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Neuromuscular diseases are caused by functional defects of skeletal muscles, directly via muscle pathology or indirectly via disruption of the nervous system. Extensive studies have been performed to improve the outcomes of therapies; however, effective treatment strategies have not been fully established for any major neuromuscular disease. Human pluripotent stem cells have a great capacity to differentiate into myogenic progenitors and skeletal myocytes for use in treating and modeling neuromuscular diseases. Recent advances have allowed the creation of patient-derived stem cells, which can be used as a unique platform for comprehensive study of disease mechanisms, in vitro drug screening, and potential new cell-based therapies. In the last decade, a number of methods have been developed to derive skeletal muscle cells from human pluripotent stem cells. By controlling the process of myogenesis using transcription factors and signaling molecules, human pluripotent stem cells can be directed to differentiate into cell types observed during muscle development. In this review, we highlight signaling pathways relevant to the formation of muscle tissue during embryonic development. We then summarize current methods to differentiate human pluripotent stem cells toward the myogenic lineage, specifically focusing on transgene-free approaches. Lastly, we discuss existing challenges for deriving skeletal myocytes and myogenic progenitors from human pluripotent stem cells.
Derivation of skeletal myocytes and matured myotubes from human iPSCs using a transgene-free protocol. Human iPSCs can be sufficiently differentiated into myogenic progenitors and myotubes in a defined culture without genetic modification using free-floating spheres (EZ spheres) [59, 67]. (a) Human iPSC-derived myotubes were labeled with multiple myogenic proteins Pax3, Pax7, MyoD, myogenin (MyoG), and myosin heavy chain (MHC), demonstrating colocalization of those proteins in the same field. Some MyoD⁺ nuclei were overlapped with MyoG⁺ nuclei and fused on MHC⁺ myotubes (double arrow: MyoD⁺/MyoG⁺). The other nuclei were not overlapping with MHC but expressed either MyoD or MyoG (arrow: MyoD⁺/MyoG⁻; or arrow head: MyoD⁻/MyoG⁺) (C). Neither Pax3⁺ nuclei (A) nor Pax7⁺ nuclei (B) showed any localization with MyoG⁺ nuclei, which mostly fused on MHC⁺ myotubes. (b) Sarcomere formation in iPSC-derived myotubes. Titin staining revealed that striated patterns were clearly visible in the myotubes at 12 weeks MHC staining in the same cell preparations used for titin labeling. (c) Ultrastructures of iPSC-derived myotubes. After 12 weeks of terminal differentiation, mature sarcomeres were observed to be assembled into myofibrils. Morphological hallmarks, including I-band of actin filaments and A-band with distinct M line across myosin filaments, were clearly visible. Sarcomere Z lines appeared to be reasonably aligned and gave rise to a striated pattern. This figure is reproduced from Jiwlawat et al. [67] (under the Creative Commons Attribution license/public domain).
… 
Derivation of skeletal myocytes and matured myotubes from human iPSCs using a transgene-free protocol. Human iPSCs can be sufficiently differentiated into myogenic progenitors and myotubes in a defined culture without genetic modification using free-floating spheres (EZ spheres) [59, 67]. (a) Human iPSC-derived myotubes were labeled with multiple myogenic proteins Pax3, Pax7, MyoD, myogenin (MyoG), and myosin heavy chain (MHC), demonstrating colocalization of those proteins in the same field. Some MyoD⁺ nuclei were overlapped with MyoG⁺ nuclei and fused on MHC⁺ myotubes (double arrow: MyoD⁺/MyoG⁺). The other nuclei were not overlapping with MHC but expressed either MyoD or MyoG (arrow: MyoD⁺/MyoG⁻; or arrow head: MyoD⁻/MyoG⁺) (C). Neither Pax3⁺ nuclei (A) nor Pax7⁺ nuclei (B) showed any localization with MyoG⁺ nuclei, which mostly fused on MHC⁺ myotubes. (b) Sarcomere formation in iPSC-derived myotubes. Titin staining revealed that striated patterns were clearly visible in the myotubes at 12 weeks MHC staining in the same cell preparations used for titin labeling. (c) Ultrastructures of iPSC-derived myotubes. After 12 weeks of terminal differentiation, mature sarcomeres were observed to be assembled into myofibrils. Morphological hallmarks, including I-band of actin filaments and A-band with distinct M line across myosin filaments, were clearly visible. Sarcomere Z lines appeared to be reasonably aligned and gave rise to a striated pattern. This figure is reproduced from Jiwlawat et al. [67] (under the Creative Commons Attribution license/public domain).
… 
Derivation of skeletal myocytes and matured myotubes from human iPSCs using a transgene-free protocol. Human iPSCs can be sufficiently differentiated into myogenic progenitors and myotubes in a defined culture without genetic modification using free-floating spheres (EZ spheres) [59, 67]. (a) Human iPSC-derived myotubes were labeled with multiple myogenic proteins Pax3, Pax7, MyoD, myogenin (MyoG), and myosin heavy chain (MHC), demonstrating colocalization of those proteins in the same field. Some MyoD⁺ nuclei were overlapped with MyoG⁺ nuclei and fused on MHC⁺ myotubes (double arrow: MyoD⁺/MyoG⁺). The other nuclei were not overlapping with MHC but expressed either MyoD or MyoG (arrow: MyoD⁺/MyoG⁻; or arrow head: MyoD⁻/MyoG⁺) (C). Neither Pax3⁺ nuclei (A) nor Pax7⁺ nuclei (B) showed any localization with MyoG⁺ nuclei, which mostly fused on MHC⁺ myotubes. (b) Sarcomere formation in iPSC-derived myotubes. Titin staining revealed that striated patterns were clearly visible in the myotubes at 12 weeks MHC staining in the same cell preparations used for titin labeling. (c) Ultrastructures of iPSC-derived myotubes. After 12 weeks of terminal differentiation, mature sarcomeres were observed to be assembled into myofibrils. Morphological hallmarks, including I-band of actin filaments and A-band with distinct M line across myosin filaments, were clearly visible. Sarcomere Z lines appeared to be reasonably aligned and gave rise to a striated pattern. This figure is reproduced from Jiwlawat et al. [67] (under the Creative Commons Attribution license/public domain).
… 
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Review Article
Current Progress and Challenges for Skeletal Muscle
Differentiation from Human Pluripotent Stem Cells Using
Transgene-Free Approaches
Nunnapas Jiwlawat,
1
Eileen Lynch,
1
Jeremy Jeffrey ,
1
Jonathan M. Van Dyke,
1
and Masatoshi Suzuki
1,2
1
Department of Comparative Biosciences, University of Wisconsin, Madison, WI, USA
2
The Stem Cell and Regenerative Medicine Center, University of Wisconsin, Madison, WI, USA
Correspondence should be addressed to Masatoshi Suzuki; masatoshi.suzuki@wisc.edu
Received 21 December 2017; Revised 11 February 2018; Accepted 18 February 2018; Published 11 April 2018
Academic Editor: Zhaohui Ye
Copyright © 2018 Nunnapas Jiwlawat et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Neuromuscular diseases are caused by functional defects of skeletal muscles, directly via muscle pathology or indirectly via
disruption of the nervous system. Extensive studies have been performed to improve the outcomes of therapies; however,
eective treatment strategies have not been fully established for any major neuromuscular disease. Human pluripotent stem
cells have a great capacity to dierentiate into myogenic progenitors and skeletal myocytes for use in treating and modeling
neuromuscular diseases. Recent advances have allowed the creation of patient-derived stem cells, which can be used as a
unique platform for comprehensive study of disease mechanisms, in vitro drug screening, and potential new cell-based
therapies. In the last decade, a number of methods have been developed to derive skeletal muscle cells from human pluripotent
stem cells. By controlling the process of myogenesis using transcription factors and signaling molecules, human pluripotent
stem cells can be directed to dierentiate into cell types observed during muscle development. In this review, we highlight
signaling pathways relevant to the formation of muscle tissue during embryonic development. We then summarize current
methods to dierentiate human pluripotent stem cells toward the myogenic lineage, specically focusing on transgene-free
approaches. Lastly, we discuss existing challenges for deriving skeletal myocytes and myogenic progenitors from human
pluripotent stem cells.
1. Introduction
Recent advances in stem cell biology hold great promise for
use in treating and modeling neuromuscular diseases [1].
Neuromuscular diseases aecting the function or develop-
ment of skeletal muscle can arise directly via muscle pathology
or indirectly via disruption of the nervous system. Despite
devastating consequences, no eective treatment strategies
exist in many cases, including muscular dystrophy. Attractive
therapeutic strategies include the replacement of aected
muscle cells with healthy myocytes or progenitor cells,
thereby restoring skeletal muscle function.
Human pluripotent stem cells (PSCs), which include
embryonic stem cells (ESCs) and induced pluripotent stem
cells (iPSCs), represent a robust cell source for developing
cell-based therapies targeting degenerating muscles as well
as modeling neuromuscular disease conditions and for
drug screening in culture. Particularly, iPSC technology
allows creation of patient-derived stem cells, which can
simulate pathophysiological conditions in vitro [2]. These
in vitro models are expected to work as a unique platform
for drug screening and allow comprehensive study of dis-
ease mechanisms.
In the last decade, a number of culture methods for myo-
genic dierentiation from human PSCs have been published
[3]. These include (1) transgene methods employing the direct
manipulation of gene expression and (2) transgene-free
methods employing pharmacologic inhibitors and agonists
Hindawi
Stem Cells International
Volume 2018, Article ID 6241681, 18 pages
https://doi.org/10.1155/2018/6241681
as well as isolated cytokines or other protein-based signals
[3]. In this review, we discuss relevant pathways and events
during skeletal muscle development which have been stud-
ied and manipulated in an eort to derive myogenic cell
types from human PSCs. We then overview recent prog-
ress of the methods for myogenic derivation from human
PSCs, specically focusing on transgene-free approaches.
Finally, we discuss the limitations and potential of these
approaches for future treatment and modeling of neuro-
muscular diseases.
2. Skeletal Muscle Development and
Molecular Networks
2.1. Embryonic Myogenesis and Terminal Dierentiation into
Myobers. During early embryogenesis, the formation of
skeletal muscle begins when the paraxial mesoderm segments
form somites in response to signals from the notochord, neu-
ral tube, and surface ectoderm [4]. The developing somite
then forms the dermomyotome, myotome, and sclerotome.
