Manifestation of Novel Social Challenges of theEuropean Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the Universityof Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
REGENERATION ANDTRANSDIFFERENTIATION OF SKELETALMUSCLE
Dr. Péter Balogh and Dr. Péter Engelmann
Transdifferentiation and regenerative medicine –Lecture 7
Manifestation of Novel Social Challenges of theEuropean Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the Universityof Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
Conditions requiringskeletal muscleregeneration
Injury leading to extensive muscle damageInjury leading to extensive muscle damage
Duchenne’s musculardystrophy:Inherited diseases – Duchenne’s musculardystrophy:
•X-linked mutation of dystrophin gene
•1:3500 males affected
•Dystrophin (2.4 Mb in size) is the largestknown mammalian gene
•Onset of the disease: DMD-afflicted patientsare diagnosed in childhood. The progressivemuscle-wasting disease affects striatedmuscle including limb muscles, diaphragm,and heart leading to cardiorespiratoryfailure, and death usually occurs in theteenage years or early 20s.
TÁMOP-4.1.2-08/1/A-2009-0011
Experimental models forstudying muscleregeneration
•Mdx mice: spontaneous mutation of thedistrophin gene (variable severities indifferent inbred mouse strains)
•Distrophin/utrophin double mutant mouse
•Canine X-linked muscular dystrophy (cxmd) isthe best representation of DMD, but thephenotype is variable.
TÁMOP-4.1.2-08/1/A-2009-0011
Embyonic development ofskeletal muscle
Myf5Myf5
Myf6Myf6
Pax3Pax3
MyoDMyoD
MyogenesisMyogenesis
Myogn Myf6, MyoDMyogn Myf6, MyoD
NTNT
NCNC
MTMT
SCSC
LimbLimb
VLLVLL
DTDT
DTDT
SCSC
Myf5Myf5
Pax3/Pax3/
Pax7Pax7
MyoDMyoD
Bmp4Bmp4
Wnt1/3Wnt1/3
NogNog
Wnt11Wnt11
Pax3Pax3
MyoDMyoD
ShhShh
Wnt7aWnt7a
NogNog
Myf5Myf5
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Transcriptional control ofmyogenic differentiation
Transit Amplifying cells
Myotube
Myoblast
DifferentiationDifferentiation
Activation/ProliferationActivation/Proliferation
Myogenic progenitor cells (MPC)
Myogenic stem cell (MSC)
QuiescentQuiescent
Cd34
Cdh15
Foxk1
Met
Pax3
Pax7
Sox8
Sdc4
Sox15
Vcam1
Myf5
Myf6
MyoD
Des
Myog
Myofiber
nuclei
InjuryInjury
FusionFusion
DifferentiationDifferentiation
MaturationMaturation
Proliferation and
self-renewal of satellite cells
Regenerating
myofiber nuclei
Satellite cell
(quiescent)
Satellite cell
(quiescent)
TÁMOP-4.1.2-08/1/A-2009-0011
Cellular sources for muscleregeneration
•Satellite cells and their precursors•Satellite cells and their precursors
•Endothelial cells associated with embryoniclimb muscles
•Mesangioblasts
•Bone marow-derived stem cells
•Pluripotent cells found within muscle-derived side population (SP) cells
•Highly active Mdr-dependent expulsion ofHoechst 33342 dye
TÁMOP-4.1.2-08/1/A-2009-0011
Tissue sources for muscleregeneration
Vascular progenitorsVascular progenitors
Interstitial cellsInterstitial cells
Bone marrow cellsBone marrow cells
MyofiberMyofiber
nucleinuclei
SatelliteSatellite
cellcell
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Muscle stem cells –satellite cells
•The satellite cells reside beneath the basallamina of muscle, closely juxtaposed tomuscle fibers
•Approximately up 2–7% of the nucleiassociated with a particular fiber
•Heterogeneous composition: fusing/non-fusingsubsets
•Ontogeny: somite/perivascular cellsexpressing Pax3/Pax7
•Surface markers
–Mouse: M-cadherin, CD34, VCAM, CD56, c-met(HGF-receptor)
–Human: CD56
TÁMOP-4.1.2-08/1/A-2009-0011
Structure and regenerationof 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 andFusion and
differentiationdifferentiation
Return toReturn to
quiescencequiescence
Myoblast
Pax7-
Myf5+MyoD+
ExpansionExpansion
(symmetric(symmetric
division)division)
AsymmetricAsymmetric
divisiondivision
ActivationActivation
Myocyte
MyoD+
TÁMOP-4.1.2-08/1/A-2009-0011
Kinetics of muscle repair
ActivationActivation
ProliferationProliferation
DifferentiationDifferentiation
MaturationMaturation
0
1
2
5
10
14
Days post injury
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Problems with myoblastregeneration in Duchenne’smuscular distrophy
•Necessity for immunosuppression
•Immunosuppressant drugs cause myoblastapoptosis
•Short migratory distance followingintramuscular injection – 100 injections/cm2(totalling up to 4,000 injections in asingle patient!)
TÁMOP-4.1.2-08/1/A-2009-0011
Non-SCs contributing tomuscle regeneration
Expansion
Commitment (if needed)
Allogeneic transplantation
Autologous transplantation
(after genetic correction)
Mesenchymal differentationMesenchymal 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 cellsiPS cells
iPS cellsiPS cells
ReprogrammingReprogramming
Dermis or other tissuesDermis or other tissues
Dermis or other tissuesDermis or other tissues
Skeletal muscle
Skeletal muscle
Bone marrow
Bone marrow
Other sources
Other sources
Blood
Blood
Vessels
Vessels
CharacterizationCharacterization
CharacterizationCharacterization
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Summary
•The prime candidates for skeletal muscleregeneration are the satellite cells, butcells from other sources (embryonic as wellas non-embryonic) may also associate/promotethe process.
(a)(b)(c)•Muscle regeneration is accomplished through(a) promoting vascular repair, (b) cellulardifferentiation from muscle stem cells and(c) possible transdifferentiation.