Effects of Hypoxia on Proliferation and Differentiation in Belgian Blue and Hanwoo Muscle Satellite Cells for the Development of Cultured Meat.

Among future food problems, the demand for meat is expected to increase rapidly, but the production efficiency of meat, which is a protein source, is very low compared to other foods. To address this problem, research on the development and production of cultured meat as an alternative meat source using muscle stem cells in vitro has recently been undertaken. Many studies have been conducted on myosatellite cells for medical purposes, but studies on alternative meat production are rare. In vitro cell culture mimics the in vivo environment for cell growth. The satellite cell niche is closer to hypoxic (2% O2) than normoxic (20% O2) conditions. The aim of this study was to investigate the efficient oxygen conditions of myosatellite cell cultures for the production of cultured meat. The bovine satellite cell counts and mRNA (Pax7, Myf5 and HIF1α) levels were higher in hypoxia than normoxia (p < 0.05). Through Hoechst-positive nuclei counts, and expression of Pax7, MyoD and myosin protein by immunofluorescence, it was confirmed that muscle cells performed normal proliferation and differentiation. Myoblast fusion was higher under hypoxic conditions (p < 0.05), and the myotube diameters were also thicker (p < 0.05). In the myotube, the number of cells was high in hypoxia, and the expression of the total protein amounts, differentiation marker mRNA (myogenin, myosin and TOM20), and protein markers (myosin and TOM20) was also high. The study results demonstrated that the proliferation and differentiation of bovine myosatellite cells were promoted more highly under hypoxic conditions than under normoxic conditions. Therefore, hypoxic cultures that promote the proliferation and differentiation of bovine myosatellite cells may be an important factor in the development of cultured meat.

Epithelial-mesenchymal transition induced by MyoD inhibits growth of high metastatic colorectal cancer.

OBJECTIVES: The study aimed to investigate the tumor-suppressing factor myogenicity differentiation factor (MyoD) against high metastatic colorectal cancer through its powerful transformation by which the tumor cells were converted into muscle cells or other cells to inhibit the malignant proliferation of tumor cells. METHODS: The roles of MyoD in colon cancer proliferation, invasion and migration were analyzed by CCK-8 assay and Transwell, and EMT by real-time PCR and Western blot. The secretion of TGFß1 was assayed by ELISA and activation of p-Smad2/3 were assayed by western blot. The effects of MyoD on intestinal cancer growth and EMT in vivo were also analyzed. RESULTS: We found MyoD inhibited the proliferation, invasion and migration of colon cancer cell. Moreover, MyoD inhibited the expression of E-cadherin and promoted the expression of vimentin and α-SMA. The secretion of TGFß1 increased and p-Smad2/3 was activated after MyoD expression. MyoD also inhibits intestinal cancer growth and promoted EMT in vivo. CONCLUSION: Our findings indicate that MyoD inhibited cancer progression and metastasis by promoting EMT through TGF-ß1/Smad2/3 activation, which provide new support for MyoD maybe as a novel anti-cancer method for the treatment of colon cancer in the future.

Tat-MyoD fused proteins, together with C2c12 conditioned medium, are able to induce equine adult mesenchimal stem cells towards the myogenic fate.

The Tat protein is able to translocate through the plasma membrane and when it is fused with other peptides may acts as a protein transduction system. This ability appears particularly interesting to induce tissue-specific differentiation when the Tat protein is associated to transcription factors. In the present work, the potential of the complex Tat-MyoD in inducing equine peripheral blood mesenchymal stem cells (PB-MSCs) towards the myogenic fate, was evaluated. Results showed that the internalization process of Tat-MyoD happens only in serum free conditions and that the nuclear localization of the fused complex is observed after 15 hours of incubation. However, the supplement of Tat-MyoD only was not sufficient to induce myogenesis and, therefore, in order to achieve the myogenic differentiation of PB-MSCs, conditioned medium from C2C12 cells was added without direct contact. Real Time PCR and immunofluorescence methods evaluated the establishment of a myogenic program. Our results suggest that TAT- transduction of Tat-MyoD, when supported by conditioned medium, represents a useful methodology to induce myogenic differentiation.

