Language：English   Publishing type：Research paper (scientific journal)  

Skeletal muscles exert remarkable regenerative or adaptive capacities in response to injuries or mechanical loads. However, the cellular networks underlying muscle adaptation are poorly understood compared to those underlying muscle regeneration. We employed single-cell RNA sequencing to investigate the gene expression patterns and cellular networks activated in overloaded muscles and compared these results with those observed in regenerating muscles. The cellular composition of the 4-day overloaded muscle, when macrophage infiltration peaked, closely resembled that of the 10-day regenerating muscle. In addition to the mesenchymal progenitor-muscle satellite cell (MuSC) axis, interactome analyses or targeted depletion experiments revealed communications between mesenchymal progenitors-macrophages and macrophages-MuSCs. Furthermore, granulin, a macrophage-derived factor, inhibited MuSC differentiation, and Granulin-knockout mice exhibited blunted muscle hypertrophy due to the premature differentiation of overloaded MuSCs. These findings reveal the critical role of granulin through the relayed communications of mesenchymal progenitors, macrophages, and MuSCs in facilitating efficient muscle hypertrophy.

DOI： 10.1016/j.celrep.2024.114052

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1016/j.bbrep.2023.101478

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41574-023-00831-6

Language：English   Publishing type：Research paper (scientific journal)  

Skeletal muscle is mainly composed of multinucleated cells called myofibers, and has excellent regenerative and adaptive abilities. These abilities are granted by muscle satellite cells (MuSCs), which are anatomically defined cells located between myofibers and basal lamina. In addition to myofibers and MuSCs, skeletal muscle contains several types of cells located in interstitial areas, such as mesenchymal progenitors. These cells are positive for platelet-derived growth factor receptor alpha and are called fibro/adipogenic progenitors (FAPs) or mesenchymal stromal cells. Although mesenchymal progenitors were originally identified as the causative cells of ectopic fat accumulation in skeletal muscles, recent studies have shed light on their beneficial roles in homeostasis, regeneration, and hypertrophy. Furthermore, the heterogeneity of mesenchymal progenitors is of great interest in understanding skeletal muscle development, homeostasis, regeneration, aging, and diseases. In this concise review, we summarize recent findings on the physiological roles of mesenchymal progenitors and their heterogeneity and discuss the remaining critical concerns.

DOI： 10.1093/stmcls/sxad023

Language：English   Publishing type：Research paper (scientific journal)  

Muscle regeneration requires the coordination of muscle stem cells, mesenchymal fibro-adipogenic progenitors (FAPs), and macrophages. How macrophages regulate the paracrine secretion of FAPs during the recovery process remains elusive. Herein, we systemically investigated the communication between CD206+ M2-like macrophages and FAPs during the recovery process using a transgenic mouse model. Depletion of CD206+ M2-like macrophages or deletion of CD206+ M2-like macrophages-specific TGF-β1 gene induces myogenesis and muscle regeneration. We show that depletion of CD206+ M2-like macrophages activates FAPs and activated FAPs secrete follistatin, a promyogenic factor, thereby boosting the recovery process. Conversely, deletion of the FAP-specific follistatin gene results in impaired muscle stem cell function, enhanced fibrosis, and delayed muscle regeneration. Mechanistically, CD206+ M2-like macrophages inhibit the secretion of FAP-derived follistatin via TGF-β signaling. Here we show that CD206+ M2-like macrophages constitute a microenvironment for FAPs and may regulate the myogenic potential of muscle stem/satellite cells.

DOI： 10.1038/s41467-022-34191-y

Language：English   Publishing type：Research paper (scientific journal)  

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).

DOI： 10.1016/j.xpro.2022.101593

Language：English   Publishing type：Research paper (scientific journal)  

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).

