Targeting specific DNA G-quadruplexes with CRISPR-guided G-quadruplex-binding proteins and ligands.

Despite the demonstrated importance of DNA G-quadruplexes (G4s) in health and disease, technologies to readily manipulate specific G4 folding for functional analysis and therapeutic purposes are lacking. Here we employ G4-stabilizing protein/ligand in conjunction with CRISPR to selectively facilitate single or multiple targeted G4 folding within specific genomic loci. We demonstrate that fusion of nucleolin with a catalytically inactive Cas9 can specifically stabilize G4s in the promoter of oncogene MYC and muscle-associated gene Itga7 as well as telomere G4s, leading to cell proliferation arrest, inhibition of myoblast differentiation and cell senescence, respectively. Furthermore, CRISPR can confer intra-G4 selectivity to G4-binding compounds pyridodicarboxamide and pyridostatin. Compared with traditional G4 ligands, CRISPR-guided biotin-conjugated pyridodicarboxamide enables a more precise investigation into the biological functionality of de novo G4s. Our study provides insights that will enhance understanding of G4 functions and therapeutic interventions.

Inhibition of skeletal muscle differentiation by calciprotein particles in human primary myoblasts.

Sarcopenia is a common complication of chronic kidney disease (CKD) and has a detrimental effect on prognosis. Previous studies have explored the role of secondary calciprotein particles (CPP2) in determining the progression of complications and poor outcomes in patients with CKD. However, no study has demonstrated that CPP2 impairs skeletal myogenesis. Our study revealed that CPP2 exposure inhibits skeletal myogenesis by suppressing myotube formation and expression of skeletal muscle-specific myosin heavy chain and actin in human primary myoblasts. Moreover, CPP2 exposure altered the expression patterns of lineage-determinative transcription factors responsible for regulating myotube differentiation marker genes. This study first demonstrated that CPP2 interferes with myoblast differentiation and myotube formation in vitro.

Unveiling the role of circRBBP7 in myoblast proliferation and differentiation: A novel regulator of muscle development.

Muscle development is a multistep process regulated by diverse gene networks, and circRNAs are considered novel regulators mediating myogenesis. Here, we systematically analyzed the role and underlying regulatory mechanisms of circRBBP7 in myoblast proliferation and differentiation. Results showed that circRBBP7 has a typical circular structure and encodes a 13 -kDa protein. By performing circRBBP7 overexpression and RNA interference, we found that the function of circRBBP7 was positively correlated with the proliferation and differentiation of myoblasts. Using RNA sequencing, we identified 1633 and 532 differentially expressed genes (DEGs) during myoblast proliferation or differentiation, respectively. The DEGs were found mainly enriched in cell cycle- and skeletal muscle development-related pathways, such as the MDM2/p53 and PI3K-Akt signaling pathways. Further co-IP and IF co-localization analysis revealed that VEGFR-1 is a target of circRBBP7 in myoblasts. qRT-PCR and WB analysis further confirmed the positive correlation between VEGFR-1 and circRBBP7. Moreover, we found that in vivo transfection of circRBBP7 into injured muscle tissues significantly promoted the regeneration and repair of myofibers in mice. Therefore, we speculate that circRBBP7 may affect the activity of MDM2 by targeting VEGFR-1, altering the expression of muscle development-related genes by mediating p53 degradation, and ultimately promoting myoblast development and muscle regeneration. This study provides essential evidence that circRBBP7 can serve as a potential target for myogenesis regulation and a reference for the application of circRBBP7 in cattle genetic breeding and muscle injury treatment.

OTUB1 regulates ferroptosis to inhibit myoblast differentiation into myotubes by deubiquitinating P62.

