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Mesoangioblast stem cells ameliorate muscle function
in dystrophic dogs
Authors:
Source:
Nature. 2006 Nov 30 :574-9.
Outlines
1. Background :
a. Human Duchenne Muscular Dystrophy ( DMD )
b. Golden retriever muscular dystrophy model ( GRMD )
c. Mesoangioblast stem cells
2. Experimental design
3. Results
4. Conclusions
Duchenne muscular dystrophy ( DMD )
Genetics:
X-linked ( Xp21 ) recessive dystrophin-deficient muscular dystrophy leading to weakened sarcolemma .
Clinical features:
a. Onset in early childhood
b. Progressive muscular weakness ,hard to run and climb stairs
c. Gower’s manoeuvre
d. Wheelchair needed in most cases by age 12
e. 20 % boys , IQ< 70
f. Epidemiology: 3 x10-4 at boys birth
g. death at cardiac involvement
Lab diagnosis:
a. Serum creatine kinase:
released by damaged muscle fibres
b.Electromyography
c.Muscle histology :
fibre size , fibre necrosis, invasion by macrophages, and replacement by fat and connective
tissue.
d. immunohistochemistry for dystrophin protein
Normal
DMD
Treatment:
a. No effective drugs
b. Gene therapy
c. Cell therapy
dystrophin
Dystrophin protein
rod-shaped structural protein, about 150 nm, 3684 amino acids ,M.W. 427 kDa
Functions:
a. connect the sarcolemmal cytoskeleton to the extra-cellular matrix
b. dissipate muscle contractile force from the intracellular cytoskeleton to the extracellular matrix
Loss of function:
membrane fragility and sarcolemma injury during contraction
Muscle membrane
Dystrophin glycoprotein complex
( DCG )
Golden retriever muscular dystrophy ( GRMD )
Human DMD animal model
display clinical signs of human DMD
Great difficulty in walk by 8 months of age and death at 1 year
GRMD dog
Mesoangioblast stem cells
a class of vessel-associated fetal stem cells
differentiate into most mesoderm cell types when exposed to certain cytokines
more than 50 passages in culture and no tumorigenesis in nude mice
Aim:
To test the efficacy of stem cell and/or gene therapy
in GRMD dogs
Experimental design
Autologous,gene therapy
Heterologous ,
wild type donor
Lentiviral vector
expressing
human
microdystrophin
Muscle-specific creatine kinase
promoter
Myosin light chain 1
fast promoter
Mesoangioblasts
rapamycin
GRMD dogs
cyclosporine
Untreated
Isolation and characterization of canine mesoangioblasts
15 days postnatal ( P15 )
Morphology
Proliferation
Wild-type
Dystrophic
Euploid Karyotype
( 78 chromosomes )
Lentiviral vector expression of canine mesoangioblasts
Mesoangioblasts with lentiviral vector
Mesoangioblasts
GFP
expression
Mesoangioblasts with lentiviral vector
+ C2C12 mouse myoblasts
co-culture
Myotubes
GFP
expression
Lentiviral vector expression of canine mesoangioblasts
Mesoangioblasts with lentiviral vector
+ MyoD (Myogenic Determination protein ) transfection
Myotubes
Myotubes
GFP
expression
MyHC
expression
( Myosin Heavy
Chain )
Merged
Conclusions
Mesoangioblasts isolated from muscle biopsies were proliferated and differentiated well
in vitro.
Lentiviral vector could be transduced and expressed in mesoangioblasts.
