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

Lea Melzener, Shijie Ding, Rui Hueber, Tobias Messmer, Guanghong Zhou, Mark J Post, Joshua E Flack

Preprint posted on 17 January 2022 https://www.biorxiv.org/content/10.1101/2022.01.14.476358v2

“Special Source”: How the donor animal origin defines applicability of cells for use in cultured meat

Selected by Alexander Ward

Categories: cell biology, genetics, zoology

Background

For many years now, a few visionary skeletal muscle biologists have been looking down the eyepieces of microscopes at cell culture dishes of muscle cells, thinking, “What if I were to eat these? What do they taste like?”. In 2013, one of the authors of this pre-print, Professor Mark Post of the University of Maastricht, made this dream a reality with the creation of the world’s first cell-cultured burger at a cost of €250,000. Since Post’s breakthrough, over 100 companies1 and dozens of academic labs around the world have begun working towards the production of cultured meat. Cultured meat (or in vitro or cultivated meat) involves the isolation, expansion and often differentiation of animal muscle cells taken from living animals, through minimally invasive biopsy techniques, to produce edible animal tissue without the need to harm a single animal (Figure 1).

 

Figure 1. Workflow for cultured meat production

 

In 2018, global meat production was more than 340 million tonnes per year2, with this figure expected to rise to 530 million tonnes by 20303. At present, meat production represents ~15% of global green-house gas emissions (including land use)4, which will likely increase as the global population swells to over 9 billion by 2050 and global demand for meat increases by 70%5. With governmental and institutional pledges to reach Net Zero by 2050, there simply has to be a change in our agricultural system if these targets are to be met6. Part of this reform can come from technologies like cultured meat, which is anticipated to be far more efficient than conventional agriculture in terms of greenhouse gas emissions, water consumption and land use5. Of course, there are caveats to the potentially transformative contributions of cultured meat, as there always will be with new technologies7. These include the use of animal products in cell culture (e.g. bovine serum), the energy demands of cultured meat production and the economic and biological scalability of the processes. However, cultured meat, together with plant-based alternatives, fermentation-produced foods and over-consumption solutions, represents an essential component of the reshaping of our food system and a critical step towards reducing its effect on our planet.

Cultured meat biotechnology can be broadly considered as aiming towards the reconstitution of mature muscle, often from beef, chicken and other livestock animals. This technology can take many forms, from 3D printing steaks to replicating salmon sashimi to exploring muscle from the most diverse species from across the globe. Traditionally, cultured meat has sought to replicate existing food products (e.g., chicken nuggets, beef burgers and sausages), but the future of cultured meat could look vastly different as companies seek to leverage cell and molecular biology to produce new and exciting foods not previously available through traditional agriculture. Biologically, cultured meat relies heavily on muscle cell-types that have the capacity to self-renew, such as skeletal muscle stem cells (satellite cells in muscle) and adipogenic stem cells (fibro-adipogenic progenitors in muscle) (Figure 1). These cells typically have the capacity to propagate significantly longer in culture than other terminally differentiated cell types.

Beyond cell type selection, the field of cultured meat brings with it some unique and fundamental biological challenges. There is a need to maximise cell density in culture, to produce the most mass in the smallest volume, while at the same time reducing the cost of growth media. The reliance on culturing cells without animal derived media components, such as foetal bovine serum, must also be overcome. Novel approaches to achieve improved differentiation, fusion and late-stage maturation of muscle cells need to be conceived. Finally, cultured meat scientists must also solve the Hayflick limit8, the point at which a stem cell undergoes irreversible senescence, in order to generate enough cellular mass to produce meat from just a small amount of starting material. These fundamental obstacles still to be overcome make the science of cultured meat an extremely exciting biological space, not only technically, but intellectually as well.

Key findings

In this preprint, Melzener and colleagues, from Mosa Meat and the University of Maastricht, took a unique approach to profile skeletal muscle stem cell (MuSC) behaviours isolated from 5 different donor cattle species. These cattle species represented a broad range of bovine phenotypes, from the common dairy cow, Holstein Friesian (Figure 2A), to the ‘double muscle’, gigantic Belgian Blue (Figure 2B). The Belgian Blue, famed for its size and muscle mass, contains a deletion in its Myostatin (MYSTN) gene, that leads to significant muscle hypertrophy. Myostatin is a member of the TGF-b family of secreted growth factors and functions to limit skeletal muscle growth9. Through selective breeding, this naturally occurring genetic variant has led the Belgian Blue to become a prized animal for meat production.

 

Figure 2. Two breeds of cattle used in the meat and dairy industry A.Holstein Freisian dairy cow. B. Belgian Blue meat cow with genetic variant in Myostatin gene.

