Flashcards in Muscle Regeneration Deck (100):
why can looking at the developmental pathways in muscle development be useful?
signalling pathways and transcription factors can be redeployed in the regenerative response
at what point in development is skeletal muscle established in the mouse?
8.5-18.5 with a further maturation period postnatally for around 2-3 weeks
what is the regeneration time in mice normally?
why are skeletal muscle such a good model for muscle regeneration?
- they are not needed to survive
- they have amazing regenerative capabilities
- they can regenerate multiple times so the exhaustion of stem cell pools can be investigated
what is the lineage progression of a stem cell normally?
stem cell to progentior to precursor to differentiated cell
when is pax3 normally important in development in mice?
early in development, it is involved in the formation of the somites and the epithelium (?) so before pax7. it is also needed for the delamination of the myoblasts from the comite to form the limbs
when is pax 7 important in mice?
in the second or third founder stem cell line. It is required for the post-natal satellite cel formation. A pax7 mutant are severely deficient in Satellite Cells.
what is the phenotype of a pax3/pax7 double mutant in mice?
lacks body muscle from the mid embryonic stages due to a loss of the founding stem/progenitor populations
what are the Myf factors?
factors involved in the determination of the myoblast identity. These are Myf5, Mrf4, MyoD and maybe Myogenin. They are thought of as the gatekeepers to entry into the myogenic lineage in mice
what is the phneotype of a MyoD/Myf5/Mrf4 triple mutant?
The mice lack myoblasts and muscle fibres but stem/progenitor cells are present in the absenceof myogenic commitment
where do all skeletal muscle in the body arise from?
how is head myogenesis generally different to body myogenesis?
they are different pathways regulating differentiation- pax3 is not expressed
what is the phenotype of a pax3;myrf5 mutant and what does this show?
It uncouples head and body myogenesis- the body does not form but the head does. This shows that there are different pathway sinvolved in myogenesis of the head and body and it also suggests that these two genes act in parallel signalling pathways (?)
where are the muscles in the head generally from?
the cranial paraxial mesoderm or the prechordal mesoderm
what do the myoblasts from the first pharyngeal arch form?
some form cardiac muscle and some form the skeletal muscle of the face
what do the extraocular myoblasts give rise to?
a particular set of 7 muscles in mice (6 in humans)
what is the general consensus about the regulatory networks and pathways involved in the formation of myoblast in development and postnatally?
there is a huge variation in the regulatory pathways involved in patterning different parts of the body and that the location from which the myoblasts descended from is important. Some SC's have greater potency that others and some populations will be able to form certain tissue types due to the different origins (an example could be the cardiac muscles that are formed from the first pharyngeal arch in the face) so these could mean that different stem cell pools can only be used to formed certain tissues due to shared pathways
what is the difference between the myogenic pathway in develpment of the myoblasts of the phayrnegal arches and in the body?
the pathway is started by pax3 is the body but by tbx1 in the arches.
what are the 3 pathways of the phraryngeal arches, body and extraocular skeletal muscles differentiation pathways?
somites: pax3 activates Myf5 and Mrf4 which both activated MyoD and myogenin. Pax3 also activates MyoD which activates Myogenin.
extraocular: does not have pax3 ut instead has pitx2 which activates Myrf5 and Mrf4.
Pharyngeal arches: has pitx2 and Tbx1 in the place of Pax3
what is a common feature of the satellite cell populations of the body?
they are heterogenous and express different levels of "stemness"
what are the roles of the 3 founder cell populations in mice?
founder cells 1: establish the primary muscle mass (mytotome) from E9. These express Pax3 mostly, but not all require pax 3 as mytome does form in pax3 mutants. these cells form the dorsal and ventral lips of the dermomytome
founder cells 2: form later, in the embryonic stage. These are released from the the central dermomyotome into the underlying mytome and express pax3 and 7.
founder cells : these cells leave the ventral dermomytoome of the somites to establish skeletal muscle int he limbs, diaphragm and tongue.
FC2 and 3 are thought to give rise to satellite cells postnatally whereas FC1 are exhausted
what do most satellite cells express in mice?
what is the WNT pathway generally thought to be involved in in development and regeneration?
enlarging the stem cell pool of satellite cells and regulating self renewal after injury
after injury, what do essentially all SCs express?
can some sat cells lose Myf5/MyoD expression after activation, if so what do they continue to express?
yes, Pax7, these go back in self renewal and replenish the stem cell pool
how was the asymmetric cell division found?
