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Cellular Physiology > Satellite cells > Flashcards

Satellite cells Flashcards

1
Q

Define satellite cells

A

= Population precursors that provide reserve capacity to replace old, differentiated, post-mitotic cells.

They are the ultimate perfect example of an adult stem cell.

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2
Q

Under what conditions do muscle cells regenerate?

A

During sporadic growth or repair

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3
Q

What is muscle regeneration dependent on?

A

A reserve population of undifferentiated cells

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4
Q

Describe the arrangement of cells in muscle fibres

A

Highly specialised

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5
Q

What is each myofibre associated with?

A

A number of satellite cells

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6
Q

When were satellite cells discovered?

A

1961

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7
Q

Where can satellite cells be found

A

Beneath the basal lamina, closely in contact with plamsalemma

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8
Q

In normal muscle cells, in what state do satellite cells exist in mitotically?

A

Quiescent (dormant)

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9
Q

When and how do satellite cells become activated

A

Following muscle damage or increased workload; become activated to divide in response to signals released after injury

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10
Q

What happens to satellite cells after activation?

A
  1. Can either fuse with each other forming new myofibers
  2. Fuse to an existing muscle fiber donating their nucleus to the fiber,
    3> or return to the quiescent state, replenishing the resident pool of satellite cells through self-renewal
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11
Q

What do satellite cells divide into?

A

Satellite cells progeny = myoblast

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12
Q

Myoblasts undergo a.___________ ___________ and are incorporated in mature muscle. Now, they are called b._________.

A

a. terminal differentiation
b. post-mitotic myonuclei cells

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13
Q

In the quiescent state of satellite cell existence, what myogenic regulatory factor is expressed?

A

Myf5

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14
Q

Do satellite cells conform to the definition of a perfect stem cell?

A
  • In the lab researchers can generate colonies of differentiation-competent progeny in vitro
  • We can do this in the lab by isolating mononucleated cells from adult skeletal muscle = enzymatic degradation
  • Plated and form colonies
  • A proportion of the cells will retain capacity to differentiate = myogenic clones
  • Cells that do not differentiate form fibroblastic type cells that are not stem cells
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15
Q

When satellite cells are activated (exercise), what myogenic regulatory/transcription factor is expressed?

A

MyoD

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16
Q

What is MyoD?

A

= belongs to a family of proteins known asmyogenic regulatory factors (transcription factor).

= one of the earliest markers of myogenic commitment.

= expressed at extremely low and essentially undetectable levels in quiescentsatellite cells.

= BUT expression of MyoD is activated in response to exercise or muscle tissue damage.

= effect of MyoD on satellite cells is dose-dependent:
*high MyoD expression represses cell renewal, promotes terminal differentiation and can induce apoptosis.

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17
Q

What is the trigger for MyoD?

A

= The release of cytokines, neurotrophic factors, growth factors, or oxygen tension (which mediates the hypoxia-inducible gene program such as Hif1α, Hif2α, NO, Vegf, etc.)

= collectively orchestrates and modulates the status of the satellite cell pool.

= Crushed muscle fibers themselves produce growth factors

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18
Q

MyoD is expressed in somites and developing muscle during embryogenesis. Explain the diagram.

A

(A) Whole-mount image of the MyoD promoter (containing the core enhancer and distal regulatory region)-GFP transgenic embryo at E11.5. Note expression in somites and limb muscles.

(B) In situ hybridization of a parasagittal section of a E13.5 mouse embryo using a 35S-labeled MyoD riboprobe. Note expression in back, intercostal, tongue, and limb muscle groups.

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19
Q

What happens to the quiescent satellite cell in response to an injury?

A
  1. Factors activate the satellite cells (Fgfs, Hgf, activated Notch or NICD, Tnf-α)
  2. MyoD is upregulated by this activation
  3. Happens within 2h of injury
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20
Q

Gene expression identifies stem cell and progenitor cell populations in adult skeletal muscle

A
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21
Q

Describe the specialised satellite cell niche (in adult skeletal muscle). Note that A=Electron micrograph of adult skeletal muscle demonstrating the myocyte nucleus (MC) and the satellite cell nucleus (SC) and B = schematic rep of A

A

satellite cell is characterized by its:
1. size (i.e., small),
2. high nuclear to cytoplasm ratio,
3. relative absence of cytoplasmic organelles, and
4. increased nuclear heterochromatin representing a quiescent state of the cell.
5. satellite cell lies between the basal lamina (black arrowhead or green line) and the sarcolemma (white arrowhead or red line).