The cells in the dermomyotome express the paired box
transcription factors Pax3 and Pax7 [47]. The dorsomedial
and ventrolateral portions of the dermomyotome give rise
to the epaxial (primaxial) and hypaxial (abaxial) myotomes,
respectively. Myf5-positive cells in the epaxial myotomes
dierentiate and form the trunk and back muscles. In con-
trast, MyoD-positive progenitors delaminate and migrate
from the hypaxial myotome into the developing limb as
the source of limb muscles. Myf5 and MyoD are expressed
in committed muscle cells and are located in the myotome,
which is formed from the maturation of dermomyotome
lips [810].
The terminal dierentiation of progenitors and myo-
blasts initiates when myogenic progenitors in the dermo-
myotome stop dividing and exit the undierentiated stage
(Figure 1). Pax3- and/or Pax7-positive proliferating progeni-
tors withdraw from the cell cycle once the dierentiation step
is initiated. These progenitors then become committed myo-
blasts expressing Myf5 and/or MyoD and form the nascent
myotubes expressing myogenin and myosin heavy chain
(MHC) (Figure 2(a)). Two waves of myotube formation
occur during skeletal muscle development, sequentially giv-
ing rise to primary and secondary myotubes [4, 11]. Primary
myotubes are generated from the fusion of early myoblasts
and are aligned between muscle tendons to form the basis
for embryonic muscle development. Late-stage myoblasts
proliferate alongside primary myotubes and fuse to form
secondary myotubes. As the secondary myotubes form,
motor axons begin to innervate the embryonic muscle [11].
Single-nucleated myoblasts then fuse with the nearby myo-
tubes to form multinucleated myotubes. Thick-myosin and
thin-actin laments within the myotube begin organizing
and form sarcomeres, the functional units of muscle contrac-
tion. Sequential chains of sarcomeres, called myobrils, align
in maturing myotubes. Mature myotubes contain well-
organized and aligned myobrils which give rise to the char-
acteristic striated pattern of skeletal myocytes (Figures 2(b)
and 2(c)).
2.2. Signaling Molecules for Myogenesis. Myogenesis is deli-
cately regulated by signaling events that inuence prolifera-
tion and dierentiation of stem cells and progenitor cells
[4]. These events are driven by paracrine and/or autocrine
signaling molecules that pattern and generate speciccel-
lular lineages. A number of signaling molecules have been
characterized to play critical roles for specication and
dierentiation from the somite to the myotomes [12, 13]. Sig-
naling molecules can also contribute to terminal dierentia-
tion of myoblasts and myotube formation. These molecules
regulate the expression of myogenic genes and proteins and
inuence the growth and fusion of MHC-positive myotubes.
This section will introduce several signaling molecules criti-
cal for myogenesis; however, this is not an exhaustive list.
Wnt signaling plays a signicant role in the development
of myogenic progenitors in the somite and the formation
of committed myoblasts in later stages of myogenesis. A
diverse family of Wnt proteins is secreted from the neural
tube and ectoderm. Wnt1 [12] and Wnt3a [14, 15] are pro-
duced in the dorsal neural tube, while Wnt7a is expressed
in the dorsal ectoderm [12], and Wnt5a is localized in the
dorsal ectoderm and limb mesenchyme [14]. Wnt ligands
bind to Frizzled (Fzd) receptors and take action through a
canonical (β-catenin) pathway or noncanonical pathways
[16]. In mouse explant cultures, Wnt1 can enhance Myf5
expression and aects epaxial muscle formation. In contrast,
Wnt7a promotes MyoD expression and inuences hypaxial
myogenesis [12, 17]. The initial expression of Pax3 and
Myf5 was decreased in mice lacking both Wnt1 and Wnt3a
[15]. A Wnt antagonist Frzb1 inhibits myogenesis in preso-
mitic mesoderm, but not in mature somites. When Frzb1
was injected in a pregnant mouse, the process of myogenesis
was disturbed by the reduction of Myf5 expression [18].
An inhibitor of Wnt/β-catenin signaling (IWR1-endo)
inhibits myotube formation in murine myotube culture
[19]. Additionally, an inhibition of glycogen synthase kinase
3β(GSK3β) can promote mesoderm dierentiation via acti-
vating Wnt pathways [2022].
Sonic hedgehog (Shh) is secreted from the notochord and
oor plate of the neural tube [23] and regulates myogenic
progenitor proliferation and dierentiation [24]. In zebrash,
the number of Pax3- and Pax7-positive cells was signicantly
increased by a knockdown of the Shh gene [24]. Shh shows
positive eects on muscle development by directing progen-
itor cells to Myf5-/MyoD-positive committed myocytes in
the myotome by downregulating Pax3/Pax7 expression
[25]. A reduced level of Myf5 expression was observed in
Shh-null mice, resulting in a loss of distal limb structures
[26]. Shh also enhances myogenic dierentiation by increas-
ing MyoD expression. An implantation experiment using
A-Gel agarose beads soaked with 100 μg/ml N-Shh in the
lumen of the neural tube showed that Shh activates both
MyoD and a sclerotomal marker, Pax1, in quail embryos
[27]. Shh also promotes sclerotome formation while inhibit-
ing dermatome formation [23].
Fibroblast growth factors (FGFs), including FGF2 (or
basic FGF, bFGF), are critical factors for controlling prolif-
eration and dierentiation of myogenic progenitors and
myoblasts during myogenesis. FGF2 is known to inhibit
2 Stem Cells International
the dierentiation of myogenic progenitors into myotubes
[28, 29], implying that FGF2 could be used to maintain the
progenitors at an immature stage. Interestingly, in murine
myoblast C2C12 cells, inhibition of the mitogen-activated
protein kinase (MAPK) pathway, which is downstream of
FGF, increased the expression of MyoD, myogenin, and
MHC and led to more myoblast fusion [29]. Both paracrine
and autocrine eects of FGFs are proposed, as myocytes have
been found to express both FGF ligands and FGF receptors.
FGF ligands can bind to four FGF receptors (FGFR14) with
varying levels of anity. FGFR14 are transmembrane tyro-
sine kinase receptors capable of activating various down-
stream signaling cascades. FGFR1, 2, and 4 are expressed in
immortalized myoblast cell lines such as mouse Sol 8 cells.
Inhibitory eects of myocyte dierentiation by FGF mole-
cules were only observed when FGFR1 and 2 were presented
in Sol 8 cells. Myogenic dierentiation was stimulated when
FGFR1 signals were inhibited by overexpressing truncated
FGFR1 molecules [28]. Another study using chromatin
immunoprecipitation-on-chip analyses demonstrated that
FGFR4 is a direct downstream target of Pax3 in mouse
embryo [30]. Further studies are necessary to elucidate which
FGFRs are involved in modulating myogenesis. In addition,
application of FGF2 or forskolin to C2C12 mouse myoblasts
resulted in phosphorylation and activation of cyclic AMP
response binding (CREB) protein. A gain-of-function muta-
tion in CREB increased myoblast proliferation [31], indicat-
ing involvement of CREB signaling in myogenesis. Loss of
CREB activity signicantly decreased Pax3, Myf5, and MyoD
expression in mouse embryos [17].
Both bone morphogenetic protein 4 (BMP4) and Notch
enhance progenitor proliferation but inhibit muscle dier-
entiation [25]. BMP4, secreted from the lateral plate meso-
derm, sustains Pax3 expression and delays Myf5 and MyoD
expression in chicken embryos [32]. An increased level of a
BMP4 inhibitor Noggin in the dorsomedial lip of the
dermomyotome of chick embryos inhibits BMP signaling
and increases medial, rather than lateral, somite patterning
[33]. Noggin-soaked bead implants promote muscle dier-
entiation in chick embryos [34]. While BMP4 works as a
secreted factor, an activation of Notch signaling requires
direct cell-cell contacts. The Notch receptor is a single-
pass transmembrane protein. Notch ligands bind to the
extracellular domain of the receptor and then lead to pro-
teolytic cleavage at the intracellular domain. After the intra-
cellular domain is released, it migrates toward nucleases
and modulates the expression of downstream genes [35].
A subset of migrating neural crest cells expresses a Notch
ligand, Delta1. When chick embryo dermomyotomal cells
transiently contact Delta1-expressing cells, expression of
Myf5 and MyoD is activated. However, a prolonged contact
with Delta1-expressing cells reverses the myogenic process
resulting in Pax7-positive progenitor cells [36]. Notch sig-
naling increases proliferation of myogenic progenitors but
inhibits muscle dierentiation by blocking MyoD transcrip-
tional activity [37].
Transforming growth factor beta (TGF-β) and a TGF-β
superfamily protein, myostatin, are known to modulate myo-
genic dierentiation. TGF-βinhibits myogenic dierentia-
tion by suppressing the activity of myogenin [38]. However,
a potent and selective inhibitor for TGF-βtype I receptor
(SB431542) and retinoic acid have been shown to rescue
the negative eect of TGF-βon MHC
+
myotube formation
in C2C12 mouse myoblasts [39]. In mouse embryonic stem
cells, a combination of TGF-βinhibitor (SB431542), a Wnt
activator (BIO), and a Shh inhibitor (erismodegib) increased
the expression of Pax7, Myf5, MyoD, and myogenin and the
number of MHC
+
myotubes [40]. Myostatin (also known as
growth and dierentiation factor-8, GDF-8) aects muscle
cell dierentiation in a manner similar to that of TGF-β.
Dorsomorphin and LDN193189, which inhibit myostatin
activity, signicantly enhance myotube formation when
Myf5 +
MyoD
MyoD+
Myogenin
+
(MyoG)
MHC+
IGF-I
Myostatin
TGF-
BMP
+
Myoblast Committed
myocyte
Nascent
myotube Myobril
Multinucleated
myotube
Myogenic
progenitor
Fusion
hypertrophy
Myober
Pax3+
Pax7+
Sarcomere
organized
myolaments
Figure 1: Skeletal muscle dierentiation in vitro. The terminal dierentiation starts when Pax3
+
and/or Pax7
+
progenitors begin to express
Myf5 or MyoD as committed myoblasts. These myoblasts gradually express myogenin (MyoG) and form single-nucleated nascent
myotubes with myosin heavy chain (MHC
+
). Insulin-like growth factor-I (IGF-I), TGF-β1 inhibitor, and myostatin inhibitors induce
myotube fusion to form multinucleated myotubes. Actin, myosin, and elastic myolaments are arranged to form organized sarcomeres
within the myotubes. Organized sarcomere structures give rise to a striated pattern in the myotubes and represent the functional
contraction unit of muscles.
