Optimisation of cell fate decisions for cultured muscle differentiation

Production of cultured meat requires defined medium formulations for the robust differentiation of myogenic cells into mature skeletal muscle fibres in vitro. Whilst such formulations can drive myogenic differentiation to an extent similar to serum-starvation based protocols, these cultures are visually heterogeneous in nature, with a significant proportion of cells not participating in myofusion, limiting the 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. By targeting the ERK, RXR and NOTCH pathways, we show that cell cycle exit can be promoted whilst simultaneously abrogating reserve cell formation. Under these improved culture conditions, fusion indices close to 100% can be robustly obtained in 2D culture. We further show that this translates to higher levels of myotube formation and muscle protein accumulation in animal-free bioartificial muscle (BAM) constructs, providing proof of principle for the generation of highly differentiated cultured muscle with excellent mimicry to traditional meat. To study transcriptional hetereogeneity during myogenic differentiation, Bovine satellite cells (SCs) were differentiated using a serum-free differentiation media, harvested on subsequent days (0 h, 24 h, 48 h, 72 h, 96 h) and analysed with single-nucleus RNA sequencing.