Muscle Cell Therapyo

Topics Addressed:
- Muscle formation and repair.
- Genetic control of myogenesis.
- How to identify satellite cells.
- Assessing satellite cell function in vivo.
- Non-satellite cells with myogenic potential.
- Relative contribution of satellite cells versus non-satellite cells to muscle regeneration.

Non-striated smooth muscle:
- Involuntary.
- Controlled by the autonomic nervous system.
- Spindle-shaped single cells.
Striated cardiac muscle:
- Involuntary.
- Controlled by the autonomic nervous system.
- Single (some binucleate), branched cells joined by intercalated discs.
Striated skeletal muscle:
- Voluntary.
- Controlled by the somatic nervous system.
- Multinucleated, un-branched muscle fibers.

Humans have approximately 639 skeletal muscles.
Skeletal muscle composition: Men average 38.4% of mass (~33 kg), women average 30.6% of mass (~21 kg). Source: Janssen et al. 2000 (doi: 10.1152/jappl.2000.89.1.81)
Roles of skeletal muscle:
- Force for movement and breathing.
- Force for postural support.
- Heat production.
- Metabolism.

Each muscle fiber is a single muscle cell.
Muscle fibers have many nuclei (myonuclei) on the periphery, controlling a common cytoplasm (syncytial cell).
Cytoplasm contains myofibrils made of repeating sarcomeres.
Rich blood supply in endomysium/perimysium.
Connective Tissue: Tendon

Vertebrate skeletal muscles originate from four main mesodermal progenitor populations:
- Presomitic mesoderm (PSM): Gives rise to most body musculature through differential contribution of the somitic derivatives; ventral somitic compartments give rise to body wall, limb, tongue, and pharyngeal muscles; dorsal somitic compartments give rise to deep back muscles.
- Lateral plate mesoderm (LPM): Contributes to some mastication muscles following migration through the pharyngeal arches and to the neck musculature.
- Posterior head mesoderm (PHM): Generates progenitors that migrate into the pharyngeal arches; those migrating through the second arch give rise to the muscles of facial expression, while those contributing to the first arch give rise to muscles of mastication and with some contribution to the extraocular muscles.
- Anterior head mesoderm: Contributes progenitors that migrate directly to the eye area to give rise to extraocular muscles.
Limb muscle progenitors migrate from somites to form limbs, tongue, and diaphragm.
- Teleosts fish: cells migrate to form appendicular fin muscle
- Sharks: direct contribution-ventral myotome via finger-like processes extending to form appendicular fin muscle

Muscle precursors (myoblasts) proliferate, differentiate and fuse to form myotubes (immature muscle fibre).
Myotube then become muscle fibres.

5-azaC causes loss of -CH3 on Cytosine in DNA - leads to activation of repressed genes: A single gene can ‘commit’ a cell to myogenesis
Myf5 and MRF4 were found by sequence homology. All are transcription factors that contain the basic helix-loop-helix DNA binding and dimerisation motif
Subtractive cDNA approach to find cDNA present in azamyoblast and C2C12 But absent in 10T1/2
MyoD able to direct 10T1/2 to myogenesis
A family of 4 transcription factors control myogenesis

Myogenic Regulatory Factors (MRFs) are Myf5, MyoD, myogenin, and MRF4.
Basic helix-loop-helix transcription factors.
Control specification and differentiation of skeletal muscle during embryogenesis, growth, and repair.
Invertebrates (e.g., Caenorhabditis elegans, Drosophila) have a single MRF gene.
Vertebrates evolved 4 MRFs to regulate gene expression in myogenesis.

Phylogenetic analysis indicates 4 vertebrate MRF genes evolved from a single ancestral MRF gene by duplication and divergent mutation.
- Myf5 is likely the ancestral gene.
- MRF4 evolved from Myf5 by gene duplication at the locus.
- Myogenin evolved from MRF4 after gene-duplication to another chromosome.
- MyoD evolved from Myf5 after gene-duplication to a third chromosome.
Myf5 or MyoD Is Required for the Formation of Skeletal Muscle
Deletion of either Myf5 or MyoD does not cause major perturbation in muscle formation. BUT deletion of both Myf5 and MyoD results in virtually no myoblasts
Myogenin needed for myogenic differentiation
Myogenin null mice have myoblasts but are severely deficient in muscle differentiation.

Myogenic Regulatory Factors expressed sequentially
Commitment makes cells myoblasts - dividing cells fated to become muscle fibres
Myf5 or MyoD are required for commitment
They can remodel chromatin and maintain their own expression
Myogenesis occurs in two distinct steps: commitment and differentiation
Terminal differentiation produces myotubes which mature into muscle fibres: post-mitotic multinucleate cells that exist for the lifetime of the animal (individual myonuclei turnover)
Myogenin and MEF2 are required for terminal differentiation, myogenin is a target of MyoD
MyoD and myogenin also re-expressed in muscle regeneration

Location of quiescent satellite cell on muscle fibre is between the basal lamina and plasmalemma
Satellite cells generate myoblasts in adult muscle

Endogenous markers are used to identify satellite cells.
- M-Cadherin: satellite cell number varies between muscles.
  - Soleus I / IIa: 26 ± 1% satellite cells, 432 ± 10 myonuclei.
  - EDL IIb / IId: 8 ± 1% satellite cells, 265 ± 4 myonuclei.
Genetic means are also used to identify satellite cells.

Stem cells are defined by their behavior:
- Self-renewal.
- Differentiation (e.g., muscle cells).
- Deregulated self-renewal can lead to tumorigenesis.
- Deregulated differentiation can lead to stem cell depletion.
A few grafted satellite cells can form large amounts of muscle.
A single satellite cell can engraft muscle.
Satellite cells can also be serially transplanted.

Principle of cell therapy: Replace stem cell pool, restore defective muscle repair, uses