L'impiego delle cellule staminali nelle malattie neuromuscolari - Prof N. Bresolin Dip. Scienze Neurologiche, Universita'di Milano Fondazione ...
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L’impiego delle cellule staminali nelle
malattie neuromuscolari
Prof N. Bresolin
Dip. Scienze Neurologiche, Universita’di Milano
Fondazione IRCCS Ospedale Maggiore Policlinico,
Mangiagalli e Regina Elena, Milano
IRCCS E. Medea, Bosisio Parini, LeccoCell transplantation and replacement Neural Stem cell therapy
GLIAL NEURONAL
degeneration Degeneration
Demyelinating disease Paracrine systems (PD)
Neurodegenerative
Neuromuscular
Diseases SELECTIVE
GLOBAL Degeneration
Degeneration ALS, HD, Ataxias,
Trauma and stroke FSH,DMD,LG etc
Cognitive and Physical
Neuroprotective Therapies
rehabilitationCellule staminali • Capacità di autorinnovarsi • Dare origine a cellule differenziate
STEM CELLS CONTINUUM
Embryonic Somatic
stem cells stem cells
Zigote
Bone
Totipotent Pluripotent marrow, skin
stem cells stem cells muscle etc
stem cellsLe cellule staminali danno origine
a cellule differenziate
Bone
Kidney osteoblasts Skin
Blood
blood cells Nervous
System
neurons
astrocytes
Vessels oligodendrocytes
endothelial cells
Liver
liver cells
Heart
cardiomyocytes
Pancreas
insulin producing cells
MuscleReprogramming of somatic stem cells
Teratoma derived from human iPS cells
Injected in SCID mice
Human Fibroblasts Induced pluripotent
stem cells (iPS)In vitro differentiation of iPS.
In vivo engraftment
iPS cell-derived neurons integrate into
the striatum of hemiparkinsonian
rats and improve behavioral deficits.Le cellule staminali possono avere effetti
terapeutici attraverso diversi meccanismi
Neuroprotezione Sostituzione cellulare
Produzione di molecole con effetto Genesi di:
neurotrofico, antiinfiammatorio,
vasogenico etc. •Nuovi neuroni
Geneticamente modificate per •Glia
produrre specifici fattoriCellule staminali neurali
SVZ
Isolation SVZ Dissociate
EGF/FGF-2
MATURE CELLS
SELF-RENEWAL
Neurons
EGF/FGF-2
Astrocytes Oligodendrocytes
Corti et alCellule staminali neuronali CD133 positive si
integrano in vivo nella corteccia
Corti et al.2007Multipotentiality,
Multipotentiali ty, homing properties and pyramidal neurogenesis of CNS
CNS--derived
LeX(ssea--1)+/CXCR4+ stem cells
LeX(ssea
S. Corti, FASEB J. 2005 Nov;19(13):1860-
Nov;19(13):1860-2
Isolamento di una
sottofrazione
staminale con
doppia positività
per LeX(Le) e
CXCR4(CX) che
possiede elevato
potenziale di
homing nel SNC
ed estesa capacità
di engraftementIsolamento di una sottofrazione cellulare Le+CX+
Adult Phase//DAPI
Phase LeX//CXCR4
LeX
murine brain SVZ
Neurospheres
LeX CXCR4
MACS selection for
LeX followed by
FACS selection for
LeX+CXCR4+
Phase//DAPI
Phase LeX//CXCR4
LeX
Evaluation of
Self--renewal
Self
DifferentationIl trapianto di cellule staminali Le+CX+ si integra in
corteccia e ricostituisce i circuiti neuronali in un
modello ischemico murino
Corti et al.2007Trapianto di cellule staminali
nelle malattie neuromuscolari:
le distrofie muscolariDistrofia Muscolare
di Duchenne
E’ una malattia geneticamente
determinata X-linked dovuta all’assenza
di distrofina
•E’ caratterizzata da distrofia muscolare
progressiva con ipostenia muscolare
ingravescente e perdita della
deambulazione intorno a 12 anni.Il trapianto di cellule staminali puo’
contribuire alla rigenerazione del
tessuto muscolare scheletricoIl nostro obiettivo: trapiantare le cellule
muscolari attraverso la circolazione sanguigna
Injected MSCs
Muscle progenitors
Rescue of muscular dystrophyIsolation of muscle-derived stem cells
(MDSCs).
