Tissue-specific functions of MSCs are linked to homeostatic muscle maintenance and alter with aging.

Mesenchymal stromal cells (MSCs), also known as fibro-adipogenic progenitors, play a critical role in muscle maintenance and sarcopenia development. Although analogous MSCs are present in various tissues, recent single-cell RNA-seq studies have revealed the inter-tissue heterogeneity of MSCs. However, the functional significance of MSC heterogeneity and its role in aging remain unclear. Here, we investigated the properties of MSCs and their age-related changes in seven mouse tissues through histological, cell culture, and genetic examinations. The tissue of origin had a greater impact on the MSC transcriptome than aging. By first analyzing age-related changes, we found that Kera is exclusively expressed in muscle MSCs and significantly down-regulated by aging. Kera knockout mice recapitulated some sarcopenic phenotypes including reduced muscle mass and specific force, revealing the functional importance of Kera in the maintenance of muscle youth. These results suggest that MSCs have tissue-specific supportive functions and that deterioration in these functions may trigger tissue aging.

Transcriptome analysis of mesenchymal stromal cells of the large and small intestinal smooth muscle layers reveals a unique gastrontestinal stromal signature.

Mesenchymal stromal cells in the muscle layer of the large intestine are essential for the regulation of intestinal motility. They form electrogenic syncytia with the smooth muscle and interstitial cells of Cajal (ICCs) to regulate smooth muscle contraction. Mesenchymal stromal cells are present in the muscle layer throughout the gastrointestinal tract. However, their area-specific characteristics remain ambiguous. In this study, we compared mesenchymal stromal cells from the large and small intestinal muscle layers. Histological analysis using immunostaining showed that the cells in the large and small intestines were morphologically distinct. We established a method to isolate mesenchymal stromal cells from wild-type mice with platelet-derived growth factor receptor-alpha (PDGFRα) as a marker on the cell surface and performed RNAseq. Transcriptome analysis revealed that PDGFRα+ cells in the large intestine exhibited increased expression levels of collagen-related genes, whereas PDGFRα+ cells in the small intestine exhibited increased expression levels of channel/transporter genes, including Kcn genes. These results suggest that mesenchymal stromal cells differ morphologically and functionally depending on gastrointestinal tract. Further investigations of the cellular properties of mesenchymal stromal cells in the gastrointestinal tract will aid in optimizing methods for the prevention and treatment of gastrointestinal diseases.

Analyses of Mesenchymal Progenitors in Skeletal Muscle by Fluorescence-Activated Cell Sorting and Tissue Clearing.

Mesenchymal progenitors, which are resident progenitor populations residing in skeletal muscle interstitial space, contribute to pathogeneses such as fat infiltration, fibrosis, and heterotopic ossification. In addition to their pathological roles, mesenchymal progenitors have also been shown to play important roles for successful muscle regeneration and homeostatic muscle maintenance. Therefore, detailed and accurate analyses of these progenitors are essential for the research on muscle diseases and health. Here, we describe a method for purification of mesenchymal progenitors based on the expression of PDGFRα, which is a specific and well-established marker for mesenchymal progenitors, using fluorescence-activated cell sorting (FACS). Purified cells can be used in several downstream experiments including cell culture, cell transplantation, and gene expression analysis. We also describe the method for whole-mount 3-dimensional imaging of mesenchymal progenitors by utilizing tissue clearing. The methods described herein provide a powerful platform for studying mesenchymal progenitors in skeletal muscle.

Whole-mount immunofluorescence staining of mesenchymal progenitors in murine plantaris muscle.

We recently demonstrated that mesenchymal progenitors play a critical role in regulating satellite cell-dependent myonuclear accretion during overload-induced muscle hypertrophy. Here, we describe the detailed protocol for whole-mount immunofluorescence staining of mesenchymal progenitors in mouse plantaris muscle. Z-stack image reconstruction provides a whole-cell image and enables examination of YAP nuclear translocation in mesenchymal progenitors induced by overload. For complete details on the use and execution of this protocol, please refer to Kaneshige et al. (2022a).

Detection of muscle stem cell-derived myonuclei in murine overloaded muscles.