3Stem Cells International
applied to primary human myotubes and murine myotubes
[41]. Follistatin, another myostatin inhibitor, increased
fusion index and myogenic protein expression (including
MyoD, Myf5, and myogenin) in C2C12 cells [42]. Another
myostatin inhibitor, growth and dierentiation factor-
associated serum factor protein 1 (GASP-1), also enhances
myogenin expression and fusion index in myotubes dieren-
tiated from C2C12 cells [43].
Insulin-like growth factor-I (IGF-I) is produced and
secreted from myogenic cells and regulates muscle dieren-
tiation and growth. Both IGF-I receptors and IGF binding
proteins are dramatically increased in mouse C2 myoblast
cells during muscle dierentiation [44]. IGF-I triggers termi-
nal dierentiation of myoblasts through the MAPK signaling
pathway and increases protein expression of myogenin in
murine C2C12 myotubes [29]. IGF-I, but not IGF-II,
Pax7 MyoG MHC
(A) (B) (C)
MyoD MyoG MHCPax3 MyoG MHC
25 m
(a)
MHC
Hoech st 10 m
Titin
Hoech st 20 m
(b)
10 m
2 m
1 m
Z
I
ZM
A
(c)
Figure 2: Derivation of skeletal myocytes and matured myotubes from human iPSCs using a transgene-free protocol.Human iPSCs can be
suciently dierentiated into myogenic progenitors and myotubes in a dened culture without genetic modication using free-oating
spheres (EZ spheres) [59, 67]. (a) Human iPSC-derived myotubes were labeled with multiple myogenic proteins Pax3, Pax7, MyoD,
myogenin (MyoG), and myosin heavy chain (MHC), demonstrating colocalization of those proteins in the same eld. Some MyoD
+
nuclei
were overlapped with MyoG
+
nuclei and fused on MHC
+
myotubes (double arrow: MyoD
+
/MyoG
+
). The other nuclei were not
overlapping with MHC but expressed either MyoD or MyoG (arrow: MyoD
+
/MyoG
; or arrow head: MyoD
/MyoG
+
) (C). Neither Pax3
+
nuclei (A) nor Pax7
+
nuclei (B) showed any localization with MyoG
+
nuclei, which mostly fused on MHC
+
myotubes. (b) Sarcomere
formation in iPSC-derived myotubes. Titin staining revealed that striated patterns were clearly visible in the myotubes at 12 weeks MHC
staining in the same cell preparations used for titin labeling. (c) Ultrastructures of iPSC-derived myotubes. After 12 weeks of terminal
dierentiation, mature sarcomeres were observed to be assembled into myobrils. Morphological hallmarks, including I-band of actin
laments and A-band with distinct M line across myosin laments, were clearly visible. Sarcomere Z lines appeared to be reasonably
aligned and gave rise to a striated pattern. This gure is reproduced from Jiwlawat et al. [67] (under the Creative Commons Attribution
license/public domain).
4 Stem Cells International
promotes myober fusion and hypertrophy in avian myo-
tubes. This hypertrophy was promoted by increased synthesis
and lower degradation of MHC proteins [45]. Interestingly,
the steroid testosterone can stimulate fusion and hypertro-
phy of primary human myotubes via the IGF-I signaling
pathway [46].
3. Derivation of Skeletal Muscle Cells from
Human PSCs
Cell signaling plays a critical role in all stages of myogenesis.
The timing of expression and the levels of signaling mole-
cules are tightly controlled in order for the dierent stages
of myogenesis to occur smoothly [12, 13]. Accumulated
knowledge of the signaling pathways guiding myogenesis
has aided the creation of a number of methods for deriving
myogenic progenitors and myocytes from human PSCs.
Current methods can be broadly categorized into two
approaches: (1) induction of myogenic dierentiation by
overexpression of myogenic genes (transgene methods) and
(2) derivation of myogenic progenitors under dened culture
using growth factors and/or signaling molecules without
transgenes (transgene-free methods).
3.1. Transgene-Based Approaches to Enhance Myogenic
Dierentiation. Selective induction of myogenic genes, such
as the overexpression of PAX3, PAX7, and MYOD1, has been
used in order to increase the eciency of myogenic dieren-
tiation [3]. As discussed above, these transcription factors
play critical roles in promoting proliferation and dierentia-
tion of myogenic progenitors and myoblasts during embry-
onic myogenesis. Dierent systems of gene expression, such
as lentiviral and piggyback-based approaches, have been
applied to transduce PAX7 [47, 48] and MYOD1 [4952]
genes into human PSCs. The transcription of myogenic genes
can also be controlled by inducible gene expression systems
such as tetracycline or tamoxifen [4752]. These progenitors
can be suciently enriched by uorescence-activated cell
sorting (FACS) if the transgene construct contains a uoro-
phore reporter gene like green uorescent protein (GFP)
and mCherry [47, 49].
One notable advantage of the transgene method is that
transgene-based approaches can secure high eciency of
progenitor preparation (more than 90% in several methods).
Typically, transgene methods yield progenitors more rapidly
than transgene-free methods. However, as these approaches
require an introduction of exogenous genes to the cells, the
resulting cells may not fully reect the normal processes of
progenitor proliferation, dierentiation, and maturation.
Additionally, genetic modication remains a regulatory con-
cern if the progenitors are to be used for cell-based therapy
in patients. As such, myogenic progenitors prepared by
transgene-free methods may be more suitable for transplan-
tation in patients.
3.2. Transgene-Free Approaches: Myogenic Derivation under
Dened Culture Conditions. Recent attempts have been made
to derive myogenic progenitors from human iPSCs and ESCs
under dened culture conditions using specic molecules
secreted as paracrine factors that play important roles in
muscle development (Table 1). These molecules control pro-
liferation, migration, and dierentiation from mesodermal
cells into somite and dermomyotome [25]. FGF2 has been
used at varying concentrations (5100 ng/ml) to direct and
enhance myogenic dierentiation [20, 5361]. Although 10
20 ng/ml FGF2 is commonly used to maintain proliferation
in cell lines or primary cells, during our recent study, we found
that a high concentration of FGF2 (100 ng/ml) signicantly
increased the number of Pax7-positive myogenic progenitors
from human PSCs [59]. Other growth factors such as
insulin-like growth factor-I (IGF-I), epidermal growth factor
(EGF), hepatocyte growth factor (HGF), and platelet-derived
growth factor (PDGF) have also been known to promote myo-
genic progenitor expansion and dierentiation in human
PSCs [57]. IGF-I can enhance myotube hyperplasia and fusion
[62, 63]. IGF-I has been used at a concentration of 250 ng/ml
to enhance terminal dierentiation [5557, 61, 64].
Small molecule inhibitors have also been used to direct
and enhance myogenic dierentiation. GSK3βinhibitors,
such as CHIR99021 [55, 61] and BIO (6-bromoindirubin-
3-oxime) [20], can promote mesoderm induction during
dierentiation by activating Wnt pathways. CHIR99021 sig-
nicantly enhances the expression of mesoderm genes such
as T,TBX6, and MSGN1 in human PSCs [54, 55, 65], indicat-
ing that this selective GSK3βinhibitor can promote meso-
derm dierentiation. While CHIR99021 has proven useful
for in vitro mesoderm dierentiation, it should be noted that
it should only be used in culture for short periods and at a low
concentration due to its toxicity [54, 55]. In fact, a longer
exposure (more than 3 mM for 4 days) or a higher concentra-
tion (10 μM for 2 days) of CHIR99021 results in toxicity in
human PSC cultures [54, 55]. By contrast, a potent and
reversible GSK3βinhibitor (BIO) demonstrates the lowest
toxicity among other GSK3βinhibitors [20]. Further, an ade-
nylyl cyclase activator, forskolin, has been used in a triple
cocktail with FGF2 and a GSK3βinhibitor (BIO) to promote
muscle dierentiation [20].
Inhibitors of BMP type I receptors or TGF-βtype I
receptors, such as LDN193189 [56, 61, 64] and SB431542
[57], have been used to enhance derivation of a myogenic
population from human PSCs. In some protocols, basal
medium supplement of insulin-transferrin-selenium (com-
monly known as ITS) has been used to induce the initial
step of mesodermal specication [53, 55, 66]. Oncostatin,
necrosulfonamide, ascorbic acid, insulin, and dexametha-
sone were recently used in combination with growth factors
and TGF-β1 inhibitors to increase skeletal myocyte deriva-
tion eciency. These small molecules promoted a high per-
centage of skeletal muscle dierentiation (up to 70% MHC
+
myotubes) and shortened the dierentiation period to less
than a month [57]. A Notch antagonist DAPT (γ-secretase
inhibitor) increased MyoD and myogenin gene expression
[65]. A combination of CHIR99021 and DAPT synergisti-
cally enhanced myogenic dierentiation [65]. Additionally,
the rescue eect of LDN193189 and SB431542 mixture was
demonstrated by the reduction of BMP4 levels and an
increase of fusion index when applied to myotubes prepared
from patient iPSCs with Duchenne muscular dystrophy [65].
5Stem Cells International
Table 1: Transgene-free methods of skeletal muscle dierentiation using human pluripotent stem cells.
Reference PSC
type PSC culture
Myogenic progenitor
derivation and
proliferation
Progenitor
purication
Terminal
dierentiation Eciency of myogenic
dierentiation In vivo engraftment Use of disease-specic
iPSCs
Culture condition Culture condition
Barberi
et al. 2007
[66]
ESCs
Feeder-
dependent
protocol
using MEF
Monolayer cells
were plated at 1 ×103
cells/cm
2
on MEF, or
3×10
3
cells/cm
2
on
feeder-free
gelatin-coated plates,
for 3-4 days under
standard ESC culture
conditions. The cells
were then switched
in DMEM/F12
supplemented with
ITS for 20 days, and
in αMEM, 10% FBS for
an additional 14 days.
FACS based on
CD73
+
/NCAM
+
.
Sorted NCAM
+
cells
were grown in
αMEM, 10% FBS
until conuence.
The cells were
dierentiated in serum-
free N2 medium.
6080% of sorted
NCAM
+
cells were
MyoD
+
. At 24 hours
after exposure
to N2 medium,
approximately 7%
and 46% of the total
cells expressed Pax7
+
and MyoG
+
,
respectively.
Upon terminal
dierentiation, MyoG,
desmin, skeletal muscle
actin, and myosin
(MHC) were
identied.
Spontaneous twitching
of myotubes was
conrmed.