Density gradient separation
Magnetic labeling using
Sca-1/CD34 microbeads
Separation with MACS
column type LS
Elution of
highly pure MDSCsMuscle homing of the Sca-1+/CD34-MDSCs after
i.m. transplantation of mdx mice
Quadriceps
PectoralisSca-1+/CD34- MDSCs express the L-selectin
adhesion moleculesMyogenic differentiation of Sca-1+/CD34-/L-selectin+ MDSCs after i.v. injection of mdx mice
Delivery of stem cells to muscle fibers via
intra-venous injection
Two months-old mdx One year-old mdxClonogenic, self-renewal and multi-potency
of AC133 positive cells from blood
VEGF
TRAP assay
CFU-C assay in methyl
celluloseExpression of muscle markers by CD133 positive cells
derived from the blood tissues.
MyHC GFP Merge
AC133+Double-blinded randomized clinical trial phase I: autologous
transplantation of muscle-derived AC133+ cells in Duchenne
Muscular Dystrophy.
Eight DMD patients were included in this study and randomized into two
groups:
Group A (n=5; subjects 003-004-005-006-007)
AC133+cells injection into left abductor digiti minimi muscles (ADM)
Group B (n=3 subjects 008-009-010)
saline solution injection into ADM
Primary outcome: Tolerance and feasibility of intramuscular
transplantation of AC133+ cells to always ensure first, the
patient’s safety and well-being, while aiming towards a treatment.
Secondary outcome: muscular strength tests by MVIC and muscle
force analysis skinned myofibers.
Torrente Y et al. Cell Transplantation 2007Autologous transplantation of muscle-derived AC133+
cells
Muscle dissotiation LIBERASE Hi In vitro serum free
culture for 48h
•RPMI +
•human albumin 20%+
Quality control,
•human insulin 100 Ul/m
Tibialis Anterior muscle (1gr) microbiology
Left abductor digiti minimi muscle (ADM)
3
Myofibers Injections of 20X10 AC133+ cells
*15ml Hamilton with a 27-G needle
*5ml of cell suspension delivered in
each injection
Injection site *Injection depth 0.5 cm,inter-
injection distances 1mm (sterile
transparent grid)Local side effects after intramuscular transplantation of
muscle-derived AC133+ cells
6
Treated Controlateral 5
4
3
2
1
0
2004- 2004- 2004- 2004- 2004- 2004- 2004- 2004-
003 004 005 006 007 008 009 010Single muscle fibre strenght increase in
DMD patients
after AC133+ local injection
Torrente Y et al. Cell Transplantation 2007
0003C 0003T
Slow Myofibers
Fast Myofibers
CD133+/CXCR4+/CD34+
CD31 vesselsIntra-arterial delivery of wild-type
mesoangioblasts
in alpha-SG null mice
i.a. α-SG KOExpression of alpha-SG in alpha-SG null mice
after intra-arterial delivery of wild-type
mesoangioblasts
Sampaolesi et al. Science 2003;301(5632):487-92Expression of α-SG and dystrophin related proteins
in α-SG null mice after intra-arterial delivery of
wild-type mesangioblasts
Sampaolesi et al. Science 2003;301(5632):487-92Morphology by Evans blue and Azan-Mallory
stainings of long-term treated α-SG null dystrophic
muscles after three consecutive i.a. of wild-type