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22
Q

Discuss the satellite cell response to myotrauma (minor or major, like Duchenne muscular dystrophy), using a diagram to explain the process.

A
  1. In response to an injury, satellite cells become activated and proliferate.
  2. Some of the satellite cells will reestablish a quiescent satellite cell pool through a process of self-renewal.
  3. Satellite cells will migrate to the damaged region and, depending on the severity of the injury, fuse to the existing myofiber or align and fuse to produce a new myofiber.
  4. In the regenerated myofiber, the newly fused satellite cell nuclei will initially be centralized but will later migrate to assume a more peripheral location.
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23
Q

Name the different discrete stages of muscle repair, including approximate amount of time in days post injury.

A
  • Activation
  • Proliferation
  • Differentiation
  • Maturation
    Schematic outlining the stages of regeneration including the activation of satellite cells (within 2 h of injury), satellite cell proliferative (or transit amplifying cell) stage (cellular proliferation peaks at days 2–3 following injury), differentiation (characterized by the formation of centronucleated myofibers), and maturation
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24
Q

Account for the difference between B and C

A

B: Following an intramuscular injection of cardiotoxin (a myonecrotic agent), ∼70%–90% of the muscle is destroyed. Regeneration by stem/progenitor cells results in the formation of small, centronucleated (slightly basophilic) myofibers between 5 and 7 d post-injury (arrowhead marks the centronucleus of the newly regenerated myofiber)
C: Hematoxylin and eosin-stained transverse section of post-injured skeletal muscle reveals restoration of the cellular architecture within ∼2 wk of injury. The nucleus occupies a peripheral position in fully mature myofibers.

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25
Q

What are the growth factors of crushed muscle fibres?

A
  • Basic-fibroblast growth factor (basic-FGF)
  • Platelet-derived growth factor
  • Transferrin
  • Hepatocyte growth factor
  • Macrophage invasion after injury which also produce factors: these = mitogenic for muscle precursor
    cells
26
Q

Which macrophage growth factors stimulate satellite cells?

A
  • Platelet-derived growth factor
  • Transforming growth factor beta (TGF-B) is a multifunctional cytokine belonging to
    the transforming growth factor superfamily
  • Basic-fibroblast growth factor
  • Leukaemia inhibitory factor (LIF) is a multifunctional cytokine belonging to the interleukin-6 family
    and has been shown to stimulate regeneration of injured skeletal muscle
27
Q

Satellite cells are influenced by a variety of molecules and other cells from various areas of body. It is thus regulated by multiple events. Discuss these events.

A
  • Skeletal muscle secretes insulin-like growth factors I and II (IGF-I and IGF-II),
    which are known to be important in the regulation of insulin metabolism
  • In addition, these growth factors are important in the regulation of skeletal muscle regeneration.
  • IGF-I and IGF-II increase satellite cell proliferation and differentiation in vitro
28
Q

Discuss inflammation within muscle regeneration.

A
  • After skeletal muscle damage, inflammatory cells are recruited to help repair and restore the damaged tissue by interacting with the stem cells resident within the muscle
  • initially neutrophils, a specialized type of white blood cell, invade the area and promote muscle
    damage by releasing reactive chemicals. However they also alleviate damage by degrading damaged muscle using specialized enzymes.
  • monocytes, another specialized type of white blood cell, also enter the tissue from the bloodstream and gives rise to M1 type macrophages
  • they release chemicals to produce inflammation
  • during this time, FAP’s and satellite cells within the muscle proliferate
  • FAPs also promote the expansion of satellite cells
  • within 48 hours of muscle damage, the neutrophils leave
  • after day four the satellite cells differentiate and stop proliferating
  • the FAP’s then begin to die due to the presence of M1 type macrophages; these macrophages then decrease in number, likely giving rise to M2 type macrophages which stimulates satellite cell
    differentiation and supports the survival of remaining FAPs
  • the satellite cells then rebuild the damaged muscle fibre, however muscle regeneration depends
    on the timely change of M1 to M2 macrophages
  • with the correct given switch between M1 to M2 macrophages, the tissue will repair itself efficiently
  • if M2 macrophages expand prematurely, excessive matrix deposition is observed as a result of FAPs survival and differentiation. The tissue is then unable to restore itself
  • researching the relationship between inflammatory cells and stem cells within the muscle is
    crucial to determine how to restore chronically damaged tissue which can often lead to death
29
Q

Tissue-engineered skeletal muscle holds promise as a source of what?

