AMERICAN PHYSIOLOGICAL SOCIETY EDUCATION COMMITTEE Refresher Course 2002:

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 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 Stem cells, growth factors and neurodegeneration Growth, differentiation and transplantation Gene transfer Neurospheres as mini-pumps Modeling degenerative disease Sources of human neural stem cel for research and cell therapy Embryonic Stem Cells

Svendsen and Smith, TINS 99 ?Isolation of neural Neural Stem Cells stem cells Skin Stem Cells, Marrow Stromal Cells ? FUSION? ? Adult Neural Stem Cells Expansion Gene Modification Neuron Oligodendrocyte Astrocyte Differentiation

10 -14 Days 10 8 EGF alone Clon-5382 10 7 Number of Spheres Chop sphere EGF FGF-2 Heparin B27 4 weeks 10 5 Sectioning Method 10 4 10 3 10 2 Normal Passaging 10 1

10 No serum No mouse feeder layers 0 56 28 84 112 140 Days of growth LIF allows neural precursor cell with stable telomere length to continually renew + - + 27 0 d

- + DIV LIF LIF 40d 140d 190d - +- + 220d - 22 0 d B C LIF 220d Telomere Length (kbp)

- + 19 0 d + 455d 14 0 d + 405d DIV LIF LIF 220d LIF 40d + 370d A + 32 0d Automated Chopping BRC-44

10 6 270d 320d 370d 405d 455d - - +- + - +- +- + Telomere length (kpb) 5kbp + LIF - LIF 9 6 3 100 10kbp Carpenter, Exp Neurol Vescovi, Exp Neurol Fricker, J. Neurosci 12 200

300 400 500 Days in vitro 12 9 6 3 25 50 75 PD's 100 Svendsen et al. A new method for the long term growth of human neural precursor cells. J Neurosci Methods (1998) 85:141-52 Human neurospheres are regionally specified A B

3 [H]thymidine (CP M) 1000 Generation of neurospheres D Mes Cer Differentiation of neurospheres E Total TuJ1+ve neurons per field Ctx Total cells per field C 750

500 250 0 Ctx Str Cer Thal Mes Ctx Str Cer Thal Mes 600 500 400 300 200 100 0 300 44% 200

40% 100 9% 0 (no TH neurons) Ctx Str 7% 4% Cer Thal Mes Ostenfeld et al, 2002 Pre-differentiation can generate committed neurons in vitro Plate onto PLL & Laminin 7Days Fix & stain Triturate and

plate as 7 days Pre-differentiation 7 days single cells TuJ1+ cells (% total cells) 75 * *** 60 45 30 * * * Control CNTF NT3 BDNF

PDGF NT4 15 0 Caldwell et al, 2001 Stem cells, growth factors and neurodegeneration Growth, differentiation and transplantation Gene transfer Neurospheres as mini-pumps Modeling degenerative disease 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 Stem cells, growth factors and neurodegeneration Growth, differentiation and transplantation Gene transfer

Neurospheres as mini-pumps Modeling degenerative disease 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 GDNF for ALS and PD Possible solutions Direct infusion into brain tissue with pump Bristol PD Infusion Trial, UK. Delivery by injecting GDNF producing virus Kordower et al (2000) Science 290: 767 Delivery using encapsulated cells secreting GDNF Emerich et al (1996) Brain Res 736: 99 Delivery using neural stem cells secreting GDNF Akerud et al (2001) J. Neurosci 21: 8108

+ GFP NS + GDNF NS GDNF secreting stem cells increase TH neuronal survival in grafts graft Total TH +ve neurons in graft Total TH neurons per (every 6th section) 150 0 Control Lenti-GFP Lenti-GDNF Control Lenti-GFP *

100 50 Lenti-GDNF 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. Stem cells, growth factors and neurodegeneration Growth, differentiation and transplantation Gene transfer Neurospheres as mini-pumps Modeling degenerative disease Approaches to disease modeling using human neural stem cells Mice are not men! I. Genetic modification of cells to express abnormal gene

ALS SOD Mutation; HD- CAG repeats; PD Syn mutation etc.. II. Deriving neural stem cells from patients Diseases: 1. Huntingtons 3. Fragile X 4. Down Syndrome 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. Using neurospheres to model Down Syndrome Fetal Cortex 653bp 517bp Control N=3

DS N=3 453bp 394bp 10weeks EGF 298bp 234bp RNA 154bp + + + - + + + - + + + - Sabina Bahn Downs Control 1 Control 2 Genetic profile of Downs neurospheres

0.75 0.50 DSCR1 DSCAM GAP43 APP ALPHA TUBULIN REST-4 REST-1 BETA TUBULIN 4 SYNAPSIN I 0.00 L1 0.25 SCG10

% mRNA Expression / GAPDH 1.00 CONT DOWNS NRSF (neuron-restrictive silencing factor) also known as REST (repressor element 1 silencing factor) SCG10 (neuron specific growth associated protein); L1 Cell adhesion molecule e Stem Cell Research Program in Neuroscience ic Science - Cell Production - Gene manipulation Clinical Trials Collaborators Cambridge Rowan Burnstein Maeve Caldwell Sidharthan Chandran Francesca Ciccolini Xiaoling He Janel Le Belle Thor Ostenfeld Anna Peters Yu Tzu Tai Sarah Stevens

Bristol James Uney Tom Harding Madison Anita Bhattacharyya Soshana Behrstock Sandra Klein Lynda Wright Rebekah Jakel Telma Schwindt Borris Haupt MSD Harlow Scott Pollack Neil Wilkie Sarah Harper Lausanne Nicole Dglon Patrik Aebischer Manchester Pedro Lowenstein Maria Graciela Castro Babraham/Hinxton Etienne Joly Fiona McLaren Piers Emson

Sabina Bahn Margaret Ryan Groningen Karen Prowse Maarten Linskens London (UCL) |Eric Jauniaux

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