Copyright: Michael G. Fehlings MD PhD

16/10/2014
Stem Cell
WORKING TOGETHER
FOR HEALTHY BRAINS
Repair and Regeneration of the Injured Brain in Cerebral Palsy with Stem Cells: Fact or Fantasy?
Michael G. Fehlings MD PhD FRCSC FACS FRSC
Professor of Neurosurgery
Hallbert Chair in Neural Repair and Regeneration
University of Toronto
Medical Director, Krembil Neuroscience Center
University Health Network
Director University of Toronto Neuroscience Program
www.neurodevnet.ca
Copyright: Michael G. Fehlings MD PhD
Key Points
• What is a stem cell?
– Different types
– Properties
• How could the pathology of CP be amenable to stem
cells (or not)?
• What trials are going on in CP?
– is this appropriate for children in my practice?
– What do I need to be aware of?
• What’s “hot” in the stem cell field regarding CP?
– Endogenous stem cells
– Neural stem cells
– Induced pluripotent stem cells (iPSCs)
– Reduction of the glial scar
• Where do I get more information? (http://drfehlings.ca/new-
Stem Cells: Definitions
• Stem Cell
Cardinal Properties
– self-renewal
– multipotent
articles-on-the-potential-of-stem-cells-for-cerebral-palsy/)
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Goals of cell-based therapy
• Neuroprotection
– Neurotrophins, growth factors, cytokines
• Enhancement of neural plasticity
• Gene therapy
• Cell replacement
– Neurons
– Oligodendrocytes
• Promotion of axonal regeneration?
• Other
– eg re-vascularization
Copyright: Michael G. Fehlings MD PhD
http://neonatology.ucsf.edu
http://www.neuropathologweb.org
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Cell-Based Therapies
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Activated macrophages (Procord )
Embryonic stem cells (ESCs)
Schwann cells
Olfactory ensheathing cells
Skin-derived precursor cells
Mesenchymal stem cells (eg umbilical cord)
Neural stem cells
Induced pluripotent stem cells
Endogenous stem cells
Copyright: Michael G. Fehlings MD PhD
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Neurogenesis in the ADULT HUMAN brain…
Gage, et al…Alvarez-Buyla et al
Ages 10 months-10 yrs
35: pUCB
36 EPO
34 control
November 1998
February 2004
The adult brain harbors a mechanism for replacing neurons
Can a patient’s own cells replace damaged neurons?
Copyright: Michael G. Fehlings MD PhD
Umbilical Cord-derived Stem cells
• Appealing
– Especially when from the patient or a family member
• Important Caveats
– The evidence that they make nerve cells is not convincing
– They have not been tested in the chronic phases of injury
– They seem to work best in the acute phases of brain injury
by protecting nerve cells and by providing growth factors
to promote endogenous repair
Meier C, Middelanis J, Wasielewski B, Neuhoff S, Roth-Haerer A, Gantert M, Dinse HR, Dermietzel R,
Jensen A. Spastic paresis after perinatal brain damage in rats is reduced by human cord blood
mononuclear cells.Pediatr Res. 2006 Feb;59(2):244-9.
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The “Default” Pathway of Neural Induction
Schematic of Fibroblast to Axonal Remyelination*
Rationale: Taking advantage of the intrinsic capacity of ES cells to acquire a neural identity may
provide a simple, eloquent means to derive a therapeutically useful cell population. May allow for
the circumvention of complex genetic manipulation which impedes clinical translation.
iPS cell
* Collaboration Michael Fehlings, Andras Nagy and Derek van der Kooy
“Default” Pathway Derived
YFP+ mES Neurospheres
Nestin Positive “Definitive” Neural
Stem Cells
Copyright: Michael G. Fehlings MD PhD
Cell Based Repair Strategies For SCI
THE FUTURE:
Induced Pluripotent Stem Cells (iPS Cells)*
Synergies between
Cerebral palsy and neurotrauma
• Patient specific pluripotent cell population from
which to produce neural or oligodendrocyte
precursor cells.
• The “Magic Four” Transcription factors: Oct3/4,
Sox2, Klf4, c-Myc (also Nanog and Lin28)
• This technology can be coupled with existing
protocols for the in vitro production of NSCs or
OPCs.
• *Lowry WE et al Proc Natl Acad Sci U S A. 2008 Feb
26;105(8):2883-8.
