Biology subjects

Melzener, L.

Publications and source records attributed to Melzener, L..

3 recordsLinked to original sources

bioRxiv2023

Optimisation of cell fate determination for cultured muscle differentiation

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.

cell biology↗

bioRxiv2022

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

Cultured 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 cells, including muscle fibres, satellite cells (SCs) and fibro-adipogenic progenitors (FAPs), and recreation of the tissue in vitro thus requires the characterisation and manipulation of a broad range of cell types. Here, we use a single-cell RNA sequencing approach to characterise cellular heterogeneity within bovine muscle and muscle-derived cell cultures over time. Using this data, we identify numerous distinct cell type, 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 time-resolved flow cytometric analysis, we characterise dynamic subpopulations and transitions between active, quiescent, and committed states of SCs, and demonstrate methods for modulation of these states during long-term proliferative cultures. This work provides an important reference for advancing our knowledge of bovine skeletal muscle biology, and its application in the development of cultured meat technologies.

cell biology↗

bioRxiv2022

Comparative analysis of cattle breeds as satellite cells donors for cultured beef

BackgroundCultured meat is a promising new field with the potential for considerable environmental and animal welfare benefits. One technological approach to cultured meat production utilises the proliferative and differentiative capacity of muscle-derived satellite cells (SCs) to produce large volumes of cultured muscle tissue from small biopsies of donor animals. Differing genotypes between cattle breeds lead to predictable phenotypic traits, resulting in breeds being favoured for their respective meat or milk production characteristics in the livestock industry. However, whilst these breeds show significant differences in muscle growth, it is unclear whether the physiological differences observed between them in vivo are reflected in differences in SC behaviour in vitro, particularly with respect to proliferation, differentiation and cellular longevity, and hence whether particular breeds might represent preferred SC donors for a cultured beef bioprocess. ResultsComparing SCs isolated from five breeds (Belgian Blue, Holstein Friesian, Galloway, Limousin and Simmental), we found that the proliferation rates were largely unaffected by the donor breed. In contrast, potentially meaningful differences were observed in the kinetics and extent of myogenic differentiation. Furthermore, whilst differentiation dropped for all breeds with increasing population doublings (PDs), SCs from Belgian Blue and Limousin cattle showed significantly longer retention of differentiation capacity over long-term passaging. ConclusionSCs from all breeds were able to proliferate and differentiate, although Limousin and (particularly) Belgian Blue cattle, both breeds commonly used for traditional meat production, may represent preferred donors for cultured beef production.

cell biology↗