Cultured meat production requires the robust differentiation of satellite cells into mature muscle fibres without the use of animal-derived components. Current protocols induce myogenic differentiation in vitro through serum starvation, that is, an abrupt reduction in serum concentration. Here we used RNA sequencing to investigate the transcriptomic remodelling of bovine satellite cells during myogenic differentiation induced by serum starvation. We characterized canonical myogenic gene expression, and identified surface receptors upregulated during the early phase of differentiation, including IGF1R, TFRC and LPAR1. Supplementation of ligands to these receptors enabled the formulation of a chemically defined media that induced differentiation in the absence of serum starvation and/or transgene expression. Serum-free myogenic differentiation was of similar extent to that induced by serum starvation, as evaluated by transcriptome analysis, protein expression and the presence of a functional contractile apparatus. Moreover, the serum-free differentiation media supported the fabrication of three-dimensional bioartificial muscle constructs, demonstrating its suitability for cultured beef production.
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Parodi, A. et al. The potential of future foods for sustainable and healthy diets. Nat. Sustain. 1, 782–789 (2018).
Post, M. J. et al. Scientific, sustainability and regulatory challenges of cultured meat. Nat. Food 1, 403–415 (2020).
Bischoff, R. Regeneration of single skeletal muscle fibers in vitro. Anat. Rec. 182, 215–235 (1975).
Collins, C. A. et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289–301 (2005).
Vandenburgh, H. et al. Tissue-engineered skeletal muscle organoids for reversible gene therapy. Hum. Gene Ther. 7, 2195–2200 (1996).
Ben-Arye, T. et al. Textured soy protein scaffolds enable the generation of three-dimensional bovine skeletal muscle tissue for cell-based meat. Nat. Food 1, 210–220 (2020).
Pirkmajer, S. & Chibalin, A. V. Serum starvation: caveat emptor. Am. J. Physiol. Cell Physiol. 301, C272–C279 (2011).
Das, M. et al. Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation. In Vitro Cell. Dev. Biol. Anim. 45, 378–387 (2009).
Guo, X. et al. In vitro differentiation of functional human skeletal myotubes in a defined system. Biomater. Sci. 2, 131–138 (2014).
Chal, J. et al. Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat. Biotechnol. 33, 962–969 (2015).
Akiyama, T. et al. Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing. Sci. Rep. 8, 1189 (2018).
Chal, J. & Pourquié, O. Making muscle: skeletal myogenesis in vivo and in vitro. Development 144, 2104–2122 (2017).
Millay, D. P. et al. Myomaker: a membrane activator of myoblast fusion and muscle formation. Nature 499, 301–305 (2013).
Bryson-Richardson, R. J. & Currie, P. D. The genetics of vertebrate myogenesis. Nat. Rev. Genet. 9, 632–646 (2008).
Tsumagari, K. et al. Gene expression during normal and FSHD myogenesis. BMC Med. Genomics 4, 67 (2011).
He, H. & Liu, X. Characterization of transcriptional complexity during longissimus muscle development in bovines using high-throughput sequencing. PLoS ONE 8, e64356 (2013).
Tripathi, A. K. et al. Transcriptomic dissection of myogenic differentiation signature in caprine by RNA-Seq. Mech. Dev. 132, 79–92 (2014).
Lee, E. J. et al. Identification of genes differentially expressed in myogenin knock-down bovine muscle satellite cells during differentiation through RNA sequencing analysis. PLoS ONE 9, e92447 (2014).
Nishiyama, T., Kii, I. & Kudo, A. Inactivation of Rho/ROCK signaling is crucial for the nuclear accumulation of FKHR and myoblast fusion. J. Biol. Chem. 279, 47311–47319 (2004).
Capkovic, K. L., Stevenson, S., Johnson, M. C., Thelen, J. J. & Cornelison, D. Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation. Exp. Cell. Res. 314, 1553–1565 (2008).
Tang, Z. et al. Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle. J. Biol. Chem. 271, 2255–2261 (1996).
Seale, P. et al. Pax7 is required for the specification of myogenic satellite cells. Cell 102, 777–786 (2000).
