Origins, potency, and heterogeneity of skeletal muscle fibro-adipogenic progenitors—time for new definitions

Abstract

Striated muscle is a highly plastic and regenerative organ that regulates body movement, temperature, and metabolism—all the functions needed for an individual’s health and well-being. The muscle connective tissue’s main components are the extracellular matrix and its resident stromal cells, which continuously reshape it in embryonic development, homeostasis, and regeneration. Fibro-adipogenic progenitors are enigmatic and transformative muscle-resident interstitial cells with mesenchymal stem/stromal cell properties. They act as cellular sentinels and physiological hubs for adult muscle homeostasis and regeneration by shaping the microenvironment by secreting a complex cocktail of extracellular matrix components, diffusible cytokines, ligands, and immune-modulatory factors. Fibro-adipogenic progenitors are the lineage precursors of specialized cells, including activated fibroblasts, adipocytes, and osteogenic cells after injury. Here, we discuss current research gaps, potential druggable developments, and outstanding questions about fibro-adipogenic progenitor origins, potency, and heterogeneity. Finally, we took advantage of recent advances in single-cell technologies combined with lineage tracing to unify the diversity of stromal fibro-adipogenic progenitors. Thus, this compelling review provides new cellular and molecular insights in comprehending the origins, definitions, markers, fate, and plasticity of murine and human fibro-adipogenic progenitors in muscle development, homeostasis, regeneration, and repair.

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R E V I E W Open Access
Origins, potency, and heterogeneity of
skeletal muscle fibro-adipogenic
progenitors—time for new definitions
Osvaldo Contreras
1,2,3*
, Fabio M. V. Rossi
4
and Marine Theret
4*
Abstract
Striated muscle is a highly plastic and regenerative organ that regulates body movement, temperature, and
metabolism—all the functions needed for an individual’s health and well-being. The muscle connective tissue’s
main components are the extracellular matrix and its resident stromal cells, which continuously reshape it in
embryonic development, homeostasis, and regeneration. Fibro-adipogenic progenitors are enigmatic and
transformative muscle-resident interstitial cells with mesenchymal stem/stromal cell properties. They act as cellular
sentinels and physiological hubs for adult muscle homeostasis and regeneration by shaping the microenvironment
by secreting a complex cocktail of extracellular matrix components, diffusible cytokines, ligands, and immune-
modulatory factors. Fibro-adipogenic progenitors are the lineage precursors of specialized cells, including activated
fibroblasts, adipocytes, and osteogenic cells after injury. Here, we discuss current research gaps, potential druggable
developments, and outstanding questions about fibro-adipogenic progenitor origins, potency, and heterogeneity.
Finally, we took advantage of recent advances in single-cell technologies combined with lineage tracing to unify
the diversity of stromal fibro-adipogenic progenitors. Thus, this compelling review provides new cellular and
molecular insights in comprehending the origins, definitions, markers, fate, and plasticity of murine and human
fibro-adipogenic progenitors in muscle development, homeostasis, regeneration, and repair.
Keywords: Mesenchymal stromal/stem cell, Fibro/adipogenic progenitor, Fibroblast, Adipocyte, Regeneration,
Single-cell RNAseq
Background
In mammals, skeletal muscle represents ~ 30–40% of the
total body mass, regulating body temperature, metabolism,
and physical activity. Comprising the musculoskeletal sys-
tem, striated muscles are responsible for voluntary and
non-voluntary movements. Skeletal muscles are recog-
nized as highly plastic tissue, illustrated by atrophic or
hypertrophic changes when disused or trained.
Mammalian adult skeletal muscle has extraordinary re-
generation capabilities upon injury, making the organ a
perfect model to study regeneration and repair, and inves-
tigate the contribution of adult stem and interstitial cells
in settings of acute or chronic injury. The muscle connect-
ive tissue (MCT) components are the extracellular matrix
(ECM) and its stromal cells, which actively produce, main-
tain, and remodel this dynamic scaffold during develop-
ment, homeostasis, and after trauma.
Among the several cell types that participate in muscle re-
generation, tissue-resident mesenchymal progenitors play a
crucial role by providing signaling cues that modulate other
muscle-resident cells’function, and actively remodel the
ECM during this process. Fibro-adipogenic progenitors
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data made available in this article, unless otherwise stated in a credit line to the data.
* Correspondence: o.contreras@victorchang.edu.au;mtheret@brc.ubc.ca
1
Developmental and Stem Cell Biology Division, Victor Chang Cardiac
Research Institute, Darlinghurst, NSW 2010, Australia
4
Biomedical Research Centre, Department of Medical Genetics and School of
Biomedical Engineering, University of British Columbia, Vancouver, BC V6T
1Z3, Canada
Full list of author information is available at the end of the article
Contreras et al. Skeletal Muscle (2021) 11:16
https://doi.org/10.1186/s13395-021-00265-6
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(FAPs) have been identified as platelet-derived growth factor
receptor alpha (PDGFRα,alsoknownasPDGFRA)express-
ing cells [1,2].Agrowingbodyofevidenceshowsthat
PDGFRα+ FAPs provide regenerative cues to control muscle
stem cell (MuSC) expansion, fate, and myogenesis after acute
damage and aging [1–7]. Furthermore, the ablation of stro-
mal cells by using mice model expressing the diphtheria
toxin receptor (DTR) under the control of the fibroblast acti-
vation protein alpha promoter (FAPα-DTR) impairs the
long-term maintenance of hematopoiesis, muscle mass, and
cachexia [8]. To note, FAPα+ cells are found in most tissues
such as bone, salivary gland, visceral adipose tissue, skeletal
muscle, and pancreas; express CD90, CD140a, and SCA-1;
and so are most likely to be mesenchymal progenitors, hence
FAPs in skeletal muscle [8]. These findings have been con-
firmed by the Rando laboratory using a knock-in PDGFRα
C-
reER
:Rosa26
DTA
mice model [7], and more recently, by
Tsuchida’s group using a similar cell ablation strategy [9]. In-
deed, genetic ablation of PDGFRα+ lineage cells leads to im-
paired MuSC expansion and leucocyte infiltration, leading to
deficient skeletal muscle regeneration after acute chemical
injury and neuromuscular defects and muscle atrophy [7,9].
In addition, following limb ischemia, proper muscle revascu-
larization and repair are lost after ablating FAPs [10]. Hence,
PDGFRα+ FAPs are required for successful muscle regener-
ation, repair, and maintenance during tissue homeostasis and
in pathological states.
Muscle-resident PDGFRα+ cells readily initiate fi-
broblastic colonies (also called fibroblast colony-
formingunits,CFU-F,(Fig.1a)) and can clonally dif-
ferentiate not only into activated fibroblasts/myofibro-
blasts and adipocytes but also into chondrogenic and
osteogenic lineages depending on the context [1,2,
10–15]. The plasticity and clonal expansion of muscle
FAPs are also seen in humans [16]. However, the ef-
fects of damage-induced signals and cues on their
plasticity, fate, and functions have only recently begun
to be explored. The development of new in vivo
lineage tracing tools used to identify and track cells
expressing specific markers in various animal and
damage models in parallel with the recent emergence
of single-cell omics have allowed the identification of
a broad spectrum of specific stromal populations and
their relative contribution to muscle homeostasis, re-
generation, and repair.
The developmental ontology of muscle-resident
mesenchymal progenitors
From the embryo to the adult: role of MCT mesenchymal
progenitors on muscle development
Adult MCT is mainly composed of ECM, largely fibrillar
collagens type 1 and 3, elastin, fibronectin and proteo-
glycans, and the supportive matrix-resident stromal cells,
also called mesenchymal progenitors or traditionally
known as muscle fibroblasts [17–19]. However, com-
pared with the ever-growing knowledge about adult
MCT, the composition and the dynamic remodeling of
embryonic MCT are poorly understood. While evidence
about the ontogeny of interstitial muscle cells exists
[20–22], only a paucity of studies have reported their
embryonic determination, and hence, the developmental
origin and role of these ECM-embedded cells are not yet
fully appreciated and understood.
Kardon and colleagues published early evidence of the
function of these cells in the formation of limb muscles
in the 2000s [23]. The authors described that a mesoder-
mal population of TCF7L2+ cells (formerly known as T-
cell factor 4 or TCF-4, a TCF/LEF transcription factor
downstream the canonical Wnt/β-catenin signaling) reg-
ulates the spatiotemporal determination and differenti-
ation of myogenic progenitors and, therefore, regulates
limb muscle development in chicks [23]. Limb TCF7L2+
precursors derive from the lateral plate mesoderm in a
muscle-specific pattern, but are different from myogenic
precursors since they do not form muscle nor express
classical myogenic markers (e.g., Pax7)[6,23,24]. Thus,
myogenic precursors are patterned by extrinsic cues,
mostly coming from the MCT, after the cells have mi-
grated through the limb rather than being embryonically
predetermined to form particular muscle anatomical
structures [23,25]. These MCT progenitors also influ-
ence the myofiber type of limb and diaphragm muscles
in a paracrine fashion [24]. Interestingly, not all limb
muscles contain TCF7L2+ cells during mouse embryo
development, which suggest a distinct patterning and
three-dimensional distribution of these cells in different
subtypes, or the existence of MCT progenitors that do
not express this marker [26]. Nevertheless, TCF7L2 la-
bels a significant proportion of mammalian stromal non-
myogenic precursors at birth and during adulthood [24,
27,28]. Additionally, MuSCs and endothelial cells also
express Tcf7l2 mRNA and protein, albeit at low levels
compared with FAPs [7,24,28].
Researchers have argued that vertebrate muscles de-
rive from several developmental sources, adding com-
plexity to our understanding of the different origins of
MCT in muscle development. For comprehensive re-
views, see: [20–22]. Like myogenic precursors, MCT
progenitors originate from different and distinct struc-
tural origins during embryonic development. In mam-
mals, these include the somites for axial-trunk muscles
[29], the lateral plate mesoderm for limb muscles [23,
30], the neural crest cells (NCCs) for head and neck
muscles [31–34], and the transient developmental
structure originating from the somites called pleuroper-
itoneal folds (PPFs) for the diaphragm [35]. Remark-
ably, Merrell and colleagues demonstrated that PPF-
resident TCF7L2
+
/GATA4
+
CT precursors regulate the
Contreras et al. Skeletal Muscle (2021) 11:16 Page 2 of 25
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development of the diaphragm and participate in the
etiology of congenital diaphragm hernias (CDH), a type
of fibroproliferative developmental disorder [35]. The
authors also demonstrated that Gata4 null mutations
in CT progenitors expressing Paired related homeobox
1(Prx1)could cause CDH during diaphragm develop-
ment. These studies indicate that the aberrant behavior
of PPFs CT progenitors can cause congenital muscle
diseases like CDH [35].
