Information

Are (muscle) satellite cells the same as muscle stem cells?


In terms of muscle: are the terms 'satellite cell' and 'muscle stem cell' interchangeable? That is, are there muscle stem cells that are not satellite cells, or vice versa?


Based on this review, satellite cells can replenish injured muscles, but among satellite cells there are satellite stem cells to maintain the pool of satellite cells.


Skeletal muscle stem cells

Satellite cells are myogenic stem cells responsible for the post-natal growth, repair and maintenance of skeletal muscle. This review focuses on the basic biology of the satellite cell with emphasis on its role in muscle repair and parallels between embryonic myogenesis and muscle regeneration. Recent advances have altered the long-standing view of the satellite cell as a committed myogenic stem cell derived directly from the fetal myoblast. The experimental basis for this evolving perspective will be highlighted as will the relationship between the satellite cell and other newly discovered muscle stem cell populations. Finally, advances and prospects for cell-based therapies for muscular dystrophies will be addressed.


Introduction

It is now over 45 years since the first description of the canonical satellite cell in adult skeletal muscle(Mauro, 1961). Satellite cells reside in a niche that lies beneath the basal lamina but outside of the associated muscle fibers (Fig. 1A) (Bischoff,1990). Satellite cells have long been believed to represent a committed muscle progenitor that is responsible for the postnatal growth and regenerative capacity of skeletal muscle(Seale et al., 2000). Typically mitotically quiescent, satellite cells are activated in response to stress that is induced by weight bearing or by trauma, such as injury(Charge and Rudnicki, 2004). The descendants of activated satellite cells, called myogenic precursor cells or skeletal myoblasts, undergo multiple rounds of division prior to terminal differentiation and fusion to form multinucleated myofibers.

Different states of the skeletal muscle satellite cell. (A)Pax7-positive satellite cells (red) at the surface of a single fiber isolated from the extensor digitorum longus (EDL) muscle of an adult mouse. Nuclei are stained with DAPI (blue). (B) During embryonic development, a Pax7-positive population of myogenic progenitors contributes to myogenesis and gives rise to satellite cells. Cryosection through a masseter muscle from an E14 mouse embryo. Differentiated muscle fibers are stained with an antibody against myosin heavy chain (cytoplasm, blue) and an antibody against myogenin(nuclei, red). Pax7-positive cells (nuclei, green) are located between the newly formed fibers. (C) Transverse cryosection of a transplanted mouse tibialis muscle, showing that transplanted YFP-positive satellite cells can fuse with host muscle fibers and contribute to the host stem cell niche. Grafted cells are labeled with an anti-GFP antibody (green) and anti-Pax7 antibody (red). Muscle fiber basal laminas are labeled with a laminin antibody(white) and nuclei with DAPI (blue). Arrow shows a grafted YFP-positive Pax7-positive cell in a satellite cell position. Images in A and B are courtesy of Fabien Le Grand image in C is courtesy of S. Kuang.

Different states of the skeletal muscle satellite cell. (A)Pax7-positive satellite cells (red) at the surface of a single fiber isolated from the extensor digitorum longus (EDL) muscle of an adult mouse. Nuclei are stained with DAPI (blue). (B) During embryonic development, a Pax7-positive population of myogenic progenitors contributes to myogenesis and gives rise to satellite cells. Cryosection through a masseter muscle from an E14 mouse embryo. Differentiated muscle fibers are stained with an antibody against myosin heavy chain (cytoplasm, blue) and an antibody against myogenin(nuclei, red). Pax7-positive cells (nuclei, green) are located between the newly formed fibers. (C) Transverse cryosection of a transplanted mouse tibialis muscle, showing that transplanted YFP-positive satellite cells can fuse with host muscle fibers and contribute to the host stem cell niche. Grafted cells are labeled with an anti-GFP antibody (green) and anti-Pax7 antibody (red). Muscle fiber basal laminas are labeled with a laminin antibody(white) and nuclei with DAPI (blue). Arrow shows a grafted YFP-positive Pax7-positive cell in a satellite cell position. Images in A and B are courtesy of Fabien Le Grand image in C is courtesy of S. Kuang.

During the course of the last decade, numerous researchers have provided evidence to support the hypothesis that a subpopulation of satellite cells exists that possess stem cell-like properties(Collins et al., 2005). In fact, it has been demonstrated recently that satellite cells represent a heterogeneous, hierarchical population that is composed of a small number of satellite stem cells and a larger number of committed myogenic progenitors(Kuang et al., 2007). Moreover, our understanding of non-conventional myogenic stem cells (side population cells, mesoangioblasts, and muscle-derived stem cells) has been clarified by the appreciation that they might be derived from a pericyte-like cell in muscle tissue (Peault et al.,2007). These and other advances in our understanding of skeletal muscle satellite and stem cell biology were discussed at the recent FASEB conference, which we review below.


Limb Muscle Satellite Cells: Establishing the Canon

Skeletal muscles of rodent hindlimbs are commonly used to study satellite cells as these muscles are easy to identify, dissect, collect, and manipulate experimentally. The skeletal muscles of the limbs and abdomen arise from somitic mesoderm and are referred to as hypaxial muscles (Figure ​ Figure1 1 ). They arise developmentally from the ventrolateral dermomyotome of the segmented paraxial mesoderm. In vivo and in vitro studies examining limb muscles provide fundamental insights into the mechanisms and regulatory pathways involved with skeletal muscle regeneration, muscle growth, and satellite cell biology.

