Cellule staminali nella patologia postraumatica midollare - Laura Calzà HST-ICIR, University of Bologna - UniSalute
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Cellule staminali nella patologia postraumatica midollare
Laura Calzà
HST-ICIR, University of Bologna
Convegno UniSalute
Bologna, 30 settembre 2011Spinal 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 therapiesNeural cells from stem cells
nestin
MCM2
ESCs
Hoechst
doublecortin
Hoechst
EBs
CNPase
Hoechst
NSCs
Fernandez, Paradisi, Lizzo, Alessandri, BaldassarroCells to replace lost cells: so simple, so unlikely…
•in vitro expansion
•differentiation
stem
SOURCE
SOURCE: transplant HOST
HOST:
systemic delivery
Embryo/fetal homing
bone marrow/adipose lodging/engrafting
adult tissues repair
iPS side-effects
Cell replacementWhich 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; mesenchymalSpinal cord injury: an evolving pathology
Acute vs chronic: 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 efficacySpinal cord repair + stem cells: 723 items Cell therapies: to focus the goal
208 review articles
Cell therapy
Which cell? Totipotent/Multipotent? Isolation, storage, expansion,
Progenitors? Differentiated cells? differentiation, transplant,
Which source? Donors? Autologous? homing,
To do what? To replace cells? To retard engrafting, efficacy, side-
degeneration? To control inflammation and scar effects…..
formation? To promote self repair? To control NOT ONLY CELL
pain? REPLACEMENTHow to deliver cells
1. Intra CNS transplant:
- most of the cells in stem/progenitor
transplants die after brain
transplantation
- intraparenchymal approaches target OX42
the site of the most extensive natural
recovery in humans: transplantation
may damage repair attempts hNSC, 70000/rat
cyclosporine+betametazon
2. Systemic delivery
- 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)
- 2D + Cultrex
Scaffolds and “drug” delivery: - 3D Cultrex
Flexible, permeable, implantable
biological reservoirs
- PLLA (polylactic acid)
- PLLA + Cultrex
Physical conditioning (non genetic):
- acellular human derma (GMP)
EMFs, laser light, mechanical stimuli……..RESCs pluripotency….
nestin
Oct4
ectoderm
BMP
mesoderm
endoderm vimentin
Oct4 ECM ECM
actin integrin alpha3
3DIV 12DIV…RESCs growth factor expression...
VEGF
AACt RESCs
700
600
500
400
300
200
100
10 Flk1
8
6
4
2
0
BDNF NGF GDNF CNTF VEGF
Alessandri, Lizzo, FernandezEMC 3D scaffold conditioning
3D cultrex
25 ***
20
15
550
HIDROGEL 3D 10
500
CULTREX 3D
450
5
*
400
350
** ***
0
300
VEGF NGF BDNF GDNF
250
200
0 1 2 3 4 5 6
DIV
Alessandri, LizzoPLLA nanofiber scaffold conditioning
Oct4
acid) electrospun
nanofiber scaffolds: 600nm
fibers, pores 5µm
poly(L-lactic
poly(L
70µm
actin
1.25
glass
1.00 PLLA
0.75
0.50
0.25
0.00
0 1 2 3 4 5 6 7 8
DIV
Lizzo, Alessandri, Focarete, GualandiRESC 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
0.25
31 D P.A.
0.00
Lizzo, Bondioli, FiniRESCs summary
long term culture VEGF NGF BDNF GDNF CNTF
PLLA ND ND
=
PLLA+Cultrex ND ND
3D Cultrex
derma =
+ - 5x
+ - 10x
+ - 15x ND, not determinedPerspective: GMP-hMSCs
10 DIV 20 DIV 2^(-AACt) NGF
spontaneous
20
20 DIV
BME
10
20 DIV
BME + RA
0
GFAP Musashi GFAP
DCX Tuj1
D: dental pulp MO/B/P: large vessels wall
A: amnios ADS: adipose tissue
B: bone marrow WJ: Warton Jelly
Bagnara&Calzà groupshMSC vs hNSC: individual variability
#M #P
#N #O
300 300 40
35
30
30
25
200 200
20
20
15
100 100
10 10
5
0 0 0 0
1 60 1.5
1.0 12.5
50
0.8 10.0
0.050
40 1.0
0.6 7.5
30
0.4 0.025 5.0
20 0.5
0.2 2.5
10
0.0 0.000 0 0.0 0.0
HSC NSC HSC NSC HSC NSC HSC NSC HSC NSC
BDNF NGF CNTF GDNF VEGF
Paradisi et al., NAN, 201017.500.000 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: very clear
SC: somewhat clear
SI: somewhat unclear
VI: very unclear“Trading on hope”
Nature Biotecnology, September 2009
Barbados Messico
Cina Panama
Costa Rica Perù
Filippine Portogallo
Florida Porto Rico
Georgia (USA) Rep Domenicana
Georgia, rep. di Russia
Germania Sud Corea
Guatemala Svizzera
India Tailandia
Isreale Turchia
Olanda UcrainaEvidence Based Medicine: bench-to-bed May 2011
May 2011
$1 bilion
10 yearsClinical Trials: Neural Stem Cell-Mediated CNS Regenerative Therapy,
Neuron, May 26, 2011, 7 studies
Geron Corp., CA, www.geron.com, Phase I: huESC-derived oligodendrocyte
Stanford Univ,/Santa Clara Valley Med Neurologically complete subacute, progenitor cells, GRNOPC1®Allogeneic
Ctr, Palo Alto, CAPI: G. Steinberg, MD, thoracic spinal cord injury.
