Differentiation Of Bovine Satellite Cells Research Articles

Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production

Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment—with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

Comparison of secreted miRNAs and proteins during proliferation and differentiation of bovine satellite cells in culture implies potential roles in regulating myogenesis

Cultivated meat is an emerging new technology to produce sustainable meat for the future. The common approach for cultivated meat, is the isolation of satellite cells from donor animals, followed by in vitro proliferation and differentiation into primitive muscle fibers. The transformation of satellite cells into myofibers is tightly orchestrated by intra-cellular signaling, while the inter-cellular signaling is less well understood. Thus, the current study was conducted to map the secretion of potential signaling molecules (MicroRNAs and proteins) during proliferation and differentiation. Primary cultures of satellite cells were grown to 50% and 80% confluence, representing the proliferative phase or serum-starved for 1 and 3 days to induce differentiation. Post incubation in FBS-free media, the media were collected and analyzed for miRNA and protein content using gene-arrays and LC-MS/MS, respectively. When comparing the miRNA secretome at 50% and 80% confluence, we observed four differentially expressed miRNA, while only five were differentially expressed when comparing Day 1 to Day 3. A subsequent in silico analysis suggested that pathways of importance for myogenesis, e.g., MAPK and AMPK signaling, could be regulated by the secreted miRNAs. In addition, >300 proteins were secreted, including insulin-like growth factor 1 binding proteins 2, 3, 4, 5 and 6. In conclusion, this study demonstrated differential secretion of several miRNAs and proteins during both proliferation and differentiation of bovine satellite cells in vitro.

Genome-wide identification of enhancers and transcription factors regulating the myogenic differentiation of bovine satellite cells

BackgroundSatellite cells are the myogenic precursor cells in adult skeletal muscle. The objective of this study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells into myotubes.ResultsChromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed to identify genomic regions where lysine 27 of H3 histone is acetylated (H3K27ac), i.e., active enhancers, from bovine satellite cells before and during differentiation into myotubes. A total of 19,027 and 47,669 H3K27ac-marked enhancers were consistently identified from two biological replicates of before- and during-differentiation bovine satellite cells, respectively. Of these enhancers, 5882 were specific to before-differentiation, 35,723 to during-differentiation, and 13,199 common to before- and during-differentiation bovine satellite cells. Whereas most of the before- or during-differentiation-specific H3K27ac-marked enhancers were located distally to the transcription start site, the enhancers common to before- and during-differentiation were located both distally and proximally to the transcription start site. The three sets of H3K27ac-marked enhancers were associated with functionally different genes and enriched with different transcription factor binding sites. Specifically, many of the H3K27ac-marked enhancers specific to during-differentiation bovine satellite cells were associated with genes involved in muscle structure and development, and were enriched with binding sites for the MyoD, AP-1, KLF, TEAD, and MEF2 families of transcription factors. A positive role was validated for Fos and FosB, two AP-1 family transcription factors, in the differentiation of bovine satellite cells into myotubes by siRNA-mediated knockdown.ConclusionsTens of thousands of H3K27ac-marked active enhancers have been identified from bovine satellite cells before or during differentiation. These enhancers contain binding sites not only for transcription factors whose role in satellite cell differentiation is well known but also for transcription factors whose role in satellite cell differentiation is unknown. These enhancers and transcription factors are valuable resources for understanding the complex mechanism that mediates gene expression during satellite cell differentiation. Because satellite cell differentiation is a key step in skeletal muscle growth, the enhancers, the transcription factors, and their target genes identified in this study are also valuable resources for identifying and interpreting skeletal muscle trait-associated DNA variants in cattle.

192 Identification of Fos and FosB as Transcriptional Regulators of Bovine Satellite Cell Differentiation

Abstract Transcription factors (TFs) are key regulators of gene expression during cell differentiation. Four TFs including Myf5, MyoD, MyoG and Myf6 have been identified as key myogenic regulatory factors (MYFs) that regulate gene transcription during myogenesis. Satellite cells (SCs) are the myogenic precursor cells in adult skeletal muscle. The objective of this study was to identify additional TFs that control the differentiation of bovine satellite cells. Bovine satellite cells (bSCs) were isolated from 4 crossbred steers and were initially cultured in growth medium for 12 days to expand and then in differentiation medium for 48 hours to differentiate. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to identify chromatin regions marked with acetylation of histone H3 on lysine 27 (H3K27ac). This ChIP-seq analysis revealed 3,348 and 38,800 H3K27ac-associated chromatin regions in bSCs before and after differentiation, respectively. A motif enrichment analysis of the H3K27ac-marked chromatin regions from the differentiated bSCs indicated the enrichment of binding sites for the 4 MYFs and many other TFs including Fos and FosB. RNA-sequencing revealed the upregulation of Fos and FosB mRNAs in bSCs from growth to differentiation. To verify the roles of Fos and FosB in bSC differentiation, their expressions in bSCs were reduced by siRNA-induced knockdown. Based on qRT-PCR analyses, expressions of MYH2, MYH3, MYOG, and CKM mRNAs, which were selected as markers of muscle cell differentiation, were increased (P < 0.05) in bSCs from growth to differentiation, but the increases in at least three of them were reversed (P < 0.05) by Fos or FosB knockdown. Taken together, these results establish Fos and FosB as transcriptional regulators of bovine satellite cell differentiation.

