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

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

Days EGF alone EGF FGF-2 Heparin B27 4 weeks Chop sphere Number of Spheres Clon-5382 BRC-44 Days of growth Normal Passaging Sectioning Method Svendsen et al. A new method for the long term growth of human neural precursor cells. J Neurosci Methods (1998) 85: No serum No mouse feeder layers Carpenter, Exp Neurol Vescovi, Exp Neurol Fricker, J. Neurosci

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

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

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

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

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

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 Diseases: 1. Huntington’s 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. 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

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

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

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

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