Mouse Myoblasts Research Articles

Augmented mitochondrial apoptotic signaling impairs C2C12 myoblast differentiation following cellular aging through sequential passaging.

Aging is associated with the steady decline of several cellular processes. The loss of skeletal muscle mass, termed sarcopenia, is one of the major hallmarks of aging. Aged skeletal muscle exhibits a robust reduction in its regenerative capacity due to dysfunction (i.e., senescence, lack of self-renewal, and impaired differentiation) of resident muscle stem cells, called satellite cells. To replicate aging in vitro, immortalized skeletal muscle cells (myoblasts) can be treated with various agents to mimic age-related dysfunction; however, these come with their own set of limitations. In the present study, we used sequential passaging of mouse myoblasts to mimic impaired differentiation that is observed in aged skeletal muscle. Further, we investigated mitochondrial apoptotic mechanisms to better understand the impaired differentiation in these "aged" cells. Our data shows that sequential passaging (>20 passages) of myoblasts is accompanied with significant reductions in differentiation and elevated cell death. Furthermore, high-passage (HP) myoblasts exhibit greater mitochondrial-mediated apoptotic signaling through mitochondrial BAX translocation, CYCS and AIFM1 release, and caspase-9 activation. Finally, we show that inhibition of mitochondrial outer membrane permeability partly recovered differentiation in HP myoblasts. Together, our findings suggests that mitochondrial apoptotic signaling is a contributing factor to the diminished differentiation that is observed in aged myoblasts.

Magnetically assembled endothelial cell-coated spheroid for vascularization

3D spheroids are used as a therapeutic strategy and transplanted in vivo owing to their ability to better mimic the microphysiological environment that is crucial for the transplants to be able to engraft and vascularize for their long-term survival and functionalization in the host tissue. Among various methods of fabricating spheroids, the utilization of magnetic force enables rapid cell aggregation and spatial regulation. Encapsulating a magnetic spheroid with endothelial cells paves the way toward vascularization. Here, we magnetically bioprinted vascular assembly by coating magnetic spheroids with the magnetic nanoparticles (MNPs)-internalized vascular endothelial cells. MNPs-internalized mouse myoblasts cells (C2C12) were magnetically aggregated by placing a neodymium magnet under a well plate and then encapsulated by MNPs-internalized human umbilical vein endothelial cells (HUVECs). Internalization of MNPs did not hinder cell viability and vascularization-related gene expression, and the transplant of the vascular assemblies could sprout and be engrafted into the host tissue within three days. As these vascular assemblies are easily fabricated by coating spheroids with MNPs-internalized endothelial cells and controlled by a magnetic force, they may be used for engraftment and vascularization in various applications with spheroids with different functions.

METTL21C mediates autophagy and formation of slow-twitch muscle fibers in mice after exercise.

Homeostasis is essential for muscle repair and regeneration after skeletal muscle exercise. This study investigated the role of methyltransferase-like 21C (METTL21C) in skeletal muscle of mice after exercise and the potential mechanism. First, muscle samples were collected at 2, 4 and 6 weeks after exercise, and liver glycogen, muscle glycogen, blood lactic acid and triglyceride were assessed. Moreover, the expression levels of autophagy markers and METTL21C in skeletal muscle were analyzed. The results showed that the expression levels of METTL21C and MYH7 in the gastrocnemius muscle of mice in the exercise group were significantly higher after exercise than those in the control group, which suggested that long-term exercise promoted the formation of slow-twitch muscle fibers in mouse skeletal muscle. Likewise, the autophagy capacity was enhanced with the prolongation of exercise in muscles. The findings were confirmed in mouse C2C12 cells. We discovered that knockdown of Mettl21c reduced the expression of MYH7 and the autophagy level in mouse myoblasts. These findings indicate that METTL21C promotes skeletal muscle homeostasis after exercise by enhancing autophagy, and also contributes to myogenic differentiation and the formation of slow muscle fibers.