A
  • graft tissue for repair of volumetric muscle loss and
  • as a model system for pharmaceutical testing.
30
Q

What are are proven methods for increased myogenesis in engineered muscle?

A
  • hepatocyte and fibroblast growth factors used to accelerate satellite cell activation and proliferation,
  • followed by addition of insulin-like growth factor as a potent inducer of differentiation
31
Q

Discuss some of the dilemmas with tissue engineered muscle.

A
  • Engineered muscle does not produced forces comparable to native muscle, limiting its potential
    for repair and for use as an in vitro model for pharmaceutical testing.
  • Dexamethasone (DEX), a glucocorticoid that stimulates myoblast differentiation and fusion into myotubes, on tissue engineered three-dimensional skeletal muscle units
  • Addition of DEX to isolated muscle satellite cells in culture can improve functional and structural
    characteristics of tissue engineered skeletal muscle when administered at optimal doses and
    timings. Addition of DEX before induction of differentiation improved myogenic proliferation of
    muscle satellite cells, which subsequently led to increased myogenic differentiation and myotube
    fusion.
32
Q

What is the cause of most myopathies?

A

a molecular mutation that affects the structural or cytoskeletal proteins in
skeletal muscle

33
Q

What is the most common and devastating myopathy?

A

Duchenne Muscular Dystrophy (DMD)

34
Q

What is DMD?

A
  • a recessive X-linked disease
  • results in a null mutation at the dystrophin locus
  • Absence of this cytoskeletal protein =
    renders muscle fibre extremely fragile
  • In response to mechanical stress associated with
    repeated contraction, there is widespread degeneration.
  • The satellite cells respond to the injury by
    repopulating the injured skeletal muscle with defective myofibers lacking dystrophin.
  • Process results in continuous degeneration-regeneration
    cycles
  • Exhausts the
    satellite cell pool.
35
Q

Explain the novel virus-delivery treatment for DMD.

A
  • Duchenne muscular dystrophy primarily affects boys (only seen walking at 18 months)
  • Over time, it destroys muscles and people rarely live past age 30
  • dogs can develop the disease and a team of researchers at the University of Missouri have
    successfully treated dogs with Duchenne
  • you have to deliver your therapeutic gene to every muscle in the body
  • the University of Missouri School of Medicine research team created a micro, unmutated version of this gene and use a common virus to deliver the therapy to every muscle in the body
  • after more than a decade of research Dhawan says clinical trials in humans are the next step
36
Q

Isolation, Culture, and Transplantation of Muscle Satellite Cells

A
  • Muscle satellite cells are a stem cell population required for postnatal skeletal muscle
    development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibres.
  • In adult muscle, satellite cells are normally mitotically quiescent.
  • Following injury, satellite cells initiate cellular proliferation to produce myoblasts, their progenies,
    to mediate the regeneration of muscle.
  • Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy
    for several regenerative diseases including muscular dystrophy, heart failure, and urological
    dysfunction.
  • Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning
    urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibres in the host
    tissues and display partial functional improvement in these diseases.
  • Therefore, the development of efficient purification methods of quiescent satellite cells from
    skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and
    transplantation methods for myoblasts, are essential for understanding the molecular
    mechanisms behind satellite cell self-renewal, activation, and differentiation.
  • Additionally, the development of cell-based therapies for muscular dystrophy and other
    regenerative diseases are also dependent upon these factors.
  • However, current prospective purification methods of quiescent satellite cells require the use of
    expensive fluorescence-activated cell sorting (FACS) machines.
  • Here, we present a new method for the rapid, economical, and reliable purification of quiescent
    satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magneticactivated
    cell sorting (MACS).
  • Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large
    numbers of myoblasts after several passages.
  • These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted
    into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of
    donor-derived cells to regenerating muscle fibres, as well as to satellite cell compartments for the
    examination of self-renewal activities.
  • The overall goal of this procedure is to transplant purified quiescent adult murine skeletal muscle
    satellite cells for the treatment of skeletal muscle injury
  • this is accomplished by first dissecting skeletal muscle from adult mice and enzymatically digesting
    skeletal muscle, and then sorting the quiescent satellite cells by magnetic beads separation
  • next the isolated cells are expanded on collagen coated culture dishes and in the final step the in
    vitro expanded cells are injected into the cardiotoxin treated skeletal muscles of adult mice
  • ultimately, the engraftment and contribution of in vitro expanded satellite cells to the
    regeneration of muscle fibres can be assessed by X gal staining
  • the isolation of quiescent satellite cell which is a muscle stem cell population is essential to the
    understanding of muscle stem cell biology and muscle regeneration, as well as the stem cell
    transplantation for therapies in muscular dystrophies such as Duchene muscular dystrophies
  • main advantage of this technique over existing method like is that our method is rapid,
    economical and reliable purification method of quiescent satellite cells from the adult mouse
    skeletal muscle
37
Q