Oral mucosal progenitor cell clones resist in vitro myogenic differentiation.

Progenitor cells derived from the oral mucosa lamina propria (OMLP-PCs) demonstrate an ability to differentiate into tissue lineages removed from their anatomical origin. This clonally derived population of neural-crest cells have demonstrated potential to differentiate along mesenchymal and neuronal cell lineages. OBJECTIVE: Significant efforts are being made to generate functioning muscle constructs for use in research and clinical tissue engineering. In this study we aimed to determine the myogenic properties of clonal populations of expanded OMLP-PCs. DESIGN: PCs were subject to several in vitro culture conditions in an attempt to drive myogenic conversion. Methodologies include use of demethylation gene-modifying reagents, mechanical conditioning of tissue culture substrates, tuneable polyacrylamide gels and a 3-dimensional construct as well as published myogenic media compositions. PCR and immunostaining for the muscle cell markers Desmin and MyoD1 were used to assess muscle differentiation. RESULTS: The clones tested did not intrinsically express myogenic lineage markers. Despite use of two and 3-dimensional pre-published in vitro culture protocols OMLP clones could not be differentiated down a myogenic lineage. CONCLUSIONS: Within the confines of these experimental parameters it was not possible to generate identifiable muscle using the clonal populations. When reviewing the previously successful reports of myogenic conversion, cells utilised have either been derived from tissues that are already 'primed' with the requisite myogenic genetic potential or have undergone specific genetic reprogramming to enhance the myogenic conversion rate. This, along with as yet unidentified stromal interplay, may therefore be required for positive myogenic differentiation to be realised.

Nanolipodendrosome-loaded glatiramer acetate and myogenic differentiation 1 as augmentation therapeutic strategy approaches in muscular dystrophy.

BACKGROUND: [Corrected] Muscular dystrophies consist of a number of juvenile and adult forms of complex disorders which generally cause weakness or efficiency defects affecting skeletal muscles or, in some kinds, other types of tissues in all parts of the body are vastly affected. In previous studies, it was observed that along with muscular dystrophy, immune inflammation was caused by inflammatory cells invasion - like T lymphocyte markers (CD8+/CD4+). Inflammatory processes play a major part in muscular fibrosis in muscular dystrophy patients. Additionally, a significant decrease in amounts of two myogenic recovery factors (myogenic differentation 1 [MyoD] and myogenin) in animal models was observed. The drug glatiramer acetate causes anti-inflammatory cytokines to increase and T helper (Th) cells to induce, in an as yet unknown mechanism. MyoD recovery activity in muscular cells justifies using it alongside this drug. METHODS: In this study, a nanolipodendrosome carrier as a drug delivery system was designed. The purpose of the system was to maximize the delivery and efficiency of the two drug factors, MyoD and myogenin, and introduce them as novel therapeutic agents in muscular dystrophy phenotypic mice. The generation of new muscular cells was analyzed in SW1 mice. Then, immune system changes and probable side effects after injecting the nanodrug formulations were investigated. RESULTS: The loaded lipodendrimer nanocarrier with the candidate drug, in comparison with the nandrolone control drug, caused a significant increase in muscular mass, a reduction in CD4+/CD8+ inflammation markers, and no significant toxicity was observed. The results support the hypothesis that the nanolipodendrimer containing the two candidate drugs will probably be an efficient means to ameliorate muscular degeneration, and warrants further investigation.

Involvement of MyoD and PEA3 in regulation of transcription activity of MDR1 gene.

