C2C12 cells (Yaffe and SAXEL, 1977b), an immortalized mouse myoblast cell line (ECACC: 91,031,101), were cultured for rapid proliferation in a “DF10” medium composed of 10% Fetal Bovine Serum (Gibco, 10270-106) and DMEM medium (nacalai tesque, 08458-45) supplemented with Penicillin-Streptomycin Mixed Solution (P/S) (1x) (nacalai tesque, 26253-84). The cells were passaged every 3-4 days with TrypLE (Gibco, 12604-013) at a split ratio of 1:5. C2C12 cells are capable of differentiating into myotubes under low serum conditions (Burattini et al., 2004), (Andrés and Walsh, 1996). For induction of differentiation, 3.0 × 10⁴ cells were seeded onto one well of a 4-well plate (Nunc) in the DF10 medium. At 2-3 days after seeding, when the cell confluency reached 90%–100%, the medium was changed to a “DH2” medium, which is a reduced-serum condition composed of 2% Horse Serum (H1138, Sigma)-contained DMEM supplemented with P/S (1x) with Methyl-β-cyclodextrin (MβCD) (Sigma-Aldrich, 332615) at a concentration of 3 mM. The solvent, sterile water, was added for the control. In the case of cultivation for 5 days, the DH2 medium was changed on day 3.

Thigh muscle tissue was dissected from 10-day-old chick embryos, typically obtained from a certified poultry farm, using fine forceps. The femurs were surgically removed from the thigh muscle tissue, and the isolated thigh muscle tissue was minced with fine forceps and washed in PBS twice. The minced tissue was then digested with 2 mg/ml collagenase Type II (Worthington Biochemical Corporation, WOR-CLS2) in HBSS (nacalai tesque, 09735-75) at 37°C for 40-60 min, depending on the detachment of cells, with pipetting every 10–15 min. The dissociated myogenic cells were passed through a 40 μm cell strainer (Corning, 431750) for single-cell suspension, and 5.0 × 10⁵ dissociated cells were plated onto one well of a 4-well plate precoated with 0.2% gelatin in DF10 medium for stable attachment. On the following day, the medium was changed to DH2 medium containing 2% horse serum in DMEM supplemented with P/S (1x) with MβCD (the solvent, sterile water, was added for control) at a concentration of 1 mM. After induction of differentiation in the DH2 medium for 1 day with or without MβCD, the cultured chick myogenic cells were further analyzed. All animal experiments were performed following ethical guidelines set by Kindai University, and animal protocols were reviewed and approved by the Kindai University Animal Care and Use Committee.

For immunofluorescence studies, cells grown on a 4-well plate were fixed with freshly prepared 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized and blocked with 1% Triton-X, 1% BSA, and 10% FBS-contained PBS for 1 h at room temperature. The cells were incubated with primary antibodies in 10% FBS, 1% BSA, and 1% Triton-X-containing PBS overnight at 4°C. On the following day, the cells were washed three times in 0.1% Triton/PBS and incubated with fluorescent-labeled IgG (H + L) secondary antibodies (Abcam, Goat anti-mouse (Alexa Fluor 488): ab150113, (Alexa Fluor 594): ab150116, Goat anti-rabbit (Alexa Fluor 594: ab150080) at 1:500 dilutions in 1% FBS, 1% BSA, and 1% Triton-X-containing PBS for 2 h at room temperature. The cells were washed three times and filled with 0.1% Triton-containing PBS, and nuclei were counter-stained with DAPI (Sigma-Aldrich, D9542) for immunofluorescence. Primary antibodies used in this study include Anti-Myosin Heavy Chain antibody (MF20) (1:500, R&D Systems, MAB4470), Anti-Myogenin antibody (1:500, Abcam, ab124800), Anti-p-Histone H3 antibody (C-2) (1:1000, Santa Cruz, sc-374669), and Anti-Cleaved Caspase-3 antibody (Asp175) (5A1E) (1:500, Cell Signaling Technology, #9664). The observation was performed under a microscope (Keyence, BZ-X710).

