Previous studies showed that the bladder extracellular matrix (B-ECM) could increase the differentiation efficiency of mesenchymal cells into smooth muscle cells (SMC). This study investigates the potential of human amniotic membrane-derived hydrogel (HAM-hydrogel) as an alternative to xenogeneic B-ECM for the myogenic differentiation of the rabbit adipose tissue-derived MSC (AD-MSC). Decellularized human amniotic membrane (HAM) and sheep urinary bladder (SUB) were utilized to create pre-gel solutions for hydrogel formation. Rabbit AD-MSCs were cultured on SUB-hydrogel or HAM-hydrogel-coated plates supplemented with differentiation media containing myogenic growth factors (PDGF-BB and TGF-β1). An uncoated plate served as the control. After 2 weeks, real-time qPCR, immunocytochemistry, flow cytometry, and western blot were employed to assess the expression of SMC-specific markers (MHC and α-SMA) at both protein and mRNA levels. Our decellularization protocol efficiently removed cell nuclei from the bladder and amniotic tissues, preserving key ECM components (collagen, mucopolysaccharides, and elastin) within the hydrogels. Compared to the control, the hydrogel-coated groups exhibited significantly upregulated expression of SMC markers (p ≤ .05). These findings suggest HAM-hydrogel as a promising xenogeneic-free alternative for bladder tissue engineering, potentially overcoming limitations associated with ethical concerns and contamination risks of xenogeneic materials.

Sestrin2 (Sesn2) has been previously confirmed to be a stress-response molecule. However, the influence of Sesn2 on myogenic differentiation remains elusive. This study was conducted to analyze the role of Sesn2 in the myogenic differentiation of C2C12 myoblasts and related aspects in mdx mice, an animal model of Duchenne muscular dystrophy (DMD). Our results showed that knockdown of Sesn2 reduced the myogenic differentiation capacity of C2C12 myoblasts. Predictive analysis from two databases suggested that miR-182-5p is a potential regulator of Sesn2. Further experimental validation revealed that overexpression of miR-182-5p decreased both the protein and mRNA levels of Sesn2 and inhibited myogenesis of C2C12 myoblasts. These findings suggest that miR-182-5p negatively regulates myogenesis by repressing Sesn2 expression. Extending to an in vivo model of DMD, knockdown of Sesn2 led to decreased Myogenin (Myog) expression and increased Pax7 expression, while its overexpression upregulated Myog levels and enhanced the proportion of slow-switch myofibers. These findings indicate the crucial role of Sesn2 in promoting myogenic differentiation and skeletal muscle regeneration, providing potential therapeutic targets for muscular dystrophy.

OBJECTIVE: Study of the role of mitochondria-generated reactive oxygen species (mtROS) and mitochondrial polarization in mitochondrial fragmentation at the initial stages of myogenesis.
METHODS: Mitochondrial morphology, Drp1 protein phosphorylation, mitochondrial electron transport chain components content, mtROS and mitochondrial lipid peroxidation levels, and mitochondrial polarization were evaluated on days 1 and 2 of human MB135 myoblasts differentiation. A mitochondria-targeted antioxidant SkQ1 was used to elucidate the effect of mtROS on mitochondria.
RESULTS: In immortalized human MB135 myoblasts, mitochondrial fragmentation began on day 1 of differentiation before the myoblast fusion. This fragmentation was preceded by dephosphorylation of p-Drp1 (Ser-637). On day 2, an increase in the content of some mitochondrial proteins was observed, indicating mitochondrial biogenesis stimulation. Furthermore, we found that myogenic differentiation, even on day 1, was accompanied both by an increased production of mtROS, and lipid peroxidation of the inner mitochondrial membrane. SkQ1 blocked these effects and partially reduced the level of mitochondrial fragmentation, but did not affect the dephosphorylation of p-Drp1 (Ser-637). Importantly, mitochondrial fragmentation at early stages of MB135 differentiation was not accompanied by depolarization, as an important stimulus for mitochondrial fragmentation.
CONCLUSIONS: Mitochondrial fragmentation during early myogenic differentiation depends on mtROS production rather than mitochondrial depolarization. SkQ1 only partially inhibited mitochondrial fragmentation, without significant effects on mitophagy or early myogenic differentiation.