Weiss, A. & Leinwand, L. A. The mammalian myosin heavy chain gene family. Annu. Rev. Cell Dev. Biol. 12, 417–439 (1996).
Pardee, A. B. A restriction point for control of normal animal cell proliferation. Proc. Natl Acad. Sci. USA 71, 1286–1290 (1974).
He, K. et al. A transcriptomic study of myogenic differentiation under the overexpression of PPARγ by RNA-Seq. Sci. Rep. 7, 15308 (2017).
Massoner, P., Ladurner-Rennau, M., Eder, I. E. & Klocker, H. Insulin-like growth factors and insulin control a multifunctional signalling network of significant importance in cancer. Br. J. Cancer 103, 1479–1484 (2010).
Chen, G. et al. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8, 424–429 (2011).
Breton, C. et al. Presence of functional oxytocin receptors in cultured human myoblasts. J. Clin. Endocrinol. Metab. 87, 1415–1418 (2002).
Nikolić, N. et al. Electrical pulse stimulation of cultured skeletal muscle cells as a model for in vitro exercise—possibilities and limitations. Acta Physiol. 220, 310–331 (2017).
Gawlitta, D., Boonen, K. J. M., Oomens, C. W. J., Baaijens, F. P. T. & Bouten, C. V. C. The influence of serum-free culture conditions on skeletal muscle differentiation in a tissue-engineered model. Tissue Eng. A 14, 161–171 (2008).
Lawson, M. A. & Purslow, P. P. Differentiation of myoblasts in serum-free media: effects of modified media are cell line-specific. Cells Tissues Organs 167, 130–137 (2000).
Shoji, E., Woltjen, K. & Sakurai, H. Directed myogenic differentiation of human induced pluripotent stem cells. Methods Mol. Biol. Clifton NJ 1353, 89–99 (2016).
Tong, H. L. et al. Transcriptional profiling of bovine muscle-derived satellite cells during differentiation in vitro by high throughput RNA sequencing. Cell. Mol. Biol. Lett. 20, 351–373 (2015).
Brunetti, A., Maddux, B. A., Wong, K. Y. & Goldfine, I. D. Muscle cell differentiation is associated with increased insulin receptor biosynthesis and messenger RNA levels. J. Clin. Invest. 83, 192–198 (1989).
Ozawa, E. Transferrin as a muscle trophic factor. Rev. Physiol., Biochem. Pharmacol. 113, 89–141 (1989).
Cox, R. D., Garner, I. & Buckingham, M. E. Transcriptional regulation of actin and myosin genes during differentiation of a mouse muscle cell line. Differ. Res. Biol. Divers. 43, 183–191 (1990).
Hansen, L. H., Abrahamsen, N. & Nishimura, E. Glucagon receptor mRNA distribution in rat tissues. Peptides 16, 1163–1166 (1995).
Uhlén, M. et al. Proteomics. Tissue-based map of the human proteome. Science 347, 1260419 (2015).
Cencetti, F. et al. Lysophosphatidic acid stimulates cell migration of satellite cells. A role for the sphingosine kinase/sphingosine 1-phosphate axis. FEBS J. 281, 4467–4478 (2014).
Yoshida, S., Fujisawa-Sehara, A., Taki, T., Arai, K. & Nabeshima, Y. Lysophosphatidic acid and bFGF control different modes in proliferating myoblasts. J. Cell Biol. 132, 181–193 (1996).
Zhang, Z. et al. Oxytocin is involved in steroid hormone-stimulated bovine satellite cell proliferation and differentiation in vitro. Domest. Anim. Endocrinol. 66, 1–13 (2019).
Denes, L. T. et al. Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation. Skelet. Muscle 9, 17 (2019).
Stephens, N. et al. Bringing cultured meat to market: technical, socio-political, and regulatory challenges in cellular agriculture. Trends Food Sci. Technol. 78, 155–166 (2018).
Furuhashi, M. et al. Formation of contractile 3D bovine muscle tissue for construction of millimetre-thick cultured steak. NPJ Sci. Food 5, 6 (2021).
