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A serum-free media formulation for cultured meat production supports bovine satellite cell differentiation in the absence of serum starvation

Abstract

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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Fig. 1: Bovine SCs undergo extensive transcriptomic changes upon serum starvation.
Fig. 2: Differential expression analysis identifies surface receptors upregulated upon serum starvation.
Fig. 3: Serum-free differentiation is induced by supplementation of ligands to upregulated receptors.
Fig. 4: Serum-free differentiation is of comparable extent to serum starvation.
Fig. 5: Transcriptional remodelling during serum-free differentiation is similar to serum starvation.
Fig. 6: Serum-free differentiation medium enables fabrication of bioartificial muscles.

Data availability

RNA-seq data has been deposited to the GEO (accession number GSE173199). Source data are provided with this paper. Further data supporting the findings of this study are available from the authors upon request.

Code availability

Analysis code is available from the authors upon request.

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

T.M., I.K., C.F., E.O., A.D. and J.E.F. performed experiments and analysis. A.D., H.C., M.J.P. and J.E.F. supervised the study. T.M., M.J.P. and J.E.F. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Joshua E. Flack.

Ethics declarations

Competing interests

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.

Additional information

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

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.

Extended Data Table 1 Media Formulations
Extended Data Table 2 RT–qPCR primers

Supplementary information

Reporting Summary

Supplementary Video 1

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.

Supplementary Table 1

Differential gene expression analysis between 0 and 96 h of serum starvation.

Supplementary Table 2

Proteomic changes during serum starvation.

Source data

Source Data Fig. 1

Uncropped blots corresponding to Fig. 1.

Source Data Fig. 4

Uncropped blots corresponding to Fig. 4.

Source Data Fig. 6

Uncropped blots corresponding to Fig. 6.

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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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