Mosa Meat B.V., Maastricht, The Netherlands.
Department of Physiology, Maastricht University, Maastricht, The Netherlands.
Commun Biol. 2024 Nov 12;7(1):1493. doi: 10.1038/s42003-024-07201-6.
Production of cultivated meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibres in vitro. Although these formulations can drive myogenic differentiation levels comparable to serum-starvation-based protocols, the resulting cultures are often heterogeneous, with a significant proportion of cells not participating in myofusion, limiting maturation of the muscle. To address this problem, we employed RNA sequencing to analyse heterogeneity in differentiating bovine satellite cells at single-nucleus resolution, identifying distinct cellular subpopulations including proliferative cells that fail to exit the cell cycle and quiescent 'reserve cells' that do not commit to myogenic differentiation. Our findings indicate that the MEK/ERK, NOTCH, and RXR pathways are active during the initial stages of myogenic cell fate determination, and by targeting these pathways, we can promote cell cycle exit while reducing reserve cell formation. This optimised medium formulation consistently yields fusion indices close to 100% in 2D culture. Furthermore, we demonstrate that these conditions enhance myotube formation and actomyosin accumulation in 3D bovine skeletal muscle constructs, providing proof of principle for the generation of highly differentiated cultivated muscle with excellent mimicry to traditional muscle.
培养肉的生产需要定义的培养基配方，以在体外将肌源性细胞有力地分化为成熟的骨骼肌纤维。尽管这些配方可以驱动与血清饥饿为基础的方案相当的肌源性分化水平，但得到的培养物通常是异质的，有相当比例的细胞不参与肌融合，限制了肌肉的成熟。为了解决这个问题，我们采用 RNA 测序以单细胞分辨率分析分化中的牛卫星细胞的异质性，鉴定出包括不能退出细胞周期的增殖细胞和不向肌源性分化作出承诺的静止“储备细胞”等不同的细胞亚群。我们的研究结果表明，MEK/ERK、NOTCH 和 RXR 途径在肌源性细胞命运决定的初始阶段是活跃的，通过靶向这些途径，我们可以促进细胞周期退出，同时减少储备细胞的形成。这种优化的培养基配方在 2D 培养中始终能产生接近 100%的融合指数。此外，我们证明这些条件可以促进 3D 牛骨骼肌构建体中的肌管形成和肌球蛋白积累，为生成具有极好的传统肌肉模拟能力的高度分化培养肉提供了原理证明。