Muscle satellite cells (MuSCs) supply nuclei to existing myofibers in response to mechanical loading. This myonuclear accretion is critical for efficient muscle hypertrophy. Herein, we present protocols for the detection of MuSC-derived new myonuclei in loaded mouse muscle, including procedures for EdU injection to stain myonuclei, followed by surgery and skeletal muscle fixation. We then describe immunostaining for EdU+ myonuclei and image acquisition for quantitative analyses. For complete details on the use and execution of this protocol, please refer to Kaneshige et al. (2022).

Tenogenic Induction From Induced Pluripotent Stem Cells Unveils the Trajectory Towards Tenocyte Differentiation.

The musculoskeletal system is integrated by tendons that are characterized by the expression of scleraxis (Scx), a functionally important transcription factor. Here, we newly developed a tenocyte induction method using induced pluripotent stem cells established from ScxGFP transgenic mice by monitoring fluorescence, which reflects a dynamic differentiation process. Among several developmentally relevant factors, transforming growth factor-beta 2 (TGF-ß2) was the most potent inducer for differentiation of tenomodulin-expressing mature tenocytes. Single-cell RNA sequencing (scRNA-seq) revealed 11 distinct clusters, including mature tenocyte population and tenogenic differentiation trajectory, which recapitulated the in vivo developmental process. Analysis of the scRNA-seq dataset highlighted the importance of retinoic acid (RA) as a regulatory pathway of tenogenic differentiation. RA signaling was shown to have inhibitory effects on entheseal chondrogenic differentiation as well as TGF-ß2-dependent tenogenic/fibrochondrogenic differentiation. The collective findings provide a new opportunity for tendon research and further insight into the mechanistic understanding of the differentiation pathway to a tenogenic fate.

Increased MFG-E8 at neuromuscular junctions is an exacerbating factor for sarcopenia-associated denervation.

Sarcopenia is an important health problem associated with adverse outcomes. Although the etiology of sarcopenia remains poorly understood, factors apart from muscle fibers, including humoral factors, might be involved. Here, we used cytokine antibody arrays to identify humoral factors involved in sarcopenia and found a significant increase in levels of milk fat globule epidermal growth factor 8 (MFG-E8) in skeletal muscle of aged mice, compared with young mice. We found that the increase in MFG-E8 protein at arterial walls and neuromuscular junctions (NMJs) in muscles of aged mice. High levels of MFG-E8 at NMJs and an age-related increase in arterial MFG-E8 have also been identified in human skeletal muscle. In NMJs, MFG-E8 is localized on the surface of terminal Schwann cells, which are important accessory cells for the maintenance of NMJs. We found that increased MFG-E8 at NMJs precedes age-related denervation and is more prominent in sarcopenia-susceptible fast-twitch than in sarcopenia-resistant slow-twitch muscle. Comparison between fast and slow muscles further revealed that arterial MFG-E8 can be uncoupled from sarcopenic phenotype. A genetic deficiency in MFG-E8 attenuated age-related denervation of NMJs and muscle weakness, providing evidence of a pathogenic role of increased MFG-E8. Thus, our study revealed a mechanism by which increased MFG-E8 at NMJs leads to age-related NMJ degeneration and suggests that targeting MFG-E8 could be a promising therapeutic approach to prevent sarcopenia.

Relayed signaling between mesenchymal progenitors and muscle stem cells ensures adaptive stem cell response to increased mechanical load.

Adaptation to mechanical load, leading to enhanced force and power output, is a characteristic feature of skeletal muscle. Formation of new myonuclei required for efficient muscle hypertrophy relies on prior activation and proliferation of muscle stem cells (MuSCs). However, the mechanisms controlling MuSC expansion under conditions of increased load are not fully understood. Here we demonstrate that interstitial mesenchymal progenitors respond to mechanical load and stimulate MuSC proliferation in a surgical mouse model of increased muscle load. Mechanistically, transcriptional activation of Yes-associated protein 1 (Yap1)/transcriptional coactivator with PDZ-binding motif (Taz) in mesenchymal progenitors results in local production of thrombospondin-1 (Thbs1), which, in turn, drives MuSC proliferation through CD47 signaling. Under homeostatic conditions, however, CD47 signaling is insufficient to promote MuSC proliferation and instead depends on prior downregulation of the Calcitonin receptor. Our results suggest that relayed signaling between mesenchymal progenitors and MuSCs through a Yap1/Taz-Thbs1-CD47 pathway is critical to establish the supply of MuSCs during muscle hypertrophy.