Embryonic mesodermal contributions to adult skeletal muscles. (A) Schematic of mesodermal origins in a 3𠄵 somite stage mouse embryo. (B) Skeletal muscles of the trunk, limb, diaphgram, and tongue arise from somitic mesoderm. In contrast, the extraocular muscles (EOMs) arise from prechordal mesoderm and cranial paraxial mesoderm of the first pharyngeal arch the masseter muscle from the first and second pharyngeal arches of the cranial paraxial mesoderm, and the pharynx from the third and fourth pharyngeal arches of the caudal paraxial mesoderm. Tongue muscles arise from both somitic and cranial mesoderm while developing within the niche of the cranial mesenchyme, which is supplied by all four pharyngeal arches.

Muscle regeneration is a robust and complex cellular process that restores injured muscle to a state that is morphologically and functionally similar to that of uninjured muscle (Figure ​ Figure2 2 Abmayr and Pavlath, 2012). Regeneration of skeletal muscle occurs in two distinct phases: a degenerative phase and a regenerative phase (Rai et al., 2014). The main characteristics of the degenerative phase involve myofiber sarcolemmal damage or myofiber necrosis, followed by an influx of mononucleated inflammatory cells and an increase in fibroblasts (Mathew et al., 2011 Murphy et al., 2011 Rai et al., 2014). Factors released from damaged myofibers initiate an inflammatory response that recruits neutrophils, macrophages, and activates fibro/adipogenic progenitors to facilitate the removal of cellular debris and regulate muscle repair (McLennan, 1996 Lescaudron et al., 1999 Joe et al., 2010 Uezumi et al., 2010 Pallafacchina et al., 2013). The basal lamina remains intact acting as a scaffold for the next phase, muscle regeneration (Schmalbruch, 1976). Several molecular signals, such as growth factors, chemokines, and cytokines, are released which activate satellite cells both locally and systemically within the first 24� h following injury (Chang and Rudnicki, 2014 Rodgers et al., 2014). Myoblasts then terminally differentiate becoming post-mitotic myocytes, which then fuse with other myocytes or myofibers to regenerate or repair damaged myofibers. Thereby, new myonuclei are added to damaged or nascent myofibers (Abmayr and Pavlath, 2012). A subset of myogenic cells repopulate the satellite cell niche, thus maintaining and replenishing the quiescent satellite cell pool for subsequent rounds of regeneration (Collins et al., 2005 Shinin et al., 2006).

Myofiber structure and cellular progression of myogenesis. Myofibers are surrounded by a basal lamina, underneath which lie satellite cells in close apposition to the myofiber. With injury, satellite cells proliferate and give rise to myoblasts, which differentiate, migrate, adhere, and fuse with one another to form multiple myotubes within the basal lamina scaffold. Myoblasts/myotubes fuse with the stumps of the surviving myofiber and myotubes also fuse with each other to repair the injured myofiber. Regenerated myofibers are identifiable by the presence of centrally located nuclei. Representative hematoxylin and eosin stained muscle cross-sections from chemically injured murine muscles are provided for each stage of muscle regeneration to illustrate the differential tissue morphology.

The role of satellite cells in postnatal growth has also been studied in limb muscles. In mice, the first 3 weeks of neonatal growth results in a threefold increase in muscle mass during which the satellite cell population undergoes a significant reduction from

30% of myonuclei per myofiber down to 5%, following fusion with neonatal muscles. Parallel increases in myonuclear numbers and cytoplasmic proteins occur up to postnatal day 21 (White et al., 2010). After postnatal day 21, satellite cells enter into a quiescent cellular state under the regulation of Notch signaling (Fukada et al., 2011), but myofiber size continues to increase without the addition of new myonuclei (White et al., 2010). Recent satellite cell ablation studies have also shown that myonuclear addition from satellite cells is dispensable for hypertrophic growth of limb muscles in the adult (McCarthy et al., 2011). Furthermore, satellite cells do not appear to be required for maintenance of most adult limb muscles. A recent satellite cell ablation study found that loss of 㺐% of adult limb satellite cells failed to alter muscle size or myofiber type in five different limb muscles with aging (Fry et al., 2015). However, myonuclear addition does occur at a basal level in uninjured postnatal limb muscles and may be required for maintenance of extensor digitorum longus myofiber size with aging (Keefe et al., 2015). Together, these studies suggest that the initial phase of postnatal muscle growth occurs with the addition of myonuclei from satellite cells, but maintenance of most adult limb muscle size is not dependent on satellite cells.

Regulatory genes involved in satellite cell biology have also been elucidated from studies of limb muscle. In adult skeletal muscle, quiescent satellite cells express Pax7, a transcription factor that specifies the myogenic lineage (Seale et al., 2000). Once activated, satellite cells exit cellular quiescence, enter the cell cycle, and begin progression through the myogenic lineage under the control of myogenic regulatory factors (MRFs), muscle-specific transcription factors of the basic-helix-loop-helix (bHLH) class, including myogenic differentiation protein (MyoD), myogenic factor 5 (Myf5), myogenic regulatory factor 4 (Mrf4), and myogenin (Weintraub et al., 1991 Olson and Klein, 1994 Chang and Rudnicki, 2014). MyoD and Myf5 are expressed during the proliferative phase and regulate myogenic differentiation (Cooper et al., 1999 Valdez et al., 2000), while Mrf4 and myogenin are expressed upon terminal differentiation and exit from the cell cycle (Chang and Rudnicki, 2014).