ESC-derived cells
(5 ×10
5
cells, CD73
+
/
NCAM
+
cells) were
transplanted into a
muscle injury model
in SCID/Beige mice.
The expression of
reporter proteins
(luciferase and GFP),
human cell-specic
nuclei, and laminin-
positive myobers
were identied in the
grafted muscles.
Awaya
et al. 2012
[53]
ESCs
and
iPSCs
Feeder-
dependent
protocol,
using human
ES cell
maintenance
medium
(hESM)
EBs were formed
by suspension in
hESM for 7 days
and then plated
onto gelatin-coated
tissue culture plates
in ITS medium for
an additional
14 days.
EBs were dierentiated
in skeletal muscle
induction medium
containing 10% FCS
and 5% HS until day
112 of dierentiation.
In some experiments,
dissociated EB cells
(3 ×10
3
cells/cm
2
) were
seeded on collagen type
I-coated plates. On day
49, the medium was
changed to ITS
medium.
xIn the cells migrating
out of the EBs, the
clusters Pax3
+
and
Pax7
+
cells were
randomly distributed at
day 21. Skeletal
myosin-positive
multinucleated
myobers had appeared
within most of the
attached EBs at day 63.
Progenitors (15×10
5
cells) were transplanted
into the muscle of
immunodecient NOG
(NOD/Shiscid/IL-
2Rγnull) mice
following cardiotoxin
injury. Four weeks after
transplant, human
cell-specic laminin
A/C-positive nuclei
were detected in the TA
muscles. Further, the
detection of human-
specic laminin a2
proved that the
transplanted cells
produced human
protein around the
muscle bers into
which they had
integrated.
6 Stem Cells International
Table 1: Continued.
Reference PSC
type PSC culture
Myogenic progenitor
derivation and
proliferation
Progenitor
purication
Terminal
dierentiation Eciency of myogenic
dierentiation In vivo engraftment Use of disease-specic
iPSCs
Culture condition Culture condition
Xu et al.
2013 [20] iPSCs
Feeder-free
protocol
using mTeSR
on Matrigel-
coated plates
EB culture in STEMdi
APEL Medium
supplemented with
10 ng/ml FGF2, 0.5 μM
GSK3βinhibitor BIO,
20 μM forskolin (triple
cocktail) for 7 days.
EB cells were
then cultured on
Matrigel-coated
plates in DMEM,
2% HS for an
additional 29 days.
Under terminal
dierentiation
procedures (day 36),
most of the cells
expressed desmin
(72%) and MyoG
(92%), forming
multinucleated
myobers. Sarcomere
structures were also
conrmed by electron
microscopy.
iPSC-derived myogenic
progenitors (1 ×10
5
cells at day 14 of
dierentiation) were
transplanted into
cardiotoxin-injured
muscles in NSG (NOD/
SCID/IL-2Rγnull)
mice. Human δ-
sarcoglycan expression
in myobers and
colocalization of
human-specic histone
H2A and Pax7 were
characterized in the
grafted muscles.
Borchin
et al. 2013
[55]
ESCs
and
iPSCs
Feeder-free
protocol
using
mTeSR1 on
Matrigel-
coated plates
PSC colonies were
cultured in ITS medium
(DMEM/F12
supplemented with
ITS) in the presence of
3μM GSK3βinhibitor
CHIR99021 for 4 days,
and then in ITS
medium containing
20 ng/ml FGF2 for an
additional 14 days. The
cells were then
maintained in ITS
medium alone for a
further 17 days of
culture in ITS medium
alone.
FACS based on the
expression of HNK,
AChR, CXCR4,
C-MET, following
the dierentiation
for 35 days.
FACS-sorted AChR
+
myocytes and CXCR4
/
C-MET
+
and CXCR4
+
/
C-MET
+
precursors
were plated onto
bronectin/laminin-
coated tissue culture
wells in ITS medium
supplemented with
10 μM ROCK inhibitor
Y-27632. The myocytes
were maintained in ITS
medium with 50 ng/ml
IGF-I. The progenitor
cells were dierentiated
in ITS medium.
In presorting cultures
of CXCR4
/C-MET
+
and CXCR4
+
/C-MET
+
cells isolated at day 35
of dierentiation, >18%
Pax3
+
/Pax7
+
and >8%
MF20
+
muscle cells
were identied. In
postsorting cultures at
day 35, 97% in
CXCR4
/C-MET
+
and
98% in CXCR4
+
/C-
MET
+
were PAX3
+
;
84% in CXCR4
/C-
MET
+
and 96% in
CXCR4
+
/C-MET
+
were
PAX7
+
. After 3 days of
culture, few cells
retained PAX7
expression, whereas all
cells expressed MYH5.
In postsorting cultures
of AChR
+
myocytes, all
AChR
+
cells were
MyoG
+
and MHC
+
at
24 hours after plating.
7Stem Cells International
Table 1: Continued.
Reference PSC
type PSC culture
Myogenic progenitor
derivation and
proliferation
Progenitor
purication
Terminal
dierentiation Eciency of myogenic
dierentiation In vivo engraftment Use of disease-specic
iPSCs
Culture condition Culture condition
Hwang
et al. 2013
[58]
ESCs
(OCT4-
GFP
reporter
line)
Feeder-
dependent
protocol
using MEF
Single cells were
cultured in suspension
on ultra-low
attachment plates to
form EBs for 9 days, in
high glucose DMEM
containing 5% FBS,
2mML-glutamine,
100 nM
dexamethasone,
100 μM
hydrocortisone, 1%
penicillin/
streptomycin, 1 mM
transferrin, 86.1 μM
recombinant insulin,
2 mM progesterone,
10.01 mM putrescine,
and 3.01 mM selenite.
The EBs were then spilt,
transferred, and
cultured on a Matrigel-
coated dish for an
additional 8 days.
The cells growing
out of the EBs were
concentrated by
FACS based on
PDGFRA and
OCT4-GFP.
PDGFRA
+
and
PDGFRA
cells
were expanded in
growth medium,
containing high
glucose DMEM,
10% FBS, 2 mM
L-glutamine, and
1% penicillin/
streptomycin.
PDGFRA
+
and
PDGFRA
cells (1 ×10
4
cells/cm
2
) were plated
on gelatin-coated
culture plates and
dierentiated in high
glucose DMEM
containing 2 mM
L-glutamine, 100 nM
dexamethasone,
100 mM
hydrocortisone, 1%
penicillin/
streptomycin, 1 mM
transferrin, 86.1 mM
insulin, 2 mM
progesterone,
10.01 mM putrescine,
and 3.01 mM selenite
with 10% FBS or
without FBS.
The morphology of
PDGFRA
+
cells
progressively became
more spindle-like and
fused and formed
multinucleated
myotubes
(approximately 30%
MHC
+
) after 14 days of
terminal
dierentiation. In
contrast, little or no
myogenic
dierentiation was
observed in PDGFRA
cells population.
ESC-derived PDGFRA
+
cells were transplanted
into the muscle of
NOD/SCID mice
following cardiotoxin
injury. Human
laminin
+
myobers
were identied after 14
days
posttransplantation.
Hosoyama
et al. 2014
[59]
ESC and
iPSCs
Feeder-
dependent
with MEF
(ESCs) or
feeder-
independent
protocols
(iPSCs)
Sphere-based culture
(EZ sphere) was
maintained in Stemline
medium containing
100 ng/ml FGF2,
100 ng/ml EGF, 5 ng/ml
heparin sulfate for 612
weeks (4284 days).
The spheres were
passaged by mechanical
chopping every week.
Monolayer culture in
high glucose DMEM,
2% B27 serum-free
supplement on poly-L-
lysine/laminin-coated
coverslips.
Before terminal
dierentiation,
progenitors were
approximately 40% and
56% Pax7
+
,
respectively. After 14
days of dierentiation,
the prevalence of Pax7
+
,
MyoD
+
,3661%
MyoG
+
, 24-25% MHC
+
after 14 days of
dierentiation.
Spontaneous
contraction and AChR
+
in myotubes were
conrmed after 25 days
of terminal
dierentiation.
The protocol
was applied to
patient-derived
iPSCs (ALS with
SOD1 or VAPB
mutation, BMD,
and SMA iPSC
lines) for
myogenic
dierentiation.
8 Stem Cells International
Table 1: Continued.
Reference PSC
type PSC culture
Myogenic progenitor
derivation and
proliferation
Progenitor
purication
Terminal
dierentiation Eciency of myogenic
dierentiation In vivo engraftment Use of disease-specic
iPSCs
Culture condition Culture condition
Shelton
et al. 2014
[54] and
2016 [22]
ESCs
Feeder-free
protocol in
E8 medium
Monolayer cells
(1.5 ×10
5
cells per well)
were plated on
Matrigel-coated dishes
in E8 medium
supplemented with
10 μM Y-27632
overnight. Cells were
grown in E6 medium
supplemented with
0.1% CHIR99021,
BMP4, or activin-A for
2 days and then
switched in
unsupplemented E6
medium until day 12.
From day 12 to 20, the
medium was replaced
with StemPro-34
media. Cells were then
returned to E6 medium
from day 20 to 35.
The cells were
dierentiated in N2
medium until the
endpoint of the
experiment.
Skeletal myocytes
were prominent
approximately 37%
Pax7
+
and 14% MHC
+
by day 40 following 5
days of growth in N2
medium. Skeletal
muscle contractions
could be observed at
this time point. When
the cells were left in N2
media until day 50, 43%
Pax7
+
and 47% MHC
+
were identied.
Chal et al.
2015 [56]
and 2016
[64]
ESCs
and
iPSCs
Feeder-free
protocol in
mTeSR1
media
Single cells from PSC
colonies were seeded on
Matrigel-coated plates
(15,00018,000 cells/
cm
2
) in mTeSR
medium supplemented
with Y-27632 for 1 day.
The medium was
changed to a DMEM-
based medium
supplement with ITS,
3μM CHIR99021, and
0.5 μM LDN193189 for
2 days. At day 3, 20 ng/
ml FGF2 was added for
an additional 3 days. At
day 6, the cells were
changed to a DMEM-
based medium
At day 8 in culture, the
medium was changed
to DMEM, 15% KSR,
supplemented with
2 ng/ml IGF-I for 4
days and then
supplemented with
both 10 ng/ml HGF and
2 ng/ml IGF-I after day
12.
After 20 days, the
cultures contained large
elds comprising
MHC
+
and MyoG
+
bers and PAX7
+
cells.
