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

2. 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

3. 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 Treatment: a. No effective drugs b. Gene therapy c. Cell therapy DMDNormal dystrophin

4. 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

5. 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

6. 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

7. Experimental design Mesoangioblasts Lentiviral vector expressing human microdystrophin GRMD dogs Autologous,gene therapyHeterologous, wild type donor Untreated Muscle-specific creatine kinase promoter Myosin light chain 1 fast promoter cyclosporinerapamycin

8. Isolation and characterization of canine mesoangioblasts 15 days postnatal ( P15 ) MorphologyProliferation Wild-type Dystrophic Euploid Karyotype ( 78 chromosomes )

9. Lentiviral vector expression of canine mesoangioblasts GFP expression Mesoangioblasts with lentiviral vector + C2C12 mouse myoblasts co-culture Mesoangioblasts with lentiviral vector MyotubesMesoangioblasts GFP expression

10. 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

11. Mesoangioblasts isolated from muscle biopsies were proliferated and differentiated well in vitro. Lentiviral vector could be transduced and expressed in mesoangioblasts. Conclusions

12. 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

13. Migration of canine mesoangioblasts into skeletal muscle i : Injected leg U : Unjected leg Qd: Quadriceps Gs: Gastrocnemius TA: Tibialis cranialis Lv: Liver Sp: Spleen Mouse GRMD dogs

14. 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 )

15. . 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

16. Mesoangioblasts Lentiviral vector expressing human microdystrophin GRMD dogs Autologous gene therapy Heterologous wild type donor Untreated Autologous V.S. Heterologous cell transplantation Muscle-specific creatine kinase promoter cyclosporine 3 injections ( 1-month intervals, 5x10 7 cells )

17. Autologous V.S. Heterologous cell transplantation AutologousHeterologous Morphology Dystrophin Laminin

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

19. Wild-type Mesoangioblasts GRMD dogs Heterologous cell transplantation cyclosporine 5 injections RapamycinRapamycin/ IL-10 3 injections Myocarditis

20. Heterologous cell transplantation 5 injections & cyclosporine Morphology Dystrophin Laminin U: unjected leg i : injected leg Biceps femoralis Sar: Sartorius Gas: Gastrocnemius TC: Tibialis cranialis BF: Biceps femoralis β- Sarcoglycan

21. After 13 months ValgusVarus 3 injections & Rapamycin 5 injections & cyclosporine

22. 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

23. Mesoangioblasts Lentiviral vector expressing human microdystrophin GRMD dogs Autologous, modified gene therapy Myosin light chain 1 fast promoter Pneumonia 5 injections

24. Morphology Dystrophin Laminin β-sarcoglycan Autologous, modified gene therapy Sar: Sartorius Gas: Gastrocnemius TC: Tibialis cranialis BF: Biceps femoralis U: unjected leg i: injected leg BeforeAfter

25. 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

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

27. Dystrophin Laminin Azor Azur The efficacy of late transplantation of donor mesoangioblasts

28. 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

29. 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 leg Force of untreated leg X 100 % Autologous GRMD dog Heterologous GRMD dog ( P75 ) Heterologous GRMD dog ( P159 )

30. 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

31. 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