Increasing evidence suggests that satellite cells within a muscle are heterogeneous (Motohashi and Asakura, 2014). Satellite cells containing high levels of Pax7 demonstrate slower proliferation rates, lower metabolism, and resistance toward differentiation, indicating a more “stem-like” phenotype compared to satellite cells with lower levels of Pax7 (Rocheteau et al., 2012). Various groups have also discovered distinct satellite cell subpopulations based on differential expression of other proteins including 㬗-integrin, 㬡-integrin, c-met, CD34, calcitonin receptor, C-X-C chemokine receptor type 4 (CXCR4), M-cadherin, Myf5, neural cell adhesion molecule 1, syndecans 3 and 4, and vascular cell adhesion molecule 1 (Rosen et al., 1992 Cornelison and Wold, 1997 Beauchamp et al., 2000 Blanco-Bose et al., 2001 Cornelison et al., 2001 Tamaki et al., 2002 Sherwood et al., 2004 Fukada et al., 2007 Ikemoto et al., 2007 Kuang et al., 2007 Kafadar et al., 2009). While the mechanisms underlying satellite cell heterogeneity are still being elucidated, growing evidence suggests that satellite cell biology is also variable in a muscle-dependent manner, as discussed below.


Results

Satellite cell clones from both slow and fast muscles spontaneously undergo myogenic terminal differentiation with a distinct timing

To determine the myogenic differentiation potential of each muscle satellite cell clone derived from slow and fast muscle, we isolated single myofibers from extensor digitorum longus (EDL fast muscle) and soleus (slow muscle) muscles, and cultured them in pmGM for up to 8 days. Muscle satellite cells were activated to proliferate, and migrated onto the bottom of culture vessels. Myogenic cells derived from satellite cells grew clonally on a collagen type I-coated substratum. Within 4 days of culture, the myogenic cells from both EDL and soleus muscles displayed a rounded shape, suggesting a weak attachment to the substratum (Fig. 1A,C). These cells are designated `round cells' in the present study. Later, more flattened cells, designated `thick cells', appeared in culture, and the number of round cells decreased in each colony(Fig. 1B,D). Thick cells were easily distinguished from fibroblastic cells, which appeared much more flattened. Terminal myogenic differentiation into myotubes occurred spontaneously after the appearance of thick cells(Fig. 1B-D). Satellite cells derived from both soleus and EDL muscles underwent the same proliferation/differentiation processes, although thick cells and myotubes appeared earlier in soleus myofiber cultures than in EDL cultures(Fig. 1C-E). Round cells continuously migrated, as suggested by their numerous filopodia,(Fig. 1F), and formed`burst'-type colonies in which cells kept their distance from each other(Fig. 1A). The number of colonies containing MHC-expressing cells increased with time in both fast and slow muscle fiber cultures (Fig. 1E). On the eighth day of culture, all satellite cell-derived clones underwent myogenic terminal differentiation, regardless of their origin. Thus, all satellite cell-derived clones kept myogenic differentiation potential under our isolation and culture conditions.

Two distinct subpopulations appear in satellite cell-derived clones. (A-D)Single myofibers were isolated from EDL (A,B) or soleus (C,D) muscle and cultured for 4 (A,C) or 6 days (B,D). Two distinct subpopulations, including cells designated round cells (white arrowheads) with a rounded shape and more flattened cells designated thick cells (black arrowheads), appeared in either EDL- or soleus-fiber culture. Images were obtained by phase-contrast microscopy. Scale bars: 50 μm. (E) The ability to undergo myogenic differentiation of satellite cell clones derived from EDL (white column) and soleus muscles (black column). Single myofiber cultures were fixed on day 4 or 8, and then subjected to immunofluorescence for MHC. Expression of MHC was determined in 45-80 colonies. (F) Ultra-microscopic view of round cells. Round cells derived from gastrocnemius muscle were observed under a scanning electron microscope. The arrow indicates a filopodium. Scale bar: 10μm.

Two distinct subpopulations appear in satellite cell-derived clones. (A-D)Single myofibers were isolated from EDL (A,B) or soleus (C,D) muscle and cultured for 4 (A,C) or 6 days (B,D). Two distinct subpopulations, including cells designated round cells (white arrowheads) with a rounded shape and more flattened cells designated thick cells (black arrowheads), appeared in either EDL- or soleus-fiber culture. Images were obtained by phase-contrast microscopy. Scale bars: 50 μm. (E) The ability to undergo myogenic differentiation of satellite cell clones derived from EDL (white column) and soleus muscles (black column). Single myofiber cultures were fixed on day 4 or 8, and then subjected to immunofluorescence for MHC. Expression of MHC was determined in 45-80 colonies. (F) Ultra-microscopic view of round cells. Round cells derived from gastrocnemius muscle were observed under a scanning electron microscope. The arrow indicates a filopodium. Scale bar: 10μm.

Conversion of round cells to thick cells occurs prior to myogenic terminal differentiation

The observation that the number of thick cells rapidly increased in colonies simultaneously with a rapid decrease in the number of round cells(Fig. 1A,B) suggests the conversion of round cells to thick cells. To reveal the sequence of events that occur during myogenic terminal differentiation in the single fiber culture, the behavior of round cells derived from gastrocnemius muscle was examined by phase-contrast, time-lapse microscopy. Fig. 2 shows the images taken between 1 and 108 minutes from time-lapse movies on day 7 of the culture. The round cell (arrowhead) migrated continuously during the culture(Fig. 2A-C). When a round cell contacted another cell, its rounded morphology flattened(Fig. 2D-I) thus, the nucleus became visible under a phase-contrast microscope(Fig. 2H-J). Then, the newly formed thick cell fused with another thick cell and differentiated into a myotube (Fig. 2K,L). In addition, expression of myogenin (Wright et al., 1989), an early marker of myogenesis, was induced in a fraction of the thick cells but in none of the round cells(Fig. 3A-D) in colonies containing myotubes. These results indicate that round cells are converted to thick cells without cell division, and that thick cells, but not round cells,undergo myogenic terminal differentiation.