PhD Shepard Ctr, Atlanta PI: D. Apple, ClinicalTrials.gov ID#NCT01217008U.S.
MD; Northwestern Univ., Chicago PI: R. Food & Drug Administration
Fessler, MD, PhD; Thomas Jefferson
Univ Hosp, Phil PI: J. Harrop, PM
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: 2010-08- stable neural stem cell lines from the
25): human hippocampus
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 post-
injury.Alessandri M.
Baldassarro V.A.
THANKS!!!
Fernandez M.
Giuliani A.
Gusciglio M. The right cell,
Lizzo G.
Lorenzini L.
in the right place,
Mangani C. for the right time
Sivilia S.
& Giardino L.
Focarete ML, UniBo
Bagnara GP, UniBo
collaborators of the past:
Bondioli E, AUSL Cesena
Paradisi M Fini M, IOR Bologna
Pozzati E, AUSL Bologna sud
Pirondi SCNS repair : what is needed? loss of glial cells: To remyelinate MS, trauma loss of NT- producing To replace NT cells: PD at target loss of a specific To replace a neuron phenotype: type and connection HD, ALS global degeneration: To replace cells trauma, and connections ischemia
Which cell? How many?
type
source
Embryonic stem cells Autologous
Fetal cells heterologous
Adult pluripotent cells Immortalized lines
iPCs amount
Therapeutic (3
treatments)Biology of the lesion: Stem cell properties:
Inflammation differentiation
Demyelination/remyelination Immune-modulation
Axon contusion/transection secretion
Self-repair attempts
Scar formationhMSCs: PC12 assay for NGF activity
+ NGF + hMSC cm
Giuliani, ManganoProof-of-concept - Humoral communication - no direct cell-host contact - “self-regulating” cells - autologous cells - tailor scaffold
NSCs and derived cells (on Cultrex)
AACt VEGF AACt BDNF
***
1.25 4
AACt NSF
1.00
3
90 0.75
2
80 0.50 ***
70 1
0.25
60
0.00 0
50 Per garantire la priv acy , è stato impedito il download automatico di questa immagine esterna. Per scaricare e v isualizzare l'immagine, fare clic su Opzioni sulla barra dei messaggi, quindi fare clic su A ttiv a contenuto esterno.
AACt NGF
10 3
***
8
6 2
4
2 1
0
BDNF NGF GDNF CNTF VEGF 0
Lizzo, ParadisiStem 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? MS
To provide neurotrasmitter at target? PD
To replace a specific neuron phenotype? HD, ALS
To replace many neuron phenotypes? Stroke, trauma
To re-establish connections? All above conditions
Main question 2: acute vs chronic degenerationSpinal cord repair + stem cells:
723 items
208 review articles Clincal studies
NINDS Facilities of
Research Excellence
There are no fully restorative in Spinal Cord Injury
therapies for SCI NINDS workshop
as yet and so prevention (for on translating
example, effective seat promising strategies
belts, weapons restrictions and for spinal cord
safety in sports) is the injury therapy
best medicine (see Foundation for
Spinal Cord Injury SCI: metodological
Prevention, Care and Cure considerationsSpinal 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: Stem cell properties:
Inflammation differentiation
Demyelination/remyelination Immune-modulation
Axon contusion/transection Paracrine properties:
•Inflammation
Self-repair attempts
•glial scarring
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