G. Engineering

Tissue
Engineering
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© Mary Ann Liebert, Inc.

DOI: 10.1089/ten.TEB.2017.0451
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Stem Cells for Skeletal Muscle Tissue Engineering

This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Molly N. Pantelic MS1 and Lisa M. Larkin PhD1,2

1
Molecular and Integrative Physiology, University of Michigan, 2025 BSRB, 109 Zina Pitcher
Place, Ann Arbor, MI, 48109‐2200
Downloaded by Tufts University package NERL from www.liebertpub.com at 03/19/18. For personal use only.
2
Biomedical Engineering, University of Michigan, 2025 BSRB, 109 Zina Pitcher Place, Ann
Stem Cells for Skeletal Muscle Tissue Engineering (DOI: 10.1089/ten.TEB.2017.0451)
Arbor, MI, 48109‐2200

Tissue Engineering

Corresponding Author: Lisa Larkin, PhD
Associate Professor
Molecular and Integrative Physiology
Biomedical Engineering
University of Michigan
Biomedical Science Research Building (BSRB)
109 Zina Pitcher Place
Room #2025
48109‐2200
Phone: (734) 936‐8181
Fax: (734) 615‐3292
e‐mail: [email protected]

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Abstract
Volumetric muscle loss (VML) is a debilitating condition wherein muscle loss
overwhelms the body’s normal physiological repair mechanism. VML is particularly
common among military service members who have sustained war injuries. Because of the
high social and medical cost associated with VML and suboptimal current surgical
treatments, there is great interest in developing better VML therapies. Skeletal muscle
tissue engineering (SMTE) is a promising alternative to traditional VML surgical treatments
that use autogenic tissue grafts, and rather uses isolated stem cells with myogenic
potential to generate de novo skeletal muscle tissues to treat VML. Satellite cells are the
native precursors to skeletal muscle tissue, and are thus the most commonly studied
starting source for SMTE. However, satellite cells are difficult to isolate and purify, and it is
presently unknown whether they would be a practical source in clinical SMTE applications.
Alternative myogenic stem cells, including adipose‐derived stem cells (ADSCs), bone
marrow‐derived mesenchymal stem cells (BM‐MSCs), perivascular stem cells (PVSCs),
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umbilical cord mesenchymal stem cells (UC‐MSCs), induced pluripotent stem cells (iPSCs)
and embryonic stem cells (ESCs) each have myogenic potential and have been identified as
possible starting sources for SMTE, though they have yet to be studied in detail for this
purpose. These alternative stem cell varieties offer unique advantages and disadvantages
that are worth exploring further to advance the SMTE field towards highly functional, safe,
and practical VML treatments. The following review summarizes the current state of
satellite cell‐based SMTE, details the properties and practical advantages of alternative
myogenic stem cells, and offers guidance to tissue engineers on how alternative myogenic
stem cells can be incorporated into SMTE research.

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Introduction
Volumetric muscle loss (VML) is skeletal muscle loss due to injury, denervation,
disease, or tumor excision that exceeds the body’s regenerative capacity. Thus, VML
cannot be cured via the endogenous repair process, and often requires surgical
intervention. VML is particularly prevalent among military service members who have
sustained war injuries, which is a general category that includes blast injuries, gunshot
wounds, and fragmentation injuries (1,2). Recent military conflicts like Operation Iraqi
Freedom have especially high rates of extremity injury and VML due to the nature of
combat (3,4), so VML cases are becoming more prevalent over time.
VML causes chronic pain, impaired function, and reduced mobility. A study by
Corona et al. (5) found that VML was directly responsible for disability in 8% of all disabled
veterans surveyed, and within a group of veterans with type III open tibia fractures, VML
was an important contributor to 65% of the permanent disability they experienced. Corona
et al. also found that the average lifetime disability cost for surveyed veterans with VML in
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the general disability population was $341,300 per person (5). Compounding this problem,
current surgical procedures to treat injury‐induced VML are suboptimal at best, and full
structural and functional recovery is rare (6).
The most common procedure that is currently used to treat VML is muscle flap
surgery (6), which involves transferring autologous tissue with intact blood and nerve
supply from a donor site into the patient's injury site. Typically, this tissue is rotated from
an adjacent donor site directly into the injury site; however, in the case of free functional
muscle transfer, autologous donor muscle is transplanted from a site not immediately
adjacent to the patient's injury, and the arteries, veins, and nerves are cut at the donor site
and sewed into the injury site (6). Muscle flap procedures are severely limited in terms of
the amount of tissue that can be taken and rarely result in full functional recovery (6).
Donor site morbidity, which refers to any complication in the patient's donor site as it
heals, is also a significant concern with these procedures (6).
Composite tissue allografts (CTAs) have been used successfully in recent years for
face, hand, larynx, and abdominal wall transplants (7–12), and they may emerge as a more
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popular surgical treatment for VML in the coming years. CTA procedures transplant
combinations of skeletal muscle, skin, bone, and tendon from an organ donor that is
typically not genetically identical to the patient. More tissue can be implanted using CTA,
but this procedure also carries significant negative side effects. Patients require lifelong
immunosuppression, and tissue rejection, malignancies, and infections are serious
concerns.
Because of the complications and inadequacies associated with current VML
surgeries, there is intense interest in devising better treatment options for VML. In more
recent years, skeletal muscle tissue engineering (SMTE) has emerged as a promising
alternative to traditional VML surgical procedures. Generally, SMTE protocols start with a
biopsy to isolate a stem cell population with myogenic potential. After the stem cells are
allowed to proliferate, they may be differentiated in vitro. Depending on the specific
protocol used, the cells can be seeded onto a scaffold for structural support (13–15) or
allowed to develop into a scaffoldless skeletal muscle tissue prior to implantation (16). The
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engineered tissue is implanted back into the VML injury site to restore structure and
function. Vascularization of engineered skeletal muscle tissues is critical for proper
functioning in vivo, and is thus a burgeoning field of study, with several published methods
to date (17–21). As summarized in a review of engineered skeletal muscle techniques by
Bian et al. (22), innervation methods are also being developed for skeletal muscle tissue
engineering, as innervation is essential for the development of functional, non‐atrophic
skeletal muscle.
As an alternative to the general SMTE process, some labs have experimented with
decellularization techniques that use biologic scaffolds composed of extracellular matrix
(ECM) proteins. When implanted into a VML site, ECM scaffolds encourage some degree of
stem cell recruitment and de novo fiber regeneration without a prior biopsy and in vitro
cell culture (23). These methods rely on ECM's ability to create an environment for wound
healing that minimizes infection, promotes moisture balance, and regulates cell behavior
to promote tissue repair (24). However, Garg et al. (25) found that using an ECM scaffold
alone without a stem cell source was unable to recruit enough host stem cells in vivo to
promote sufficient de novo fiber regeneration for functional recovery, and most SMTE
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laboratories currently use stem cells in their protocols.
Developing a successful SMTE protocol relies on choosing the proper stem cell
starting source, and making this determination requires a detailed understanding of the
properties of myogenic stem cells. Ideally, a stem cell population for SMTE should be easy
to isolate, easy to purify, easy to expand in vitro without sacrificing myogenic potential,
and able to properly fuse with existing skeletal muscle tissue in vivo to contribute to
structural and functional VML recovery.
Because satellite cells are the native stem cell precursor to skeletal muscle, they
are the most common starting source for SMTE. However, there are difficulties associated
with using satellite cells. Satellite cell isolations are laborious, time‐consuming, and
variable between research labs. There is no consensus as to whether purification is
required, but in protocols that use some degree of purification, the purification process
can be challenging, and currently used methods are inconsistent, wasteful, or impractical.
Looking towards personalized medicine applications, there are concerns as to how an
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adequate satellite cell population could be obtained. Acquiring enough satellite cells from
a small skeletal muscle biopsy to produce a sufficiently sized tissue is challenging, yet a
large skeletal muscle biopsy would not be clinically feasible because of the potential
irreversible injury at the biopsy site. Senescence of satellite cells as they are expanded in
vivo causes them to lose proliferative capacity, which reduces their ability to contribute to
myogenesis over time (26,27). More generally, growing skeletal muscle in vitro is also
challenging because there is not a devoted collection of growth factors whose only
purpose is to induce skeletal muscle growth, whereas, for instance, bone and cartilage
tissue engineering methods have had great success using bone morphogenic proteins
(BMPs) (28).
To overcome the problems currently associated with satellite cell‐based SMTE,
tissue engineers should investigate the best stem cell source for SMTE. Alternative
varieties of myogenic stem cells, including adipose‐derived stem cells, bone marrow‐
derived mesenchymal stem cells, perivascular stem cells, and induced pluripotent stem
cells may offer attractive practical advantages over satellite cells, and should thus be
explored in future SMTE studies in addition to satellite cells to advance SMTE towards
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highly functional, safe, and clinically feasible VML treatments. Before discussing these
alternative stem cells, it is first important to understand satellite cell properties and the
current state of satellite cell‐based SMTE.