By 4 weeks, ~22%
nuclei were MyoG
+
and
23% of nuclei were
Pax7
+
. The muscle
bers showed
sarcomeres, as
demonstrated by titin
and fast MHC staining.
These striated bers
exhibited spontaneous
twitching. The diameter
of the muscle bers was
~3.5 μm.
9Stem Cells International
Table 1: Continued.
Reference PSC
type PSC culture
Myogenic progenitor
derivation and
proliferation
Progenitor
purication
Terminal
dierentiation Eciency of myogenic
dierentiation In vivo engraftment Use of disease-specic
iPSCs
Culture condition Culture condition
supplemented with
10 ng/ml HGF, 2 ng/ml
IGF-I, 20 ng/ml FGF-2,
and 0.5 mM
LDN193189 for 2 days.
Caron et al.
2016 [57]
ESCs
and
iPSCs
Feeder-free
protocol in
serum-free
M2 medium
Monolayer culture at
2500 cells/cm
2
on
collagen-coated plates
and maintained in
skeletal muscle
induction medium
containing 5% HS,
3μM CHIR99021,
2μM Alk5 inhibitor,
10 ng/ml EGF, 10 μg/ml
insulin, 0.4 μg/ml
dexamethasone, and
200 μM ascorbic acid
for 10 days.
At day 10, cells were
dissociated with trypsin
and replated at 2500
cells/cm
2
onto
collagen-coated plates
and maintained for 8
days in skeletal
myoblast medium
containing 5% HS,
10 mg/ml insulin,
10 ng/ml EGF, 20 ng/ml
HGF, 10 ng/ml PDGF,
20 ng/ml FGF2, 20 mg/
ml oncostatin, 10 ng/ml
IGF-I, 2 μM SB431542,
and 200 μM ascorbic
acid. After 18 days of
dierentiation, cells
were maintained in
myotube medium,
containing 10 μg/ml
insulin, 20 μg/ml
oncostatin, 50 mM
necrosulfonamide, and
200 μM ascorbic acid,
for 7 days.
After 10 days in
skeletal muscle
induction medium,
80% Pax3
+
, 20%
Pax7
+
, and
3040% CD56
+
cells
were identied. At
day 18 (after the second
step of dierentiation),
5060% of the cells
were MyoD1
+
and 20%
desmin
+
. At day 26
(after the third and nal
stage of the process),
5080% of the cells
formed elongated and
multinucleated
myotubes that stained
positive for MyoG,
MHC, dystrophin,
and α-actinin.
FSHD1- or BMD-
aected ESCs were
dierentiated into
MHC
+
myotubes
expressing MyoG.
Patient-derived iPSCs
with FSHD1 were also
tested.
Choi et al.
2016 [65]
ESCs
and
iPSCs
Feeder-
dependent
protocol
using MEF
At day 0, nonadherent
cells were plated on a
gelatin-coated dish (at
1.5 ×10
5
cells per well
of a 24-well plate), in
MEF-conditioned N2
media containing
10 ng/ml FGF2 and
10 μM Y-27632. At day
1, N2 media with 3 μM
To determine the
presence of fusion
component myoblasts,
the dissociated cells
from the CHIR99021-
DAPT culture (days
2530) were also
replated.
At day 30 in the
CHIR99021-DAPT
culture, approximately
63% of cells were
MHC
+
and 61% were
MyoG
+
. The culture
resulted in
dierentiation of
myoblasts into
multinucleated and
The dissociated
CHIR99021-DAPT
culture cells (13×10
6
cells) were transplanted
into the injured TA
muscle of NRG mice.
At 6 weeks after
transplantation, human
nuclei (human-specic
lamin A/C
+
) and
Disease-specic
cellular characteristics
were characterized in
the myotubes from
patient-derived iPSC
lines (FSHD, ALS with
C9orf72 repeats, and
DMD).
10 Stem Cells International
Table 1: Continued.
Reference PSC
type PSC culture
Myogenic progenitor
derivation and
proliferation
Progenitor
purication
Terminal
dierentiation Eciency of myogenic
dierentiation In vivo engraftment Use of disease-specic
iPSCs
Culture condition Culture condition
GSK3βinhibitor
CHIR99021 was added.
At day 4, N2 media
with 10 mM γ-secretase
inhibitor DAPT were
added until day 12. The
resulting cells
(CHIR99021-DAPT
culture) were
maintained in dened
N2 media until day 30.
spontaneously
contractile myotubes
with sarcomere
structures. When the
cells from the
CHIR99021-DAPT
culture were replated,
the attached and
surviving cells were
mono-nucleated at day
2 after replating and
then formed
multinucleated
myotubes at day 10
after replating with
typical striations and
expression of 35%
dystrophin
+
, 37% titin
+
,
and 40% α-actinin
+
.
human-specic
laminin
+
myobers
were detected in the
grafted muscles.
Swartz
et al. 2016
[60]
iPSCs
Feeder-free
culture on
vitronectin-
coated plates
in TeSR-E8.
When iPSC colonies
were ~250400 μmin
diameter (day 1), 1.5%
DMSO in TeSR-E8
medium was added. On
day 0, cells were
cultured in chemically
dened medium
(CDM) supplemented
with 20 ng/ml FGF2,
10 μM LY294002,
10 ng/ml BMP4, 10 μM
CHIR99021 (FLyBC)
for 36 hours. Cells were
then cultured in CDM
supplemented with
20 ng/ml FGF-2 and
10 μM LY294002 (FLy)
for an additional
5.5 days. On day 7, cells
were cultured in MB-1
and 15% FBS for 6 days.
After 10 days of fusion
medium (22 days total
from the start of
dierentiation), the
cells were changed to
N2 medium (DMEM/
F12 supplemented with
1% N2 supplement and
1% ITS).
At day 5, <5% of the
total cells were Pax3
+
mesodermal
progenitors. At day 36,
up to 64% (median
44.8%) of nuclei were
MyoG
+
. A mix of
intermediate- and late-
stage muscle cells as
demonstrated by
desmin
+
and MHC
+
.
After 63 total days in
fusion medium, brief
and spontaneous
contractions in a small
set of myotubes were
observed. Seven to 10
days after the addition
of N2 medium, robust
spontaneous
contractions
throughout the cell
11Stem Cells International
Table 1: Continued.
Reference PSC
type PSC culture
Myogenic progenitor
derivation and
proliferation
Progenitor
purication
Terminal
dierentiation Eciency of myogenic
dierentiation In vivo engraftment Use of disease-specic
iPSCs
Culture condition Culture condition
On day 12, the cells
were cultured in fusion
medium (2% HS in
DMEM).
cultures were observed.
Titin
+
striation was
displayed.
Xi et al.
2017 [61]
ESCs
and
iPSCs
Feeder-free
culture on
Matrigel-
coated plates
in mTeSR1
medium.
On day 1, single cells
from PSC colonies
(25,000 cells/cm
2
) and
seeded on Matrigel-
coated plates in
mTeSR1 medium
containing 10 μM
Y-27632. On day 0, cells
were switched to basal
dierentiation medium
(BDM; DMEM/F12, 1%
ITS and 0.5%
penicillin
streptomycin)
supplemented with
3μM CHIR99021 for 2
days. On day 2, cells
were switched to BDM
supplemented with
200 nM LDN193189
and 10 μM SB431542
for another 2 days. On
day 4, culture medium
was changed to BDM
supplemented with
3μM CHIR99021 and
20 ng/ml FGF2 for 2
days. On day 6,
medium was switched
to a KSR/HGF/IGF-I-
based dierentiation
medium (DMEM,
0.5% penicillin
streptomycin and 15%
KSR, 10 ng/ml HGF,
2 ng/ml IGF-I) for
1421 days.
At day 29, cell
suspension was ltered
through cell strainers to
exclude cell aggregates.
Filtered cells were
resuspended in SkGM2
medium supplemented
with 20 ng/ml FGF-2
and replated at 15,000
20,000 cells onto
Matrigel-coated plates.
Cells were cultured for
710 days until
reaching >70%
conuency, and then
medium was switched
to N2 medium (BDM
containing 1% N2
supplement) for 5 days.
At day 2, ~80% cells
were Pax3
+
. Expression
of myogenic markers
was gradually increased
toward day 20. At day
27, large areas of MHC
+
cells emerged
throughout the culture,
and the majority also
expressed titin. A high
proportion of Pax7
+
,
MyoD
+
, and MyoG
+
was also identied. At
day 44, approximately
58% MHC
+
myocytes
and myotubes were
identied, as well as
cells outside MHC
+
area (6.5% Pax7
+
/
MyoD
, 9.1% Pax7
/
MyoD
+
, and 4.9%
Pax7
+
/MyoD
+
).
12 Stem Cells International
Table 1: Continued.
Reference PSC
type PSC culture
Myogenic progenitor
derivation and
proliferation
Progenitor
purication
Terminal
dierentiation Eciency of myogenic
dierentiation In vivo engraftment Use of disease-specic
iPSCs
Culture condition Culture condition
Hicks et al.
2017 [79]
ESCs
and
iPSCs
Feeder-free
culture on
Matrigel-
coated plates
in mTeSR1
medium.
For direct
dierentiation from
PSCs, two published
protocols (Shelton et al.
2014 [54] and Chal
et al. 2015 [56], listed in
Table 1) were used.
Myogenic progenitors
from day 50 culture
were dissociated and
ltered through
100 mm meshes.
FACS based on
HNK
/NCAM
+
or
ERBB3
+
/NGFR
+
were performed.
Sorted cells could be
grown in SkBM-2
media.
Myogenic progenitors
were induced to
dierentiate in N2
media for 7 days. In
some experiments,
TGF-βinhibitors
(SB431542 or A83-01)
were added in
dierentiation media.
HNK
/NCAM
+
enrichment increased
PAX7 and MYF5
expression by ~1.7-fold
in comparison to
unsorted SMPCs.
When dierentiated in
culture, the number of
MHC
+
cells was
increased in HNK
/
NCAM
+
cells
compared to replated/
unsorted cells. ERBB3
+
/
NGFR
+
progenitors
were enriched for PAX7
and MYF5 by 20-fold in
comparison to
ERBB3
/NGFR
cells.
ERBB3
+
/NGFR
+
progenitors could form
homogenous myotubes
following terminal
dierentiation. TGF-β
inhibitors (SB431542 or
A83-01) signicantly
facilitated myotube
fusion and maturation,
as demonstrated by
MHC protein levels
(MYH1 and MYH8)
and sarcomere
formation.
