1. Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

2. REGENERATION AND TRANSDIFFERENTIATIO N OF SKELETAL MUSCLE Dr. Péter Balogh and Dr. Péter Engelmann Transdifferentiation and regenerative medicine – Lecture 7 Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

3. TÁMOP-4.1.2-08/1/A-2009-0011 Conditions requiring skeletal muscle regeneration Injury leading to extensive muscle damage Duchenne’s muscular dystrophy: Inherited diseases – Duchenne’s muscular dystrophy: X-linked mutation of dystrophin gene 1:3500 males affected Dystrophin (2.4 Mb in size) is the largest known mammalian gene Onset of the disease: DMD-afflicted patients are diagnosed in childhood. The progressive muscle-wasting disease affects striated muscle including limb muscles, diaphragm, and heart leading to cardiorespiratory failure, and death usually occurs in the teenage years or early 20s.

4. TÁMOP-4.1.2-08/1/A-2009-0011 Experimental models for studying muscle regeneration Mdx mice: spontaneous mutation of the distrophin gene (variable severities in different inbred mouse strains) Distrophin/utrophin double mutant mouse Canine X-linked muscular dystrophy (cxmd) is the best representation of DMD, but the phenotype is variable.

5. TÁMOP-4.1.2-08/1/A-2009-0011 Embyonic development of skeletal muscleMyf5Myf6Pax3 MyoD Myogenesis Myogn Myf6, MyoD NT NC MT SC Limb VLL DT DT SC Myf5 Pax3/Pax7 MyoD Bmp4 Wnt1/3 Nog Wnt11 Pax3 MyoD Shh Wnt7a Nog Myf5

6. TÁMOP-4.1.2-08/1/A-2009-0011 Transcriptional control of myogenic differentiation Transit Amplifying cells MyotubeMyoblast Differentiat ion Activation/Proliferation Myogenic progenitor cells (MPC)Myogenic stem cell (MSC) Quiescent Cd34 Cdh15 Foxk1 Met Pax3 Pax7 Sox8 Sdc4 Sox15 Vcam1 Myf5 Myf6 MyoD Des Myog Myofiber nuclei InjuryFusion Differentiation Maturation Proliferation and self-renewal of satellite cells Regenerating myofiber nuclei Satellite cell (quiescent) Satellite cell (quiescent)

7. TÁMOP-4.1.2-08/1/A-2009-0011 Cellular sources for muscle regeneration Satellite cells and their precursorsSatellite cells and their precursors Endothelial cells associated with embryonic limb muscles Mesangioblasts Bone marow-derived stem cells Pluripotent cells found within muscle- derived side population (SP) cells Highly active Mdr-dependent expulsion of Hoechst 33342 dye

8. TÁMOP-4.1.2-08/1/A-2009-0011 Tissue sources for muscle regeneration Vascular progenitors Interstitial cells Bone marrow cells Myofibernuclei Satellitecell

9. TÁMOP-4.1.2-08/1/A-2009-0011 Muscle stem cells – satellite cells The satellite cells reside beneath the basal lamina of muscle, closely juxtaposed to muscle fibers Approximately up 2–7% of the nuclei associated with a particular fiber Heterogeneous composition: fusing/non-fusing subsets Ontogeny: somite/perivascular cells expressing Pax3/Pax7 Surface markers –Mouse: M-cadherin, CD34, VCAM, CD56, c-met (HGF-receptor) –Human: CD56

10. TÁMOP-4.1.2-08/1/A-2009-0011 Structure and regeneration of skeletal muscle Myofibril Hematopoietic cells Pericyte Endothelial cell Arteriole and capillaries Interstitial cell Basal lamina Satellite cell (SC) Muscle fiber Myonucleus Quiescent SC Pax7 + Activated SC Pax7 + Myf5 + MyoD + Fusion and differentiation Return to quiescence Myoblast Pax7 - Myf5 + MyoD + Expansion(symmetricdivision) Asymmetricdivision Activation Myocyte MyoD +

11. TÁMOP-4.1.2-08/1/A-2009-0011 Kinetics of muscle repairActivation Proliferation Differentiation Maturation 01251014 Days post injury

12. TÁMOP-4.1.2-08/1/A-2009-0011 Problems with myoblast regeneration in Duchenne’s muscular distrophy Necessity for immunosuppression Immunosuppressant drugs cause myoblast apoptosis Short migratory distance following intramuscular injection – 100 injections/cm 2 (totalling up to 4,000 injections in a single patient!)

13. TÁMOP-4.1.2-08/1/A-2009-0011 Non-SCs contributing to muscle regeneration Expansion Commitment (if needed) Allogeneic transplantation Autologous transplantation (after genetic correction) Mesenchymal differentation Adipose-derived stem cells MyoD-converted cells Adipose-derived stem cells MyoD-converted cells HSCs Side population Mesenchymal stem cells MAPCs HSCs Side population Mesenchymal stem cells MAPCs SCs and subpopulations MDSCs CD133 + stem cells SCs and subpopulations MDSCs CD133 + stem cells HSCs Side population CD133 + stem cells HSCs Side population CD133 + stem cells MABs/pericytes Myoendothelial cells EPCs MSCs MABs/pericytes Myoendothelial cells EPCs MSCs iPS cells Reprogramming Dermis or other tissues Skeletal muscle Bone marrow Other sources Blood Vessels CharacterizationCharacterization

14. TÁMOP-4.1.2-08/1/A-2009-0011 Summary The prime candidates for skeletal muscle regeneration are the satellite cells, but cells from other sources (embryonic as well as non-embryonic) may also associate/promote the process. (a)(b) (c)Muscle regeneration is accomplished through (a) promoting vascular repair, (b) cellular differentiation from muscle stem cells and (c) possible transdifferentiation.