Satellite Cells
Satellite cells are located in all skeletal muscle tissues beneath the basal lamina and
outside the surrounding myofiber plasma membrane (29,30) and are the primary
contributors to skeletal muscle regeneration. In fact, Lepper et al. (31) showed that
satellite cells are absolutely required for normal muscle regeneration in vivo. Normal
satellite cell myogenesis (Figure 1) in response to injury begins when satellite cells are
activated by local signals and upregulate the transcription factor myogenic differentiation
1 (MyoD) and myogenic regulatory factor 5 (Myf5), thereby committing to a myoblastic
lineage and becoming myoblasts (32,33) in a process termed induction. Myoblasts
proliferate and differentiate into myocytes, which fuse into multinucleated myotubes after
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upregulating the transcription factor myogenin in a process called terminal differentiation.
Alternatively, myoblasts can directly fuse with damaged myofibers in vivo. After terminal
differentiation, myotubes can mature into myofibers.
The essential satellite cell marker is paired box protein‐7 (Pax7), which is a
transcription factor that is required for normal satellite cell function, and is expressed by
both quiescent and proliferating satellite cells (34). Beyond Pax7, there is significant
heterogeneity within the satellite cell population along with differences in satellite cell
marker expression depending on the anatomical location of their niche (35).
The anatomical location of the satellite cell niche also affects satellite cell
differentiation potential. For instance, as demonstrated by Collins et al. (36), satellite cells
from mouse extensor digitorum longus and soleus muscles contribute significantly more to
regeneration in vivo than tibialis anterior (TA) muscle satellite cells. These differences are
caused by local stem cell niche and injury microenvironment (37), and are also affected by
differences in cellular composition of the niche that arise from two distinct satellite cell
populations that Shultz (38) identified: a reserve population and a responsive population.
The responsive satellite cell population comprises roughly 80% of total satellite cells and is
the population that readily divides to contribute to de novo myofiber formation (38). The
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remaining 20% of satellite cells are part of a reserve population that divides slowly and
only in response to an intensive need for muscle regeneration (38).

Satellite Cell Isolation
All satellite cell‐based SMTE protocols must create an in vitro approximation of the
in vivo stem cell niche and injury environment to induce proper induction, proliferation,
terminal differentiation, and tissue formation, and each protocol must begin with proper
satellite cell isolation. Satellite cell isolations begin with a skeletal muscle biopsy to obtain
skeletal muscle tissue. Skeletal muscle biopsies are painful, and only a small amount of
tissue can be taken before a permanent functional deficit is introduced. After biopsy, most
satellite cell isolation methods fall into one of two general categories: single fiber explant
culture or enzymatic digestion.
Explant culture is a general category of cell isolation wherein pieces of tissue are
plated and cells are allowed to egress out of the tissue and onto the cell culture dish.
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Bischoff et al. (39) developed the first single fiber explant culture isolation protocol for
satellite cell isolation in rats, and it was later improved upon by Rosenblatt et al. (26). In
their current state, single fiber explant culture methods (Figure 2, top) (40) are highly pure
but low yield due to the nature of isolation (40). In contrast, enzymatic digestion methods
(Figure 2, bottom) (40) are less pure but yield larger satellite cell populations, and are thus
the most common isolation method currently used in SMTE (40). Enzymatic digestion
protocols involve dissecting muscle biopsies, removing any connective tissue by hand,
mincing the muscle, and then performing enzymatic digestion, using enzymes like trypsin,
Pronase, dispase, and collagenase to reduce the tissue into individual cells (40). Enzymatic
digestion is laborious, and efficiency varies widely between labs.

Satellite Cell Purification
After enzymatic digestion, the resulting isolate is typically purified to remove debris
and other abundant cell types, including epithelial cells and fibroblasts. Fluorescent
Activated Cell Sorting (FACS) (Figure 3A) (33) is a purification technique that uses
fluorophore conjugated cell markers to dye cells and applies electrical charges to samples
to sort cells by surface marker (41,42). Determining appropriate positive and negative
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satellite cell markers can be challenging due to satellite cell heterogeneity. Although
satellite cells have successfully been purified using FACS (43–46), applying electrical
charges to live cells can alter gene expression (47) and reduce proliferative capacity (48),
which could limit their SMTE applications.
Magnetic Activated Cell Sorting (MACS) (Figure 3B) (33) is an alternative to FACS
that works by incubating cells with antibodies for surface markers that are conjugated to
magnetic microbeads. A magnetic field is then applied to sort the cells out of the cell
suspension. However, appropriate markers must also be determined for MACS, and the
tight antibody‐antigen association can sometimes cause the magnetic microbeads to be
endocytosed by the sorted cells (40), rendering the beads impossible to remove prior to
implantation. Although MACS and FACS are powerful experimental techniques, given their
limitations, it is presently unclear whether they will ever be clinically useful purification
techniques for SMTE.
In contrast, the pre‐plate protocol (Figure 3C) (33) developed by Richler and Yaffe
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(49) is a simple, fast method to remove fibroblasts and epithelial cells by repeatedly
plating and recollecting the cell suspension obtained from enzymatic dissociation over
time. Lavasani et al. (50) later developed the modified pre‐plate technique, which coats
pre‐plates in collagen I to purify cells by their affinity for collagen I. Pre‐plating is attractive
for its simplicity, but it is variable and results in significant loss of satellite cells on pre‐
plates. Recently, Syverud et al. (51) published a novel, promising purification method that
uses a microfluidic sorting device termed a "Labyrinth" to obtain significantly purer
satellite cell populations using label‐free, inertial separation; the higher‐purity population
was then used to grow skeletal muscle constructs able to produce significantly greater
tetanic forces than unsorted controls.

Satellite Cell Preservation
To date, satellite cell preservation is not well studied, and the field has yet to
comprehensively address the best method for satellite cell cryopreservation or
cryopreservation's effects on satellite cell viability, proliferation, marker expression, and
differentiation potential. Bian et al. (52) found that satellite cells isolated from Luxi cattle
embryo could be successfully cryopreserved after fifteen passages and retained an average
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viability of 97.90 ± .96% after cryopreservation. Though it is perhaps likely that
cryopreservation would be an effective method for satellite cell preservation, more
research must be done to assess the most effective satellite cell preservation techniques
and any negative effects of the cryopreservation process.

