Oleic Acid and Transferrin Synergistically Induce Serum-Free Adipogenic Differentiation of Porcine Preadipocytes via the SEPTIN4/PPARγ Axis

Cultured meat represents an emerging frontier in cellular agriculture, garnering increasing interest due to its potential benefits regarding sustainability, animal welfare, and food safety. However, its development is hampered by challenges in flavor modulation and sensory quality, primarily due to the limited biosynthesis of fat-derived flavor compounds. Although adipose tissue engineering has been extensively studied, its industrial-scale production is hampered by serum dependency and low differentiation efficiency. Therefore, the establishment of serum-free, efficient strategies for regulating lipid synthesis is urgently needed. In this study, we developed a serum-free adipogenic induction system and investigated its underlying regulatory mechanisms. We demonstrated that Serum-Free Differentiation Medium 1 (SFM-1) initiated the differentiation program and induced intracellular lipid deposition in preadipocytes (~10% by Day 8). Serum-free differentiation medium 2 (SFM-2), which supplied oleic acid (OA) as a lipid substrate and signaling activator, markedly enhanced lipid droplet accumulation and differentiation efficiency. Ultimately, serum-free differentiation medium 3 (SFM-3), leveraging the synergistic action of oleic acid (OA) and transferrin (TRF), successfully activates the expression of SEPTIN4, which in turn regulates a core adipogenic network—including the master transcription factors PPARγ and CEBPα, as well as downstream functional genes. Mechanistically, the OA/TRF combination in SFM-3 upregulates SEPTIN4, unveiling a previously unrecognized regulatory axis that activates the PPARγ signaling pathway, thereby synchronizing the proliferation and differentiation of precursors and guiding them from initiation to functional maturity. Our study presents a chemically defined, scalable platform for the serum-free adipogenic differentiation of porcine adipocytes, offering a promising strategy for the controllable production of fat components in cultured meat and supporting its industrialization.