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Cellule staminali nella patologia postraumatica midollare
Laura Calzà
HST-ICIR, University of Bologna
Convegno UniSalute
Bologna, 30 settembre 2011
Spinal cord injury: numbers
• USA: 2.5 million people live with spinal cord injury (SCI), with more than
130,000 new injuries reported each year (International Campaign for Cures of
Spinal Cord Injury Paralysis)
• China: more than 80 million people enrolled in the Chinese Spinal Cord Injury
Association
• Italy: 60-70mila people, but the there is no registry of spinal injuries, yet
(initiative launched in April 2011)
There are no fully restorative therapies for SCI
Thus: new hopes from cell therapies
ESCs
Neural cells from stem cells
nestin
MCM2
Hoechst
EBs
doublecortin
Hoechst
NSCs
CNPase
Hoechst
Fernandez, Paradisi, Lizzo, Alessandri, Baldassarro
Cells to replace lost cells: so simple, so unlikely…
•in vitro expansion
•differentiation
stem
SOURCE
SOURCE:
Embryo/fetal
bone marrow/adipose
adult tissues
iPS
transplant
systemic delivery
Cell replacement
HOST
HOST:
homing
lodging/engrafting
repair
side-effects
Which cells, to do what: state of the art for CNS
To generate neural cells
Endogenous NSCs:
• cell lines from donated fetal CNS tissue (eg GRPs)
• olfactory ensheathing cells
• skin-derived multipotent precursors (ectoderm)
• activation of endogenous NSCs
Pluripotent and Induced:
• cell lines from human embryonic stem
•iPCs
Controversial non-neural sources of neural cells:
Bone marrow; mesenchymal
Acute vs chronic:
Axonal damage
Neuronal degeneration
Demyelination
Scar formation
Inflammation
Immune reaction
CSF flow disorder
Motor impairment:
loss-of-function
gain-of-function
Sensory impairment:
loss-of-function
gain-of-function: pain
Need for a better monitoring of the spontaneous
recovery and of treatments efficacy
Spinal cord injury: an evolving pathology
acute vs chronic
Cell therapies: to focus the goal
Spinal cord repair + stem cells: 723 items
208 review articles
Cell therapy
Which cell? Totipotent/Multipotent?
Progenitors? Differentiated cells?
Which source? Donors? Autologous?
To do what? To replace cells? To retard
degeneration? To control inflammation and scar
formation? To promote self repair? To control
pain?
Isolation, storage, expansion,
differentiation, transplant,
homing,
engrafting, efficacy, sideeffects…..
NOT ONLY CELL
REPLACEMENT
How to deliver cells
1.
2.
Intra CNS transplant:
-
most of the cells in stem/progenitor
after brain
transplants die
transplantation
-
intraparenchymal approaches target
the site of the most extensive natural
recovery in humans: transplantation
may damage repair attempts
Systemic delivery
-
OX42
hNSC, 70000/rat
cyclosporine+betametazon
Very poor homing and engrafting
….THUS….
-Scaffolds to maintain cells in the lesion side for the right time
-The right cell in the right place: paracrine properties
-Personalized medicine: autologous source (bone marrow, adipose tissue)
The right cell in the right place for the right time
Rat Embryonic Stem Cells (but also
human Mesenchymal Stem Cells):
- standard (2D glass and/or plastic)
Scaffolds and “drug” delivery:
Flexible, permeable, implantable
biological reservoirs
Physical conditioning (non genetic):
EMFs, laser light, mechanical stimuli…
- 2D + Cultrex
- 3D Cultrex
- PLLA (polylactic acid)
- PLLA + Cultrex
- acellular human derma (GMP)
…..RESCs pluripotency….
nestin
Oct4
ectoderm
BMP
mesoderm
endoderm
Oct4
actin
ECM
vimentin
ECM
integrin alpha3
3DIV
12DIV
…RESCs growth factor expression...