Fermented Yak-Kong using Bifidobacterium animalis derived from Korean infant intestine effectively relieves muscle atrophy in an aging mouse model.

Yak-Kong (YK) is a small black soybean widely cultivated in Korea. It is considered to have excellent health functionality, as it has been reported to have better antioxidant efficacy than conventional black or yellow soybeans. Since YK has been described as good for the muscle health of the elderly in old oriental medicine books, this study sought to investigate the effect of fermented YK with Bifidobacterium animalis subsp. lactis LDTM 8102 (FYK) on muscle atrophy. In C2C12 mouse myoblasts, FYK elevated the expression of MyoD, total MHC, phosphorylated AKT, and PGC1α. In addition, two kinds of in vivo studies were conducted using both an induced and normal aging mouse model. The behavioral test results showed that in the induced aging mouse model, FYK intake alleviated age-related muscle weakness and loss of exercise performance. In addition, FYK alleviated muscle mass decrease and improved the expression of biomarkers including total MHC, myf6, phosphorylated AKT, PGC1α, and Tfam, which are related to myoblast differentiation, muscle protein synthesis, and mitochondrial generation in the muscle. In the normal aging model, FYK consumption did not increase muscle mass, but did upregulate the expression levels of biomarkers related to myoblast differentiation, muscle hypertrophy, and muscle function. Furthermore, it mitigated age-related declines in skeletal muscle force production and functional limitation by enhancing exercise performance and grip strength. Taken together, the results suggest that FYK has the potential to be a new functional food material that can alleviate the loss of muscle mass and strength caused by aging and prevent sarcopenia.

Electric field-induced self-assembly of zein for constructing a bilayer microporous membrane with potential for cell culture

The persistent challenge of membrane contamination, primarily stemming from the extensive usage of non-decomposable materials, stands as a significant barrier limiting the application of microporous membranes in fields like food packaging and biomaterials. This study focused on utilizing indium tin oxide (ITO) conductive glass coated with polylactic acid (PLA) as the electrode. Through electrical induction, zein was prompted to self-assemble, resulting in the creation of porous zein bilayer microporous membranes (PLA/Zein-BMMs). These membranes underwent comprehensive characterization of their physicochemical properties and structure. The outcomes revealed that an electric field voltage of 100 V, an electrode distance of 25 mm, a current density of 15 A/m2, and a protein concentration of 0.05 g/mL, were most suitable for generating microporous membranes featuring a pore diameter of 2 μm and a thickness of 8 μm. During this stage, the microporous membrane exhibited an interconnected three-dimensional network structure, achieving a tensile strength of 49 MPa. Simultaneously, it showcased a surface porosity of 8.15%, a water contact angle measuring 74.28°, and a water vapor transmission rate of 3.2 × 10-3 g/m2•h•KPa. These attributes confer significant application potential in the realms of food packaging and preservation. Additionally, owing to its porous structure and hydrophilic nature, it exhibited favorable biocompatibility when cultured with mouse myoblasts (C2C12), indicating its promising value in the field of biomaterials as well.

Skeletal Muscle Spheroids as Building Blocks for Engineered Muscle Tissue.

