Hidetoshi Sakurai

Dilated cardiomyopathy (DCM) is a genetically heterogeneous disorder, characterized by ventricular dilatation and impaired systolic function, leading to heart failure and sudden cardiac death. Despite advances in genomic technologies, the genetic cause of DCM remains unidentified in more than half of the cases. Here, we performed an integrative analysis of genomic and transcriptomic data from patient-derived cardiac tissue to identify causative variants in genetically undiagnosed DCM. This approach enabled us to identify a homozygous splice-site variant (c.243+6T>A) in the sarcoglycan gene SGCB , which results in exon 2 skipping. This variant was significantly enriched in patients with DCM compared with the general population, with consistent genotype–phenotype correlations observed across multiple families. Protein-level analysis of cardiac tissue from homozygous individuals revealed loss of β-sarcoglycan, the protein product of SGCB , and destabilization of the sarcoglycan complex. Although SGCB has been previously associated with limb-girdle muscular dystrophy, these homozygous individuals showed no biochemical or clinical signs of skeletal muscle involvement, indicating an absence of myopathy. Compared with variant-negative patients with DCM, homozygous individuals also had a higher risk of early-onset adverse cardiac events. Together, these findings identify c.243+6T>A in SGCB as a cause of isolated DCM associated with unfavorable clinical outcomes.

Oculopharyngodistal myopathy (OPDM) is characterized by ptosis, ophthalmoparesis, dysphagia, and distal weakness. Myopathological features include rimmed v

Facioscapulohumeral muscular dystrophy (FSHD) is a rare genetic disease with an estimated prevalence of no more than 1 in 8000; however, it is among the most common myopathies affecting global populations. This condition is classically categorised into two genetic types, FSHD1 (MIM: 158900) and FSHD2 (MIM: 158901), which, although have different genetic causes, are phenotypically indistinguishable, manifesting as progressive muscle weakness primarily affecting the face and periscapular muscles, as well as other muscle groups in later stages. The intense efforts of clinical and basic studies to understand this disease have revealed the critical necessity for disease manifestation: ectopic activation of the embryogenic and germline gene DUX4 (double homeobox 4, MIM: 606009) in skeletal muscles and the genetic and epigenetic backgrounds allowing DUX4 expression. Thus, the potential target therapies of FSHD include silencing DUX4 transcription or blocking its translation. Although the central role of DUX4 in FSHD pathology has almost reached a consensus, the mechanism of its activation remains largely unclear. Notably, the clinical dissection of genotype–epigenotype-phenotype observations, including non-penetrant and asymptomatic carriers of permissive genetic backgrounds, highlights the yet unsolved clinical diversity with potential additional layers of DUX4 regulation or other disease-modifying factors. This review provides an overview of essential findings with potential implications for further understanding the mechanisms underlying diverse clinical cases of FSHD and endogenous DUX4 activation in FSHD pathology.

Patient-derived disease-specific induced pluripotent stem cells (iPSCs) have opened the door to recreating pathological conditions in vitro using differentiation into diseased cells corresponding to each target tissue. To investigate muscular diseases, we have established a myogenic differentiation protocol mediated by inducible MYOD1 expression that drives human iPSCs into myocytes. This highly reproducible differentiation protocol yields a homogenous skeletal muscle cell population, reaching efficiencies as high as 90%. Such high efficiency enables us to evaluate the efficacy of exon skipping in disease-specific myocytes. These disease-specific iPSC-derived myocytes can be applied not only for the validation of the therapeutic efficacy of specific antisense oligonucleotide, but also for the screening of exon skipping chemicals combined with the multi-well differentiation system. Although we previously presented the protocol in 2018, we have updated the myogenic differentiation protocol to the current version.

The reproducibility and robustness of many directed differentiation protocols from human induced pluripotent stem cells (hiPSCs) remain low, and the long differentiation induction period significantly limits protocol optimization. To address this, we developed an early and non-destructive prediction system for the differentiation induction efficiency of hiPSCs using bioimage informatics. We employed a directed differentiation protocol for muscle stem cells (MuSCs), a promising cell source for the regenerative therapy of muscular dystrophy. Biological analyses suggested that days 14–38 are positive for forecasting the induction efficiency on day 82. Therefore, we conducted six independent experiments, inducing MuSC differentiation in a total of 34 wells, and captured a total of 5,712 phase contrast cell images between days 14 and 38. We selected Fast Fourier transform (FFT) as the feature extraction method and confirmed that it captures the characteristics of cells during differentiation. By classifying images on each day using extracted features and machine learning, we found that samples with high and low induction efficiency could be predicted at approximately 50 days before the end of induction. This system is expected to contribute to regenerative therapy through effective protocol optimization.

This work established a human induced pluripotent stem (iPS) cell-based model for statin-induced myopathy (SIM) and demonstrated that blocking the mevalonate pathway disrupts the balance between protein synthesis and degradation, leading to myopathy. Furthermore, the present study showed that pharmacological inhibition of the transcription factor FOXO1 prevents SIM in human iPS cell-derived myocytes, suggesting that FOXO1 is a key mediator of SIM and a potential target for its prevention.