The studies of Logan’s group have also helped to ad-
vance our understanding of the developmental role of
MCT precursors in muscle morphogenesis. Initially,
through a combination of conditional deletion and ad-
vanced imaging techniques, they demonstrated the cru-
cial participation of T-box transcription factors, Tbx4
and Tbx5, in determining the formation of muscles and
tendons of the musculoskeletal system [36]. Interest-
ingly, they found that the myoblast-specific loss of Tbx5
does not affect the correct positioning of myogenic pre-
cursors. However, genetic deletion of Tbx5 and Tbx4 in
the mesenchyme (paired related homeobox (Prx) ex-
pressing lineage), resulted in the perturbation of MCT
organization, and therefore, caused mispatterned muscle
limbs. Although the authors observed no changes in the
expression of Tcf7l2 in the absence of Tbx4/5, the lack
of these transcription factors impaired the spatiotempo-
ral distribution of TCF7L2+ cells [36]. Remarkably, the
Holt-Oram syndrome, known for leading to skeletal ab-
normalities and congenital heart disease, is caused by
mutations in the Tbx5 gene [37]. The study of Hasson
Fig. 1 aIllustration of FAP cellular properties, including the high expression of PDGFRα, quiescency, CFU-F, and mesenchymal/stromal cell
multipotency. Skeletal muscle fibro-adipogenic progenitors form clonal CFU-F following in vitro cell culture. bZ-stack confocal images showing
the localization of PDGFRα-EGFP
+
cells in tibialis anterior muscle sections of adult PDGFRαH2BEGFP/+ knock-in mice. Pictures show different
skeletal muscle anatomical locations of muscle FAPs. Laminin (magenta) and nuclei (Hoechst, blue) were also stained. Scale bars: 50μm
Contreras et al. Skeletal Muscle (2021) 11:16 Page 3 of 25
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et al. reinforces the model in which MCT gives rise to
muscle pre-patterned structures to guide myogenic pre-
cursors during development and further demonstrated
that extrinsic MCT-derived cues are critical for muscle
morphogenesis. Without surprise, the Transforming
Growth Factor beta (TGF-β) signaling pathway is in-
volved in this process. Indeed, Kutchuk et al. demon-
strated that embryonic myofibers and C2C12 myoblasts
express Lysyl Oxidase (Lox, an enzyme required for
cross-linkage formation in elastin and collagen) and that
its deletion upregulates the TGF-βsignaling. Lox
−/−
mu-
tants display MCT disorganization and delayed myogen-
esis [38]. Thus, this study illustrates the homeostatic
cross-talk between MCT and muscle cells during limb
musculoskeletal system development.
The above-proposed model was recently corroborated
in detail by Besse and colleagues [39]. These authors
took full advantage of an array of labeling and imaging-
based studies, mouse genetics, and transcriptomic ana-
lyses to establish how individual muscle bundles are gen-
erated and established, shedding profound lights on the
role of MCT precursors on muscle morphogenesis at
unprecedented resolution. They provided a compelling
demonstration that muscle morphogenesis is primarily
orchestrated by CT mesenchymal progenitors via the se-
cretion of matrix-modifying proteoglycans [39]. Thereby,
through the expression and the secretion of a myriad of
chemoattractants, ECM components, and growth fac-
tors, these stromal cells promote a variety of responses
in myogenic precursors, including repulsion, attraction,
migration, and patterning [20]. Hence, the MCT creates
a developmental pre-pattern that orientates and controls
the positioning of myogenic precursors that differentiate
into myofibers forming muscle bundles and, conse-
quently, will serve to define the size and shape of mus-
cles, the orientation of its myofibers, and points of origin
and insertion on bones [23,25,36,39,40].
The notion that MCT cells participate in muscle
morphogenesis leads to wonder what determines the
spatiotemporal dynamics and positional information of
MCT precursors. Hox genesareasetofgenescoding
for transcription factors that specify segment identity
and provide positional information during animal de-
velopment [41]. Among them, the caudal Hox11 genes
participate in determining the proximal-distal axis of
the musculoskeletal system of limbs [42–45]. Hoxa11 is
broadly expressed through the distal primordium of
limb buds at E10.5, but later on, at E14.5, it is exclu-
sively expressed in the CT of tendons, perichondrium,
and TCF7L2+ cells, but not in endothelial cells, chon-
drocytes, osteocytes, nor myogenic precursors [46].
Genetic deletion of Hoxa11/Hoxd11 paralogs, which
have a prominent role in patterning bones during de-
velopment, leads to severe defects in the pattern and
regionalization of muscles and tendons, independently
of bone defects [46]. Altogether, these results not only
demonstrate a previously unappreciated function of
Hox genes for proper patterning and integration of
muscles, tendons, and bones but also illustrated that
CT spatiotemporal dynamics participate in the integra-
tion of the musculoskeletal system as a whole. Further
studies should detail how, when and what factor(s)
modulate the spatiotemporal dynamics and positional
fate of muscle connective tissue cells.
Searching for cell-type-specific markers of muscle stromal
fibro-adipogenic progenitors
In adult tissue, two studies characterized a population of
interstitial muscle-resident progenitors with spontaneous
mesenchymal stem/stromal cell (MSC) potential towards
fibrous myofibroblast and fatty differentiation [1,2].
Using fluorescence-activated cell sorting (FACS) of
digested mouse skeletal muscle, our laboratory identified
and named these cells as fibro-adipogenic progenitors
(FAPs) based on their spontaneous differentiation along
these lineages [1]. We characterized these progenitors as
lineage-negative (Lin−, not expressing hematopoietic
(CD45), endothelial (CD31, also known as PECAM-1) or
myogenic markers (α7-INTEGRIN) and positive for
Stem cell antigen-1 (SCA-1) and CD34 cell-surface anti-
gen expression. Interestingly, while quiescent MuSCs,
endothelial cells, and a subset of hematopoietic cells ex-
press CD34, its genetic deletion impairs MuSC but not
FAP proliferation [47]. We also demonstrated that most
Lin-/α7 INTEGRIN-/SCA-1+ cells express high levels of
the receptor tyrosine kinase PDGFRα[1]. Similarly,
Uezumi et al. characterized the same population using a
different gating strategy. They used CD45, CD31, and
Sm/C2.6 (MuSC marker) as a negative selection and
CD140a (PDGFRα) as positive. They showed that Lin-
PDGFRα+ cells express a low level of PDGFRβand can
differentiate in adipocytes, myofibroblasts, and chondro-
cytes in vitro [2]. They also observed that muscle PDGF
Rα+ cells were perivascular but did not co-localize with
NG2, suggesting that PDGFRα+ cells are not pericytes
[2].
PDGFRα+ cells reside in the muscle interstitium and
are more abundant in the epimysium and perimysium
than in the endomysium. Although most muscle-
resident PDGFRα+ progenitors are in close association
with blood vessels [1,2,48], they are distinct from peri-
cytes. Indeed, pericytes are embedded within the endo-
thelium basement membrane, but PDGFRα+ cells reside
outside of vessels. The localization of FAPs is evident
around large blood vessels, in which they adopt an ad-
ventitial position. With rare exceptions in organs other
than muscle, PDGFRαcells do not express defining peri-
cyte markers like Cspg4 (NG2), Rgs5,Pdgfrβ,orMcam
Contreras et al. Skeletal Muscle (2021) 11:16 Page 4 of 25
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(CD146) [2,48,49]. Notably, while FAPs were initially
described in murine muscles, growing evidence indi-
cates that human FAPs have a similar phenotype and
functions to mouse FAPs [16,50–54]. In summary, FAPs
(historically called fibroblasts) and the ECM they actively
secrete and modify are both significant constituents of the
interstitium and perivascular CT.
Distinct subpopulations of CT progenitors exist and
express an array of proteins and transcription factors, al-
beit at variable levels. In the mouse embryo, CT progeni-
tor markers include PDGFRα, TCF7L2, TBX3/4/5,
HOX11, and the Odd-skipped transcription factors
OSR1 and OSR2 [21,23,26,40,55,56] (Table 1). In
murine adult muscles, the large majority of CT fibro-
adipogenic progenitors express PDGFRα, SCA-1 (also
known as Ly6A/E), CD90 (THY1), CD34, TCF7L2,
HIC1, VIMENTIN, DECORIN, and ADAM12 but few of
these markers are specific and unique for this heteroge-
neous population of cells (discussed below) [1,11,12,
28,57,59,61,63,66] (Table 1). Of note, murine adult
muscle PDGFRα+ FAPs express low levels of Osr1,
which increases upon acute injury in a small subset of
FAPs, suggesting the participation of regulatory mecha-
nisms that tightly turn on the expression of Osr1 resem-
bling developmental-like programs [67]. Remarkably,
damage-activated OSR1+ FAPs proliferate faster com-
pared with OSR1- FAPs [67], suggesting that either
OSR1 modulates the expansion and functions of FAPs,
or it represents an activation marker whose expression
increases in proliferating cells.
Recently, Gli1 (also known as glioma-associated oncogene
1) expression has been shown to label a subpopulation of
muscle FAPs with higher clonogenicity and reduced adipo-
genic differentiation than Gli negative FAPs [65]. Perivascu-
lar cells expressing the zinc finger protein Gli1 undergo
proliferative expansion and generate myofibroblasts after
kidney, lung, liver, and heart injury [68]andheartinjury
[6], suggesting that Gli1+ cells are likely a FAP subpopula-
tion as recently shown in skeletal muscles [69].
In humans, cell-surface markers like PDGFRα, CD201,
CD166, CD105, CD90, CD73, and CD15 identify skeletal
muscle FAPs (Table 2)[16,51,53,54,64,71,72]. Remark-
ably, the expression of SCA-1 defines a particular cluster of
stromal cells within the murine FAP population with differ-
ent potency and properties in vivo and in vitro, both in the
skeletal muscle and heart [66,73]. However, as SCA-1 does
not have a human homolog, its use to identify FAPs is lim-
ited by the absence of this antigen in humans.
Recently, we showed that the majority of cells express-
ing the protein-coding gene Hypermethylated in Cancer
1(Hic1) correspond to quiescent muscle-resident FAPs
in mice [48]. In adult muscles, HIC1+ progenitors reside
in the interstitial space and the myotendinous junctions.
In addition to FAPs, small subsets of pericytes (SCA-1−,
RSG5+ cells) and tenogenic cells (SCA-1−, SCX+ and
FMOD+ cells) express HIC1. Therefore, the expression
of Hic1 comprises a larger proportion of mesenchymal
stromal progenitors compared with the expression of PDGF
Rα, which is limited to FAPs [48]. Along with others, we
have confirmed that cardiac PDGFRα+ cells also exhibit
Table 1 Summary of endogenous murine skeletal muscle fibro-adipogenic progenitors
Murine
cell
Canonical
Markers
Alternative
markers
Negative
markers
Localization Differentiation potential Additional comments References
Embryonic-
fetal FAPs
PDGFRα
TCF7L2/
TCF4
Osr1
Osr2
Hox11
Tbx3
Tbx4
Tbx5
Sca-1
a
CD34
a
Adam-12
Tie-2
a
CD45
CD31
Ter119
α7-
Integrin
Muscle-
associated
connective
tissue and
muscle
interstitium
Robust in vitro adipogenic
and fibrogenic differentiation
but low chondrogenic and
no detectable osteogenic or
myogenic potential. Osr1+
progenitors also give rise to
embryonic fibroblast-like cells
in the dermis and FABP4+
adipocytes in white fat pads
Little is known about their
origin, fate, gene
regulation, function,
stemness, and self-
renewal
[24]; [26]; [57];
Adult FAPs PDGFRα
SCA-1
Hic1
CD90
Decorin
(Dcn)
PDGFRβ
b
Col1a1
b
TCF7L2/
TCF4
b
CD34
b
Adam-12
c
Tie-2
c
Gli1
d
CD45
CD31
Ter119
α7-
Integrin
NG2/
Cspg4
Rsg5
Fascia,
epimysium,
perimysium, and
endomysium;
abundant as
perivascular cells
Adipocytes, myofibroblasts,
osteocytes, and
chondrocytes after muscle
injury and in vitro, with no
myogenic potential
Required for adult skeletal
muscle regeneration and
homeostasis; cellular and
molecular dysfunction in
pathology and disease
[1]; [11,12,27,28];
[13]; [14]; [58]; [58];
[59]; [60]; [61]; [62],
[2,15,53,63,64];
a
These markers have not been studied in the embryo with detail
b
These markers are also expressed by different cell types, including satellite cells, pericytes, and endothelial cells
c
Adam-12 and Tie-2 expression appears to be restricted for a subpopulation of FAPs
d
Gli1 defines a subpopulation of murine muscle FAPs with pro-myogenic and anti-adipogenic functions [65]
Contreras et al. Skeletal Muscle (2021) 11:16 Page 5 of 25
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multilineage properties in vivo and in vitro [11,12,73–75].
