Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs Authors: Source: Nature. 2006 Nov 30 :574-9.

Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs

Authors:

Source: Nature. 2006 Nov 30 :574-9.

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

Outlines

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 proteinTreatment: a. No effective drugs b. Gene therapy c. Cell therapy

DMDNormal

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

Dystrophin glycoprotein complex ( DCG )

Muscle membrane

Human DMD animal model

display clinical signs of human DMD

Great difficulty in walk by 8 months of age and death at 1 year

Golden retriever muscular dystrophy ( GRMD )

GRMD dog

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

Mesoangioblast stem cells

Experimental design

Mesoangioblasts

Lentiviral vector expressing human microdystrophin

GRMD dogs

Autologous,gene therapy Heterologous , wild type donor

Untreated

Muscle-specific creatine kinase promoter

Myosin light chain 1 fast promoter

cyclosporinerapamycin

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

GFP expression

Mesoangioblasts with lentiviral vector + C2C12 mouse myoblasts

co-culture

Mesoangioblasts with lentiviral vector

MyotubesMesoangioblasts

GFP expression

Merged

GFP expression

MyHC expression

( Myosin Heavy Chain )

Mesoangioblasts with lentiviral vector + MyoD (Myogenic Determination protein ) transfection

Myotubes

Lentiviral vector expression of canine mesoangioblasts

Myotubes

Mesoangioblasts isolated from muscle biopsies were proliferated and differentiated well in vitro.

Lentiviral vector could be transduced and expressed in mesoangioblasts.

Conclusions

Migration of canine mesoangioblasts into skeletal muscle

Mouse mesoangioblasts ( GFP-expressed lentiviral vector )

GRMD mesoangioblasts( GFP-expressed lentiviral vector )

SCID mice’s femoral artery ( Serve combined immunodeficiency )

Isolation of several muscles

Real-time PCR analysis for GFP expression

6 hours

Migration of canine mesoangioblasts into skeletal muscle

i : Injected legU : Unjected leg

Qd: QuadricepsGs: GastrocnemiusTA: Tibialis cranialisLv: LiverSp: Spleen

Mouse

GRMD dogs

Muscle fibres reconstitution of canine mesoangioblasts

※ 21 days

Lamin A-C

DAPI

Mouse muscle fibres

Dystrophin

Mouse muscle fibres

Laminin

( Nuclear Envelope Marker )

( Structural protein in membrane )

. 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

Conclusions

Mesoangioblasts

Lentiviral vector expressing human microdystrophin

GRMD dogs

Autologousgene therapy

Heterologous wild type donor

Untreated

Autologous V.S. Heterologous cell transplantation

Muscle-specific creatine kinase promoter

cyclosporine

3 injections ( 1-month intervals, 5x107 cells )

Autologous V.S. Heterologous cell transplantation

Autologous Heterologous

Morphology

Dystrophin

Laminin

Modified treatment: a. Increase injections to five b. Use stronger myosin light chain 1F promoter

Conclusions

Wild-type Mesoangioblasts

GRMD dogs

Heterologous cell transplantation

cyclosporine

5 injections

Rapamycin Rapamycin/ IL-10

3 injections 3 injections

Myocarditis

Heterologous cell transplantation

5 injections&

cyclosporine

Morphology

Dystrophin

Laminin

U: unjected leg i : injected leg

Biceps femoralis

Sar: SartoriusGas: GastrocnemiusTC: Tibialis cranialisBF: Biceps femoralis

β- Sarcoglycan

After 13 months

Valgus Varus

3 injections &

Rapamycin

5 injections&

cyclosporine

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.

Conclusions

Mesoangioblasts

Lentiviral vector expressing human microdystrophin

GRMD dogs

Autologous, modified gene therapy

Myosin light chain 1 fast promoter

Pneumonia

5 injections

Morphology

Dystrophin

Laminin

β-sarcoglycan

Autologous, modified gene therapy

Sar: SartoriusGas: GastrocnemiusTC: Tibialis cranialisBF: Biceps femoralis

U: unjected leg i: injected leg

Before After

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.

Conclusions

To test less effective results obtained with autologous cells was due to the later onset of the treatment

Dystrophin

Laminin

Azor

Azur

The efficacy of late transplantation of donor mesoangioblasts

Even with a later onset of treatment, heterologous cell transplantation seems to produce a greater amelioration of muscular dystrophy than is produced by autologous dystrophin- expressing cells .

Conclusions

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

Untreated GRMD dog

Autologous GRMD dog ( P113 )

Heterologous GRMD dog ( P75 )

Heterologous GRMD dog ( P159 )

Force of treated legForce of untreated leg X 100 %

Autologous GRMD dog

Heterologous GRMD dog ( P75 )

Heterologous GRMD dog ( P159 )

Heterologous GRMD dog ( P159 )

Force of contraction on isolated single muscle fibres in vitro

Dystrophin expression

Heterologous

Specific force

Immunostaing by dystrophin antibody

Analysis of enhancement of contraction force in heterologous GRMD dogs

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.

Conclusions