Satellite Cell Proliferation and Differentiation
After isolation and purification, most SMTE protocols proliferate satellite cells by
suspending them in a muscle growth medium with high serum and high glucose content
(40). A selection of biomolecules are typically added to enhance proliferation and
myogenic potential in culture, including fibroblast growth factor (FGF), hepatocyte growth
factor (HGF), platelet‐derived growth factor (PDGF), and insulin‐like growth factor 1 (IGF‐1)
(39,53–55). Weist et al. (56) found that when added to muscle medium, transforming
growth factor beta (TGF‐β1) enhances contractility and extracellular matrix organization in
scaffoldless, tissue‐engineered skeletal muscle tissues. Syverud et al. (57) found that the
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glucocorticoid dexamethasone (DEX) is a useful addition to muscle media because it
enhances the myogenic potential of rat satellite cells in culture, thereby improving the
terminal differentiation process by increasing myotube fusion and thickness and enhancing
sarcomeric organization.
After being suspended in appropriate media, satellite cells are often seeded onto
tissue culture plastic that is coated with an adhesion protein. Laminin, fibronectin,
matrigel, and polylysine have all been successfully used in various protocols (53,55,58),
and there is no consensus as to which adhesion protein works best. Alternatively,
hydrogels, which are gelatinous mixtures of proteins and water, are sometimes used in lieu
of protein‐coated, stiff glass dishes. Pollot et al. (59) recently compiled a useful review of
natural polymeric hydrogels for SMTE. It has been hypothesized that hydrogels can
promote growth in a way that is more similar to a stem cell niche by mimicking the
stiffness of the extracellular matrix (ECM) niche in vivo. Indeed, Engler et al. (60) found
that increasing or decreasing hydrogel stiffness outside of physiologically‐normal range for
skeletal muscle significantly reduces myogenic differentiation in mouse myoblasts, which
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underscores the importance of engineering an in vitro environment that is as similar to the
stem cell niche as possible.
After allowing the cells to proliferate into myoblasts, SMTE protocols induce
myogenic differentiation via a muscle differentiation medium (MDM). MDMs typically
contain low serum content, because myoblasts interpret serum deprivation in the local
environment as a differentiation signal (61). A wide variety of growth factors, including
IGF‐1, are also known to promote skeletal muscle differentiation and increase skeletal
muscle mass in vivo (62). Kang et al. (63) found that adding recombinant netrin, a secreted
axonal guidance protein that associates with the cell membrane and ECM (64,65), to
mouse myoblasts in vitro can induce myotube production and increase myotube size via
nuclear factor of activated T cell (NFAT) activation. Arachidonic acid enhances myotube
growth in vitro (66), as do cytokines via the Janus kinase/Signal Transducer and Activator
of Transcription (JAK/STAT) pathway (67).
The presence of ECM also encourages myogenic differentiation due to interactions
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between integrins in the ECM and myoblasts that induce signaling events to promote
myogenesis. A study by Stern et al. (68) found that adding ECM extracts from the thigh
muscles of adult rats to myoblasts enhances myoblast differentiation in vitro. Mechanical
stimulation can also be used to encourage myogenic differentiation. In vivo, mechanical
stimulation is required to prevent muscle atrophy, and this has been attributed to the
effects that mechanical stimulation has on the mammalian target of rapamycin (mTOR)
pathway to regulate gene and protein expression (69); it is thought that muscle behaves
similarly in vitro.
Because proper alignment of skeletal muscle fibers is crucial for function, tissue
engineers must also develop strategies to align skeletal muscle myotubes as they fuse in
vitro. Methods to align cells in vitro include topography methods (70,71), surface
patterning methods (72), mechanical stimulation (73–75), and electrical and magnetic
stimulation (76,77). Topography methods use surface features to manipulate cell behavior.
For instance, growing myoblasts on parallel, micropatterned grooves on tissue culture
plates promotes myoblast alignment (78) and myotube fusion in organized, end‐to‐end
configurations (79). Custom bioreactors (80), which contain lined, cylindrical microwells,
can be used to promote cell alignment and myoblast fusion using topographical principles.
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The same concepts are applied in surface patterning. Surface patterning is an umbrella
term for a variety of techniques that promote cell alignment, including soft lithography
(81). ECM proteins, including collagen, fibronectin, and laminin can be printed onto cell
surfaces via soft lithography to promote proper myotube alignment (61). As summarized in
several review articles(82,83), mechanical stimulation can also be used to promote
myotube alignment in vitro by using custom, dynamic bioreactors to add cyclic strain,
which aligns myotubes parallel to the direction of strain (84). As summarized in a useful
review by Ahadian et al. (85), electrical stimulation works by applying an electric field to
muscle cells in vitro; because these cells are electroactive, applying an electric field
regulates cell behavior and induces muscle tissue alignment parallel to the direction of an
applied electric field (86,87). Similarly, magnetic stimulation applies a magnetic field to
align myoblasts and elongates them as they differentiate along the axis of the applied
magnetic poles (88); however, it must be noted that magnetic stimulation is presently a
clinically infeasible process, as it requires that cells be electroporated with magnetic
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nanoparticles.

Satellite Cells in SMTE for VML
Several studies have tested satellite cell‐based SMTE in VML animal models (89,90).
In 2011, Machingal et al. (13) created and tested the first tissue‐engineered muscle repair
(TEMR) construct. The researchers created the TEMR by performing an enzymatic
dissociation isolation of rat muscle to obtain muscle progenitor cells (MPCs), which is a
more general category of Pax7+ cells that includes satellite cells. They cultured the MPCs to
obtain rat myoblasts, and seeded the myoblasts onto a porcine bladder acellular matrix
(BAM) scaffold to produce the TEMR. Afterwards, Machingal et al. used a custom
bioreactor for a week after seeding to provide mechanical stretch, thereby promoting
differentiation and alignment within the TEMR. Fluorescent microscopy and scanning
electron microscopy analysis of the TEMR after bioreactor preconditioning confirmed that
the scaffold contained aligned, multinucleated myotubes. After implanting final TEMRs
into a murine model for VML injury in the lattissimus dorsi (LD) muscle and allowing the
mice to recover for 2 months, they measured isometric tetanic force and observed
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significant functional recovery of the injured LD in mice treated with TEMRs compared to
no repair, although function was not returned to pre‐injury levels.
This TEMR protocol was further refined by Corona et al. (91) by experimenting with
three different varieties of TEMR. The first type of TEMR, termed TEMR‐1SP, was seeded
with rat‐derived MPCs and allowed to proliferate for only a short period of time without
bioreactor preconditioning. The second TEMR variety, called TEMR‐1SPD, was given a
longer cellular proliferation and differentiation period and was conditioned with a
bioreactor; this is the TEMR variety that was used in the original Machingal et al. (13)
study. The third TEMR variety, named TEMR‐2SPD, was given similar treatment to TEMR‐
1SPD but additional MDCs were seeded onto the scaffold during bioreactor
preconditioning to simulate functional recovery during exercise, wherein MPCs are
typically recruited to injured fibers. After implanting these TEMRs into a murine model for
VML injury, they measured in vitro isometric tetanic and twitch force. Results showed that
TEMR‐1SPs and TEMR‐1SPDs conferred similar functional recovery, while TEMR‐2SPDs
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induced functional recovery that had twice the magnitude of TEMR‐1SPs and TEMR‐1SPDs.
Corona et al. (14) later used the same protocol used to generate TEMR‐1SPDs in a
Lewis rat model of VML. When VML rats were treated with TEMRs, results were binary; the
rats were either responsive or nonresponsive to the tissue repair. Nonresponsive rats with
TEMR implants had no significant improvements in structural or functional recovery
compared to untreated rats, while responsive rats had significant structural and functional
recovery compared to untreated rats, as assessed by histology and in vivo torque
measurements. The researchers noted that responsive and nonresponsive rats had
different immune responses in their injury site that could have contributed to the
observed variability.
VanDusen et al. (16) published a method for engineering scaffoldless, three‐
dimensional, functional skeletal muscle units (SMUs) and tested them in a rat VML model.
The researchers used enzymatic digestion of rat soleus muscles to isolate satellite cells,
cultivated them using muscle growth medium, and differentiated them in vitro using a
medium with low serum content. After the muscle monolayers rolled up into a 3‐D skeletal
muscle tissue due to passive tension, they were pinned to the plate and engineered
bone/tendon anchors were added to either end, generating the final SMU. In this specific
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study, the researchers measured isometric tetanic force of their SMUs to ensure
functionality. The SMUs were then implanted into a VML site in the rat's TA muscle. After
28 days of recovery, they observed a significant increase in isometric tetanic force for VML
muscles treated with SMUs compared to rats with VML receiving no treatment, and a
force per unit mass for SMU‐treated muscles that was equivalent to native muscle.

Satellite Cell Limitations
Satellite cell‐based SMTE is undoubtedly a promising field with demonstrated
potential as a VML treatment, but using satellite cells has significant limitations. A skeletal
muscle biopsy is painful and invasive, and only a small biopsy would be clinically feasible.
Enzymatic dissociation is the only isolation method that is able to achieve sufficient
satellite cell yields, yet it is a laborious process and produces impure populations of
satellite cells. With respect to purification, FACS, MACS and pre‐plating all have significant
practical limitations, while microfluidic inertial separation is a promising, yet nascent
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method. Satellite cell preservation is not well studied, and satellite cell self‐renewal is not
unlimited, and satellite cell differentiation potential declines rapidly in vivo. To address
these issues, it is important to consider alternative stem cell sources, including adipose‐
derived stem cells, bone marrow‐derived mesenchymal stem cells, umbilical cord‐derived
mesenchymal stem cells, perivascular stem cells, induced pluripotent stem cells, and
embryonic stem cells.

