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Stem cells in Myopahies
Farzad Fatehi
Stem Cells in Muscles
Stem cell types based on division
potentiation
Differentiation Potential
Number of cell types
Example of stem cell
Cell types resulting from differentiation
Totipotential
All
Zygote (fertilized egg), blastomere
All cell types
Pleuripotential
All except cells of the
embryonic membranes
Cultured human ES cells
Cells from all three germ layers
Multipotential
Many
Hematopoietic cells
skeletal muscle,cardiac muscle, liver cells, all
blood cells
5 types of blood cells (Monocytes,
macrophages, eosinophils, neutrophils,
erythrocytes)
Oligopotential
Few
Myeloid precursor
Quadripotential
4
Mesenchymal progenitor cell
Tripotential
3
Glial-restricted precursor
Bipotential
2
Bipotential precursor from murine
fetal liver
B cells, macrophages
Unipotential
1
Mast cell precursor
Mast cells
Nullipotential
None
Terminally differentiated cell e.g.
Red blood cell
No cell division
Cartilage cells, fat cells, stromal cells, boneforming cells
2 types of astrocytes, oligodendrocytes
Stem Cells in Muscles
• Role of stem cells in muscle diseases:
– Deliver normal genomes to myofibers
• When a donor myogenic cell fuses with a host
myofiber:
– This myofiber will express proteins coded by the
donor and host nuclei, now being called a hybrid
myofiber.
Stem Cells in Muscles
• This allows the expression of a protein whose
deficiency in the myofiber cause a disease, as
observed for dystrophin in patients with Duchenne
muscular dystrophy and in mdx mice (a model of
DMD), for merosin in dy/d mice (a model of congenital
muscle dystrophy), and for dysferlin in SJL mice (a
model of dysferlinopathies; Jackson Laboratory,
Maine).
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Mendell JR, Kissel JT, Amato AA, et al.: Myoblast transfer in the treatment of Duchenne's muscular
dystrophy. N Engl J Med 1995, 333:832-838.
Partridge TA, Morgan JE, Coulton GR, et al.: Conversion of mdx myofibres from dystrophin-negative
to -positive by injection of normal myoblasts. Nature 1989, 337:176-179.
Vilquin JT, Kinoshita I, Roy B, et al.: Partial laminin alpha2 chain restoration in alpha2 chain-deficient
dy/dy mouse by primary muscle cell culture transplantation. J Cell Biol 1996, 133:185-197.
Leriche-Guerin K, Anderson LV, Wrogemann K, et al.: Dysferlin expression after normal myoblast
transplantation in SCID and in SJL mice. Neuromuscul Disord 2002, 12:167-173.
Stem Cells in Muscles
• Some mouse experiments suggested that, in
addition, donor myoblasts can provide a
permanent source of precursor cells in the
host muscles.
Donor cell choice
• Muscle Precursor Cells From Muscular
Sources
– Satellite cells
– Multi-potent stem cells
– Fibroblasts
– Bone Marrow stem cells
– Mesenchymal Stem cells
Satellite cells
• The satellite cell is the most obvious source of
donor cells for the treatment of skeletal
muscle, and it is the most frequently used.
• Studies have suggested that satellite cells are
not the only source of myoblasts in the
skeletal muscle, the other being vascularassociated cells.
Satellite cells
• Myosatellite cells or satellite cells are small
mononuclear progenitor cells with virtually no
cytoplasm found in mature muscle.
• They are found sandwiched between the
basement membrane and sarcolemma (cell
membrane) of individual muscle fibers, and
can be difficult to distinguish from the subsarcolemmal nuclei of the fibers.
Satellite cells
• Satellite cells are able to:
–differentiate and fuse to augment
existing muscle fibers
–to form new fibers
Satellite cells
• In undamaged muscle, the majority of satellite
cells are quiescent;
– They neither differentiate nor undergo cell
division.
• Mechanical strain  satellite cells activated
Satellite cells
• Upon minimal stimulation 
• satellite cells in vitro or in vivo will undergo a
myogenic differentiation program.
• Unfortunately, transplanted satellite cells
have a limited capacity for migration
• And are only able to regenerate muscle in the
region of the delivery site.
Satellite cells
– As such systemic treatments or even the
treatment of an entire muscle in this way is
impossible.
– However, other cells in the body such as pericytes
and hematopoietic stem cells have all been
shown to be able to contribute to muscle repair in
a similar manner to the endogenous satellite cell.
Mesoangioblasts
• The advantage of using these cell types
(pericytes, mesoangioblasts) for therapy in
muscle diseases is that they can be systemically
delivered, autonomously migrating to the site of
injury.
• Particularly successful recently has been the
delivery of mesoangioblast cells into the Golden
Retriever dog model of Duchenne muscular
dystrophy, which effectively cured the disease.
– Amini-Nik S, Glancy D et al. (2011). "Pax7 expressing cells contribute to
dermal wound repair, regulating scar size through a β-catenin
mediated process". Stem Cells 9 (29): 1371-9.