Inthemurineheart,HIC1+progenitors represent a signifi-
cant proportion of cardiac FAPs [73]. HIC1 (also known as
ZBTB29) is a transcription factor involved in quiescence and
cell cycle control [76,77]. Consistent with these roles, the
conditional deletion of Hic1 induces aberrant cell activation
and proliferation of FAPs, impairing muscle regeneration fol-
lowing acute damage and leading to spontaneous develop-
ment of arrhythmogenic cardiomyopathy-like pathology and
signs in the mouse heart [48,73]. Thus, the unrestrained ac-
tivation of these progenitor cells and the consequent gener-
ation of differentiated progeny are potential pathological
drivers of disease.
We claim that the heterogeneity of FAP markers
makes sense in a context where the upregulation and
downregulation of cell-specific makers participate in
modulating the commitment of FAPs into a transitional
cell state or differentiation process during lineage pro-
gression in response to injury or in disease states. Hence,
FAP heterogeneity might contribute to restricting or
priming the multipotency of PDGFRα+ FAPs. These are
important issues to explore in future research.
Adult muscle connective tissue and PDGFRα+
progenitors
Adult skeletal muscle contains several cell types that
work in unison under tightly regulated conditions to
maintain homeostasis. Adult mammalian muscle is a re-
markable exception to the low regenerative potential of
several organs and tissues like the heart. Before we start
highlighting the contribution of endogenous PDGFRα+
cells to mammalian skeletal muscle homeostasis, regen-
eration, and repair, it is worth revisiting the terminology
in this area. Muscle regeneration is defined as the spe-
cific substitution or replacement of lost tissue, eventually
leading to full restoration of muscle strength. This re-
generative capacity relies on resident adult unipotent
stem cells (also known as satellite cells), which are quies-
cent but activate to rebuild this tissue upon injury [6,
78–80].
In comparison, skeletal muscle repair aims to safe-
guard the remaining function of muscle following solu-
tions of continuity after partial loss of tissue due to
massive traumas or chronic insults such as repetitive in-
juries, disease, and aging. Thus, muscle repair often en-
tails replacing lost myofibers with scar tissue, which acts
as a bridge between areas still capable of contraction (for
review of tissue regeneration and repair see [81–83].
Therefore, while repair restores muscle integrity, regen-
eration accounts for restoring tissue function. As ob-
served in other mammalian regenerative tissues such as
the liver [81,84], the form and periodicity of damage
can impair the ability of the skeletal muscle to return to
homeostasis [59,85,86]. Therefore, the current estab-
lished dual role that PDGFRα+ cells play in acute (re-
generative) and chronic damage (reparative/
degenerative) suggests that the organization of their
intracellular signaling network may integrate opposite
complementary signals whose relative strength mainly
depends on the type, extension, and frequency of the
Table 2 Summary of endogenous human skeletal muscle fibro-adipogenic progenitors
Human
cell
Canonical
Markers
Alternative
markers
Negative
markers
Localization Differentiation potential Additional
comments
References
Embryonic-
fetal FAPs
PDGFRA DCN
FN1
LUM
OSR1
POSTN
FAP
THY1/CD90
VIM
NT5E/CD73
COL1A1
COL1A2
COL3A1
PTN
OGN
FBLN5
PAX3
PAX7
Similar to what is
found in mouse
development,
although not
evaluated in detail
Not evaluated but probably
similar to what is found in
mouse development
No information
about their origin,
gene regulation,
function, and
potency
[70]
a
Adult FAPs PDGFRA, CD34
(when negative
for CD56, CD31
and CD45)
CD201
CD166
CD105
CD90
CD73
CD34
CD15
COL1A1
TCF7L2/
TCF4
CD31
CD45
CD56
α7-
Integrin
NG2/
CSPG4
RSG5
Fascia, epimysium,
perimysium, and
endomysium;
abundant as
perivascular cells
Adipocytes, myofibroblasts,
osteocytes, and chondrocytes
in diseased states and in vitro.
Lack of myogenic potential
Increased numbers
in diverse
pathologies
[16]; [51];
[71]; [72];
[52]; [53,
54,64]
a
These other alternative markers suggested by Pyle and colleagues are based on scRNAseq data ([70])
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injury. For this review, we redefine the fate and hetero-
geneity of muscle-resident PDGFRα+ progenitors and
explain their multilineage potentials.
The adult muscle connective tissue
The muscle environment is complex in structure and
several heterogeneous cell types co-exist within it to
regulate its function and structure. Although skeletal
muscles have an intricate network of blood vessels and
nervous tissue, most of their mass is comprised of myo-
fibers. In adults, MCT, which accounts for 1–20% of the
total dry mass of muscles, surrounds, protects, and inter-
connects these primary components [19,87,88]. The
amount of CT varies significantly from one muscle to
another, depending on the anatomical location and
physiological function of particular muscles [89]. The
adult MCT follows the nomenclature of fascia, epimy-
sium, perimysium, and endomysium accordingly to its
location and arrangement within the tissue [90,91] (Fig.
1b). The topological organization of the covering con-
nective tissue from the outside is described as follows:
the fascia, the CT outside the epimysium that surrounds
and separates the muscles; the epimysium, which sur-
rounds each muscle group, linking them to the tendons
at the myotendinous junctions; the perimysium, which
consists of collagen-rich structures that surround the
fascicles and interconnect with the epimysium; and the
endomysium, which represents a modified basement
membrane unsheathing individual myofibers and inter-
connects to the perimysium [19,91,92] (Fig. 1b). These
four levels of stromal organization describe the intercon-
nected ECM compartments within muscles. Although
each compartment is distinguished by its anatomical
position, it is difficult to discriminate each of these ECM
compartments in terms of their protein and cellular
composition. Remarkably, MCT not only determines the
macro and microstructures of embryonic and adult mus-
cles but also connects the myofibers to produce and
transmit force. As a result, it increases not only the effi-
ciency of force generation but also protects myofibers
from excessive stretching, supporting muscle regener-
ation and cellular mechanosensation [17–19].
Muscle-resident fibro-adipogenic progenitors: definitions
and identity
Historically, the observation of ECM proteins, such as
collagens, being produced and deposited in skeletal
muscle suggested the existence of a resident collagen-
producing cell within the tissue [17,18,90]. Later, nu-
merous observations of CT hyperplasia and interstitial
proliferation associated with healing scars in skeletal
muscle diseases, including congenital muscular dystro-
phies, immobilized muscles, and neuromuscular disor-
ders (e.g., amyotrophic lateral sclerosis and denervation)
radically increased the attention paid to MCT [93–100].
In order to understand MCT development, establish-
ment, and remodeling, it is crucial to consider the stro-
mal cells that participate in these processes. A critical
and challenging step towards a complete understanding
of MCT biology has been the identification of a hetero-
geneous population as the primary effector of ECM de-
position and remodeling [11,12,17,18,48,62].
Increasing evidence suggests that there are distinct sub-
sets of stromal cells located in discrete yet similar ana-
tomical positions during muscle development and into
adulthood [20–22]. This stromal cellular diversity and
heterogeneity have been an obstacle to attributing the
primary role for matrix deposition to a specific subset of
stromal cells.
Jackson and colleagues reported the existence of
tissue-resident mesenchymal progenitors with multiline-
age differentiation capabilities in damaged human
muscle over a decade ago [101]. Today, thanks to the
great effort of many researchers, we know that adult
MCT is mainly produced by muscle-resident PDGFRα+
cells with multilineage progenitor properties and a
fibroblast-like phenotype, called FAPs. Increasing evi-
dence suggests that these muscle-resident cells are the
primary cellular source of regenerative matrix deposition
as well as scarring following muscle injury, disease,
neuromuscular disorders, or aging [1,2,5,9,11,12,14,
15,27,53,54,59,61,102–105]. Vallecillo-García and
colleagues showed that the source of developmental
ECM in limb muscles is a heterogeneous population of
PDGFRα-expressing progenitors called embryonic FAPs,
closely resembling the population of adult stromal cells
we have described, along with other groups [1,2,6,23,
24,26]. These findings led to some confusion in the no-
menclature, with some publications distinguishing be-
tween FAPs and fibroblasts, some using the term FAPs
as better representing their predominant fibrogenic and
adipogenic developmental potential, and some remaining
faithful to the historical term fibroblast, which are also
known for being heterogeneous and plastic cells. Here,
we propose that these muscle-resident multipotent pro-
genitors, whether called FAPs or fibroblasts, are the
same cells.
From this point on, the term PDGFRα+ FAPs will
refer to muscle-resident CT mesenchymal progenitor
with multilineage developmental properties. As dis-
cussed below, recent advances in single-cell RNA se-
quencing demonstrated that FAPs comprise multiple
sub-populations, some of which could be bona fide dif-
ferentiated cells with little developmental potential left
[16,48,62,106–109]. This may create a problem with
nomenclature diversity, speculation, and high cellular
heterogeneity within the adult stromal lineage [110].
FAP heterogeneity is also known to increase following
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injury and disease, which also complicates their classifi-
cation and nomenclature [48,62,66,108,111].
The muscle community has historically described
interstitial cells with MSC capability (i.e., fibrogenic, adi-
pogenic, chondrogenic, and osteogenic potency). In
addition to PDGFRα+ cells, muscle-resident pericytes
have also been proposed to be MSCs that have adapted
to the specialized functions required by their adjacent
vascular niche. However, although PDGFRα+ FAPs be-
have as and present defined canonical MSC properties,
FAPs are different from tissue-resident pericytic “MSCs.”
Indeed, pericytes' cell-surface profile is CD34-/CD45-
and CD146+ [112,113]. Remarkably, Bianco and col-
leagues revisited the MSC origins and differentiation po-
tential using a broad set of human MSC-like cells (HLA
class I, CD73, CD90, CD105, and CD146 positive cells).
The authors showed that the cell surface phenotype of
“MSCs”isolated from bone marrow, skeletal muscle,
periosteum, and cord blood, although quite identical, did
not reflect these cells’cell transcriptomic identity, func-
tion, and therefore, their differentiation properties. Thus,
“MSCs”are separated from each other, as the authors
defined it, by a developmental origin factor [113]. Not-
ably, the authors also showed that CD146+ pericytes are
not true MSCs in most of the analyzed tissues, with the
possible exception of the bone marrow, where they in-
herently form bone and bone marrow stroma but lack
chondrogenic potential in vivo or myogenic in vitro. On
the contrary, in skeletal muscle, CD146+ perivascular
pericytes are rather inherently myogenic than skeleto-
genic [113]. Remarkably, skeletal muscle pericytes are a
distinct cell type from MuSCs (CD56+/CD146-) and
CD34+/CD146+ endothelial cells that possess a latent
myogenic gene signature and potential, and hence,
muscle pericytes are committed myogenic progenitors
[113,114]. These pivotal studies have challenged the
loose and non-specific MSC nomenclature. However,
further studies with lineage tracing and clonal assays are
needed to deeply understand stromal cell dynamics in
development, homeostasis, and injury and, therefore, to
finally faithfully unify their markers, nomenclatures, and
definitions.
The abundance of collagen, especially the most abun-
dant protein in animals, type I collagen, determines the
stiffness of mammalian tissues [115]. Notably, increased
production and deposition of type I collagen fibrils are
found after muscle damage. Several cell sources have
been suggested as producers of collagen proteins. Using
a murine model of increased of increased muscle fibro-
sis, Chapman et al. corroborated that at least three dif-
ferent muscle-resident cell populations express collagen
I, among them PDGFRα+ FAPs. However, muscle pro-
genitors (α7-INTEGRIN)+ and SCA-1+ cells also ex-
press the mRNA for this fibrillar matrix protein [17,18].
These results further confirm our idea that muscle FAPs
cannot be solely identified using collagen I reporter
mice, but as previously suggested, we strongly recom-
mend employing PDGFRαexpression. Since most of the
work related to FAPs biology refers to models of single,
or repeated rounds of injury, we believe that further
studies will likely uncover the role of PDGFRα+ cells in
atrophy-related pathologies such as aging-related sarco-
penia, cachexia, myasthenia gravis, polytrauma, and
neuromuscular disorders. Further research is needed to
clarify the existence of subtle differences within stromal
cells that might have functional impacts and conse-
quences in muscle physiology, not only during mainten-
ance but also in pathological and disease states.