Adipose‐Derived Stem Cells (ADSCs)
Adipose‐derived stem cells (ADSCs) are an abundant, adult, multipotent,
mesenchymal stem cell (MSC) variety with myogenic potential. MSCs are a general
category of non‐hematopoietic stem cells with specific cluster of differentiation (CD)
protein surface markers that reside in many tissue types, including adipose tissue. MSCs
have the capacity to differentiate into tissues of mesodermal lineage, including cartilage,
bone, adipose tissue, and skeletal muscle (92–94). A useful ADSC tissue engineering review
by Zuk (95) contains a comprehensive discussion of human ADSC surface markers.
ADSCs are attractive to tissue engineers not just for their abundance, but because
cultured ADSCs express very low levels of major histocompatibility complex class I (MHC I)
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and do not express major histocompatibility complex class II (MHC II) (96). Thus, ADSCs
from allogeneic or xenogeneic donors might be able to evade the patient's immune system
after implantation, potentially eliminating the need for lifelong immunosuppression in
patients (96). It is important to note that the low levels of MHC I and MHC II observed in
ADSCs are present before differentiation; there has not yet been a study that investigates
MHC I and MHC II levels in ADSC‐derived cells after myogenic differentiation.

ADSC Isolation and Purification
ADSC isolation protocols begin with an adipose tissue biopsy (97). As noted in the
Forcales (98) review of ADSCs for muscle regenerative therapies, adipose tissue is a highly‐
abundant, cosmetically favorable tissue type to biopsy in most individuals, and a simple
liposuction procedure to collect lipoaspirate typically suffices. Moreover, ADSC isolations
are generally efficient, with an average of 404,000 ± 206,000 cells isolated per milliliter of
lipoaspirate (99). Importantly, ADSC yield does not depend on the donor site (100), so fat
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can be taken from the most convenient location possible. Thus, ADSCs offer significant
advantages over satellite cells with respect to biopsy.
After biopsy, ADSC isolations typically use enzymatic digestion of the biopsied
adipose tissue or lipoaspirate with collagenase type 1A (98). After digestion, the mixture is
centrifuged to obtain the stromal vascular fraction (SVF) pellet, which contains many cell
types, including preadipocytes, pericytes, fibroblasts, red blood cells, and smooth muscle
cells (98). The SVF is plated in an ADSC media, which can vary by protocol but typically
contains Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS).
The cells that adhere to the plate are considered to be ADSCs (98). FACS and MACS can be
used to purify ADSC populations, but since ADSCs lack a universal marker, these processes
are complicated and imperfect. Cryopreservation is a suitable method for ADSC
preservation, as summarized in a review by Miyagi‐Shiohira et al. (101).
Several specific ADSC isolation methods may offer advantages over satellite cell
enzymatic dissociation. Like satellite cells, explant culture can be used for ADSC isolations,
and Priya et al. (102) developed an explant culture method that achieved equivalent to
superior human ADSC yields when compared to enzymatic dissociation. Because explant
culture tends to result in purer cell populations than enzymatic dissociation (40), using
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explant culture could eliminate the need for further purification. There is also a novel
protocol developed by Francis et al. (103) to isolate human ADSCs in just 30 minutes.
Future SMTE studies could consider using one of these protocols to obtain a starting ADSC
population.

ADSC Induction
There are several different methods for inducing ADSCs into myoblasts, including
using an appropriate myogenic media with myogenic biochemical factors, co‐culturing
ADSCs with myoblasts, adding biophysical stimuli, and genetically manipulating the ADSCs.
Interestingly, ADSC myogenesis is similar to satellite cell myogenesis in terms of when
Pax7, Myf5, and MyoD are expressed (104).
Di Rocco et al. (105) observed that a small proportion of mouse ADSCs are able to
sporadically convert into a myogenic lineage, which suggests that certain ADSCs do have
inherent myogenic potential. They also experimented with 50‐50 co‐cultures of myoblasts
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and ADSCs, and found that ADSCs will fuse into myotubes when cultured with myoblasts,
but only when the ADSCs and myoblasts are in direct contact. Afterwards, they tested
ADSC regenerative capacity in vivo, and found that when uncultured ADSCs were injected
into a mouse with ischemic skeletal muscle, they were able to fuse with muscle fibers to
contribute to structural recovery in vivo. Thus, simply placing ADSCs into the site of injury
can induce some degree of induction and differentiation towards a myogenic lineage,
though it is unknown whether this would contribute to meaningful functional recovery.
Biophysical stimuli can also induce ADSCs to a myogenic lineage, and Huri et al.
(106) found that adding cyclic strain to human ADSCs (hADSCs) treated with muscle
induction media not only encourages proper cell alignment, but also enhances myogenic
differentiation into myotubes. In fact, after adding cyclic strain, the researchers observed a
4.5‐fold increase in nuclei per cell, which suggests enhanced myogenesis. They also
observed significantly higher myogenic protein expression compared to cultures not given
cyclic strain.
Altering gene expression has also been explored as a method to differentiate
ADSCs, and Goudenege et al. (107) developed an ADSC differentiation protocol that uses a
mouse MyoD lentiviral vector to forcibly induce MyoD expression, thereby causing
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myogenic induction. MyoD is an essential myogenic regulatory factor that induces an open
chromatin structure at myogenic genes (108). The researchers found that using their
protocol to induce MyoD expression in hADSCs was a fairly efficient way to achieve
myogenic induction, with 30% of treated hADSCs expressing myogenic markers, as
compared to just 2% of untreated hADSCs. Notably, Goudenege et al. also found that
when injected into TA cryoinjuries in a murine model, the transfected hADSCs were
capable of fusing with regenerating fibers in the injury site to contribute to structural and
functional recovery in vivo. Sung et al. (109) tested a safer method for induction by
engineering and applying MyoD protein to hADSC cultures to induce myogenesis in vitro.
From a clinical perspective, applying MyoD protein is a far more practical and safe method
than altering MyoD gene expression, and would be a more feasible method to try in SMTE.

ADSCs in SMTE for VML
In 2016, Kesireddy (110) published the first comparative study that used rat ADSCs
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to create a TEMR construct to treat VML injury in a murine model and compared it to MPC
TEMRs. To make a TEMR, he isolated ADSCs and MPCs from donor rats, cultivated them,
and seeded either ADSCs or MPCs into BAM scaffolds. Afterwards, he implanted them into
a murine VML injury model in the LD muscle. Histological analysis of the injury site 2
months post‐repair revealed that ADSC and MPC TEMRs displayed similar regeneration
potential, and both showed signs of neotissue formation, which is characterized by
scaffold remodeling, fiber formation, and cell fusion in the VML site (110).
Kesireddy also did qualitative observation of lentivirus‐Cherry‐labeled donor cells
to determine the origin of the structural recovery that was observed, and discovered that
ADSCs contributed fairly little to new myofiber formation compared to donor MPCs.
However, ADSCs contributed greatly to vascularization in the injury site, and histological
images revealed greater vascularization after being treated with ADSCs than MPCs (110),
which is a positive sign of healing. Unfortunately, this experiment did not conduct force
testing to assess functional recovery, but the results nevertheless suggest that ADSCs
could be useful for treating VML.

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Bone Marrow‐Derived Mesenchymal Stem Cells (BM‐MSCs)
Bone marrow‐derived mesenchymal stem cells (BM‐MSCs) are another notable
MSC variety with myogenic potential. BM‐MSCs are a multipotent, adult stem cell
population that resides in the bone marrow (BM) on the abluminal side of vessels and can
differentiate into mesodermal tissues like skeletal muscle, bone, and cartilage (111). It is
important to note that BM‐MSCs are distinct from BM hematopoietic stem cells and do
not directly participate in hematopoiesis; rather, BM‐MSCs play an immunomodulatory
role, and provide microenvironment support for hematopoietic stem cells in vivo (112).
BM‐MSCs are a heterogeneous stem cell population, and individual subsets have
significant differences in markers, as detailed in a review by Boxall et al. (113). Because of
the similarities between BM‐MSCs and ADSCs, which are both MSCs with myogenic
potential that are found in vessel walls in their respective tissues of origin, Bayati et al.
(114) compared rat BM‐MSCs and ADSCs, and discovered that the two cell populations had
differential surface marker expression but did not significantly differ in terms of
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proliferative capacity or myogenic potential. Also like ADSCs, BM‐MSCs have poor ability to
stimulate an in vitro allogeneic T cell response due to absent MHC II and low MHC I
expression (115), which makes them well‐suited to transplantation.