Multipotent stem cells
• They have been considered by some authors
to be precursors of the satellite cells.
• They have:
• A high proliferative capacity
• A potential advantage to be used for
autotransplantation
• Some of their properties may also favor a
potential delivery through the circulatory
system.
Multipotent stem cells
• It was reported that the iM injection of
normal muscle-derived stem cells in mdx
mice produced 10-fold more dystrophinpositive myofibers than the injection of
myoblasts derived from satellite cells.
•
Qu-Petersen Z, Deasy B, Jankowski R, et al.: Identification of a novel
population of muscle stem cells in mice: potential for muscle regeneration. J
Cell Biol 2002, 157:851-864.
Muscle Precursor Cells of Nonmuscular Sources
• Fibroblasts were transformed into myoblasts
either by introducing MyoD1, a master
regulator gene for myogenesis, and by
treating the cells with galectin-1, a lectin
secreted by myoblasts and myotubes.
Bone Marrow-Derived Circulating Cells
• Recent interest in these cells has been based
not only on the search for an alternative
source of myogenic cells but on the hope of
developing a systemic treatment for DMD, ie,
a bone marrow transfusion producing the
passage of myogenic precursors from the
blood to the myofibers in a sufficient number
to be therapeutic.
• Previous evidence in mice indicated that the role
of non-muscular cells for muscle regeneration is
null or negligible.
• However, some hope was raised after dystrophin
expression was observed in some myofibers after
the intravenous infusion of normal hematopoietic
cells in mdx mice and some participation of
donor cells in muscle regeneration after bone
marrow transplantation in normal mice.
Mesenchymal Stem Cells
• The stromal cells that participate in the
supporting structures of bone marrow were
reported to differentiate in vitro into a
mesodermal phenotype, such as skeletal
muscle, and for this reason were called
mesenchymal stem cells.
• Preferential differentiation into skeletal
muscle was reported under specific
conditions in mice.
Mesenchymal stem cells
• It was reported that mesenchymal stem cells
isolated from the synovial membrane of adult
humans have the potentiality to fuse with
myofibers and to remain as satellite cells after
the intramuscular implantation in mice.
Mesenchymal stem cells
• It was also suggested that they have the
capacity to participate in muscle regeneration
after IV injection in the same model.
Route of Donor Cell Delivery
Donor Cell Delivery
• Two routes have been tested for the skeletal
muscle:
– Local
– Systemic
• The challenge is important in myopathies
because the target tissue accounts for more
than half of the body.
Local Delivery
• iM injection is the most frequent method of
delivering donor cells to skeletal muscles.
–It ensures sufficient numbers of donor
cells into the muscle.
–It produces tissue damage 
• ↑ uptake of the donor myoblasts by
regenerating myofibers.
Local Delivery
• However, donor cells do not diffuse
significantly.
 cells fuse mainly with the myofibers reached
by the injection  A narrow track of hybrid
myofibers.
iM injection
• For better fusion  multiple injections
close to one another.
Increasing the Fusion of Donor
Myoblasts With Host Fibers
• Increasing the number of regenerating
myofibers  increases the uptake of the
donor myoblasts into hybrid myofibers, and
inhibiting the participation of host satellite cells
further favors donor myoblasts.
Ways to inhibit host satellite cells
• Ionizing radiation at high doses: the most
method used
• iM injection of myotoxins from snake venom
in mice and in monkey experiments.
• Local anesthetics in mouse experiments.
• Cryodamage  muscle necrosis  killing the
host satellite cells
Systemic delivery
Systemic Delivery
• Donor cell delivery by the bloodstream  it
would be ideal
– Myogenic cells would be distributed through all
skeletal muscles, including those not appropriate
for local injections, such as the diaphragm.
Systemic Delivery
– However, IV and intraperitoneal injections of
myoblasts were unsuccessful, even after extensive
muscle injury.
• Intraarterial injection of a myoblast was more
successful, but only after mechanical injury of
the muscle.
Systemic Delivery
• The systemic delivery of hematopoietic stem
cells, unfractionated bone marrow also
produced fusion with host myofibers, mainly
after muscle injury.
– Ferrari G, Cusella-De Angelis G, Coletta M, et al.: Muscle regeneration by bone
marrow-derived myogenic progenitors. Science 1998, 279:1528-1530.
– Ferrari G, Stornaiuolo A, Mavilio F: Failure to correct murine muscular
dystrophy. Nature 2001, 411:1014-1015.
Donor Cell Survival
Donor Cell Survival
• If an optimal donor cell is appropriately
delivered to the target organ, it remains to
ensure the posttransplantation survival of
those cells.
– Early survival
• During the immediate hours or days after
transplantation, before the acute rejection reaction.
– Long-term survival
– Defining long-term as the period needed for a
clinical benefit, which ideally must be lifelong.
Early Survival
• Many donor cells die rapidly after
transplantation
– During the first 1 to 3 days
• This does not prevent the success of myoblast
transplantation
– Because the phenomenon is limited and the
proliferation of the surviving cells compensates for
the cell death.