Total RNAs were extracted using RNeasy Mini Kit (QIAGEN, 74104) according to the manufacturer’s instructions. RNAs were reverse-transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO, FSQ-201), and real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix (TOYOBO, QPS-201) in MIC qPCR (bio molecular systems). Expression levels of each gene were normalized according to each purpose to Gapdh or Hprt expression and calculated using the comparative CT method. The primer sequences are shown below: Gapdh-F (AGG​TCG​GTG​TGA​ACG​GAT​TTG), Gapdh-R (TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA), Hprt-F (CAG​TCC​CAG​CGT​CGT​GAT​TA), Hprt-R (AGC​AAG​TCT​TTC​AGT​CCT​GTC), MyoD-F (GAT​GGC​ATG​ATG​GAT​TAC​AGC​GGC), MyoD-R (GTG​GAG​ATG​CGC​TCC​ACT​ATG​CTG), Myogenin-F (CCC​TAT​TTC​TAC​CAG​GAG​CCC​CAC), Myogenin-R (GCG​CAG​GAT​CTC​CAC​TTT​AGG​CAG), Myh7-F (ATG​CTG​ACA​GAT​CGG​GAG​AA), Myh7-R (GGT​TGG​CTT​GGA​TGA​TTT​GA), Mymk-F (CCT​GCT​GTC​TCT​CCC​AAG), Mymk-R (AGA​ACC​AGT​GGG​TCC​CTA​A), Srebf2-F (ACC​TAG​ACC​TCG​CCA​AAG​GT), Srebf2-R (GCA​CGG​ATA​AGC​AGG​TTT​GT), Hmgcs1-F (GCC​GTG​AAC​TGG​GTC​GAA), Hmgcs1-R (GCA​TAT​ATA​GCA​ATG​TCT​CCT), Hmgcr-F (CTT​GTG​GAA​TGC​CTT​GTG​ATT​G), Hmgcr-R (AGC​CGA​AGC​AGC​ACA​TGA​T), Pmvk-F (AAA​ATC​CGG​GAA​GGA​CTT​CGT), Pmvk-R (AGA​GCA​CAG​ATG​TTA​CCT​CCA), Cyp51-F (GAC​AGG​AGG​CAA​CTT​GCT​TTC), Cyp51-R (GTG​GAC​TTT​TCG​CTC​CAG​C).

For modulating the amount of membrane cholesterol, solubilized cholesterol (Sigma, C8667) (as a complex of MβCD and cholesterol in EtOH) was added at 15 µM (the solvent, EtOH, was added for control) as an optimized concentration (Supplementary Figure S4) in the induction of differentiation of C2C12 cells.

Cells were grown on rounded cover glass (MATSUNAMI, 83-0232) treated with Poly-L-lysine coating solution (COSMO BIO, SPL01) in a 4-well plate and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature to label membrane cholesterol. The cells were then washed three times in PBS and incubated with 1.5 mg/ml glycine in PBS for 10 min at room temperature to quench the paraformaldehyde. After that, cells were stained with filipin working solution (0.05 mg/ml filipin complex (Sigma-Aldrich, F9765) in 10% FBS-contained PBS) for 2 h at room temperature and washed three times in PBS. Finally, images were collected using a confocal microscope (Olympus, FV-3000).

Cells were disassociated using 0.25% Trypsin/EDTA (nacalai tesque, 35554-64), washed with 1% BSA/PBS, and fixed in 1% PFA on ice for 1 h. After washing with 1% BSA/PBS, the cells were permeabilized/blocked with 1% Triton-X-, 1% BSA- and 10% FBS-contained PBS for 1.5 h at room temperature. They were then incubated overnight at 4°C with appropriate amounts of primary antibodies, including the Anti-p-Histone H3 antibody (C-2) (1:100, Santa Cruz, sc-374669) and Mouse mAb IgG_2b Isotype Control monoclonal antibody (1:100, Santa Cruz, sc-3879) in 1% FBS-, 1% BSA- and 0.1% Triton-X-contained PBS. On the following day, after washing with 0.1% Triton/PBS, the cells were incubated with fluorescent-labeled IgG (H + L) secondary antibodies (Abcam, Goat anti-mouse, Alexa Fluor 594) at 1:500 dilution in 1% FBS-, 1% BSA- and 0.1% Triton-X-contained PBS for 2 h at room temperature. Immediately after washing with 0.1% Triton/PBS, the cells were resuspended in an ice-cold Sheath fluid (Sysmex, CG-974-836) at 1 × 10⁶ cells/mL and analyzed on a flow cytometer. FACS analysis was performed using Sysmex RF-500 with FlowJo (BD Biosciences), and a minimum of 10,000 events were recorded for each sample. Fluorescence minus control was used to gate the subpopulations.