Multipotency of muscle-resident PDGFRα+ fibro-
adipogenic progenitors
In healthy adult muscles, we and others have demon-
strated that PDGFRα+ cells represent between ~ 5–15%
of the total nuclei and ~ 20–30% of the interstitial mono-
nuclear cells at homeostasis [11,12,106,107,116]. Stro-
mal PDGFRα+ FAPs display MSC properties and can
spontaneously differentiate into adipocytes (rounded,
single-vacuole lipid-rich cells, perilipin+ and peroxisome
proliferator-activated receptor gamma+ (PPARγ)), acti-
vated fibroblasts (long-shaped contractile cells with
fibroblast-like morphology, αSMA
+
(Acta2), and highly
producing ECM cells), as well as chondrocytes/osteoblasts
when bulk cultured, and in clonal assays in vitro and
in vivo [1,2,11–13 15,50,51,59,104,117]. Notably,
HGFA, an injury-induced systemic cue, activates muscle
FAPs, priming these cells to transition from quiescence
into a cellular state with enhanced regenerative potential
also known as G alert state [118]. In the following chap-
ters, we discuss FAP multipotency (Fig. 2).
Fibrogenic potential of PDGFRα+ FAPs
When FAPs are cultured in vitro using standard growth
media and 20% oxygen, a large proportion of them will
spontaneously differentiate into activated fibroblasts
with αSMA
+
stress fibers [1,11,12,15] (Fig. 2). This
demonstrates that FAPs have intrinsic capabilities to dif-
ferentiate, which is unleashed following their activation
and makes in vitro studies easily feasible. However, the
mechanisms regulating the fibrogenic potential of FAPs
remain underexplored.
Transforming growth factor-beta signaling
One of the most studied signaling pathways in regulating
the behavior and fate of muscle FAPs is the transforming
growth factor-beta (TGF-β) signaling pathway. The
TGF-βsub-family of cytokines (TGF-β1, TGF-β2, and
TGF-β3) are secreted proteins that participate in cell-
and tissue-specific biological processes such as wound
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healing, angiogenesis, immune regulation, apoptosis,
tumorigenesis, and proliferation. In pathological condi-
tions, they strongly associate with tissue damage, dys-
function, and fibrosis and are notably mis-expressed
(Burks & Cohn, [119,120]). The complexity of the TGF-
βpathway is exemplified by its pleiotropic effects, indu-
cing growth arrest in some cell types but promoting the
proliferation of others [121,122]. TGF-βenhances the
proliferation and differentiation of several cell types, in-
cluding stromal cells (for review, see [123,124].
When secreted, TGF-βassociates non-covalently to a
large complex consisting of the latency-associated pep-
tide (LAP) and latent TGF-β-binding protein (LTBP)
proteins [125]. Extracellular TGF-βis activated after its
release from the LAP-LTBP complex, which can occur
via proteolytic rupture or through ECM-cell forces gen-
erated by cell traction via the integrin complexes [126–
129]. After release, TGF-βbinds to its heteromeric
serine/threonine kinase type 1 and 2 receptors
[TGFBR1/ALK5 and TGFBR2, respectively], and
TGFBR3 (also known as betaglycan) co-receptor on the
cell surface of the target cell. Of interest, while TGF-
βfamily ligands can bind TGFBR3, this receptor does
not have signaling activity on its own, but it modifies the
affinity of TGFBR1 and 2 to TGF-βligands [130]. In-
deed, TGFBR3 acts as a co-receptor, amplifying TGF-β
signaling activation [131]. TGFBR3 also binds other
TGF-β-family ligands such as ACTIVINS, INHIBINS,
Fig. 2 Skeletal muscle FAPs are quiescent cells with multipotency to differentiate towards all the mesenchymal lineages, depending on the
degree of activation and tissue damage. Tissue injury and its associated biochemical cues and cell-secreted factors activate muscle FAPs.
Activated FAPs act as immunomodulatory stromal cells and signaling hubs before their commitment to more specialized cells. Usually, muscle
injury induces the differentiation of them into activated fibroblasts and adipocytes. Severe damage and chronic pathologies tip their
differentiation also into chondrogenic and osteogenic lineages. The figure also shows different molecules and factors as well as ligands that
regulate their differentiation potential and fate. Notably, many of these molecules hold several steps of FAP life. As quiescent FAPs find their way
into activation and cell differentiation, they lose the expression of quiescence markers and their FAP identity but gain cell differentiation markers
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and bone morphogenetic proteins (BMPs), which we
know are primordial proteins for ECM remodeling in
skeletal muscle [132,133]. This co-receptor can also be
soluble (by a mechanism called shedding [134]) and
could, in some cases, act as an inhibitor of the TGF-β
signaling by sequestration of its various ligands [131,
135]. Nevertheless, the function of TGFBR3 in FAP or
mesenchymal progenitor behavior has not been studied
yet. Its modulation could be a powerful tool as TGFBR3
misexpression is associated with cancer and metastasis
[136], in which ECM remodeling is known to be highly
active.
Then TGF-βcanonical downstream effectors SMAD2
and SMAD3 (R-SMADs) are phosphorylated throughout
TGFBR1/ALK5 kinase activity and form a cytoplasmic
heteromeric complex with SMAD4 (co-SMAD) [121,
122]. This ternary protein complex translocates to the nu-
cleus where it recognizes SMAD-binding elements (SBE)
in the DNA to regulate the expression of diverse target
genes [123,137]. In parallel, SMAD6 and SMAD7 act as
inhibitors (also called I-SMADs). Their model of action
can be various (via TGFBR1 or SMAD4), but their activa-
tion is often a result of a negative feedback loop aiming to
downregulate the TGF-βor the BMP signaling pathway
[138–140]. TGF-βalso activates non-canonical down-
stream signaling pathways such as ABL, PI3K-AKT, RHO,
TAK1, ERK1/2, JNK, and p38-MAPK [124,141]. In vitro
and in vivo experiments suggest that both canonical and
non-canonical TGF-βpathways are involved in fibroblast
proliferation and myofibroblast differentiation, and
thereby modulate TGF-β-induced fibrosis and ECM re-
modeling [11,12,120,126,128,141–143]. However, the
specific role of TGF-βcanonical and non-canonical path-
ways in regulating muscle-resident PDGFRα+ FAP plasti-
city and fate remains underexplored.
In response to muscle injury, TGF-βis produced and
secreted by macrophages, FAPs, and regenerating myofi-
bers [11,12,59,144,145]. Muscle FAPs express the
three TGF-βisoforms (TGF-β1, TGF-β2, and TGF-β3)
and TGF-βreceptors (TGFBR1, TGFBR2, and TGFBR3)
[11,12]. TGF-βligands through TGFBRs induce FAP-
myofibroblast differentiation and ECM production [11,
12,144,146]. In addition, TGF-βinhibits the adipogenic
priming of muscle FAPs [11], and is pro-mitogenic, and
hence, stimulates the proliferation of PDGFRα+ FAPs
[11,12,59,144] (Fig. 2). TGF-βsignaling pathway acti-
vation also seems to be required for FAP survival since
the in vivo treatment of mice with SB431542—a selective
and potent ALK4, ALK5, and ALK7 receptor inhibitor—
reduced the number of expanded FAPs following rotator
cuff tear injury [147] (Table 3). Remarkably, we also
showed that TGFBR1 and the p38-MAPK protein are re-
sponsible for TGF-β-mediated downregulation of PDGF
Rα[11], associated with a decrease in TCFL2 expression
in vitro and in vivo [28]. Thus, as these cells activate,
proliferate, and differentiate they lose or reduce the ex-
pression of their progenitor state markers (Fig. 2).
Wnt/β-catenin signaling
The Wnt/β-catenin pathway relies on the binding of
Wnt ligands to Frizzled receptors and the co-receptors
LRP5 and LRP6 at the cell surface to initiate a cascade
that regulates the intracellular proteostasis of β-catenin
(for recent reviews about the Wnt/β-catenin signaling
see [156,157]. At steady state, the β-catenin pool that is
not participating in cell adhesion is bound to a destruc-
tion complex, where it becomes phosphorylated and tar-
geted for degradation in a process mediated by the
ubiquitin-proteasome system (UPS) [158]. The Wnt
ligand-mediated destabilization of the β-catenin destruc-
tion complex leads to the accumulation of activated β-
catenin (unphosphorylated). Accumulated cytoplasmatic
β-catenin subsequently translocates to the nucleus and
associates with DNA-binding T-cell factor (TCF) or
lymphoid enhancer factor (LEF)–TCF/LEF−transcrip-
tion factors (TFs) [159]. The binding of β-catenin and
TCF/LEF recruits transcriptional partners and chromatin
remodeling complexes to regulate the expression of
TCF/LEF target genes [160,161].
Despite the increasing knowledge about the Wnt sig-
naling pathway, the participation of Wnt proteins and
signaling in modulating FAP fate has not been investi-
gated until recently. Skeletal muscle SCA-1+ cells (FAPs)
are abundant in the muscles of the mdx mice (model of
the Duchenne Muscular Dystrophy (DMD)), and
WNT3a treatment promotes their proliferation and col-
lagen expression both in vitro and in vivo [162]. Interest-
ingly, the treatment of dystrophic mice with DKK1
(Dickkopf 1, a WNT inhibitor) reduced β-catenin pro-
tein levels and muscle fibrosis [162]. On the other hand,
increased canonical Wnt/β-catenin signaling regulates
satellite cell fate and fibrogenic commitment via cross-
talk with TGF-β2 in dystrophic mdx muscles [163]. Ac-
cordingly, we also observed increased β-catenin protein
levels upon acute glycerol muscle injury [28]. Xiang and
colleagues showed that the conditional genetic loss of β-
catenin in heart fibroblast (Transcription factor 21
(TCF21) + cells) and activated fibroblasts and myofibro-
blasts (Periostin+) lineages reduces fibrosis and amelio-
rates cardiac hypertrophy induced by pressure overload
[164]. In agreement, the sole transgenic overexpression
of canonical WNT10B is sufficient to induce fibrosis
in vivo [165]. Overall, the Wnt/β-catenin pathway regu-
lates the expression of several ECM genes in fibroblasts
from different tissues and organs following injury and
disease [28,164–166].
The outcomes of Wnt/β-catenin signaling depend on
the TCF/LEF TFs. However, the potential roles of them
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in muscle FAPs are underexplored. These TFs recognize
TCF/LEF-binding elements and regulatory regions of
target genes to regulate gene expression. In this context,
we showed the expression of the four Wnt TCF/LEF
members in MSC and fibroblast cell lines, as well as
tissue-resident FAPs from skeletal muscle and cardiac
tissues [28]. We observed that Tcf7l2 and Tcf7l1 were
the two most highly expressed members, whereas the
fibroblast lineage, including FAPs, express Tcf7 and Lef1
at lower levels. Moreover, treatment with TGF-βde-
creases both the mRNA and protein levels of TCF7L2 in
PDGFRα+ cells. We described that this regulatory mech-
anism requires the transcriptional regulation activity of
histone deacetylases (HDACs) and the participation of
the UPS [28].
Interestingly, TGF-βactivates the canonical Wnt/β-ca-
tenin cascade and induces nuclear accumulation of β-
catenin, which in turn reduced the expression of the
WNT inhibitor DKK1 [165]. In agreement with our
most recent results showing that TGF-βreduces the ex-
pression of several TCF7L2 target genes, whereas it pro-
motes the expression of ECM remodeling genes in
idiopathic pulmonary fibrosis and heart fibroblasts [28].
Hence, our work confirms the cross-talk between the
Wnt and TGF-βpathways that controls the fate of
PDGFRα+ cells and potentially fibrosis (Table 3). In
summary, the Wnt cascade modulates TGF-β-mediated
effects in fibroblasts, and vice versa [28,167–169].