Measurement of Lateral Transmission of Force in the Extensor Digitorum Longus Muscle of Young and Old Mice.

The main function of skeletal muscles is to generate force. The force developed by myofiber contraction is transmitted to the tendon. There are two pathways of force transmission from myofibers to tendons: longitudinal transmission that depends on tension elicited via the myotendinous junction and lateral transmission that depends on shear elicited via the interface between the myofiber surface and surrounding connective tissue. Experiments using animal muscle and mathematical models indicated that lateral transmission is the dominant pathway in muscle force transmission. Studies using rat muscle showed that the efficiency of lateral force transmission declines with age. Here, the lateral transmission of force was measured using the extensor digitorum longus muscle from young and old mice. Dependence on longitudinal transmission increased in the old muscle, and there was a trend for lower efficiency of lateral force transmission in the old muscle compared to the young muscle. There was a noticeable increase in the connective tissue volume in the old muscle; however, there was no significant change in the expression of dystrophin, a critical molecule for the link between the myofiber cytoskeleton and extracellular matrix. This study demonstrates the measurement of lateral force transmission in mouse muscles and that alteration in force transmission property may underlie age-related muscle weakness.

Transgenic Expression of Bmp3b in Mesenchymal Progenitors Mitigates Age-Related Muscle Mass Loss and Neuromuscular Junction Degeneration.

Skeletal muscle is a vital organ for a healthy life, but its mass and function decline with aging, resulting in a condition termed sarcopenia. The etiology of sarcopenia remains unclear. We recently demonstrated that interstitial mesenchymal progenitors are essential for homeostatic muscle maintenance, and a diminished expression of the mesenchymal-specific gene Bmp3b is associated with sarcopenia. Here, we assessed the protective function of Bmp3b against sarcopenia by generating conditional transgenic (Tg) mice that enable a forced expression of Bmp3b specifically in mesenchymal progenitors. The mice were grown until they reached the geriatric stage, and the age-related muscle phenotypes were examined. The Tg mice had significantly heavier muscles compared to control mice, and the type IIB myofiber cross-sectional areas were preserved in Tg mice. The composition of the myofiber types did not differ between the genotypes. The Tg mice showed a decreasing trend of fibrosis, but the degree of fat infiltration was as low as that in the control mice. Finally, we observed the preservation of innervated neuromuscular junctions (NMJs) in the Tg muscle in contrast to the control muscle, where the NMJ degeneration was conspicuous. Thus, our results indicate that the transgenic expression of Bmp3b in mesenchymal progenitors alleviates age-related muscle deterioration. Collectively, this study strengthens the beneficial role of mesenchymal Bmp3b against sarcopenia and suggests that preserving the youthfulness of mesenchymal progenitors may be an effective means of combating sarcopenia.

Liver fibrosis-induced muscle atrophy is mediated by elevated levels of circulating TNFα.

Liver cirrhosis is a critical health problem associated with several complications, including skeletal muscle atrophy, which adversely affects the clinical outcome of patients independent of their liver functions. However, the precise mechanism underlying liver cirrhosis-induced muscle atrophy has not been elucidated. Here we show that serum factor induced by liver fibrosis leads to skeletal muscle atrophy. Using bile duct ligation (BDL) model of liver injury, we induced liver fibrosis in mice and observed subsequent muscle atrophy and weakness. We developed culture system of human primary myotubes that enables an evaluation of the effects of soluble factors on muscle atrophy and found that serum from BDL mice contains atrophy-inducing factors. This atrophy-inducing effect of BDL mouse serum was mitigated upon inhibition of TNFα signalling but not inhibition of myostatin/activin signalling. The BDL mice exhibited significantly up-regulated serum levels of TNFα when compared with the control mice. Furthermore, the mRNA expression levels of Tnf were markedly up-regulated in the fibrotic liver but not in the skeletal muscles of BDL mice. The gene expression analysis of isolated nuclei revealed that Tnf is exclusively expressed in the non-fibrogenic diploid cell population of the fibrotic liver. These findings reveal the mechanism through which circulating TNFα produced in the damaged liver mediates skeletal muscle atrophy. Additionally, this study demonstrated the importance of inter-organ communication that underlies the pathogenesis of liver cirrhosis.