Overexpression of multidrug resistance 1 (MDR1) in cancer remains one of the major causes for the failure of chemotherapy. In the present study, we found that MyoD and PEA3 could activate P-glycoprotein (P-gp) expression in SGC7901 cells. Knockdown of MyoD and PEA3 attenuated MDR1 expression and increased the sensitivity of multidrug resistant cancer cells to cytotoxic drugs that were transported by P-gp in SGC7901/VCR cells. MyoD or PEA3 could bind to the E-box and PEA3 sites on the MDR1 promoter and activate its transcription. The regulation of MDR1 expression by MyoD and PEA3 may provide potential ways to overcome MDR in cancer treatment.

Desmin immunolocalisation in autosomal dominant Emery-Dreifuss muscular dystrophy.

Autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) is one of a number of allelic disorders caused by mutations in the nuclear lamina proteins, lamins A and C. The disorder is characterised by the early onset of skeletal muscle weakness and joint contractures and later, by dilated cardiomyopathy and cardiac arrythmias. Although the pathophysiology is not understood, one theory suggests that disordered structural organisation at weakened nuclei in contractile cells may underlie the disease. Previous work shows that mice deficient in lamin A/C develop similar skeletal and cardiac muscle signs to patients with AD-EDMD and ultrastructural examination of muscle from these mice shows abnormal localisation of desmin. We hypothesised therefore that desmin localisation may be abnormal in muscle or cells from patients with AD-EDMD and/or in cells expressing mutant lamins. In order to evaluate this, desmin immunolocalisation was determined in skeletal muscle biopsy sections from patients with AD-EDMD and cell lines including MyoD-transfected fibroblast-derived myotubes from AD-EDMD patients and murine embryonic stem cell-derived cardiomyocytes stably transfected with mutant human lamin A. Ultrastructural examination of patient muscle was also performed. Desmin was expressed and localised normally in patient muscle and cell lines and ultrastructural examination was similar to controls. These results fail to provide any evidence that dominant mutations in lamin A/C lead to a disorganisation of the desmin associated cytoskeleton.

[In vitro differentiation of rat mesenchymal stem cells into skeletal muscle cells induced by myoblast differentiation factor and 5-azacytidine].

OBJECTIVE: To explore the in vitro differentiation of the rat mesenchymal stem cells (MSCs) into the skeletal muscle cells induced by the myoblast differentiation factor (MyoD) and 5-azacytidine. METHODS: The MSCs were taken from the rat bone marrow and the suspension of MSCs was made and cultured in the homeothermia incubator which contained 5% CO2 at 37 degrees C. The cells were observed under the inverted phase contrast microscope daily. The cells spreading all the bottom of the culture bottle were defined as one passage. The differentiation of the 3rd passage of MSCs was induced by the combination of 5-azacytidine, MyoD, transforming growth factor beta1, and the insulin-like growth factor 1. Nine days after the induction, the induced MSCs were collected, which were analyzed with the MTT chromatometry, the flow cytometry, and the immunohistochemistry. RESULTS: The primarily cultured MSCs grew as a colony on the walls of the culture bottle; after the culture for 5-7 days, the cells were shaped like the fibroblasts, the big flat polygonal cells, the medium-sized polygonal cells, and the small triangle cells; after the culture for 12 days, the cells were found to be fused, spreading all over the bottle bottom, but MSCs were unchanged too much in shape. After the induction by 5-azacytidine, some of the cells died, and the cells grew slowly. However, after the culture for 7 days, the cells grew remarkably, the cell volume increased gradually in a form of ellipse, fusiform or irregularity. After the culture for 14 days, the proliferated fusiform cells began to increase in a great amount. After the culture for 18-22 days, the myotubes increased in number and volume, with the nucleus increased in number, and the newly-formed myotubes and the fusiform myoblst grew parallelly and separately. The immunohistochemistry for MSCs revealed that CD44 was positive in reaction, with the cytoplasm in a form of brown granules. And the nucleus had an obvious border, and CD34 was negative. The induced MSCs were found to be positive for desmin and specific myoglobulin of the skeletal muscle. The flow cytometry showed that most of the MSCs and the induced MSCs were in the stages of G0/G1, accounting for 79.4% and 62.9%, respectively; however, the cells in the stages of G2/S accounted for 20.6% and 36.1%. The growth curve was drawn based on MTT,which showed that MSCs were greater in the growth speed than the induced MSCs. The two kinds of cells did not reach the platform stage,having a tendency to continuously proliferate. CONCLUSION: In vitro, the rat MSCs can be differentiated into the skeletal muscle cells with an induction by MyoD and 5-azacytidine, with a positive reaction for the desmin and the myoglobulin of the skeletal muscle. After the induction, the proliferation stage of MSCs can be increased, with a higher degree of the differentiation into the skeletal muscle.