Round cells are converted to thick cells prior to myogenic terminal differentiation. The sequence of events that occur during myogenic terminal differentiation of satellite cell-descendants was observed by phase-contrast,time-lapse microscopy. (A-L) The images were taken at the indicated time points. Arrowheads indicate the position near the nucleus of the same cell in A through L. Scale bar: 10 μm.

Round cells are converted to thick cells prior to myogenic terminal differentiation. The sequence of events that occur during myogenic terminal differentiation of satellite cell-descendants was observed by phase-contrast,time-lapse microscopy. (A-L) The images were taken at the indicated time points. Arrowheads indicate the position near the nucleus of the same cell in A through L. Scale bar: 10 μm.

Thick cells, but not round cells, express myogenin. Myofibers obtained from gastrocnemius muscle were cultured in pmGM for 7 days. Then, satellite cell-derived colonies were subjected to immunostaining with anti-myogenin antibody. (A,B) A fraction of thick cells (arrowheads) around a myotube(asterisk) expressed myogenin (green in B). Scale bar: 50 μm. (C,D) One of the thick cells (arrowhead), but none of the round cells (within the square),expressed myogenin (D). Scale bar: 20 μm. Nuclei were visualized with DAPI(blue in B and D). (A,C) Phase contrast microscopy images of the same fields as those shown in B and D.

Thick cells, but not round cells, express myogenin. Myofibers obtained from gastrocnemius muscle were cultured in pmGM for 7 days. Then, satellite cell-derived colonies were subjected to immunostaining with anti-myogenin antibody. (A,B) A fraction of thick cells (arrowheads) around a myotube(asterisk) expressed myogenin (green in B). Scale bar: 50 μm. (C,D) One of the thick cells (arrowhead), but none of the round cells (within the square),expressed myogenin (D). Scale bar: 20 μm. Nuclei were visualized with DAPI(blue in B and D). (A,C) Phase contrast microscopy images of the same fields as those shown in B and D.

Satellite cell clones from both slow and fast muscles retain the ability to respond to BMP2

To determine whether each muscle satellite cell-derived clone retains the ability to undergo osteogenic differentiation or not, we exposed the single fiber culture to BMP2 for 2 days during either an early (days 2-4) or a late(days 7-9) period in vitro. ALP, an early marker of osteogenic differentiation, was detectable in only 3.6% of EDL-derived colonies that were exposed to BMP2 on days 2-4 (Fig. 4A,C). By contrast, ALP expression occurred in 86.4% of EDL-derived colonies that were exposed to BMP2 on days 7-9(Fig. 4B,C). BMP2 markedly inhibited myogenic terminal differentiation but did not affect the conversion of round cells to thick cells.

A distinct subpopulation responds to BMP2 and undergoes osteogenic differentiation. (A,B) Satellite cell-derived colonies obtained in EDL-fiber culture were exposed to BMP2 on days 2-4 (A) or days 7-9 (B). They were then subjected to staining for ALP. Scale bar: 20 μm. (C,D) Analysis of the ability to respond to BMP2 by satellite cell-descendants derived from EDL- (C)or soleus- (D) fiber cultures. Satellite cell-derived colonies were exposed to BMP2 on days 2-4 (BMP d2-4) or days 7-9 (BMP d7-9), or were cultured for the same periods in the absence of BMP2. Light blue indicates colonies that contain neither thick cells nor ALP-positive cells (ALP– Thick–). Yellow indicates colonies that contain thick cells but no ALP-positive cells(ALP– Thick cell+). Dark red indicates colonies that contain ALP-positive cells but no thick cells (ALP+ Thick cell–). Dark blue indicates colonies that contain both ALP-positive cells and thick cells (ALP+Thick cell+). The numbers of colonies examined were 52-109 in control cultures without exposure to BMP2 and 107-199 in cultures exposed to BMP2.

A distinct subpopulation responds to BMP2 and undergoes osteogenic differentiation. (A,B) Satellite cell-derived colonies obtained in EDL-fiber culture were exposed to BMP2 on days 2-4 (A) or days 7-9 (B). They were then subjected to staining for ALP. Scale bar: 20 μm. (C,D) Analysis of the ability to respond to BMP2 by satellite cell-descendants derived from EDL- (C)or soleus- (D) fiber cultures. Satellite cell-derived colonies were exposed to BMP2 on days 2-4 (BMP d2-4) or days 7-9 (BMP d7-9), or were cultured for the same periods in the absence of BMP2. Light blue indicates colonies that contain neither thick cells nor ALP-positive cells (ALP– Thick–). Yellow indicates colonies that contain thick cells but no ALP-positive cells(ALP– Thick cell+). Dark red indicates colonies that contain ALP-positive cells but no thick cells (ALP+ Thick cell–). Dark blue indicates colonies that contain both ALP-positive cells and thick cells (ALP+Thick cell+). The numbers of colonies examined were 52-109 in control cultures without exposure to BMP2 and 107-199 in cultures exposed to BMP2.

The ability of soleus-derived clones to respond to BMP2 developed similarly, although the incidence of ALP-positive colonies in the soleus fiber culture was lower than that in the EDL fiber culture following BMP2 exposure at 7-9 days in vitro (Fig. 4D). Given that the ability of myogenic cells to respond to BMP2 is lost when they undergo myogenic terminal differentiation(Wada et al., 2002), the relatively low incidence of osteogenic differentiation (62.3% versus 86.4% of colonies) in soleus myofiber culture may be due to the more rapid progression of myogenesis (Fig. 1E). Indeed, continuous exposure to BMP2 from day 0 to day 9 in vitro prevented myogenic differentiation and increased the incidence of osteogenic differentiation of soleus-derived colonies up to 86.2% of 87 independent colonies.