Mesenchymal Bmp3b expression maintains skeletal muscle integrity and decreases in age-related sarcopenia.

Age-related sarcopenia constitutes an important health problem associated with adverse outcomes. Sarcopenia is closely associated with fat infiltration in muscle, which is attributable to interstitial mesenchymal progenitors. Mesenchymal progenitors are nonmyogenic in nature but are required for homeostatic muscle maintenance. However, the underlying mechanism of mesenchymal progenitor-dependent muscle maintenance is not clear, nor is the precise role of mesenchymal progenitors in sarcopenia. Here, we show that mice genetically engineered to specifically deplete mesenchymal progenitors exhibited phenotypes markedly similar to sarcopenia, including muscle weakness, myofiber atrophy, alterations of fiber types, and denervation at neuromuscular junctions. Through searching for genes responsible for mesenchymal progenitor-dependent muscle maintenance, we found that Bmp3b is specifically expressed in mesenchymal progenitors, whereas its expression level is significantly decreased during aging or adipogenic differentiation. The functional importance of BMP3B in maintaining myofiber mass as well as muscle-nerve interaction was demonstrated using knockout mice and cultured cells treated with BMP3B. Furthermore, the administration of recombinant BMP3B in aged mice reversed their sarcopenic phenotypes. These results reveal previously unrecognized mechanisms by which the mesenchymal progenitors ensure muscle integrity and suggest that age-related changes in mesenchymal progenitors have a considerable impact on the development of sarcopenia.

Methods for Accurate Assessment of Myofiber Maturity During Skeletal Muscle Regeneration.

Adult skeletal muscle has a remarkable ability to regenerate. Regeneration of mature muscle fibers is dependent on muscle stem cells called satellite cells. Although they are normally in a quiescent state, satellite cells are rapidly activated after injury, and subsequently proliferate and differentiate to make new muscle fibers. Myogenesis is a highly orchestrated biological process and has been extensively studied, and therefore many parameters that can precisely evaluate regenerating events have been established. However, in some cases, it is necessary to evaluate the completion of regeneration rather than ongoing regeneration. In this study, we establish methods for assessing the myofiber maturation during muscle regeneration. By carefully comparing expression patterns of several muscle regeneration-related genes, we found that expression of Myozenin (Myoz1 and Myoz3), Troponin I (Tnni2), and Dystrophin (Dmd) is gradually increased as muscle regeneration proceeds. In contrast, commonly used regeneration markers such as Myh3 and Myh8 are transiently upregulated after muscle injury but their expression decreases as regeneration progresses. Intriguingly, upregulation of Myoz1, Myoz3 and Tnni2 cannot be achieved in cultured myotubes, indicating that these markers are excellent indicators to assess myofiber maturity. We also show that analyzing re-expression of Myoz1 and dystrophin in individual fiber during regeneration enables accurate assessment of myofiber maturity at the single-myofiber level. Together, our study provides valuable methods that are useful in evaluating muscle regeneration and the efficacy of therapeutic strategies for muscle diseases.

The CalcR-PKA-Yap1 Axis Is Critical for Maintaining Quiescence in Muscle Stem Cells.

Quiescence is a fundamental property of adult stem cells. Recent evidence indicates that quiescence is not a default state but requires active signaling that prevents accidental or untimely activation of stem cells. The calcitonin receptor (CalcR) is critical for sustaining quiescence in muscle satellite (stem) cells (MuSCs). However, the molecular mechanisms by which CalcR signaling regulates quiescence in MuSCs are enigmatic. Here, we demonstrate that transgenic expression of the catalytic domain of protein kinase A (PKA) restores the quiescence of CalcR-mutant MuSCs and delays MuSC activation. Mechanistically, CalcR-activated PKA phosphorylates Lats1/2, the main effector of Hippo signaling, thereby inhibiting the nuclear accumulation of Yap1, which prevents expression of Hippo-target genes, including cell-cycle-related molecules. Importantly, genetic inactivation of Yap1 in CalcR-mutant MuSCs reinstates quiescence in CalcR-mutant MuSCs, indicating that the CalcR-PKA-Lats1/2-Yap1 axis plays a critical role in sustaining MuSC quiescence.