DOI： 10.1016/j.xpro.2022.101307

Language：Others   Publishing type：Research paper (scientific journal)  

<title>Abstract</title><sec>
<title>Background</title>
Mesenchymal stromal cells (MSCs) function as supportive cells on skeletal muscle homeostasis through several secretory factors including type 6 collagen (COL6). Several mutations of <italic>COL6A1</italic>, <italic>2</italic>, and <italic>3</italic> genes cause Ullrich congenital muscular dystrophy (UCMD). Skeletal muscle regeneration deficiency has been reported as a characteristic phenotype in muscle biopsy samples of human UCMD patients and UCMD model mice. However, little is known about the COL6-dependent mechanism for the occurrence and progression of the deficiency. The purpose of this study was to clarify the pathological mechanism of UCMD by supplementing COL6 through cell transplantation.


</sec><sec>
<title>Methods</title>
To test whether COL6 supplementation has a therapeutic effect for UCMD, in vivo and in vitro experiments were conducted using four types of MSCs: (1) healthy donors derived-primary MSCs (pMSCs), (2) MSCs derived from healthy donor induced pluripotent stem cell (iMSCs), (3) COL6-knockout iMSCs (COL6KO-iMSCs), and (4) UCMD patient-derived iMSCs (UCMD-iMSCs).


</sec><sec>
<title>Results</title>
All four MSC types could engraft for at least 12 weeks when transplanted into the tibialis anterior muscles of immunodeficient UCMD model (<italic>Col6a1</italic>KO) mice. COL6 protein was restored by the MSC transplantation if the MSCs were not COL6-deficient (types 1 and 2). Moreover, muscle regeneration and maturation in <italic>Col6a1</italic>KO mice were promoted with the transplantation of the COL6-producing MSCs only in the region supplemented with COL6. Skeletal muscle satellite cells derived from UCMD model mice (<italic>Col6a1</italic>KO-MuSCs) co-cultured with type 1 or 2 MSCs showed improved proliferation, differentiation, and maturation, whereas those co-cultured with type 3 or 4 MSCs did not.


</sec><sec>
<title>Conclusions</title>
These findings indicate that COL6 supplementation improves muscle regeneration and maturation in UCMD model mice.


</sec>

DOI： 10.1186/s13287-021-02514-3

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.3390/ijms222212356

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.3390/ijms221910246

Language：English   Publishing type：Research paper (scientific journal)  

Muscle stem cells, also called muscle satellite cells (MuSCs), are responsible for skeletal muscle regeneration and are sustained in an undifferentiated and quiescent state under steady conditions. The calcitonin receptor (CalcR)-protein kinase A (PKA)-Yes-associated protein 1 (Yap1) axis is one pathway that maintains quiescence in MuSCs. Although CalcR signaling in MuSCs has been identified, the critical CalcR signaling targets are incompletely understood. Here, we show the relevance between the ectopic expression of delta-like non-canonical Notch ligand 1 (Dlk1) and the impaired quiescent state in CalcR-conditional knockout (cKO) MuSCs. Dlk1 expression was rarely detected in both quiescent and proliferating MuSCs in control mice, whereas Dlk1 expression was remarkably increased in CalcR-cKO MuSCs at both the mRNA and protein levels. It is noteworthy that all Ki67+ non-quiescent CalcR-cKO MuSCs express Dlk1, and non-quiescent CalcR-cKO MuSCs are enriched in the Dlk1+ fraction by cell sorting. Using mutant mice, we demonstrated that PKA-activation or Yap1-depletion suppressed Dlk1 expression in CalcR-cKO MuSCs, which suggests that the CalcR-PKA-Yap1 axis inhibits the expression of Dlk1 in quiescent MuSCs. Moreover, the loss of Dlk1 rescued the quiescent state in CalcR-cKO MuSCs, which indicates that the ectopic expression of Dlk1 disturbs quiescence in CalcR-cKO. Collectively, our results suggest that ectopically expressed Dlk1 is responsible for the impaired quiescence in CalcR-cKO MuSCs.