Histochemical analysis indicated that ALP expression was induced exclusively in thick cells but not in round cells(Fig. 4A,B). The temporal change of BMP2 responsiveness of satellite cell descendants in the myofiber culture paralleled the time course of the round cell to thick cell conversion(Fig. 4C,D). The conversion occurred earlier in the single-fiber culture of soleus muscle(Fig. 1B). Actually, even when exposed to BMP2 on days 2-4, ALP was induced in certain soleus colonies containing thick cells (13.3% Fig. 4D).

The present results suggest that round cells represent activated satellite cells, whereas thick cells represent multipotent progenitor cells(multiblasts) (Wada et al.,2002). Taken together, the results suggest that round cells are converted to thick cells prior to both myogenic and osteogenic terminal differentiation.

Basic FGF and LIF synergistically suppress both round cell to thick cell conversion and myogenic terminal differentiation

Differences in responsiveness to differentiation signals between round cells and thick cells support the notion that the efficiency of their contributions to muscle regeneration in vivo on transplantation differs in the two cell types of satellite cell descendants. However, we were unable to obtain a sufficient number of round cells to test this because round cells are spontaneously converted into thick cells during culture. To achieve continuous clonal expansion of round cells without conversion to thick cells, we cultured isolated myofibers from gastrocnemius muscles of adult mice in pmGM supplemented with various growth factors. Basic FGF (bFGF) or LIF markedly inhibited the expression of MHC (Fig. 5A-C). When combined, bFGF and LIF synergistically inhibited myogenic differentiation (Fig. 5D,E). In addition, bFGF, but not LIF, markedly enhanced the proliferation of round cells (Fig. 5E).

Basic FGF and LIF synergistically suppress the expression of MHC. (A-D)Single myofibers isolated from gastrocnemius muscle were cultured in pmGM (A),or in pmGM supplemented with bFGF alone (B), LIF alone (C), or bFGF plus LIF(D), for 6 days. Cells were then subjected to immunostaining for MHC (red). Nuclei were visualized by DAPI. Scale bars: 100 μm. (E) Quantitative analysis of inhibition of MHC expression by growth factors. Percentages of nuclei in MHC-expressing cells to total nuclei (bars) and total numbers of nuclei in each field (0.55 mm 2 ) (open circles) were calculated in four independent samples. Averages and standard errors are shown.

Basic FGF and LIF synergistically suppress the expression of MHC. (A-D)Single myofibers isolated from gastrocnemius muscle were cultured in pmGM (A),or in pmGM supplemented with bFGF alone (B), LIF alone (C), or bFGF plus LIF(D), for 6 days. Cells were then subjected to immunostaining for MHC (red). Nuclei were visualized by DAPI. Scale bars: 100 μm. (E) Quantitative analysis of inhibition of MHC expression by growth factors. Percentages of nuclei in MHC-expressing cells to total nuclei (bars) and total numbers of nuclei in each field (0.55 mm 2 ) (open circles) were calculated in four independent samples. Averages and standard errors are shown.

In the absence of additional bFGF and LIF, round cells were spontaneously converted to thick cells and then differentiated into myotubes, resulting in small colonies (Fig. 6B,C). By contrast, round cells continued to proliferate for at least 7 days without conversion to thick cells in medium containing both bFGF and LIF(Fig. 6A,B compare the size of the colony in A with that in B at the same magnification). The inhibition of the round cell to thick cell conversion by the combination of bFGF and LIF resulted in extensive growth of round cells. We routinely obtained at least 10,000 purified round cells when 120-160 myofibers from a pair of gastrocnemius muscles were cultured under such conditions. However, the present culture conditions cannot prevent the conversion during a prolonged culture period.

Basic FGF and LIF synergistically enhance the clonal growth of round cells. Myogenic cell clones derived from satellite cells in gastrocnemius muscle were cultured in pmGM alone (B,C), or in pmGM supplemented with bFGF plus LIF(A,D), for 7 days. Images were obtained by phase-contrast microscopy. Scale bars: 1 mm in A,B, 100 μm in C,D.

Basic FGF and LIF synergistically enhance the clonal growth of round cells. Myogenic cell clones derived from satellite cells in gastrocnemius muscle were cultured in pmGM alone (B,C), or in pmGM supplemented with bFGF plus LIF(A,D), for 7 days. Images were obtained by phase-contrast microscopy. Scale bars: 1 mm in A,B, 100 μm in C,D.

Round cells contribute to reconstruction of myofibers more efficiently than thick cells

After development of the culture system that enables clonal expansion of round cells, we transferred round cells to regenerating gastrocnemius muscles to determine their possible contribution to muscle reconstruction in vivo. We routinely obtained 10,000 round cells in a myofiber culture derived from a pair of mouse gastrocnemius muscles, which avoided physiological damage by minimizing the time spent in cell preparation. Five thousand round cells obtained from a single-fiber culture of GFP-transgenic mice were injected into gastrocnemius muscles of C57Bl/6 mice that had received an intramuscular injection of CTX on the day before transplantation. Both round cells and thick cells had formed few GFP-positive myofibers at 14 days after transplantation to host muscles (Table 1). After 28 days, more GFP-positive myofibers were found in cryosections of host muscles injected with 5000 round cells(Fig. 7A, Table 1). Peripherally located nuclei in several GFP-positive myofibers suggest the completion of myofiber reconstruction (Fig. 7B). By contrast, very few, if any, GFP-positive myofibers were found at 28 days after transplantation in host muscles injected with 5000 thick cells(Fig. 7C). However, when 1,000,000 thick cells were transferred, they were capable of greatly contributing to myofiber formation (Fig. 7D, Table 1). Therefore, thick cells still retain the capacity to reconstitute myofibers in vivo, although their efficiency in myofiber formation is comparatively much lower than that of round cells (approximately 2-5%). We cannot exclude the possibility that a thick cell culture contains a small number of round cells or cells similar to round cells. However, round cells will be easily lost from the thick cell culture through successive passages because these cells divide slowly.