mesangioblasts
Functional properties of single muscle
fibres of long-term treated a-SG null
dystrophic muscles
Sampaolesi et al. Science 2003;301(5632):487-92Three-dimensional visualization of injected
stem cells labeled with iron oxide
nanoparticles after their intra-arterial
transplantation
Torrente Y et al., FEBS Lett. 2006;580(24):5759-64.DMD genotypes for exon- skipping of AC133+ stem cells Exon phasing around exon 51 : DMD genotypes selected
Lentivirus-mediated exon-skipping
Lentivirus U7exon51 map : Characteristics :
. Pseudotype : VSV-G
. Title : 2.109 ip/ml
. Transduction : 106 to 108 ip/ml
U7SmOPT Downstream
Promoteur U7 (267 Pb)
(85 pb) Sequences (116 pb)
Exon-skipping efficiency in vitro :
GGGUCUAGAUAACAACAUAGGAGCUGUGAU
UGGCUGUUUUCAGCCAAUCAGCACUGACUC
CCCAAUUUCACUGGU
CUACAAUGAAAGCAA tested on human myoblasts Δ52
AUUUGCAUAGCCUUUACAAGCGGUCACAAAC AACAGUUCUCUUCCC
UCAAGAAACGAGCGGUUUUAAUAGUCUUUUA CGCUCCCCGGUGUG
GAAUAUUGUUUAUCGAACCGAAUAAGGAACU UGAGAGGGGCUUUG 1 2 3 4
GUGCUUUGUGAUUCACAUAUCAGUGGAGGG AUCCUUCUCUGGUUU A 1 2 3 4
GUGUGGAAAUGGCACCUUGAUCUCACCCUC CCUAGGAAACGCGUA T 30 50
AUCGAAAGUGGAGUUGAUGUCCUUCCCUGG UGUGGCUAGCUUU
CUCGCUACAGACGCACUUCCGCAA
Skipped band
UC
U G
CG
AU Legend :
GC B
UA 1 : Myob Δ52 no transduced
CG
Ex50 Ex53
Site de liaison 2 : Transduction (106 ip/ml)
UA
aux protéines UA 3 : Transduction (107 ip/ml)
h51AON2 h51AON1 SM (OPT) UA
UA 4 : H2O
GC
5’ CCUCUGUGAUUUUAUAACUUGAU/UCAAGGAAGAUGGCAUUUCUAAUUUUUGGAGCAG CCCU 3’Human dystrophin expression in scid/mdx mice
after transplantation of Delta 48-50 DMD exon
skipped blood-derived AC133+ stem cells
8 weeks after i.m. injection of skipped DMD D48-50
blood-derived AC133+ cells (2.104 cells/TA)
Genotype D48-50
Skipping exon 51
340 bp
SM 1 2
340 bpHuman dystrophin expression in scid/mdx mice after transplantation of Delta 48-50 DMD exon skipped blood-derived AC133+ stem cells
Lou Stefano
Sclerosi Laterale Amiotrofica
Transplantation of LeX+/CXCR4+ Adult Neural Stem Cells in the Spinal
Cord of a Murine Model of Amyotrophic Lateral Sclerosis
C57Bl6 SOD1-
SOD1-
G93A (treated n=24
control n=24)
70 days
Transplantation
into spinal cord
20 000 cells
Primed Le+CX+
Donor: β-actinGFP
Hb9GFPLeX+CX+ cells share the properties of stem cells
and produce MN protective cytokinesAcquisizione di un fenotipo colinergico motoneuronale
HB9eGFP/HB9
HB9eGFP HB9 HB9eGFP/Isl1
HB9eGFP Isl1
30
% of HB9eGFP cells
25
20
15
10
5
0
0 1 10 100 1000
Shh
HB9eGFP/ChAT
HB9eGFP ChAT HB9eGFP/ChAT
HB9eGFP ChAT HB9eGFP/BTX
HB9eGFP BTXIl trapianto di cellule Le+CX+ migliora la funzione
neuromuscolare e la sopravvivenza in topi SOD1
Survival Plot (PL estimates)
Survivor
1,00
1
GFP
2
ctr11
3
Hb9 0,75
4
ctr12
0,50
0,25
250
0,00
T im e to fall (s)
120 140 160 180 200
200 GFP Times
150 HB9
100 CTR1
50 CTR2
0
10 11 12 13 14 15 16 17 18 19 20 21 22
age (weeks)La sopravvivenza dei motoneuroni è incrementata