The enriched cells
(1 ×106 cells per 5 μl,
injected 510 μlof
cells) were transplanted
into the injured or
irradiated TA muscle of
mdx-NRG mice. At 30
days after
transplantation,
human-specic lamin
A/C
+
, spectrin
+
, and
dystrophin
+
myobers
were detected in the
grafted muscle. HNK
/
NCAM
+
sorted cells
survived in the grafted
muscle but did not
improve engraftment in
comparison to unsorted
cells. ERBB3
+
/NGFR
+
progenitors
signicantly increased
the number of
engrafted myobers in
comparison to NCAM
+
sorted cells.
Disease-specic iPSCs
from DMD patients
were used in this study.
The mutation in
DMD-iPSC lines was
corrected by CRISPR-
Cas9 gene editing,
which could restore
dystrophin expression.
AChR: acetylcholine receptor; αMEM: alpha minimum essential medium; ALS: amyotrophic lateral sclerosis; BMD: Becker muscular dystrophy; CXCR4: C-X-C chemokine receptor 4; DMD: Duchenne muscular
dystrophy; DMEM: Dulbeccos modied Eagles medium; DMSO: dimethyl sulfoxide; EB: embryoid body; EGF: epidermal growth factor; ERBB3: receptor tyrosine-protein kinase erbB-3; SC: embryonic stem cell;
FACS: uorescence-activated cell sorting; FBS: fetal bovine serum; FCS: fetal calf serum; FGF-2: broblast growth factor 2; FSHD: facioscapulohumeral muscular dystrophy; FTD: frontotemporal dementia; GFP:
green uorescent protein; GSK3β: glycogen synthase kinase 3b; HNK: human natural killer; HS: horse serum; HGF: hepatocytegrowth factor; ITS: insulin-transferrin-selenium; iPSC: induced pluripotent stem cell;
KSR: knockout serum replacement; MEF: mouse embryonic broblasts; MHC: myosin heavy chain; MYH1, MYH5, or MYH8: myosin heavy chain type 1, 5, or 8; MyoG: myogenin; NCAM: neural cell adhesion
molecule (or CD56); NGFR: nerve growth factor receptor; NOD: nonobese diabetic; PDGF: platelet-derived growth factor receptor; PDGFRA: platelet-derived growth factor receptor-α; PSC: pluripotent stem cell;
SCID: severe combined immunodeciency; SMA: spinal muscular atrophy; SOD1: superoxide dismutase 1; TA: tibialis anterior; TGF-β: transforming growth factor beta; VAPB: vesicle-associated membrane
protein/synaptobrevin-associated membrane protein B.
13Stem Cells International
4. Challenges for the Derivation of Skeletal
Myocytes from Human
PSCs Using Transgene-Free Methods
The evaluation of dierentiation eciency and myocyte
maturity has been inconsistent between studies that focus
on dierentiating skeletal myocytes from stem cells. It would
be of great benet to the eld to establish standards for these
evaluations in order to more directly compare dierentiation
methods. Another challenge facing the eld is that in vitro
stem cell-derived skeletal myocytes often have an embryonic
or perinatal phenotype. Additional bioengineering methods
may be necessary in order to achieve skeletal muscle that is
fully mature and therefore more physiologically relevant to
in vivo skeletal muscle. In this section, we will discuss existing
concerns of the current methods for preparing skeletal myo-
cytes and myogenic progenitors from human PSCs, speci-
cally related to transgene-free methods. However, several
concerns are also applicable to transgene methods.
4.1. Dierentiation Eciency. Compared to when using
transgene protocols, dierentiation eciency of skeletal
myocytes overall still remains low when using transgene-
free approaches. In order for the eld to move forward
toward goals of disease modeling, drug testing, and thera-
peutic development, dierentiation eciency should be
improved. Currently, there is a wide range of reported e-
ciencies due to dierences in reporting methods and the def-
initions used to describe the maturity of myogenic cell types.
It is common to use stains for myogenic markers such as
Pax7, MyoD, myogenin, and MHC. However, there is varia-
tion in how these stains are used to determine eciency.
Some protocols claim a very high eciency rate of myogenic
dierentiation but often use a pooled percentage of Pax7,
MyoD, myogenin, and/or MHC-positive cells. Others with
lower eciency may only be using one of the markers, which
could be dierent from the marker chosen in another study.
Along with the usage of immunocytochemistry for MHC,
the counting of MHC
+
cells in a eld of view, the number
of nuclei per myocyte, and the percentage of nuclei within
myocytes (fusion index) have all been used to evaluate dif-
ferentiation eciency. Often, myocyte density and/or dier-
entiation eciency varies across a culture. Therefore, it is
important to report the number of elds counted and how
they were selectedspecically noting how bias was con-
trolled. Overall, there is a need to standardize methods of
calculating dierentiation eciency in order to facilitate
comparisons between dierentiation protocols.
4.2. Dening and Measuring the Extent of Myotube
Maturation. In recent years, there have been a number of
culture methods developed that yield MHC-positive skeletal
myocytes from human pluripotent cells. Many of them
require an extended culture period in comparison to methods
for deriving other cell types. A method yielding myogenic
progenitors or mature myocytes after a relatively short time
would be of high signicance to the eld. However, it is also
important to evaluate the maturity of cells yielded from rapid
preparations. To date, it is dicult to compare the maturation
state of myocytes generated by dierent methods due to dif-
ferences in how each study denes maturity. Some focus on
anatomical features, while others examine physiological func-
tionality. Ideally, both aspects should be considered when
evaluating myotube maturity. Studies taking an anatomical
approach tend to use immunocytochemistry or electron
microscopy to evaluate sarcomere formation and myobril
alignment as indicators of myotube maturity. Immunocyto-
chemistry using antibodies against MHC or titin is a relatively
accessible method to detect striations (Figure 2(b)); however,
electron microscopy makes it possible to visualize sarcomeres
at an ultrastructural level and examine sarcomeric organiza-
tion and alignment (Figure 2(c)). It should be noted that
in some preparations of maturing human PSC-derived
myocytes, the results of immunocytochemical labeling of
sarcomeric proteins (such as titin) may not correlate with
ultrastructural results obtained through electron micros-
copy [67].
Some, but not all, studies take a physiological approach to
determine myocyte maturation by examining the functional-
ity of the cells. One method is to measure the frequency and
coordination of spontaneous contractions observed in the
dierentiating myocytes. Contractions can be stimulated by
a calcium ux or with addition of acetylcholine to the culture.
Spontaneous contractions can also be observed shortly after
culture medium changes [59, 67]. To properly examine spon-
taneous contractions, it should be taken into consideration
when the cells were last given fresh media or were supple-
mented with the compounds that can promote contractions.
Electrophysiology has also been used to monitor contractions
and record contraction frequency and strength in cultured
myotubes [48, 68]. Further, calcium imaging using dyes such
as Fluo-3AM can be applied as an alternative method to elec-
trophysiology. With a wide variety of methods currently
being used, it is necessary to establish a preferred method
of assessing physiological maturity of in vitro myocytes to
better compare derivation methods. Another aspect of myo-
cyte maturity is the ber type expressed. During myogenesis,
embryonic and slow type I MHC are expressed rst. Then at
later stages of maturation, myobers develop glycolytic fast
twitch MHC types IIa, IIb, and IIx [24, 69]. Commonly,
MHC expression is examined using an antibody that reacts
to all isoforms of MHC (such as MF20 clone), but a more
detailed evaluation of MHC type would be useful for describ-
ing derived myocyte maturation.
In order to use human PSC-derived myocytes for in vitro
modeling for adult-onset neuromuscular diseases, it is neces-
sary to generate fully matured myotubes. However, iPSC-
derived skeletal myocytes prepared using current methods
typically are of an embryonic or perinatal phenotype. In
addition to better understanding signaling molecules and
the timing required for generating mature myocytes, bioengi-
neering techniques will be needed to create surfaces recog-
nized by human PSC-derived myocytes as appropriate for
growth and maturation. Dierentiation eciency can likely
be improved by controlling features such as surface coatings,
adhesion ligands, and/or growth surfaces that encourage
directionality and elongation. For instance, micropatterned
surfaces can give myocytes much needed directionality [70].
14 Stem Cells International
It is likely that most two-dimensional culture environments
are not similar enough to in vivo and that three-dimensional
constructs will become necessary to encourage further stages
of maturation [67]. Cocultures with motor neurons may
support myotube maturation, as stimulation is required for
proper contractility in vivo [71].In the absence of motor
neurons, myotubes can be chemically stimulated to contract
by adding acetylcholine to the culture [72]. Electrical stimu-
lation can also induce contractions and enhance maturation
of myotubes [49, 73]. Further, mechanical stimulation may
accelerate muscle dierentiation and maturation [74].
4.3. Cell Enrichment and Large-Scale Expansion. While the
way we report the eciency of myogenic dierentiation is
valuable, it is also important to improve upon current
methods to gain a pure population of myogenic progenitors
and skeletal myocytes in culture. When prepared by a
transgene-free method, the cultures commonly contain a
heterogeneous cell population with myocytes and other cell
types. Such heterogeneity inuences the eciency of in vivo
engraftment following transplantation [75]. In order to
improve dierentiation eciency, there is a need for more pre-
cise denition of which signal molecules to use and the timing
of their use, but improved cell sorting techniques will also be
necessary to further enrich derived myocytes. Fluorophore-
labeled progenitors can easily be puried by FACS, if genetic
modication is used [47, 49]. Also, several combinations of
specic cell surface markers can be used to enrich myogenic
progenitors and skeletal myocytes [55, 7678]. Examples
include combinations of CD54
+
/integrin α9β1
+
/SDC2
+
[76],
CD45
/CD11b
/GlyA
/CD31
/CD34
/CD56
int
/ITGA7
hi
[77],
CD56
+
/CD15
[78], CXCR4
+
/C-MET
+
[55], and HNK
/
NCAM (CD56)
+
[65, 79]. The most recent study indicated
that a combination of two surface markers (ERBB3 and
NGFR) can be applied to suciently purify a specic cellular
population of human PSC-derived myogenic progenitors by
FACS [79].
Another important consideration when developing deri-
vation methods is whether they are adaptable to a large-scale
expansion of myogenic progenitors and skeletal myocytes.