 

Firstly, the authors showed that isolation of MuSCs across distinct cattle species was comparable, with some slight differences. For example, Galloway cattle showed a slightly higher proportion of MuSCs in comparison to other breeds, which, according to the authors, may be related to a lower muscle fibre diameter. Next, the authors investigated how MuSCs behaved after differentiation induction. Briefly, MuSC differentiation to mature muscle, or myogenesis, occurs through differentiation into myocytes, followed by fusion into multi-nucleated immature myotubes; myotubes next undergo a maturation stage, involving significant hypertrophy and organisation of myofibrils (the contractile unit of muscle), to form a mature muscle fibre. The speed and differentiation capacity of MuSCs, measured by myotube fusion index and qualitative assessment of differentiation, was then assessed. MuSCs from the Belgian Blue breed showed the fastest differentiation, reaching close to maximal capacity after only 3 days. After 6 days, other breeds reached a similar maximal differentiation capacity as the Belgian Blue, highlighting that it may not simply be the overall amount of muscle formed that accounts for the breeds impressive muscularity, but the speed at which myogenesis occurs.

Critically in the context of cultured meat, the authors next assessed the differentiation capacity of MuSCs from different breeds across 40 population doublings (PDs), to determine whether certain breeds showed improved myogenic potential after the expansion phase (for the production of cell mass or high cell numbers). All breeds showed a reduced differentiation capacity over time. But, again, the Belgian Blue breed showed the highest differentiation potential after 30 PDs, suggesting that the natural MYSTN gene deletion not only increases the speed of myogenesis, but also its longevity, a feature essential for the scalable production of cultured meat. After 40 PDs, however, the differentiation capacity of even Belgian Blue MuSCs was comparable to other breeds, indicating that the breed is not protected from the effects of stem cell senescence.

What I liked about this preprint

The isolation and characterisation of MuSCs from donor animals selectively bred for meat-specific qualities is a unique approach to identifying the optimal cell-types for use in cultured meat. This is especially relevant with the inclusion of the Belgian Blue breed. This breed showed consistently increased myogenic potential, and a clear genotype-phenotype correlation in vitro that can underline the suitability of MuSCs from specific, selectively bred animals as excellent cellular candidates for cultured meat. Due to the need to produce cultured meat without any genetic manipulation, the approach used here represents an interesting one that exploits the natural and selective variation of the donor system for cultured meat applicability. This type of genotype-phenotype driven donor selection in cultured meat could one day be used to exploit the innate longevity of giant tortoises or the fast twitch muscle of a cheetah together with the myogenic capacity and size of an African elephant. Imagine a world like that, where food possibilities are limitless, the forest is steadily recovering and not a single animal was needlessly killed in the process. This will one day be a reality and this preprint, and other research like it, represents one of the first steps this scientific, ethical and environmental journey.

Tags: cultured meat

Posted on: 28 February 2022

doi: https://doi.org/10.1242/prelights.31498

Read preprint (1 votes)

Author's response

Joshua E Flack shared

  1. Given the plateauing of differentiation capacity at higher population doublings and the similarity between breeds at these later stages, do you think that these findings can be applied at the vast scales needed for production?

This is a great question. Right now, we haven’t identified the culture conditions that retain the cells in something close to their ’native’ in vivo state, that much is very clear from our data. This certainly means that by the time we get around to differentiation, the cells have lost something of their original identity. As we improve our understanding of the cells and culture conditions, we suspect that we will retain more of the original biology, and be achieving robust differentiation at higher and higher PDs, which is obviously important for upscaling. We already have quite a lot of ideas on this since we performed these breed comparison experiments. So overall, yes, we think that donor characteristics will be important at the production scale.

  1. How heterogeneous are satellite cells isolated from each of these breeds, in terms of their myogenic capacity? And do you see a spread of maximal differentiation abilities across single isolated satellite cells?

This is a problem we are very interested in tackling. We think every cell theoretically retains the capacity for myogenic differentiation. Actually, executing that differentiation pathway in a large culture of SCs is a different story of course; we know that there are certain pathways that specify ‘reserve cells’ that do not differentiate, but return to a quiescent phenotype. Whether this process differs between the breeds, or underlies some of the differences we observed in this study, is an open question at this point.

  1. Did you observe differences in myotube size and hypertrophy in the Belgian Blue-derived myocytes?

We didn’t observe major differences in myotube size for the Belgian Blue-derived cells, or between any of the other breeds. This is probably limited by the relatively short timeline of our differentiation assays, since the myotubes begin to detach from a 2D surface as they assemble a contractile apparatus. Would be interesting to compare differentiation and myotube characteristics between breeds in a more sophisticated assay system that retains the myotubes for a longer time, and thus allows a greater degree of maturation to be reached.

  1. Do you anticipate large differences in longevity of differentiation capacity to be driven by the longevity of the donor animal?

Another great question. This comparison of breeds is of course only one small aspect when it comes to assessing donor characteristics. We are particularly interested in comparing cells derived from donors of different ages within the same breed, where we would most obviously expect the remaining ‘longevity’ of the donor to vary in a (somewhat) controlled fashion. We’re also interested in comparing between sexes, and of course the location the sample is taken from. Overall though it’s rather hard at this stage to say exactly how the cellular ageing process within the animal corresponds to what is happening in our in vitro system, and hence to make predictions of cellular longevity based on donor characteristics.

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