10% of pax7 expressing cells had never expressed the flr protein linked with Myrf5 expression so they had never expressed myf5?
what did transplantation experiments using pax7+/Myf5- and pax7+/myf5+ cells show?
pax7+/Myf5- cells were able to repopulate a niche 3-4 times more efficiently and the myf5+ cells had a grearer propensity to differentiate
what factors are involved in activating satellite cells following injury?
TGF-ebat family: insuilin like growth factor 1 and 2, hepatocyte growth factors and interleukin. some of these are also involved in developmental myogenesis
what are some differences between the factors implicated in development and those needed for regeneration?
- inactivation of Myogenin in the adult has no effect but it is implicated in myogenesis in development. Myogenin also has different downstream targets in development and in adults.
- pax3/7 inactivation in adults does not hinder regeneration however these are definitley needed in development for myotome formationa nd satellite cell formation
once it was shown that pax3 and 7 were not needed for regeneration, which factors have been shown to be needed?
Foxk1 and sox15
what is the difference between development and regen in terms of scaffolding and what has been sugegsted?
in development the first founder cells form the scaffolding m yotome but this is not formed in regeneration. it has been suggested that the basement membrane provides guidance and and alignment for the regenerating muscle as this is still intact
what is the idea of satellite cells molecular signatures?
it is thought that different satellite cells have a different molecular signature that is determined by heir developmental history- this could affect the remodelling ability of the cells.
what is the satellite cell developmental niche thought to be?
- it is asymmetric in nature due to one being in contact with the plasmalemma and the other with the basement membrane
- endothelial cells or perciytes of the vasculature might play a key role as over 9%% of staellite cells are subjacent to a vessel
- Sf/Hgf may regulate myoblat prolfieration and differentiation via Met receptor
- the quiscent cells remain unresponsive to the FGF2and FGFr4 that is being presented by the muscle fibre.
what is the general role of notch to myoblasts thought to be? what experimental evidence supports this?
a role in expansion of the myoblast pool. notch mutants show a redcued pax3/pax7 stem/progenitor pool. FUrthermore, in ageing muscle there is decreased notch signalling du to its ligand delta. ... in ageing the number of sat cells decreases but their ability to regen remains the same.
what is the specific role for WNT7a thought to be and what is the evidence for this?
null WNT7a have fewer satellite cells and symmetrical type cell divisions on isolated myofibers. Overexpression results in more satellite cells being produced. WNT7a is part of the non-canonical, cell polarity pathway. The basal lamina of the basement mem brane and the orientati on of the mitotic spindle [ probably play critical roles in mediating the distribution of cell fate regulators selectively to daughter cells in the adult, and perhaps also in the embryo.
because there is no single satellite cell marker, what is the best way to find them?
by using a combination of markers.
what is a reliable SC marker in humans?
give 2 successful experiments on the success of myoblast transplantation?
a normal myoblast was transplanted into an Mdx mouse which lacks dystrophin, this resulted in fusion with the host fibers and increase in dystrophin production
2. an entire myofiber containing 7 myoblasts was transplanted into a mouse and it led to an increase number of new sat cells whch generated more than 100 new muscle fibres.
what are 3 reasons for the failure in success of myoblast transplantation?
low migratory rate, low surival rate, immune response
what happens to pure myoblasts that are kept in vitro to try to increase numbers and what is an explanation for this?
- the stem-like fractions die once kept in culture or become more differeniated in culture
what makes the delivery of myoblast transplants hard?
they cant cross the endothelial wall with makes systemic delivery impossible and prevents possible healing of the diaphragm and cardiac muscle which are critical for patientn survival
what is good about being able to find other myogenic stem cell sources that arent satellite cells?
it would ideaaly be good to find a cell type that can be administered systemically and will then be ablel to be myogenic, unlike stalleite cells which cant cross the endothelial wall
name 6 other myogenic stem cells other than satellite cells
- hamaetopoeitc stem cells
-mesodermal derived cells such as mesoangioblasts
- muscle derived stem cells
what is good about being able to use hameotpoeitc stem cells as a source of myoblasts?
they can be delivered systemically but the likely hood of thisis very low
what cell subpopulation was found to be the population which was myogenic from the hameotpoetic cell pool?
what experiment has suggested CD133+ cells as a potential myogenic cell source?
when injected into the circulation of dystrophic mdx mice, they have been found to contribute to muscle repair, recovery of force and replenishment of the SC pool.