In vivo myofiber formation by myogenic cells at different stages of maturation/differentiation

. . . Number of GFP(+) fibers . Required cell number .
Cell type . Number of transplanted cells . Days after transplantation . Gastrocnemius . GFP(+) fiber .
Round cell 5000 14 4.8±7.5 * (5) † , ‡ 1041 §
5000 28 15±5 (4) 333
Thick cell 5000 14 2.7±4.3(6) ‡ 1851
5000 28 0.3±0.5 (6) 16667
10 6 28 138±46 (4) 7246
. . . Number of GFP(+) fibers . Required cell number .
Cell type . Number of transplanted cells . Days after transplantation . Gastrocnemius . GFP(+) fiber .
Round cell 5000 14 4.8±7.5 * (5) † , ‡ 1041 §
5000 28 15±5 (4) 333
Thick cell 5000 14 2.7±4.3(6) ‡ 1851
5000 28 0.3±0.5 (6) 16667
10 6 28 138±46 (4) 7246

Average and standard deviation

Number of samples examined

Expression level of GFP in GFP-positive myofibers after 14 days was relatively low compared with that apparent in GFP-positive myofibers after 28 days

Estimated values based on [number of transplanted cells]/[number of GFP-positive fibers]

Round cells enhance muscle regeneration more efficiently than thick cells. Five thousand round cells (A,B) or thick cells (C), or 1,000,000 thick cells(D) derived from gastrocnemius muscles of GFP-transgenic mice were transplanted into gastrocnemius muscles of congenic C57Bl/6 mice pre-treated with CTX. The muscles were removed and cryosectioned 28 days after transplantation. Images in A-D were obtained by epifluorescence microscopy with a GFP filter. Scale bars: 100 μm in A,C,D 50 μm in B.

Round cells enhance muscle regeneration more efficiently than thick cells. Five thousand round cells (A,B) or thick cells (C), or 1,000,000 thick cells(D) derived from gastrocnemius muscles of GFP-transgenic mice were transplanted into gastrocnemius muscles of congenic C57Bl/6 mice pre-treated with CTX. The muscles were removed and cryosectioned 28 days after transplantation. Images in A-D were obtained by epifluorescence microscopy with a GFP filter. Scale bars: 100 μm in A,C,D 50 μm in B.

To compare the ability of quiescent satellite cells to form new myofibers with that of their cultured descendant cells, we transplanted ten or thirty single myofibers isolated from gastrocnemius muscles of GFP-transgenic mice into host gastrocnemius muscles that were pre-treated with CTX. The fact that GFP-positive myofibers were not observed 24 hours after transplantation implies that the transplanted single myofibers were physically damaged during the process of transplantation. Transplanted quiescent satellite cells located on single myofibers formed GFP-positive myofibers within two weeks: the average number of GFP-positive myofibers from three independent experiments was 0.52±0.16/transplanted myofiber. Single myofibers of mouse gastrocnemius muscle were associated with 3.3±1.3 satellite cells (T.M. and N.H., unpublished). Thus, only seven quiescent satellite cells are sufficient for the formation of a single new myofiber by intramuscular transplantation. These results suggest that quiescent satellite cells have an extremely high ability to reconstitute myofibers in vivo. The efficiency of muscle regeneration by cells belonging to the satellite cell lineage might be altered as a result of isolation and/or tissue culture(Smythe et al., 2001).

Round cells have the ability to restore dystrophin in myofibers of mdx mice

To determine whether round cells have the capacity to restore dystrophin in the muscle of mdx nude mice, which lack functional dystrophin due to a genetic mutation (Sicinski et al.,1989), 5000 round cells derived from GFP-transgenic mice were injected into gastrocnemius muscles of mdx nude mice that had received an intramuscular injection of CTX on the day before transplantation. We did not find any revertant fibers in the gastrocnemius muscles in this series of experiments (Fig. 8A). After 28 days, GFP-positive myofibers (approximately 10-30/gastrocnemius muscle) were identified in cryosections of host muscles(Fig. 8B). Immunofluorescence analysis shows that dystrophin was restored in approximately 10% of GFP-positive myofibers (Fig. 8C). Thus, round cells representing immediate descendants of quiescent satellite cells have the capacity to restore dystrophin in the muscle of mdx nude mice.

Round cells retain the capacity to restore dystrophin in myofibers of mdx nude mice. Five thousand round cells derived from GFP-transgenic mice were transplanted into gastrocnemius muscles of mdx nude mice pre-treated with CTX. The muscles were removed 28 days after transplantation and subjected to immunofluorescence analysis with antibodies to GFP (B) and dystrophin (C). Immunofluorescence analysis with antibodies to dystrophin revealed the absence of revertant myofibers in muscles that were treated with CTX alone (A). Scale bars: 20 μm.

Round cells retain the capacity to restore dystrophin in myofibers of mdx nude mice. Five thousand round cells derived from GFP-transgenic mice were transplanted into gastrocnemius muscles of mdx nude mice pre-treated with CTX. The muscles were removed 28 days after transplantation and subjected to immunofluorescence analysis with antibodies to GFP (B) and dystrophin (C). Immunofluorescence analysis with antibodies to dystrophin revealed the absence of revertant myofibers in muscles that were treated with CTX alone (A). Scale bars: 20 μm.