Spheroids exhibit enhanced cell-cell interactions that facilitate improved survival and mimic the physiological cellular environment in vivo. Cell spheroids have been successfully used as building blocks for engineered tissues, yet the viability of this approach with skeletal muscle spheroids is poorly understood, particularly when incorporated into three-dimensional (3D) constructs. Bioprinting is a promising strategy to recapitulate the hierarchical organization of native tissue that is fundamental to its function. However, the influence of bioprinting on skeletal muscle cell spheroids and their function are yet to be interrogated. Using C2C12 mouse myoblasts and primary bovine muscle stem cells (MuSCs), we characterized spheroid formation as a function of duration and cell seeding density. We then investigated the potential of skeletal muscle spheroids entrapped in alginate bioink as tissue building blocks for bioprinting myogenic tissue. Both C2C12 and primary bovine MuSCs formed spheroids of similar sizes and remained viable after bioprinting. Spheroids of both cell types fused into larger tissue clusters over time within alginate and exhibited tissue formation comparable to monodisperse cells. Compared to monodisperse cells in alginate gels, C2C12 spheroids exhibited greater MyHC expression after 2 weeks, while cells within bovine MuSC spheroids displayed increased cell spreading. Both monodisperse and MuSC spheroids exhibited increased expression of genes denoting mid- and late-stage myogenic differentiation. Together, these data suggest that skeletal muscle spheroids have the potential for generating myogenic tissue via 3D bioprinting and reveal areas of research that could enhance myogenesis and myogenic differentiation in future studies.

Generation of highly pure pluripotent stem cell-derived myogenic progenitor cells and myotubes

Driving efficient and pure skeletal muscle cell differentiation from pluripotent stem cells (PSCs) has been challenging. Here, we report an optimized protocol that generates skeletal muscle progenitor cells with high efficiency and purity in a short period of time. Human induced PSCs (hiPSCs) and murine embryonic stem cells (mESCs) were specified into the mesodermal myogenic fate using distinct and species-specific protocols. We used a specific maturation medium to promote the terminal differentiation of both human and mouse myoblast populations, and generated myotubes associated with a large pool of cell-cycle arrested PAX7+ cells. We also show that myotube maturation is modulated by dish-coating properties, cell density, and percentage of myogenic progenitor cells. Given the high efficiency in the generation of myogenic progenitors and differentiated myofibers, this protocol provides an attractive strategy for tissue engineering, modeling of muscle dystrophies, and evaluation of new therapeutic approaches invitro.

Piezo1 Is Required for Myoblast Migration and Involves Polarized Clustering in Association with Cholesterol and GM1 Ganglioside.

A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca2+ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.

All-trans retinoic acid and dexamethasone regulate phagocytosis-related gene expression and enhance dead cell uptake in C2C12 myoblast cells

Extensive mechanical stress frequently causes micro-traumas in skeletal muscle, followed by a regeneration period. The effective removal of dead myofibers is a prerequisite for proper regeneration, and several cell types, including professional phagocytes, were reported to be active in this process. Myoblasts express several molecules of the phagocytic machinery, such as BAI1, stabilin-2, and TAM (Tyro3, Axl, Mertk) tyrosine kinase receptors, but these molecules were reported to serve primarily cell fusion and survival, and their role in the phagocytosis was not investigated. Therefore, we aimed to investigate the in vitro phagocytic capacity of the C2C12 mouse myoblast cell line. RNA sequencing data were analyzed to determine the level and changes of phagocytosis-related gene expression during the differentiation process of C2C12 cells. To study the phagocytic capacity of myoblasts and the effect of dexamethasone, all-trans retinoic acid, hemin, and TAM kinase inhibitor treatments on phagocytosis, C2C12 cells were fed dead thymocytes, and their phagocytic capacity was determined by flow cytometry. The effect of dexamethasone and all-trans retinoic acid on phagocytosis-related gene expression was determined by quantitative PCR. Both undifferentiated and differentiated cells engulfed dead cells being the undifferentiated cells more effective. In line with this, we observed that the expression of several phagocytosis-related genes was downregulated during the differentiation process. The phagocytosis could be increased by dexamethasone and all-trans retinoic acid and decreased by hemin and TAM kinase inhibitor treatments. Our results indicate that myoblasts not only express phagocytic machinery genes but are capable of efficient dead cell clearance as well, and this is regulated similarly, as reported in professional phagocytes.