Platelet-derived growth factor signaling
The platelet-derived growth factor (PDGF) signaling
pathway regulates not only vascular development and
angiogenesis [170] but also plays crucial roles during de-
velopment, stem cell fate, migration, and proliferation.
PDGF receptors (PDGFRs) are the cell membrane-
bound tyrosine kinase receptors for PDGF ligands [171–
174]. PDGFs were initially described as serum-derived
mitogens essential for fibroblast and smooth muscle cell
growth [175,176]. PDGFs ligands are four gene products
consisting of five dimeric isoforms: the homodimers
PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and the
PDGF-AB heterodimer [177]. PDGFs are known for be-
ing released from α-granules of platelets and are potent
chemoattractants and mitogens for cells of mesenchymal
Table 3 Summary of drug strategies to target muscle fibro-adipogenic progenitor differentiation and fate
Therapy Target Cell
survival
Proliferation Cell death/
apoptosis
Fibrogenesis Adipogenesis References
AG1296 PDGFR kinase activity
inhibitor
Not
evaluated
Reduced? Not evaluated Reduced Not evaluated [11]
AICAR AMPK activator Reduced Not
evaluated
Induced Not
evaluated
Reduced [148]
Azathioprine Immunosuppressant Not
affected
Reduced Not affected Not affected Reduced [149]
Batimastat MMPs inhibitor
(including MMP14)
Not
affected
Not affected Not affected Not affected Reduced [14][104]
BMS493 Pan-retinoic acid
receptor (RAR) inverse
agonist
Not
evaluated
Reduced Not evaluated Reduced Induced
spontaneous
differentiation
[69]
Dexamethasone Glucocorticoid
receptor
Induced Induced Not affected Not
evaluated
Induced [150]
HDAC inhibitors
a
(TSA and
Pracinostat)
HDACs Not
evaluated
Not
evaluated
Not evaluated Reduced Reduced [28][151]
[152];
LY2090314 & other GSK
inhibitors
GSK3 inhibitors Slightly
decreased
Not affected Not affected Mixed results Reduced [153]
Metformin AMPK activator Not
evaluated
Reduced Not evaluated Not
evaluated
Reduced [16]
Molsidomine NO donating molecule Reduced? Reduced? Not evaluated Reduced Reduced [154]
Promethazine hydrochloride H1 histamine receptor Not
affected
Not affected Not affected Not
evaluated
Reduced [72]
SB525334/SB431542 TGFBR kinase activity
inhibitor
Reduced Reduced Induced after
long
treatment
Reduced Not evaluated [11,12][147]
TKIs (imatinib, nilotinib,
crenolanib, sorafenib, and
masitinib)
Abl, PDGFRs, Kit, DDRs,
p38
Reduced Reduced Induced Reduced Reduced and/or
Induced
[11,12];
b
[16];
[59]; [155];
[146];
a
HDACs-mediated effects on FAP fate are seen only in young mdx but not aged mdx mice
b
[16] reported that imatinib enhances the amount of perilipin+ FAP-derived adipocytes in vitro
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origin [178]. However, several other cell types express
and secrete these ligands, such as inflammatory cells
(e.g., macrophages) and fibroblasts [179]. Post-
translational proteolytic processing of PDGFs is neces-
sary for their activation. It occurs extracellularly for
PDGF-C and PDGF-D but intracellularly for PDGF-A,
PDGF-B, and PDGF-AB [178,179]. A biologically active
PDGF ligand is a dimer of two single PDGF chains,
which binds one PDGFR.
PDGFRs genes (PDGFRA and PDGFRB) encode
single-pass transmembrane receptors with an extracellu-
lar portion of five immunoglobulin-like domains, a
transmembrane segment, a juxtamembrane segment, a
tyrosine kinase domain, and a carboxy-terminal tail
[180]. PDGFRs are monomeric before exposure to PDGF
[181]. Its ligand binding-induced dimerization causes
their activation, and therefore, later PDGFR de-
repression and activation of the receptor's tyrosine kin-
ase activity [180,182,183]. Three known functional
dimer forms of the receptors exist. They consist of the
PDGFRα/αand PDGFRβ/βhomodimers and the PDGF
Rα/βheterodimer [179,180]. PDGF-AA, PDGF-AB,
PDGF-BB, and PDGF-CC promote PDGFRα/αhomodi-
mer formation, PDGF-BB, PDGF-CC, PDGF-DD, and
PDGF-AB promote PDGFRα/βheterodimer assembly.
PDGFRβ/βhomodimer can only be induced by PDGF-
BB and PDGF-DD isoforms [177,178,180]. Although
the precise role of PDGF and its receptors in vivo in
muscle-resident FAPs is unknown, PDGF signaling
seems to regulate FAP survival, activation, proliferation,
migration, and fate. In this review, we focused on PDGF
ligands and PDGFRαin skeletal muscle health and
pathophysiology.
Treatment of ex vivo FAPs with PDGF-AA and PDGF-
BB ligands activates the PDGF cascade inducing FAP ac-
tivation and proliferation (Fig. 2)[11,53]. In addition,
upregulated expression of ECM genes and activated
downstream ERK1/2, PI3K-AKT, and SMAD2/3 signal-
ing pathways is observed in ex vivo FAPs in response to
PDGF-AA treatment [53,184]. By utilizing a pharmaco-
logical inhibitor of PDGFR signaling, Mendias and col-
leagues showed that PDGFR signaling modulates muscle
ECM remodeling and angiogenesis upon synergist abla-
tion surgery to induce postnatal muscle growth or
hypertrophy [185]. In addition, the treatment with
PDGF-AA induces the phosphorylation of PDGFRαand
the proliferation of PDGFRα+ cells (Fig. 2)[53]. The au-
thors also suggested, using pharmacological inhibitors,
that both PI3K-Akt and MEK2-MAPK signaling path-
ways are necessary for PDGFRα-induced proliferation
[53,54]. However, persistent PDGF ligand exposure and
enhanced PDGFRαsignaling levels can cause patho-
logical muscle fibrosis [53,54,155,184]. We have re-
cently shown that PDGF-BB treatment activates
proliferative and differentiation-related downstream sig-
naling pathways such as PI3K-AKT, ERK1/2, p38-
MAPK, and STAT3 in PDGFRαexpressing cells [11,
12]. Recently, Farup et al. showed that PDGF-AA treat-
ment increases the expression of collagen type I in FAPs,
whereas it reduces their adipogenic differentiation (Fig.
2). Notably, the PDGF-AA-mediated fibrogenic fate of
FAPs associates with a metabolic switch that promotes
enhanced glucose consumption [16]. Hence, PDGF sig-
naling could regulate the potency and fate of skeletal
muscle FAPs (Fig. 2).
In the heart, Asli et al. showed that PDGF-AB treat-
ment promotes colony formation and self-renewal of
cardiac fibroblast, whereas the PDGFR inhibitor,
AG1296, suppressed these activities [186]. Interestingly,
activated PDGFRαH2BEGFP-mid fibroblasts formed at
the expense of resting PDGFRαH2BEGFP-high fibro-
blasts [73,186]. These results are in agreement with our
recent findings where the expression of PDGFRα
changes dynamically during muscle regeneration and re-
pair [11]. Moreover, in vivo PDGF-AB treatment of un-
injured hearts did not cause fibroblast activation;
however, it increased the number of PDGFRαH2BEGFP-
mid fibroblasts after myocardial infarction [186]. There-
fore, PDGF-AB isoform targets tissue-resident fibroblasts
by increasing the activated fibroblast pool after injury.
Interestingly, genetic loss of Pdgfra in the resident car-
diac fibroblast lineage (TCF21+ cells) results in an over-
all reduction in the fibroblast population in adult hearts,
demonstrating that PDGFRαregulates fibroblast main-
tenance and homeostasis [187]. Consistently, lineage-
specific deletion of Pdgfra in tubulin polymerization-
promoting protein family member 3 expressing cell
population (Tppp3+ tendon stem cells) caused impaired
tendon regeneration, and therefore, corroborates the cell
requirements of PDGFRαsignaling for proper tendon
healing [188]. Remarkably, the passaging of plastic ad-
herent FAPs obtained from muscles reduces the protein
levels of PDGFRα, which associates with their differenti-
ation [12]. Thus, cellular PDGFRαbioavailability may be
a modulating factor in PDGF-mediated responses of
FAP lineage during survival, fate decisions, and damage-
associated behaviors.
Adipogenic potential of PDGFRα+ FAP cells
Infiltration and deposition of fatty adipose tissue are
hallmarks of several skeletal muscle pathologies. How-
ever, the cellular and molecular mechanisms underlying
fatty infiltration of muscles have not been extensively in-
vestigated compared with the ever-growing research in
muscle fibrosis. A better understanding of such a
discrete fat compartment between myofibers and fascia,
also called intra/intermuscular adipose tissue (IMAT),
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may allow for the targeting of these adipogenic progeni-
tors to increase muscle regeneration and repair.
The lack of reliable cell-specific markers for fat pre-
cursor cells has been the main limitation of studying
IMAT. As described above, the studies of Joe et al. and
Uezumi et al. helped to clarify many aspects of the
muscle adipogenic precursor cells. One major focus of
these approaches was determining whether IMAT-
associated adipocytes were in vivo derived from pre-
existent muscle-resident PDGFRα+ cells, other muscle-
resident cells, or circulating cells. The work led by Liu
et al. in murine skeletal muscle is a classic example of
these efforts. The authors suggested that IMAT derives
from a lineage of cells not expressing Pax3 (i.e., non-
myogenic). They also showed that the genetic ablation
of intramuscular adipogenic progenitors based on Ap2
(also known as fatty acid-binding protein 4 (FABP4)) ex-
pression leads to impaired skeletal muscle regeneration,
suggesting for the first time that damage-induced fatty
tissue may support efficient regeneration upon acute in-
jury [189]. However, AP2/FABP4 expression is com-
monly thought to be restricted to committed or
differentiated adipocytes than progenitor cells [190],
questioning the interpretation of the results. Marinkovic
et al. [111] showed that Notch signaling is a pivotal
pathway regulating FAP adipogenesis in wild-type cells
and that dystrophic FAPs are insensitive to Notch-
mediated adipogenic inhibition compared with acute
injury-derived FAPs [111]. Hence, these results demon-
strate that wild-type and dystrophic muscle PDGFRα+
FAPs are in different functional states, which influences
their fate and responsiveness to extracellular cues, as
previously suggested [62].
Human PDGFRα+ FAPs exist in healthy and DMD
pathological muscles, being bona fide counterparts of
the PDGFRα+ cells found in mouse muscles [16,50,51,
53,54,109]. Remarkably, FACS-isolated human FAPs
(CD15+/PDGFRα+/CD56−) differentiate towards fully
mature adipocytes, phenocopying the in vitro differenti-
ation kinetic and potential of adipose stromal cells ob-
tained from subcutaneous adipose tissue depots [51].
Moreover, when transplanted into a glycerol-damaged
muscle (an injury model that promotes adipogenesis)
[191,192], murine FAPs readily differentiate into adipo-
cytes. In concordance to the in vitro report of Liu and
colleagues using mouse muscle samples, Arrighi et al.
also showed that FAP-derived adipocytes from human
muscle biopsies are white rather than beige/brown fat
cells. In contrast, Gorski et al. showed increased expres-
sion of UCP1, a brown/beige fat cell marker [193], in
muscle as well as in FAP cultures following induction of
IMAT by glycerol injection [194]. Throughout the body,
white fat cells store energy in large, often single, oily
droplets. Obesity causes these white adipose tissue cells
to multiply and hypertrophy [195,196]. On the other
hand, brown fat cells are equipped with smaller droplets
and large mitochondria concentration, giving the tissue
its chestnut hue. Hence, in brown adipose tissue, mito-
chondria produce heat using these fatty droplets, a
process also known as thermogenesis [197]. The role of
FAP-derived fat cells, whether brown/beige or white, in
skeletal muscle health, regeneration, and disease is
unknown.
