Integrated Multi-Omics Characterization of CDK4/hTERT-Immortalized Ovine Satellite Cells for Cultivated Meat Applications

Cultivated meat production requires stable, scalable cell lines that maintain essential biological characteristics. Here, we establish and comprehensively characterize CDK4/hTERT immortalized ovine satellite cells using flow cytometry-based isolation and integrated transcriptomic-proteomic analysis. Flow cytometry identified highly purified satellite cells which were successfully immortalized and maintained stable proliferation (14-22h doubling time) through 67 passages. Multi-omics analysis revealed progressive molecular evolution with 754-1180 differentially expressed genes and 960 differentially expressed proteins, demonstrating coordinated transcriptional and post-transcriptional adaptations. Immortalization triggered species-specific extracellular matrix remodeling and inflammatory activation while preserving essential myogenic markers and apoptotic machinery. Immunofluorescence analysis demonstrated that key proteins, including PAX7, remained detectable despite transcriptional downregulation and absence of corresponding signals in mass spectrometry. Pathway analysis confirmed non-oncogenic profiles with enhanced metabolic flexibility and preserved differentiation capacity. These findings provide the first comprehensive molecular characterization of immortalized ovine satellite cells, revealing species-specific responses crucial for cultivated meat applications and establishing a framework for systematic cell line safety assessment.