Culture Method and Regulation Mechanisms of Stages of C2C12 Cell Line Myogenesis

INTRODUCTION: C2C12 is an immortalized mouse myoblast cell line actively used as in vitro experimental models to study skeletal muscle metabolism in biomedical research. C2C12 cells can differentiate into myocytes under appropriate culture conditions. It is important to understand the specifics of each stage of myogenesis and its regulatory mechanisms for the targeted exposure and development of medical drugs, taking into account the mechanisms of pathogenesis of various diseases.
 AIM: To describe the method of culturing С2С12 cell line and to study regulatory mechanisms of С2С12 cell line myogenesis.
 MATERIALS AND METHODS: The study was conducted on C2C12 cell line. Differentiation of myoblasts was induced in the nutrient medium containing 2% horse serum. The differentiation stages were evaluated by studying the cells on the 1st, 4th and 7th days. Cells before differentiation were used as a comparison group. At each differentiation stage, the myoblast fusion index (IM) was evaluated with preliminary staining of the cells by Romanowsky–Giemsa method. The cell differentiation mechanism was evaluated by the level of myosin, a-actin, myogenic differentiation protein (MyoD), myogenin (MyoG) using Western blot method.
 RESULTS: On the 1st day of C2C12 cells culturing, formation of myotubes was observed (IM = 0.15 ± 0.05), on the 4th day — fusion of myoblasts with the formation of binucleated cells accompanied by an increase in the amount of MyoD and α-actin protein (IM = 0.44 ± 0.14). By the 7th day of differentiation, the fusion of cells increased with the formation of myotubes containing more than two nuclei; the content of MyoD and α-actin did not differ from the control, and the amount of MyoG and myosin increased (IM = 0.77 ± 0.04).
 CONCLUSION: The described method of culturing C2C12 cell line in Dulbecco’s Modified Eagle’s Medium with a high glucose content (4500 mg/l), containing 2% horse serum, L-glutamine (4 mМ), 100 Un/ml and 100 µg/ml of penicillin and streptomycin, is suitable for the formation of muscle cells, where MyoD and MyoG participate in the regulation of the increase in the amount of specific muscle proteins — α-actin and myosin.

Expression and electrophysiological characteristics of VGSC during mouse myoblasts differentiation

Voltage-gated sodium channels (VGSC) are essential for triggering and relaying action potentials (AP), which perform critical functions in a variety of physiological processes, such as controlling muscle contractions and facilitating the release of neurotransmitters. In this study, we used a mouse C2C12 cell differentiation model to study the molecular expression and channel dynamics of VGSC and to investigate the exact role of VGSC in the development of muscle regeneration. Immunofluorescence, Real-time quantitative polymerase chain reaction, Western blot, and whole-cell patch clamp were employed for this purpose in mouse myoblasts. The findings revealed an increase in intracellular sodium concentration, NaV1.4 gene expression, and protein expression with the progress of differentiation (days 0, 1, 3, 5 and 7). Furthermore, VGSC dynamics exhibit the following characteristics: ① The increase of sodium current (INa); ② The decrease in the activation threshold and the voltage trigger maximum of INa; ③ A positive shift in the steady-state inactivation curve; ④ The recovery of INa during repolarization is delayed, the activity-dependent decay rate of INa was accelerated, and the proportionate amount of the fraction of activated channels was reduced. Based on these results, it is postulated that the activation threshold of AP could be decreased, and the refractory period could be extended with the extension of differentiation duration, which may contribute to muscle contraction. Taken together, VGSC provides a theoretical and empirical basis for exploring potential targets for neuromuscular diseases and other therapeutic muscle regeneration dysfunctions.