With the current environmental impact of large-scale animal production and societal concerns about the welfare of farm animals, researchers are questioning whether we can cultivate animal cells for the purpose of food production. This review focuses on a pivotal aspect of the cellular agriculture domain: cells. We summarised information on the various cell types from farm animals currently used for the development of cultured meat, including mesenchymal stromal cells, myoblasts, and pluripotent stem cells. The review delves into the advantages and limitations of each cell type and considers factors like the selection of the appropriate cell source, as well as cell culture conditions that influence cell performance. As current research in cultured meat seeks to create muscle fibers to mimic the texture and nutritional profile of meat, we focused on the myogenic differentiation capacity of the cells. The most commonly used cell type for this purpose are myoblasts or satellite cells, but given their limited proliferation capacity, efforts are underway to formulate myogenic differentiation protocols for mesenchymal stromal cells and pluripotent stem cells. The multipotent character of the latter cell types might enable the creation of other tissues found in meat, such as adipose and connective tissues. This review can help guiding the selection of a cell type or culture conditions in the context of cultured meat development.

Nano-plastics (NPs) have emerged as a significant environmental pollutant, widely existing in water environment, and pose a serious threat to health and safety with the intake of animals. Skeletal muscle, a vital organ for complex life activities and functional demands, has received limited attention regarding the effects of NPs. In this study, the effects of polystyrene NPs (PS-NPs) on skeletal muscle development were studied by oral administration of different sizes (1 mg/kg) of PS-NPs in mice. The findings revealed that PS-NPs resulted in skeletal muscle damage and significantly hindered muscle differentiation, exhibiting an inverse correlation with PS-NPs particle size. Morphological analysis demonstrated PS-NPs caused partial disruption of muscle fibers, increased spacing between fibers, and lipid accumulation. RT-qPCR and western blots analyses indicated that PS-NPs exposure downregulated the expression of myogenic differentiation-related factors (Myod, Myog and Myh2), activated PPARγ/LXRβ pathway, and upregulated the expressions of lipid differentiation-related factors (SREBP1C, SCD-1, FAS, ACC1, CD36/FAT, ADIPOQ, C/EBPα and UCP-1). In vitro experiments, C2C12 cells were used to confirm cellular penetration of PS-NPs (0, 100, 200, 400 μg/mL) through cell membranes along with activation of PPARγ expression. Furthermore, to verify LXRβ as a key signaling molecule, silencing RNA transfection experiments were conducted, resulting in no increase in the expressions of PPARγ, LXRβ, SREBP1C, FAS, CD36/FAT, ADIPOQ, C/EBPα and UCP-1 even after exposure to PS-NPs. However, the expressions of SCD-1and ACC1 remained unaffected. The present study evidenced that exposure to PS-NPs induced lipid accumulation via the PPARγ/LXRβ pathway thereby influencing skeletal muscle development.

Bisphenol A (BPA), a typical environmental endocrine disruptor, has raised concerns among researchers due to its toxicological effects. Whether neohesperidin (NEO) can intervene in the toxic effects of BPA remains unknown. This study aims to investigate the effects and mechanisms of NEO on the myogenic differentiation of umbilical cord-derived mesenchymal stem cells (UC-MSCs) exposed to BPA. Sheep UC-MSCs were isolated, characterized, and induced to myogenic differentiation. BPA decreased cell viability, cell migration, and the expressions of myogenic marker genes, leading to myogenic differentiation inhibition, which were reversed by NEO. Network pharmacology suggested the IGF1R/AKT1/RHOA pathway as potential targets of BPA and NEO regulating muscle development. Western blot results showed that NEO could reverse the down-regulation of the pathway proteins induced by BPA, and counteract the effects of picropodophyllin (PPP) or MK-2206 dihydrochloride (MK-2206) in the myogenic differentiation of sheep UC-MSCs. Additionally, the expression levels of (p-) IGF1R, AKT1, and RHOA were positively correlated. Taken together, the mechanisms of NEO resistance to BPA involved the IGF1R/AKT1/RHOA signaling pathway. These findings provide a scientific basis for alleviating BPA toxicity, preventing and treating muscular dysplasia, and promoting muscle damage repair.