Total RNAs were extracted using the RNeasy Mini Kit (QIAGEN, 74104) following the manufacturer’s instructions. For each experiment, two independent biological replicates were used for transcriptomic analysis. Sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB) and were sequenced on an Illumina Novaseq platform to generate 150 bp paired-end reads. The adaptors of the obtained raw sequence data were trimmed using Trim Galore (v0.6.3), and quality control using FastQC (v0.11.7). Processed reads were aligned to the mouse reference genome (UCSC mm10) using STAR (v2.7.0c) (Dobin et al., 2013). FeatureCounts v1.5.2 was used to generate the read-counting data for each gene. Differential gene expression analysis was performed using TCC (Sun et al., 2013), (Tang et al., 2015). PCA analysis and Heatmap clustering were performed using the princomp, and heatmap packages from R. Gene Ontology (GO) analysis was performed using the clusterProfiler (v4.4.4) package from R (Yu et al., 2012).

Statistical analysis was performed using one-way ANOVA, when significant, group differences were evaluated using a Student’s t-test. p values <0.05 were statistically significant.

To inhibit myoblast differentiation, we examined MβCD, a derivative of parent β-cyclodextrin, as a potential component (Gonzalez Pereira et al., 2021), (Mortensen et al., 2016). MβCD was selected because it has been shown to be the most effective agent among the various agents used for cholesterol depletion from cells (Gotoh et al., 2014) as well as a food additive. It is a widely accepted fact that when cells are depleted of cholesterol, they lose their ability to fuse myoblasts, and also re-introduction of cholesterol to the culture medium can restore it, as demonstrated in previous studies (Mukai et al., 2009), (Taulet et al., 2009). The addition of MβCD to C2C12 cells cultured under serum-reduced conditions resulted in a significant reduction in the appearance of elongated cells, a characteristic of differentiating myotubes, compared to the control group where elongated cells were evident (Figure 1B, indicated by white arrowheads). To investigate the impact of MβCD on myogenic differentiation, we evaluated the expression of key transcription and protein markers of myogenic differentiation (Figures 1C, D). The results of quantitative PCR analysis and immunofluorescence showed that cells cultured under MβCD-containing conditions had significantly lower expression levels of myotube differentiation markers compared to the control group (Figures 1C, D). Furthermore, the inhibitory effect of MβCD on myotube differentiation was observed in a dose-dependent manner (Supplementary Figure S1). These findings suggest that MβCD can inhibit myotube differentiation in C2C12 cells under serum-reduced conditions.

We conducted a gene profiling analysis using RNA-sequence to evaluate the inhibitory effects of MβCD on differentiation (Figures 2A, B). Our results from the PCA analysis indicated that C2C12 myoblast cells differentiate into myotubes with DH2 medium and PC1 represents the extent of differentiation (Figure 2A). Interestingly, the plots of day 3 in differentiation with MβCD were positioned close to day 1 plots, indicating that MβCD has potent inhibitory effects on differentiation. We used hierarchical clustering heatmap analysis to categorize the group of molecules associated with GO_BP into five clusters based on the differentially expressed genes (DEGs) (Figure 2B). At day 3 differentiation, the expression of molecules associated with cell fusion-related processes genes, lipid metabolism, peptidases, and other proteolytic molecules, and molecules related to extracellular matrix and structure were significantly reduced in MβCD-contained conditions (Cluster 1). Additionally, the expression of muscle differentiation-related genes, such as muscle differentiation markers and molecules related to mitochondrial respiration, was significantly reduced (Cluster 2). The effect of cholesterol depletion of MβCD was evident in the cellular response, with a marked increase in cholesterol biosynthesis-related molecules from day 1 after the addition of MβCD (d1_MβCD and d3_MβCD) (Cluster 3). Notably, the high expression of cell cycle- and cell division-related molecules seen before differentiation induction was markedly reduced immediately after differentiation induction (d1_Control & d1_MβCD) and almost absent by day 3 under control conditions, whereas a certain amount of expression could still be seen even on day 3 of differentiation with MβCD (d3_MβCD) (Cluster 4) (Figure 2B).

