Single-cell analysis of bovine muscle-derived cell types for cultured meat production

Cultured' or 'cultivated' meat technologies leverage the proliferation and differentiation of animal-derived stem cells ex vivo to produce edible tissues for human consumption in a sustainable fashion. However, skeletal muscle is a dynamic and highly complex tissue, involving the interplay of numerous mono- and multinucleated cell types, including muscle fibres, satellite cells (SCs) and fibro-adipogenic progenitors (FAPs), and accurate recreation of the entire tissue thus requires the identification, purification, proliferation and characterisation of a broad range of cell types. Here, we use a single-cell RNA sequencing approach to characterise transcriptional heterogeneity within bovine muscle and derived in vitro cultures over time. Using this data, we identify numerous distinct cell types, and develop robust protocols for the easy purification and proliferation of several of these populations. We note overgrowth of undesirable cell types within heterogeneous proliferative cultures as a barrier to efficient cultured meat production, and use transcriptomics to identify conditions that favour the growth of SCs in the context of serum-free medium. Combining RNA velocities computed in silico with functional data, we characterise dynamic subpopulations and transitions between active, quiescent and committed substates of SCs, and demonstrate methods for modulation of these states during long-term proliferative cultures. This work represents an important resource for advancing our knowledge of bovine skeletal muscle biology, and its application in the development of cultured meat technologies. Overall design: Five timepoints throughout long-term in vitro culture were selected for single-cell RNA sequencing (scRNA-seq), for each of which cells from all genotypes (donor animals) were pooled in equal ratios and washed with 1% BSA in PBS prior to injection. Timepoints 1 to 5 corresponded to unsorted cells directly after isolation ('Muscle') and after 72 h ('passage 0'), and to cultured cells (unsorted cells (genotypes 1, 2, 9), SCs (genotypes 5, 6, 10) and FAPs (genotypes 4, 7, 8) at passages 2, 5, and 8 respectively. Please note that processed data files generated from both run 1 and run2 are linked to the corresponding* run1 sample records.