Permissive roles of phosphatidyl inositol 3-kinase and Akt in skeletal myocyte maturation.

Skeletal muscle differentiation, maturation, and regeneration are regulated by interactions between signaling pathways activated by hormones and growth factors, and intrinsic genetic programs controlled by myogenic transcription factors, including members of the MyoD and myocyte enhancer factor 2 (MEF2) families. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo, and in the maintenance and hypertrophy of mature muscle in the adult, but the precise signaling pathways responsible for these effects remain incompletely defined. To study mechanisms of IGF action in muscle, we have developed a mouse myoblast cell line termed C2BP5 that is dependent on activation of the IGF-I receptor and the phosphatidyl inositol 3-kinase (PI3-kinase)-Akt pathway for initiation of differentiation. Here, we show that differentiation of C2BP5 myoblasts could be induced in the absence of IGF action by recombinant adenoviruses expressing MyoD or myogenin, but it was reversibly impaired by the PI3-kinase inhibitor LY294002. Similar results were observed using a dominant-negative version of Akt, a key downstream component of PI3-kinase signaling, and also were seen in C3H 10T1/2 fibroblasts. Inhibition of PI3-kinase did not prevent accumulation of muscle differentiation-specific proteins (myogenin, troponin T, or myosin heavy chain), did not block transcriptional activation of E-box containing muscle reporter genes by MyoD or myogenin, and did not inhibit the expression or function of endogenous MEF2C or MEF2D. An adenovirus encoding active Akt could partially restore terminal differentiation of MyoD-expressing and LY294002-treated myoblasts, but the resultant myofibers contained fewer nuclei and were smaller and thinner than normal, indicating that another PI3-kinase-stimulated pathway in addition to Akt is required for full myocyte maturation. Our results support the idea that an IGF-regulated PI3-kinase pathway functions downstream of or in parallel with MyoD, myogenin, and MEF2 in muscle development to govern the late steps of differentiation that lead to multinucleated myotubes.

Reciprocal inhibition between MyoD and STAT3 in the regulation of growth and differentiation of myoblasts.

The development of myoblasts is regulated by various growth factors as well as by intrinsic muscle-specific transcriptional factors. In this study, we analyzed the roles for STAT3 in the growth and differentiation of myoblasts in terms of cell cycle regulation and interaction with MyoD using C2C12 cells. Here we found that STAT3 inhibited myogenic differentiation induced by low serum or MyoD as efficiently as the Ras/mitogen-activated protein kinase cascade. As for this mechanism, we found that STAT3 not only promoted cell cycle progression through the induction of c-myc but also inhibited MyoD activities through direct interaction. STAT3 inhibited not only DNA binding activities of MyoD but also its transcriptional activities. However, the inhibited transcriptional activities were restored by the supplement of p300/CBP and PCAF, suggesting that STAT3 might deprive MyoD of these transcriptional cofactors. In addition, we found that MyoD inhibited DNA binding activities of STAT3, thereby inhibiting STAT3-dependent cell growth and survival of Ba/F3 cells. These results suggest that the development of muscle cells is regulated by the coordination of cytokine signals and intrinsic transcription factors.

Transactivation of capn2 by myogenic regulatory factors during myogenesis.