As the largest organ in the human body, skeletal muscle is essential for breathing support, movement initiation, and maintenance homeostasis. It has been shown that programmed cell death (PCD), which includes autophagy, apoptosis, and necrosis, is essential for the development of skeletal muscle. A novel form of PCD called ferroptosis is still poorly understood in relation to skeletal muscle. In this study, we observed that the activation of ferroptosis significantly impeded the differentiation of C2C12 myoblasts into myotubes and concurrently suppressed the expression of OTUB1, a crucial deubiquitinating enzyme. OTUB1-silenced C2C12 mouse myoblasts were used to investigate the function of OTUB1 in ferroptosis. The results show that OTUB1 knockdown in vitro significantly increased C2C12 ferroptosis and inhibited myogenesis. Interestingly, the induction of ferroptosis resulting from OTUB1 knockdown was concomitant with the activation of autophagy. Furthermore, OTUB1 interacted with the P62 protein and stabilized its expression by deubiquitinating it, thereby inhibiting autophagy-dependent ferroptosis and promoting myogenesis. All of these findings demonstrate the critical role that OTUB1 plays in controlling ferroptosis, and we suggest that focusing on the OTUB1-P62 axis may be a useful tactic in the treatment and prevention of disorders involving the skeletal muscle.

Novel Circular RNA CircSLC2A13 Regulates Chicken Muscle Development by Sponging MiR-34a-3p.

The skeletal muscle is the major muscle tissue in animals, and its production is subject to a complex and strict regulation. The proliferation and differentiation of myoblasts are important factors determining chicken muscle development. Circular RNAs (circRNAs) are endogenous RNAs that are widely present in various tissues of organisms. Recent studies have shown that circRNA plays key roles in the development of skeletal muscles. The solute carrier (SLC) family functions in the transport of metabolites such as amino acids, glucose, nucleotides, and essential nutrients and is widely involved in various basic physiological metabolic processes within the body. In this study, we have cloned a novel chicken circular RNA circSLC2A13 generated from the solute carrier family 2 member 13 gene (SLC2A13). Also, circSLC2A1 was confirmed by sequencing verification, RNase R treatment, and reverse transcription analysis. Currently, our results show that circSLC2A13 promoted the proliferation and differentiation of chicken myoblasts. The double luciferase reporter system revealed that circSLC2A13 regulated the proliferation and differentiation of myoblasts by competitive binding with miR-34a-3p. In addition, results indicated that circSLC2A13 acts as a miR-34a-3p sponge to relieve its inhibitory effect on the target SMAD3 gene. In summary, this study found that chicken circSLC2A13 can bind to miR-34a-3p and weaken its inhibitory effect on the SMAD family member 3 gene (SMAD3), thereby promoting the proliferation and differentiation of myoblasts. This study laid foundations for broiler industry and muscle development research.

Muscle-specific pyruvate kinase isoforms, PKM1 and PKM2, regulate mammalian SWI/SNF proteins and histone 3 phosphorylation during myoblast differentiation.

Pyruvate kinase is a glycolytic enzyme that converts phosphoenolpyruvate and ADP into pyruvate and ATP. There are two genes that encode pyruvate kinase in vertebrates; Pkm and Pkl encode muscle- and liver/erythrocyte-specific forms, respectively. Each gene encodes two isoenzymes due to alternative splicing. Both muscle-specific enzymes, PKM1 and PKM2, function in glycolysis, but PKM2 also has been implicated in gene regulation due to its ability to phosphorylate histone 3 threonine 11 (H3T11) in cancer cells. Here, we examined the roles of PKM1 and PKM2 during myoblast differentiation. RNA-seq analysis revealed that PKM2 promotes the expression of Dpf2/Baf45d and Baf250a/Arid1A. DPF2 and BAF250a are subunits that identify a specific sub-family of the mammalian SWI/SNF (mSWI/SNF) of chromatin remodeling enzymes that is required for the activation of myogenic gene expression during differentiation. PKM2 also mediated the incorporation of DPF2 and BAF250a into the regulatory sequences controlling myogenic gene expression. PKM1 did not affect expression but was required for nuclear localization of DPF2. Additionally, PKM2 was required not only for the incorporation of phosphorylated H3T11 in myogenic promoters but also for the incorporation of phosphorylated H3T6 and H3T45 at myogenic promoters via regulation of AKT and protein kinase C isoforms that phosphorylate those amino acids. Our results identify multiple unique roles for PKM2 and a novel function for PKM1 in gene expression and chromatin regulation during myoblast differentiation.