what are the positives concerrning using mesoangioblasts as myogenic cell source?
they can be injected systemically and are able to corss the vessel wall, they are easily transduced with lentiviral vectors so can be easily corrected to produce host specific cells.
what are embyroid bodies?
tridimensional ( 3 cell type) sturctures formed when ES cells are grown in the absence of an embyonic fibroblast feeder layer. they express skeletal heavy chain genes and intramuscular injection of mouse mebryoid bodies cocultures with mdx muscle derived progenitors in mdx mice led to the production of a few donor derived dystrophin positive fibers.
what are the issues with using iPS cells?
postnatally (perinatally) what do satellite cells do?
proliferate and contribute to adult muscle
what part of the somites form the dermomyotome?
what part of the dermomyotome will form the muscle?
the dorsal and ventral lips (check)
from which lip do pax3 expressing cells deliminate to form the limb muslce?
what is the relationship between otch and MyoD?
notch suppresses it- prevent differentiation during pool expansion
what are the 2 factors impliacted in myogenic precursor expansion?
what do Myf5, Myogenin, Mrf4 have the ability to do?
induce myoblast traits in non-muscle cell lines
what does MyoD have the ablity to do?
to transform a selection of cell types such as fibroblats into cells that are capable fo fusing into myotomes
what is the first Myogenic regulatory factor to be expressed in development?
Myf5 but then it is quickly downregulated before being upregaulted in concert withthe other MRFs during the formation of the myotome
what was found from looking at MyoD and Myf5 knockouts?
not all myoblasts express both of them, some only express one or the other and when one cell type is lost, the other type can proliferate and compensate.
where is pax 7 expressed in the dermomytome?
centrally rather than in the lips
what is thought to be the relationship ebtween pax3 and pax7?
they are thought to be able to compensate for eachother
what is the general role of the satellite stem cell niche?
supprot selfrenewal while support quisceence and prevent differentiation
what has proved that the satellite stem cell niche is so important?
attempts to culture progenitor cells have had low survival rates and use in therapy for diease.
what happens when you try to culture and expand satellite cells in vitro?
they become myeoblasts and express the MRFs
the addition of what to ES cells and iPS cells has been shwon to turn them myogneic?
what happens when pax 7 satellite cells are ablated from the stem cell pool?
nothing replenishes them or to repair the tissue. This shows that pax-7 expressing sat cells are the only cells involved in muscle regen
what have WNTs been implicated in?
WNT7a in symm division and expansion of renewable cells that are myf5-. Wnt3a is thought to be expressed after notch signalling and is thought to be required for myoblast differentiation and muscle regeneration/
how have miRNAs been implciated in development?
MiR1 and MirR-20 are involved in the differentiation of myoblasts
what type of cell fusion can occur followinf injruy, during regeneration?
myoblast- myoblast (which can be visualised by the centralisation of the nuclei and the later peripheralisation during myofiber maturation) or myoblast- myofiber fusion
what is regeneration in the adult muscle characterised by/
the rexpression of the embryonic and neonatal myosin heavy chain
how can satellite cells be distinguished?
They are spindle shaped and elongate along the axis of the myofiber, their nuclei are smaller than myonuclei and contain more condensed chromatin. In the adult the S population if quiescent unlike it embyronic counterpart. wwhen activated they migrate along the myofiber
what expression do myoblasts lose during differentaition
what are the characteristics of more stem like satellite cells?
no MRFS, and pax 7 expression is upreg
what experiment involving 2 mice support notch role in terms of stallite cells and ageing?
parabiotic mice whereby young blood and mix with old blood causes an increase in sat cells caused by increased notch signallign brought on by the new blood
what is good about muscle derived stem cells?
they have been shown to engraft more easily than stellite cellsowing ot a better survival and better ability to promote angiogenesis
what is the mdx mouse?
has a sponatenous stop codon n exon 23 and shown muscular dystrophy until around 5 at a critical point, but then they regenerate and are fine and do not die early ( so maybe not a great model for chronic human diseases)
how can you make themdx mouse more similar to human DMD diseaase?
cross with a mouse with mutated satellite cell functioning
what causes duschenne muscular dystrophy?
a mutation in dystrophin
what happens when dystrophin is mutated/ how isits functioning perturbed?
dystrophin stabilises the sarcolemmea during muscle contraction, linking the cytokeleton and extracellular matrix. The dystrophin n-terminal binds to the cytoskeleton actin whereas the c-terminal end binds to the dystrophin-associated glycoportein complex at the sarcolemma. An absence of reduction in dystrophon expression causes disruption of the DGC , leading to contraction induced breakdown of muscle membrane and tissue degen
what are the two main aim for therapies for DMD?
to replace the expression of the mutated gene or repair the mutated gene
what are repair approaches of DM therapy?
