Optimisation of cell fate determination for cultured muscle differentiation

Abstract

Production of cultured meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibers in vitro. Whilst such formulations can drive myogenic differentiation to an extent similar to serum-starvation based protocols, these cultures are invariably heterogeneous in nature, with a significant proportion of cells not participating in myofusion, limiting maturation of the muscle. Here, we use RNA sequencing to characterise this heterogeneity at single-nucleus resolution, identifying distinct cellular subpopulations, including proliferative cells that fail to exit the cell cycle, and reserve cells that do not commit to myogenic differentiation. We show that the ERK, NOTCH and RXR pathways act during the first stages of myogenic cell fate determination, and by targeting these pathways, cell cycle exit can be promoted whilst abrogating reserve cell formation. Under these improved culture conditions, fusion indices close to 100% can be robustly obtained in 2D culture. Finally, we demonstrate that this translates to higher levels of myotube formation and muscle protein accumulation in animal component-free bioartificial muscle constructs, providing proof of principle for the generation of highly differentiated cultured muscle with excellent mimicry to traditional muscle.