Tbx1 regulates inherited metabolic and myogenic abilities of progenitor cells derived from slow- and fast-type muscle.

Skeletal muscle is divided into slow- and fast-type muscles, which possess distinct contractile and metabolic properties. Myogenic progenitors associated with each muscle fiber type are known to intrinsically commit to specific muscle fiber lineage during embryonic development. However, it is still unclear whether the functionality of postnatal adult myogenic cells is attributable to the muscle fiber in which they reside, and whether the characteristics of myogenic cells derived from slow- and fast-type fibers can be distinguished at the genetic level. In this study, we isolated adult satellite cells from slow- and fast-type muscle individually and observed that satellite cells from each type of muscle generated myotubes expressing myosin heavy chain isoforms similar to their original muscle, and showed different metabolic features. Notably, we discovered that slow muscle-derived cells had low potential to differentiate but high potential to self-renew compared with fast muscle-derived cells. Additionally, cell transplantation experiments of slow muscle-derived cells into fast-type muscle revealed that slow muscle-derived cells could better contribute to myofiber formation and satellite cell constitution than fast muscle-derived cells, suggesting that the recipient muscle fiber type may not affect the predetermined abilities of myogenic cells. Gene expression analyses identified T-box transcriptional factor Tbx1 as a highly expressed gene in fast muscle-derived myoblasts. Gain- and loss-of-function experiments revealed that Tbx1 modulated muscle fiber types and oxidative metabolism in myotubes, and that Tbx1 stimulated myoblast differentiation, but did not regulate myogenic cell self-renewal. Our data suggest that metabolic and myogenic properties of myogenic progenitor cells vary depending on the type of muscle from which they originate, and that Tbx1 expression partially explains the functional differences of myogenic cells derived from fast-type and slow-type muscles.

Interleukin-1beta (IL-1ß)-induced Notch ligand Jagged1 suppresses mitogenic action of IL-1ß on human dystrophic myogenic cells.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive muscle disorder caused by mutations in the dystrophin gene. Nonetheless, secondary processes involving perturbation of muscle regeneration probably exacerbate disease progression, resulting in the fatal loss of muscle in DMD patients. A dysfunction of undifferentiated myogenic cells is the most likely cause for the reduction of regenerative capacity of muscle. To clarify molecular mechanisms in perturbation of the regenerative capacity of DMD muscle, we have established several NCAM (CD56)-positive immortalized human dystrophic and non-dystrophic myogenic cell lines from DMD and healthy muscles. A pro-inflammatory cytokine, IL-1ß, promoted cell cycle progression of non-dystrophic myogenic cells but not DMD myogenic cells. In contrast, IL-1ß upregulated the Notch ligand Jagged1 gene in DMD myogenic cells but not in non-dystrophic myogenic cells. Knockdown of Jagged1 in DMD myogenic cells restored the IL-1ß-promoted cell cycle progression. Conversely, enforced expression of Jagged1-blocked IL-1ß promoted proliferation of non-dystrophic myogenic cells. In addition, IL-1ß prevented myogenic differentiation of DMD myogenic cells depending on Jagged1 but not of non-dystrophic myogenic cells. These results demonstrate that Jagged1 induced by IL-1ß in DMD myogenic cells modified the action of IL-1ß and reduced the ability to proliferate and differentiate. IL-1ß induced Jagged1 gene expression may be a feedback response to excess stimulation with this cytokine because high IL-1ß (200-1000 pg/ml) induced Jagged1 gene expression even in non-dystrophic myogenic cells. DMD myogenic cells are likely to acquire the susceptibility of the Jagged1 gene to IL-1ß under the microcircumstances in DMD muscles. The present results suggest that Jagged1 induced by IL-1ß plays a crucial role in the loss of muscle regeneration capacity of DMD muscles. The IL-1ß/Jagged1 pathway may be a new therapeutic target to ameliorate exacerbation of muscular dystrophy in a dystrophin-independent manner.