What are the potential uses of human stem cells and what are the obstacles to overcome?

A
  • Various intrinsic changes in muscle cell regeneration process during ageing
  • This prevents muscle cell from regenerating
  • Cause detrimental effects to ageing individual
38
Q

Dysregulated signalling in ageing are linked concepts. Explain the impact of dysregulated signalling on satellite cells and pathways that are affected.

A
  • Satellite cells in an aged niche are exposed to
    dysregulated signals that lead to:
    a. loss of quiescence and
    b. impaired function to regenerate.
  • The 2 pathways, Notch and Wnt, are
    aberrantly/atypically regulated in ageing muscle
  • work together to maintain cell function and allow
    satellite cells time to repair and regenerate
39
Q

Discuss the role of the Notch pathway in young, healthy muscle.

A

critical for satellite cell activation and self-renewal

40
Q

What happens to the Notch pathway as we age? What are the effects of this?

A
  • As we age, there is a decreased expression of the Notch ligand Delta-1 (on the myofibre cell membrane)
  • The Notch protein is a transmembrane protein (part of it faces intracellularly, part of it faces extracellularly)
  • Ligand proteins, that bind to the extracellular domain of Notch, induce proteolytic cleavage and release of the
    intracellular domain of Notch (NICD), which enters the nucleus to modify gene expression.
41
Q

When does Notch signalling activation occur?

A
  • When the transmembrane Notch receptor interacts with one of its cell-membrane-anchored DSL ligands:
    1. Delta/Jagged,
    2. Serrate, or
    3. Lag2
  • Then, it undergoes a series of cleavages from
    metalloproteases and gamma-secretases –> result: intercellular domain of Notch (NICD) translocates to the nucleus
42
Q

What are metalloproteases? Give and example and its function.

A

= protease enzyme whose catalytic mechanism involves a metal
= Example: Meltrin (plays a
significant role in the fusion of muscle cells during embryo development, in a process known as myogenesis)

43
Q

The Notch pathway in the nucleus interacts with transcriptional repressors. Discuss this.

A
  • active NICD
    interacts with transcriptional repressors of the CSL family (CBF1/RBP-J, Suppressor of Hairless (Su[H]), and Lag-
    1)
  • converts them to transcriptional activators (NICD-CSL).
  • With involvement of other proteins, the CSL-NICD complex regulates target genes (Hey and Hes genes),
  • These encode proteins known to be involved in
    myogenesis
  • Within hours to days post injury there = increased expression of Notch signalling components (Delta-1, Notch-1 and active Notch) on activated satellite cells and neighbouring
    muscle fibres
  • Notch and its ligands are overexpressed in breast cancer, and one method of effectively blocking Notch activity is preventing its cleavage at the cell surface with gamma-secretase inhibitors
44
Q

The Wnt pathway has a role in stem cell regeneration too. Discuss what happens to the Wnt pathway as we age.

A
  • In aging = increased circulating Wnt3a (wingless-type MMTV integration site family member 3a)
  • Circulating Wnt3a stimulates beta-catenin signalling = induces aberrant/atypical fibrogenic differentiation of muscle stem cells and progenitor cells
45
Q

Other than stimulating beta-catenin signalling, what is the other role of Wnt?

A

Wnt antagonizes Notch signalling (critical for satellite cell activation and self-renewal)

46
Q

Discuss Wnt ligands

A
  1. large family of secreted glycoproteins
  2. cysteine-rich
  3. highly hydrophobic
47
Q

Discuss the function(s) of beta-catenin

A

Dual function protein: involved in regulation and coordination of cell-cell adhesion and proliferation

48
Q

Discuss the Wnt signalling cascade

A
  • The Wnt signalling cascade needs soluble Wnt ligands to interact with Frizzled receptors and low density
    lipoprotein receptor-related protein co-receptors (LRP).
  • This coordination stimulates phosphorylation of Dishevelled and inactivates GS3Kß’s
    phosphorylation of ß catenin.
49
Q

List the proteins involved in the Wnt signalling cascade.