BM‐MSC Isolation and Purification
BM‐MSC isolation begins with a BM biopsy, which is a painful and invasive process;
however, it is still advantaged over satellite cell biopsies in that more tissue can be taken
without introducing a functional deficit. After a biopsy, typical BM‐MSC isolations use
enzymatic digestion, just like ADSC and satellite cell protocols (116). BM isolates are
filtered and washed, and enzymes are used to digest the BM isolate to retrieve the BM‐
MSCs. Similarly to ADSCs, explant culture has also more recently been proposed as an
efficient method for BM‐MSC isolation (117). Generally speaking, BM‐MSC isolation from
BM samples is a fairly low‐yield process, because there are less than .01% BM‐MSCs
present in total BM cells (92). FACS and MACS can be used to purify BM‐MSC populations if
that is deemed necessary, and cryopreservation methods for BM‐MSCs have been
developed (118,119).

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BM‐MSC Induction
BM‐MSC induction methods are similar to what was already discussed for ADSCs
and satellite cells; most involve muscle induction media, co‐culture, biophysical stimuli, or
some combination of these methods. Treatment with 5‐azacytidine is a common method
for induction of BM‐MSCs to a myogenic lineage in vitro, though this is not unique to BM‐
MSCs, and can also be used to for induction of ADSCs (120). Although using 5‐azacytidine
for myogenic induction is a useful research technique, it is known that 5‐azacytidine is a
carcinogen (121), and is therefore impractical for SMTE. A study by Stern‐Straeter et al.
(122) evaluated the efficacy of six different muscle induction media formulations for
hADSCs and human BM‐MSCs (hBM‐MSCs) to offer guidance on which induction medias
work best. Beier et al. (123) perfected a rat BM‐MSC/myoblast co‐culture method by using
basic fibroblast growth factor (bFGF) and dexamethasone to maximize myogenic potential.
Egusa et al. (124) were the first to discover that adding cyclic strain to mouse BM‐MSCs
accelerates myogenic induction and promotes proper fiber alignment during the terminal
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differentiation process.
An important study by Meligy et al. (125) compared proliferation and myogenic
differentiation capacity between ADSCs, BM‐MSCs, and skeletal muscle tissue
mesenchymal stem cells (MC‐MSCs), which are found in skeletal muscle but have a MSC
phenotype and are distinct from satellite cells. After isolating and cultivating rat MSCs
from their respective tissues of origin, they treated the cells with 5‐azacytidine to promote
induction to a myogenic lineage. ADSCs had the highest observed proliferation rate and
BM‐MSCs had the lowest proliferation rate (125), though all were fairly high. The
researchers measured differentiation potential by myogenin expression, and observed
that BM‐MSCs had higher myogenic differentiation capacity than ADSCs, though both were
inferior to MC‐MSCs (125). From this, Meligy et al. concluded that ADSCs were the most
easily accessible and highly proliferative MSC. Although MC‐MSCs had the highest
observed myogenic differentiation potential, the researchers noted that MC‐MSCs isolated
from a skeletal muscle biopsy are no more accessible than satellite cells, suggesting that
ADSCs and BM‐MSCs could still be a viable starting source for SMTE regardless of their
lower myogenic differentiation potential.

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BM‐MSCs in SMTE
There has never been a published study that used BM‐MSCs to engineer skeletal
muscle tissue specifically for VML. However, there are several studies that have used BM‐
MSC injections to treat skeletal muscle injuries, and they offer clues as to how BM‐MSCs
would behave in SMTE specifically for VML. Natsu et al. (126) isolated BM‐MSCs from
green fluorescent protein (GFP) rats and injected them into lacerated TA muscles of
Sprague‐Dawley (SD) rats after implanting a fibrin scaffold. One month after
transplantation, the rats treated with BM‐MSCs showed significant functional recovery
that was almost that of pre‐injury levels. Interestingly, histological analysis did not indicate
that the GFP BM‐MSCs actually differentiated or fused into myofibers in the SD rats, which
suggests that BM‐MSCs may play an indirect role in healing, possibly by promoting a
healing immune environment in the injury site.
Merritt et al. (127) repaired skeletal muscle injury in a rat model by implanting an
extracellular matrix (ECM) scaffold into injured lateral gastrocnemius muscles and injecting
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BM‐MSCs into a group of rats treated with the ECM scaffold a week after surgery. After 42
days, the injured muscles treated with ECM and BM‐MSCs displayed significant structural
recovery (127) and partial functional recovery that was superior to muscles treated with
ECM alone (127). BM‐MSC‐injected muscles also had more blood vessels and regenerating
fibers. From this, the researchers concluded that BM‐MSCs could be a viable therapy for
skeletal muscle injury in the future. Andrade et al. (128) found that BM‐MSCs administered
via intramuscular injection into a rat model of successive injury significantly increased
maximal skeletal muscle contraction, muscle fiber cross‐sectional area, and the number of
muscle fibers in the injury site 28 days after injection.
Helal et al. (129) found that BM‐MSCs modulate the local immune system
environment in the site of VML to encourage recovery. Specifically, administration of BM‐
MSCs via intramuscular injection into a VML injury in a rat was shown to improve
structural and functional recovery by suppressing inflammatory cytokines, promoting anti‐
inflammatory cytokines, and also by directly contributing to myogenesis and angiogenesis.
BM‐MSCs also downregulate transforming growth factor beta (TGF‐β) in the injury site,
which prevents fibrosis. As this study demonstrates, BM‐MSCs show promise for SMTE
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applications not just because of their myogenic potential, but also because of the role they
play in immunomodulation to promote proper healing.

Umbilical Cord‐Derived Mesenchymal Stem Cells (UC‐MSCs)
Umbilical cord‐derived mesenchymal stem cells (UC‐MSCs) are derived from
compartments of the umbilical cord, with UC‐MSCs isolated from the cord‐placenta
junction region showing particularly high differentiation potential (130). UC‐MSCs are
more similar to embryonic stem cells than adult stem cells, and they are capable of faster
self‐renewal than adult MSCs (131). Collecting UC‐MSCs is also a painless process, and UC‐
MSCs lack MHC II, which is useful for transplantation (132). UC‐MSCs can be isolated via
enzymatic digestion (133) or explant culture methods (130,134). A good manufacturing
practice (GMP) protocol for clinical‐grade human UC‐MSC isolation was recently published
(135), and a useful summary of UC‐MSC markers, characteristics, and isolation methods is
included in a review by Nagamura et al. (136).
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Several myogenic induction protocols exist for UC‐MSCs. Gang et al. (137)
demonstrated that UC‐MSCs are capable of being induced into a myogenic lineage by
cultivating them in a myogenic media that contained 5% horse serum, 0.1 μM
dexamethasone, and 50 μM hydrocortisone for six weeks. Kocaefe et al. (132) transfected
human UC‐MSCs with MyoD to force induction into a myoblastic lineage. They found that
the resulting cells were capable of undergoing terminal differentiation by fusing with rat
myoblasts to form myotubes, which suggests that UC‐MSCs could be a viable starting
source for SMTE in the future.
To date, UC‐MSCs are best studied in the context of muscular dystrophy therapies.
A study by Zucconi et al. (138) found that undifferentiated UC‐MSCs injected into mouse
and dog muscular dystrophy models did not differentiate into skeletal muscle in vivo, yet
dystrophic animals injected with them displayed significant functional recovery compared
to untreated controls. In contrast, a different study by Nunes et al. (139) was able to
observe some degree of structural recovery, with a small number of mouse UC‐MSCs able
to differentiate into dystrophin‐expressing myotubes in vivo when transplanted into mdx
mice. While the exact reason for these conflicting results is not clear, it was shown in a
study by Koponen et al. (140) that UC‐MSCs are able to promote skeletal muscle
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regeneration in lacerated mouse skeletal muscle not by directly fusing with myofibers, but
by promoting a healing cytokine environment. While it appears that UC‐MSCs could be a
useful source for clinical skeletal muscle treatments in the future, more research must be
done to understand the mechanism and extent of healing in traumatic injury applications.

