Project description:In this study we analyzed the bovine satellite cells with single-cell RNA sequencing (scRNA-seq). We isolated muscle satellite cells from a male calf, cultured them in growth medium for a week, and then constructed two scRNA-seq libraries from them using the 10x Genomics platform. Next generation sequencing yielded 860 million reads. Cell calling analyses revealed that these reads represented 15 cell clusters that differed in gene expression profile from a total of 19,096 individual cells.
Project description:In this study we isolated satellite cells from skeletal muscle from two Angus-crossbred steers, initially cultured them in growth medium for proliferation, and then in differentiation medium to induce them to differentiate and fuse into myotubes. We used ChIP-seq to identify enhancers marked with H3K27ac, i.e., active enhancers, from these cells immediately before and two days after induction of differentiation. Nearly 20,000 and 50,000 H3K27ac-marked enhancers were consistently identified from two biological replicates of before- and during-differentiation bovine satellite cells, respectively.
Project description:Satellite cells are myogenic precursor cells in adult skeletal muscle and play a crucial role in skeletal muscle repair, remodeling, and maintenance. Like myoblasts, satellite cells have the ability to proliferate, differentiate, and fuse to form multinucleated myofibers. In this study we aimed to identify the enhancers and the transcription factors binding to these enhancers that control gene expression in proliferating and differentiating bovine satellite cells. Using ChIP-seq, we identified 56,973 and 54,470 active enhancers, genomic regions marked with both H3K4me1 and H3K27ac, and 50,956 and 59,174 repressed enhancers, genomic regions marked with H3K27me3, in proliferating and differentiating bovine satellite cells, respectively. Motif enrichment analyses revealed many transcription factors including the AP-1 family transcription factors that regulate gene expression in proliferating bovine satellite cells and many transcription factors besides MyoG that regulate gene expression in differentiating bovine satellite cells.
Project description:Production of cultured meat requires the robust differentiation of satellite cells into mature muscle fibers without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, an abrupt reduction in serum concentration. Here, we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of mature three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production.
Project description:Production of cultured meat requires the robust differentiation of satellite cells into mature muscle fibers without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, an abrupt reduction in serum concentration. Here, we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of mature three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production.
Project description:Cultured' or 'cultivated' meat technologies leverage the proliferation and differentiation of animal-derived stem cells ex vivo to produce edible tissues for human consumption in a sustainable fashion. However, skeletal muscle is a dynamic and highly complex tissue, involving the interplay of numerous mono- and multinucleated cell types, including muscle fibres, satellite cells (SCs) and fibro-adipogenic progenitors (FAPs), and accurate recreation of the entire tissue thus requires the identification, purification, proliferation and characterisation of a broad range of cell types. Here, we use a single-cell RNA sequencing approach to characterise transcriptional heterogeneity within bovine muscle and derived in vitro cultures over time. Using this data, we identify numerous distinct cell types, and develop robust protocols for the easy purification and proliferation of several of these populations. We note overgrowth of undesirable cell types within heterogeneous proliferative cultures as a barrier to efficient cultured meat production, and use transcriptomics to identify conditions that favour the growth of SCs in the context of serum-free medium. Combining RNA velocities computed in silico with functional data, we characterise dynamic subpopulations and transitions between active, quiescent and committed substates of SCs, and demonstrate methods for modulation of these states during long-term proliferative cultures. This work represents an important resource for advancing our knowledge of bovine skeletal muscle biology, and its application in the development of cultured meat technologies.
Project description:Cultured meat is an emergent technology with the potential for significant environmental and animal welfare benefits. Accurate mimicry of traditional meat requires fat tissue; a key contributor to both the flavour and texture of meat. Here, we show that fibro-adipogenic progenitor cells (FAPs) are present in bovine muscle, and are transcriptionally and immunophenotypically distinct from satellite cells. These two cell types can be purified from a single muscle sample using a simple fluorescence-activated cell sorting (FACS) strategy. FAPs demonstrate high levels of adipogenic potential, as measured by gene expression changes and lipid accumulation, and can be proliferated for a large number of population doublings, demonstrating their suitability for a scalable cultured meat production process. Crucially, FAPs reach a mature level of adipogenic differentiation in three-dimensional, edible hydrogels. The resultant tissue accurately mimics traditional beef fat in terms of lipid profile and taste, and FAPs thus represent a promising candidate cell type for the production of cultured fat.
Project description:The objective of this study was to investigate the role of serine/arginine-rich splicing factor 1 (SRSF1) in the myogenic differentiation of bovine skeletal muscle satellite cells and its regulatory relationship with fibroblast growth factor 16 (FGF16). Bovine skeletal muscle satellite cells were subjected to SRSF1 knockdown and overexpression to evaluate their proliferation and differentiation capacities. RNA sequencing and subsequent validation experiments were performed to identify potential downstream targets of SRSF1, while functional assays were conducted to determine the role of FGF16 in myogenesis. RNA immunoprecipitation (RIP) and actinomycin D treatment were further used to investigate RNA-level interactions and transcript stability, and changes in PI3K/AKT signaling activity were assessed by western blotting. The results showed that SRSF1 expression dynamically increased during myogenic differentiation and significantly promoted myotube formation without markedly affecting cell proliferation. Transcriptomic analysis and validation identified FGF16 as a potential downstream target of SRSF1, and functional analyses demonstrated that FGF16 promoted myogenic differentiation. RIP assays suggested an association between SRSF1 mRNA and FGF16-associated complexes, while modulation of SRSF1 expression altered FGF16 expression and the phosphorylation status of PI3K and AKT. Collectively, these findings suggest that SRSF1 may promote the myogenic differentiation of bovine skeletal muscle satellite cells through post-transcriptional regulation associated with FGF16 and modulation of PI3K/AKT signaling activity, providing new insights into RNA-binding protein-mediated regulatory mechanisms involved in bovine skeletal muscle development.
Project description:To understand the effect of LncMyod knockdown on cultured satellite cells, we isolated satellite cells from mouse hindlimb and cultured them in vitro with siRNA treatment targeting LncMyod. Following RNA-seq showed that loss of LncMyod leads to difficiencies in myogenic differentiation.