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Development and proteome characterisation of muscle cell culture derived from Genetically improved farmed tilapia, Oreochromis niloticus (Linnaeus,1758)

ABSTRACT: A quantitative proteomics approach was used to investigate in Oreochromis niloticus cell line across two passages P10, and P20. A cell line developed from the muscle tissue of O. niloticus (ONM) was utilized to understand the proteome in-depth. Comparative proteome analysis was performed for ONM muscle cells between two passages (10 and 20) using label free quantitative proteomics. High-resolution mass spectrometry was used for data using label-free quantification. Data were further analysed using a statistical tool across passages of rohu muscle cell line. Proteomic analysis across two passages (P10 and P20) revealed pathways involved in myogenesis were striated muscle contraction, muscle contraction, insulin pathway, regulation of actin cytoskeleton, hedgehog on state, VEGFA-VEGFR2 and signalling by WNT. The cell line generated can have wide range of application in cultivated meat as well as in in vitro application.

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ORGANISM(S): Oreochromis Niloticus

TISSUE(S): Cell Culture, Muscle Cell Proliferation, Fibroblast

SUBMITTER: Nevil Pinto

LAB HEAD: Mukunda Goswami

PROVIDER: PXD060219 | Pride | 2026-04-23

REPOSITORIES: Pride

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Project description:BACKGROUND: Periodontal ligament mesenchymal stem cells (PDLSCs) are a promising cell resource for cell-based regenerative medicine in dentistry. PDLSCs inevitably acquire a senescent phenotype after prolonged in vitro expansion, and the key regulators of cells during replicative senescence remain unclear.METHODS: We cultured periodontal ligament stem cells to passages 4, 10 and 20 (P4, P10, P20). The senescent phenotypes, proliferation and migration ability of PDLSCs (P4, P10, P20) were detected, and non-targeted metabonomic sequencing was performed. We treated PDLSCs with AICAR and detected the expression of FOXO1, FOXO3a, FOXO6 and AMPK phosphorylation (p-AMPK) levels of replicating senescent PDLSCs to explore the correlation between the metabolic changes and the AMPK pathway.RESULTS: Immunofluorescence staining of γ-H2AX, β-galactosidase staining, cell scratch test and qPCR were performed to confirm the occurrence of replicative senescence in PDLSCs during passaging. Three groups of cells at passage 4 (P4), passage 10 (P10) and passage 20 (P20) were collected for non-targeted metabolomics analysis. Metabonomic sequencing showed that the metabolism of replicative senescence in PDLSCs varied significantly. In particular, the content of fatty acid metabolites decreased with senescence, including capric acid, stearic acid, myristic acid and dodecanoic acid. KEGG pathway analysis showed that the AMPK signaling pathway was closely related to AMP levels. The AMP:ATP ratio increased in senescent PDLSCs; however, the levels of p-AMPK and the profile of FOXO1 and FOXO3a, which are downstream of the AMPK signaling pathway, decreased with senescence. We treated PDLSCs with AICAR, an activator of the AMPK pathway, and the phosphorylated AMPK level at P20 PDLSCs was partially restored. CONCLUSION: In summary, our study suggests that the metabolic process of PDLSCs is active in the early stage of senescence, prefers to consume fatty acids, and is attenuated in the later stages of senescence. AMP accumulates in replicative senescent PDLSCs; however, the sensitivity of AMPK phosphorylation sites is impaired, causing senescent PDLSCs to fail to respond to changes in energy metabolism. Our findings provide a new basis for the clinical application of periodontal ligament stem cells.

Project description:Because of its high plasticity and rapid growth rate, turkey skeletal muscle is an important model for studying mechanisms responsible for vertebrate skeletal muscle development and function. Skeletal muscle is composed of metabolically heterogeneous myofibers that exhibit high plasticity at both the morphological and transcriptional levels. The objective of this study was to employ microarray analysis to elucidate the differential gene expression between the tonic- “red” Anterior latissimus dorsi (ALD) muscle, the phasic- “white” Posterior latissimus dorsi (PLD), and “mixed”-phenotype Biceps femoris (BF) in 1-week and 19-week old male turkeys. A total of 170 differentially expressed genes were identified in the analyzed muscle samples (P<0.05). Gene Go analysis software was utilized to identify top gene networks and metabolic pathways involving differentially expressed genes. The largest differences were observed between ALD and PLD muscles, where 32 genes were over-expressed and 82 genes were under-expressed in ALD1-PLD1 comparison; and 70 genes were over-expressed and 70 under-expressed in ALD19-PLD19 comparison. The largest number of genes over-expressed in ALD muscles as compared to other muscles, code for extracellular matrix proteins such as dystroglycan and collagen. Furthermore, a number of genes involved in glycolytic metabolism were under-expressed in ALD muscles as compared to BF and PLD muscles. The gene analysis revealed that phenotypically “red” BF muscle has high expression of glycolytic genes usually associated with “white” muscle phenotype. Muscle-specific differences were observed in expression levels of genes coding for proteins involved in mRNA processing and translation regulation, proteosomal degradation, apoptosis, and insulin resistance. The study utilized Nicholas turkey males at two different stages of growth (1 week old: high satellite cell mitotic activity; 19 weeks old: low satellite cell mitotic activity, hypertrophy, market age). Six 1-week-old and six 19-week-old Nicholas turkey males (Meleagris gallopavo) were randomly selected from a single flock. The experiments were designed to compare genes characterizing muscle fiber phenotype at each time point, and to elucidate age-related differences in gene expression levels. Each analyzed cDNA sample was derived from six pooled RNA samples, resulting with six cDNA samples per muscle (one cDNA per bird). To minimize dye bias, the cDNA samples were labeled either with Cy3 or Cy5. A total of 17 Turkey Skeletal Muscle Long Oligo (TKSMLO) microarrays were utilized. Birds were randomly assigned to each array and the cDNA hybridizations we performed at random. The array data was Log2 transformed and then normalized by using locally weighed regression and smoothing within each array and across all arrays. Mixed model ANOVA with Bonferroni correction of P=0.05 for multiple testing was performed to increase the sensitivity of detected differences in gene expression levels.

			Action	DRS
	ONM-P13.raw	Raw
	ONM-P20.raw	Raw
	UP000005207_8128.fasta	Fasta
	mzTab.mzTab	Mztab
	txt.zip	Other

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Development and proteome characterisation of muscle cell culture derived from Genetically improved farmed tilapia, Oreochromis niloticus (Linnaeus,1758)

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