growth cone BDNF Protein synthesis exocytosis ECM GABA GABAA GABAB Propagated waves [Ca2+]I release [Ca2+]I phosphorylation Protein production release Jelitai, Madarasz; 2006 Synapses can be formed and maintained between synchronuosly active partners „Fire together, wire together” Corpus geniculatum laterale: Layering disappears, if the n. optici are synchroniously stimulated Giant depolaririzing potentials (GDP) Ben-Ari, 2007 - Generated in divers spots of the developig brain Spread along fasciculated processes Help to generate synapses of syncronuosly active axons Play fundamental role in the formation of large projecting fibre tracks ES cells, embryonic tissue stem cells and IPSCs are not to be used for direct cell therapy! High probability of non-controlled proliferation → tumour formation; Unknown tissue instructions → unknown routes of cell development RA 9 Schlett, 1997. J. Neurosci.Res. synaptophysin E9 D 21 * kamra SVZ NeuN-bIII tubulin 40 mm Demeter et al., 2004. Exp. Neurol. Brain injury → neural stem cell proliferation Transient and „abortive”: Does not result in neural tissue regeneration Zheng et al., J Neurotrauma. 2013 neuron fromation starts, but..... Neurogenesis and neuronal regeneration in status epilepticus Rotheneichner et al., Epilepsia, 2013 The conditions for neural tissue formation are missing in the adult/diseased brain IPSCs Reprogramming Indukált pluripotens őssejtek (IPSC) Kfl, Oct4, Nanog, Sox2, Myc Epigenetic effects: growth factors (?) ES-like cells, with ES-like troubles IPSC Terminally non-differentiated! IPSCs derived from different tissue progenitors posses equal developmental potential? Advanteges: Derivation from the own body; Personal cytogenetics → improved drug-testing; Far in future, potential own tissue implantation, cell therapy The formation and maintenance of the functional nervous tissue resulted by continouos selection mechanisms Cell genesis: surviving cells are selected from an excess of generated cells Cell migration: permissive, attractive and repulsive signals select further the survivng progenitors and ingrowing axons Signals of the functioning tissue (secreted molecules and cell to cell connections) keep alive the cells The environment selects ! Only defined regions (Hyppocampus g. dentatus granule cell layer, bulbus olfactorius, SVZ) of the adult CNS provides conditions for survival and development of stem cells and novel neuronal circuits The environmental conditions for survival and proper development of neural stem cells are far from known, yet. For the time being, neural or other stem cells can not be used for (neural) cell replacement therapy Stem cells can be used for: In vitro • drug testing; • assessing individual drug reaction of own-derived iPSC generated neurons • academic studies of neuronal development and circuit formation • investigation of conditions for neurite regeneration • surface optimalization of intracerebral prostheses In vivo • dampening of local inflammatory processes • stimulating inherent regenerative processes Mesenchymal stem cells (MSCs) – in promissing clinical trials MSCs from cordblood, cord, bonemarrow Not cell replacement ! → inhibition of inflammation, stimulation of inherent tissue regeneration http://www.clinicaltrials.gov Thank you for your attention !