Dasatinib promotes muscle differentiation and disrupts normal muscle regeneration.

Dasatinib is one of the second-generation tyrosine kinase inhibitors used to treat chronic myeloid leukemia and has a broad target spectrum, including KIT, PDGFR, and SRC family kinases. Due to its broad drug spectrum, dasatinib has been reported at the basic research level to improve athletic performance by eliminating senescent cell removal and to have an effect on muscle diseases such as Duchenne muscular dystrophy, but its effect on myoblasts has not been investigated. In this study, we evaluated the effects of dasatinib on skeletal muscle both under normal conditions and in the regenerating state. Dasatinib suppressed the proliferation and promoted the fusion of C2C12 myoblasts. During muscle regeneration, dasatinib increased the gene expressions of myogenic-related genes (Myod, Myog, and Mymx), and caused abnormally thin muscle fibers on the CTX-induced muscle injury mouse model. From these results, dasatinib changes the closely regulated gene expression pattern of myogenic regulatory factors during muscle differentiation and disrupts normal muscle regeneration. Our data suggest that when using dasatinib, its effects on skeletal muscle should be considered, particularly at regenerating stages.

Knockdown of NFIC Promotes Bovine Myoblast Proliferation through the CENPF/CDK1 Axis.

As cellular transcription factors and DNA replicators, nuclear factor I (NFI) family members play an important role in mammalian development. However, there is still a lack of research on the muscle regeneration of NFI family members in cattle. In this study, the analysis of NFI family factors was conducted on their characterization, phylogenetics, and functional domains. We found that NFI family members were relatively conserved among different species, but there was heterogeneity in amino acid sequences, DNA coding sequences, and functional domain among members. Furthermore, among NFI family factors, we observed that NFIC exhibited highly expression in bovine muscle tissues, particularly influencing the expression of proliferation marker genes in myoblasts. To investigate the influence of NFIC on myoblast proliferation, we knocked down NFIC (si-NFIC) and found that the proliferation of myoblasts was significantly promoted. In terms of regulation mechanism, we identified that si-NFIC could counteract the inhibitory effect of the cell cycle inhibitor RO-3306. Interestingly, CENPF, as the downstream target gene of NFIC, could affect the expression of CDK1, CCNB1, and actively regulate the cell cycle pathway and cell proliferation. In addition, when CENPF was knocked down, the phosphorylation of p53 and the expression of Bax were increased, but the expression of Bcl2 was inhibited. Our findings mainly highlight the mechanism by which NFIC acts on the CENPF/CDK1 axis to regulate the proliferation of bovine myoblasts.

Saururus chinensis (Lour.) Baill. extract promotes skeletal muscle cell differentiation by positively regulating mitochondrial biogenesis and AKT/mTOR signaling in vitro.

Promotion of myoblast differentiation by activating mitochondrial biogenesis and protein synthesis signaling pathways provides a potential alternative strategy to balance energy and overcome muscle loss and muscle disorders. Saururus chinensis (Lour.) Baill. extract (SCE) has been used extensively as a traditional herbal medicine and has several physiological activities, including anti­asthmatic, anti­oxidant, anti­inflammatory, anti­atopic, anticancer and hepatoprotective properties. However, the effects and mechanisms of action of SCE on muscle differentiation have not yet been clarified. In the present study, it was investigated whether SCE affects skeletal muscle cell differentiation through the regulation of mitochondrial biogenesis and protein synthesis in murine C2C12 myoblasts. The XTT colorimetric assay was used to determine cell viability, and myosin heavy chain (MyHC) levels were determined using immunocytochemistry. SCE was applied to C2C12 myotube at different concentrations (1, 5, or 10 ng/ml) and times (1,3, or 5 days). Reverse transcription­quantitative PCR and western blotting were used to analyze the mRNA and protein expression change of factors related to differentiation, mitochondrial biogenesis and protein synthesis. Treatment of C2C12 cells with SCE at 1,5, and 10 ng/ml did not affect cell viability. SCE promoted C2C12 myotube formation and significantly increased MyHC expression in a concentration­ and time­dependent manner. SCE significantly increased the mRNA and protein expression of muscle differentiation­specific markers, such as MyHC, myogenic differentiation 1, myogenin, Myogenic Factor 5, and ß­catenin, mitochondrial biosynthesis­related factors, such as peroxisome proliferator­activated receptor­gamma coactivator­1α, nuclear respirator factor­1, AMP­activated protein kinase phosphorylation, and histone deacetylase 5 and AKT/mTOR signaling factors related to protein synthesis. SCE may prevent skeletal muscle dysfunction by enhancing myoblast differentiation through the promotion of mitochondrial biogenesis and protein synthesis.

Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment.

The ability of cells to organize into tissues with proper structure and function requires the effective coordination of proliferation, migration, polarization, and differentiation across length scales. Skeletal muscle is innately anisotropic; however, few biomaterials can emulate mechanical anisotropy to determine its influence on tissue patterning without introducing confounding topography. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topography, C2C12 myoblasts collectively polarize in the stiffest direction. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of the same chemical formulation. These findings provide valuable insights for designing biomaterials that mimic anisotropic microenvironments and underscore the importance of stiffness anisotropy in orchestrating tissue morphogenesis.

Mechanistic of AMPK/ACC2 regulating myoblast differentiation by fatty acid oxidation of goat.

Myoblast differentiation depends on fatty acid oxidation (FAO)，and its rate-limiting enzyme acetyl-CoA carboxylase 2 (ACC2) participate in the regulation skeletal muscle development. However, the precise regulatory mechanism is still unknown. Using previous RNA-sequencing data from our laboratory, we explored the effect of ACC2 on myoblast differentiation, as a candidate gene, since its expression is higher in myoblasts of lamb (first day of age) than that of the fetus (75th day of pregnancy). Our findings show that siACC2 inhibited myoblast proliferation, promoted differentiation, and boosted mitochondrial and fatty acid oxidation activities. The effect of ACC2 on goat muscle cell differentiation was modulated by Etomoxir, a CPT1A inhibitor. Notably, the AMPK/ACC2 pathway was found to regulate fatty acid oxidation and goat muscle cell differentiation. Inhibiting the AMPK/ACC2 pathway significantly reduced CPT1A expression. These findings indicate that AMPK/ACC2 regulate goat myoblast differentiation via fatty acid oxidation, contributing to understanding the mechanism of goat skeletal muscle development.

MTM1-mediated production of phosphatidylinositol 5-phosphate fuels the formation of podosome-like protrusions regulating myoblast fusion.

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.

Genipin Activates Autophagy and Promotes Myoblast Differentiation by Activating AMPK Pathway.

Cultured meat technology is expected to solve problems such as resource shortages and environmental pollution, but the muscle fiber differentiation efficiency of cultured meat is low. Genipin is the active compound derived from Gardenia jasminoides Ellis, which has a variety of activities. Additionally, genipin serves as a noncytotoxic agent for cross-linking, which is suitable as a foundational scaffold for in vitro tissue regeneration. However, the impact of genipin on myoblast differentiation remains to be studied. The research revealed that genipin was found to improve the differentiation efficiency of myoblasts. Genipin improved mitochondrial membrane potential by activating the AMPK signaling pathway of myoblasts, promoting mitochondrial biogenesis, and mitochondrial network remodeling. Genipin activated autophagy in myoblasts and maintained cellular homeostasis. Autophagy inhibitors blocked the pro-differentiation effect of genipin. These results showed that genipin improved the differentiation efficiency of myoblasts, which provided a theoretical basis for the development of cultured meat technology.

PHF2 regulates sarcomeric gene transcription in myogenesis.