Round cells retain stem cell-like properties and express Pax7 at high levels

The high ability of round cells to form new myofibers after transplantation suggests that these cells correspond to a stem-like subpopulation in myoblast culture (Beauchamp et al.,1999). Time-lapse recording revealed that round cells grew more slowly than thick cells (Fig. 9A). The maximum growth rate of round cells, which is estimated from the slope of their growth curve, was significantly lower than that of thick cells (9.3±0.7 versus 16.7±1.5 cells/hour). A serial recording also showed a single round cell generating two round-shaped daughter cells that migrated away soon after cell division(Fig. 9B). These results indicate that round cells retain stem cell-like characteristics: slow division, self-renewal for new stem-like cells, and generation of a progeny committed to terminal differentiation.

Round cells divide slowly and generate new round cells. (A) The behavior of round cells on day 6, 7 or 8 of myofiber cultures (black symbols), and thick cells at early passages (white symbols), was recorded by time-lapse microscopy. Cell numbers in the same fields were counted every 6 hours. Three independent cultures of round cells or thick cells were analyzed. (B) A round cell on day 6 of the fiber culture (arrowhead) generated two daughter cells(asterisks) displaying a rounded shape. The images were taken at the indicated time points. Scale bar: 10 μm.

Round cells divide slowly and generate new round cells. (A) The behavior of round cells on day 6, 7 or 8 of myofiber cultures (black symbols), and thick cells at early passages (white symbols), was recorded by time-lapse microscopy. Cell numbers in the same fields were counted every 6 hours. Three independent cultures of round cells or thick cells were analyzed. (B) A round cell on day 6 of the fiber culture (arrowhead) generated two daughter cells(asterisks) displaying a rounded shape. The images were taken at the indicated time points. Scale bar: 10 μm.

To clarify the nature and diversity of round and thick cells, the expression of satellite cell lineage markers and stem cell markers were determined by immunofluorescence analysis. Both round cells and thick cells expressed MyoD, M-cadherin, desmin and nestin, but neither Sca1 nor CD34 (data not shown). Furthermore, all round cells expressed Pax7, an essential transcription factor for satellite cell specification(Seale et al., 2000), at high levels (Fig. 10A,B). Undifferentiated thick cells also expressed Pax7, but at a lower level than that apparent in round cells (Fig. 10C,D), whereas differentiating thick cells expressed myogenin,but not Pax7 (Fig. 10E,F). The high level of Pax7 expression in round cells suggests that Pax7 is a possible molecular marker for identification of stem-like cells in myoblast culture.

Round cells express Pax7 at high levels. Myofibers from EDL muscle were cultured in pmGM for 6 days. (A,B) Round cells expressed Pax7 at high levels.(C,D) Undifferentiated thick cells expressed Pax7 at a reduced level. (E,F)Differentiating thick cells (asterisks) expressed myogenin (green in F) but not Pax7 (red in F). A round cell (arrow) expressed Pax7 but not myogenin.(A,C,E) Phase contrast microscopy images of the same fields as those shown in B, D and F. Scale bars: 20 μm.

Round cells express Pax7 at high levels. Myofibers from EDL muscle were cultured in pmGM for 6 days. (A,B) Round cells expressed Pax7 at high levels.(C,D) Undifferentiated thick cells expressed Pax7 at a reduced level. (E,F)Differentiating thick cells (asterisks) expressed myogenin (green in F) but not Pax7 (red in F). A round cell (arrow) expressed Pax7 but not myogenin.(A,C,E) Phase contrast microscopy images of the same fields as those shown in B, D and F. Scale bars: 20 μm.


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Correcting DNA at the source

The strategy pursued by the Wagers Lab aims to fully correct the genetic template for dystrophin at its source, in the DNA of stem cells (satellite cells) that create and regenerate muscle cells. Combining cutting-edge CRISPR/Cas9 genome editing technologies with a deep knowledge of stem cell science and regenerative biology, this approach if successful might offer a permanent restoration of muscular function.

“In skeletal muscle, muscle fibers are terminally post-mitotic, meaning they cannot divide and they cannot reproduce themselves,” Wagers explains. “If you lose muscle fibers, the only way to produce new muscle is from stem cells, specifically the satellite cells. The satellite cells are self-renewing, self-repairing, and ready to spring into action to create new muscle fibers. So we expect that a satellite cell with the corrected DMD gene would quite quickly and continuously propagate the edited gene throughout the muscle tissue.”

At present, research conducted in mice has shown promising results. In 2019, the Wagers Lab published the results of editing stem cells in vivo, demonstrating that stem cell genes can be edited in living systems, not only in a dish. In that work, Wagers and her team delivered genome editing molecules to the cells using adeno-associated viruses (AAVs). Her lab has also successfully used gene editing in heart, muscle, and satellite cells to partially restore the function of the DMD gene that encodes dystrophin, by chopping out faulty sequences of code that are disrupting the proper reading frame.


