Satoru Masuda

Molecular Signature of Quiescent Satellite Cells in Adult Skeletal Muscle

Skeletal muscle satellite cells play key roles in postnatal muscle growth and regeneration. To study molecular regulation of satellite cells, we directly prepared satellite cells from 8‐ to 12‐week‐old C57BL/6 mice and performed genome‐wide gene expression analysis. Compared with activated/cycling satellite cells, 507 genes were highly upregulated in quiescent satellite cells. These included negative regulators of cell cycle and myogenic inhibitors. Gene set enrichment analysis revealed that quiescent satellite cells preferentially express the genes involved in cell‐cell adhesion, regulation of cell growth, formation of extracellular matrix, copper and iron homeostasis, and lipid transportation. Furthermore, reverse transcription‐polymerase chain reaction on differentially expressed genes confirmed that calcitonin receptor (CTR) was exclusively expressed in dormant satellite cells but not in activated satellite cells. In addition, CTR mRNA is hardly detected in nonmyogenic cells. Therefore, we next examined the expression of CTR in vivo. CTR was specifically expressed on quiescent satellite cells, but the expression was not found on activated/proliferating satellite cells during muscle regeneration. CTR‐positive cells reappeared at the rim of regenerating myofibers in later stages of muscle regeneration. Calcitonin stimulation delayed the activation of quiescent satellite cells. Our data provide roles of CTR in quiescent satellite cells and a solid scaffold to further dissect molecular regulation of satellite cells.

Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection a role for osteopontin

To examine the roles of cytokines in muscle regeneration, we injected cardiotoxin into mouse tibialis anterior muscle and examined the expression profiles of cytokines and related genes in the regeneration process. Expression of 40, 64, and 7 genes among 522 genes spotted on a cytokine expression array were increased more than fivefold at 48 hours, 96 hours, and 7 days after toxin injection, respectively, when compared with those of the control muscle. Especially the levels of mRNA for chemokines and chemokine receptors, many of which are potent regulators of macrophages, were highly elevated 48 hours after injury. The expression of osteopontin (OPN), a versatile regulator of inflammation and tissue repair, was up-regulated more than 118-fold in regenerating muscle at 48 hours after injury. Northern blotting confirmed that the expression of OPN was highest at 48 hours after cardiotoxin injection and declined sharply thereafter. Immunohistochemistry showed that OPN was detected both in the cytoplasm of macrophages and in necrotic muscle infiltrated with macrophages. Our studies suggest OPN may serve as an adhesion molecule that promotes macrophage binding to necrotic fibers and may be an important mediator in the early phase of muscle regeneration.

Autologous Transplantation of SM/C-2.6+ Satellite Cells Transduced with Micro-dystrophin CS1 cDNA by Lentiviral Vector into mdx Mice

Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations in the dystrophin gene. Transplantation of autologous myogenic cells genetically corrected ex vivo is a possible treatment for this disorder. In order to test the regenerative efficiency of freshly isolated satellite cells, we purified quiescent satellite cells from limb muscles of 8-12-week-old green fluorescent protein-transgenic (GFP-Tg) mice using SM/C-2.6 (a recently developed monoclonal antibody) and flow cytometry. Freshly isolated satellite cells were shown to participate in muscle regeneration more efficiently than satellite cell-derived myoblasts passaged in vitro do, when transplanted into tibialis anterior (TA) muscles of 8-12-week-old cardiotoxin-injected C57BL/6 mice and 5-week-old dystrophin-deficient mdx mice, and analyzed at 4 weeks after injection. Importantly, expansion of freshly isolated satellite cells in vitro without passaging had no detrimental effects on their regenerative capacity. Therefore we directly isolated satellite cells from 5-week-old mdx mice using SM/C-2.6 antibody and cultured them with lentiviral vectors expressing micro-dystrophin CS1. The transduced cells were injected into TA muscles of 5-week-old mdx mice. At 4 weeks after transplantation, the grafted cells efficiently contributed to regeneration of mdx dystrophic muscles and expressed micro-dystrophin at the sarcolemma. These results suggest that there is potential for lentiviral vector-mediated ex vivo gene therapy for DMD.

Slow-dividing satellite cells retain long-term self-renewal ability in adult muscle

Satellite cells are muscle stem cells that have important roles in postnatal muscle growth and adult muscle regeneration. Although fast- and slow-dividing populations in activated satellite cells have been observed, the functional differences between them remain unclear. Here we elucidated the relationship between proliferation behaviour and satellite cell function. To assess the frequency of cell division, satellite cells isolated from mouse EDL muscle were labelled with the fluorescent dye PKH26, stimulated to proliferate and then sorted by FACS. The vast majority of activated satellite cells were PKH26low fast-dividing cells, whereas PKH26high slow-dividing cells were observed as a minority population. The fast-dividing cells generated a higher number of differentiated and self-renewed cells compared with the slow-dividing cells. However, cells derived from the slow-dividing population formed secondary myogenic colonies when passaged, whereas those from the fast-dividing population rapidly underwent myogenic differentiation without producing self-renewing cells after a few rounds of cell division. Furthermore, slow-dividing cells transplanted into injured muscle extensively contributed to muscle regeneration in vivo. Id1, a HLH protein, was expressed by all activated satellite cells, but the expression level varied within the slow-dividing cell population. We show that the slow-dividing cells retaining long-term self-renewal ability are restricted to an undifferentiated population that express high levels of Id1 protein (PKH26highId1high population). Finally, genome-wide gene expression analysis described the molecular characteristics of the PKH26highId1high population. Taken together, our results indicate that undifferentiated slow-dividing satellite cells retain stemness for generating progeny capable of long-term self-renewal, and so might be essential for muscle homeostasis throughout life.

