lecture 29: muscle stem cells and treating muscular dystrophy

Question 1

What are skeletal muscles?

Answer 1

• ~40% of body weight
• over 600 different types
• movement, posture, breathing
• metabolism
• thermoregulation

Question 2

What is formation of new myofibre from myoblasts?

Answer 2

• postnatal
• myoblasts → proliferation → differentiation and fusion → myotube → mature fibre with miyonuclei, sarcolemma, satellite cell and basement membrane

Question 3

What is duchenne muscular dystrophy?

Answer 3

• 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

Question 4

What is the Mdx mouse model for DMD?

Answer 4

• 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
  *

Question 5

What is myoblast transfer therapy (MTT)?

Answer 5

• 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

Question 6

What are issues for MTT (as gene therapy)?

Answer 6

• 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

Question 7

Where are satellite cells located?

Answer 7

• satellite cells = myoblasts
• on surface of myofibre

Question 8

What happens if you injure muscle?

Answer 8

• proliferation of myoblasts
• widely considered that satellite cells are the major source of myoblasts in damaged muscle fibre → proliferate → fuse to form myotubes → myofibre

Question 9

What are myogenic stem cells?

Answer 9

• “magic” cells that can come from other sources than the satellite cell
• plasticity

Question 10

What are cell types in skeletal muscle?

Answer 10

• 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

Question 11

What are markers for donor myonuclei?

Answer 11

• 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

Question 12

What is the use of a Y-chromosome specific DNA clone to identify donor cells in situ?

Answer 12

• 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

Question 13

What are sliced muscle grafts?

Answer 13

• 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

Question 14

What is rapid death of injected myoblasts in myoblast transfer therapy?

Answer 14

• 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

Question 15

What are cultured primary myoblasts?

Answer 15

• 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)

Question 16

What was seen in Y-1 in situ hybridisation after MTT?

Answer 16

• 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

Question 17

What was seen after intramuscular injection?

Answer 17

• 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

Question 18

What PCR detection of Y-1 DNA after MTT?

Answer 18

• quantification of all donor (male) nuclei in tissue after intramuscular injection
• confirms rapid death of most cultured donor myoblasts within 1 week
• 1. death of culture cells in vivo
     * quantitate remaining male DNA
     * detect number and area of dystrophin positive fibres
• 2. mouse cells can readily transform and become tumorigenic
• shows importance of having at least two markers

Question 19

What does intramuscular injection of cultured myoblasts result in?

Answer 19

• massive and rapid loss of donor nuclei, more than 80% dead by 3 days
• reason: adverse effects of tissue culture (e.g. trypsin) on survival of donor myoblasts in vivo
• applies to many types of culture cells injected in vivo
• (in contrast, grafted whole muscles, muscle segments, and isolated myofibres survive)

Question 20

What are bone-marrow derived stem cells for MTT?

Answer 20

• 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

Question 21

What are cells that can contribute to skeletal muscle repair?

Answer 21

• mesangioblasts
• pericytes
• AC133+ cells, DC133+
• bone marrow MSCs, HSC, ESC
• (fibroblasts, myoblasts, synovial MSCs)

Question 22

What are stem cells to treat DMD?

Answer 22

• translation from animal models to boys
• heterologous (foreign) - muscle fibres will express dystrophin → immune rejection
• autologous (self) → no dystrophin
• autologous (self) → genetically modify to make dystrophin: dystrophin minigene (viral insertion), U7 RNA to skip exons in gene for dystrophin

Question 23

What was seen in golden retriever muscular dystrophy (GRMD)?

Answer 23

• treated with mesangioblasts via the blood to deliver dystrophin gene
• progressive severe disease
• dogs have a very variable phenotype
• Mesoangioblasts to treat dystrophic dogs (2006) →
• intra-arterial delivery of cells
• heterologous (normal donor dogs) → host immunosupression
• autologous → genetically corrected
• problems in interpretation
  
  • no controls to test for beneficial effect of immunosuppressant
  • functional improvement correlation?
  • huge biological variation between dogs
  • form satellite (stem) cells?

Question 24

What are circulating (stem) cells to make muscle?

Answer 24

• bone marrow stem cells (poor efficacy)
• cells associated with blood vessels/endothelial cells
  
  • mesangioblasts derived from blood vessels
  • pericutes
  • CD133+ cells give rise to endothelial and muscle cells

Question 25

What about stem cells from cadavers?

Answer 25

• long live the stem cell: the use of stem cells isolated from post mortem tissues for translational strategies
• Hodgetts S et all (2014)

Question 26

What are strategies to ‘generate’ stem cells?

Answer 26

• inducible pluripotent stem cells (iPS) as an alternative to ESC or somatic nuclear transfer
• which of these 3 can be autologous?
• Nobel Prize in 2012 → Shinya Yamanaka for iPS cells
• ~50 years: special gurdon isssue in Differentiation (2014)
• H. Blau - sir john gurdon: father of nuclear reprogramming
• m buckingham: interactions with john gurdon - muscle as a mesodermal read-out and the community effect
• human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in Dystrophic Mice (2012)
• this study provides Proof of Principle
• intramuscular injection of converted human cells into muscles of (NOD/SCID gamma c) immunodeficient mdx mice

Question 27

What are stem cell problems to solve for translational MTT purposes?

Answer 27

• beware the word ‘potential’
• source of the cells (capacity to expand population)
• heterologous or autologous (gene correction)
• amount of muscle formed
• functional improvement of muscle
• longevity of donor nuclei (repeat treatment)
• functional satellite cells
• systemic delivery (ideal)
• cancers from stem cells