Myogenesis is regulated mainly by transcription factors known as Myogenic Regulatory Factors (MRFs), and the transcription is affected by epigenetic modifications. However, the epigenetic regulation of myogenesis is poorly understood. Here, we focused on the epigenomic modification enzyme, PHF2, which demethylates histone 3 lysine 9 dimethyl (H3K9me2) during myogenesis. Phf2 mRNA was expressed during myogenesis, and PHF2 was localized in the nuclei of myoblasts and myotubes. We generated Phf2 knockout C2C12 myoblasts using the CRISPR/Cas9 system and analyzed global transcriptional changes via RNA-sequencing. Phf2 knockout (KO) cells 2 d post differentiation were subjected to RNA sequencing. Gene ontology (GO) analysis revealed that Phf2 KO impaired the expression of the genes related to skeletal muscle fiber formation and muscle cell development. The expression levels of sarcomeric genes such as Myhs and Mybpc2 were severely reduced in Phf2 KO cells at 7 d post differentiation, and H3K9me2 modification of Mybpc2, Mef2c and Myh7 was increased in Phf2 KO cells at 4 d post differentiation. These findings suggest that PHF2 regulates sarcomeric gene expression via epigenetic modification.

Immunohistochemical properties of embryonic telocytes in a myogenic microenvironment.

Telocytes are a unique interstitial cell type that functions in adulthood and embryogenesis. They have characteristic immunohistochemical phenotypes while acquiring different immunohistochemical properties related to the organ microenvironment. The present study aims to investigate the immunohistochemical features of embryonic telocytes during myogenesis and describe their morphology using light microscopy and TEM. Telocytes represent a major cellular constituent in the interstitial elements. They had distinguished telopodes and podoms and formed a 3D interstitial network in the developing muscles. They formed heterocellular contact with myoblasts and nascent myotubes. Telocytes also had distinctive secretory activity. Telocytes identified by CD34. They also express CD68 and MMP-9 to facilitate the development of new tissues. Expression of CD21 by telocytes may reveal their function in immune defense. They also express VEGF, which regulates angiogenesis. In conclusion, the distribution and immunological properties of telocytes in the myogenic tissue indicate that telocytes provide biological and structural support in the development of the myogenic tissue architecture and organization.

Molecular cloning of TPM3 gene in qinchuan cattle and its effect on myoblast proliferation and differentiation.

Tropomyosin 3 (TPM3) plays a significant role as a regulatory protein in muscle contraction, affecting the growth and development of skeletal muscles. Despite its importance, limited research has been conducted to investigate the influence of TPM3 on bovine skeletal muscle development. Therefore, this study revealed the role of TPM3 in bovine myoblast growth and development. This research involved conducting a thorough examination of the Qinchuan cattle TPM3 gene using bioinformatics tools to examine its sequence and structural characteristics. Furthermore, TPM3 expression was evaluated in various bovine tissues and cells using quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that the coding region of TPM3 spans 855 bp, with the 161st base being the T base, encoding a protein with 284 amino acids and 19 phosphorylation sites. This protein demonstrated high conservation across species while displaying a predominant α-helix secondary structure despite being an unstable acidic protein. Notably, a noticeable increase in TPM3 expression was observed in the longissimus dorsi muscle and myocardium of calves and adult cattle. Expression patterns varied during different stages of myoblast differentiation. Functional studies that involved interference with TPM3 in Qinchuan cattle myoblasts revealed a very significantly decrease in S-phase cell numbers and EdU-positive staining (P < 0.01), and disrupted myotube morphology. Moreover, interference with TPM3 resulted in significantly (P < 0.05) or highly significantly (P < 0.01) decreased mRNA and protein levels of key proliferation and differentiation markers, indicating its role in the modulation of myoblast behavior. These findings suggest that TPM3 plays an essential role in bovine skeletal muscle growth by influencing myoblast proliferation and differentiation. This study provides a foundation for further exploration into the mechanisms underlying TPM3-mediated regulation of bovine muscle development and provides valuable insights that could guide future research directions as well as potential applications for livestock breeding and addressing muscle-related disorders.