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Copyright: Michael G. Fehlings MD PhD
Demyelination and Myelin Repair
• Demyelination is an early consequence of spinal cord Injury observed in
surviving injured axons
Normal Spinal cord
Injured Spinal cord
Remyelination as a Strategy for Repair
and Regeneration in CP
•Abnormal properties of surviving axons largely reflects the effects
of demyelination
Normal myelinated axons
Dysmyelinated axons
Kv1.2 NF200
Fehlings lab: Nashmi et al, 2000, Karimi-Abdolrezaee et al., 2004, Eftekharpour et al., 2005;
Sinha et al 2006; Eftekharpour et al 2007
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Adult neural stem cells for myelin repair after spinal cord injury
Stem Cells for Spinal Cord Repair
GFP- NSCs in the Spinal cord of a Subacutely Injured Rat Six weeks after
Transplantation
Rostral
Caudal
4 mm
*
*
GM
After transplantation into the injured spinal cord,
adult neural stem cells generate oligodendrocytes (myelin-forming cells, green),
ensheath the injured axons (blue) and restore myelin sheath (red) around the axons.
WM
From the laboratory of Dr. Michael Fehlings at Toronto Western Research Institute.
Data from Karimi S, Eftekharpour E, Wang J, Morshead C, Fehlings MG Journal of
Neuroscience 2006: 29;26(13):3377-89.
Karimi S, Eftekharpour E, Wang, J. Morshead C, Fehlings MG Journal of Neuroscience 2006
Copyright: Michael G. Fehlings MD PhD
Transplantation of adult neural precursor cells into injured
spinal cord improved neurological functional recovery
YFP-NPCs derived Oligodendrocytes Generate Myelin Basic Protein
and Ensheath the injured Axons of Spinal Cord
YFP
MBP
merged
BBB
14
*
12
YFP MBP NF200
Grid Walking
*
*
*
10
8
6
plain injured
Control
4
2
0
NPCs
Transplantation
2
3
4
5
6
7
8
Plain injured
Time (weeks after injury)
Control
15
*
NPCs
*
10
5
0
Karimi S, Eftekharpour E, Wang, J. Morshead C, Fehlings MG Journal of Neuroscience 2006
Karimi-Abdolrezaee et al., J. Neuroscience, 2006
2
3
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Time (weeks after transplantation
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Footprint Analysis
Uninjured
Plain injured
Control
NPC- transplanted
Eftekharpour et al., J. Neuroscience 2007, P0+12w
Plain injured
Control
NPCs
*
3
Plain injured
Control
NPCs
30
*
2
20
1
10
0
*
0
normal
2
3
4
5
6
normal
2
3
4
5
6
Time (weeks after transplantation)
Time (weeks after transplantation)
Karimi-Abdolrezaee et al., J. Neuroscience, 2006
Copyright: Michael G. Fehlings MD PhD
NPC-derived Oligodendrocyte Ensheath Spinal Cord Axons of Shiverer Mice
Dysmyelinated Shiverer Mice:
•Spontaneous mutation of Myelin Basic Protein (MBP)
YFP MBP NF200
•Dysmyelination/ Hypomyelination of Central Nervous System
•Constant shivering, seizures and pre-mature death (13-15 weeks)
YFP MBP
Sinha et al J Neurophysiol 2006
PNS
CNS
Eftekharpour et al Journal of Neuroscience 2007
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Transplanted aNPCs localize to the perinatal white matter, incorporating into the CC, fimbria
of the hippocampus, and periventricular parenchyma.
Ruff C A et al. J. Neurosci. 2013;33:11899-11915
©2013 by Society for Neuroscience
Copyright: Michael G. Fehlings MD PhD
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1
Olig2
Immune Privilege
Clinical Relevance to CP
P0~ preemie
P7~ newborn
P21~toddler
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K+
Na+
Nav1.6 and Caspr
aNPC
• aNPC
transplantation
significantly alters
potassium channel
distribution
**P<0.01 and ***P<0.001 in one-way ANOVA with Tukey’s post-hoc analysis.
WT
Shi
Density/100µm2
Shi
Density/100µm2
Kv1.2 and Caspr
WT
aNPC
• aNPC
transplantation does
not significantly
affect sodium
channel distribution
Copyright: Michael G. Fehlings MD PhD
Electrophysiological evidence of myelination and enhancement in axonal conductance after
aNPC transplantation into shiverer CC. A, Dual recording of CAPs from the CC in shiverer
mouse brain slices.
Na+
Caspr:
WT
Caspr
NF200
5μm
Shi Caspr
aNPCCaspr
NF200
NF200
5μm
5μm
Caspr length (μm)
CASPR
K+
*
*
•aNPC transplantation does not alter node length
•Reduced node length can account for 10% of the decrease in
axonal conduction found in shi/shi mice
Ruff C A et al. J. Neurosci. 2013;33:11899-11915
©2013 by Society for Neuroscience
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Myelin reveals normalized axonal profile: activation threshold, refractoriness, and HFS. A,
Myelination lowers the threshold for axon activation.
