Vitamin C and N-acetylcysteine promotes bovine AD-MSCs proliferation and differentiation via Akt/mTOR/P70S6K signaling pathway for cultured meat production.

Objective: Traditional meat production is insufficient to meet the increasing protein requirements; necessitating cultured meat, which is safe, worthwhile, and scalable. Fat is crucial for making cultured meat more acceptable to consumers by enhancing flavour and providing a natural appearance. Mesenchymal stem cells from adipose tissue are a promising source for this purpose, but in vitro expansion of cells decreases their proliferation ability and increases cellular senescence. The objective of this study was to improve the proliferation and differentiation abilities of adipose-derived mesenchymal stem cells (AD-MSCs).

Methods: In this study, vitamin C (VC) and N-acetylcysteine (NAC) antioxidants were used to treat AD-MSCs from Yanbian cattle testicles. CCK8, EdU staining, RT-qPCR, and western blot were used to test the cell viability and proliferation ability of AD-MSCs, ORO staining, triglycerides assay, and adipogenic specific markers expression were determined to analyse the adipogenic differentiation ability. Furthermore, oxidative stress parameters and activation of the Akt/mTOR/P70S6K signalling pathway were also studied.

Results: Results showed that VC and NAC both increased proliferation and differentiation ability of AD-MSCs by increasing the expression of cell cycle regulatory and differentiation genes and proteins expression, and decreasing the expression of cell cycle inhibitory factors, and up-regulating stemness markers expression, while co-treatment showed enhanced effect. Oxidative stress was reduced by decreased ROS production, malondialdehyde content, and enhanced glutathione activity, as well as declined cellular senescence. Subsequently, the Akt/mTOR/P70S6K signalling pathway was activated by VC and VC+NAC treatment in AD-MSCs, while NAC only activates Akt expression, indicating its role in controlled cell growth.

Conclusion: This research concludes that NAC (2 mM) and VC (200 μM) improved the proliferation, differentiation potential, and stemness by decreasing oxidative stress and senescence, parallelly activating Akt/mTOR/P70S6K signalling pathway, while combined treatment (NAC+VC) enhanced these effects, providing bases for their utilisation to culture fat in cultivated meat production.