- exon skipping
- readthrough strategies for nonsense mutation suppression
- gene targetting and endonucleases
what does exon skipping involve?
- exon skipping is a form of RNA splicing used to cause cells to “skip” over faulty or misaligned sections of genetic code, leading to a truncated but still functional protein despite the genetic mutation. exon skipping is used to restore the reading frame within a gene. exon slipping is used to restore the reding frame within a gene. During splicing the introns are removed and the exons aere joined. The mechanisms behind exon skipping is a mutation specific antisense oligonucleotide . An AON is a synthesized short nucleic acid polymer, around 50 base pairs in length that will bind to the mutation site in the pre-messenger RNA to induce exon skipping. the AON binds to the mutated exon so that when the gene is then translated from the mature RNA, it is skipped over, thus restoring the disurpted reading frame. This allows for the genration of an internally deleted, but largely functional protein.
- around 70% of the mutatuons responsible for DMD lead to a disrupted reading frame, reuslting in a truncated non-functional dystrophin protein. Another, more mild form of the idsease exists called Becker’s muscular dystrophy, which allows the translation of a smaller but partially functional dystrophin. Therefore, exon skipping aims to convert DMD into BMD. this results in the mRBA eing inframe so that a functional but truncated pritein can be made
when cant exon skipping be used?
- However, exon skipping can’t be used for patients that have large deletions of the gene or those with mutations in the regulatory regions or N-/c-terminal regions of dystrophin. moreover, AONs are rapidly cleared from the circulation, meaning that constant therapy may be required. To address this people have looked into in situ production of AONs. Accordingly, chimeric small nuclear (snRNAs) have been designed to shuttle AONs that omit exon 51 viral vector-mediated U1 and U1snRNA expression showed a lon-lasting restoration of dystrophin in vitro and in vivio.
give an exmaple of exon skipping working in practise
- exon 51 skipping underwent clinical experimtation and other trials are currently being developed, including exon skipping of exons other than exon 51. So far it has shown to be safe and can increase by 19% compared to controls
what is the readthrough treatment?
- readthrough The continuation of transcription of DNA beyond a normal stop signal, or terminator sequence, due to failure of RNA polymerase to recognize the signal. Readthrough can also occur in translation, when a mutation has converted a normal stop codon into one encoding an amino acid. This results in extension of the polypeptide chain until the next stop codon is reached, producing a so-called readthrough protein.
- read-through for nonsence mutations is based upon the administration of small molecules that are able to introduce a conformational change in the mRNA structure, thus allowing the ribosomal subunits to substitute a mutation-induced stop codon with a single amino acid. This results in an increased read-through of the premature stop and the production of a full-length preotin. To date two drugs have undergone pre-clinical and clinical investigations: gentamicin and ATaluren. The trials for these have so far been fairly successful
what 2 drugs have been used for read through?
: gentamicin and ATaluren
what is the gene targetting endonuclease approach to therapy?
- can use endonucleases engineered to induce double-strand breaks in specific DNA sequences. Endonucleases are divided into three main groups: meganucleases (MGN), znecfingers and Tal effector nucleases. As a consequence of these, double strand breaks can be made and repaired via homolgous recombination which will use a wild type copy of the gene to replace the gene already within the mutated loci. OR double stranded breaks can be made and repaired by non- homolgpous end joining which will result in micro-insertions/deletions at the sites of the breaks, which eventually restore the reading frame. It has been shown that both MGN and zinc fingers can target the dystrophin gene producing insertions/deletions which restore the reading frame.
what are the gene replacement approahces to therapy?
vector-mediated gene therapy
human artificial chromosomes
what is the vector-mediated gene therapy approach?
an additional functional copy of a named gene is put into a virus by homolgous recmbonation which occurs in a cotransfected cell. This vrus then inserts the gene into the targets cells and causes the expression of the correct coding gene
what are the pros and cons or adeno-associated viral vectors?
- you cant put long sequences in as a vector (limited cloning capacity)
- DNA is not inserted into the genome meaning that expression is transients and diluted out
- BUT you can use micro or mini dystrophons which cause truncated proein expression buta less extreme phenotype.