Perhaps the most serious disadvantage of these studies
is that they do not directly address the in vivo adipo-
genic differentiation potential of adult PDGFRα+ FAPs.
The definitive proof that muscle PDGFRα+ cells are the
main, if not the only, source of injury-induced adipo-
cytes came from lineage tracing experiments using
Pdgfra
CreERT
:Rosa26
EYFP
transgenic mice [14]. The au-
thors demonstrated that seven days after acute intramus-
cular injury, a large proportion of perilipin+ adipocytes
derived from PDGFRα+ FAPs, indicating that PDGFRα
expressing progenitors are the major source of damage-
induced fat cells in normal muscle regeneration and in
muscular dystrophy. Indeed, using similar lineage tracing
strategies we have demonstrated that cardiac PDGFRα+
FAPs can cause fibrofatty infiltration within the myocar-
dium in an arrhythmogenic cardiomyopathy mouse
model driven by the conditional deletion of the
quiescence-associated factor Hic1 in heart FAPs [73].
Intriguingly, PDGFRα+ FAPs are ciliated cells and thus
possess primary cilium. Conditional deletion of a gene
required for ciliogenesis, Ift88, in FAPs impaired the
injury-induced formation of adipocytes [14]. Mechanis-
tically, the cilia-dependent modulation of FAP adipogen-
esis involves the participation of Sonic Hedgehog (SHH)
signaling, which is repressed in the absence of cilia. In-
deed, constitutive activation of the Shh-pathway via gen-
etic deletion of the repressor Ptch1 was sufficient to
block adipocytes’generation following injury [14]. Re-
markably, elimination of the primary cilium in PDGF
Rα+ FAPs led to enhanced regeneration of myofibers by
reducing fatty degeneration of dystrophic muscles,
which was also associated with increased myofiber
size. The authors also showed that tissue inhibitor of
metalloproteinase 3 (TIMP3), an ECM modifier, in-
hibits adipocyte formation by muscle PDGFRα+pro-
genitors. Interestingly, aiming to mimic TIMP3
activity, the authors utilized batimastat and showed
that the treatment with this pharmacological inhibitor
of metalloproteinases prevented injury-induced adipo-
genesis in vivo [14](Table3).
In a different study, Jaiswal and colleagues showed that
the treatment with Batimastat prevented FAP spontan-
eous adipogenesis and reduced fat in dysferlinopathic
muscle of dysferlin-deficient (B6A/J) mice [104]. Hence,
the authors suggested that the accumulation and
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adipogenic differentiation of FAP are critical contribu-
tors to limb-girdle muscular dystrophy type 2B. Surpris-
ingly, the authors observed no changes in either FAP
accumulation, proliferation, or fibrosis as a result of bati-
mastat treatment (Table 3). Nevertheless, the batima-
stat’s off-targets on other tissue-resident cells such as
MuSCs, myofibers, endothelial cells, pericytes, or infil-
trating CD45+ cells have not been evaluated yet.
Altogether, these findings suggest novel strategies to
combat fatty degeneration of chronically damaged mus-
cles by targeting the adipogenic conversion of PDGFRα
expressing FAPs to inhibit the deposition of injury- and
disease-induced intramuscular fat.
To date, there is not a single clinically approved drug
used to prevent IMAT accumulation in muscle disease.
However, significant pre-clinical advances have been
made. In vivo treatment of mdx mice with molsido-
mine—a nitric oxide (NO) donating molecule—reduced
muscle pathology, IMAT accumulation, and fibrosis
[154]. These improvements were at least in part medi-
ated by the inhibition of NO-mediated FAP adipogenesis
(Table 3). Hence, altered synthesis of NO, a typical find-
ing in DMD, could contribute to enhanced fat depos-
ition. On the search for adipogenic inhibitors, Uezumi
and colleagues found that promethazine hydrochloride
inhibits, through binding to the H1 histamine receptor,
the in vitro and in vivo formation of ectopic adipocytes
derived from PDGFRα+ lineage cells in the muscle [72]
(Table 3). Promethazine hydrochloride is a first-
generation antagonist of the H1 histamine receptor, and
therefore, this family of drugs emerge as attractive novel
therapeutics against ectopic fat formation in muscle
pathologies.
Histone deacetylation leads to the repression of gene
expression, and histone deacetylase inhibitors (HDACi,
like trichostatin A) provide an exciting means to treat
DMD. HDACi have been used in both pre-clinical and
clinical studies to improve muscle regeneration and re-
pair in DMD [151,198–200]. As HDACi treatment in-
hibits fibro-fatty differentiation of PDGFRα+ FAPs, it
reduces the dystrophic pathology through increasing
muscle regeneration [151]. Remarkably, in dystrophic
FAPs, an HDAC–myomiR–BAF60 molecular network
regulates FAP fate, and old FAPs become resistant to
HDACi-induced chromatin remodeling compared with
young FAPs [201]. Also, HDACi restore the dystrophic-
mediated loss of intercellular communication between
PDGFRα+ FAPs and myogenic progenitors required for
proper muscle regeneration [151], and as recently sug-
gested through an extracellular vesicle-mediated transfer
of miRNAs [200]. Interestingly, aging and DMD disease
progression limit HDACi-mediated effects [151], which
suggests that aging affects the fate of FAPs, as recently
detailed by Lukjanenko and colleagues [5]. Recently,
Feeley and colleagues showed that rotator cuff tears en-
hanced HDAC activity in FAPs and trichostatin A inhib-
ited it. HDAC inhibition prevented FAP-mediated fatty
infiltration in supraspinatus muscles. Also, trichostatin
A regulates muscle FAP adipogenesis by promoting FAP
browning (Table 3)[152].
These studies demonstrated that HDACs-mediated
pharmacological intervention might counter DMD pro-
gression and chronic muscle injury by increasing regen-
eration by inhibiting fibro-fatty degeneration while
favoring the interplay and communication between FAPs
and myogenic progenitors. Recently, we have shown that
two well-characterized pan-HDACi reduce TGF-β-
induced ECM gene expression and also block TGF-β-
mediated downregulation of Tcf7l2 expression [28].
Mechanistically, histone deacetylase inhibitors modulate
TGF-β-mediated changes in the expression of TCF7L2
transcription factor target genes of the Wnt pathway
[28]. Further investigations should unravel the mechan-
ism by which HDACs regulate the fate of FAPs and how
could this be used to target muscle-associated diseases.
In a recent study, Reggio and colleagues used a large
drug library screen with pharmacological approaches to
demonstrate that the inhibition of the cytoplasmic sig-
naling protein, glycogen synthase kinase 3 (GSK3), re-
duces PDGFRα+ FAP adipogenesis in vitro, while also
repressing muscle glycerol-induced fatty degeneration
[153] (Table 3). GSK3 is composed of 2 isoforms (αand
β) and is part of the destruction complex of β-catenin,
which we showed earlier to play a modulatory role in
FAP fate (see the “Wnt/βsignaling”section). Mechanis-
tically, the authors suggested that UPS-targeted β-
catenin degradation causes an imbalance in the adipo-
genic fate of dystrophic mdx FAPs. The authors also
exploited single-cell data and in silico modeling to show
that PDGFRα+ FAPs compose the core of the stromal
cells in the muscle cell niche by expressing Wnt compo-
nents and also for being the primary source of Wnt li-
gands. FAPs seem to actively communicate with
endothelial cells, tenocytes, and MuSCs through the
production of Wnt ligands. Among the Wnt ligands,
they observed that dystrophic FAPs downregulate
Wnt5a expression compared with wild-type cells.
Moreover, WNT5a treatment reduced FAP-induced
adipogenesis in vitro by repressing PPARγexpression
throughout the activation of β-catenin, suggesting
that the Wnt signaling modulates the adipogenic
commitment of FAPs in dystrophic muscles (Fig. 2).
On the other hand, Zhao and colleagues recently de-
scribed that the supplementation of retinoic acid (RA)
enhances the proliferation of FAPs at the expense of
inhibiting their adipogenic and fibrogenic differentiation
[69]. Additionally, treatment of isolated FAPs with a
pan-retinoic acid receptor antagonist, BMS493, blocked
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the RA-mediated effects. Notably, the authors also
showed that RA treatment rescued obesity-impaired
skeletal muscle regeneration. These findings showed a
FAP-type specific effect of RA signaling that regulates
skeletal muscle regeneration and repair by means of pre-
serving their progenitor state. Taken together, these
findings suggest a novel potential retinoic acid-based
strategy to combat chronic skeletal muscle fibro-fatty
degeneration of obese patients.
On the contrary, several factors positively regulate
muscle FAP adipogenesis. For instance, the matricellular
protein CCN family member 1 (CCN1/CYR61) is ele-
vated in the serum and sarcopenic muscles of a murine
model of chronic kidney disease and induces FAP adipo-
genesis [202]. In vivo treatment of mice with the gluco-
corticoid dexamethasone enhanced IMAT deposition
following acute injury (Dong et al., 2014 [150]). Dexa-
methasone also induces FAP proliferation while increas-
ing their adipogenesis, possibly involving the reduction
of IL-4 expression (Dong et al., 2014 [150]). Remarkably,
IL-4 administration reduces dexamethasone-induced
FAP-derived adipocyte formation, suggesting a novel
therapeutic use of IL-4 to reduce IMAT accumulation
due to glucocorticoid use in DMD patients (Fig. 2). Per-
petuini et al. showed that the glucocorticoid-related
molecules, dexamethasone, and budesonide, inhibited
the insulin-induced adipocyte formation from mdx-
derived FAPs. However, both drugs have a pro-
adipogenic impact when the adipogenic mix contains
factors that increase the concentration of cyclic AMP.
The authors also showed that, only in anti-adipogenic
conditions, budesonide suppresses the expression of
Pparg, a master adipogenic regulator, via the
glucocorticoid-induced-leucine-zipper (GILZ/TSC22D3),
and the glucocorticoid antagonist mifepristone alleviates
such inhibitory effect [203] (Table 3). This study may
shed light on some of the mechanisms underlying the
use of glucocorticoids in DMD patients under this kind
of treatment. The use of glucocorticoids to treat DMD
patients is so far the most common treatment available
to delay muscle necrosis and degeneration up to date
[204–206]. Finally, the same group, using a similar
chemical library-based approach, identified an immuno-
suppressant drug, azathioprine, that negatively perturbs
the intrinsic adipogenic fate, also via PPARγrepression,
of wild type and mdx PDGFRα+ FAPs (Table 3).
On the other hand, we recently showed that TGF-β
treatment negatively affects FAP differentiation to adipo-
cytes while inducing FAP-to-myofibroblast commitment
(Fig. 2). TGF-β1 impairs basal PDGFRα+ FAP differenti-
ation into the adipogenic lineage, by reducing the
steady-state percentage of adipocytes but increasing the
number of myofibroblasts [11,207]. Mechanistically,
TGF-βtreatment reduces the expression of Pparγand
Adiponectin in skeletal muscle FAPs [11,12]. We also
showed that the adipogenic differentiation of FAPs re-
presses the expression of PDGFRα[11]. Taken together,
these studies demonstrate that IMAT-associated adipo-
cytes can derive from pre-existent muscle-resident fibro-
adipogenic progenitors.
Osteogenic differentiation of PDGFRα+ FAP cells
Muscle PDGFRα+ FAPs have osteogenic potential
in vitro [2] and when transplanted can successfully en-
graft and form calcification-rich structures using an
in vivo heterotopic ossification (HO) model [208]. HO is
a musculoskeletal disorder distinguished by the patho-
logic formation of extraskeletal bone in muscle, tendon,
ligaments, and fascia [209]. BMP2 promotes intramuscu-
lar HO regardless of damage; however, BMP9-induced
HO requires skeletal muscle injury [210] (Fig. 2). The
authors described that intramuscular HO might involve
a population of Lin-SCA-1+ cells—likely FAPs [210].