The calcium-activated cysteine protease m-calpain plays a pivotal role during the earlier stages of myogenesis, particularly during fusion. The enzyme is a heterodimer, encoded by the genes capn2, for the large subunit, and capn4, for the small subunit. To study the regulation of m-calpain, the DNA sequence upstream of capn2 was analyzed for promoter elements, revealing the existence of five consensus-binding sites (E-box) for several myogenic regulatory factors and one binding site for myocyte enhancer factor-2 (MEF-2). Transient transfections with reporter gene constructs containing the E-box revealed that MyoD presents a high level of transactivation of reporter constructs containing this region, in particular the sequences including the MEF-2/E4-box. In addition, over-expression of various myogenic factors demonstrated that MyoD and myogenin with much less efficiency, can up-regulate capn2, both singly and synergistically, while Myf5 has no effect on synthesis of the protease. Experiments with antisense oligonucleotides directed against each myogenic factor revealed that MyoD plays a specific and pivotal role during capn2 regulation, and cannot be replaced wholly by myogenin and Myf5.

Alpha-tropomyosin gene expression in Xenopus laevis: differential promoter usage during development and controlled expression by myogenic factors.

Tropomyosins (TMs) constitute a group of contractile proteins encoded by a multigene family showing distinct cell-type-specific and developmental expression patterns. In mammals and birds, the alpha-TM gene is the most complex and can produce several muscle and non-muscle isoforms. We report here the characterization of the 5' region of the Xenopus laevis alpha-TM gene and its developmental expression. The 5' region of the gene is structurally related to the avian and mammalian cognates and presents two promoters flanking a pair of alternatively spliced exons, 2a/2b, where exon 2a is a smooth-muscle-specific exon. The internal promoter is used to generate a non-muscle low molecular weight TM whilst muscle TM isoforms originate from the distal promoter. RNase protection analysis shows that the two promoters have distinct temporal programs of activation. The internal promoter is activated early in oogenesis and non-muscle transcripts are found throughout oogenesis, embryogenesis and in adult tissues. Only low molecular weight non-muscle TM-encoding mRNAs are expressed in oogenesis. The distal promoter is silent during oogenesis, and the skeletal muscle alpha-TM transcripts accumulate from stage 15 in the embryo and are expressed in adult striated muscle tissues. In situ hybridization indicates that these transcripts are expressed in both the somites and heart of the embryo. Ectopic expression of myogenic factors, but not the MEF2 myocyte-specific enhancer factor 2 factors SL1 and SL2, can induce the expression of the alpha-TM gene suggesting that the gene is a direct target for myogenic but not for MEF2 factors. The amphibian alpha-TM gene constitutes a gene marker for studying the developmental control expression of muscle genes in the different myogenic lineages.

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures.

We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.

MyoD induces retinoblastoma gene expression during myogenic differentiation.

In skeletal muscle cells permanent withdrawal from the cell cycle is a prerequisite for terminal differentiation. The muscle-specific transcription factor MyoD can activate downstream muscle structural genes and myogenic conversion in many different cell types. It has been demonstrated that the product of the retinoblastoma susceptibility gene, with its growth-suppressive activity, is involved in the myogenic function of MyoD (Caruso et al., 1993; Gu et al., 1993). The present study characterises the modulation of retinoblastoma (Rb1) mRNA levels during myogenic differentiation of the murine C2 cell line and provides evidence that the muscle-specific regulatory factor MyoD enhances Rb1 gene transcription. We demonstrate that MyoD mediates the transactivation of a CAT construct whose expression is driven by the human Rb1 gene promoter, and that this is not a consequence of direct binding of MyoD to an E-box DNA sequence motif present in the Rb1 promoter sequences. In addition we have tested the capability of several MyoD mutant proteins of inducing the Rb1 promoter CAT construct. Our results indicate that the MyoD function required for induction of Rb1 promoter activity is distinct from its myogenic function.