Effects of theasaponin E1 on the regulationglucose uptake of C2C12 myoblasts PI3K/Akt/mTOR signaling pathway

ABSTRACT Targeting disorders of glucose metabolism and the occurrence of hyperglycemia provides a potential therapeutic strategy for balancing energy to reduce insulin resistance such as type 2 diabetes. The present study investigated whether theasaponin E1 regulate energy metabolism in skeletal muscle cells. C2C12 mouse myoblasts were differentiated into myotubes . An MTT colorimetric assay verified cell viability. Theasaponin E1 (30, 45 or 60 mg/ ml), or metformin (2.5 mM), insulin (150 nM) for reference were used to treat the C2C12 myotubes. The expressions of energy metabolism were measured by western blotting in C2C12 myotubes. 2-NBDG kit to detect the glucose uptake capacity. Theasaponin E1 Treatment increases glucose uptake,GSK 3 β, as well as increases to the glycogen synthesis through the PI3K/AKT/mTOR signaling pathway, while reducing AMPK and ACC activities, and reducing the adipose synthesis pathway. These results suggest that theasaponin E1 may be increases glucose uptake by improving muscle energy metabolism.

TMED10 mediates the trafficking of insulin-like growth factor 2 along the secretory pathway for myoblast differentiation.

The insulin-like growth factor 2 (IGF2) plays critical roles in cell proliferation, migration, differentiation, and survival. Despite its importance, the molecular mechanisms mediating the trafficking of IGF2 along the secretory pathway remain unclear. Here, we utilized a Retention Using Selective Hook system to analyze molecular mechanisms that regulate the secretion of IGF2. We found that a type I transmembrane protein, TMED10, is essential for the secretion of IGF2 and for differentiation of mouse myoblast C2C12 cells. Further analyses indicate that the residues 112-140 in IGF2 are important for the secretion of IGF2 and these residues directly interact with the GOLD domain of TMED10. We then reconstituted the release of IGF2 into COPII vesicles. This assay suggests that TMED10 mediates the packaging of IGF2 into COPII vesicles to be efficiently delivered to the Golgi. Moreover, TMED10 also mediates ER export of TGN-localized cargo receptor, sortilin, which subsequently mediates TGN export of IGF2. These analyses indicate that TMED10 is critical for IGF2 secretion by directly regulating ER export and indirectly regulating TGN export of IGF2, providing insights into trafficking of IGF2 for myoblast differentiation.

Nutritionally enhanced fibrous scaffolds by rolling droplet-interfacial polyelectrolyte complexation (RD-IPC)

The challenge of producing protein in a more sustainable manner than what is currently afforded by meat from livestock has led to the advent of cell-based or cultured meat. One method of obtaining whole-cuts of meat would be to culture the relevant cells on fibrous scaffolds to obtain meat-like textures. In the present work, a novel process of rolling droplet interfacial polyelectrolyte complexation (RD-IPC) is used to make aligned fibrous constructs that can be incorporated with nutritional components. Fiber is drawn by a falling droplet consisting of two oppositely charged polyelectrolytes that are continuously delivered by means of a syringe pump. The fibers are collected individually as they form and accumulate on a rotating collecting device to form planar constructs. Scaffolds incorporating gelatin, soybean flour, potato starch and garlic could be made by dispersing the respective powders into the polyelectrolyte solutions. Following the formation of the constructs, a heating step was found to improve their mechanical properties, in particular the potato starch-incorporated constructs, which exhibited higher tensile moduli due to a gelatinization process. Scaffolds imbued with gelatin and RGD moieties supported the adhesion, alignment, and differentiation of mouse myoblasts, while scaffolds incorporating gelatin alone sufficed for the differentiation of preadipocytes. RD-IPC provides for a scalable, continuous process that allows the incorporation of nutritional components into scaffolds possessing good mechanical and cell-adhesive properties. These fibrous scaffolds are constructed using edible polyelectrolyte components and aqueous solutions at relatively mild process conditions, underlining their promise for the nutritional enhancement of cultured meat.

A synergistic blend of Garcinia mangostana fruit rind and Cinnamomum tamala leaf extracts enhances myogenic differentiation and mitochondrial biogenesis in vitro and muscle growth and strength in mice.

