lecture 29: muscle stem cells and treating muscular dystrophy Flashcards
1
Q
What are skeletal muscles?
A
- ~40% of body weight
- over 600 different types
- movement, posture, breathing
- metabolism
- thermoregulation
2
Q
What is formation of new myofibre from myoblasts?
A
- postnatal
- myoblasts → proliferation → differentiation and fusion → myotube → mature fibre with miyonuclei, sarcolemma, satellite cell and basement membrane
3
Q
What is duchenne muscular dystrophy?
A
- an X-linked lethal muscle disease
- due to defects in dystrophin / loss of dystrophin
- affects ~1 in 3,000 boys
- progressive muscle necrosis and loss of skeletal muscle
- repeated muscle necrosis → loss of myofibres
- manifests in young boys
- wheelchair by ~12 years
- die by ~20 years
- increase myofibre fragility = increased damage
- increased myofibre necrosis and regeneration
- then muscle replaced by fatty fibrous tissue
4
Q
What is the Mdx mouse model for DMD?
A
- equivalent gene defect with lack of dystrophin
- milder phenotype for limb muscles than DMD
- therapy: ideally replace the defective dystrophin protein or gene
- use molecular (e.g. exon skipping), gene or Cell therapy
*
5
Q
What is myoblast transfer therapy (MTT)?
A
- aims to introduce normal myonuclei (with dystrophin gene) into dystrophic muscle cells, to replace the defective dystrophin within multinucleated myofibres
- takes advantage of myoblast fusion during myogenesis
- unique as gene therapy, since donor and host nuclei within same cell
6
Q
What are issues for MTT (as gene therapy)?
A
- source of donor myoblasts: muscle or other cells
- tracking fate of donor myoblasts: markers for donor myonuclei
- delivery of donor myoblasts: into muscle OR via the circulation
7
Q
Where are satellite cells located?
A
- satellite cells = myoblasts
- on surface of myofibre
8
Q
What happens if you injure muscle?
A
- proliferation of myoblasts
- widely considered that satellite cells are the major source of myoblasts in damaged muscle fibre → proliferate → fuse to form myotubes → myofibre
9
Q
What are myogenic stem cells?
A
- “magic” cells that can come from other sources than the satellite cell
- plasticity
10
Q
What are cell types in skeletal muscle?
A
- the myofibre: satellite cells (and stem cells?)
- outside the myofibre:
- interstitial stem cells (mesenchymal stem cells)
- blood vessel associated cells (mesangioblasts, pericytes)
- circulating cells (bone-marrow derived stem cells, CD133+)
- need to be mindful of this when extracting cells from muscle
11
Q
What are markers for donor myonuclei?
A
- dystrophin protein: made by normal donor myonuclei (1986)
- Y-chromosome probe: identifies male donor nulcei (1991)
- reporter genes: transgenic (LacZ or GFP) donor nulcei (1994)
- we started out myoblast transplantation studies in 1978 - not many good markers around then
12
Q
What is the use of a Y-chromosome specific DNA clone to identify donor cells in situ?
A
- Y-chromosome specific (Y1) probe: nuclear marker
- only in the nucleus of a male cell
- stable
- identifies donor male nuclei
- not tissue specific
- can be used in all strains of mice
- gene expression not required
- used the Y-1 probe to track donor (male) cells and tissues transplanted into female host mice in many papers until 2005
13
Q
What are sliced muscle grafts?
A
- a potential alternative strategy for myoblast transfer therapy
- female host + male graft from skeletal muscle + Y-1 probe analysis
- excellent survival of male donor mpc in muscle grafts (1 year) → striking contrast with rapid death of same donor myoblasts after tissue culture
- limited migration of donor (male) cells into host tissue
14
Q
What is rapid death of injected myoblasts in myoblast transfer therapy?
A
- normal (male) myoblasts isolated from muscles of donor normal male mice
- myoblasts expanded in tissue culture, collected by trypsin (protease)
- donor myoblasts injected into dystrophic host female muscles
- muscles sampled at 12 time-points for up to one year
15
Q
What are cultured primary myoblasts?
A
- female host
- primary myoblasts from skeletal muscle of male donor
- injected myoblasts
- Y-1 probe analysis
- in situ hybridisation on muscle sections (localisation), or
- total DNA extraction and PCR quantification (total donor male nuclei)
16
Q
What was seen in Y-1 in situ hybridisation after MTT?
A
- showing number and location of donor (male) nuclei
- donor cells initially in interstitial space
- Y-1 probe labelled with digoxigenin and detected by antibody/alk phos
17
Q
What was seen after intramuscular injection?
A
- massive and rapid loss of donor cells
- few remain by 1 week
- almost non at 1 month +
- 292 female host muscles examined
- 6 months - the rare male donor nuclei appear to be within myofibres - 2 fields from one muscle
18
Q
What PCR detection of Y-1 DNA after MTT?
A
- quantification of all donor (male) nuclei in tissue after intramuscular injection
- confirms rapid death of most cultured donor myoblasts within 1 week
- death of culture cells in vivo
* quantitate remaining male DNA
* detect number and area of dystrophin positive fibres
- death of culture cells in vivo
- mouse cells can readily transform and become tumorigenic
- shows importance of having at least two markers
20
Q
What are bone-marrow derived stem cells for MTT?
A
- instead of intramuscular injection of cultured donor myoblasts
- bone-marrow derived stem cells to give rise to myoblasts
- great attraction is delivery through the circulation to ALL muscles
- skeletal muscle precursors do not arise from bone marrow cells (1983)
- 15 years later Cossu and colleagues showed that bone-marrow cells COULD become mpc, using LacZ and lineage specific myogenic MLC promoter
- many studies followed using reporter genes and transgenic mice as cell markers for donor cells (circulating)
- often with greatly inflated claims of success
- limitations of these markers
- the vast majority of bone-marrow derived cells integrated into mdx muscle fibres are silent despite long term engraftment
- bone marrow derived stem cells can convert to muscle nuclei in vivo BUT it is a rare event: very low efficiency (less than 1%) → not useful clinically