Perivascular Stem Cells (PVSCs)
Perivascular stem cells (PVSCs) are yet another MSC population that can be
subdivided into pericytes and adventitial cells (141). Adventitial cells are found in the
tunica adventitia (Figure 4, right) (142), which is the outermost layer of arteries and veins
(143), and they are defined as positive for CD34 and negative for CD31, CD146, and CD45
(144). Although adventitial cells share developmental features and general MSC properties
with pericytes, they are a phenotypically and genotypically distinct population (143). Their
myogenic potential has not yet been studied, whereas pericytes have been studied in
greater detail, discussed below.
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Pericytes
Pericytes are a stem cell population found in capillary and microvessel walls (142)
underneath the basal lamina (Figure 4, left) (142) in a variety of tissues, including skeletal
muscle (145) and adipose tissue (141). Pericytes isolated from skeletal muscle, white
adipose tissue, pancreas, and placenta have myogenic potential (146), and adipose tissue
in particular is an attractive biopsy site to target. Dellavale et al. (147) discovered that
mouse pericytes actually participate in normal postnatal skeletal muscle development by
differentiating into muscle fibers and infiltrating the satellite cell niche to generate satellite
cells. Thus, pericyte myogenesis is a natural process that occurs during development and
increases in response to acute injury. Pericytes are defined as positive for CD146 and
negative for CD34 and CD45 (141). Most pericyte isolations involve enzymatic digestion,
though explant culture has also been used (148), and Bryan et al. (149) wrote a useful
review of methods to isolate and culture pericytes. After isolation, pericytes can be
purified using FACS or MACS.
An important study by Dellavalle et al. (148) first discovered the promise of
pericytes for skeletal muscle therapies. Dellavale et al. began by isolating pericytes from
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human skeletal muscle biopsies with and without Duchenne Muscular Dystrophy (DMD),
and then expanded them out to 20 population doublings, which allowed them to obtain at
least 1 x 109 cells from a single biopsy, an amount they considered sufficient for clinical
DMD therapy. After culturing the pericytes, they promoted induction to a myogenic
lineage and differentiation using both a myogenic differentiation medium and co‐culture
with myoblasts, and observed that pericytes formed differentiated myotubes
spontaneously with high efficiency using both differentiation methods (148). In fact, the
differentiation potential they observed for pericytes was strikingly high, and up to an order
of magnitude higher than most other myogenic stem cells they compared them to,
excluding satellite cells. Dellavale et al. also injected cultured human pericytes into a
severe combined immune deficient–X‐linked, mouse muscular dystrophy (scid–mdx)
mouse model, and found that pericytes could successfully engraft with dystrophic muscle
fibers to contribute to structural recovery in vivo. The qualities of pericytes Dellavale et al.
observed ‐ adequate proliferative capacity, high myogenic potential, ability to contribute
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to fiber regeneration in vivo ‐ coupled with the fact that pericytes can be isolated from
other, more convenient tissue types to biopsy, suggests that pericytes could be a highly
useful starting cell for SMTE. Moreover, pericytes do not express MHC II or essential T‐cell
activating costimulatory molecules like CD80 and CD86, which suggests that pericytes are a
promising candidate for allotransplantation (150).
To date, pericytes have never been used in a SMTE study specifically for VML;
however, a study by Fuoco et al. (151) offers guidance to tissue engineers looking to
develop a pericyte SMTE protocol. Fuoco et al. isolated skeletal muscle‐derived pericytes
(MPs) from young and old pigs using enzymatic digestion with collagenase II and induced
myogenesis via a media with low serum content. Myogenic potential of pericytes declines
with age, and the researchers found that old MPs had reduced ability to form myotube
and capillary‐like structures in vitro on their own. However, when the old MPs were
cultured on mixtures of polyethylene glycol, fibrinogen, and water called PF hydrogels,
they displayed enhanced myogenic potential in vitro and in vivo (152). PF hydrogels are
believed to rejuvenate myogenic potential because they better mimic the stem cell niche
in vivo, and they could be a useful scaffold material to try in a pericyte‐based SMTE
protocol in the future.
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Induced Pluripotent Stem Cells (iPSCs)
Induced pluripotent stem cells (iPSCs) are produced in the lab by culturing adult
cells and forcing them to upregulate transcription factors that induce pluripotency.
Because they are pluripotent, they are capable of unlimited self‐renewal in culture, which
is a major advantage over satellite cells. Nishikawa et al. (153) wrote a useful review of the
promises and present limitations of iPSCs.

Making iPSCs
Technically, any tissue type could be used to make iPSCs; however, in practice,
making iPSCs for use in skeletal muscle therapies (Figure 5) (154) typically begins with a
simple skin biopsy to isolate epithelial cells and fibroblasts (154). These cell types are
chosen for their abundance and convenience; thus, iPSC biopsy offers a clear advantage
over satellite cell biopsy. After biopsy, pluripotency must be induced in the cells. Takahashi
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et al.'s (155) pioneering paper determined four essential transcription factors, termed
Yamanaka factors, that must be transduced into starting cells using a viral vector to
reprogram the cells into iPSCs: KLF4, c‐MYC, OCT4, and SOX2. Although fibroblasts and
epithelial cells are abundant, iPSC generation is not efficient, with the percentage of adult
cells successfully reprogramming to pluripotency often somewhere in the range of 0.1‐3%
(155,156). This is partially attributable to the fact that cells retain epigenetic markers from
their tissue of origin that make them resistant to reprogramming (157,158).
In the years since Yamanaka's pioneering study, other scientists have worked to
develop more efficient iPSC protocols. Warren et al. (159) optimized the original iPSC
method using synthetic modified mRNA to transfect cells, causing them to be
reprogrammed to pluripotency at a significantly higher efficiency, while Yulin et al. (160)
and Kunisato et al. (161) have developed more efficient iPSC generation protocols that use
BM‐MSCs as a starting source; given that BM‐MSCs have inherent myogenic differentiation
potential, it is presently unclear whether these would have any relevance to SMTE. Malik
(162) compiled a more comprehensive review of human iPSC generation methods.
Regardless, the development of more efficient iPSC protocols suggests that iPSCs could
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eventually be a more practical therapeutic option in SMTE applications. After iPSC
generation, cryopreservation is an efficient method for iPSC storage (163).

iPSC Induction
iPSCs must be properly induced into a myogenic lineage prior to implantation. A
variety of different protocols have been developed to induce myogenesis in iPSCs, and
most of these methods involve inducing myogenic gene expression. Shoji et al. (164)
developed a reliable myogenic induction protocol for iPSCs that works by delivering a
tetracycline‐inducible MyoD piggyBac (PB) vector to human iPSCs, achieving robust
differentiation efficiency that is as high as 70‐90% in vitro. Chal et al. (165) developed a
serum‐free method for inducing iPSCs into muscle fibers and satellite‐like cells by
mimicking embryonic signaling events to induce myogenesis in vitro. Maffioletti et al. (166)
demonstrated a complex, thorough method for myogenic induction and differentiation of
human embryonic stem cells (hESC) and iPSCs that used a four‐stage system with different
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culture densities, media combinations, and protein coatings on the cell culture dish. First,
they drove ES and iPSCs to a lineage that was similar to mesoangioblasts, and then they
induced MyoD1 expression via a lentivirus vector with MyoD1 cDNA fused to an estrogen
receptor, thereby driving the cells to a myogenic lineage. Darabi et al. (167) developed a
protocol that makes iPSCs from fibroblasts derived from inducible Pax7 mice and then
induces Pax7 expression to induce the iPSCs into myogenic progenitors.
It is important to note that iPSCs are ultimately only useful in SMTE if they are
clinically safe. Reprogramming iPSCs to pluripotency and then to a myogenic lineage can
induce genetic instability and abnormalities that cause cancers, and iPSCs are known to be
tumorigenic (168,169). More emphasis should be placed on developing iPSC myogenic
induction and differentiation methods that do not require genetic modification, and
researchers have already begun this work. Hosoyama et al. (170) derived myogenic
progenitors from human iPSCs by using a medium with fibroblast growth factor‐2 (FGF‐2)
and epidermal growth factor to form free‐floating spherical cultures that contained
myogenic progenitors after 6 weeks. After culturing these progenitors in a muscle
differentiation medium for 2 weeks, the researchers observed multinucleated myotubes.
Xu et al. (171) identified three specific myogenic factors: a glycogen synthase kinase 3β
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(GSK3β) inhibitor called 6‐bromo‐indirubin‐3'‐oxime (BIO), FGF, and forskolin. By treating
human iPSCs with a combination of these three factors, they were able to produce
myogenic progenitors that could engraft into mdx mouse dystrophic muscle fibers in vivo.
Any of these methods could be tried on iPSCs in a SMTE study.

