Transcript Document

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 cells
for research and cell therapy
Svendsen and Smith, TINS 99
Embryonic Stem Cells
? 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
BRC-44
Number of Spheres
10 6
Chop sphere
EGF
FGF-2
Heparin
B27
4 weeks
10 5
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
+ 320d
Automated Chopping
Sectioning Method
270d 320d 370d 405d 455d
- - + - + - + - + - +
12
+ LIF
- LIF
9
6
3
100
10kbp
200
300
400
500
Carpenter, Exp Neurol
Vescovi, Exp Neurol
Fricker, J. Neurosci
5kbp
Telomere
length (kpb)
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
750
500
250
0
Generation of neurospheres
D
Mes
Cer
Differentiation of neurospheres
E
Ctx
Str
Cer Thal Mes
Ctx
Str
Cer Thal Mes
600
500
400
300
200
100
0
Total TuJ1 +ve
neurons per field
Ctx
Total cells per field
C
300
44%
40%
200
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
7 days
Pre-differentiation
cells
7 days
as single
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
Control
Lenti-GFP
Lenti-GDNF
Control
Lenti-GFP
*
100
50
0
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. 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.
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
Down’s
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
The Stem Cell Research Program – in Neuroscience
Basic 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 Déglon
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