The challenge of the chronic
injury
http://www.neuropathologweb.org
Ruff C A et al. J. Neurosci. 2013;33:11899-11915
• Gliosis
• Loss of neural tissue/cavitation
©2013 by Society for Neuroscience
Copyright: Michael G. Fehlings MD PhD
Elevated level of CSPG- NG2 in chronically injured SC
NG2
NG2 DAPI
NG2
GFAP
Merged
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CSPG/GFAP positive astrocytic processes surround
non-viable YFP-NPCs following transplantation in chronic SCI
Surviving adult NPCs at 8 weeks post transplantation
in chronically injured spinal cord pre-treated with ChABC and supplemented with GFs
NPC DAPI
lateral
Medial
200 μm
NPC CGRP DAPI
NPC BTIII DAPI
dorsal
20 μm
ventral
200 μm
Copyright: Michael G. Fehlings MD PhD
NPC-derived oligodendrocytes ensheath and remyelinate
the chronically injured axons
(8 weeks post transplantation)
Treatment with ChABC leads to degradation of CSPGs in
chronically injured spinal cord
A
NPC B
NF200
C
MBP
D
NPC NF200 MBP
Injured- Vehicle treated 6 weeks post-injury
20 µm
Injured- ChABC treated 6 weeks post-injury
Journal of Neuroscience Feb 2010
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QL6 Self-Assembling Peptides
QL6 promotes axonal connectivity – BDA corticospinal tract
•The synthetic self-assembling peptides can self-assemble into nanofiber in situ
under physiological conditions
• Injectable; non-immunogenic; provide a bridging scaphold
Injured control
QL6 injected
K2(QL)6K2 (QL6)
Primary structures of the (QL)6 series
of peptides (From Dong et al., J. AM.
CHEM. SOC. 2007, 129, 12468-12472)
Copyright: Michael G. Fehlings MD PhD
QL6 leads to reduced astrogliosis in chronic SCI
Are stem cells right for my child?
Fold increase in GFAP immunointensity
(normalized to uninjured value)
GFAP
Injured control
3.5
QL6 injected
Control
3
QL6
*
2.5
2
**
1.5
1
0.5
0
0mm
1mm
1 wk
2mm
0mm
1mm
2mm
8 weeks after SCI, GFAP immunointensity
revealed a significant difference between
QL6 treated and control animals. Data are
mean ± SEM (bars) values, n=6/group; **
p<0.01, *p<0.05. Images were taken from
the injury epicenter
8 wks
Distance to injury centre
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Stem Cell Tourism
• Families should be critical and informed reviewers of clinics
offering these treatments.
• Critically examine the information
•
Without objective evidence, patient testimonials cannot be trusted.
•No trustworthy stem cell treatment for CP currently exists.
•It is best to wait for proof that these treatments work.
•Once stem cells are put in, they cannot be taken out.
Key Points
• What is a stem cell?
– Different types
– Properties
• How could the pathology of CP be amenable to stem
cells (or not)?
• What trials are going on in CP?
– is this appropriate for children in my practice?
– What do I need to be aware of?
• What’s “hot” in the stem cell field regarding CP?
– Endogenous stem cells
– Neural stem cells
– Induced pluripotent stem cells (iPSCs)
– Reduction of the glial scar
• Where do I get more information?
Copyright: Michael G. Fehlings MD PhD
(http://drfehlings.ca/new-articles-on-the-potential-ofstem-cells-for-cerebral-palsy/)
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Stem Cell Facts: Where to Look
NeuroDevNet
www.neurodevnet.ca/kt/stem-cells
International Society for Stem Cell Research (ISSCR)
http://www.isscr.org/public
http://www.closerlookatstemcells.org/
Canadian Institutes of Health Research
http://www.cihr-irsc.gc.ca/e/15255.html
National Institutes of Health Research
http://www.ninds.nih.gov/disorders/cerebral_palsy
http://stemcells.nih.gov/
Clinical Trials
http://clinicaltrials.gov
Copyright: Michael G. Fehlings MD PhD
Crystal Ruff
James Austin
David Cadotte
Gregory Hawryluk
Spyros Karadimas
Yang Liu
Ryan Salewski
Sasha Velumian
Hui Ye
Behzad Azad
Sarah Figley
Patrick Hislop
Alina Karpova
Eun Su Moon
Reaz Vawda
Jian Wang
Nicole Forgione
Stuart Faulkner
Julio Furlan
Karina Goncharenko
Sukhvinder Kalsi-Ryan
Wenru Yu
Kajana Satkunendrarajah
Jared Wilcox
Alex Laliberte
Michelle Legasto
Natasha Stribbell
Anna Artymowicz
Collaborators:
Jerome Yager
Liang Zhang
Warren Foltz
Derek van der Kooy
Cindi Moreshead
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