Moreover, Lin
−
/TIE2+/PDGFRα+ progenitors respond
to BMP2-stimulated osteogenic commitment and con-
tribute to HO in mice [211]. Additionally, muscle-
derived MSCs contribute to fracture repair in a tumor
necrosis factor-alpha (TNFα) dependent manner [212].
The above findings are consistent with a recent study of
Goldhamer’s group, where the authors employed and
characterized a transgenic mouse model that recapitu-
lates a rare autosomal-dominant disorder called fibro-
dysplasia ossificans progressiva (FOP), which results
from a single activating mutation in ACVR1; the type I
BMP receptor also known as ACVR1/ALK2. The Tie2-
driven expression of the mutation Acvr1 R206H is suffi-
cient to phenocopy the spectrum of HO observed in
FOP patients [60]. Moreover, they also showed that
intramuscular transplantation of mutant Acvr1R206H/+
FAPs into immunodeficient mice resulted in the forma-
tion of HO in an Activin A-dependent fashion. Overall,
these data established TIE2+/SCA-1+/PDGFRα+ FAPs
as the predominant cell-of-origin and driver of patho-
logical HO. However, it has been suggested that TIE2 is
a nonspecific marker for a subset of PDGFRα+ cells
since its expression overlaps with other cell populations
like endothelial cells, MuSCs, and subsets of
hematopoietic cells [2,213,214]. Hence, the precise
mechanisms and the populations of cells involved in the
formation and remodeling of HO remained unknown
until then. We recently took advantage of a novel PDGF
Rαlineage tracing reporter mouse (Pdgfrα-CreERT2-
TdTomato) to further explore the cellular source of
muscle ossification [13]. Using a model of BMP2-
stimulated intramuscular HO, we showed that a large
proportion (~80%) of differentiated osteogenic cells were
TdTomato+ after 21 days of muscle injury. Thus, the
cell-source responsible for forming ectopic bone in
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muscle is a subpopulation of muscle-resident PDGFRα+
progenitors [13]. Overall, these studies demonstrate that
FAPs are a significant cellular source of chondrogenic
cells and osteogenic cells in severely damaged muscles.
Remarkably, intramuscular calcium deposits serve as a
pathohistological feature of DMD [215]. Notably, the de-
gree of osteogenic commitment of FAPs appears to
match the model of muscle damage and degeneration/
regeneration used. Using the severe D2-mdx (DBA/2J-
mdx) dystrophic mice, which better recapitulates the hu-
man characteristics of DMD myopathology, Mázala et al.
demonstrated that PDGFRα+ FAPs accumulate within
calcified deposits in degenerative muscles [117]. Also,
the in vitro osteogenic differentiation of these cells posi-
tively correlates with the degree and extension of muscle
degeneration and TGF-βlevels, which supports previous
studies showing that FAPs vastly expand and accumulate
accordingly with the extension of damage, TGF-βlevels,
and fibrosis [11,12,15,27,53,54,59,103,117]. In sum-
mary, FAP activity and responses are highly contextual,
which suggests that signals emanating from the local
niche determine their phenotypic multi-lineage-fate.
Why are different muscle groups affected to a different
extent in muscular dystrophy or neuromuscular disor-
ders? Although several hypotheses might explain this, in-
cluding muscle fiber type, muscle fiber innervation,
muscle of origin, calcium homeostasis, and muscle activ-
ity, we still lack information of the role that FAPs play in
these processes.
Fibro-adipogenic cell diversity: Single-cell omics
unveil stromal populations in muscles
The recent revolution in single-cell omics technologies,
including single-cell RNA sequencing (scRNAseq),
single-cell epigenomics (e.i. scATACseq), and single-cell
mass cytometry (e.i., CyTOF) has helped to uncover the
mysteries of muscle cellular composition and heterogen-
eity as well as to faithfully recreate a more precise cellu-
lar atlas of murine and human adult skeletal muscle in
homeostasis, regeneration, and repair [48,58,62,106–
109,111,216] (Fig. 3). Muscle single-cell analyses faith-
fully recapitulate key cellular events involved in skeletal
muscle regeneration and repair, derived from studies
over many years. Such tools and information led us to
realize that a complex array of non-myogenic cells (tis-
sue-resident and non-tissue-resident) engage in active
cross-talk between each other and with MuSCs to re-
store tissue function following damage. Single-cell stud-
ies evaluate molecular signatures and expression levels
of genes or cell surface protein abundance in large num-
bers of individual cells. They aim to describe at an un-
precedented resolution the total interstitial populations
of cells in a resting state and to understand their flux in
response to injury and disease. Owing to the ability of
single-cell omics technologies to refine our understand-
ing of cell heterogeneity by using a plethora of genes
and proteins to identify a particular cluster or subpopu-
lation of cells, they are significantly more accurate com-
pared with the use of a single marker to identify cell
types.
The classical view of cellular muscle composition is
that most of the non-myogenic cells play a positive role
and generate a pro-regenerative transitional niche,
which, among other functions, support MuSC-driven
myogenesis following acute damage [217]. These popula-
tions of non-myogenic cells include endothelial cells
(CD31+) [218,219], FAPs (PDGFRα+) [1,2], connective
tissue fibroblasts TCF7L2+ (significantly overlapping
with FAPs) [6,24,27,28], pericytes (NG2+, RGS5+)
[48], mesoangioblasts [220,221], tenocytes (TNMD+,
SCX+), glial cells (PIP1+, KCNA1+) [48,58], and a com-
plex array of immune cells [222–224].
With single-cell omics technologies, the transcriptional
identity in homeostasis and lineage trajectories of
muscle-resident FAPs during the regenerative response
and in disease states have started to be discovered. Male-
cova and colleagues were the first to initially show the
existence of cellular heterogeneity in muscle FAPs using
single-cell RT-PCR and showed that it increases in re-
generating muscles [62] (Table 4). The authors showed
that a specific subpopulation of vascular cell adhesion
molecule VCAM-1+ FAPs vastly expands and drives
muscle fibrosis in acute damaged muscles and adult dys-
trophic muscles of the mdx mice [62]. Notably, the
VCAM-1+ subFAPs are absent in uninjured muscles,
suggesting that Vcam1 may be an activation marker. Re-
cently, a population of quiescent HIC1+ mesenchymal
progenitor cells has been described. This population
contains precursor cells for several mesenchymal line-
ages including muscle FAPs (Pdgfra+, Ly6a+), tenocytes
(Tnmd+), pericytes (Rgs5+), and a new subset of cells
called myotenocytes (Col22a1+) [48]. However, further
functional analyses will be required to confirm if the
myotenocyte cells present at the regenerated myotendi-
nous junction represent an independent subpopulation
of HIC1+ progenitors with a specific function or a differ-
entiated cell state of tenocyte progenitors in the myoten-
dinous niche. Even though HIC1 is a broad stromal
progenitor marker, FAP gene signatures segregate from
other interstitial populations of mesenchymal cells like
pericytes and tenocytes [48]. Remarkably, the gene sig-
nature of muscle PDGFRα+ FAPs is heterogeneous and
progresses over time after acute muscle injury, which re-
veals their dynamic role in regeneration [48,108].
Therefore, FAPs acquire a unique plastic transcriptome
that changes as the inflammation progresses and damage
resolves through regeneration. Concerning mouse mus-
cles, two major FAP populations have been described
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Fig. 3 aSingle-cell RNA sequencing analyses to map muscle-resident FAP mononuclear landscape in murine (left graph) and human (right
graph) skeletal muscle tissue. Three different studies, utilizing mice, agree with the existence of at least two principal muscle FAP subpopulations
(here shown as FAPs 1 and FAPs 2; see text for details). On the other hand, FAP clustering and FAP subpopulations greatly vary in human
muscles. Two different bioinformatic techniques for the presentation of large scRNA-seq datasets and their dimensionality reduction are shown:
uniform manifold approximation and projection (UMAP) algorithm and t-Distributed Stochastic Neighbor Embedding (t-SNE). Colored dots
represent individual FAP cells. Dotted lines illustrate the different studies discussed in this review. bFAP cell trajectories are based on the gene
signatures of single cells following damage [48,108]. The transcriptomes of FAPs indicate high cellular heterogeneity within the FAP populations
in response to injury. In mouse muscles, two major FAP subpopulations (Dpp4 FAPs and Cxcl14 FAPs) are present in homeostatic conditions (for
detailed markers, see Table 4). Analysis of the pseudotime trajectory of different FAP subpopulations suggests that FAP cells follow a continuum
and diverge into two major subclusters upon damage
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(FAP1 and FAP2) in undamaged muscles (Fig. 3a). FAP1
associates with the ECM gene signatures, such as colla-
gens Col4,Col6 and Col15,Lum,Sparcl1,Podn,Smoc2,
Mgp,Cxcl14,and Bgn. On the other hand, the FAP2
subpopulation expresses genes involved in cell signaling
and migration, including Sfrp4,Igfbp5,Sema3c,Dpp4,
Tgfrb2, and Wnt2 [48] (Table 4). In contrast, another
study described no segregation of the FAP population,
primarily based on the expression of a few FAP markers
[216]. These subtle differences might be explained by a
number of technical factors such as muscle groups used
for scRNAseq, tissue digestion and cell-type enrichment
methods, single-cell RNA sequencing platform—al-
though most of them used Chromium 10× Genomics,
the number of cells captured and recovered after se-
quencing, and downstream data processing (e.g., version
of Seurat R package used) and interpretation.
Oprescu et al. [108] reported that murine CXCL14+
FAPs (also expressing Smoc2,Ccl11,Gsn,and Dcn) and
DPP4+ (expressing Pi16,Igfbp5,Igfbp6,Fbn1, and Ugdh)
FAPs represent two different FAP subtypes present in
non-injured murine tibialis anterior muscle, suggesting
that FAPs could represent two distinct subpopulations
of interstitial cells in resting conditions, as it was previ-
ously shown by us [48] (Table 4). However, in response
to injury, the two populations follow a linear trajectory
into a single population of activated FAPs (highly
expressing chemokine genes like Cxcl5,Cxcl3,Ccl7, and
Ccl2) at 0.5 and 2-day post-injury (DPI), then progres-
sing into WISP1+ FAPs at 3.5 and 5 DPI (highly ex-
pressing Postn,Csrp2,Sfrp2,Ptn,Cilp, and Cthrc1),
followed by DLK1+ FAPs at 10 DPI (expressing Itm2a,
B830012L14Rik,Meg3,Airn,Peg3,Zim1,H19, and Igf2),
and finally two FAP subpopulations at day 21 DPI,
OSR1+ (expressing Gsn,Ccl1,Bmp4,Bmp5, and Wnt5a)
and fibroblast FAPs (expressing Col3a1,Col1a1,Col1a2,
Col6a3, and Meg3)[108] (Table 4and Fig. 3b). Notably,
the authors showed that a proportion of the OSR1+
FAPs at 21 DPI diverge into the two populations ob-
served in undamaged muscle: DPP4+ FAPs and
CXCL14+ FAPs (Fig. 3b). Therefore, the gene expression
of single-cell FAPs is highly diverse, representing a con-
tinuum state during skeletal muscle regeneration (Fig.
3b). Owing to the high degree of FAPs diversity, we
speculate that FAP subpopulations have adapted to play
supportive and distinct roles during regeneration. These
data also suggest that the transcriptional diversity of
PDGFRα+ FAPs at the single-cell level might reflect
their differential developmental potential.