Costantini, M. et al. Microfluidic-enhanced 3D bioprinting of aligned myoblast-laden hydrogels leads to functionally organized myofibers in vitro and in vivo. Biomaterials 131, 98–110 (2017).
Lk, L. et al. A pilot study on pain and the upregulation of myoglobin through low-frequency and high-amplitude electrical stimulation-induced muscle contraction. J. Phys. Ther. Sci. 26, 985–988 (2014).
Melzener, L., Verzijden, K., Buijs, A., Post, M. & Flack, J. Cultured beef: from small biopsy to substantial quantity. J. Sci. Food Agric. 101, 7–14 (2020).
Ding, S. et al. Maintaining bovine satellite cells stemness through p38 pathway. Sci. Rep. 8, 10808 (2018).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Yates, A. D. et al. Ensembl 2020. Nucleic Acids Res. 48, D682–D688 (2020).
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
The Gene Ontology Consortium. The Gene Ontology Resource: 20 years and still GOing strong. Nucleic Acids Res. 47, D330–D338 (2019).
Zhang, X. et al. Proteome-wide identification of ubiquitin interactions using UbIA-MS. Nat. Protoc. 13, 530–550 (2018).
We thank K. Derks and D. Tserpelis (Genome Services Maastricht UMC+) for their support in the acquisition and analysis of RNA-seq data, R. Mohren (Imaging Mass Spectrometry, M4I, Maastricht University) for proteomics data, and H. Duimel and C. López Iglesia (Microscopy CORE Lab, M4I, Maastricht University) for SEM. We also thank J. Melke and D. Remmers (Mosa Meat BV) for their assistance with confocal microscopy.
T.M., I.K., C.F., E.O., A.D., H.C. and J.E.F. are employees of Mosa Meat BV. M.J.P. is co-founder and stakeholder of Mosa Meat BV. The study was funded by Mosa Meat BV. Mosa Meat BV has patents pending on serum-free proliferation medium (PCT/P125933PC00) and serum-free differentiation medium (JBB/P126144NL00). All authors declare no other competing interests.
Peer review information Nature Food thanks Deepak Choudhury, Laura Domigan and Min Du for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Gene expression during myogenic differentiation induced by serum starvation (related to Fig. 1).
a, RNA-seq-derived median fold changes of selected muscle-related genes during serum starvation compared to 0 h. b, Mean fold changes of genes shown in a), determined via RT–qPCR; error bars indicate SD, n = 3. c, Median fold changes of selected strongly upregulated myogenic genes compared to 0 h, determined via RNA-seq. d, Median fold changes of selected downregulated genes compared to 0 h, determined via RNA-seq; boxes indicate IQR, whiskers show 1.5 × IQR.
Extended Data Fig. 2 Transcriptomic and proteomic characterisation of serum starvation (related to Fig. 2).
a, Volcano plot showing differentially expressed genes between 0 h (yellow) and 96 h (red) of serum starvation. Selected differentially expressed muscle, stem cell or cell cycle-related genes are indicated. b, Scatter plot showing correlation of log2-fold changes of overlapping genes from bovine (x-axis) with C2C12 (y-axis) with indicated Pearson correlation coefficient (R). Colours indicate upregulation (red) or downregulation (yellow) in bovine gene expression, shapes indicate whether differentially expressed genes are simultaneously up/downregulated in both species (squares) or significantly up/downregulated in one species while inversely regulated in the other (triangles). c, Median fold changes of muscle-related protein levels from 0 h to 72 h post serum starvation, normalised against 0 h; boxes indicate IQR, whiskers show 1.5 × IQR. d, Scatter plot showing the Pearson correlation of log2-fold changes of genes from RNA-seq (y-axis) and corresponding proteins from mass spectrometry (x-axis) upon serum starvation with indicated correlation coefficient (R). Colours indicate upregulation (red) or downregulation (yellow) while shapes indicate significantly regulated proteins (points), genes (triangles), or both (squares). e, Mean fold gene expression changes of differentially regulated surface receptors, determined by RT–qPCR; error bars indicate SD, n = 3.
