Differentiation Of Skeletal Cells Research Articles

Effect of IGF1 on Myogenic Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells Through PI3K/AKT Signaling Pathway

Effect of IGF1 on Myogenic Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells Through PI3K/AKT Signaling Pathway

Bioactive Coatings on 3D Printed Polycaprolactone Scaffolds for Bone Regeneration: A Novel Murine Femur Defect Model for Examination of the Biomaterial Capacity for Repair

AbstractBone tissue engineering seeks to develop treatment approaches for nonhealing and large bone defects. An ideal biodegradable scaffold will induce and support bone formation. The current study examines bone augmentation in critical‐sized bone defects, using functionalized scaffolds, with the hypothesized potential to induce skeletal cell differentiation. 3D printed, porous poly(caprolactone) trimethacrylate (PCL‐TMA900) scaffolds are applied within a murine femur defect, stabilized by a polyimide intramedullary (IM) pin. The PCL‐TMA900 scaffolds are coated with i) elastin‐like polypeptide (ELP), ii) poly(ethyl acrylate) (PEA)/fibronectin (FN)/bone morphogenetic protein‐2 (PEA/FN/BMP‐2), iii) both ELP and PEA/FN/BMP‐2, or iv) Laponite nanoclay binding BMP‐2. Sequential microcomputed tomography (µCT) and histological analysis are performed. PCL‐TMA900 is robust and biocompatible and when coated with the nanoclay material Laponite and BMP‐2 induce consistent, significant bone formation compared to the uncoated PCL‐TMA900 scaffold. Critically, the BMP‐2 is retained, due to the Laponite, producing bone around the scaffold in the desired shape and volume, compared to bone formation observed with the positive control (collagen sponge/BMP‐2). The ELP and/or PEA/FN/BMP‐2 scaffolds do not demonstrate significant or consistent bone formation. In summary, Laponite/BMP‐2 coated PCL‐TMA900 scaffolds offer a biodegradable, osteogenic construct for bone augmentation with potential for development into a large scale polymer scaffold for clinical translation.

Identification of novel transcription factors regulated by H3K27 acetylation in myogenic differentiation of porcine skeletal muscle satellite cells.

H3K27 acetylation (H3K27ac) is crucial in muscle development as it regulates gene expression. Dysregulation of H3K27ac level has been linked to muscle-related diseases such as Duchenne muscular dystrophy, yet the mechanisms through which H3K27ac influences myogenic differentiation are not fully understood. Here, we utilized the SGC-CBP30 drug, a CBP/p300 bromodomain inhibitor, to reduce H3K27ac level and investigated its effect on myogenic differentiation of porcine skeletal muscle satellite cells. The results demonstrated an increased H3K27ac level during normal muscle satellite cell differentiation. We found that the addition of SGC-CBP30 resulted in a reduced level of H3K27ac based on ATAC-seq and CUT&Tag data. Our analysis revealed that a cluster characterized by reduced levels of H3K27ac and increased levels of H3K27me3 was enriched with motifs corresponding to Bach2, MafK, and Fosl2 transcription factors. Furthermore, knockdown of Bach2, MafK, and Fosl2 produced a similar suppression effect on myogenic differentiation. Taken together, our study contributes to a better understanding of how H3K27ac influences myogenic differentiation.

Effect of NR1D1 on the proliferation and differentiation of yak skeletal muscle satellite cells.

The severe conditions at high altitudes, where yaks inhabit, contribute to delayed muscular growth and compromised tenderness of their muscle tissue. Myosatellite cells are responsible for the growth and regeneration of skeletal muscle after birth and have the potential to proliferate and differentiate, its development is closely related to meat quality, and the nuclear receptor gene NR1D1 is involved in muscle formation and skeletal muscle regulation. Therefore, in order to understand the effect of NR1D1 on muscle satellite cells, we identified the mRNA expression levels of marker genes specifically expressed in muscle satellite cells at different stages to determine the type of cells isolated. Eventually, we successfully constructed a primary cell line of yak muscle satellite cells. Then we constructed NR1D1 overexpression vector and interference RNA, and introduced them into isolated yak skeletal muscle satellite cells. We performed qPCR, CCK8, and fluorescence-specific to detect the expression of genes or abundance of proteins as markers of cell proliferation and differentiation. Compared with those in the control group, the expression levels of proliferation marker genes KI-67, CYCLIND1, and CYCLINA were significantly inhibited after NR1D1 overexpression, which was also supported by the CCK-8 test, whereas differentiation marker genes MYOD, MYOG, and MYF5 were significantly inhibited. Fluorescence-specific staining showed that KI-67 protein abundance and the number of microfilaments both decreased, while the opposite trend was observed after NR1D1 interference. In conclusion, we confirmed that NR1D1 inhibited the proliferation and differentiation of yak skeletal muscle satellite cells, which provides a theoretical basis for further research on the effect of NR1D1 on improving meat quality traits and meat production performance of yaks.