In addition to these studies, Rubenstein et al. [109] de-
scribed two human FAP subpopulations as LUMICAN
(LUM)+ FAP and FIBRILLIN 1 (FBN1)+ FAP subtypes
(Table 4and Fig. 3a). Interestingly, both FAP subpopula-
tions showed specific differences in the expression of
Table 4 scRNA-seq gene signatures used for FAP identification and clustering in muscle homeostasis
Genes/markers FAP subpopulations Species Reference
Sca1,Cd34,Pdgfra SubFAPs: Tie2
low
(Tek), Vcam1
low
Mouse [62]
SubFAPs: Tie2
high
(Tek), Vcam1
high
Ly6a (Sca1), Ly6e,Pdgfra,Dcn Not determined Mouse [58]
Pdgfra,Ly6a (Sca1), Hic1 FAP1: Cxcl14,Col4,Col6,Col15,Lum,Sparcl1,Podn,Smoc2,Mgp, and Bgn Mouse [48]
FAP2: Dpp4,Sfrp4,Igfbp5,Sema3c,Tgfrb2, and Wnt2
Pdgfra,Ly6a (Sca1), Dcn,Cd34 Not determined Mouse [216]
Pdgfra,Col3a1,Dcn, and Gsn Not determined Mouse [107]
Pdgfra,Ly6a (Sca1), Cd34 FAP1: Cxcl14,Enpp2 (Autotaxin), Crispld2,Hsd11b1,Smoc2,Ccl11,Gsn, and Dcn Mouse [108]
FAP2: Dpp4,Pi16,Wnt2,Igfbp5,Igfbp6,Fbn1, and Ugdh
PDGFRA,CD34,COLLAGEN 1,
COLLAGEN 3,and COLLAGEN 6
LUMICAN (LUM) FAP: LUM,DCN,CXCL14,COLLAGEN 4, and COLLAGEN 15,SMOC2, and GSN Mouse
and
human
[109]
FIBRILLIN 1 (FBN1) FAP: FNB1,MFAP5,LOXL1, PRG4, ELN,IGFBP5, and FSTL1
PDGFRA FAP1 (fibroblasts 1): COL1A1,SFRP4,SERPINE1, and CCL2 Human [106]
FAP2 (Fibroblast 2): FBN1,MFAP5, and CD55
FAP3 (Fibroblast 3): SMOC2,ADH1B, and ABC18
PDGFRA,CD34,COL1A1,COL6A3,
TCF7L2
FAP1: PCOLCE2,MFAP5,IGFBP6,ENNP1,CD55, and AXL Human [16]
a
FAP2: LUM,MYOC,CCL2,ADH1B,SFRP2,CXCL14, and MGP
FAP3: TNXB,C3,COL15A1,SMOC2,ABCA8,COL6A1, and HMCN2
FAP4: IGF1,CRLF1,SCN7A,ITIH5,PTGDS and NOV
FAP5: SEMA3C,PRG4,DEFB1,CCDC80,LINC01133, and IGFBP5
a
By re-clustering the FAP population, the authors described the existence of 7 different FAP subpopulations in human muscles [16]
Contreras et al. Skeletal Muscle (2021) 11:16 Page 18 of 25
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Collagen types. The authors also validated the existence
of these two distinct FAP subtypes by using scRNAseq
of mouse quadriceps and diaphragm muscles [109].
ECM gene expression was also consistent among mouse
and human muscles, with COLLAGEN 1,COLLAGEN 3,
and COLLAGEN 6 broadly expressed across FAP subpop-
ulations. In contrast, LUM+ FAPs express COLLAGEN 4,
and COLLAGEN 15 predominantly compared with
FBN1+ FAPs (Table 4). Interestingly, the authors reported
differences in the gene expression of the precursor gene of
TIE2 protein among the two species, which was expressed
only in the FBN1+ FAP subtype found in mouse muscle
but none of the FAP subtypes found in human muscles.
The meaning of these subtle differences in gene expres-
sion across mouse and human muscle FAP subtypes
should be address in future research.
De Micheli and collegues collected and integrated ~
22,000 single-cell transcriptomes generating for the first
time a consensus cell atlas of human skeletal muscles
[106] (Table 4and Fig. 3a). The authors described three
subpopulations of fibroblasts (likely FAPs) in which
COLLAGEN 1,SFRP4,SERPINE1 and CCL2 are highly
expressed by fibroblast 1; FBN1+,MFAP5, and CD55 are
expressed by fibroblast 2, whereas fibroblast 3 highly ex-
presses SMOC2 [106,107] (Table 4).
Recently, Farup and colleagues described 5 subpop-
ulations of human muscle FAPs (Table 4and Fig. 3a).
However, by sub-setting the FAP population and re-
clustering, the number of clusters increased to 7. The
authors reported that the expression of THY1/CD90
is enriched in cluster 4, whereas PDGFRA gene ex-
pression is broadly distributed among the FAP sub-
populations (Table 4). Remarkably, the CD90+
subpopulation of FAPs is associated with increased
fibro-fatty infiltration and seems to drive the muscle
degeneration found in obese and type-2 diabetes pa-
tients [16]. Although the composition of each cellular
interstitial compartment changes dramatically after in-
jury and in disease settings in mice, there is no infor-
mation about how the FAP population behaves
following injury or how degenerative diseases alter its
activities in humans. Nonetheless, we and others have
detected a diverse range of mesenchymal stromal cells
including quiescent subsets, which rapidly expand fol-
lowing injury and secrete cytokines modulating in-
flammation, trophic factors, and regenerative cues to
promote skeletal muscle maintenance, MuSC renewal,
and regeneration.
In conclusion, further studies should focus on un-
derstanding the mechanisms by which FAP cell het-
erogeneity arises. We aim to understand the lineage
restriction of FAPs by gene regulatory networks and
epigenetic factors that, in combination with the ex-
trinsic effects of the spatial context could regulate
their fate and plasticity within muscles. Although
there has been encouraging progress in understanding
FAP phenotypic variability and activities, future re-
search should look to translating this knowledge into
efficient medical applications.
Future perspectives
Skeletal muscle requires a complex orchestra of special-
ized populations of cells to perform its crucial functions.
The origin, behavioral activities, lineage potency, and ex-
pression of markers associated with stem or progenitor
cell states define these specialized cell types. Here, we
have focused on the unappreciated role of PDGFRα+
FAPs in muscle biology, health, structure, and regener-
ation. Apart from the accepted structural part that the
connective tissue provides for proper muscle develop-
ment, the complex cues and matrix that stromal cells
produce are essential to sustain myogenesis and support
proper muscle morphogenesis. FAPs are implicated in
muscle scarring, disease, and pathology. Although sub-
stantial progress has been made in understanding FAP
behavior, they remain poorly characterized, and the rela-
tionships with other stromal cells are not well under-
stood. PDGFRα+ FAPs and their descendant lineages,
including activated-fibroblasts/myofibroblasts, adipo-
cytes, chondrogenic and osteogenic cells, modulate
muscle regeneration and repair. These plastic cells play
broad roles as sentinels, stress sensors, immune regula-
tors, cellular hubs, and paracrine factories, which are still
under active research in multiple pathological settings.
As discussed above, lineage tracing technologies com-
bined with single-cell sequencing strategies should by-
pass the significant limitations that historically
prevented us from deconvolving the complexity of stro-
mal cell populations. The diverse fibroblast nomencla-
ture has periodically led to confusing claims in muscle
biology and ensuing turmoil in the literature. Thus, re-
solving the regenerative vs. reparative dichotomy of
muscle-resident mesenchymal progenitors, and distin-
guishing true lineage heterogeneity from the diverse
functional states that these cells can dynamically and re-
versibly acquire remains a high-priority issue for the
field. Despite these various uncertainties, in this review,
we establish a baseline for the contribution of fibro-
adipogenic progenitors to muscle development, homeo-
stasis, regeneration, and repair.
Conclusions
In this review, we document new insights about the vari-
ous properties of muscle-resident PDGFRα+ FAPs and
discuss the current state of knowledge on their origins
and lineage capabilities. Here we propose to define a cell
as FAP if they present the following characteristics: 1.
Express PDGFRαat the gene and protein level. 2. It is
Contreras et al. Skeletal Muscle (2021) 11:16 Page 19 of 25
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

located in the tissue's interstitium and behaves as a peri-
vascular cell but not residing in the blood vessel cavity.
3. It can form colonies in vitro. 4. Can differentiate into
activated fibroblasts, adipocytes, chondrocytes, and oste-
ocytes in vitro and in vivo.
We illustrated their importance in maintaining proper
muscle function and critical role during the onset and
establishment of scarring in pathology and disease.
Growing evidence shows that PDGFRα+ cells are hetero-
geneous and act as signaling hubs by providing regenera-
tive cues and integrating these signals in the muscle
niche. Thus, FAPs influence other populations of cells
within skeletal muscle and vice versa. Ultimately, by un-
derstanding and manipulating the complexity and vari-
ability of the stromal compartment, specifically the FAP
lineage, we aim to develop novel therapeutics to treat
several scar-forming pathologies. It remains plausible to
foresee a future where clinical leaps could be made
based on these cells and where severe muscle injury
could be treated without prolonged myodegeneration
and muscle malfunctioning.
Abbreviations
aSMA: Alpha smooth muscle actin; BMP: Bone morphogenetic protein;
CFU: Colony-forming unit; CDHs: Congenital diaphragmatic hernias;
CT: Connective tissue; DMD: Duchenne muscular dystrophy; DTA: Diphteria
toxin A; DTR: Diphteria toxin receptor; ECM: Extracellular matrix;
FAPα: Fibroblast activation protein alpha; FAPs: Fibro-adipogenic progenitors;
FOP: Fibrodysplasia ossificans progressiva; GSK: Glycogen Synthase Kinase-3;
HO: Heterotopic ossification; IL: Interleukin; IMAT: Intermuscular adipose
tissue; LGMD: Limb-girdle muscular dystrophy; Lox: Lysyl oxidase;
MSCs: Mesenchymal stem cells; MCT: Muscle connective tissue;
MuSCs: Muscle stem cells; NCCs: Neural crest cells; PDGFRα: Platelet-derived
growth factor receptor alpha; PDGFRβ: Platelet-derived growth factor
receptor beta; PDGF: Platelet-derived growth factor; PPFs: Pleuroperitoneal
folds; OSR: Odd-skipped-related; Shh: Sonic Hedgehog signaling; Sca-1: Stem
cell antigen-1; Tbx: T-box transcription factor; TIMP3: Tissue inhibitor of
metalloproteinases 3; TCF21: Transcription factor 21; TGF-β: Transforming
growth factor beta; UPS: Ubiquitin-proteasome system
Acknowledgements
The authors acknowledge Yen Tran and Ralph Patrick for helpful suggestions
to the single-cell omics chapter, and Lucas Rempel for his inputs improving
the review. Figures were created using Illustrator (Adobe Inc.) and Key-
note (Apple Inc.) for macOS.
Authors’contributions
O.C. and M.T. drafted the review and figures. F.M.V.R. revised and reviewed
the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by Comisión Nacional de Ciencia y Tecnología
CONICYT Beca Doctorado Nacional 2014 folio 21140378 “National Doctorate
Fellowship”, by Centro Basal de Excelencia en Envejecimiento y
Regeneración (CONICYT-AFB 170005), and by the Victor Chang Cardiac
Research Institute to O.C.; by Fondation pour la Recherche Médicale (FRM,
40248), by the European Molecular Biology Organization (EMBO, ALTF 115-
2016), by the Association contre les myopathies (AFM, 22576), and by Mi-
chael Smith Foundation for Health Research (MSFHR, 18351) to M.T.; and by
the Canadian Institutes of Health Research (CIHR-FDN-159908) to F.M.V.R. The
funding agencies had no role in the design of the study, data collection and
analysis, the decision to publish, or preparation of the manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this
published article.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Developmental and Stem Cell Biology Division, Victor Chang Cardiac
Research Institute, Darlinghurst, NSW 2010, Australia.
2
St. Vincent’s Clinical
School, Faculty of Medicine, UNSW Sydney, Kensington 2052, Australia.
3
Departamento de Biología Celular y Molecular and Center for Aging and
Regeneration (CARE-ChileUC), Facultad de Ciencias Biológicas, Pontificia
Universidad Católica de Chile, 8331150 Santiago, Chile.
4
Biomedical Research
Centre, Department of Medical Genetics and School of Biomedical
Engineering, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
Received: 22 January 2021 Accepted: 22 March 2021
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