iPSCs in Skeletal Muscle Therapies
To date, iPSCs have never been used in a SMTE study to treat VML, but iPSC
injection therapy for DMD is better studied, and offers clues as to how iPSCs might behave
in SMTE. Darabi et al. (172) found that human iPSC‐derived myogenic progenitors restore
dystrophin and improve contractility upon transplantation in dystrophic mice, and Xu et al.
(171) found that human iPSC‐derived myogenic precursors can engraft with mdx mouse
muscle fibers in vivo. Quattrocelli et al. (173) found that iPSCs that have been induced to a
mesodermal lineage are able to contribute to functional recovery of cardiac and skeletal
muscle in a mdx mouse and dystrophic canines. Goudenege et al. (174) found that
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myoblasts derived from human iPSCs are able to efficiently fuse with existing muscle fibers
following transplantation. These positive results suggest that iPSCs could integrate with
existing tissue in an injury site in SMTE for VML.

Embryonic Stem Cells (ESCs)
Embryonic stem cells (ESCs) are pluripotent stem cells isolated from the inner cell
mass of a blastocyst. Beyond the ethical challenges that using ESCs poses (175), ESCs are
difficult and inefficient to isolate, and the isolation process requires high specialization and
training (176). ESCs are also tumorigenic (177). Partially because of these factors, ESCs
have not been used in muscle therapy studies to any meaningful degree.

Discussion
Although satellite cells remain the best‐studied cell type in SMTE for VML, they can
be difficult to isolate, purify, and biopsy. Alternative stem cells have characteristics that
may provide solutions to these problems, although much is presently unknown about how
alternative stem cell varieties will perform in SMTE studies.
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Judging on the biopsy alone (Table 1), ADSCs and pericytes are highly attractive
starting sources that offer advantages over satellite cells, because both can be isolated
from adipose tissue. Adipose tissue is the most promising location to target for pericyte
isolation not just for convenience, but also because pericytes make up 15% of the SVF that
is obtained during normal ADSC isolation (178), but constitute less than .5% of cells
isolated from bone marrow. Adventitial cells make up 20% of the SVF (144), but their
myogenic potential is currently unknown, and should be explored in future studies. BM‐
MSCs are superior to satellite cells in that a larger biopsy can be taken without introducing
a functional deficit. Yet, a BM biopsy is inferior to an adipose tissue biopsy, and BM‐MSCs
make up less than .01% of total BM cells biopsied (92). UC‐MSCs can be easily obtained
after birth, but unless allotransplanted UC‐MSCs become viable, UC‐MSCs will not be an
option for many current VML patients. iPSCs can be obtained from a convenient skin
biopsy, but iPSC reprogramming is not highly efficient.
With respect to isolation (Table 1), ADSCs and BM‐MSCs may offer advantages over
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satellite cells in SMTE. ADSC isolation in particular is highly efficient (98), and in recent
years, explant culture has been developed as an efficient method for ADSC and BM‐MSC
isolation that results in purer cell populations than enzymatic dissociation. Explant culture
may also decrease the cost, processing time, and labor required for cell isolation (179). The
Francis et al. (103) protocol to isolate ADSCs in 30 minutes is a unique and promising
method that also does not use enzymatic dissociation, and it is far faster and simpler than
satellite cell isolation. Pericytes can also use explant culture; although yields are fairly low,
pericytes can be expanded for at least 20 population doublings in culture (148). Notably, a
clinically safe GMP protocol for UC‐MSC isolation has also been developed (135).
No alternative stem cell is inherently easier to purify than satellite cells, and FACS
and MACS remain the most common purification techniques (Table 2) across cell types.
Alternative stem cell varieties have significant marker heterogeneity, so FACS and MACS
can be particularly challenging, in addition to the inherent limitations of these methods.
However, it must also be noted that purification is more commonly required when
enzymatic dissociation is performed, because the resulting isolate is less pure. If explant
culture methods to isolate ADSCs, BM‐MSCs, or pericytes could be used effectively in
SMTE, it is perhaps less likely that purification would be required, which is an important
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potential advantage that these cell types offer. Notably, a microfluidic inertial sorting
method was recently developed to obtain purer satellite cell populations (51); this method
is advantaged in that it is label‐free, but it has yet to be widely experimented with.
In terms of proliferative capacity and myogenic differentiation potential (Table 2),
it is difficult to say which alternative stem cell is best, though pericytes have particular
promise. As Dellavale et al. (148) noted, pericytes have higher innate myogenic potential
than most other stem cells, and their proliferative capacity is sufficient for clinical therapy.
Pierantozzi et al. (180) found that pericytes have similar proliferative capacity to ADSCs,
and are able to differentiate towards myocytes more efficiently than ADSCs. Pericytes'
proliferative capacity and myogenic potential compared to UC‐MSCs and BM‐MSCs should
be determined in future studies. UC‐MSCs also have great promise. It is known that UC‐
MSCs have higher proliferative capacity than adult MSCs like ADSCs and BM‐MSCs (181),
but the myogenic differentiation ability of UC‐MSCs compared to ADSCs and BM‐MSCs is
not well understood. Between BM‐MSCs and ADSCs, it is difficult to determine which cell
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type is superior. Meligy et al. (125) found that ADSCs were more highly proliferative than
BM‐MSCs, but BM‐MSCs had a higher myogenin expression than ADSCs, which correlates
to myogenic differentiation potential. Conflicting with this finding, Bayati et al. (114)
observed that ADSCs and BM‐MSCs had similar differentiation potentials, though, as Bayati
et al. noted, their protocol differed from Meligy et al.'s in terms of the rat species and
differentiation method used. At this point, pericytes, ADSCs, UC‐MSCs, and BM‐MSCs all
appear worthy of future exploration in SMTE VML studies, and many of them have already
been tested to some extent in muscle therapy studies (Table 3).
iPSCs and ESCs are presently less promising than other cell types. iPSCs can be
generated from an abundant tissue like skin, can be patient‐matched, have unlimited self‐
renewal, and can have high myogenic differentiation capacity in culture. Yet, inducing
pluripotency is not efficient, iPSCs are tumorigenic, and most differentiation methods rely
on gene overexpression, which is not clinically feasible. Moreover, unlike pericytes, ADSCs,
and BM‐MSCs, iPSCs do not have immunological characteristics that are useful for
allotransplantation (96,150). Although iPSCs are useful in experimental settings, and offer
some potential advantages over satellite cells, they have more significant barriers to
overcome than other stem cells before they can be used in SMTE. ESCs have many of the
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same limitations as iPSCs, but they are also harder to obtain than iPSCs, carry enormous
ethical and legal burdens, and are not patient‐specific.
Although there is no perfect alternative to satellite cells, based on what is currently
known, ADSCs, BM‐MSCs, pericytes, and UC‐MSCs are particularly promising candidates
for SMTE studies. Each offers potential advantages over satellite cells with respect to
biopsy, isolation, and purification while also having high proliferative capacities and
significant myogenic differentiation potentials. Future studies should consider using these
cell types to develop new SMTE protocols and should compare them with current satellite
cell‐based engineering methods. In the process, it will be important to optimize isolation
and differentiation methods across cell types, to work towards clinical grade SMTE
protocols that do not use gene overexpression, exogenous DNA, or media that contain
animal products. Ensuring proper myogenic differentiation of alternative stem cells either
in vitro or in vivo will be particularly crucial, because, unlike satellite cells, they are capable
of differentiating into multiple tissue types. Despite the challenges ahead, alternative stem
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cells may be the impetus that advances SMTE towards highly functional and practical VML
treatments, to give VML patients the quality of life they deserve.