Long-Term Survival
• The long-term success of normal myoblast
transplantation is threatened by acute rejection.
• Acute rejection is the mechanism by which donor
alloantigen-reactive cytolytic T lymphocytes
destroy the allograft by a response implicating
recognition of MHC I on the donor cells.
• Acute rejection can be clinically controlled by
immunosuppressants and experimentally avoided
by the development of immune tolerance or by
autotransplantation.
Stem cells in Inherited
Myopathies
Primary Myopathies
• Primary myopathies are characterized by
– Progressive wasting of skeletal muscle that leads to
deterioration of movements and, in the most severe
cases, such as in Duchenne’s muscular dystrophy
(DMD), to complete paralysis and death.
• Most myopathies in which the molecular defect
has been identified are due to mutations
affecting proteins that form a supramolecular link
between the cytoskeleton and the extracellular
matrix, such as dystrophin, the mutated protein
in DMD.
Current treatment
• The current therapeutic approaches to DMD
involve pharmacological suppression of the
inflammatory and immune responses, which
usually provides only modest and temporary
beneficial effects.
Future treatment
• Future approaches depend on:
– Cell therapy
– Gene therapy
• These range from the design of efficient,
nonantigenic gene transfer vectors for in vivo
gene therapy, to pharmacological upregulation of
the synthesis of utrophin, a related protein that
compensates for the loss of dystrophin , to
myoblast transplantation, the focus of this
Perspective.
In Duchenne:
• In 1989, Partridge and his collaborators
showed that iM injection of C2C12 cells, an
immortal myogenic cell line derived from
adult satellite cells, could efficiently
reconstitute dystrophin-positive, apparently
normal fibers in dystrophic mdx mice.
– Yao SN, Kurachi K: Implanted myoblasts not only fuse with myofibers but also survive as
muscle precursor cells. J Cell Sci 1993, 105:957-963.
In Duchenne:
• This finding inspired a number of problematic attempts
in the early 1990s to apply this strategy clinically, but
using non-immortal myoblasts in patients.
• Myogenic cells isolated from immune-compatible
donors were expanded in vitro and injected into
specific muscles of DMD patients.
• All trials failed, for a number of reasons, some of
which could have been predicted.
• C2C12 cells have unlimited life-spans and are syngeneic
with mdx mice, features that are lacking in normal
human donor cells.
In Duchenne: cells succumb soon
• Other difficulties :
• We now know that most myoblasts (up to
99%) succumb soon after injection, due first to
an inflammatory and then to a cell-mediated
immune response.
In Duchenne: Not further migration
• We also know that the cells that survive this
initial catastrophe do not migrate more than a
few millimeters away from the injection site,

– countless injections would be required to provide
a significant distribution of donor cells.
These protocols 
Better designed clinical trials on the
horizon
Clearer definitions of clinically relevant
endpoints
Inflammatory Myopathies
Polymyositis (PM) and Dermatomyositis (DM)
• Polymyositis (PM) and dermatomyositis (DM)
are idiopathic inflammatory muscle disorders
characterised by muscle weakness and fatigue
and by skin involvement in DM.
• Complications can be life-threatening.
• Corticosteroids and immunosuppressant
agents remain the mainstay of treatment, but
there are still a significant percentage of nonresponders and clinical relapses.
PM and DM
• Haematopoietic stem cell transplantation is
performed in patients with refractory PM with
satisfactory clinical efficacy, but the
conditioning regimen for the procedure has
many side effects. So newer and less toxic
treatments are urgently needed for patients
with refractory DM/PM.
• Henes JC, Heinzelmann F, Wacker A, et al. Antisignal recognition particlepositive polymyositis successfully treated with myeloablative autologous
stem cell transplantation. Ann Rheum Dis 2009;68:447–8 .
PM and DM
• In a recent study, the efficacy of mesenchymal
stem cell was checked.
• Dandan Wang; Huayong Zhang; Mengshu Ca. Efficacy of Allogeneic
Mesenchymal Stem Cell Transplantation in Patients with Drug-resistant
Polymyositis and Dermatomyositis. Ann Rheum Dis. 2011;70(7):12851288.
Methods
• A single-arm trial involving 10 patients with
DM/PM who were either refractory to
standard treatment, or had severe systemic
involvement.
• All patients consented and underwent
allogeneic MSCT.
• Clinical and laboratory manifestations were
compared before and after MSCT.
Methods
• Baseline observations included a complete medical history
and detailed physical and laboratory examination.
• Follow-up evaluation was conducted at 1, 2, 3 and 6
months after transplantation, then 6 monthly thereafter.
• Transplantation-related mortality included all deaths
associated with transplantation of MSC, except those
related to recurrence of underlying disease.
• Disease activity was measured by CK, CK-MB, manual
muscle test of eight muscle groups as performed previously
by the same rheumatologist.
• Patient's global assessment of the overall impact of disease
on well-being was rated on a 0–10 visual analogue scale
(VAS).
Conclusion
• Stem cell therapy may be considered as an
option for refractory inflammatory
myopathies.