Migration of canine mesoangioblasts into skeletal muscle
Mouse mesoangioblasts
GRMD mesoangioblasts
( GFP-expressed lentiviral vector )
( GFP-expressed lentiviral vector )
SCID mice’s femoral artery
( Serve combined immunodeficiency )
6 hours
Isolation of several muscles
Real-time PCR analysis for GFP expression
Migration of canine mesoangioblasts into skeletal muscle
Mouse
GRMD dogs
i : Injected leg
U : Unjected leg
Qd: Quadriceps
Gs: Gastrocnemius
TA: Tibialis cranialis
Lv: Liver
Sp: Spleen
Muscle fibres reconstitution of canine mesoangioblasts
※ 21 days
Mouse muscle fibres
Lamin A-C
Mouse muscle fibres
Dystrophin
( Nuclear Envelope Marker )
DAPI
Laminin
( Structural protein
in membrane )
Conclusions
. Canine mesoangioblasts migrated from the femoral artery to the downstream muscles with
an efficiency similar to that of their wild-type mouse counterparts
Canine mesoangioblasts had the ability to reconstitute muscle fibres
Autologous V.S. Heterologous cell transplantation
Autologous
gene therapy
Heterologous
wild type donor
Lentiviral vector
expressing human
microdystrophin
Muscle-specific creatine kinase
promoter
Mesoangioblasts
3 injections
( 1-month intervals, 5x107 cells )
cyclosporine
GRMD dogs
Untreated
Autologous V.S. Heterologous cell transplantation
Autologous
Morphology
Dystrophin
Laminin
Heterologous
Conclusions
Modified treatment:
a. Increase injections to five
b. Use stronger myosin light chain 1F promoter
Heterologous cell transplantation
Wild-type
Mesoangioblasts
cyclosporine
Rapamycin
Rapamycin/ IL-10
GRMD dogs
5 injections
3 injections
3 injections
Myocarditis
Heterologous cell transplantation
5 injections
&
cyclosporine
U: unjected leg
i : injected leg
Morphology
Sar: Sartorius
Gas: Gastrocnemius
TC: Tibialis cranialis
BF: Biceps femoralis
Dystrophin
β- Sarcoglycan
Laminin
Biceps femoralis
After 13 months
5 injections
&
cyclosporine
Valgus
3 injections
&
Rapamycin
Varus
Conclusions
a.
The clinical motility of GRMD dogs was improved by 5 cell injections .
b.
The Immuno-supression did not show significant differences between cyclosporine and rapamycin.
c.
The heterologous GRMD dogs expressed well-preserved morphology and dystrophin protein .
d.
The expression of β-sarcoglycan indicated reconstitution of the dystrophin-associated complex.
Autologous, modified gene therapy
Lentiviral vector
expressing
human
microdystrophin
Myosin light chain 1
fast promoter
Mesoangioblasts
5 injections
GRMD dogs
Pneumonia
Autologous, modified gene therapy
Before
Morphology
After
U: unjected leg
i: injected leg
Sar: Sartorius
Gas: Gastrocnemius
TC: Tibialis cranialis
BF: Biceps femoralis
β-sarcoglycan
Dystrophin
Laminin
Conclusions
Vampire
All three dogs treated with autologous ,genetically corrected cells performed poorly ,even though two of them
showed amelioration of morphology and expression of dystrophin protein.
To test less effective results obtained with autologous cells was due to the later onset of the
treatment
The efficacy of late transplantation of donor mesoangioblasts
Dystrophin
Azur
Laminin
Azor
Conclusions
Even with a later onset of treatment, heterologous cell transplantation seems to produce a
greater amelioration of muscular dystrophy than is produced by autologous dystrophinexpressing cells .
Analysis of enhancement of contraction force in heterologous GRMD dogs
A. Tetanic force of skeletal muscles in vivo
B. Force of contraction on isolated single muscle fibres in vitro
Normal dog
Autologous GRMD dog ( P113 )
Heterologous GRMD dog ( P75 )
Heterologous GRMD dog ( P159 )
Untreated GRMD dog
Force of treated leg
Force of untreated leg
X 100 %
Heterologous GRMD dog ( P75 )
Heterologous GRMD dog ( P159 )
Heterologous GRMD dog ( P159 )
Autologous GRMD dog
Analysis of enhancement of contraction force in heterologous GRMD dogs
Force of contraction on isolated single muscle fibres in vitro
Immunostaing by dystrophin antibody
Specific
force
Dystrophin
expression
Heterologous
Conclusions
The transplantation of mesoangioblasts into dystrophic cells could obtain an extensive reconstitution of
fibres expressing dystrophin ,an improvement in the contraction force and a preservation of walking ability.
Donor wild-type mesoangioblasts seemed to be more efficient than autologous ,genetically corrected cells.
A different onset of treatment should not be crucial.
Mesoangioblasts were a good candidates for future stem cell therapy for Duchenne patients.