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muscle tissue engineering. Front Physiol 5, 203, 2014.
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Table 1. Biopsy, Isolation Summary
Cell Tissue of Origin Biopsy Characteristics Isolation

Characteristics
Satellite Cells Skeletal muscle Painful, invasive, lower Enzymatic dissociation is
(29,30) volume tissue yield higher yield but less pure,
while single fiber explant
culture is highly pure but low
yield (40).
ADSCs White adipose Less painful, less Enzymatic dissociation is
tissue (97) invasive, higher volume common (98) and efficient

tissue yield (98) (99); explant culture can
achieve equivalent yield with
greater inherent purity (102)
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BM‐MSCs Bone marrow Painful, invasive, Enzymatic dissociation is

(111) higher volume tissue common (116), but explant
yield culture has been shown to
achieve equivalent yields
(117). However, overall yield
is low, as BM‐MSCs make up
.01% of total BM cells (92).
UC‐MSCs Umbilical cord, Painless, not invasive, Enzymatic dissociation is
cord‐placenta temporally constrained, common (133), and explant
junction shows not everyone will have culture methods have also
particular promise access to an autogeneic been developed (130, 134).
(130) source
Pericytes White adipose For adipose tissue Explant culture is a higher
tissue, skeletal biopsy: less painful, less purity, low yield method that
muscle, bone invasive, higher volume may still be feasible given
marrow, and tissue yield pericytes' high proliferative
pancreas (146) capacity (148, 149).
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iPSCs All tissues, though Less painful, less Enzymatic dissociation is
skin is typically invasive, higher volume followed by inducing
preferred (154) tissue yield pluripotency (154,155).

Reprogramming is not highly
efficient (155,156) but has
been improved (159‐162), and
the process introduces
genetic instability (168,169).
ESCs Inner cell mass of Painless, ethical Isolation is generally

blastocyst challenges (175), inefficient and requires highly

allogeneic skilled, experienced
researchers (176).

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Table 2. Proliferative Capacity and Myogenic Differentiation Potential Summary
Cell Type Proliferative Capacity Myogenic Differentiation

Potential
Satellite Cells Have been expanded to at least 50 Usually highest, but senescence
population doublings in culture (26) as cells are expanded contributes
to decline in myogenesis in vivo
(26, 27).
ADSCs Similar to pericytes (180). Lower than satellite cells, MC‐

Higher or equivalent to BM‐MSCs MSCs, and pericytes (125,180).
(125). Unknown whether BM‐MSCs
Higher than MC‐MSCs (125) have higher myogenic
differentiation potential.
BM‐MSCs Less than ADSCs (125). Lower than satellite cells and
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MC‐MSCs (125). Unknown
whether ADSCs have higher
myogenic differentiation
potential (114, 125).
UC‐MSCs Higher than adult MSCs (181). Relative myogenic potential
unknown.
Pericytes Similar to ADSCs (180). Determined to be an order of
magnitude higher than most
alternative myogenic stem cells,
excluding satellite cells (134).
iPSCs, ESCs Unlimited Can be high when gene
overexpression is used (164‐
167), but these methods are not
clinically feasible. Using medium,
relative myogenic potential
compared to other types is
unknown.
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Table 5. Muscle Therapy Summary
Cell Type Muscle Therapy Applications To Date
Satellite Cells Used in SMTE studies specifically for VML.
Both scaffold (13‐15) and scaffoldless (16) constructs are able to
partially restore structure and function in VML rats and mice.
ADSCs Used in one SMTE study specifically for VML (110).
Undifferentiated ADSC scaffold construct was able to partially
restore structure in a VML rat (110). Functional recovery is
presently unknown.
BM‐MSCs Administered undifferentiated via injection to treat skeletal muscle
injuries in several SMTE studies (126‐128) that are not strictly
confined to VML. Able to partially restore structure and function,
and may also play an immunomodulatory role in healing (129).
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UC‐MSCs UC‐MSC injections promoted functional recovery in DMD mice and
dogs (138). UC‐MSC injections promoted both structural and
functional recovery (139) in a separate study. UC‐MSC injections
were able to promote skeletal muscle regeneration in lacerated
mouse muscle by promoting a healing cytokine environment, rather
than directly fusing with fibers (140).
Pericytes After systemic injection, pericytes can fuse with dystrophic mouse
fibers to contribute to structural recovery in a DMD mouse model
(148).
iPSCs After systemic injection, human iPSCs can fuse with dystrophic
fibers to contribute to structural and functional recovery in
dystrophic mice and canines (171‐174).
ESCs Not well‐studied due to technical and ethical challenges (175‐177)
as well as their tumorigenicity (178).

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Figure 1. Pathway of normal satellite cell myogenesis. Satellite cells express express
paired box protein‐7 (Pax7) and can commit to myoblasts in a process called induction or
can self‐renew. Myoblasts express Pax7, transcription factor myogenic differentiation 1
(MyoD), myogenic regulatory factor 5 (Myf5), and the myogenic protein desmin, and can
Tissue Engineering
differentiate into myocytes, which express MyoD, desmin, and the transcription factor
myogenin. In a process termed terminal differentiation, myocytes fuse into
multinucleated, differentiated myotubes that express the myogenic proteins alpha actinin,
desmin, and myosin heavy chain. Afterwards, myotubes can further mature into
myofibers. Figure adapted from (33).

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Tissue Engineering
Figure 2. Methods for satellite cell isolation. Single fiber isolation (top) typically involves
digesting a whole muscle and dissecting out individual fibers. Fibers have attached satellite
cells that are exposed during the digestion process, and the fibers are plated, allowing the
satellite cells to egress onto tissue culture plates. Depending on the protocol, fibers may
later be removed from the plates to obtain a more pure satellite cell population. Enzymatic
dissociation (bottom) involves mincing a muscle biopsy, performing an enzymatic digestion
to obtain a heterogeneous population of isolated cells that is typically less pure than the
product from single fiber isolation, and then typically purifying the population to obtain an
isolated satellite cell product. Figure adapted from (40).

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Figure 3. Methods for satellite cell purification. FACS (A), MACS (B), and pre‐plating (C)
methods to purify satellite cells. FACS uses fluorophore‐conjugated antibody dyes to sort
cells into groups by electrical charge using a laser, which causes the cells to deflect to
specific regions depending on their charge. MACS uses magnetic microbeads coated in
antibody to sort cells by using a magnet. First, a magnet is applied. Cells that aren't
attracted to the magnet are negative for antibody markers and will thus be collected first.
Then, the magnet is removed to elute cells that are attracted to the magnet. Pre‐plating is
done by serially plating a satellite cell suspension over time to purify satellite cells, and
works because other cell types like fibroblasts and epithelial cells tend to adhere to the
plate first. Figure adapted from (42).

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Figure 4. Visual summary of the relative locations and basic properties of the two varieties
of perivascular stem cells: pericytes and adventitial cells. Pericytes are located in capillary
and microvessel walls and are defined as positive for CD146 and negative for CD34 and
CD45. Adventitial cells are located in the walls of large vessels (veins and arteries) and are
positive for CD34 and negative for CD31, CD145, and CD45. Figure adapted from (142).

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Figure 5. Visual summary of induced pluripotent stem cell (iPSC) generation for muscle
therapy applications with a comparison to satellite cell methods. Making iPSCs starts with
taking a skin biopsy to isolate fibroblasts. The four Yamanaka transcription factors (Oct4,
Sox2, KLF4, and c‐Myc) are added to induce pluripotency. iPSC cells can become muscle
precursor cells by inducing muscle‐specific genes like Pax7 and Pax3. Then, the myogenic
precursors can be transplanted into a diseased patient. Unlike iPSCs, satellite cells are
isolated from skeletal muscle biopsy and must be expanded in culture, but do not have the
capacity for unlimited self‐renewal. After expansion, satellite cells can be transplanted
back into a patient. Figure from (154).

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Tissue

Cell Tissue of Origin Biopsy Characteristics Isolation

tissue (97) invasive, higher volume common (98) and efficient

BM‐MSCs Bone marrow Painful, invasive, Enzymatic dissociation is

preferred (154) tissue yield pluripotency (154,155).

ESCs Inner cell mass of Painless, ethical Isolation is generally

blastocyst challenges (175), inefficient and requires highly

Cell Type Proliferative Capacity Myogenic Differentiation

ADSCs Similar to pericytes (180). Lower than satellite cells, MC‐

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