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
REGENERATION AND
TRANSDIFFERENTIATIO N OF SKELETAL
MUSCLE
Dr. Péter Balogh and Dr. Péter Engelmann
Transdifferentiation and regenerative medicine – Lecture 7
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
Conditions requiring skeletal muscle regeneration
Injury leading to extensive muscle damage
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.
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.
Embyonic development of skeletal muscle
Myf5 Myf6 Pax3
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
Transcriptional control of myogenic differentiation
Transit Amplifying cells
Myotube Myoblast
Differentiation 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
Injury Fusion Differentiation Maturation
Proliferation and
self-renewal of satellite cells
Regenerating myofiber nuclei Satellite cell
(quiescent)
Satellite cell (quiescent)
Cellular sources for muscle regeneration
• Satellite 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
Tissue sources for muscle regeneration
Vascular progenitors Interstitial cells
Bone marrow cells Myofiber
nuclei Satellite
cell
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
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 (symmetric division) Asymmetric
division Activation
Myocyte MyoD+
Kinetics of muscle repair
Activation
Proliferation
Differentiation
Maturation
0 1 2 5 10 14
Days post injury
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/cm2 (totalling up to 4,000 injections in a single patient!)
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
HSCs Side population Mesenchymal stem cells
MAPCs
SCs and subpopulations MDSCs
CD133+ stem cells HSCs
Side population CD133+ stem cells
MABs/pericytes Myoendothelial cells
EPCs MSCs
iPS cells Reprogramming Dermis or other tissues
Skeletal muscle
Bone marrow
Other sources Blood Vessels
Characterization
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.
• Muscle regeneration is accomplished through (a) promoting vascular repair, (b) cellular differentiation from muscle stem cells and (c) possible transdifferentiation.