While C2C12 cells are commonly used as a model system to study skeletal muscle differentiation, they have also been shown to have multipotential capabilities such as osteogenesis and adipogenesis by specific stimuli (Katagiri et al., 1994), (Teboul et al., 1995), leading to a possibility that they differentiate into those cell types other than myogenesis in response to MβCD-treatment. However, no changes in gene expression calculated from RNA-seq data were detected that would support that possibility (Supplementary Figure S3).

Through PCA analysis, it was observed that there was no apparent difference between the control and MβCD-treated cells on day 1 differentiation, but a significant difference was observed on day 3. The control cells exhibited advanced muscle differentiation, while the MβCD-treated cells remained at a stage similar to day 1 differentiation, suggesting that the addition of MβCD inhibits the early stages of differentiation (Figures 2A, B). To determine at what stage MβCD inhibits the differentiation of C2C12 cells, the expression of various muscle differentiation markers was analyzed. It was found that the percentage of MyoD-positive cells, an early marker of muscle differentiation, was significantly increased in the MβCD-contained condition compared to the control on day 3 after differentiation induction. In contrast, the percentage of Myogenin-positive cells, a mid-to-late-stage marker, was significantly decreased. Moreover, transcriptional analysis also revealed that the differentiation progression was arrested at the MyoD-positive stage (Figures 2C, D).

Given the fact that cell fusion between myoblasts accompanies their differentiation process, the effect of MβCD on myoblast cell fusion was investigated. Considering that cell fusion of myoblasts is observed after 1 day of differentiation in C2C12 cells induced by the DH2 condition, it was hypothesized that MβCD might have inhibitory effects on myoblast cell fusion. Myomaker (Mymk), a Myoblast-specific molecule that plays a crucial role in cell fusion between Myoblasts, was studied (Bi et al., 2017). The results of quantitative PCR analysis demonstrated that Mymk expression was mainly downregulated in MβCD-treated conditions (Figure 2E). Correspondingly, MHC-positive cells with a single nucleus were predominant in the MβCD-treated conditions (Figure 2F). These observations indicate that MβCD inhibits the progression of C2C12 cell differentiation at the MyoD-positive stage of muscle differentiation, and part of this inhibitory effect is due to its interference with cell fusion between myoblasts.

To evaluate the effectiveness of MβCD in reducing membrane cholesterol levels in differentiating C2C12 myoblast cells, filipin fluorescence staining was utilized to label cholesterol. We observed reduced cholesterol signals in the membrane of C2C12 myoblast cells cultured in DH2 medium for 44 h with MβCD treatment compared to the control (Figures 3A, B). Furthermore, intense linearized signals of membrane cholesterol were found in cells exhibiting elongated cell morphology, which is a characteristic of myotubes (white arrowheads in Figure 3A). Consistent with gene profiling analysis using RNA-sequence (Figure 2B), we observed significant upregulation of cholesterol biosynthesis-related genes in cells cultured with MβCD for 1 day, which is the primary response of cholesterol-depleted cells to compensate, while no change was observed in the control compared to cells cultured in DF10 as day 0 (Figure 3C). To investigate the possibility that MβCD-induced cellular cholesterol depletion inhibits C2C12 myoblast cell differentiation, we performed rescue experiments by administering cholesterol for myotube differentiation (Figure 3D). The optimized concentration of cholesterol solution for myotube differentiation of C2C12 myoblast cells without cytotoxicity was determined to be 15 µM (Supplementary Figure S4). Immunofluorescence for MHC demonstrated that the inhibitory effect of MβCD on myotube differentiation was significantly compensated by the administration of cholesterol (Figure 3D). Additionally, administration of cholesterol alone was found to increase differentiation efficiency compared to the control. These results suggest that cellular cholesterol in the plasma membrane of C2C12 myoblast cells is essential for differentiation into myotubes and that MβCD-induced reduction of cholesterol levels causes inhibition of myotube differentiation in serum-reduced conditions.