Skeletal muscle is a highly regenerative tissue in the vertebrate body. This skeletal muscle regenerative capacity is mediated by muscle satellite cells (MuSCs), a stem cell population within skeletal muscle [1,2]. While MuSCs display some non-muscle differentiation potential, such as adipocytes, osteocytes, and fibroblasts in vitro or in some diseased environments [3], their primary differentiation fate in normal muscle regeneration is skeletal muscle cells. MuSCs usually display a mitotically quiescent state and reside on the myofibers. However, during muscle regeneration, MuSCs are activated, exit the quiescent state, and give rise to myogenic precursor cells. After several rounds of cell proliferation, these myogenic precursor cells exit the cell cycle and fuse with each other to contribute to the newly formed myofibers. MuSC-derived myogenic precursor cells or myoblasts expanded ex vivo can be utilized for myoblast-mediated cell therapy for skeletal muscle diseases, including Duchenne muscular dystrophy (DMD) when injected into damaged myofibers via integration into myofibers [4][5][6][7]. However, there are significant limitations to treatment in humans. The number of MuSCs and myoblasts available from human biopsies is limited. In addition, low cell viability and low contribution of transplanted cells have hindered their practical use in patients [4,6,7].Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells originally derived from adult somatic cells. These cells are genetically initialized to an embryonic stem cell (ESC)-like pluripotent state by expressing genes and factors that are essential for maintaining the characteristic properties of pluripotent stem cells. hiPSCs can be generated from human somatic cells from various tissues [8][9][10]. They are capable of self-renewal and can give rise to any cell type, including skeletal muscle cells and MuSCs, both in vitro and in vivo. Therefore, hiPSCderived myogenic cells are a source of myogenic precursor cells that repair injured or diseased or aged skeletal muscle in patients after transplantation [11]. In addition, the ability to capture the genetic diversity of different inherent muscle diseases and age-related muscle atrophy in an accessible culture system makes hiPSCs an attractive model source for generating myogenic cells for drug screening [11]. This research topic aims to highlight the progress of research on disease modeling using iPSCs and iPSC-based cell therapy in the field of skeletal muscle diseases. In particular, collecting technical tips for successful induction protocols of iPSC-derived skeletal muscle [12][13][14] suitable for regenerative medicine and in vitro iPSC-based disease modeling is critical for understanding the molecular and cellular mechanisms of disease pathogenesis as well as drug screening.Five primary research or review manuscripts were published on this research topic. In the first manuscript, Hamer and Rossi summarized the protocols to improve the generation, purification, and maturation of iPSC-derived myogenic cells for novel therapeutic applications [Hamer and Rossi]. Fibro/adipogenic progenitors (FAPs) residing in skeletal muscle play essential roles in skeletal muscle homeostasis and regeneration via secreted molecules and remodeling of extracellular matrix [15]. In diseased environments, FAPs give rise to adipogenic, osteogenic, or fibroblastic cells that significantly modulate normal muscle function. The second manuscript, by Zhao and Ikeya, summarized the current progress of the developmental origins and functions of FAPs as stromal cells of the muscle connective tissue (MCT) and the FAP-mediated heterotopic ossification causing the rare genetic disorder fibrodysplasia ossificans progressive (FOP) [16;Zhao and Ikeya]. In addition, this manuscript provides novel insights into the heterotopic ossification from patient-derived iPSCs. The third manuscript, by Xie et al., uncovered the molecular mechanism by which leukemia inhibitory factor (LIF) promotes the self-renewal and proliferation of MuSCs [Xie et al.]. Treatment with LIF also enhances the engraftment capability of MuSCs following injection into the skeletal muscle of DMD model mdx mice, suggesting the beneficial effect of LIF for hiPSC-derived MuSC transplantation therapy. The mutations of superoxide dismutase 1 (SOD1) gene have been known as a genetic cause of familial amyotrophic lateral sclerosis (ALS), a progressive neuromuscular disease without cure [17]. In the fourth manuscript, Couturier et al. reported that hiPSC-derived myogenic cells carrying the SOD1 D90A mutation display a reduction of several myogenic markers, including modulation of nuctic acetylcholine receptor (nAChR) cluster expression and the function, causing the morphological alteration of sarcomeres in myotubes [Couturier et al.]. Therefore, the authors introduced the novel hiPSC-derived neuromuscular model for ALS [18,19]. As humans age, changes in the composition of blood circulating factors are thought to contribute to the decline in muscle mass and strength. In the fifth manuscript, Tey et al. reported that supplementation with serum from aged rats significantly reduced cell proliferation, myotube formation, and myofiber maturation, significantly increased cell death, and induced transcriptomic changes in both ESC-and iPSCderived myogenic cells, providing therapeutic applications to prevent age-related muscle weakness [Tey et al.]. This series of five articles highlights the high quality of manuscripts published in the research topic of the Stem Cell Research section of Frontiers in Cell and Developmental Biology and reveals a research field that is moving forward at a remarkable pace.

