Transcript Document

Stem cells in Myopahies
Farzad Fatehi
Stem Cells in Muscles
Stem Cells in Muscles
• To potentially achieve a therapeutic objective in
inherited myopathies, cell therapies offer two
complementary actions:
– Deliver normal genomes to myofibers
– Increase the myogenic capital of wasted muscles
• 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).
–
–
–
–
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 and to form
new fibers.
• These cells represent the oldest known adult
stem cell niche, and are involved in the normal
growth of muscle, as well as regeneration
following injury or disease.
Satellite cells
• In undamaged muscle, the majority of satellite
cells are quiescent; they neither differentiate
nor undergo cell division.
• In response to mechanical strain, satellite cells
become activated.
• Activated satellite cells initially proliferate as
skeletal myoblasts before undergoing
myogenic differentiation.
Satellite cells
• Upon minimal stimulation, satellite cells in vitro or in
vivo will undergo a myogenic differentiation program.
• Unfortunately, it seems that transplanted satellite cells
have a limited capacity for migration, and are only able
to regenerate muscle in the region of the delivery site.
– As such systemic treatments or even the treatment of an
entire muscle in this way is not possible.
– 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
• In addition, there has been recent interest in
multipotent stem cells isolated from mouse skeletal
muscles and called muscle-derived stem cells.
• They have been considered by some authors to be
precursors of the satellite cells.
• Those cells were reported to have a high proliferative
capacity, a potential advantage to be used for
autotransplantation of genetically corrected myoblasts.
• Some of their properties may also favor a potential
delivery through the circulatory system.
Multipotent stem cells
• It was indeed reported that the iM injection of
normal muscle-derived stem cells in mdx mice
produced 10-fold more dystrophin-positive
myofibers than the injection of myoblasts
derived from satellite cells, although other
researchers did not observe any difference.
•
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. 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.
Bone marrow Transplant
• A recent analysis of a DMD patient who
received a bone marrow transplantation at
the age of 1 year to treat an X-linked severe
combined immunodeficiency showed that 13
years thereafter only 0.5 to 0.9% of the
myofibers presented donor nuclei.
– Gussoni E, Bennett RR, Muskiewicz KR, et al.: Long-term persistence of
donor nuclei in a Duchenne muscular dystrophy patient receiving bone
marrow transplantation. J Clin Invest 2002, 110:807-814.
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 intravenous injection in the same model.
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, favoring the uptake of
the donor myoblasts by regenerating myofibers.
Local Delivery
• However, donor myoblasts do not diffuse
significantly from their site of implantation.
• Because those cells fuse mainly with the
myofibers reached by the injection, each
single myoblast injection in the primate leads
a narrow track of hybrid myofibers.
iM injection
• For better fusion  multiple injections
close to one another.
• To reach 50% of hybrid fibers in monkeys
 interinjection distance of 1 mm and
3×107 myoblasts per cm3 of muscle are
needed.
Increasing the Fusion of Donor
Myoblasts With Host Fibers
• Increasing the number of regenerating
myofibers during myoblast transplantation
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.
– However, intravenous and intraperitoneal injections of
myoblasts were unsuccessful, even after extensive
muscle injury.
• Intraarterial injection of a myoblast cell line
produced fusion with the myofibers in those
muscles irrigated by the arteries, but only after
mechanical injury of the muscle.
Systemic Delivery
• The systemic delivery of hematopoietic stem
cells, unfractionated bone marrow, and
putative muscle-derived stem cells 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.
Systemic Delivery
• These observations showed that myogenic
cells infused by the bloodstream fuse with
myofibers mainly at the sites where an injury
creates a physical route from the vessels to
the regenerating myofibers.
• In addition, the intravenous route was less
efficient than the intraarterial route.
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.
Early Survival
• Early survival is not well understood.
• Some observations have implicated immune
cells (lymphocyte function associated antigen
[LFA]-1-expressing) in killing many donor
myoblasts.
• Inflammatory cells (neutrophils and
macrophages) may be responsible for killing
the donor cells.
Early Survival
• NK cells and CD8+ cells kill most donor
myoblasts immediately after implantation.
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 major histocompatibility complex
class 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 intramuscular 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,
indicating that countless injections would be
required to provide a significant distribution of
donor cells into the patient’s muscles.
In Duchenne
• Through the years, the work of several
laboratories has focused on these problems
and has produced a stepwise, progressive
increase in the recovery, survival, and
colonization efficiency of injected myoblasts in
mouse models.
In Duchenne: Solutions
• Solutions:
– Partial immune suppression,
– Injection of neutralizing antibodies directed
against surface molecules of infiltrating cells (e.g.,
– Pretreatment of myoblasts in vitro with growth
factors
– Modification of the muscle connective tissue
• All contributed to improved myoblast survival upon
injection in vivo.
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
• Mesenchymal stem cells (MSC) can suppress
the activity of various immune cells and,
moreover, have no or limited immunogenicity.
• Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal
stromal cells. Blood 2007;110:3499–506 .
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).
Results
• Serum CK for eight patients, with abnormal levels at
baseline, decreased significantly by 6 months.
–
–
–
–
–
–
–
–
–
–
baseline: 2958±700 U/l;
1 month: 1274±266 U/l;
2 months: 599±155 U/l;
3 months: 109±20 U/l; 6 months: 401±203 U/l, all p<0.05 vs
baseline value, figure 1A);
in parallel with the amelioration of serum CK-MB (mean±SEM,
baseline: 145±45 U/l;
1 month: 96±34 U/l;
2 months: 67±22 U/l;
3 months: 28±5 U/l;
6 months: 36±10 U/l, all p<0.05 vs baseline value, figure 1B).
Results
• Six patients, who had elevated serum CK and
CK-MB at baseline, completed a 12-month
visit, with significant improvements in serum
CK.
– CK: 708±412 U/l
– CK-MB: 51±25 U/l vs 151±61 U/l
• Although two of these six patients (patient 1
and 3) had disease relapse 6 months after
MSCT.
Results
• Patient 2 had a common cold 3 months after the second MSCT and
his clinical status deteriorated.
• Muscle strength was 1/5 in the lower extremities and 2/5 in the
upper extremities. He developed dyspnoea.
• Disease progressed rapidly and his CK levels increased to thousans
U/l despite treatment with high doses of methylprednisolone (500
mg/day) and intravenous immunoglobulin for immunosuppression.
• The patient died of severe myocarditis.
• Patient 4 had an upper respiratory tract infection 6 months after
MSCT, the disease deteriorated and she developed severe
hydropericardium and hydrothorax, and became dyspnoeic and
died of heart failure. No other adverse event was seen in other
patients during the follow-up period.
Conclusion
• Stem cell therapy may be considered as an
option for refractory inflammatory
myopathies.