dopo il trapianto di cellule LeX+CX+
35
no. motor neurons
30
25
20
15
10
5
0
wt GFP HB9 SOD1- SOD1-
transplanted untransplanted untransp.1 untransp.2
wt SOD1 SOD1
1200
1000
no. axons
800
600
400
200
transplanted untransplanted 0
wt GFP HB9 SOD- SOD-
wt SOD1 SOD1 untrasp.1 untrasp.2Il trapianto di cellule LeX+CXCR4+ incrementa
la produzione di growth factors
% o f IG F B 5 h ig h p o s itiv e M N
% of IGF1-R positive MNs
5 100 100
4 80 80
wt
3 60 60
Treated
2 Untreated 40 40
1 20 20
0 0 0
GDNF VEGF IGF wt tr-SOD1 untr-SOD1 wt tr-SOD1 untr-SOD1
Transplanted Untransplanted
Wild type
SOD1G93A SOD1G93A
IGFBP5
IGF--1R β
IGFIl trapianto di cellule LeX+CXCR4+ modifica il signalling di IGF1 nel midollo spinale dei topi SOD1
Cellule staminali nelle malattie del motoneurone
Neuroprotezione Sostituzione cellulare
Normal Intermediate End Stage
stageAtrofie Muscolari Spinali:
SMA e SMARD1Atrofia Muscolare Spinale (SMA) SMN expression
SMARD1 is due to mutations in the IGHMBP2, a RNA/DNA Helicase
Neuroni ALDH proiettano lunghi assoni e formano giunzioni neuromuscolari
Differenziamento delle cellule
staminali
Nestina
GFP CD15 Merge GFP/ChAT
+ Neurobasal
+ EGF/FGF RA+Shh
ES NSCs Motoneurons
- Feeder (CD15+Nestin+)
- LIF
Corti et al.2008Disegno Sperimentale
GFP/Nestina
Predifferenziamento
TOPO SMA
TRATTATO
TOPO SMA
GFP/ChATAnalisi del fenotipo SMA dopo il trapianto
GFP/DAPI Midollo spinale di topo trapiantato
Cellule trapiantate si differenziano in motoneuroni
GFP ChAT
Neu-N MergeEffetti del trapianto sui motoneuroni del midollo spinale
Il trapianto
aumenta il numero
di motoneuroni e
il loro diametroEffetti del trapianto sulle miofibre muscolari
Il trapianto aumenta il
numero, il diametro
delle miofibre e l’area
muscolareUtilizzo di sostanze per promuovere la crescita degli
assoni verso i muscoli (GDNF e rolipram)Dino Ferrari Centre,
Department of Neurological Sciences,
University of Milan
IRCCS Foundation “Ospedale
Maggiore Policlinico Mangiagalli
and Regina Elena”, Milan
Stem Cell Lab
Yvan Torrente
Lab of Biochemistry and Genetics Marzia Belicchi
Andrea Farini
Giacomo P. Comi Roberto Del Bo Mirella Meregalli
Stefania Corti Francesco Fortunato Manuela Gavina
Dimitra Papadimitriou Andreina Bordoni Federica Colleoni
Domenico Santoro Sabrina Lucchiari
Di Fonzo Alessio Sabrina Salani
Francesca Magri Chiara Donadoni
Isabella Ghione Martina Nardini
Marinella Carpo Serena Pagliarani
Dario Ronchi Domenica Saccomanno
Monica Nizzardo Francesca Saladino
Serena GhezziCollaborations
Stem Cell Research Institute University of Pavia
DIBIT-HSR, MILAN Bottinelli R
Cossu G D’Antona G
Sampaolesi M IRCCS E. Medea
Tonlorenzi R University of Verona Bosisio Parini
Costantin G
UMR,CNRS 7000 Rossi B D’Angelo MG
Paris
Butler Browne G
Sironi M
University of Laval
Mouly V Sante Foy, Canada Cagliani R
Tremblay J
University of Paris
Pauline D
GENETHON
Garcia L
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