Six family genes control the proliferation and differentiation of muscle satellite cells.

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4(+/-)Six5(-/-) mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.

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

Summary 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.

Participation of bone marrow-derived cells in fibrotic changes in denervated skeletal muscle.

In denervated skeletal muscle, mononuclear interstitial cells accumulate in the perisynaptic regions before fibrotic change occurs. These cells are currently considered to be fibroblasts that originate from muscle tissue. However, when we denervated hind limbs of GFP-bone marrow chimeric mice by excising the sciatic nerve unilaterally, many bone marrow-derived cells (BM-DCs) infiltrated the interstitial spaces and accumulated in the perisynaptic regions, peaking 14 days after denervation. They accounted for nearly one-half of the increase in mononuclear interstitial cells. Although BM-DCs did not incorporate into satellite cells, immunohistochemical and FACS analyses revealed that BM-DCs were both CD45 and CD11b positive, indicating that they were of macrophage/monocyte lineage. BrdU staining showed inactive proliferation of BM-DCs. Reverse transcriptase-polymerase chain reaction of mononuclear cells isolated by FACS revealed that BM-DCs did not express type I collagen or tenascin-C; however, they did express transforming growth factor-beta1, suggesting that they regulate the fibrotic process. In contrast, muscle tissue-derived interstitial cells expressed type I collagen and tenascin-C, suggesting that these populations were the final effectors of fibrosis. These findings identify elementary targets that may regulate the migration, homing, differentiation, and function of BM-DCs, leading to amelioration of the excessive fibrosis of denervated skeletal muscle.

Systemic administration of the antisense oligonucleotide NS-065/NCNP-01 for skipping of exon 53 in patients with Duchenne muscular dystrophy

NS-065/NCNP-01, an antisense oligonucleotide that enables exon 53 skipping in the dystrophin gene, showed a favorable safety profile and promising pharmacokinetics in 10 patients with Duchenne muscular dystrophy. Exon skipping to treat DMD Duchenne muscular dystrophy (DMD) is an inherited muscle disorder that is ultimately fatal. A deficiency in normal dystrophin, a structural protein that is indispensable for muscle cell function, causes severe damage to muscle cells. This dystrophin deficiency is due to mutations in the gene encoding dystrophin. Komaki et al. have now developed a morpholino antisense oligonucleotide, NS-065/NCNP-01, designed to recover dystrophin function and halt muscle damage by skipping exon 53 in the dystrophin gene. These authors report the results of a phase 1 clinical trial of NS-065/NCNP-01 conducted in 10 patients with DMD. The drug showed a favorable safety profile and pharmacokinetics, and the authors demonstrated that it effectively skipped exon 53 in the dystrophin gene, suggesting that a phase 2 trial of the drug is warranted. Duchenne muscular dystrophy (DMD) is a lethal hereditary muscle disease caused by mutations in the gene encoding the muscle protein dystrophin. These mutations result in a shift in the open reading frame leading to loss of the dystrophin protein. Antisense oligonucleotides (ASOs) that induce exon skipping correct this frame shift during pre-mRNA splicing and partially restore dystrophin expression in mouse and dog models. We conducted a phase 1, open-label, dose-escalation clinical trial to determine the safety, pharmacokinetics, and activity of NS-065/NCNP-01, a morpholino ASO that enables skipping of exon 53. Ten patients with DMD (6 to 16 years old), carrying mutations in the dystrophin gene whose reading frame would be restored by exon 53 skipping, were administered NS-065/NCNP-01 at doses of 1.25, 5, or 20 mg/kg weekly for 12 weeks. The primary endpoint was safety; the secondary endpoints were pharmacokinetics and successful exon skipping. No severe adverse drug reactions were observed, and no treatment discontinuation occurred. Muscle biopsy samples were taken before and after treatment and compared by reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence, and Western blotting to assess the amount of exon 53 skipping and dystrophin expression. NS-065/NCNP-01 induced exon 53 skipping in dystrophin-encoding mRNA in a dose-dependent manner and increased the dystrophin/spectrin ratio in 7 of 10 patients. Furthermore, the amount of exon skipping correlated with the maximum drug concentration in plasma (Cmax) and the area under the concentration-time curve in plasma (AUC0-t). These results indicate that NS-065/NCNP-01 has a favorable safety profile and promising pharmacokinetics warranting further study in a phase 2 clinical trial.

Induction of Pluripotent Stem Cells from a Manifesting Carrier of Duchenne Muscular Dystrophy and Characterization of Their X-Inactivation Status

Three to eight percent of female carriers of Duchenne muscular dystrophy (DMD) develop dystrophic symptoms ranging from mild muscle weakness to a rapidly progressive DMD-like muscular dystrophy due to skewed inactivation of X chromosomes during early development. Here, we generated human induced pluripotent stem cells (hiPSCs) from a manifesting female carrier using retroviral or Sendai viral (SeV) vectors and determined their X-inactivation status. Although manifesting carrier-derived iPS cells showed normal expression of human embryonic stem cell markers and formed well-differentiated teratomas in vivo, many hiPS clones showed bi-allelic expression of the androgen receptor (AR) gene and loss of X-inactivation-specific transcript and trimethyl-histone H3 (Lys27) signals on X chromosomes, suggesting that both X chromosomes of the hiPS cells are in an active state. Importantly, normal dystrophin was expressed in multinucleated myotubes differentiated from a manifesting carrier of DMD-hiPS cells with XaXa pattern. AR transcripts were also equally transcribed from both alleles in induced myotubes. Our results indicated that the inactivated X chromosome in the patients fibroblasts was activated during reprogramming, and XCI occurred randomly during differentiation.

Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.