Teacher note: Further background information of relevance to this powerpoint
presentation can be found in pages 183-209 of the outreach manual
Muscle and Stem Cell Regenerative Medicine Power Point Presentation
Notes
Slide 1
The expanding field of Regenerative Medicine offers new hope to patients
suffering from severely compromised tissue structure or function. Muscle and other
connective tissues remain as relevant targets for such therapy. In the Western
Pennsylvania area, the Growth and Development Laboratory is seeking new means of
addressing muscle maladies and of generating novel, effective adult stem cell populations
to enhance a number of regenerative therapies.
Slide 2
A large staff of scientists, clinicians, and support staff is dedicated to
translating basic science into effective clinical applications. Addressing muscle
regeneration remains the major focus, though the application of muscle derived stem cells
(MDSC’s) to additional tissue maladies is also being explored.
Slide 3
Skeletal muscles are the major means by which we interface with the
world, and they are capable of generating amazing force.
Slide 4
Muscles are complex organs consisting of various cell and tissue types
such as nerves, vasculature, myofibers, and connective tissue. Within the muscle
compartment itself, multiple cell types have been identified, some of which are now
being evaluated as therapeutic agents. Within a given myofiber (contractile cell), a
number of molecular complexes have been recognized as essential elements of
contraction and cell structual stability.
Slides 5-6
Contractile force is typically transferred from the internal sarcomere
complexes to the sarcolemma, through the various connective tissue compartments
(endomysium, perimysium, and epimysium), tendon, periosteum, and ultimately to a
skeletal component.
Slides 7-8
These cross sections of skeletal muscle reveal the parallel organization of
bundles of myofibers known a fascicles. The parallel arrangement of these fibers allows
a unidirectional transfer of force.
Slide 9
The transfer of force, and maintenance of cellular integrity in response to
such force, is aided by a complex array of molecules associated with the contractile
apparatus and the sarcolemma. It is reasonable to assume that a disruption of this
network could produce a variety of structural and functional problems. One classic and
devastating variation, aberrant dystrophin molecules, has been identified as the
underlying cause of Duchenne Muscular Dystrohpy (DMD).
Slide 10
Rationale for muscle therapy
Slide 11
As indicated in slide 9, a disruption of the molecular stability apparatus
can have devastating consequences. DMD is a sex linked disorder, and thus
overwhelmingly, a male disorder. The abnormal structure and function of the dystrophin
gene product results in a progressive deterioration of skeletal muscle fibers, leading to
early onset of paralysis, usually culminating in death by the early 20’s. Muscle cross
sections stained with a dystophin-specific fluorescent marker are shown on the right.
Dystrophin is observed in the outer membrane as bright green. Note the relative paucity
of dystrophin in the DMD muscle section (below) compared to a normal section (above).
Slide 12
There are a variety of conditions that could potentially compromise
muscle seriously enough to warrant therapy. Various types of physical traumas and a
host of inherent biological disorders can overwhelm the regenerative capacities of
muscle.
Slide 13
Any injury to muscle typically results in the following repair cascade.
During the first few days, degeneration of muscle fibers occurs, often exacerbated by
characteristic inflammation and other immune system responses. Regeneration follows
for about 2 weeks. Growth factors are thought to influence satellite cell and myoblast
activity, promoting the repair, and sometimes formation of, myofibers. Throughout the
last phase, the formation of scar tissue (fibrosis) limits the extent of functional recovery
and often increases susceptibility to future injury.
Slide 14
Response to trauma.
Slide 15
Associated muscle cell populations such as satellite cells, myoblasts, and
perhaps muscle stem cells allow for the repair and regeneration of myofibers.
Unfortunately, as seen in the next slide, fibrosis remains a limiting factor.
Slide 16
This is a section of a muscle that has partially recovered from the physical
trauma of laceration. Notice the large amount of scar connective tissue, a feature which
can limit the function and future prospects of this muscle.
Slide 17
DMD injury.
Slide 18
DMD presents a constant assault on the muscles of the afflicted individual.
Eventually, this continuing assault on the repair cascade leads to a complete breakdown
of various muscles, causing early death.
Slides 19-20 Two sections of dystrophic muscle. Note the large amount of fibrotic
tissue.
Slide 21
How are compromised muscles treated?
Slide 22
Considering the healing cascade, it is tempting to develop therapies that
limit or reduce degeneration, inflammation, and fibrosis, or to develop means to enhance
regeneration.
Slide 23
Historically, treatments have been of a conservative nature, typically
addressing the need to minimize inflammation and to later promote muscle functionality.
Slide 24
Recently, laboratories such as the Growth and Development Lab have
begun to investigate new types of therapies. These range from new pharmaceuticals to
gene or cellular therapies.
Slide 25
Survey of current therapies and research avenues
Slide 26
Growth factors are being explored for their effects on myoblast
proliferation and fusion (differentiation into myofibers).