Limited scalability seems to be a continued challenge among
methods [75], which limits practical application and transla-
tion to patients as cell-based therapies. Often, cells are main-
tained in small quantities as a monolayer culture that is not
always suitable for passaging. A recent study indicated that
animal serum could promote cell expansion in PSC-derived
myogenic progenitors, but the culture condition remained
less dened [56]. However, a sphere-based culture may work
to overcome this concern [59, 67]. As demonstrated in our
recent study, human PSC-derived spherical cultures can be
expanded for several weeks with specic signaling molecule
supplementation in the medium [59, 67].
5. Conclusions
Valuable knowledge regarding the dierentiation of myo-
genic progenitors and myotubes from human PSCs has been
gradually accumulating [1, 3, 8082]. Signaling molecules
signicantly contribute to generating a sucient number of
myogenic progenitors and myocytes from human ESCs and
iPSCs without genetic modication. In addition to directing
and enhancing dierentiation of myogenic cells using signal-
ing molecules, recent bioengineering approaches such as
two-dimensional or three-dimensional culture, micropat-
terning, controlled stiness, and mechanical, chemical, or
electrical stimulation have enabled us to more accurately
mimic the physiological environment of cultured cells while
improving throughput, accuracy, and eciency of in vitro
analyses. A combination of signaling molecules and bioengi-
neering approaches may further enhance the dierentiation
and maturation of human PSCs-derived myotubes for use in
disease modeling, drug testing, and therapeutic development.
Finally, in vitro cell models should represent similar morpho-
logical and physiological characteristics compared to tissues
in vivo. In the skeletal muscle, fully mature myotubes have
well-organized sarcomeres and the ability to contract in
response to stimulation. In order to assess the maturity of
human PSC-derived myotubes, it will be necessary to evaluate
them using both anatomical and functional approaches.
Conflicts of Interest
The authors declare that there is no conict of interest
regarding the publication of this paper.
Acknowledgments
The rst author (Nunnapas Jiwlawat) would like to thank
the Royal Thai Government Scholarship for the nancial
support. This work was supported by grants from the ALS
Association (15-IIP-201, Masatoshi Suzuki), NIH/NINDS
(R01NS091540, Masatoshi Suzuki), and the University of
Wisconsin Foundation (Masatoshi Suzuki).
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... Several biochemical protocols for the myogenic induction of iPSCs have been proposed. Some recent reviews analyze and compare the different approaches pursued [6][7][8][9][10] . However, in almost all of them, the focus is only on the biochemical stimuli affecting stem cell fate. ...
... High levels of differentiation, for example, can be obtained from the pooled percentage of cells expressing PAX7 or MYOD1, thus preventing a comparison with protocols that do not follow this procedure. Several groups are therefore debating how to establish standards for the evaluation of muscle cell maturation and consequent protocol differentiation efficiency 8,64 . ...
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Although skeletal muscle repairs itself following small injuries, genetic diseases or severe damages may hamper its ability to do so. Induced pluripotent stem cells (iPSCs) can generate myogenic progenitors, but their use in combination with bioengineering strategies to modulate their phenotype has not been sufficiently investigated. This review highlights the potential of this combination aimed at pushing the boundaries of skeletal muscle tissue engineering. First, the overall organization and the key steps in the myogenic process occurring in vivo are described. Second, transgenic and non-transgenic approaches for the myogenic induction of human iPSCs are compared. Third, technologies to provide cells with biophysical stimuli, biomaterial cues, and biofabrication strategies are discussed in terms of recreating a biomimetic environment and thus helping to engineer a myogenic phenotype. The embryonic development process and the pro-myogenic role of the muscle-resident cell populations in co-cultures are also described, highlighting the possible clinical applications of iPSCs in the skeletal muscle tissue engineering field.
... Thanks to the study on embryonic myogenesis that pointed out the key small molecules affecting skeletal muscle development, scientists have been able to generate skeletal myotubes in vitro with higher efficiency (Jiwlawat et al. 2018). In recent years, the significant small molecules that play critical roles in myogenesis such as GSK3ß inhibitor or Wnt agonists, TGF-ß inhibitors, and various growth factors and chemicals have been tested and have shown a positive effect on muscle development by increasing the myogenic cell population (Abujarour et al., 2014;Caron et al., 2016a;Chal et al., 2016;Chal et al., 2015;Choi et al., 2016a;Xu et al., 2013). ...
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The limited amount of available human muscle cells, especially patient-specific muscle cells, causes slow progress towards finding a new treatment for neuromuscular diseases. Here we propose an easy culture system using small molecule mixtures to generate myogenic progenitors derived from human pluripotent stem cells (hPSCs). Without genetic modification or cell sorting, the lifted hPSCs formed cell aggregates (EZ spheres) and were maintained as a suspension in media with a high concentration of FGF2 and EGF for 38 days. The first 10 days also had the addition of TGF-ß1 inhibitor, Wnt activator, and adenylyl cyclase activator. EZ spheres exponentially expanded and were passaged weekly using a non-enzymatic chopping method. Upon EZ sphere dissociation and differentiation, a higher percentage (approximately 30%) of Myosin heavy chain (MHC)+ myotubes was identified than in the original protocol. With these small molecule mixtures, such myotubes presented a morphological and functional appearance resembling myotubes found in adult skeletal muscles. This updated protocol would provide an increased amount of mature muscle cells derived from hPSCs in a shorter time, which would be valuable for further research into disease mechanisms and pharmacological studies.
... Muscle systems development, as any type of adult tissue differentiation, is a stepwise process during which cells of restricted potential gradually advance toward complete specification. In the case of striated muscle, three types of cells, myogenic progenitors (Expressing the PAX3 and PAX7 factors), myoblasts (Expressing MYF5 and MYOD1) and myocytes (Expressing MYOG1) precede the formation of more complex structures, the myotubes, myofibrils and myofibers, expressing the Major Histocompatibility Complex I (MHC-I) (Jiwlawat et al., 2018) (Figure 42A). When myocytes differentiation is directed from human iPSCs, the process takes up to 6 weeks in transgene-free conditions, reclaiming use of several pharmacological inhibitors and growth factors, and reclaims up to the double in duration to obtain myofibers (Jiwlawat et al., 2017;Shelton et al., 2016). ...
Thesis
Pluripotency is defined by the ability of a cell to self-renew and to differentiate in to the three primary germ layers, mesoderm, ectoderm and endoderm. In the mouse, two types of pluripotency have been defined: naïve and primed, the former originating from the naïve epiblast of the pre-implantation embryo, and the latter from the post-implantation epiblast. These two states, despite sharing the core characteristics of pluripotency, differ in the molecular pathways and transcriptional networks underpinning their regulation. In primates, naïve pluripotency as defined in mice cannot be captured in vitro. In spite of the many protocols established to reprogram primed human cells to a “naïve-like” state, the issue remains unsolved, suggesting that other regulators of primates’ naïve pluripotency exist and remain to be identified. In the mouse, it has recently been shown that Netrin-1, a protein belonging to the Laminins superfamily, is a regulator of naïve pluripotency. In this work, we thus undertook to characterize Netrins family function in primates’ pluripotency regulation. We demonstrated that, unlike for the mouse and macaque, NTN1 expression is not associated with pluripotency in human, but rather triggers differentiation of naïve-like human pluripotent stem cells (PSCs) to mesodermal lineages. NTN1 antagonist DRAXIN, on the contrary, is enriched in the human pre-implantation epiblast and shields PSCs against NTN1-induced differentiating, therefore constituting a new potential regulator of pluripotency in human.
... The above observations provided an impetus to determine whether the NKA α1 CBMmediated control of Wnt/β-catenin revealed by our recent report [12] is a key molecular determinant of skeletal muscle cell specification and differentiation. To test this functionally, we used an approach based on human iPSC differentiation in vitro, which allowed us to transpose early signaling events of mesoderm specification in the vertebrate embryo to a system that is more amenable to genetic and pharmacological interventions [30,[33][34][35]]. ...
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The N-terminal caveolin binding motif (CBM) in Na/K-ATPase (NKA) α1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared to the parental wild type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated iSkm (induced Skm) cells. Another NKA functional mutant, A420P, which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells’ ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (T) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of β-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/β-catenin signaling.
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Background Reports of clinical improvement following mesenchymal stromal cell (MSC) infusions in refractory lupus patients at a single centre in China led us to perform an explorative phase I trial of umbilical cord derived MSCs in patients refractory to 6 months of immunosuppressive therapy. Methods Six women with a SLEDAI >6, having failed standard of care therapy, received one intravenous infusion of 1×10 ⁶ MSCs/kg of body weight. They maintained their current immunosuppressives, but their physician was allowed to adjust corticosteroids initially for symptom management. The clinical endpoint was an SRI of 4 with no new British Isles Lupus Activity Guide (BILAG) As and no increase in Physician Global Assessment score of >0.3 with tapering of prednisone to 10 mg or less by 20 weeks. Results Of six patients, five (83.3%; 95% CI 35.9% to 99.6%) achieved the clinical endpoint of an SRI of 4. Adverse events were minimal. Mechanistic studies revealed significant reductions in CD27IgD double negative B cells, switched memory B cells and activated naïve B cells, with increased transitional B cells in the five patients who met the endpoint. There was a trend towards decreased autoantibody levels in specific patients. Two patients had increases in their Helios+Treg cells, but no other significant T cell changes were noted. GARP-TGFβ complexes were significantly increased following the MSC infusions. The B cell changes and the GARP-TGFβ increases significantly correlated with changes in SLEDAI scores. Conclusion This phase 1 trial suggests that umbilical cord (UC) MSC infusions are very safe and may have efficacy in lupus. The B cell and GARP-TGFβ changes provide novel insight into mechanisms by which MSCs may impact disease. Trial registration number NCT03171194 .
Thesis
Dans le muscle squelettique, des invaginations du sarcolemme appelées cavéoles et leur composant principal Cav-3 seraient impliqués dans la formation des tubules transverses, des structures musculaires permettant de propager le potentiel d'action dans la fibre musculaire. Pourtant, ce mécanisme demeure à ce jour inconnu. L’importance des cavéoles et de Cav-3 est accentuée par l’existence de défauts dans l’organisation et la fonction des cavéoles dans le cas de cavéolinopathies, des maladies neuromusculaires autosomiques dominantes dues à des mutations dans le gène CAV-3 et dont les mécanismes physiopathologiques sont à ce jour incompris. L’objectif de mon projet était de comprendre le rôle des cavéoles dans la formation précoce des tubules-T. Une technique de microscopie corrélative combinant de la fluorescence à super résolution et de la microscopie électronique sur répliques de métal a permis d’examiner en détails les composants moléculaires des cavéoles et des tubules-T dans des myotubes extensivement différenciés. J’ai ainsi montré l'organisation des cavéoles sur des plateformes de Bin1 formant ainsi une nouvelle structure en anneaux semblant optimiser la tubulation de la membrane afin d’initier la formation des tubules-T. Ces anneaux ainsi que la tubulation des membranes par Bin1 sont altérés dans le cas de défauts d’expression de Cav-3 et dans les myotubes de patients cavéolinopathes. Mes travaux suggèrent que les anneaux de cavéoles constituent le site d’initiation des tubules-T et apportent les bases d’une caractérisation de la biogénèse des tubules-T dans le muscle squelettique et dans la physiopathologie des cavéolinopathies.