A proprietary combination of Garcinia mangostana fruit rind and Cinnamomum tamala leaf extracts (LI80020F4, CinDura®) improved the physical performance and muscle strength of resistance-trained adult males. This study assessed the underlying mechanisms of the ergogenic potential of LI80020F4 in in vitro and in vivo models. The individual extracts and their combination (LI80020F4) were assessed for nitrite production in EAhy926 human endothelial cells. Subsequent experiments evaluated the effect of LI80020F4 in myotube formation in C2C12 mouse myoblasts, expression of mammalian target of rapamycin (mTOR) signaling proteins, myogenic factors, and mitochondrial functions in L6 rat myoblasts.Moreover, adult male ICR mice were randomly assigned (n = 15) into vehicle control (G1), exercise alone (G2), oxymetholone-16 mg/kg body weight (bw) (G3), and 75 (G4)-, 150 (G5)-, or 300 (G6) mg/kg bw of LI80020F4, orally gavaged for 28 days. G1 and G2 mice received 0.5% carboxymethylcellulose sodium. Following completion, muscle strength and physical performance were assessed on forelimb grip strength and forced swimming test (FST), respectively. Gastrocnemius (GA), tibialis anterior (TA) muscle weights, muscle fiber cross-sectional area (CSA), levels of muscle, and serum protein markers were also determined. LI80020F4 increased nitrite production in EAhy926 cells in a dose-dependent manner. LI80020F4 induced C2C12 myotube formation, increased mitochondrial biogenesis, upregulated the expressions of activated mTOR and other mitochondria and myogenic proteins, and mitigated H2O2-induced mitochondrial membrane depolarization in the myoblast cells. In the animal study, 75, 150, and 300 mg/kg bw LI80020F4 doses significantly (P < 0.05) increased the animals' forelimb grip strength. Mid- and high-dose groups showed increased swimming time, increased muscle weight, CSA, muscle growth-related, and mitochondrial protein expressions in the GA muscles. LI80020F4 increases nitric oxide production in the endothelial cells, mitochondrial biogenesis and function, upregulates skeletal muscle growth-related protein expressions and reduces oxidative stress; together, it explains the basis of the ergogenic potential of LI80020F4.

Dielectrophoretic Movement of Cell Passing Between Surface Electrodes in Flow Channel

Abstract In this study, cell behavior in a microchannel was tracked for the application of dielectrophoresis to biological cell sorting. A pair of titanium surface electrodes was fabricated inside the microchannel by photolithographic techniques: a triangular electrode with a tip angle of 0.26 rad and a rectangular electrode. A periodic alternating current of square wave with a period of 1 μs was introduced between the electrodes to induce an asymmetric electric field perpendicular to the mainstream direction. The behavior of mouse myoblasts (C2C12: mouse myoblast cell line) was measured in vitro while the suspension was flowing. The relationship between cell shift motion near the electrode and cell shape on the two-dimensional projection plane was investigated. Experimental results showed that cell movement in the direction perpendicular to the mainstream increased with geometries away from the circle in the two-dimensional projection plane. This method can be applied to sort cells according to their degree of shape deviation from a sphere. The dielectrophoretic effect can be applied to sort cells not only by cell size but also by cell deformation.

Myogenesis Effects of RGX365 to Improve Skeletal Muscle Atrophy.

Age-related skeletal muscle atrophy and weakness not only reduce the quality of life of those afflicted, but also worsen the prognosis of underlying diseases. We evaluated the effect of RGX365, a protopanaxatriol-type rare ginsenoside mixture, on improving skeletal muscle atrophy. We investigated the myogenic effect of RGX365 on mouse myoblast cells (C2C12) and dexamethasone (10 µM)-induced atrophy of differentiated C2C12. RGX365-treated myotube diameters and myosin heavy chain (MyHC) expression levels were analyzed using immunofluorescence. We evaluated the myogenic effects of RGX365 in aging sarcopenic mice. RGX365 increased myoblast differentiation and MyHC expression, and attenuated the muscle atrophy-inducing F-box (Atrogin-1) and muscle RING finger 1 (MuRF1) expression. Notably, one month of oral administration of RGX365 to 23-month-old sarcopenic mice improved muscle fiber size and the expression of skeletal muscle regeneration-associated molecules. In conclusion, rare ginsenosides, agonists of steroid receptors, can ameliorate skeletal muscle atrophy during long-term administration.