Extended Data Fig. 3 Serum-free differentiation and serum starvation are similar with respect to cell age and coating (related to Fig. 4).
a, Normalised nuclei counts of SCs differentiating on indicated coatings after 72 h in SFB, SFDM and serum starvation as percentage against SFB; statistical significance is indicated for each media against respective Matrigel control, error bars indicate SD, n = 3. b, Normalised nuclei counts of SCs after 72 h of SFB, SFDM or serum starvation at early (left), medium (centre), and late (right) passages with indicated PDs, as percentage of low PDs in SFB; asterisks directly above bars indicate statistical significance against SFB; error bars indicate SD, n = 4. c, Representative fluorescence images of differentiating SCs at early (top), medium (middle) or late (bottom row) passages after 72 h in SFB (left), SFDM (centre) or serum starvation (right), corresponding to Fig. 4e, Extended Data Fig. 3b; green, desmin; blue, Hoechst. Scale bar, 500 µm. *P < 0.05, **P < 0.005, ***P < 0.001.
Extended Data Fig. 4 Extent of serum-free differentiation varies between donor animals (related to Fig. 4).
a, Normalised nuclei counts of SCs from different donor animals after 72 h of myogenic differentiation as percentage of SFB with statistical significance indicated between SFDM and serum starvation respectively for each donor; error bars indicate SD, n = 4. b, Mean fusion indices of SCs from different donor animals after 72 h of serum-free or serum starvation induced differentiation, normalised against respective SFB condition. Statistical significance is indicated between SFDM and serum starvation respectively for each donor; error bars indicate SD, n = 4. c, Representative fluorescence images of myogenic differentiation of SCs from different donor animals after 72 h in SFB (top), SFDM (middle) and serum starvation (bottom row); green, desmin; blue, Hoechst. Scale bar, 500 µm. *P < 0.05, **P < 0.005, ***P < 0.001.
Extended Data Fig. 5 2D serum-free differentiation can be achieved with different basal media (related to Fig. 6).
a, Representative fluorescence images of SCs after 72 h in SFB, SFDM with DMEM/F-12 and DMEM base, and upon serum starvation; green, desmin; blue, Hoechst. Scale bar, 500 µM. b, Normalised nuclei counts from a) as percentage of SFB with statistical significance indicated against SFDM (DMEM/F-12); error bars indicate SD, n = 4. c, Mean fusion indices derived from a) with statistical significance performed against SFDM (DMEM/F-12); error bars indicate SD, n = 4. d, Scatter plot indicating correlation of log2-fold changes between SFGM and DMEM/F-12-based SFDM (x-axis) against log2-fold changes between SFGM and DMEM-based SFDM (y-axis) with Pearson correlation coefficient indicated. *P < 0.05, **P < 0.005, ***P < 0.001.
Extended Data Fig. 6 Acetylcholine supplementation improves myogenic fusion in bioartificial muscles (related to Fig. 6).
a, Ultrastructure of BAMs after 192 h in SFB or SFDM (with DMEM/F-12 or DMEM basal media) or serum starvation. Scale bar, 100 µm. b, Representative fluorescence images of BAMs after 192 h in DMEM-based SFDM without (left) and with (right) 10 µM acetylcholine (ACh); pink, desmin; red, 𝛂-actin; green, myosinHC; blue, Hoechst. Scale bars, 100 µm. c, Ultrastructure (top), wide (middle) and close-up (bottom) scanning electron microscopy images of BAMs after 192 h in DMEM-based SFDM with (right) and without (left) 10 µM acetylcholine. Scale bars, 100 µm.
Electrical pulse stimulation of serum-free myotubes (related to Fig. 4). SCs were differentiated in SFDM for 192 h before electrical pulse stimulation by a C-PACE EP stimulator at 12 V, 1.0 ms pulse width and indicated frequencies.
Differential gene expression analysis between 0 and 96 h of serum starvation.
Proteomic changes during serum starvation.
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Messmer, T., Klevernic, I., Furquim, C. et al. A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation. Nat Food 3, 74–85 (2022). https://doi.org/10.1038/s43016-021-00419-1
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