Slide 27
The effects of growth factors are being characterized in both in vitro and
in vivo systems. Some have been found to be powerful agents of myofiber differentiation
and growth, and are considered to be among the promising future therapies. An
unfortunate side effect of such research is now being considered. Their dramatic effects
could make them attractive candidates for abuse, especially among athletes, who might
attempt to use them to bolster performance.
Slide 28
Reducing fibrosis is an attractive area of research.
Slide 29
As shown in this muscle healing time course, agents such as TGF-B1 are
being investigated for their ability to reduce the damaging effects of fibrosis.
Slide 30
Various experiments have yielded insights into the role of TGF-B1 in the
fibrotic response.
Slides 31-32 Complex equipment and genetic engineering has been used to produce and
manipulate cell lines for further studies on muscle regeneration. Experiments involving
the addition of a TGF-B1 gene to myoblast lines helped establish the potent role of this
growth factor in fibrosis.
Slide 33
A number of antifibrotic agents are now under investigation.
Slide 34-35 Many of these are designed to modulate the complex TGF-B1 stimulation
or inhibition signal transduction cascades. One example is suramin, a TGF-B1 receptor
antagonist.
Slides 36-37 In an in vivo mouse study, suramin therapy was employed 2 weeks after
calf muscle laceration. Histology studies were performed to assess the number of
regenerating myofibers, and physiologic strength parameters were also evaluated.
Slides 38-39 The data from this experiment clearly showed that increasing amounts of
suramin resulted in lower fibrosis.
Slide 40-41 In another study, the anti-fibrotic agent relaxin also demonstrated success
in limiting fibrosis after muscle injury.
Slide 42
The treatment of biological injury remains a more formidable challenge
for researchers. Multiple muscles and systems are typically effected, necessitating
techniques that address the problem on the cellular or molecular scales.
Slide 43
Myoblast transplantation experiments, utilizing an unpurified population
of muscle associated cells, have been attempted for about 10 years.
Slide 44
Myoblast transplantation has been considered for biology injury such as
DMD. It is hoped that transplanted myoblasts might aid in repair of damaged fibers and
direct the development of fibers containing viable dystrophin genes.
Slide 45
Though myoblast transplantation demonstrated the feasibility of cellular
implantation, therapeutic success was quite limited.
Slide 46
The response of the immune system to implanted cells remains a major
obstacle to this therapy, evidenced by the CD4 and CD8 related immune cell infiltration.
Cells were engineered to express a marker gene (lac Z, yielding blue color if functional in
presence of appropriate substrate). These were implanted into mdx (dystrophic model)
mice and mdx/SCID mice (minus a functional immune response). Increased dystrophin
expression in mdx/SCID mice correlated with a lack of CD4 and CD8 cells infiltrating
into the tissue. This helped demonstrate the role of immune rejection in myoblast
transplantation. In addition, notice that the mdx/SCID mice appeared to possess a much
larger population of surviving implanted cells, evidenced by the larger amount of both lac
Z and dystrophin expression.
Slide 47
The hunt continues for a transplantable cell type with more suitable
biological characteristics. Another population of muscle associated cells has been the
subject of intense investigation. These satellite-like cells have been found to
characteristics of stem cells and have been dubbed “muscle- derived stem cells”, or
MDSC’s.
Slide 48
A technique has been developed for the further purification of these cells
from the other populations associated with the muscle compartment. Known as the
preplate technique, it relies on the enhanced adherence and proleferative properties of the
MDSC’s in comparison to other muscle cell populations.
Slide 49-50
MDSC’s have shown a number of improved features in transplantation
capacity compared to myoblasts. One study in dystrophic mice resulted in the formation
of significantly more dystrophin positive cells when MDSC’s were transplanted.
Slide 51
Fluorescent staining revealed cell markers characteristic of stem cells,
suggesting that these cells were a self-renewing and pluripotent cell line.
Slide 52
MDSC’s have been shown to capable of differentiating into functional
myofibroblasts in injured muscle.
Slide 53-55 MDSC’s or Adult Muscle cells have demonstrated the capacity to
differentiate into cells of the three major lineages, making them an attractive therapeutic
agent for a multitude of maladies.
Slide 56
These cells continue to demonstrate the advantageous features of stem
cells, making them among the most attractive of cell therapeutics.
Slide 57
Gene therapy is another avenue to address the systemic problems of
biological muscle injury. It has the advantage of permanently altering cells to express
therapeutic agents or bioactive proteins to promote muscle regeneration.
Slide 58
Genes can be shuttled into target tissues by various means. In addition,
the vectors can be introduced directly into the body (in vivo), or the vectors can transfect
target cells outside of the body to be later transplanted.
Slide 59
It is thought that MDSC’s could be used in combination with other
therapies to enhance muscle regeneration.
Slide 60-61
Summary
Slide 62-63
Acknowledgment of contributors