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Transcriptome profiles of bovine muscle stem cells cultured in serum-containing condition and small molecule–supplemented serum-free media by RNA-seq analysis

ABSTRACT: Cultured meat, as an emerging sustainable food production technology, offers multiple advantages. However, its large-scale application still faces critical technical bottlenecks, most notably the strong reliance on fetal bovine serum (FBS). In recent years, serum-free culture systems for bovine muscle stem cells (bMuSCs) have made certain progress, such as the Beefy-9 system and its subsequent optimized versions; nevertheless, existing serum-free formulations remain insufficient to fully replace traditional serum-containing culture conditions in both cost control and functional performance. Meanwhile, the use of small-molecule compounds to precisely regulate cell proliferation and fate determination has gradually emerged as a potentially effective strategy to reduce culture costs, simplify culture systems, and enhance functional stability. In this study, through a functional screening of proliferation activators in bMuSCs, we identified forskolin and a p38 pathway inhibitor SB202190 to robustly promote cell proliferation and maintain myogenic capacity of bMuSCs under serum-free conditions. Based on this, RNA-sequencing analysis was committed to systematically elucidate the molecular differences between serum-free small-molecule regulatory strategies and conventional serum-containing conditions in bMuSCs.

ORGANISM(S): Bos taurus

PROVIDER: GSE316044 | GEO | 2026/01/14

REPOSITORIES: GEO

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Project description:Macrophages are central to innate immunity and defining the mechanisms by which these cells mediate inflammation is critical to understanding in situ homeostatic and pathophysiologic roles. Human monocyte derived macrophages (hMDM) are widely used to examine these processes. Stimuli that influence macrophage function could influence these studies, including differences in culture environment that have been suggested significantly impact macrophage function. We examined baseline hMDM activity and the response to the TLR4 agonist, lipopolysaccharide (LPS) in different culture media in the presence and absence of serum. We focused on the most common macrophage medias, DMEM and RPMI 1640 Medium +/- fetal bovine serum (FBS), as well as Macrophage Serum Free Media (M-SFM), a media specifically formulated for serum free culture. Cells cultured in these five distinct media were treated with vehicle (H20) or LPS and evaluated for changes in morphology, transcriptomic profile, and alterations in inflammatory response. Culture in M-SFM resulted in the greatest morphological differences (area, length-to-width, perimeter-to-area ratio) and while there were few transcriptomic differences between DMEM and RPMI with FBS, both the absence of FBS supplementation and culture in M-SFM significantly altered the baseline transcriptome. Culture in M-SFM also significantly reduced macrophage sensitivity to LPS as measured by NF-kB activation. All hMDM conditions showed a dose dependent phagocytic response and elicited cytokine and chemokine secretion in response to LPS stimulation, but the magnitude of these effects varied between conditions, with different trends depending on the cytokine. These findings suggest that culture media and the presence of serum alters baseline activation and the response to stimulus in macrophages, potentially obscuring or driving artifactual results. These studies confirm and expand on previous findings showing that in vitro microenvironment is not a benign component of experimental design and demonstrate the need to optimize experimental conditions for such plastic cells.

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Transcriptome profiles of bovine muscle stem cells cultured in serum-containing condition and small molecule–supplemented serum-free media by RNA-seq analysis

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