PSIX-28 Examining the effects of estradiol, trenbolone acetate, or polyamines on bovine satellite cell differentiation

Abstract The mechanism through which anabolic implants improve skeletal muscle growth of beef animals is incompletely understood. Polyamines (PA) are bioactive amino acid derivatives that act as potent growth stimulants. The objective of this study was to determine effects of anabolic implants, PA and their precursors on bovine satellite cell (BSC) differentiation. Primary BSC were cultured to approximately 80% confluency, at which time they were induced to differentiate in the presence of 3% horse serum (Con) and treated with 10nM TBA, 10 nM E2, or 10nM TBA and 10 nM E2 (ETBA), 10 mM methionine (Met), 8 mM ornithine (Orn), 2 mM putrescine (Put), 1.5 mM spermidine (Spd) or 0.5 mM spermine (Spe). Total mRNA was isolated 0, 2, 4, 8, 12, 24, or 48 h post-treatment and abundance of paired box transcription factor 7 (Pax7) and myogenic differentiation factor (MyoD) were analyzed. Treatment with the hormones (TBA, E2, or ETBA) and PA (Orn, Put, Spd, and Spe) increased (P < 0.05) abundance of MyoD 4 h post-treatment when compared to Con cultures. However, 24 h post-treatment, MyoD abundance was decreased in the presence of hormone treatments when compared to the Con, while the PA treatments increased (P < 0.05) abundance of MyoD when compared to the Con cultures. Treatment with either the hormones or PA had no effect (P > 0.05) on Pax7 abundance at 2, 4, 8, 12, 24, or 48 h post-treatment when compared to Con cultures (P > 0.05). These results indicate that treatment with PA or hormones increases abundance of MyoD, though temporally different indicating that these two classes of growth promoters impact differentiation via alternate physiological pathways. Additional research is underway in order to determine the effects of both PA and hormones on differentiation of primary BSC.

Oleic acid Stimulates the Formation of Adipocyte-Like Cells from Bovine Satellite Cells via <i>G-Protein Coupled Receptor 43</i> and <i>CCAAT/Enhancer-Binding Protein Beta</i>

Numerous physiological and pathological processes are controlled by free fatty acids, which act as signaling molecules in mammals. We hypothesized that oleic acid (Ole) might stimulate the formation of satellite-cell-derived intramuscular adipose tissue. The role of Ole as a ligand of G-protein-coupled receptor 43 (GPR43) was previously identified. Thus, the objective of the current study was to determine the effect of Ole on GPR43 and factors related to the adipogenic differentiation of bovine satellite cells (BSC). Treatments of 100 μM and 500 μM Ole tended to induce greater (P CCAAT/enhancer-binding protein β (C/EBPβ) compared to all other doses. The mRNA abundance of peroxisome proliferator-activated receptor γ (PPARγ) was not altered (P > 0.10) by treatment. The addition of 100 μM and 500 μM of Ole upregulated (P GPR43 mRNA expression. Protein level of GPR43 was increased (P C/EBPβ in response to Ole might suppress myogenic differentiation. After the treatment of cells with Ole, increased expression of GPR43 could lead to phosphorylation of 5’ AMP-activated protein kinase α (AMPKα). Altogether, the results indicated that increased Ole might stimulate adipose tissue accumulation within the skeletal muscle of cattle by promoting adipogenic differentiation and activation of GPR43 in satellite cells.

PSXIV-32 Oleic acid stimulate the formation of adipocyte-like cells from bovine satellite cells via G Protein Coupled Receptor 43 and CCAAT/Enhancer Binding Protein Beta

Abstract Numerous physiological and pathological processes are controlled by free fatty acids (FFAs), which act as a signaling molecule in mammals. We hypothesized that oleic acid (Ole) may stimulate the formation of satellite cell-derived intramuscular adipose tissue. The objective of the current study was to determine the effect of Ole on GPR43 and factors related to the adipogenic differentiation of bovine satellite cells. Bovine satellite cells were isolated from the semimembranosus of two 14-month-old crossbreed steers. The isolated muscle satellite cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) solution with 10% Fetal Bovine Serum. Upon reaching 80 to 90% confluence, the growth medium was replaced with differentiation medium composed of DMEM and 2% horse serum, 10μg/mL insulin, 10μg/mL hydrocortisone, 10μM ciglitizone, and 1×antibiotic-antimycotic with dose of: 0, 1, 10, 100, or 500 μM of oleic acid (Ole). Addition of Ole on BSC induced transdifferentiation of myogenic lineage into adipocyte-like cells which formed lipid droplets within cells. Use of 100 μM and 500 μM Ole doses tended to result in a greater (P < 0.1) amount of mRNA gene expression of C/EBPβ compared to all other doses. This might suppress myogenic differentiation. Expression of PPARγ was not altered (P > 0.1) by treatment. The addition of 100 μM and 500 μM upregulated (P < 0.05) mRNA gene expression of GPR43 and 100 μM of Ole increased protein level of GPR43 (P < 0.05) and phosphorylated AMPKα (P < 0.05).