Disuse Atrophy Accompanied by Intramuscular Ectopic Adipogenesis in Vastus Medialis Muscle of Advanced Osteoarthritis Patients.

Muscle dysfunction is the most important modifiable mediating factor in primary osteoarthritis (OA) because properly contracting muscles are a key absorber of forces acting on a joint. However, the pathological features of disuse muscle atrophy in OA patients have been rarely studied. Vastus medialis muscles of 14 female patients with OA (age range, 69 to 86 years), largely immobile for 1 or more years, were obtained during arthroplastic surgery and analyzed histologically. These were compared with female patients without arthritis, two with patellar fracture and two with patellar subluxation. Areas occupied by myofibers and adipose tissue were quantified. Large numbers of myofibers were lost in the vastus medialis of OA patients. The loss of myofibers was a possible cause of the reduction in muscle strength of the operated on knee. These changes were significantly correlated with an increase in intramuscular ectopic adipose tissue, and not observed in knees of nonarthritic patients. Resident platelet-derived growth factor receptor α-positive mesenchymal progenitor cells contributed to ectopic adipogenesis in vastus medialis muscles of OA patients. The present study suggests that significant loss of myofibers and ectopic adipogenesis in vastus medialis muscles are common pathological features of advanced knee OA patients with long-term loss of mobility. These changes may be related to the loss of joint function in patients with knee OA.

Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle.

Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases.

Dysregulation of the Bmi-1/p16(Ink4a) pathway provokes an aging-associated decline of submandibular gland function.

Bmi-1 prevents stem cell aging, at least partly, by blocking expression of the cyclin-dependent kinase inhibitor p16(Ink4a) . Therefore, dysregulation of the Bmi-1/p16(Ink4a) pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi-1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi-1/p16(Ink4a) pathway occurs during aging in vivo. Using real-time in vivo imaging of p16(Ink4a) expression in Bmi-1-KO mice, we uncovered a novel function of the Bmi-1/p16(Ink4a) pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16(Ink4a) expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging-related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly.

Pro-Insulin-Like Growth Factor-II Ameliorates Age-Related Inefficient Regenerative Response by Orchestrating Self-Reinforcement Mechanism of Muscle Regeneration.

Sarcopenia, age-related muscle weakness, increases the frequency of falls and fractures in elderly people, which can trigger severe muscle injury. Rapid and successful recovery from muscle injury is essential not to cause further frailty and loss of independence. In fact, we showed insufficient muscle regeneration in aged mice. Although the number of satellite cells, muscle stem cells, decreases with age, the remaining satellite cells maintain the myogenic capacity equivalent to young mice. Transplantation of young green fluorescent protein (GFP)-Tg mice-derived satellite cells into young and aged mice revealed that age-related deterioration of the muscle environment contributes to the decline in regenerative capacity of satellite cells. Thus, extrinsic changes rather than intrinsic changes in satellite cells appear to be a major determinant of inefficient muscle regeneration with age. Comprehensive protein expression analysis identified a decrease in insulin-like growth factor-II (IGF-II) level in regenerating muscle of aged mice. We found that pro- and big-IGF-II but not mature IGF-II specifically express during muscle regeneration and the expressions are not only delayed but also decreased in absolute quantity with age. Supplementation of pro-IGF-II in aged mice ameliorated the inefficient regenerative response by promoting proliferation of satellite cells, angiogenesis, and suppressing adipogenic differentiation of platelet derived growth factor receptor (PDGFR)α(+) mesenchymal progenitors. We further revealed that pro-IGF-II but not mature IGF-II specifically inhibits the pathological adipogenesis of PDGFRα(+) cells. Together, these results uncovered a distinctive pro-IGF-II-mediated self-reinforcement mechanism of muscle regeneration and suggest that supplementation of pro-IGF-II could be one of the most effective therapeutic approaches for muscle injury in elderly people.