Asparagine synthetase regulates the proliferation and differentiation of chicken skeletal muscle satellite cells.

Asparagine synthetase (ASNS) is an aminotransferase responsible for the biosynthesis of aspartate by using aspartic acid and glutamine. ASNS is highly expressed in fast-growing broilers, but few studies have reported the regulatory role of ASNS in muscle development. To explore the function of ASNS in chicken muscle development, the expression of ASNS in different chicken breeds and tissues were first performed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Then, using real-time quantitative RT-PCR, western blot, EdU assay, cell cycle assay and immunofluorescence, the effects of ASNS on the proliferation and differentiation of chicken skeletal muscle satellite cell (SMSC) were investigated. Finally, potential mechanisms by which ASNS influences chicken muscle fiber differentiation were identified through RNA-Seq. The mRNA expression pattern of ASNS in muscles mirrors trends in muscle fiber cross-sectional area, average daily weight gain, and muscle weight across different breeds. ASNS knockdown inhibited SMSC proliferation, while overexpression showed the opposite. Moreover, ASNS attenuated SMSC differentiation by activating the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway. Additionally, 5-aminoimidazole4-carboxamide1-β-D-ribofuranoside (AICAR) treatment suppressed the cell differentiation induced by siRNA-ASNS. RNA-Seq identified 1,968 differentially expressed genes (DEGs) during chicken SMSC differentiation when overexpression ASNS. Gene ontology (GO) enrichment analysis revealed that these DEGs primarily participated in 8 biological processes, 8 cellular components, and 4 molecular functions. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis identified several significantly enriched signaling pathways, such as the JAK-STAT signaling pathway, tumor necrosis factor signaling pathway, toll-like receptor signaling pathway, and PI3K-Akt signaling pathway. ASNS promotes proliferation while inhibits the differentiation of chicken SMSCs. This study provides a theoretical basis for studying the role of ASNS in muscle development.

Integrative analysis of whole genome bisulfite and transcriptome sequencing reveals the effect of sodium butyrate on DNA methylation in the differentiation of bovine skeletal muscle satellite cells

Butyric acid as a short-chain fatty acid (SCFA) is one of the key microbial metabolites of ruminants. Numerous studies indicate that butyrate is crucial in muscle growth and development, and plays an important molecular regulatory role mainly by inhibiting histone deacetylation. DNA methylation, a major epigenetic modification, is involved in cell differentiation. Butyrate, in addition to its role in acetylation modifications, can alter the DNA methylation status of cells. However, the impact of butyrate on the DNA methylation of bovine skeletal muscle satellite cells (SMSCs) remains unclear. In this study, we developed a differentiation model of SMSCs and employed RNA sequencing (RNA-seq) alongside whole genome bisulfite sequencing (WGBS) to explore the effects of butyrate treatment on DNA methylation status and its relationship with gene expression. Treatment of SMSCs with sodium butyrate (NaB) at 1.0 mM for 2 days significantly inhibited the expression of DNA methyltransferases (DNMT1, DNMT2, DNMT3A) at the mRNA and protein levels while promoting the expression of demethylases (TET1, TET2, TET3) at mRNA levels. WGBS identified 4292 differentially methylated regions (DMRs), comprising 2294 hypermethylated and 1998 hypomethylated regions. These DMRs were significantly enriched in the MAPK, cAMP, and Wnt signaling pathways, all of which are implicated in myogenesis and development. Combining RNA-seq and WGBS data revealed a total of 130 overlapping genes, including MDFIC, CREBBP, DMD, LTBP2 and KLF4. These genes are predominantly involved in regulating the FoxO, MAPK, PI3K-Akt, and Wnt signaling pathways. This study provides new insights into the effects of butyrate-mediated DNA methylation on SMSC development and enhances our understanding of butyrate as an epigenetic modifier beyond its role in acetylation.

Effects of rumen metabolite butyric acid on bovine skeletal muscle satellite cells proliferation, apoptosis and transcriptional states during myogenic differentiation

Butyric acid, a pivotal short-chain fatty acid in rumen digestion, profoundly influences animal digestive and locomotor systems. Extensive research indicates its direct or indirect involvement in the growth and development of muscle and fat cells. However, the impact of butyric acid on the proliferation and differentiation of bovine skeletal muscle satellite cells (SMSCs) remains unclear. This study aimed to elucidate the effects of butyrate on SMSCs proliferation and differentiation. After isolating, SMSCs were subjected to varying concentrations of sodium butyrate (NaB) during the proliferation and differentiation stages. Optimal treatment conditions (1 mM NaB for 2 days) were determined based on proliferative force, cell viability, and mRNA expression of proliferation and differentiation marker genes. Transcriptome sequencing was employed to screen for differential gene expression between 1 mM NaB-treated and untreated groups during SMSCs differentiation. Results indicated that lower NaB concentrations (≤1.0 mM) inhibited proliferation while promoting differentiation and apoptosis after a 2-day treatment. Conversely, higher NaB concentrations (≥2.0 mM) suppressed proliferation and differentiation and induced apoptosis. Transcriptome sequencing revealed differential expression of genes(ND1, ND3, CYTB, COX2, ATP6, MYOZ2, MYOZ3, MYBPC1 and ATP6V0A4,etc.) were associated with SMSCs differentiation and energy metabolism, enriching pathways such as Oxidative phosphorylation, MAPK, and Wnt signaling. These findings offer valuable insights into the molecular mechanisms underlying butyrate regulation of bovine SMSCs proliferation and differentiation, as well as muscle fiber type conversion in the future study.

The proliferation and differentiation of skeletal muscle stem cells are enhanced in a bioreactor.

Skeletal muscle (SKM) is the largest organ in mammalian body and it can repair damages by using the residential myogenic stem cells (MuSC), but this repairing capacity reduces with age and in some genetic muscular dystrophy. Under these circumstances, artificial amplification of autologous MuSC in vitro might be necessary to repair the damaged SKM. The amplification of MuSC is highly dependent on myogenic signals, such as sonic hedgehog (Shh), Wnt3a, and fibroblast growth factors, so formulating an optimum myogenic kit composed of specific myogenic signals might increase the proliferation and differentiation of MuSC efficiently. In this study, various myogenic signals have been tested on C2C12 myoblasts and primary MuSC, and a myogenic kit consists of insulin, lithium chloride, T3, and retinoic acid has been formulated, and we found it significantly increased the fusion index and MHC expression level of both C2C12 and MuSC myotubes. A novel bioreactor providing cyclic stretching (CS) and electrical stimulation (ES) has been fabricated to enhance the myogenic differentiation of both C2C12 and MuSC. We further found that coating the bioreactor substratum with collagen gave the best effect on proliferation and differentiation of MuSC. Furthermore, combining the collagen coating and physical stimuli (CS + ES) in the bioreactor can generate more proliferative primary MuSC cells. Our results have demonstrated that the combination of myogenic kit and bioreactor can provide environment for efficient MuSC proliferation and differentiation. These MuSC and mature myotubes amplified in the bioreactor might be useful for clinical grafting into damaged SKM in the future.

ADAR1 Promotes Myogenic Proliferation and Differentiation of Goat Skeletal Muscle Satellite Cells.

As one of the most important economic traits for domestic animal husbandry, skeletal muscle is regulated by an intricate molecular network. Adenosine deaminase acting on RNA (ADAR1) involves various physiological processes and diseases, such as innate immunity and the development of lung adenocarcinoma, breast cancer, gastric cancer, etc. However, its role in skeletal muscle growth requires further clarification. Here, we explored the functions of ADAR1 in the myogenic process of goat skeletal muscle satellite cells (MuSCs). The ADAR1 transcripts were noticeably enriched in goat visceral tissues compared to skeletal muscle. Additionally, its levels in slow oxidative muscles like the psoas major and minor muscles were higher than in the fast oxidative glycolytic and fast glycolytic muscles. Among the two common isoforms from ADAR1, p110 is more abundant than p150. Moreover, overexpressing ADAR1 enhanced the proliferation and myogenic differentiation of MuSCs. The mRNA-seq performed on MuSCs' knockdown of ADAR1 obtained 146 differentially expressed genes (DEGs), 87 upregulated and 59 downregulated. These DEGs were concentrated in muscle development and process pathways, such as the MAPK and cAMP signaling pathways. Furthermore, many DEGs as the key nodes defined by protein-protein interaction networks (PPI), including STAT3, MYH3/8, TGFβ2, and ACTN4, were closely related to the myogenic process. Finally, RNA immunoprecipitation combined with qPCR (RIP-qPCR) showed that ADAR1 binds to PAX7 and MyoD mRNA. This study indicates that ADAR1 promotes the myogenic development of goat MuSCs, which provides a useful scientific reference for further exploring the ADAR1-related regulatory networks underlying mammal skeletal muscle growth.

Role of circPAPD7 in regulating proliferation and differentiation of goat skeletal muscle satellite cells

The circular RNA (circRNA) plays a crucial role in various biological processes, particularly posttranscriptional regulation. However, the role of circRNA in the development of goat skeletal muscle has not been thoroughly explored. Here, we identified circPAPD7, which is a novel circular RNA that is preferentially expressed in the skeletal muscle. Functional assays demonstrated that circPAPD7 promoted proliferation and inhibited differentiation in goat skeletal muscle satellite cells (MuSCs). Mechanistically, it was discovered that circPAPD7 interacts with miR-26a-5p. Moreover, the rescue experiments indicated that the overexpression of circPAPD7 may reverse the inhibitory impact of miR-26a-5p on myoblast proliferation and the accelerated effects on differentiation. Furthermore, we provided evidence that circPAPD7 functions as a sponge for miR-26a-5p, thereby facilitating the upregulation of EZH2 expression in goat MuSCs. Together, the results revealed that circPAPD7 promote proliferation and inhibit differentiation of goat MuSCs via the miR-26a-5p/EZH2 pathway.

Role and Regulatory Mechanism of circRNA_14820 in the Proliferation and Differentiation of Goat Skeletal Muscle Satellite Cells.

Skeletal muscle satellite cells (SMSCs), a type of myogenic stem cell, play a pivotal role in postnatal muscle regeneration and repair in animals. Circular RNAs (circRNAs) are a distinct class of non-coding RNA molecules capable of regulating muscle development by modulating gene expression, acting as microRNAs, or serving as protein decoys. In this study, we identified circ_14820, an exonic transcript derived from adenosine triphosphatase family protein 2 (ATAD2), through initial RNA-Seq analysis. Importantly, overexpression of circ_14820 markedly enhanced the proliferation of goat SMSCs while concomitantly suppressing their differentiation. Moreover, circ_14820 exhibited predominant localization in the cytoplasm of SMSCs. Subsequent small RNA and mRNA sequencing of circ_14820-overexpressing SMSCs systematically elucidated the molecular regulatory mechanisms associated with circ_14820. Our preliminary findings suggest that the circ_14820-miR-206-CCND2 regulatory axis may govern the development of goat SMSCs. These discoveries contribute to a deeper understanding of circRNA-mediated mechanisms in regulating skeletal muscle development, thereby advancing our knowledge of muscle biology.

The L-ascorbic acid increases proliferation and differentiation of Yanbian cattle skeletal muscle satellite cells by activating the Akt/mTOR/P70S6K signaling pathway

The L-ascorbic acid increases proliferation and differentiation of Yanbian cattle skeletal muscle satellite cells by activating the Akt/mTOR/P70S6K signaling pathway

Rankl genetic deficiency and functional blockade undermine skeletal stem and progenitor cell differentiation

BackgroundSkeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation.MethodsTo dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl−/−) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs).ResultsWe found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl−/− mice as compared to wild-type. Rankl−/− mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl−/− SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures.ConclusionsWe provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.

Role of ubiquitination in the occurrence and development of osteoporosis (Review).

The ubiquitin (Ub)‑proteasome system (UPS) plays a pivotal role in maintaining protein homeostasis and function to modulate various cellular processes including skeletal cell differentiation and bone homeostasis. The Ub ligase E3 promotes the transfer of Ub to the target protein, especially transcription factors, to regulate the proliferation, differentiation and survival of bone cells, as well as bone formation. In turn, the deubiquitinating enzyme removes Ub from modified substrate proteins to orchestrate bone remodeling. As a result of abnormal regulation of ubiquitination, bone cell differentiation exhibits disorder and then bone homeostasis is affected, consequently leading to osteoporosis. The present review discussed the role and mechanism of UPS in bone remodeling. However, the specific mechanism of UPS in the process of bone remodeling is still not fully understood and further research is required. The study of the mechanism of action of UPS can provide new ideas and methods for the prevention and treatment of osteoporosis. In addition, the most commonly used osteoporosis drugs that target ubiquitination processes in the clinic are discussed in the current review.

IFRD2, a target of miR-2400, regulates myogenic differentiation of bovine skeletal muscle satellite cells via decreased phosphorylation of ERK1/2 proteins.

The proliferation and differentiation of skeletal muscle satellite cells is a complex physiological process involving various transcription factors and small RNA molecules. This study aimed to understand the regulatory mechanisms underlying these processes, focusing on interferon-related development factor 2 (IFRD2) as a target gene of miRNA-2400 in bovine skeletal MuSCs (MuSCs). IFRD2 was identified as a target gene of miRNA-2400 involved in regulating the proliferation and differentiation of bovine skeletal MuSCs. Our results indicate that miR-2400 can target binding the 3'UTR of IFRD2 and inhibit its translation. mRNA and protein expression levels of IFRD2 increased significantly with increasing days of differentiation. Moreover, overexpression of the IFRD2 gene inhibited proliferation and promoted differentiation of bovine MuSCs. Conversely, the knockdown of the gene had the opposite effect. Overexpression of IFRD2 resulted in the inhibition of ERK1/2 phosphorylation levels in bovine MuSCs, which in turn promoted differentiation. In summary, IFRD2, as a target gene of miR-2400, crucially affects bovine skeletal muscle proliferation and differentiation by precisely regulating ERK1/2 phosphorylation.

Bioinformatics Study on Mechanism of Postnatal Development of Craniofacial Bone.

The postnatal development of craniofacial bone plays a crucial role in shaping the overall structure and functionality of the skull and face. Understanding the underlying mechanisms of this intricate process is essential for both clinical and research purposes. In this study, the authors conducted a bioinformatics analysis using the Gene Expression Omnibus database to investigate the molecular pathways and regulatory networks involved in the postnatal development of craniofacial bone. In this study, the online Gene Expression Omnibus microarray expression profiling data set GSE27976 was used to identify differentially expressed genes (DEGs) in different age groups. Protein-Protein Interaction network analyses, functional enrichment, and hub genes analysis were performed. The differences in immune infiltration and microenvironment among different types of cells were also analyzed. In total, 523 DEGs, including 287 upregulated and 236 downregulated genes, were identified. GO and KEGG analysis showed that the DEGs were significantly enriched in multiple signaling pathways, such as skeletal system morphogenesis, osteoblast differentiation, and stem cell differentiation. Immune infiltration and microenvironment characteristics analysis showed that there were significant differences in fibroblasts, mesenchymal stem cell, osteoblast, stroma score, and microenvironment score between the two groups. Five hub genes, including IGF1, IL1B, ICAM1, MMP2 , and brain-derived neurotrophic factor, were filled out. The findings of this study showed a significant shift in gene expression towards osteogenesis during the first 12 months after birth. These findings emphasize the critical role of the postnatal period in craniofacial bone development and provide valuable insights into the molecular mechanisms underlying this process.

Effect of LncRNA LOC106505926 on myogenesis and Lipogenesis of porcine primary cells

BackgroundSkeletal muscle development and fat deposition have important effects on meat quality. The study of regulating skeletal muscle development and fat deposition is of great significance in improving the quality of carcass and meat. In the present study, whole transcriptome sequencing (including RNA-Seq and miRNA-Seq) was performed on the longissimus dorsi muscle (LDM) of Jinfen White pigs at 1, 90, and 180 days of age.ResultsThe results showed that a total of 245 differentially expressed miRNAs were screened in any two comparisons, which may be involved in the regulation of myogenesis. Among them, compared with 1-day-old group, miR-22-5p was significantly up-regulated in 90-day-old group and 180-day-old group. Functional studies demonstrated that miR-22-5p inhibited the proliferation and differentiation of porcine skeletal muscle satellite cells (PSCs). Pearson correlation coefficient analysis showed that long non-coding RNA (lncRNA) LOC106505926 and CXXC5 gene had strong negative correlations with miR-22-5p. The LOC106505926 and CXXC5 were proven to promote the proliferation and differentiation of PSCs, as opposed to miR-22-5p. In terms of mechanism, LOC106505926 functions as a molecular sponge of miR-22-5p to modulate the expression of CXXC5, thereby inhibits the differentiation of PSCs. In addition, LOC106505926 regulates the differentiation of porcine preadipocytes through direct binding with FASN.ConclusionsCollectively, our results highlight the multifaceted regulatory role of LOC106505926 in controlling skeletal muscle and adipose tissue development in pigs and provide new targets for improving the quality of livestock products by regulating skeletal muscle development and fat deposition.

MiR-214 promotes the activation, proliferation and differentiation of skeletal muscle satellite cells by modulating Hedgehog signaling in Chinese perch

Fish exhibit indeterminate growth by recruiting new muscle fibers (hyperplasia) and increasing the size of already existing fibers (hypertrophy) to promote muscle growth. However, the molecular mechanism by which muscle fibers maintain hyperplasia and hypertrophy during the posthatching period in fish remains unclear. microRNAs have been reported to participate in the proliferation and differentiation of myoblasts in fish. In this study, we found that overexpression of miR-214 in Chinese perch (Siniperca chuatsi) by injecting agomiR-214 in vivo resulted in an increase in the diameter of myofibers, the number of satellite cells and proliferating myoblasts as well as the expression of Pax7, Myomaker and myogenic regulatory factors. However, inhibiting miR-214 by injecting antagomiR-214 in vivo significantly decreased the diameter of myofibers, the number of proliferating myoblasts and the expression of Myf5 and MyoD. In addition, inhibition of the Hedgehog pathway showed results similar to those of inhibition of miR-214. The in silico analysis and dual luciferase reporter assay revealed that Sufu, a negative regulator of the Hedgehog (Hh) signaling pathway, is a direct target of miR-214. Taken together, our results reveal that miR-214 induces the activation of satellite cells by regulating Hh signaling activity, and subsequently stimulates the proliferation, differentiation and fusion of myoblast, thus promoting hyperplasia and hypertrophy of skeletal muscle in Chinese perch.

Retinoic acid signalling inhibits myogenesis by blocking MYOD translation in pig skeletal muscle cells

Vitamin A is an essential nutrient in animals, playing important roles in animal health. In the pig industry, proper supplementation of vitamin A in the feed can improve pork production performance, while deficiency or excessive intake can lead to growth retardation or disease. However, the specific molecular mechanisms through which vitamin A operates on pig skeletal muscle growth as well as muscle stem cell function remain unexplored. Therefore, in this study, we isolated the pig primary skeletal muscle stem cells (pMuSCs) and treated with retinoic acid (RA), the natural metabolite of vitamin A, and then examined the myogenic capacity of pMuSCs via immunostaining, real-time PCR, CCK8 and western-blot analysis. Unexpectedly, the RA caused a significant decrease in the proliferation and differentiation of pMuSCs. Mechanistically, the RA addition induced the activation of retinoic acid receptor gamma (RARγ), which inhibited the myogenesis through the blockage of protein translation of the master myogenic regulator myogenic differentiation 1 gene (MYOD). Specifically, RARγ inactivate AKT kinase (AKT) signalling and lead to dephosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (eIF4EBP1), which in turn repress the eukaryotic translation initiation factor 4E (eIF4E) complex and block mRNA translation of MYOD. Inhibition of AKT could rescue the myogenic defects of RA-treated pMuSCs. Our findings revealed that retinoid acid signalling inhibits the skeletal muscle stem cell proliferation and differentiation in pigs. Therefore, the vitamin A supplement in the feedstuff should be cautiously optimized to avoid the potential adverse consequences on muscle development associated with the excessive levels of retinoic acid.

Glutenin-chitosan 3D porous scaffolds with tunable stiffness and systematized microstructure for cultured meat model

A glutenin (G)-chitosan (CS) complex (G-CS) was cross-linked by water annealing with aim to prepare structured 3D porous cultured meat scaffolds (CMS) here. The CMS has pore diameters ranging from 18 to 67 μm and compressive moduli from 16.09 to 60.35 kPa, along with the mixing ratio of G/CS. SEM showed the porous organized structure of CMS. FTIR and CD showed the increscent content of α-helix and β-sheet of G and strengthened hydrogen-bondings among G-CS molecules, which strengthened the stiffness of G-CS. Raman spectra exhibited an increase of G concentration resulted in higher crosslinking of disulfide-bonds in G-CS, which aggrandized the bridging effect of G-CS and maintained its three-dimensional network. Cell viability assay and immuno-fluorescence staining showed that G-CS effectively facilitated the growth and myogenic differentiation of porcine skeletal muscle satellite cells (PSCs). CLSM displayed that cells first occupied the angular space of hexagon and then ring-growth circle of PSCs were orderly formed on G-CS. The texture and color of CMS which loaded proliferated PSCs were fresh-meat like. These results showed that physical cross-linked G-CS scaffolds are the biocompatible and stable adaptable extracellular matrix with appropriate architectural cues and natural micro-environment for structured CM models.