Introduction

Representing 30–40% of our body mass, skeletal muscle is a highly organized tissue made up of a large number of syncytial cells, known as myofibers, which are formed by the fusion of myogenic progenitor cells. Despite the post-mitotic nature of its myofibers, skeletal muscle has a robust regenerative capacity in response to injury. This relies on resident muscle stem cells (MuSCs), also called “satellite cells” because of their unique anatomical position at the periphery of the myofibers. MuSCs typically exist in a quiescent state but may enter the cell cycle following injury in order to regenerate the skeletal muscle tissue and replenish the stem cell pool for future needs. Several transcription factors have been identified as markers and key regulators of the quiescent state as well as of activation and progression to the myogenic lineage. Among them, the paired homeobox factors PAX3 and PAX7 as well as the so-called Myogenic Regulatory Factors – MRFs (MYF5, MYOD, MYOGENIN, MRF4) stand out for their unique and important roles in muscle formation, specification, homeostasis, and repair (for more details the reader may refer to ref. 1,2 ). PAX7 is commonly used as a marker of MuSCs, and a subset of them co-expresses PAX3 in adult muscle 3,4 . The MRFs regulate the progression of MuSCs towards myogenic determination, differentiation, and fusion to form multinucleated myofibers 2 .

The renewal of the MuSC cellular compartment requires a tightly regulated balance between quiescence and activation that is associated with many transcriptional changes in MuSCs. Activation is accompanied by metabolic reprogramming, reinforcing the evidence of a strict interplay between MuSC function and metabolic status. Moreover, recent studies show that MuSCs are a heterogeneous stem cell population, with different abilities to support tissue regeneration. The dynamic changes in MuSC behavior are regulated by the microenvironment and by distinct tissue resident cells of the niche that provide molecular cues to regulate MuSC fate. Here, we review novel findings that have challenged our knowledge of MuSC biology, discussing the molecular mechanisms regulating MuSC quiescence and activation states and heterogeneity. Moreover, we describe the latest advances that enhance our understanding of how MuSC metabolism adapts to quiescence and differentiation, and the role of the microenvironmental niche in regulating MuSC behavior and function. Finally, we present new insights into the pathological conditions associated with MuSC dysfunction, such as muscular dystrophies and aging, showing how the deregulation of MuSCs can lead to an exacerbation of pathology.


Non-satellite cell types with myogenic potential

A variety of cells different from satellite cells possess myogenic potential (Table 1). Some of these atypical myogenic cell types are considered as potential therapeutics for muscular dystrophy. Most promising amongst these are mesoangioblast-like cells/pericytes. These cells are perivascular mesenchymal-like progenitors that can differentiate into various cell types of mesodermal origin, including skeletal muscle fibers and cardiac muscle 77 , 78 . Such cells have been isolated from embryonic and postnatal aorta, bone marrow, cardiac, and skeletal muscle, as well as other tissues 79 . The ease of transduction with viral vectors and the ability of these cells to cross the endothelial wall in the presence of inflammation, as in the case of muscular dystrophy, makes them very interesting therapeutic candidates for systemic delivery 80 . Recent reports revealed that human pericytes as well as genetically corrected dystrophin deficient murine pericytes can not only fuse to muscle fibers but generate cells in the satellite cell position 81 , 82 . Taken together, this atypical myogenic cell type holds outstanding therapeutic promise and translational potential for the treatment of muscular dystrophy.

Several other cell types with varying myogenic potential that could be of therapeutic relevance have been described. (i) A rare population of cells expressing CD133 or Prominin-1 that is present in human skeletal muscle and circulating in adult blood has myogenic potential 83-85 . In co-culture with myogenic cells, CD133 positive progenitors differentiate into myotubes. Upon intramuscular application or injection into the bloodstream these cells are able to home to the satellite cell niche and to generate fibers expressing human dystrophin in immunocompromised mdx mice more efficiently than human myoblasts 85 . Interestingly, local injection of this cell type seems to promote muscle regeneration through increased vasculogenesis 86 . These studies suggest that the systemic delivery of CD133 positive cells from immunologically matched healthy donors or genetically corrected cells from patient blood could be a feasible strategy for the treatment of muscular dystrophy. (ii) In human muscles a population of myoendothelial cells that co-express myogenic and endothelial cell markers (CD56, CD34, CD144) have been described to extensively contribute to regeneration upon transplantation into cardiotoxin injured skeletal muscle of SCID mice 87 . Interestingly, this cell type can be cultured clonally for long periods while retaining its myogenic properties. (iii) Another population of muscle resident cells has been shown to express aldehyde dehydrogenase (ALDH) but not CD34 88 . These cells can participate in muscle formation and populate the satellite cell compartment upon injection into injured muscle of immunocompromised mice. ALDH positive and CD34 negative cells appear to have an outstanding capacity for proliferation upon transplantation. (iv) The Sassoon laboratory has discovered muscle resident PW1+ interstitial cells (PICs) which possess Pax7 dependent myogenic activity during postnatal muscle growth, contribute to skeletal muscle regeneration and are able to generate satellite cells 89 . (v) Asakura et al. have described a fraction of Sca-1 positive SP cells found in muscle which undergo myogenic specification after co-culture with myoblasts 90 . If injected into regenerating muscle of SCID mice, SP cells give rise to both myocytes and satellite cells. (vi) Several groups have shown that bone marrow-derived cells, such as hematopoietic and mesenchymal stem cells, can participate in the regeneration of muscle, albeit with a very low efficiency 91-95 . (vii) Last but not least, embryonic (ES) and induced pluripotent (iPS) stem cells are currently explored as potential candidates for cell therapy of muscular diseases. Barberi et al. were able to derive engraftable myoblasts from human ES cells 96 . Gene therapies with patient derived corrected iPS cells offer an advantage over ES cells by providing a genetic match and thereby decreasing the likelihood of immunorejection. Darabi et al. reported the generation of functional skeletal muscle from mouse ES and iPS cells by ectopic expression of Pax3/7 and the engraftment of these cells into the satellite cell niche in dystrophic mice 97 , 98 . Because of the possible development of teratomas, the use of ES and iPS cells will have to be carefully monitored in a clinical setting 99 .


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