LncRNA H19 promotes the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1

BackgroundH19 is a well-characterized Long noncoding RNA (lncRNA) that has been proven to promote myoblast differentiation in humans and mice. However, its mechanism of action is still not fully interpreted.MethodsUsing RT-qPCR, we examined H19 RNA levels in various tissues from 1-week, 1-month, 6-month and 36-month old male cattle (i.e., newborn, infant, young and adult). The protein and mRNA levels of MyoG, MyHC, Sirt1 and FoxO1 in the satellite and C2C12 cells with an H19 silencing or overexpression vector were respectively detected using western blot and real-time qPCR.ResultsH19 was highly expressed in skeletal muscle at all the studied ages. High expression of H19 was required for the differentiation of bovine satellite cells. Knockdown of H19 caused a remarkable increase in the myoblast-inhibitory genes Sirt1/FoxO1, suggesting that H19 suppresses Sirt1/FoxO1 expression during myogenesis. Western blotting analysis of co-transfection of Sirt1 or FoxO1 expression vectors with pcDNA-H19 indicated that Sirt1/FoxO1 overexpression neutralized the promotion of myoblast differentiation through transfection of pcDNA-H19.ConclusionH19 promoted the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1.

Identification of the STAC3 gene as a skeletal muscle-specifically expressed gene and a novel regulator of satellite cell differentiation in cattle.

Recent studies in mice and zebrafish suggest that the unannotated Src homology 3 and cysteine rich domain 3 (STAC3) gene plays an important role in skeletal muscle development and contraction. The objective of this study was to determine the tissue specificity of the bovine STAC3 gene and its potential role in the proliferation and differentiation of bovine satellite cells. The STAC3 mRNA was detected only in skeletal muscle among 18 bovine tissues examined by reverse transcription PCR. Western blotting revealed the expression of STAC3 protein in bovine skeletal muscle and the absence of it in 6 bovine tissues analyzed. Transfection of the bovine satellite cells with a pool of 2 STAC3 small interfering RNA (siRNA) caused a 90% reduction in STAC3 mRNA. Cell proliferation assays revealed that STAC3 knockdown had no effect on the proliferation rate of bovine satellite cells. Approximately 60% of bovine satellite cells transfected with STAC3 siRNA formed myotubes by 72 h of differentiation, whereas that percentage was 40% for those transfected with negative control siRNA (P < 0.05). At 24, 48, and 72 h of differentiation, bovine satellite cells transfected with STAC3 siRNA had greater mRNA expression of myogenin, myosin heavy chain 3, and myosin heavy chain 7, markers of myotubes, than those transfected with negative control siRNA (P < 0.05). These results suggest that the STAC3 gene is a negative regulator of the differentiation and fusion of bovine satellite cells into myotubes. However, STAC3 expression was increased during the differentiation of bovine satellite cells into myotubes. This suggests that STAC3 might have different functions in bovine myotubes than in bovine satellite cells.

Proteomic Analysis of Bovine Muscle Satellite Cells during Myogenic Differentiation

The aim of this study was to analyze the proteome expression of bovine satellite cells from longissimus dorsi (LD), deep pectoral (DP) and semitendinosus (ST) muscle depots during in vitro myogenic differentiation. Proteomic profiling by twodimensional gel electrophoresis and mass spectrometry of differentiating satellite cells revealed a total of 38 proteins that were differentially regulated among the three depots. Among differentially regulated proteins, metabolic proteins like lactate dehydrogenase (LDH), malate dehydrogenase (MDH) were found to be up regulated in ST, while alpha-enolase (NNE) in LD and DP depot satellite cells were down regulated. Also, our analysis found that there was a prominent up regulation of cytoskeletal proteins like actin, actincapping protein and transgelin along with chaperone proteins like heat shock protein beta 1 (HSPB 1) and T-complex protein 1 (TCP-1). Among other up regulated proteins, LIM domain containing protein, annexin 2 and Rho GDP-dissociation inhibitor 1 (Rho GDI) are observed, which were already proven to be involved in the myogeneis. More interestingly, satellite cells from ST depot were found to have a higher myotube formation rate than the cells from the other two depots. Taken together, our results demonstrated that, proteins involved in glucose metabolism, cytoskeletal modeling and protein folding plays a key role in the myogenic differentiation of bovine satellite cells.

Influence of insulin and triiodothyronine on the proliferation and differentiation of bovine satellite cells in primary culture

Influence of insulin and triiodothyronine on the proliferation and differentiation of bovine satellite cells in primary culture