- there is a high efficiency of transduction of skeeltal and cardiac muscle with AAV- making it one of the best viral vectors of gene therapy of MD
- can do a dual vector system in which the dystrophin is split into two smaller cDNAs which are then joined once expressed- but the delivery effiency is lower- there can be immune reaction to the virus
what are - adenoviruses and herpes simplex virus type-1 based vectors
adenoviral vectord and herpes simplex virus type-1-based vectors can carry large cassettes and have been used to transfer the entire dystrophin cDNA in cells and muscle. However, their use is hampered by their immunogenicity, the stability of transgene expression in vivo and the physical impediment of the myofibre basal lamina [89–94]. More recently, capsid-modified helper-dependent adenoviral vectors have attracted some interest because of their enhanced safely profile and large cloning capacity. Indeed, dystrophic mice showed the stable expression of a marker gene together with improved motor performance and life span upon delivery of the full-length dystrophin cDNA
how can lenitvurises be used?
- lentiviral vectors: have relativley large size and capacity. although their direct injection into muscle also targets progenitor cells, the overall efficiency is limited. lenitvirus vectors are currently being used to genetically modify myogenic stem cells ex vivo, which can then be transplanted into pre-clinical animal models of muscular dyst. However there is a posibble rish of tumorigenicity as a result o insertional mutagenesis
why would non viral gene therapy be preferable?
non-viral gene therapy is based mainly on the use of non-integrating gene delivery tools. Thus one of the manu advnatges of this system over conventional viral vectors is to avoid any risk of immine response owing to viral capsids or other viral proteins and insertional mutagenesis. AMong the non-integrating vectors, the most commonly used vector s are plasmids, human artificial chromosomes and transposons. all of them have large cloning capacity.
how can plasmids be used?
The application of plasmids coding for both truncated and full-length dystrophin is certainly attractive because they are produced more easily and faster than viral vectors. Indeed, previous studies based upon the direct intramuscular injection of plasmids have high- lighted the feasibility of this approach, as well as the need to increase gene-transfer efficiency and to perform long-term analyses [61,106]. Therefore, transvenous high pressure increases the efficiency when
delivering plasmids carrying the dystrophin gene into rodents and primates [107,108]. In addition, the safety of this technique has been recently tested using saline in dystrophic patients . However, this method is less efficient than the systemic injection of AAV vectors and can only be used for certain muscles. An alternative system for improving plasmid transfer into muscle is electroporation [110,111] and transgene expression by local plasmid electroporation into muscle has been demonstrated [99,112]. However, in some cases, this can result in tissue damage and its use is hard to envisage in a large muscle
how can HACs be used?
- they present several advantages as gene therapy tools, mainly owing to their peculiar sturcture which, by mimicking endogenous chromosomes, allows table episomal maintenance in host cells, together with an accurate copy number control. Importantly, they have unlimited cloning capacity (as long as you want). A HAC containing the entire 2.4 mb dystrophin locus including its regulatory elements and its native promoters has been generated.
- the transfer of HAC leads to the genetic correctoion of dystrophin mutations in dystrophic-induced pluirpotent and mesoangioblasts stem cells. In the later case, HAC-corrected dystrophic mesangioblasts have been transplanted into scid/mdx mice, with a significant amerlioration of morphology and function og dystrophic muscles. Recently, mesoangioblasts were also formed from DYS-HAC-corrected DMD iPS cells from the host. Moreover, to apply this strategy to primary human muscle-derived mesoangioblasts, these cells will require an extension of their proliferative capability (i.e. to ensure survival during clonal expansion after HAC transfer). To achieve this, we are currently developing a platform for engineering DMD mesoangioblasts with excisable lentiviral vectors expressing immortalizing genes
why are mesangioblasts good?
- can cross the vessel wall so can be delivered via the blood
- pre clinical models ahve been good
what is the evidence of the potential use of neural stem cels?
- neural stem cells have been shown to differentiate into skeletal muscle when cocultured with skeletal myoblasts or transplanted into regenerating skeletal muscle
what evidence is there for HSC being a potential candidate?hat are the down sides of HSCs ?
transplantation of bone marrow into a transgenic line resulted in b-ga being seen in the injured muscles of the mouse via the circulation
- the frequency of them being recruited is very low
what evidence is there against mesangioblasts?
- although they have been shown to differentiate in culture, they were unable to rescue mdx mice functionality