A

KRM = transmembrane kremen protein
DKK1= Dickkopf1
SFRP = secreted frizzled-related protein
Dapper1 = inhibits activation by dishevelled protein

50
Q

What is the consequence of Dapper overexpression

A

= overexpression of dapper increases axin and GSK-3, results in decreased soluble beta-catenin

51
Q

Discuss this diagram: UNSTIMULATED

A
  1. LRP5/6 and frizzled present on muscle cell membrane
  2. Inside cell: beta catenin degradation complex which tightly binds beta catenin in complex and can see nucleus with target genes
52
Q

Discuss this diagram: STIMULATED

A
  1. Wnt binds to LRP5/6 and frizzled and two membrane receptors move closer to each other
  2. Dishevelled molecule interacts and binds beta catenin complex to the receptor proteins
  3. Beta catenin = disentangled from degradation complex and it can move into nucleus to act on target genes
53
Q

Discuss this diagram: INHIBITED WNT/B-CATENIN SIGNALS

A
  1. Wnt = bound by ligands; prevents binding to receptors
  2. LRP5/6 can be associated with inhibition molecule; prevents frizzled from associating closely with LRP5/6
  3. DAPA 1 binds to dishevelled and prevents it from integrating with the receptors AND prevents beta catenin degradation complex to lose beta catenin.
  4. Complex stays intact, thus inhibited.
54
Q

Compare and contrast skeletal muscle in the young and in the aged

A

Young:
1. Notch = involved in allowing myoblast proliferation
2. Wnt = involved in myotube formation
3. Net effect = allows an eventual perfect myofibre to form

Old:
1. Notch = downregulated; fewer myoblasts being formed (less proliferation)
2. Wnt = upregulated; causes smaller, atypical myotubes to form
3. Net effect = Myofibres are poorly formed, atypical and damaged; muscle will not be as strong as young, thus prone to tears and injury

55
Q

Muscle cells require both the Notch and Wnt pathways to function optimally. What happens during ageing?

A

The ageing cell niche results in dysregulated Notch and Wnt signalling. Notch is downregulated (less myoblast proliferation) and Wnt is upregulated (smaller myotubes formed). This results in injury-prone, defective muscle tissue.

56
Q

Describe the Wnt/B-catenin pathway in the ABSENCE of Wnt.

A
  1. When Wnt is absent, B-catenin protein is bound by a “destruction complex”, which includes protein kinases CK1gamma and GSK-3beta
  2. The protein kinases phosphorylate B-catenin; this targets the protein for ubiquitination and degradation in the proteosome (thus no B-catenin = free to move into nucleus)
  3. In the absence of B-catenin, transcriptional co-repressors bind to TCF transcription factors
  4. This prevents the expression of certain genes
57
Q

Describe the Wnt/B-catenin pathway in the PRESENCE of Wnt.

A
  1. The Wnt protein binds to the transmembrane receptor protein, Frizzled
  2. This causes a signal to be transmitted across the membrane by Frizzled and LRP, thus activating them
  3. Activation of Frizzled and LRP causes protein kinases CK1gamma and GSK-3beta to associate with the membrane (same as “destruction complex”)
  4. The protein kinases then phosphorylate the tail of the activated LRP
  5. Then, the intracellular signalling protein Dishevelled and the protein Axin = recruited to the cytoplasmic tails of LRP and Frizzled
  6. This recruitment prevents the formation of the “destruction complex”, beaning B-catenin accumulates in the cytoplasm
  7. B-catenin then moves into nucleus
  8. B-catenin binds to TCF, displacing the co-repressors
  9. This enables the target genes to be expressed.
58
Q

Defin B-catenin

A

= subunit of the cadherin protein complex.
= It is a dual function protein, involved in
regulation and coordination of cell-cell adhesion and gene transcription.

59
Q

What is Frizzled?

A

G protein-coupled receptor protein

60
Q

What is Dishevelled (Dsh)?

A

cytoplasmic phosphoprotein

61
Q

What does LRP stand for?

A

Lipoprotein receptor-related protein

62
Q

What is TCF and LEF?

A

TCF (transcription factor) = T-cell factor = LEF = lymphoid enhancer factor

Cellular Physiology (4 decks)

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