Whole transcriptome profiling reveals a lncMDP1 that regulates myogenesis by adsorbing miR-301a-5p targeting CHAC1.

Myoblast proliferation and differentiation are essential for skeletal muscle development. In this study, we generated the expression profiles of mRNAs, long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) in different developmental stages of chicken primary myoblasts (CPMs) using RNA sequencing (RNA-seq) technology. The dual luciferase reporter system was performed using chicken embryonic fibroblast cells (DF-1), and functional studies quantitative real-time polymerase chain reaction (qPCR), cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry cycle, RNA fluorescence in situ hybridization (RNA-FISH), immunofluorescence, and western blotting assay. Our research demonstrated that miR-301a-5p had a targeted binding ability to lncMDP1 and ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). The results revealed that lncMDP1 regulated the proliferation and differentiation of myoblasts via regulating the miR-301a-5p/CHAC1 axis, and CHAC1 promotes muscle regeneration. This study fulfilled the molecular regulatory network of skeletal muscle development and providing an important theoretical reference for the future improvement of chicken meat performance and meat quality.

NMR-based comparative metabolomics of quiescent muscle cells.

Adult muscle tissue largely comprised of differentiated myofibers also harbors quiescent muscle-resident stem cells (MuSCs) that are responsible for its maintenance, repair and regeneration. Emerging evidence suggests that quiescent MuSCs exhibit a specific metabolic state, which is regulated during physiological and pathological alterations. However, a detailed understanding of the metabolic state of quiescent MuSCs and its alteration during activation and repair is lacking. Direct profiling of MuSCs in vivo is challenging because the cells are rare and dispersed, while isolation and enrichment leads to their activation and loss of quiescence. In this study, we employed 1H-nuclear magnetic resonance (NMR) spectroscopy to profile metabolites in an established culture model of quiescent MuSC-derived myoblasts and compared with activated, proliferative and differentiated muscle cells to determine the state-specific metabolome. We report that the proliferating and differentiated cells are highly enriched in metabolites involved in energy generation, the quiescent state is enriched in metabolites related to phospholipid catabolism (glycerophosphocholine and choline) and depleted for phosphocholine which is enriched in proliferating cells. We propose that the ratio of these metabolites may be useful as a biomarker of MuSC quiescence.

CRISPR Screen Identifies the RNA-Binding Protein Eef1a1 as a Key Regulator of Myogenesis.

Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.

Generation of Chicken Contractile Skeletal Muscle Structure Using Decellularized Plant Scaffolds.

Cultured meat is a meat analogue produced by in vitro cell culture, which can replace the conventional animal production system. Tissue engineering using myogenic cells and biomaterials is a core technology for cultured meat production. In this study, we provide an efficient and economical method to produce skeletal muscle tissue-like structures by culturing chicken myoblasts in a fetal bovine serum (FBS)-free medium and plant-derived scaffolds. An FBS-free medium supplemented with 10% horse serum (HS) and 5% chick embryo extract (CEE) was suitable for the proliferation and differentiation of chicken myoblasts. Decellularized celery scaffolds (Decelery), manufactured using 1% sodium dodecyl sulfate (SDS), were nontoxic to cells and supported myoblast proliferation and differentiation. Decelery could support the 3D culture of chicken myoblasts, which could adhere and coagulate to the surface of the Decelery and form MYH1E+ and F-actin+ myotubes. After 2 weeks of culture on Decelery, fully grown myoblasts completely covered the surface of the scaffolds and formed fiber-like myotube structures. They further differentiated to form spontaneously contracting myofiber-like myotubes on the scaffold surface, indicating that the Decelery scaffold system could support the formation of a functional mature myofiber structure. In addition, as the spontaneously contracting myofibers did not detach from the surface of the Decelery, the Decelery system is a suitable biomaterial for the long-term culture and maintenance of the myofiber structures.