正常ヒト人工多能性幹細胞（induced pluripotent stem cells，以下iPS細胞と略記）を用いた筋ジストロフィー（muscular dystrophy，以下MDと略記）への細胞治療を開発している．まず，正常ヒトiPS細胞から骨格筋幹細胞を分化誘導し，デュシェンヌ型MDモデルマウスへの移植により，ジストロフィン陽性線維の再生を証明した．また運動機能への効果については，生体内での最大収縮力よりも主に筋肉疲労耐性を改善することを明らかにした．次に正常ヒトiPS細胞から間葉系間質細胞（mesenchymal stromal cell，以下MSCと略記）を誘導する分化法も開発し，ウルリッヒ型MDモデルマウスに移植すると，6型コラーゲンの補充により，筋再生が促進された．興味深いことに，骨髄由来MSCや脂肪由来MSCでは，移植によって6型コラーゲンを補充するが治療効果に乏しく線維化を誘発した．

Ullrich congenital muscular dystrophy (UCMD) is caused by a deficiency in type 6 collagen (COL6) due to mutations in COL6A1, COL6A2, or COL6A3. COL6 deficiency alters the extracellular matrix structure and biomechanical properties, leading to mitochondrial defects and impaired muscle regeneration. Therefore, mesenchymal stromal cells (MSCs) that secrete COL6 have attracted attention as potential therapeutic targets. Various tissue-derived MSCs exert therapeutic effects in various diseases. However, no reports have compared the effects of MSCs of different origins on UCMD pathology. To evaluate which MSC population has the highest therapeutic efficacy for UCMD, in vivo (transplantation of MSCs to Col6a1-KO/NSG mice) and in vitro experiments (muscle stem cell [MuSCs] co-culture with MSCs) were conducted using adipose tissue-derived MSCs, bone marrow-derived MSCs, and xeno-free-induced iPSC-derived MSCs (XF-iMSCs). In transplantation experiments on Col6a1-KO/NSG mice, the group transplanted with XF-iMSCs showed significantly enhanced muscle fiber regeneration compared to the other groups 1 week after transplantation. At 12 weeks after transplantation, only the XF-iMSCs transplantation group showed a significantly larger muscle fiber diameter than the other groups without inducing fibrosis, which was observed in the other transplantation groups. Similarly, in co-culture experiments, XF-iMSCs were found to more effectively promote the fusion and differentiation of MuSCs derived from Col6a1-KO/NSG mice than the other primary MSCs investigated in this study. Additionally, in vitro knockdown and supplementation experiments suggested that the IGF2 secreted by XF-iMSCs promoted MuSC differentiation. XF-iMSCs are promising candidates for promoting muscle regeneration while avoiding fibrosis, offering a safer and more effective therapeutic approach for UCMD than other potential therapies.

Duchenne muscular dystrophy (DMD) is an incurable neuromuscular disease leading to progressive skeletal muscle weakness and fatigue. Cell transplantation in murine models has shown promise in supplementing the lack of the dystrophin protein in DMD muscles. However, the establishment of novel, long-term, relevant methods is needed to assess its efficiency on the DMD motor function. By applying newly developed methods, this study aimed to evaluate the functional and molecular effects of cell therapy-mediated dystrophin supplementation on DMD muscles. Dystrophin was supplemented in the gastrocnemius of a 5-week-old immunodeficient DMD mouse model (Dmd-null/NSG) by intramuscular xenotransplantation of healthy human immortalized myoblasts (Hu5/KD3). A long-term time-course comparative study was conducted between wild-type, untreated DMD, and dystrophin supplemented-DMD mouse muscle functions and histology. A novel GO-ATeam2 transgenic DMD mouse model was also generated to assess in vivo real-time ATP levels in gastrocnemius muscles during repeated contractions. We found that 10.6% dystrophin supplementation in DMD muscles was sufficient to prevent low values of gastrocnemius maximal isometric contraction torque (MCT) at rest, while muscle fatigue tolerance, assessed by MCT decline after treadmill running, was fully ameliorated in 21-week-old transplanted mice. None of the dystrophin-supplemented fibers were positive for muscle damage markers after treadmill running, with 85.4% demonstrating the utilization of oxidative metabolism. Furthermore, ATP levels in response to repeated muscle contractions tended to improve, and mitochondrial activity was significantly enhanced in dystrophin supplemented-fibers. Cell therapy-mediated dystrophin supplementation efficiently improved DMD muscle functions, as evaluated using newly developed evaluation methods. The enhanced muscle fatigue tolerance in 21-week-old mice was associated with the preferential regeneration of damage-resistant and oxidative fibers, highlighting increased mitochondrial activity, after cell transplantation. These findings significantly contribute to a more in-depth understanding of DMD pathogenesis.