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Engineering models of human skeletal muscle tissue provides unique translational opportunities to investigate and develop therapeutic strategies for acute muscle injuries, and to establish personalised and precision medicine platforms for in vitro studies of severe neuromuscular and musculoskeletal disorders. Several myogenic and non-myogenic cell types can be isolated, generated, amplified and combined with scaffolds and biomaterials to achieve this aim. Novel bio-fabrication strategies, which include exogenous stimuli to enhance tissue maturation, promise to achieve an ever-increasing degree of tissue functionalisation both in vivo and in vitro. Here we review recent advances, current challenges and future perspectives to build human skeletal muscle tissue “in a dish”, focusing on the cellular constituents and on applications for in vitro disease modelling. We also briefly discuss the impact that emerging technologies such as 3D bioprinting, organ-on-chip and organoids might have to circumvent current technical hurdles in future studies.
Chapter
Insulin resistance (IR) precedes the development of type 2 diabetes (T2D) and the metabolic syndrome and increases cardiovascular disease risk. IR is a silent pandemic of considerable proportions given that cardiometabolic diseases are the leading cause of death in the world. Although genome-wide association studies (GWASs) have uncovered new loci associated with T2D over the last years, their contribution to explain the mechanisms leading to decreased insulin sensitivity has been very limited. Thus, novel cellular systems are required to investigate the genetic basis of IR. Induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to model human disease, especially for complex polygenic diseases affecting tissues of difficult access. In this chapter, we explore the current knowledge surrounding the genetic basis of IR and potential causal tissues, along with the most current up-to-date differentiation protocols and established disease models for monogenic and common forms of IR.
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One major challenge in realizing cell-based therapy for treating muscle-wasting disorders is the difficulty in obtaining therapeutically meaningful amounts of engraftable cells. We have previously described a method to generate skeletal myogenic progenitors with exceptional engraftability from pluripotent stem cells via teratoma formation. Here, we show that these cells are functionally expandable in vitro while retaining their in vivo regenerative potential. Within 37 days in culture, teratoma-derived skeletal myogenic progenitors were expandable to a billion-fold. Similar to their freshly sorted counterparts, the expanded cells expressed PAX7 and were capable of forming multinucleated myotubes in vitro. Importantly, these cells remained highly regenerative in vivo. Upon transplantation, the expanded cells formed new DYSTROPHIN⁺ fibers that reconstituted up to 40% of tibialis anterior muscle volume and repopulated the muscle stem cell pool. Our study thereby demonstrates the possibility of producing large quantities of engraftable skeletal myogenic cells for transplantation.
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The neuromuscular junction (NMJ) is a specialized cholinergic synaptic interface between a motor neuron and a skeletal muscle fiber that translates presynaptic electrical impulses into motor function. NMJ formation and maintenance require tightly regulated signaling and cellular communication among motor neurons, myogenic cells, and Schwann cells. Neuromuscular diseases (NMDs) can result in loss of NMJ function and motor input leading to paralysis or even death. Although small animal models have been instrumental in advancing our understanding of the NMJ structure and function, the complexities of studying this multi-tissue system in vivo and poor clinical outcomes of candidate therapies developed in small animal models has driven the need for in vitro models of functional human NMJ to complement animal studies. In this review, we discuss prevailing models of NMDs and highlight the current progress and ongoing challenges in developing human iPSC-derived (hiPSC) 3D cell culture models of functional NMJs. We first review in vivo development of motor neurons, skeletal muscle, Schwann cells, and the NMJ alongside current methods for directing the differentiation of relevant cell types from hiPSCs. We further compare the efficacy of modeling NMDs in animals and human cell culture systems in the context of five NMDs: amyotrophic lateral sclerosis, myasthenia gravis, Duchenne muscular dystrophy, myotonic dystrophy, and Pompe disease. Finally, we discuss further work necessary for hiPSC-derived NMJ models to function as effective personalized NMD platforms.
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The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Here, we derive induced myogenic progenitor cells (iMPCs) via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. In 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells endogenously expressing Pax7. Under optimized 3D culture conditions, iMPCs derived from multiple hPSC lines reproducibly form functional skeletal muscle tissues (iSKM bundles) containing aligned multi-nucleated myotubes that exhibit positive force-frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. During 1-month culture, the iSKM bundles undergo increased structural and molecular maturation, hypertrophy, and force generation. When implanted into dorsal window chamber or hindlimb muscle in immunocompromised mice, the iSKM bundles survive, progressively vascularize, and maintain functionality. iSKM bundles hold promise as a microphysiological platform for human muscle disease modeling and drug development.
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Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. We observed that hPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Using RNA sequencing, we found that the cell surface receptors ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of transforming growth factor-β signalling during differentiation improved fusion efficiency, ultrastructural organization and the expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibres after engraftment of CRISPR-Cas9-corrected Duchenne muscular dystrophy human induced pluripotent stem cell-SMPCs. The work provides an in-depth characterization of human myogenesis, and identifies candidates that improve the in vivo myogenic potential of hPSC-SMPCs to levels that are equal to directly isolated human fetal muscle cells.
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Human induced-pluripotent stem cells (iPSCs) are a promising resource for propagation of myogenic progenitors. Our group recently reported a unique protocol for the derivation of myogenic progenitors directly (without genetic modification) from human pluripotent cells using free-floating spherical culture. Here we expand our previous efforts and attempt to determine how differentiation duration, culture surface coatings, and nutrient supplements in the medium influence progenitor differentiation and formation of skeletal myotubes containing sarcomeric structures. A long differentiation period (over 6 weeks) promoted the differentiation of iPSC-derived myogenic progenitors and subsequent myotube formation. These iPSC-derived myotubes contained representative sarcomeric structures, consisting of organized myosin and actin filaments, and could spontaneously contract. We also found that a bioengineering approach using three-dimensional (3D) artificial muscle constructs could facilitate the formation of elongated myotubes. Lastly, we determined how culture surface coating matrices and different supplements would influence terminal differentiation. While both Matrigel and laminin coatings showed comparable effects on muscle differentiation, B27 serum-free supplement in the differentiation medium significantly enhanced myogenesis compared to horse serum. Our findings support the possibility to create an in vitro model of contractile sarcomeric myofibrils for disease modeling and drug screening to study neuromuscular diseases.
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Pluripotent stem (PS)-cell-derived cell types hold promise for treating degenerative diseases. However, PS cell differentiation is intrinsically heterogeneous; therefore, clinical translation requires the development of practical methods for isolating progenitors from unwanted and potentially teratogenic cells. Muscle-regenerating progenitors can be derived through transient PAX7 expression. To better understand the biology, and to discover potential markers for these cells, here we investigate PAX7 genomic targets and transcriptional changes in human cells undergoing PAX7-mediated myogenic commitment. We identify CD54, integrin α9β1, and Syndecan2 (SDC2) as surface markers on PAX7-induced myogenic progenitors. We show that these markers allow for the isolation of myogenic progenitors using both fluorescent- and CGMP-compatible magnetic-based sorting technologies and that CD54+α9β1+SDC2+ cells contribute to long-term muscle regeneration in vivo. These findings represent a critical step toward enabling the translation of PS-cell-based therapies for muscle diseases.
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Skeletal muscle is the largest tissue in the body and loss of its function or its regenerative properties results in debilitating musculoskeletal disorders. Understanding the mechanisms that drive skeletal muscle formation will not only help to unravel the molecular basis of skeletal muscle diseases, but also provide a roadmap for recapitulating skeletal myogenesis in vitro from pluripotent stem cells (PSCs). PSCs have become an important tool for probing developmental questions, while differentiated cell types allow the development of novel therapeutic strategies. In this Review, we provide a comprehensive overview of skeletal myogenesis from the earliest premyogenic progenitor stage to terminally differentiated myofibers, and discuss how this knowledge has been applied to differentiate PSCs into muscle fibers and their progenitors in vitro.
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Recent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes, they have not demonstrated scalability, safety, and in vivo engraftment, which are key aspects for their future clinical application. Here we recapitulate one prominent protocol, and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation, the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle, we incorporated the inducible PAX7 lentiviral system into this protocol, which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7(+) myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases.
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Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD). Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.
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Somites form during embryonic development and give rise to unique cell and tissue types, such as skeletal muscles and bones and cartilage of the vertebrae. Using somitogenesis-stage human embryos, we performed transcriptomic profiling of human presomitic mesoderm as well as nascent and developed somites. In addition to conserved pathways such as WNT-β-catenin, we also identified BMP and transforming growth factor β (TGF-β) signaling as major regulators unique to human somitogenesis. This information enabled us to develop an efficient protocol to derive somite cells in vitro from human pluripotent stem cells (hPSCs). Importantly, the in-vitro-differentiating cells progressively expressed markers of the distinct developmental stages that are known to occur during in vivo somitogenesis. Furthermore, when subjected to lineage-specific differentiation conditions, the hPSC-derived somite cells were multipotent in generating somite derivatives, including skeletal myocytes, osteocytes, and chondrocytes. This work improves our understanding of human somitogenesis and may enhance our ability to treat diseases affecting somite derivatives.
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Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches.
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Significance: Protocols to produce skeletal myotubes for disease modeling or therapy are scarce and incomplete. The present study efficiently generates functional skeletal myotubes from human induced pluripotent stem cells using a small molecule-based approach. Using this strategy, terminal myogenic induction of up to 64% in 36 days and spontaneously contractile myotubes within 34 days were achieved. Myotubes derived from patients carrying the C9orf72 repeat expansion show no change in differentiation efficiency and normal TDP-43 localization after as many as 120 days in vitro when compared to unaffected controls. This study provides an efficient, novel protocol for the generation of skeletal myotubes from human induced pluripotent stem cells that may serve as a valuable tool in drug discovery and modeling of musculoskeletal and neuromuscular diseases.