Apoptotic cell death has been reported to play a role in promoting myoblast fusion through contact-dependent signaling with neighboring cells to encourage fusion among healthy differentiating myoblasts (Hochreiter-Hufford et al., 2013). During our observations of myotube differentiation with MβCD, we discovered that cell death of myoblasts, which consistently occurs in serum-reduced medium conditions, was significantly reduced in MβCD-contained conditions (Figure 4A). This led us to hypothesize that the depletion of cellular cholesterol by MβCD could potently block the induction of cholesterol-dependent apoptotic cell death, impair myoblast fusion, and consequently hinder myotube differentiation. We carefully observed the appearance of dead cells (Figure 4A) and validated apoptotic cell death by immunofluorescence for cleaved Caspase-3 (Figure 4B). These results suggest that MβCD effectively blocks apoptotic cell death, and cholesterol significantly restores it. Furthermore, cholesterol alone significantly induced cleaved Caspase-3-positive cells (Figure 4B). Thus, the blockage of apoptotic cell death in C2C12 myoblast cells during differentiation caused by depletion of cholesterol with MβCD is one of the potential mechanisms by which MβCD inhibits myotube differentiation in C2C12 cells since dead cells by apoptosis promote adjacent myoblasts to differentiate into myotubes by inducing myoblast cell fusion (Hochreiter-Hufford et al., 2013).

Using hierarchical clustering heatmap analysis, we observed higher expression of cell cycle-related genes in cells treated with MβCD compared to the control (Figure 2B). Thus, we investigated the effects of MβCD on cell proliferative capacity in serum-reduced conditions by preparing C2C12 myoblast cells at low density (40%–50% confluency) for further cell growth capacity, as high cell density (90%–100% confluency) is typically required for efficient differentiation (Figure 5A). We first determined whether MβCD could have a comparable inhibitory effect on the differentiation of C2C12 myoblast cells at low cell density (Figure 5B) as it does at high cell density (Figure 1). The optimized concentration of MβCD was found to be 0.8 mM without cytotoxicity at low cell density (Supplementary Figure S5). After 3 days of cell growth under serum-reduced conditions, we observed that not only the relative number of cells but also the frequency of cells positive for phosphorylated histone H3, a mitosis marker, significantly increased in MβCD-treated conditions compared to the control (Figures 5C–E). These “mitogenic” effects of MβCD on C2C12 myoblast cells were only observed under serum-reduced conditions of DH2, but not under serum-rich conditions of DF10 (Supplementary Figure S6), which strongly supports our assumption that the mitogenic effect of MβCD on C2C12 myoblast cells is due to its inhibitory effect on myoblast differentiation into myotubes, rather than directly enhancing cell proliferation of myoblasts.

To enable the industrialization of cultured meat production, it is important to investigate the potential of MβCD in livestock animals through ex vivo analysis. Previous studies have shown that short-term treatment with MβCD for 30 min in primary chick myogenic cells promotes myoblast proliferation (Portilho et al., 2012), but its long-term effects on proliferation and differentiation inhibition have not been established. In our experiments, primary cultures of chick myogenic cells were used to assess MβCD competence, as seen in the C2C12 myoblast cells mentioned above. The chick thigh muscle cells were obtained from 10-day-old chick embryos and plated. On day 1 under serum-reduced conditions, we evaluated the effects of MβCD on myotube differentiation and cell growth. The addition of MβCD resulted in the blocked maturation of MHC-positive myotubes with a narrower width and a smaller number of nuclei per cell (Figure 6B). However, MβCD led to an increase in the number of cells under serum-reduced conditions (Figure 6C), due to its ability to enhance cell proliferative capacity, as evidenced by the increased number of p-Histone H3-positive cells (Figure 6D).