VEGF
AACt RESCs
700
600
500
400
300
200
100
10
8
6
4
2
0
Flk1
BDNF
Alessandri, Lizzo, Fernandez
NGF
GDNF CNTF
VEGF
EMC 3D scaffold conditioning
3D cultrex
25
***
20
15
550
10
HIDROGEL 3D
500
CULTREX 3D
450
*
5
400
350
**
***
NGF
BDNF
0
300
VEGF
250
200
0
1
2
3
DIV
Alessandri, Lizzo
4
5
6
GDNF
PLLA nanofiber scaffold conditioning
poly(L
poly(L-lactic
acid) electrospun
nanofiber scaffolds: 600nm
fibers, pores 5µm
Oct4
70µm
actin
1.25
glass
PLLA
1.00
0.75
0.50
0.25
0.00
0
1
2
3
4
DIV
Lizzo, Alessandri, Focarete, Gualandi
5
6
7
8
RESC scaffold conditioning
Alessandri, Lizzo
Giuliani, Alessandri, Lizzo
human GMP dermis scaffold conditioning
AACt VEGF
5
***
4
3
***
2
1
0
AACt CNTF
1.25
1.00
0.75
0.50
31 D P.A.
0.25
0.00
Lizzo, Bondioli, Fini
RESCs summary
long term culture
VEGF NGF
BDNF GDNF CNTF
PLLA
=
PLLA+Cultrex
3D Cultrex
derma
=
+ - 5x
+ - 10x
+ - 15x
ND, not determined
ND
ND
ND
ND
Perspective: GMP-hMSCs
2^(-AACt) NGF
20 DIV
spontaneous
10 DIV
20
BME
20 DIV
10
BME + RA
20 DIV
GFAP
DCX
0
Musashi
Tuj1
Bagnara&Calzà groups
GFAP
D: dental pulp
MO/B/P: large vessels wall
A: amnios
ADS: adipose tissue
B: bone marrow
WJ: Warton Jelly
hMSC vs hNSC: individual variability
#M
#N
300
#P
#O
300
40
35
30
30
200
25
200
20
20
15
100
100
10
10
5
0
0
1
1.0
60
0.050
1.5
12.5
50
0.8
0
0
10.0
40
1.0
0.6
7.5
30
0.4
0.025
5.0
20
0.2
2.5
10
0.0
HSC
NSC
BDNF
Paradisi et al., NAN, 2010
0.0
0
0.000
HSC
NSC
NGF
0.5
HSC
NSC
CNTF
0.0
HSC
NSC
GDNF
HSC
NSC
VEGF
17.500.000 per cell therapy
: regenerative medicine
23.900.000 per cell therapy.com.
QuickTime™ e un
decompressore
sono necessari per visualizzare quest'immagine.
Impressione “olistica” dal sito, relativamente a
indicazioni, rischi, benefici e disponibilità
VC:
SC:
SI:
VI:
very clear
somewhat clear
somewhat unclear
very unclear
“Trading on hope”
Nature Biotecnology, September 2009
Barbados
Cina
Costa Rica
Filippine
Florida
Georgia (USA)
Georgia, rep. di
Germania
Guatemala
India
Isreale
Olanda
Messico
Panama
Perù
Portogallo
Porto Rico
Rep Domenicana
Russia
Sud Corea
Svizzera
Tailandia
Turchia
Ucraina
Evidence Based Medicine: bench-to-bed
May 2011
May 2011
$1 bilion
10 years
Clinical Trials: Neural Stem Cell-Mediated CNS Regenerative Therapy,
Neuron, May 26, 2011, 7 studies
Geron Corp., CA, www.geron.com,
Stanford Univ,/Santa Clara Valley Med
Ctr, Palo Alto, CAPI: G. Steinberg, MD,
PhD Shepard Ctr, Atlanta PI: D. Apple,
MD; Northwestern Univ., Chicago PI: R.
Fessler, MD, PhD; Thomas Jefferson
Univ Hosp, Phil PI: J. Harrop, PM
Phase I:
Neurologically complete subacute,
thoracic spinal cord injury.
ClinicalTrials.gov ID#NCT01217008U.S.