DOI： 10.1002/stem.3312

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: The infiltration of fat tissue into skeletal muscle, a condition referred to as muscle fatty infiltration or fatty degeneration, is regarded as an irreversible event that significantly compromises the motor function of skeletal muscle. PURPOSE: To investigate the effect of retinoic acid receptor (RAR) agonists in suppressing the adipogenic differentiation of fibroadipogenic progenitors (FAPs) in vitro and fatty infiltration after rotator cuff tear in mice. STUDY DESIGN: Controlled laboratory study. METHODS: FAPs isolated from mouse skeletal muscle were cultured in adipogenic differentiation medium in the presence or absence of an RAR agonist. At the end of cell culture, adipogenic differentiation was evaluated by gene expression analysis and oil red O staining. A mouse model of fatty infiltration-which includes the resection of the rotator cuff, removal of the humeral head, and denervation the supraspinatus muscle-was used to induce fatty infiltration in the supraspinatus muscle. The mice were orally or intramuscularly administered with an RAR agonist after the surgery. Muscle fatty infiltration was evaluated by histology and gene expression analysis. RESULTS: RAR agonists effectively inhibited the adipogenic differentiation of FAPs in vitro. Oral and intramuscular administration of RAR agonists suppressed the development of muscle fatty infiltration in the mice after rotator cuff tear. In accordance, we found a significant decrease in the number of intramuscular fat cells and suppressed expression in adipogenic markers. RAR agonists also increased the expression of the transcripts for collagens; however, an accumulation of collagenous tissues was not histologically evident in the present model. CONCLUSION: Muscle fatty infiltration can be alleviated by RAR agonists through suppressing the adipogenic differentiation of FAPs. The results also suggest that RAR agonists are potential therapeutic agents for treating patients who are at risk of developing muscle fatty infiltration. The consequence of the increased expression of collagen transcripts by RAR agonists needs to be clarified. CLINICAL RELEVANCE: RAR agonists can be used to prevent the development of muscle fatty infiltration after rotator cuff tear. Nevertheless, further studies are mandatory in a large animal model to examine the safety and efficacy of intramuscular injection of RAR agonists.

DOI： 10.1177/0363546520984122

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1038/s41419-020-03353-5

Language：English   Publishing type：Research paper (scientific journal)  

Heterotopic ossification (HO) is a pathological condition in which ectopic bone forms within soft tissues such as skeletal muscle. Human platelet-derived growth factor receptor α positive (PDGFRα+) cells, which were proved to be the original cells of HO were incubated in osteogenic differentiation medium with Food and Drug Administration-approved compounds. Alkaline phosphatase activity was measured as a screening to inhibit osteogenic differentiation. For the compounds which inhibited osteogenic differentiation of PDGFRα+ cells, we examined dose dependency of its effect using alizarin red S staining and its cell toxicity using WST-8. In addition, regulation of bone morphogenetic proteins (BMP)-Smad signaling which is the major signal of osteogenic differentiation was investigated by Western blotting to elucidate the mechanism of osteogenesis inhibitory effect by the compound. In vivo experiment, complete transverse incision of Achilles tendons in mice was made and mice were fed the compound by mixing with drinking water after operation. Ten weeks after operation, we assessed and quantified HO by micro-computed tomography scan. Intriguingly, we discovered desloratadine inhibited osteogenic differentiation of PDGFRα+ cells using the drug repositioning method. Desloratadine inhibited osteogenic differentiation of the cells dose dependently without cell toxicity. Desloratadine suppressed phosphorylation of Smad1/5/8 induced by BMP2 in PDGFRα+ cells. In Achilles tenotomy mice model, desloratadine treatment significantly inhibited ectopic bone formation compared with control. In conclusion, we discovered desloratadine inhibited osteogenic differentiation using human PDGFRα+ cells and proved its efficacy using Achilles tenotomy ectopic bone formation model in vivo. Our study paved the way to inhibit HO in early clinical use because of its guaranteed safety.

DOI： 10.1002/jor.24625

Event date： 2023.6

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2023.5

Language：English   Presentation type：Symposium, workshop panel (public)  

Country：Japan  

Event date： 2023.2

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan