Proliferation and differentiation of primary bovine myoblasts using Chlorella vulgaris extract for sustainable production of cultured meat.

Recently, cultured meat obtained from livestock-derived cells is being considered as a sustainable food source that reduces the use of natural resources. This study aimed to show that nutrients extracted from Chlorella vulgaris were beneficial in the culture of primary bovine myoblasts (PBMs), a major cell source for cultured meat production. Nutrients (glucose, amino acids, and vitamins) present in the animal-cell culture media were effectively recovered from C. vulgaris using acid hydrolysis treatment. On culture in nutrient-free inorganic salt solution, cell death was induced in most PBMs after 6 days of cultivation. However, the addition of C. vulgaris extract (CVE) significantly improved PBM viability, which was comparable to the viability in conventional culture medium (Dulbecco's modified Eagle's medium). Furthermore, by adding horse serum to induce differentiation, the formation of myotubes was confirmed when CVE were used. Together, the results showed that CVE could be used as an alternative to the conventional culture medium for PBMs. These findings will not only lower the environmental risks associated with the establishment of this eco-friendly cell culture system, but also highlight microalgae as a potent nutrient source that can replace conventional grain-dependent nutrient sources.

MicroRNA bta-miR-365-3p inhibits proliferation but promotes differentiation of primary bovine myoblasts by targeting the activin A receptor type I.

BACKGROUND: MicroRNAs act as post-transcriptional regulators that repress translation or degrade mRNA transcripts. Each microRNA has many mRNA targets and each mRNA may be targeted by several microRNAs. Skeletal muscles express a plethora of microRNA genes that regulate muscle development and function by controlling the expression of protein-coding target genes. To expand our understanding of the role of microRNA, specifically bta-miR-365-3p, in muscle biology, we investigated its functions in regulating primary bovine myoblast proliferation and differentiation. RESULTS: Firstly, we found that bta-miR-365-3p was predominantly expressed in skeletal muscle and heart tissue in Chinese Qinchuan beef cattle. Quantitative PCR and western blotting results showed that overexpression of bta-miR-365-3p significantly reduced the expression levels of cyclin D1 (CCND1), cyclin dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) but stimulated the expression levels of muscle differentiation markers, i.e., MYOD1, MYOG at both mRNA and protein level. Moreover, downregulation of bta-miR-365-3p increased the expression of CCND1, CDK2 and PCNA but decreased the expression of MYOD1 and MYOG at both mRNA and protein levels. Furthermore, flow cytometry, EdU proliferation assays and immunostaining results showed that increased levels of bta-miR-365-3p suppressed cell proliferation but promoted myotube formation, whereas decreased levels of bta-miR-365-3p resulted in the opposite consequences. Finally, we identified that activin A receptor type I (ACVR1) could be a direct target of bta-miR-365-3p. It was demonstrated that bta-miR-365-3p can bind to the 3'UTR of ACVR1 gene to regulate its expression based on dual luciferase gene reporter assays. Consistently, knock-down of ACVR1 was associated with decreased expressions of CDK2, CCND1 and PCNA but increased expression of MYOG and MYOD1 both at mRNA and protein level. CONCLUSION: Collectively, these data suggested that bta-miR-365-3p represses proliferation but promotes differentiation of bovine myoblasts through several biological mechanisms involving downregulation of ACVR1.

Integrated Analysis of mRNA and MicroRNA Co-expressed Network for the Differentiation of Bovine Skeletal Muscle Cells After Polyphenol Resveratrol Treatment.

Resveratrol (RSV) has been confirmed to benefit human health. Resveratrol supplemented in the feeds of animals improved pork, chicken, and duck meat qualities. In this study, we identified differentially expressed (DE) messenger RNAs (mRNAs) (n = 3,856) and microRNAs (miRNAs) (n = 93) for the weighted gene co-expression network analysis (WGCNA) to investigate the co-expressed DE mRNAs and DE miRNAs in the primary bovine myoblasts after RSV treatment. The mRNA results indicated that RSV treatments had high correlations with turquoise module (0.91, P-value = 0.01) and blue module (0.93, P-value < 0.01), while only the turquoise module (0.96, P-value < 0.01) was highly correlated with the treatment status using miRNA data. After biological enrichment analysis, the 2,579 DE genes in the turquoise module were significantly enriched in the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The top two GO terms were actin filament-based process (GO:0030029) and actin cytoskeleton organization (GO:0030036). The top two KEGG pathways were regulation of actin cytoskeleton (bta04810) and tight junction (bta04530). Then, we constructed the DE mRNA co-expression and DE miRNA co-expression networks in the turquoise module and the mRNA-miRNA targeting networks based on their co-expressions in the key module. In summary, the RSV-induced miRNAs participated in the co-expression networks that could affect mRNA expressions to regulate the primary myoblast differentiation. Our study provided a better understanding of the roles of RSV in inducing miRNA and of the characteristics of DE miRNAs in the key co-expressed module in regulation of mRNAs and revealed new candidate regulatory miRNAs and genes for the beef quality traits.