BMP9 functions as a negative regulator in the myogenic differentiation of primary mouse myoblasts.

BMP9, a member of the TGF-β superfamily, reveals the great translational promise for it has been shown to have the strong effect of osteogenic activity in vitro and in vivo. However, the implantation of certain BMPs (bone morphogenetic proteins) into muscular tissues induces ectopic bone formation. BMPs induce osteoblastic differentiation in skeletal muscle, suggesting that myogenic stem cells, such as myoblasts, are the potential progenitors of osteoblasts during heterotopic bone differentiation. Here, we investigate the role of BMP9 during primary mouse myoblasts differentiation. We found BMP9 enhanced cell proliferation and reduced myogenic differentiation of primary mouse myoblasts. In addition, adenovirus-mediated overexpression of BMP9 delayed muscle regeneration after BaCl2-induced injury. ALK1 knockdown reversed the inhibition of myoblast differentiation induced by BMP9. Our data indicate that BMP9 inhibits myogenic differentiation in primary mouse myoblasts and delays skeletal muscle regeneration after injury.

Fungus-derived protein particles as cell-adhesive matrices for cell-cultivated food

Cell-adhesive factors mediate adhesion of cells to substrates via peptide motifs such as the Arg–Gly–Asp (RGD) sequence. With the onset of sustainability issues, there is a pressing need to find alternatives to animal-derived cell-adhesive factors, especially for cell-cultivated food applications. In this paper, we show how data mining can be a powerful approach toward identifying fungal-derived cell-adhesive proteins and present a method to isolate and utilize these proteins as extracellular matrices (ECM) to support cell adhesion and culture in 3D. Screening of a protein database for fungal and plant proteins uncovered that ~5.5% of the unique reported proteins contain RGD sequences. A plot of fungi species vs RGD percentage revealed that 98% of the species exhibited an RGD percentage > = 1%. We observed the formation of protein particles in crude extracts isolated from basidiomycete fungi, which could be correlated to their stability towards particle aggregation at different temperatures. These protein particles were incorporated in 3D fiber matrices encapsulating mouse myoblast cells, showing a positive effect on cell alignment. We demonstrated a cell traction stress on the protein particles (from Flammulina velutipes) that was comparable to cells on fibronectin. A snapshot of the RGD-containing proteins in the fungal extracts was obtained by combining SDS-PAGE and mass spectrometry of the peptide fragments obtained by enzymatic cleavage. Therefore, a sustainable source of cell-adhesive proteins is widely available in the fungi kingdom. A method has been developed to identify candidate species and produce cell-adhesive matrices, applicable to the cell-cultivated food and healthcare industries.

Behavior of a Confluent Layer of Myoblasts Under Shear Flow

Abstract To investigate the effect of shear stress direction on the direction of cell activity in a confluent layer, the migration and deformation of cells oriented parallel or perpendicular to the direction of the shear flow field were optically tracked in vitro. A Couette-type shear flow between parallel walls was formed between the lower stationary culture dish and the upper rotating disk. Shear stress (&amp;lt;2 Pa) was set by adjusting the rotational speed of the upper disk. Myoblasts (C2C12: mouse myoblast cell line) were cultured in an incubator equipped with an inverted phase-contrast microscope under continuous shear flow for 7 days until confluency. Deformation and migration of each cell were tracked in time-lapse images. Analysis of these images showed that cells deform and migrate along their major axis even at confluency (whether the major axis of the cell is parallel or perpendicular to the shear stress field). As a result, the orientation of the major axis of the cell remains parallel or perpendicular to the shear stress field. This observation may be used to improve the development of engineered muscle tissue.