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AMERICAN PHYSIOLOGICAL SOCIETY EDUCATION COMMITTEE Refresher Course 2002:

AMERICAN PHYSIOLOGICAL SOCIETY EDUCATION COMMITTEE Refresher Course 2002: Recent Advances in Neuroscience. Stem Cells, Growth Factors and Neurodegeneration Dr. Clive Svendsen Waisman Mental Retardation & Human Development Center Madison, Wisconsin.

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AMERICAN PHYSIOLOGICAL SOCIETY EDUCATION COMMITTEE Refresher Course 2002:

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  1. AMERICAN PHYSIOLOGICAL SOCIETY EDUCATION COMMITTEE Refresher Course 2002: Recent Advances in Neuroscience Stem Cells, Growth Factors and Neurodegeneration Dr. Clive Svendsen Waisman Mental Retardation & Human Development Center Madison, Wisconsin

  2. We are born with a certain number of brain cells which decrease with age. Everything must die in the brain or spinal cord - nothing can regenerate. Cajal 1902

  3. Stem cells, growth factors and neurodegeneration • Growth, differentiation and transplantation • Gene transfer • Neurospheres as mini-pumps • Modeling degenerative disease

  4. Sources of human neural stem cells for research and cell therapy Svendsen and Smith, TINS 99 Embryonic Stem Cells ? Isolation of neural stem cells Neural Stem Cells Skin Stem Cells, Marrow Stromal Cells ? ? Adult Neural Stem Cells Expansion Gene Modification Neuron Differentiation Oligodendrocyte Astrocyte FUSION?

  5. 10 -14 Days EGF alone Chop sphere EGF FGF-2 Heparin B27 4 weeks 8 10 Clon -5382 7 10 BRC-44 6 10 5 Sectioning Method 10 4 10 Number of Spheres 3 10 2 10 Normal Passaging 1 10 10 0 56 140 28 84 112 Days of growth No serum No mouse feeder layers Carpenter, Exp Neurol Vescovi, Exp Neurol Fricker, J. Neurosci • Svendsen et al. A new method for the long term growth of human neural precursor cells.J Neurosci Methods (1998) 85:141-52

  6. Human neurospheres are regionally specified (no TH neurons) Ostenfeld et al, 2002

  7. Plate onto PLL & Laminin Fix & stain 7 days Triturate and plate as single cells 7 days Pre-differentiation Control BDNF CNTF PDGF NT3 NT4 7Days Pre-differentiation can generate committed neurons in vitro Caldwell et al, 2001

  8. Stem cells, growth factors and neurodegeneration • Growth, differentiation and transplantation • Gene transfer • Neurospheres as mini-pumps • Modeling degenerative disease

  9. Future strategies for using human neural stem cells for neuronal replacement • We can now generate normal human neurons • They survive transplantation • We can genetically modify them to produce dopamine • Transplant studies in PD models are underway However…… • We have no idea how to promote accurate axonal outgrowth to long distance targets

  10. Stem cells, growth factors and neurodegeneration • Growth, differentiation and transplantation • Gene transfer • Neurospheres as mini-pumps • Modeling degenerative disease

  11. Combining stem cell and gene therapy. GDNF for ALS and PD • GDNF is neurotrophic for dopamine and motor neurons However….. • Large peptide. Cannot enter brain from blood • Does not efficiently enter brain from CSF • Needs to be directly delivered to tissue

  12. GDNF for ALS and PDPossible solutions • Direct infusion into brain tissue with pump Bristol PD Infusion Trial, UK. • Delivery by injecting GDNF producing virus Kordower et al (2000) Science290: 767 • Delivery using encapsulated cells secreting GDNF Emerich et al (1996) Brain Res736: 99 • Delivery using neural stem cells secreting GDNF Akerud et al (2001) J. Neurosci21: 8108

  13. GDNF secreting stem cells increase TH neuronal survival in grafts + GDNF NS + GFP NS

  14. Total TH +ve neurons in graft (every 6th section)

  15. Future directions for GDNF secreting stem cells… • Inducible GDNF vectors and 6-OHDA lesions • Primate MPTP model of PD prior to clinical trial • New rat model of ALS Other potential uses of human neural stem cells….

  16. Stem cells, growth factors and neurodegeneration • Growth, differentiation and transplantation • Gene transfer • Neurospheres as mini-pumps • Modeling degenerative disease

  17. Approaches to disease modeling using human neural stem cells Mice are not men! • Genetic modification of cells to express abnormal geneALS – SOD Mutation; HD- CAG repeats; PD – Syn mutation etc.. • Deriving neural stem cells from patients Generate neural stem cells from human tissues. These carry the genetic deficit. They can be studied in vitro and following grafting Down stream targets of gene deficit can be identified with chip/diff display methods. Diseases: 1. Huntington’s 3. Fragile X 4. Down Syndrome

  18. Fetal Cortex Control N=3 DS N=3 10weeks EGF RNA Using neurospheres to model Down Syndrome Sabina Bahn

  19. Genetic profile of Downs neurospheres NRSF (neuron-restrictive silencing factor) also known as REST (repressor element 1 silencing factor) SCG10 – (neuron specific growth associated protein); L1 – Cell adhesion molecule

  20. The Stem Cell Research Program – in Neuroscience Basic Science - Cell Production - Gene manipulation – Clinical Trials

  21. Madison Anita Bhattacharyya Soshana Behrstock Sandra Klein Lynda Wright Rebekah Jakel Telma Schwindt Borris Haupt

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