Decellularized plant scaffolds have been used to develop edible scaffolds for cell cultured meat because of their natural structures similar to that of mammalian tissues. However, their diverse three-dimensional (3D) porous structures may lead to differences in myogenic differentiation of skeletal muscle cells. In this study, parsley plant tissues were decellularized and modified by type A gelatin and transglutaminase while retaining, respectively, longitudinal fibrous and transverse honeycomb pore structures. The effects of the structure of the decellularized parsley scaffold on the proliferation and myogenic differentiation of C2C12 cells were investigated and the quality of cell cultured meat was evaluated. The results showed that fibrous pore structure guided cells to be arranged in parallel, whereas honeycomb pore structure connected cells in a circular pattern. After induced differentiation, the fibrous scaffolds were more inclined to form multinucleated myotubes with higher expression of myogenic genes and proteins, and the final cell-based meat contained higher total protein content. Decellularized plant scaffolds with fibrous pore structure were more suitable for myogenic differentiation of C2C12 cells, providing support to the development of edible scaffolds for cultured meat. PRACTICAL APPLICATION: This study investigated the different three-dimensional (3D) pore structure of parsley parenchyma to gain insight into how the 3D pore structure of decellularized plant scaffolds regulates myogenic differentiation, which is expected to address the unstable myogenic differentiation of skeletal muscle cells on decellularized plant scaffolds in cell culture meat production.

Cellular agriculture, an alternative and innovative approach to sustainable food production, has gained momentum in recent years. However, there is limited research into the production of cultivated seafood. Here, we investigated the ability of fish mackerel cells (Scomber scombrus) to adhere to plant, algal and fungal-based biomaterial scaffolds, aiming to optimize the cultivation of fish cells for use in cellular agriculture. A mackerel cell line was utilized, and metabolic assays and confocal imaging were utilized to track cell adhesion, growth, and differentiation on the different biomaterials. The mackerel cells adhered and grew on gelatin (positive control), zein, and soy proteins, as well as on alginate, chitosan, and cellulose polysaccharides. The highest adhesion and growth were on the zein and chitosan substrates, apart from the gelatin control. These findings provide a blueprint to enhance scaffold selection and design, contributing to the broader field of cellular agriculture through the development of scalable and eco-conscious solutions for meeting the growing global demand for seafood.

Pericytes are a distinct type of cells interacting with endothelial cells in blood vessels and contributing to endothelial barrier integrity. Furthermore, pericytes show mesenchymal stem cell properties. Muscle-derived pericytes can demonstrate both angiogenic and myogenic capabilities. It is well known that regenerative abilities and muscle stem cell potential decline during aging, leading to sarcopenia. Therefore, this study aimed to investigate the potential of pericytes in supporting muscle differentiation and angiogenesis in elderly individuals and in patients affected by Ullrich congenital muscular dystrophy or by Bethlem myopathy, two inherited conditions caused by mutations in collagen VI genes and sharing similarities with the progressive skeletal muscle changes observed during aging. The study characterized pericytes from different age groups and from individuals with collagen VI deficiency by mass spectrometry-based proteomic and bioinformatic analyses. The findings revealed that aged pericytes display metabolic changes comparable to those seen in aging skeletal muscle, as well as a decline in their stem potential, reduced protein synthesis, and alterations in focal adhesion and contractility, pointing to a decrease in their ability to form blood vessels. Strikingly, pericytes from young patients with collagen VI deficiency showed similar characteristics to aged pericytes, but were found to still handle oxidative stress effectively together with an enhanced angiogenic capacity.

HDAC11 is an epigenetic repressor of gene transcription, acting through its deacetylase activity to remove functional acetyl groups from the lysine residues of histones at genomic loci. It has been implicated in the regulation of different immune responses, metabolic activities, as well as cell cycle progression. Recent studies have also shed lights on the impact of HDAC11 on myogenic differentiation and muscle development, indicating that HDAC11 is important for histone deacetylation at the promoters to inhibit transcription of cell cycle related genes, thereby permitting myogenic activation at the onset of myoblast differentiation. Interestingly, the upstream networks of HDAC11 target genes are mainly associated with cell cycle regulators and the acetylation of histones at the HDAC11 target promoters appears to be residue specific. As such, selective inhibition, or activation of HDAC11 presents a potential therapeutic approach for targeting distinct epigenetic pathways in clinical applications.