Food & Drug Administration
huESC-derived oligodendrocyte
progenitor cells, GRNOPC1®Allogeneic
hESC-derived oligodendrocyte progenitor cells that have demonstrated remyelinating and nerve growth
stimulating properties leading to restoration of function in animal models of acute spinal cord injury (Journal of
Neuroscience, Vol. 25, 2005)
Neuralstem, Inc, www.neuralstem.com/
Regulatory submission (FDA: 2010-0825):
16 long-term, or chronic, spinal cord
injury patients, with an American Spinal
Injury Association (ASIA) Grade A level
of impairment, one-to-two years postinjury.
stable neural stem cell lines from the
human hippocampus
Alessandri M.
Baldassarro V.A.
THANKS!!!
Fernandez M.
Giuliani A.
Gusciglio M.
Lizzo G.
Lorenzini L.
Mangani C.
The right cell,
in the right place,
for the right time
Sivilia S.
& Giardino L.
collaborators of the past:
Paradisi M
Pirondi S
Focarete ML, UniBo
Bagnara GP, UniBo
Bondioli E, AUSL Cesena
Fini M, IOR Bologna
Pozzati E, AUSL Bologna sud
CNS repair : what is needed?
loss of
glial cells:
MS, trauma
To remyelinate
loss of NTproducing
cells: PD
To replace NT
at target
loss of a
specific
phenotype:
HD, ALS
To replace a neuron
type and connection
global
degeneration:
trauma,
ischemia
To replace cells
and connections
Which cell? How many?
type
Embryonic stem cells
Fetal cells
Adult pluripotent cells
iPCs
source
Autologous
heterologous
Immortalized lines
amount
Therapeutic (3
treatments)
Biology of the lesion:
Inflammation
Demyelination/remyelination
Axon contusion/transection
Self-repair attempts
Scar formation
Stem cell properties:
differentiation
Immune-modulation
secretion
hMSCs: PC12 assay for NGF activity
+ NGF
Giuliani, Mangano
+ hMSC cm
Proof-of-concept
- Humoral communication
- no direct cell-host contact
- “self-regulating” cells
- autologous cells
- tailor scaffold
NSCs and derived cells (on Cultrex)
AACt VEGF
1.25
AACt NSF
4
1.00
90
80
70
60
50
***
3
0.75
2
0.50
0.25
0.00
***
1
0
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AACt NGF
10
8
6
4
2
0
3
2
1
BDNF
Lizzo, Paradisi
AACt BDNF
NGF
GDNF CNTF
VEGF
0
***
Stem cells for brain repair: to do what?
• Tissue (mature) grafting in the late 19th century
• 1917: neuron survival and growth (neonatal)
• since early 1970s: neural grafting for Parkinson disease
• clinical trials.gov around 1000 studies using stem cells
Main question 1: what we expect from cell therapies in CNS?
To remyelinate?
To provide neurotrasmitter at target?
To replace a specific neuron phenotype?
To replace many neuron phenotypes?
To re-establish connections?
Main question 2: acute vs chronic degeneration
MS
PD
HD, ALS
Stroke, trauma
All above conditions
Spinal cord repair + stem cells:
723 items
208 review articles
There are no fully restorative
therapies for SCI
as yet and so prevention (for
example, effective seat
belts, weapons restrictions and
safety in sports) is the
best medicine (see Foundation for
Spinal Cord Injury
Prevention, Care and Cure
Clincal studies
NINDS Facilities of
Research Excellence
in Spinal Cord Injury
NINDS workshop
on translating
promising strategies
for spinal cord
injury therapy
SCI: metodological
considerations
Spinal cord repair + stem cells:
723 items
208 review articles
Isolation, storage, expansion, differentiation, transplant, homing,
engrafting, efficacy, side-effects…..
NOT ONLY CELL REPLACEMENT
Biology of the lesion:
Inflammation
Stem cell properties:
differentiation
Demyelination/remyelination
Immune-modulation
Axon contusion/transection
Paracrine properties:
Self-repair attempts
Scare formation
•Inflammation
•glial scarring