Enhancement of myogenic differentiation and myotube formation by stanozolol in porcine muscle satellite cells for cultured meat production

Objective: Recent studies on cultured meat have shown that muscle satellite cells can be induced to form muscle-like tissues through proliferation and myogenic differentiation.Optimizing these processes is essential for reproducing the structure and texture of conventional meat; however, limited myogenic differentiation remains a major technical challenge.Stanozolol (STZ), a synthetic anabolic steroid derived from testosterone, is known to stimulate muscle protein synthesis and hypertrophy, but its effects on livestock-derived muscle cells for cultured meat production remain unclear.This study aimed to evaluate whether STZ enhances myogenic differentiation and myotube formation in primary porcine muscle satellite cells (PMSCs).Methods: PMSCs were treated with STZ at concentrations of 0.1, 0.5, and 1 μM.Cell viability and proliferation were assessed to determine the non-cytotoxic concentration range.Myogenic differentiation was evaluated by measuring the expression of myogenic differentiation 1 (MyoD), myogenin (MyoG), myosin heavy chain (MyHC), and myostatin (MSTN).Myotube formation was analysed by immunofluorescence staining, and the underlying mechanisms were investigated by examining PI3K/AKT/mTOR, Smad2/3, and forkhead box O3a (FoxO3a) signaling.Results: STZ showed no cytotoxicity and did not significantly affect PMSC proliferation at 0.1-1 μM.In differentiation medium, STZ significantly increased the mRNA and protein expression of myogenic markers, including MyoD, MyoG, and MyHC, in a concentration-dependent manner.Notably, 1 μM STZ promoted the formation of larger multinucleated myotubes.In addition, STZ decreased MSTN expression, activated PI3K/AKT/mTOR hypertrophic signaling, and suppressed Smad2/3 phosphorylation and FoxO3a activity. Conclusion:STZ promoted myogenic differentiation and myotube formation in PMSCs by enhancing myogenic marker expression and modulating hypertrophy-and atrophy-associated signaling pathways.These findings suggest that STZ may serve as an effective culture medium supplement for improving PMSC differentiation in cultured meat production.